HK1210499B - Culture medium composition, and method for culturing cell or tissue using said composition - Google Patents

Description

Culture medium composition and method for culturing cells or tissues using the same

Technical Field

The present invention relates to a culture medium composition containing a structure capable of suspending cells or tissues, and a method for culturing cells or tissues using the culture medium composition. The culture medium composition and the cell culture method using the culture medium composition of the present invention can be preferably used to culture animal or plant cells and/or tissues, particularly in suspension.

Background Art

In recent years, the technology of various organs, tissues and cells that play different roles in animals and plants has been developed for in vitro proliferation or maintenance. In vitro proliferation or maintenance of organs and tissues are respectively referred to as organ culture and tissue culture, and in vitro proliferation, differentiation or maintenance of cells separated from organs or tissues are referred to as cell culture. Cell culture is the technology of in vitro proliferation, differentiation or maintenance of separated cells in culture medium, and is necessary for detailed analysis of the in vivo function and structure of various organs, tissues and cells. In addition, cells and/or tissues cultivated by this technology are used in following multiple fields: effectiveness and toxicity evaluation of chemical substances, medicines, etc., large-scale production of useful substances such as enzymes, cell growth factors, antibodies, etc., regenerative medicine supplements organs, tissues and cells lost due to disease and deficiency, improves plant brands, produces genetically recombinant products, etc.

Animal-derived cells are generally divided into non-adherent cells and adherent cells based on their characteristics. Non-adherent cells are cells that do not require a scaffold for growth and proliferation, while adherent cells are cells that require a scaffold for growth and proliferation. Most of the cells that constitute a living body are the latter, i.e., adherent cells. As the culture method of adherent cells, monolayer culture, dispersed culture, embedded culture (embedded culture), microcarrier culture, ball culture (sphere culture) etc. are known.

Monolayer culture is a method of culturing target cells as a monolayer using culture vessels made of glass or synthetic polymer materials that have undergone various surface treatments, or support cells called feeder cells, as a scaffold. This method is generally the most common. For example, culture methods have been developed that use culture vessels of various shapes or properties, such as polystyrene, that have been subjected to various surface treatments (plasma treatment, corona treatment, etc.), coated with cell adhesion factors such as collagen, fibronectin, and polylysine, or pre-plated with feeder cells. However, monolayer culture has the following challenges: cells cannot maintain their specific functions in the body for long periods of time. This is because the two-dimensional culture environment is completely different from the in vivo environment, cells cannot reconstruct tissues similar to in vivo tissues, and the number of cells per constant area is limited, making it unsuitable for large-scale cell culture (Patent Document 1). Furthermore, methods of culturing target cells on feeder cells sometimes face the problem of separating the target cells from the feeder cells (Non-Patent Document 1).

Dispersion culture is a method for culturing adherent cells in suspension. It involves seeding cells in a culture medium and shaking the medium in a culture vessel with a surface treatment that inhibits cell adhesion to inhibit cell adhesion. However, adherent cells cultured using this method are unable to adhere to a scaffold, and therefore, the method cannot be applied to cells that fundamentally require adhesion to a scaffold for cell proliferation. Furthermore, cells are continuously disrupted by shear forces, preventing them from demonstrating their adherent cell function, and as a result, functional cells are sometimes unable to be cultured in large quantities (Non-Patent Document 2).

Embedded culture is a method of culturing cells by embedding and fixing them in a solid or semisolid gel matrix such as agar, methylcellulose, collagen, gelatin, fibrin, agarose, or alginate. Because this method allows cells to be cultured three-dimensionally in a state closer to that of the in vivo environment, and the gel matrix itself sometimes promotes cell proliferation and differentiation, cells can be cultured at high densities while maintaining cellular function, compared to monolayer and dispersed cultures (Patent Documents 2 and 3). Furthermore, methods for culturing cells have been developed, including forming microcapsules ranging in size from 100 to 300 μm by embedding cells in a gel matrix, and culturing cells in an aqueous culture medium while dispersing the microcapsules (Non-Patent Document 3). However, these methods have the following problems: continuous observation of cultured cells is impossible unless visible light penetrates the gel matrix; recovering cells from the culture medium requires complex, cell-damaging procedures such as enzyme treatment (for example, collagenase treatment in the case of collagen gels); and, due to the high viscosity of the culture medium and the gel matrix containing the microcapsules, medium replacement, necessary for long-term culture, is difficult. In recent years, technologies have been developed to recover cells from gel substrates by treating them with heat, shear forces, and other factors. However, heat, shear forces, and other factors may adversely affect cell function, and the safety of gel substrates for living organisms has not yet been elucidated (Patent Documents 4 and 5, Non-Patent Documents 4, 5, 6, and 7). Furthermore, in the food industry, sol foods have been developed to prevent sedimentation and allow the suspension of evenly dispersed and suspended food particles, such as fruits and vegetables, to float. However, sol foods do not allow for the recovery of dispersed food particles, and whether cells and tissues can be cultured in suspension has not been examined (Patent Document 6).

Microcarrier culture is such a method, it is by propagating on the surface of slightly heavier fine particles (hereinafter also referred to as microcarriers) than water with the cells of monolayer to culture cells in a suspended state, and stirs the fine particles in culture vessels such as flasks etc. Usually, the microcarrier used for this method is a spherical particle with a diameter of 100-300 μm, a surface area of 3000-6000 ^cm / g, a specific gravity of 1.03-1.05, and is made of materials such as dextran, gelatin, alginic acid, polystyrene etc. Collagen, gelatin or charged groups such as dimethylaminoethyl etc. can also be provided to the microcarrier surface to facilitate the adhesion of cells. Because this method can significantly increase the culture area, it is therefore applied to a large number of cultures of cells (patent documents 7, 8). However, it is difficult that the target cells are almost evenly attached to all microcarriers, and because the shear force in the stirring process, the damage on the cell etc. can cause problems such as cell dissociation from the microcarrier (non-patent document 8).

Sphere culture is a culture method that includes forming an aggregate (hereinafter also referred to as a sphere) consisting of several dozen to several hundred target cells and culturing the aggregate in a culture medium with static or shaking. Compared with monolayer culture and dispersion culture methods, spheres are known to have high cell density, reconstruct cell-cell interactions and cell structures close to those in the in vivo environment, and can be cultured while maintaining cell function for a longer period of time (non-patent literature 9, 10). However, sphere culture cannot form large spheres because when the size of the sphere is too large, the nutrient supply and waste discharge inside the sphere are difficult. In addition, since the formed spheres need to be cultured in a dispersed state on the bottom of the culture container, the number of spheres per given volume cannot be easily increased, and it is not suitable for mass culture. In addition, as methods for forming spheres, hanging drop culture, culture on a cell non-adhesive surface, culture in micropores, rotation culture, culture using a cell scaffold, and coagulation by centrifugal force, ultrasonic treatment, electric field or magnetic field are all known. However, these methods have problems in that the operation is complicated, the recovery of spheres is difficult, size control and large-scale production are difficult, the effects on cells are unknown, specific proprietary containers and equipment are required, etc. (Patent Document 9).

On the other hand, for plant, cell, protoplast or organ, tissue, plant such as leaf, stem, root, growing point, seed, embryo, pollen etc. without cell wall callus can also be grown by culturing with aseptic state.Use the culture technology for such plant tissue and cell, the brand promotion (brand improvement) of plant and the production of useful substances have become possible.As the method for a large number of plant cells and tissues in a short time, the suspension culture method of plant cells and tissues in liquid culture medium is known (non-patent literature 11).In order to realize its good propagation, supply enough oxygen, maintain uniform mixed state, prevent cell damage etc. is important.Oxygen supply and suspension cells and tissues in culture medium can be carried out by combining ventilation and mechanical stirring or individually ventilating.The former can cause impaired propagation due to stirring the damage to cells and tissues, and the problem of the latter is that even if the shearing of cells and tissues is less, due to the uniform mixed state may be difficult to maintain in high density culture, so cells and tissues form precipitation, which can reduce propagation efficiency etc.

Furthermore, in the research and development of anticancer drugs for cancer treatment, or in the selection of suitable anticancer drugs, the anticancer activity of a drug against cancer cells is evaluated in vitro by culturing cancer cells in a culture medium containing a candidate drug or anticancer drug. However, existing methods for evaluating anticancer activity suffer from issues such as a gap between in vitro evaluation results and actual clinical efficacy. To address this issue, methods have been developed that evaluate activity under cell culture conditions that closely replicate the in vivo environment. For example, methods have been developed that involve embedding cancer cells in a support such as soft agar, collagen gel, or hydrogel to allow for culturing of cancer cells in an environment that inhibits adhesion to the culture vessel, and then evaluating anticancer drugs (Patent Document 10, Non-Patent Documents 12 and 13). Furthermore, methods have been developed that involve culturing cancer cells in a cohesive state (spheroid culture) by coating the surface of the culture vessel with a material that inhibits cell adhesion or applying a special surface treatment to inhibit cell adhesion, and then evaluating anticancer activity (Patent Documents 11 and 12).

However, those cancer cell culture methods have various problems in that the production method of culture vessels and the cell culture operation are complicated, the operation of recovering cells from supports such as collagen and then evaluating anticancer activity is complicated, when the supports are animal-derived components, the supply of supports is sometimes limited due to their high cost, and cell aggregates (spheres) may have an excessively large size, thereby reducing cell survival rate and reproducibility, etc. In addition, when screening for anticancer drugs, convenient cancer cell culture methods that can process a large number of consistent samples and have high reproducibility are desired.

Furthermore, various activities of drug candidates and drugs on hepatocytes have been evaluated by culturing hepatocytes in vitro in culture medium containing the drug candidates or drugs. However, because the functions inherently exhibited by hepatocytes in vivo may be lost during in vitro culturing, existing hepatocyte culture methods have the following problems: accurate evaluation of drug candidates and drugs is impossible, and evaluation of many samples is difficult. To overcome these problems, methods have been developed that perform activity evaluation under cell culture conditions that closely replicate the in vivo environment. For example, methods have been developed that involve culturing hepatocytes on extracellular matrices such as collagen, laminin, and Matrigel (registered trademark) to maintain hepatocyte function (Patent Document 13, Non-Patent Documents 14 and 15). Furthermore, methods have been developed that involve forming hepatocyte aggregates (spheres) through treatments, such as coating the surface of the culture vessel with a material that inhibits cell adhesion, applying special surface treatments, or vibrating the culture vessel to inhibit cell adhesion, thereby maintaining hepatocyte function (Patent Documents 14 and 15, Non-Patent Documents 16 and 17).

However, those hepatocyte culture methods have various problems in that the production method of culture vessels and the cell culture operation are complicated, the operation of recovering cells from supports such as collagen and evaluating hepatocyte function is complicated, when the support is an animal-derived component, the support supply is sometimes limited due to its high cost, and the cell aggregates (spheres) may be too large, thereby reducing cell survival rate and reproducibility. In addition, when screening for drug candidates or pharmaceuticals, a convenient hepatocyte culture method that can process a large number of consistent samples and has high reproducibility is desired.

Reference List

Patent Literature

[Patent Document 1] JP-A-2001-128660

[Patent Document 2] JP-A-S62-171680

[Patent Document 3] JP-A-S63-209581

[Patent Document 4] JP-A-2009-29967

[Patent Document 5] JP-A-2005-60570

[Patent Document 6] JP-A-8-23893

[Patent Document 7] JP-A-2004-236553

[Patent Document 8] WO2010/059775

[Patent Document 9] JP-A-2012-65555

[Patent Document 10] JP-A-2008-11797

[Patent Document 11] JP-A-2008-61609

[Patent Document 12] JP-A-2012-249547

[Patent Document 13] WO2005/028639

[Patent Document 14] WO2010/079602

[Patent Document 15] JP-A-2009-50194

Non-patent literature

[Non-patent document 1] Klimanskaya et al., Lancet 2005, 365:1636-1641

[Non-patent document 2] King et al., Curr Opin Chem Biol. 2007, 11: 394-398

[Non-patent document 3] Murua et al., J. of Controlled Release 2008, 132:76-83

[Non-patent document 4] Mendes, Chemical Society Reviews 2008, 37:2512-2529

[Non-patent document 5] Moon et al., Chemical Society Reviews 2012, 41: 4860-4883

[Non-patent document 6] Pek et al., Nature Nanotechnol. 2008, 3: 671-675

[Non-patent document 7] Liu et al., Soft Matter 2011, 7:5430-5436

[Non-patent document 8] Leung et al., Tissue Engineering 2011, 17:165-172

[Non-patent document 9] Stahl et al., Biochem.Biophys.Res.Comm.2004, 322:684-692

[Non-patent document 10] Lin et al., Biotechnol J. 2008, 3:1172-1184

[Non-patent document 11] Weathers et al., Appl Microbiol Biotechnol 2010, 85:1339-1351

[Non-patent document 12] Takamura et al., Int. J. Cancer 2002, 98:450-455

[Non-patent document 13] Yang et al., Proc. Natl. Acad. Sci. USA 1979, 76: 3401-3405

[Non-patent document 14] Bissell et al., J. Clin. Invest. 1987, 79: 801-812

[Non-patent document 15] LeCluyse et al., Critical Reviews in Toxicology 2012, 42:501-548

[Non-patent document 16] Brophy et al., Hepatology 2009, 49:578-586

[Non-patent document 17] Franziska et al., World J Hepatol 2010, 2:1-7.

SUMMARY OF THE INVENTION

Problems to be solved by the present invention

An object of the present invention is to solve the above-mentioned problems of the prior art and to provide a culture medium composition for culturing animal or plant cells and/or tissues, especially in a three-dimensional or suspended state, and a method for culturing animal or plant cells and/or tissues by using the culture medium composition.

Furthermore, an object of the present invention is to solve the above-mentioned problems of the prior art and to provide a medium composition for culturing cell aggregates (spheres) of cancer cells in a three-dimensional environment and a method for testing cancer cells by using the medium composition.

Alternatively, an object of the present invention is to solve the above-mentioned problems of the prior art and provide a medium composition for culturing cell aggregates (spheres) of hepatocytes in a three-dimensional environment and a method for testing cancer cells by using the medium composition.

Furthermore, an object of the present invention is to provide a medium additive that promotes the proliferation of cancer cells in cultured cancer cells and a medium additive that suppresses the decrease in the number of hepatocytes in cultured hepatocytes.

Solutions to the Problem

The present inventors have conducted extensive research on various compounds and their effects on suspending cells and/or tissues in liquid culture media containing them, and have successfully discovered a structure capable of uniformly dispersing cells and/or tissues in a suspended state while not substantially increasing the viscosity of the liquid culture medium. They have discovered that by using a culture medium composition containing at least this structure, cells and/or tissues can be proliferated, differentiated, or maintained while remaining in a suspended state. Furthermore, they have discovered that cultured cells and/or tissues can be easily recovered from the culture medium composition, which led to the completion of the present invention.

The present inventors have also conducted extensive research on the effects of various compounds and liquid culture media containing them on cancer cell aggregates (spheroids), and have successfully discovered a culture medium composition that prevents spheroid aggregation and provides a uniform dispersion. They have found that using this culture medium composition, spheroids can be cultured with high survival rates and the activity of anticancer drugs against cancer cells can be effectively evaluated with good sensitivity. Furthermore, they have found that the cultured spheroids can be easily recovered from the culture medium composition and evaluated, leading to the completion of the present invention.

Furthermore, the present inventors have extensively studied the effects of various compounds and liquid culture media containing them on hepatocyte aggregates (spheroids), and have successfully discovered a culture medium composition that prevents spheroid aggregation and provides a uniform dispersion. They have found that using this culture medium composition, spheroids can be cultured with high survival rates while maintaining hepatocyte function, and the effects of drug candidates or drugs on hepatocytes can be efficiently evaluated with good sensitivity. Furthermore, they have found that the cultured spheroids can be easily recovered from the culture medium composition and evaluated, leading to the completion of the present invention.

Furthermore, the present inventors have found that the proliferation of cancer cells can be significantly promoted by adding deacylated gellan gum or a salt thereof to a culture medium containing cancer cells, which led to the completion of the present invention.

Furthermore, the present inventors have found that a decrease in the number of hepatocytes can be suppressed by adding deacylated gellan gum or a salt thereof to a culture medium containing hepatocytes, which led to the completion of the present invention.

Therefore, the present invention provides the following:

(1) A culture medium composition comprising a structure capable of culturing cells or tissues by suspending them.

(2) The culture medium composition of (1), which allows for exchange of the culture medium composition during culture and recovery of cells or tissues after completion of culture.

(3) The culture medium composition of (1), which does not require any temperature change, chemical treatment, enzyme treatment and shear force during the recovery of cells or tissues from the culture medium composition.

(4) The medium composition of (1), which has a viscosity of not more than 8 mPa･s.

(5) The culture medium composition of (1), wherein the above-mentioned structure has a size that allows it to pass through a filter having a pore size of 0.2 μm to 200 μm when it passes through the filter.

(6) The culture medium composition of (1), wherein the above-mentioned structure contains a polymer compound.

(7) The culture medium composition of (6), wherein the above-mentioned polymer compound includes a polymer compound having an anionic functional group.

(8) The culture medium composition of (6), wherein the above-mentioned polymer compound is a polysaccharide.

(9) The culture medium composition of (7), wherein the above-mentioned anionic functional group is at least one selected from carboxyl group, sulfonyl group and phosphate group.

(10) The culture medium composition of (8), wherein the polysaccharide is at least one selected from hyaluronic acid, gellan gum, deacylated gellan gum, rhamsan gum, diutan gum, xanthan gum, carrageenan, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin, heparin sulfate, keratin sulfate, chondroitin sulfate, dermatan sulfate, rhamsan sulfate and its salts.

(11) The culture medium composition of (10), wherein the polysaccharide is at least one selected from hyaluronic acid, deacylated gellan gum, dithia gum, xanthan gum, carrageenan and their salts.

(12) The culture medium composition of (10) or (11), wherein the polysaccharide is deacylated gellan gum or a salt thereof.

(13) The culture medium composition of (12), wherein the final concentration of the above-mentioned deacylated gellan gum or its salt in the culture medium composition is 0.001-1.0% (weight/volume).

(14) The medium composition of (13), further comprising a polysaccharide other than deacylated gellan gum or a salt thereof.

(15) The culture medium composition of (14), wherein the polysaccharide is at least one selected from xanthan gum, alginic acid, carrageenan, dithianth gum and their salts.

(16) The culture medium composition of (14), wherein the polysaccharide is at least one selected from methylcellulose, locust bean gum and salts thereof.

(17) The medium composition according to any one of (1) to (16), further comprising metal ions.

(18) The culture medium composition of (17), wherein the above-mentioned metal ions are divalent metal ions.

(19) The culture medium composition of (18), wherein the metal ion is at least one selected from calcium ion, magnesium ion, zinc ion, ferrous ion and copper ion.

(20) The culture medium composition of (19), wherein the above-mentioned metal ions are calcium ions.

(21) The medium composition of (20), further comprising metal ions other than calcium ions.

(22) The culture medium composition of (21), wherein the metal ion is at least one selected from magnesium ion, sodium ion and potassium ion.

(23) The culture medium composition according to any one of (1) to (22), further comprising an extracellular matrix and/or cell adhesion molecules.

(24) The culture medium composition of (23), wherein the extracellular matrix is at least one selected from collagen, hyaluronic acid and proteoglycan.

(25) The culture medium composition of (23), wherein the cell adhesion molecule is at least one selected from cadherin, laminin, fibronectin and vitronectin.

(26) The medium composition according to any one of (1) to (25), which is used for cell culture.

(27) The culture medium composition of (26), wherein the above-mentioned cells are adherent cells or non-adherent cells.

(28) The culture medium composition of (27), wherein the above-mentioned adherent cells are attached to microcarriers.

(29) The culture medium composition of (27), wherein the above-mentioned adherent cells are embedded in a carrier.

(30) The culture medium composition of (27), wherein the above-mentioned adherent cells are spheres.

(31) The culture medium composition of (27), wherein the above-mentioned adherent cells are selected from pluripotent stem cells, cancer cells and hepatocytes.

(32) A cell or tissue culture comprising the medium composition of any one of (1) to (31) and cells or tissues.

(33) A method for culturing cells or tissues, comprising culturing cells or tissues in the culture medium composition according to any one of (1) to (31).

(34) The culture method of (33), wherein the above-mentioned cells are selected from pluripotent stem cells, cancer cells and hepatocytes.

(35) A method for recovering cells or tissues, comprising isolating the cells or tissues from the culture of (32).

(36) The recovery method of (35), wherein the above separation is carried out by filtration, centrifugation or magnetic separation.

(37) A method for producing spheres, comprising culturing adherent cells in the medium composition of any one of (1) to (31).

(38) A method for screening anticancer drugs, comprising:

(a) a step of culturing cancer cells in the medium composition according to any one of claims 1 to 31 in the presence and absence of a test substance, and

(b) Steps for analyzing changes in cancer cell proliferation.

(39) The method of (38), further comprising the step of selecting a substance that inhibits cancer cell proliferation more effectively than in the absence of the test substance as a candidate substance.

(40) A method for screening candidate drug substances that act on hepatocytes, comprising:

(a) a step of culturing hepatocytes in the medium composition according to any one of claims 1 to 31 in the presence and in the absence of a test substance, and

(b) Steps for analyzing changes in the physiological functions of hepatocytes.

(41) The method of (40), further comprising the step of selecting a substance that inhibits or increases the physiological function of hepatocytes more than in the absence of the test substance as a candidate substance.

(42) A method for evaluating the efficacy or toxicity of a drug candidate substance acting on hepatocytes, comprising:

(a) a step of culturing hepatocytes in the medium composition according to any one of claims 1 to 31 in the presence and in the absence of a test substance, and

(b) Steps for analyzing changes in the physiological functions of hepatocytes.

(43) A culture medium additive for preparing a culture medium composition capable of culturing cells or tissues by suspending them, comprising a polymer compound dissolved or dispersed in a solvent.

(44) (43) culture medium additives, which are in a sterile state.

(45) The culture medium additive of (43) or (44), wherein the above-mentioned polymer compound is a polymer compound having an anionic functional group.

(46) The culture medium additive of (43) or (44), wherein the above-mentioned polymer compound is deacylated gellan gum or a salt thereof.

(47) A method for producing a culture medium composition capable of culturing cells or tissues by suspension, comprising mixing a polymer compound and a culture medium.

(48) The method of (47), which comprises mixing the culture medium additive of any one of (43) to (46) and a culture medium.

(49) The method of (48), wherein the above-mentioned culture medium is dissolved or dispersed in a solvent.

(50) The method of (47), wherein the above-mentioned polymer compound is a polymer compound having an anionic functional group.

(51) The method of (50), wherein the polymer compound is deacylated gellan gum or a salt thereof.

(52) The method of (47), wherein the above-mentioned polymer compound and culture medium are mixed with water.

(53) The method of (52), which comprises heating at 80-130°C after mixing with water.

(54) The method of (53), which comprises heating at 100-125°C.

(55) The method of (47), which includes filtration sterilization.

(56) The method of (55), wherein the filtration sterilization comprises passing through a 0.1-0.5 μm filter.

(57) An additive for a cancer cell culture medium, comprising deacylated gellan gum or a salt thereof, or diterpenoid gum or a salt thereof.

(58) An additive of (57) that promotes the proliferation of cancer cells in cultured cancer cells.

(59) (57) additives for evaluating the anticancer activity of anticancer drugs.

(60) A culture medium composition for cancer cells, comprising the additive according to any one of (57) to (59).

(61) A method for culturing cancer cells, comprising culturing cancer cells in the presence of the additive of any one of (57) to (59) or in the medium composition of (60).

(62) A method for evaluating the activity of an anticancer drug against cancer cells, comprising culturing cancer cells in the presence of the additive of any one of (57) to (59) or in the medium composition of (60).

(63) The method of (61) or (62), wherein the cancer cells form cell aggregates in a culture medium composition for cancer cells.

(64) The method of (61) or (62), wherein the culture container for culturing cancer cells inhibits the attachment of cancer cells.

(65) An additive for a hepatocyte culture medium, comprising deacylated gellan gum or a salt thereof, or diterpenoid gum or a salt thereof.

(66) An additive of (65) that inhibits the decrease in hepatocyte number in cultured hepatocytes.

(67) (65) additives for use in evaluating the effects of drugs and drug candidates on hepatocytes.

(68) A culture medium composition for hepatocytes, comprising the additive according to any one of (65) to (67).

(69) A method for evaluating the activity of a drug or drug candidate on hepatocytes, comprising culturing hepatocytes in the presence of the additive of any one of (65) to (67) or in the medium composition of (68).

(70) The method of (69), wherein the hepatocytes form cell aggregates in a culture medium composition for hepatocytes.

(71) The method of (69), wherein the culture container for culturing hepatocytes inhibits attachment of the hepatocytes.

Effects of the Invention

The present invention provides a culture medium composition containing a specific compound structure (hereinafter referred to as the specific compound), particularly a polymer compound having anionic functional groups. Using this culture medium composition, cells and/or tissues can be cultured in a suspended state without requiring manipulations (such as shaking or rotation) that risk damaging or losing function. Furthermore, using this culture medium composition, the culture medium can be easily exchanged during the culture process, and the cultured cells and/or tissues can be easily recovered. The culture method of the present invention can be applied to cells and/or tissues collected from animals or plants, enabling large-scale production of target cells and/or tissues without impairing their function. Cells and/or tissues obtained using this culture method can be used for evaluating the efficacy and toxicity of chemical substances and pharmaceuticals, for large-scale production of useful substances such as enzymes, cell growth factors, and antibodies, and for regenerative medicine to replenish organs, tissues, and cells lost due to disease and deficiency. In particular, culture medium compositions prepared using deacylated gellan gum are superior and possess the following characteristics: Because the concentration at which these properties are expressed is extremely low (one order of magnitude or less), the effects on culture medium components can be minimized. Since clumps are not easily formed when dissolved in water, large-scale production is easy without trouble. In addition, since the viscosity in the concentration range where the properties are expressed is low, operability such as recovery of cells and/or tissues is very good.

Furthermore, the medium composition of the present invention can inhibit the binding of cancer cell aggregates (spheroids), allowing spheroids to be cultured in a dispersed state, thereby promoting cancer cell proliferation. Furthermore, when using the medium composition to evaluate anticancer drugs, the anticancer drug can be easily added to the culture medium, and detection reagents for evaluating cell proliferation can also be easily added. Furthermore, since cultured cancer cells can be recovered, functional evaluation of the recovered cells can also be easily performed. The present invention can be advantageously utilized when evaluating and screening the efficacy of chemical substances, anticancer drugs, and the like using cancer cells obtained through culture methods.

When cultured in the medium composition of the present invention, cancer cells exhibit increased sensitivity to HB-EGF (heparin-binding epidermal growth factor), a growth factor that promotes carcinogenesis, due to minimal influence from the non-in vivo environment in two-dimensional culture, with only cell-cell adhesion occurring. Furthermore, sensitivity to inhibitors of MEK and Akt, important signaling pathways for scaffold-dependent cancer cell proliferation, is enhanced.

Alternatively, using the culture medium composition of the present invention, the binding of hepatocyte aggregates (spheres) can be inhibited, and the spheres can be cultured in a dispersed state. Consequently, hepatocyte survival and cell function are maintained in vitro. Furthermore, when using the culture medium composition for evaluating drug candidates or pharmaceuticals, the drug candidates or pharmaceuticals can be easily added to the culture medium, and detection reagents for evaluating cell function can also be easily added. Since cultured hepatocytes can be recovered, functional evaluation of the recovered cells can be easily performed. The present invention can be advantageously utilized when evaluating and screening the efficacy and toxicity of chemical substances, anticancer drugs, and the like using hepatocytes obtained through culture methods.

Furthermore, when cancer cells are cultured, the culture medium additive containing deacylated gellan gum or a salt thereof of the present invention can significantly promote the proliferation of cancer cells.

Furthermore, when culturing hepatocytes, the culture medium additive containing deacylated gellan gum or a salt thereof of the present invention can suppress a decrease in the number of hepatocytes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing that when spheres of HepG2 cells are cultured in the medium composition of the present invention, the spheres are uniformly dispersed and can be cultured in a suspended state.

FIG. 2 is a graph showing that when spheres of HeLa cells were cultured in the medium composition of the present invention, the spheres were uniformly dispersed and could be cultured in a suspended state.

FIG. 3 is a graph showing that when spheres of HeLa cells were cultured in the medium composition of the present invention and observed with a microscope, the binding of the spheres could be suppressed compared to the existing medium.

FIG. 4 is a graph showing that when pluripotent stem cells were cultured in the medium composition of the present invention, no toxicity to the cells was observed.

FIG. 5 is a graph showing that when spheres of pluripotent stem cells are cultured in the medium composition of the present invention, the spheres are uniformly dispersed and in a suspended state.

FIG. 6 is a graph showing that when spheres of pluripotent stem cells are cultured in the medium composition of the present invention, pluripotent stem cells proliferate efficiently.

FIG. 7 is a graph showing that pluripotent stem cells cultured in the medium composition of the present invention remain undifferentiated.

FIG. 8 is a graph showing that pluripotent stem cells maintain a normal karyotype after suspension static culture in the medium composition of the present invention.

FIG. 9 is a graph showing that pluripotent stem cells cultured in the medium composition of the present invention remain undifferentiated.

FIG. 10 is a graph showing that when microcarriers to which HepG2 cells are attached are cultured in the medium composition of the present invention, the HepG2 cells are able to proliferate on the microcarriers.

FIG. 11 is a graph showing that when spheres of HeLa cells were added to the medium composition of the present invention, the spheres were uniformly dispersed and in a suspended state.

FIG. 12 is a graph showing that spheres of HeLa cells can be formed in the medium composition of the present invention.

FIG. 13 is a diagram showing a film which is one embodiment of the structure of the present invention, wherein the concentration of deacylated gellan gum in the medium composition is 0.02% (weight/volume).

FIG. 14 is a graph showing that spheres of HepG2 cells can be formed in the medium composition of the present invention.

FIG. 15 is a diagram showing the suspended state of laminin-coated GEMs to which HepG2 cells are attached when they are cultured in the medium composition of the present invention.

FIG. 16 is a graph showing the suspended state of HepG2 cells embedded in alginate beads when they are cultured in the medium composition of the present invention.

FIG. 17 is a graph showing the suspended state of HepG2 cells embedded in collagen gel capsules when they are cultured in the medium composition of the present invention.

FIG. 18 is a diagram showing the suspended state of rice-derived callus when cultured in the medium composition of the present invention.

FIG. 19 is a graph showing that when spheres of HeLa cells were cultured in the medium composition of the present invention, the spheres were uniformly dispersed and could be cultured in a suspended state.

FIG. 20 is a graph showing that when spheres of A549 cells and HCT116 cells were cultured in the medium composition of the present invention, the spheres were uniformly dispersed and could be cultured in a suspended state.

FIG. 21 is a graph showing that when human primary hepatocytes were cultured in the medium composition of the present invention, spheres were formed and could be cultured.

FIG. 22 is a graph showing that when cynomolgus monkey primary hepatocytes were cultured in the medium composition of the present invention, spheres were formed and could be cultured.

FIG. 23 is a graph showing MCF-7 cell aggregates after culturing MCF-7 cells in the medium composition of the present invention for 5 days.

FIG. 24 is a graph showing aggregates of A375 cells and MNNG/HOS cells after culturing in the medium composition of the present invention for 4 days.

FIG. 25 is a graph showing aggregates after culturing MIAPaCa-2 cells in the medium composition of the present invention for 6 days.

Implementation Plan Description

The present invention is described in more detail below.

The terms used in this specification are defined as follows.

The cell in the present invention is the most basic unit constituting animals and plants, and as its elements, it has cytoplasm within a cell membrane and various organelles. In this case, the cell nucleus, which encapsulates DNA, may or may not be contained within the cell. For example, the animal-derived cells of the present invention include germ cells such as sperm and oocytes, somatic cells constituting a living organism, stem cells (such as pluripotent stem cells), progenitor cells, cancer cells isolated from a living organism, cells isolated from a living organism that have acquired immortality and are stably maintained in vitro (cell lines), cells isolated from a living organism that have been artificially genetically modified, and cells isolated from a living organism in which the cell nucleus has been artificially replaced. Examples of somatic cells constituting a living organism include, but are not limited to, fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, erythrocytes, platelets, macrophages, monocytes, osteocytes, bone marrow cells, pericytes, dendritic cells, keratinocytes, adipocytes, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatocytes, chondrocytes, cumulus cells, nervous system cells, glial cells, neurons, oligodendrocytes, microglia, astrocytes, cardiac cells, esophageal cells, muscle cells (e.g., smooth muscle cells or skeletal muscle cells), pancreatic β cells, melanocytes, hematopoietic progenitor cells (e.g., umbilical cord blood-derived CD34-positive cells), monocytes, and the like. Somatic cells include cells collected from any tissue, such as skin, kidney, spleen, adrenal gland, liver, lung, ovary, pancreas, uterus, stomach, colon, small intestine, large intestine, bladder, prostate, testis, thymus, muscle, bone tissue (bond tissue), bones, joints, vascular tissue, blood (including umbilical cord blood), bone marrow, heart, eye, brain, neural tissue, etc. Stem cells are cells that have both the ability to self-replicate and the ability to differentiate into multiple other lineages. Examples include, but are not limited to, embryonic stem cells (ES cells), embryonic tumor cells, embryonic germ stem cells, artificial pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, liver stem cells, pancreatic stem cells, muscle stem cells, germ stem cells, intestinal stem cells, cancer stem cells, hair follicle stem cells, etc. Among the above-mentioned stem cells, examples of pluripotent stem cells include ES cells, embryonic germ stem cells, and iPS cells. Progenitor cells are cells that are in the process of differentiating from the above-mentioned stem cells into specific somatic cells or germ cells. Cancer cells are cells derived from somatic cells and have acquired the ability to proliferate indefinitely. Cell lines are cells that have acquired the ability to proliferate indefinitely through in vitro artificial manipulation.

Examples of cancerous tissue include, but are not limited to, tissue from the following: stomach cancer, esophageal cancer, large intestine cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, bone marrow cancer, renal cell carcinoma, urinary tract cancer, liver cancer, bile duct cancer, cervical cancer, endometrial cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial cancer, craniopharyngioma, laryngeal cancer, tongue cancer, fibrosarcoma, mucosal sarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovial sarcoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, seminoma, Wilms' tumor, glioma, astrocytoma, myeloma, meningioma, melanoma, neuroblastoma, medulloblastoma, retinoblastoma, malignant lymphoma, as well as blood derived from a cancer patient, etc. Examples of cancer cell lines include, but are not limited to, HBC-4, BSY-1, BSY-2, MCF-7, MCF-7/ADR RES, HS578T, MDA-MB-231, MDA-MB-435, MDA-N, BT-549, T47D as human breast cancer cell lines, HeLa cells as human cervical cancer cell lines, A549, EKVX, HOP-62, HOP-92, NCI-H23, NCI-H226, NCI-H322M, NCI-H460, NCI-H522, DMS273, DMS114 as human lung cancer cell lines, Caco-2, COLO-205, HCC-2998, HCT-15, HCT-116, HT-29, KM-12, SW-620, WiDr as human colorectal cancer cell lines, DU-145, PC- 3. LNCaP as human prostate cancer cell line, U251, SF-295, SF-539, SF-268, SNB-75, SNB-78, SNB-19 as human central nervous system cancer cell lines, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, SK-OV-3, IGROV-1 as human ovarian cancer cell lines, RXF-631L, ACHN, UO-31, SN-12C, A498, CAKI-1, RXF-393L, 786-0 and TK-10 as human renal cancer cell lines, MKN45, MKN28, St-4, MKN-1, MKN-7, and MKN-74 as human gastric cancer cell lines, LOX-IMVI, LOX, MALME-3M, SK-MEL-2, SK-MEL-5, SK-MEL-28, UACC-62, UACC-257, and M14 as skin cancer cell lines, CCRF-CRM, K562, MOLT-4, HL-60TB, RPMI8226, SR, UT7/TPO, and Jurkat as leukemia cell lines, A431 as a human epithelioid cancer cell line, A375 as a human melanoma cell line, MNNG/HOS as a human osteosarcoma cell line, and MIAPaCa-2 as a human pancreatic cancer cell line, etc. Examples of cell lines include, but are not limited to, HEK293 (human embryonic kidney cells), MDCK, MDBK, BHK, C-33A, AE-1, 3D9, Ns0/1, NIH3T3, PC12, S2, Sf9, Sf21, High Five (registered trademark), Vero, and the like.

Examples of hepatocytes in the present invention include primary hepatocytes collected from liver tissue, hepatocyte strains established by passage culture under conditions optimized for in vitro culture, and hepatocytes differentiated and induced in vitro from cells derived from tissues other than the liver, pluripotent stem cells such as iPS cells, ES cells, mesenchymal stem cells, stem cells derived from peripheral blood, bone marrow stem cells, adipose stem cells, liver stem cells, liver progenitor cells, etc. Liver tissue is the liver collected from humans, rats, mice, guinea pigs, hamsters, rabbits, pigs, cattle, horses, dogs, cats, monkeys, etc., which can be normal livers or cancerous livers. Although primary hepatocytes can be separated and recovered from such livers by perfusion using collagenase, they can be purchased from reagent companies such as Primarycell, Japan Becton Dickinson and Company, Takara Bio Inc., Hokkaido System Science Co., Ltd., Lonza Japan, Veritas Ltd., Life Technologies Japan Corporation, etc. The purchased hepatocytes can be in a frozen state or attached to a carrier such as collagen. Examples of hepatocyte cell lines include, but are not limited to, HepG2, Hep3B, HepaRG (registered trademark), JHH7, HLF, HLE, PLC/PRF/5, WRL68, HB611, SK-HEP-1, HuH-4, HuH-7, and the like.

Although the function of the hepatocyte in the present invention is not particularly limited, it includes the expression of the activity of cytochrome P450 (also known as CYP) such as CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, etc. and the metabolism of drugs, etc. by these enzymes, the conjugation of drugs by glucuronic acid, glutathione, sulfate, glycine, etc., the production of useful proteins such as albumin, apolipoprotein, thrombopoietin, etc., the secretion of bilirubin, the synthesis of urea, the synthesis of bile acids and fatty acids, the transport of drugs, etc. by transport proteins, and the like. In an embodiment of the present invention, hepatocytes preferably maintain the activity of cytochrome P450, albumin production, and/or transport of drugs and the like through transport proteins (e.g., uptake of carboxydichlorofluoresceindiacetate, tetraethylammonium bromide, taurocholate, rosvastatin, and excretion of carboxydichlorofluorescein) among the above functions.

The term "drug" in the present invention includes any substance used for medical purposes. Drug candidates are substances that have been searched for, developed, or studied as drug candidates, and include synthetic compounds, proteins, nucleic acids, carbohydrates, and naturally occurring substances.

The anticancer drugs of the present invention include drugs that act directly on cancer cells and inhibit the proliferation and function of cancer cells, as well as drugs that do not act directly on cancer cells but inhibit the proliferation or function of cancer cells, or kill cancer cells by synergistically acting with immune cells or other drugs in the body. Examples of anticancer drugs include, but are not limited to, alkylating agents, platinum derivatives, metabolic antagonists represented by anticancer drugs such as 5-FU, topoisomerase inhibitors, microtubule inhibitors, anticancer antibiotics represented by epirubicin, molecular targeted drugs represented by gefitinib, trastuzumab, cetuximab, erlotinib, panitumumab, lapatinib, sirolimus, everolimus, ipilimumab, vandetanib, crizotinib, ruxolitinib, and trametinib, and the like. Examples of target molecules for molecularly targeted drugs include, but are not limited to, various kinases, HER2, EGFR (epidermal growth factor receptor), PI3K (phosphatidylinositol 3-kinase), mTOR (mammalian target of rapamycin), Akt, CDK (cyclin-dependent kinase), VEGFR (vascular endothelial cell proliferation factor receptor), PDGFR (platelet-derived growth factor receptor), FGFR (fibroblast growth factor receptor), c-Met, Raf, p38 MAPK, CTLA-4, ALK, JAK, MEK (MAPK/ERK kinase), Hsp90, histone deacetylase, etc. In addition, candidate synthetic compounds, proteins, nucleic acids, carbohydrates, and natural products for drugs with such effects are also included in the anticancer drugs of the present invention.

The plant-derived cells in the present invention also include cells isolated from various tissues of a plant body and protoplasts obtained by artificially removing cell walls from cells.

The tissue in the present invention is a structural unit in which cells having various characteristics and functions are assembled in a certain manner, and examples of animal tissues include epithelial tissue, bone tissue, muscle tissue, nervous tissue, etc. Examples of plant tissues include meristem, epidermal tissue, assimilated tissue, mesophyll tissue, conductive tissue, mechanical tissue, parenchyma tissue, dedifferentiated cell groups (callus tissue), etc.

When cells and/or tissues are cultured by the method of the present invention, the cells and/or tissues to be cultured can be arbitrarily selected and cultured from the cells and/or tissues described above. Cells and/or tissues can be directly recovered from animals or plants. Cells and/or tissues can be induced, grown or transformed from animals or plants by applying special treatments and then collecting. In this case, the treatment can be in vivo or in vitro. Examples of animals include insects, fish, amphibians, reptiles, birds, crustaceans, hexapods, mammals, etc. Examples of mammals include, but are not limited to, rats, mice, rabbits, guinea pigs, squirrels, hamsters, voles, platypuses, dolphins, whales, dogs, cats, goats, cattle, horses, sheep, pigs, elephants, common marmosets, squirrel monkeys, macaques, chimpanzees and humans. Plants are not specifically limited, as long as the collected cells and/or tissues can be applied to liquid culture. Examples include, but are not limited to, plants (e.g., ginseng, Catharanthus roseus, Hyoscyamus scopolamine, Coptis chinensis, Belladonna etc.) that produce crude drugs (e.g., saponins, alkaloids, berberine, scopolamine, phytosterols, etc.), plants (e.g., blueberry, safflower, madder, saffron etc.) that produce dyes or polysaccharides (e.g., anthocyanins, safflower dyes, madder dyes, saffron dyes, flavonoids, etc.) that are used as starting materials for cosmetics or foods, plants (e.g., blueberry, safflower, madder, saffron etc.) that produce pharmaceutical drugs, plants to be used for feed or as food (rice, corn, wheat or barley etc.), and the like.

In the present invention, the suspension of cells and/or tissues refers to a state in which the cells and/or tissues do not adhere to the culture container (non-adhesive). In addition, in the present invention, when cells and/or tissues proliferate, differentiate, or maintain, the state in which the cells and/or tissues are uniformly dispersed and suspended in the liquid culture medium composition in the absence of pressure from the outside or from shaking, rotating operations, or vibration of the liquid culture medium composition is referred to as "suspension standing", and the cultivation of cells and/or tissues under such conditions is referred to as "suspension standing culture". In "suspension standing", the suspension period includes at least 5-60 minutes, 1 hour to 24 hours, 1 day to 21 days, although this period is not limited thereto as long as the suspension state is maintained.

The culture medium composition of the present invention is a composition comprising a structure capable of culturing cells or tissues in suspension (preferably capable of static suspension culture) and a culture medium.

The culture medium composition of the present invention is preferably a composition that allows for exchange and processing of the culture medium composition during the culture process and for recovering cells or tissues from the culture medium composition after completion of the culture. More preferably, it is a composition that does not require any temperature change, chemical treatment, enzyme treatment, or shearing force during the recovery of cells or tissues from the culture medium composition.

The structures of the present invention are formed from specific compounds and exhibit the function of uniformly suspending cells and/or tissues. More specifically, they include polymer compounds assembled via ions, three-dimensional networks formed by polymer compounds, and the like. Polysaccharides are known to form microgels via metal ions (e.g., JP-A-2004-129596), and the structures of the present invention also include such microgels as an embodiment.

One embodiment of the ionically assembled polymer compound is a thin film structure. Such a thin film is shown in Figure 13 as an example.

The structure of the present invention is preferably sized to pass through a filter having a pore size of 0.2 μm to 200 μm. The lower limit of the pore size is more preferably greater than 1 μm, and in consideration of stable suspension of cells or tissues, it is more preferably greater than 5 μm. The upper limit of the pore size is more preferably less than 100 μm, and in consideration of the size of cells or tissues, it is more preferably less than 70 μm.

The specific compound herein refers to a compound that, when mixed with a liquid culture medium, forms an indeterminate structure that uniformly disperses in the liquid, substantially retains cells and/or tissues without substantially increasing the viscosity of the liquid, and prevents their precipitation. "Without substantially increasing the viscosity of the liquid" means that the viscosity of the liquid does not exceed 8 mPa·s. In this case, the viscosity of the liquid (i.e., the viscosity of the culture medium composition of the present invention) is no more than 8 mPa·s, preferably no more than 4 mPa·s, and more preferably no more than 2 mPa·s. The specific compound is not limited in terms of chemical structure, molecular weight, or other properties, as long as it forms this structure in the liquid culture medium and uniformly suspends (preferably allows for static suspension) cells and/or tissues without substantially increasing the viscosity of the liquid.

The viscosity of the liquid containing this structure can be measured, for example, by the method described in the Examples described below. Specifically, it can be measured at 37°C using an E-type viscometer (manufactured by Toki Sangyo Co., Ltd., TV-22 type viscometer, model: TVE-22L, rotator: standard rotor 1°34'×R24, rotation speed 100 rpm).

Examples of specific compounds to be used in the present invention include, but are not limited to, polymer compounds, preferably polymer compounds having an anionic functional group.

As the anionic functional group, there may be mentioned a carboxyl group, a sulfo group, a phosphoric acid group and salts thereof, preferably a carboxyl group or a salt thereof.

As the polymer compound to be used in the present invention, a polymer compound having one or more selected from the above-mentioned anionic functional groups can be used.

Preferred specific examples of the polymer compound to be used in the present invention include, but are not limited to, polysaccharides in which not less than 10 monosaccharides (e.g., triose, tetroses, pentoses, hexoses, heptoses, etc.) are polymerized, more preferably, acidic polysaccharides having anionic functional groups. The acidic polysaccharide herein is not particularly limited as long as it has anionic functional groups in its structure, and includes, for example, polysaccharides having uronic acid (e.g., glucuronic acid, iduronic acid, galacturonic acid, mannuronic acid), polysaccharides having a sulfate group or a phosphate group in a part of its structure, and polysaccharides having both structures, and includes not only naturally obtained polysaccharides but also polysaccharides produced by microorganisms, polysaccharides produced by genetic engineering, and polysaccharides artificially synthesized using enzymes. More specifically, examples thereof include polymer compounds composed of one or more selected from the group consisting of hyaluronic acid, gellan gum, deacylated gellan gum (hereinafter sometimes referred to as DAG), rhamnosaurus gum, dithia gum, xanthan gum, carrageenan, xanthan gum, ascorbic acid, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin, heparin sulfate, keratin sulfate, chondroitin sulfate, dermatan sulfate, rhamnosaurus sulfate, and salts thereof. The polysaccharide is preferably hyaluronic acid, DAG, dithia gum, xanthan gum, carrageenan, or salts thereof, and most preferably DAG, as it can be used at low concentrations to suspend cells or tissues and allows for easy recovery of cells or tissues.

The salts herein include, for example, alkali metal salts such as lithium, sodium, potassium, salts with alkaline earth metals such as calcium, barium, magnesium, and salts with aluminum, zinc, copper, iron, ammonium, organic bases and amino acids.

The weight average molecular weight of these polymer compounds (polysaccharides, etc.) is preferably 10,000-50,000,000, more preferably 100,000-20,000,000, still more preferably 1,000,000-10,000,000. For example, the molecular weight can be measured by gel permeation chromatography (GPC) based on pullulan.

As described in the Examples mentioned below, phosphorylated DAG can also be used. Phosphorylation can be performed by known methods.

In the present invention, multiple (preferably two) of the above-mentioned polysaccharides can be used in combination. The type of polysaccharide combination is not specifically limited, as long as the above-mentioned structure is formed in the liquid composition and cells and/or tissues can be uniformly suspended (preferably suspended and allowed to stand) without substantially increasing the viscosity of the liquid. Preferably, the combination includes at least DAG or a salt thereof. That is, a preferred combination of polysaccharides contains DAG or a salt thereof, and polysaccharides other than DAG and their salts (e.g., xanthan gum, alginic acid, carrageenan, dimethicone, methylcellulose, locust bean gum or its salts). Examples of specific polysaccharide combinations include, but are not limited to, DAG and rhamnosaurus gum, DAG and dimethicone, DAG and xanthan gum, DAG and carrageenan, DAG and xanthan gum, DAG and locust bean gum, DAG and κ-carrageenan, DAG and sodium alginate, DAG and methylcellulose, etc.

More preferred specific examples of the specific compound to be used in the present invention include hyaluronic acid, deacylated gellan gum, dimethicone, carrageenan, and xanthan gum and salts thereof. Most preferred examples include deacylated gellan gum and salts thereof, because the viscosity of the culture medium composition can be reduced and cells or tissues can be easily recovered.

The deacylated gellan gum of the present invention is a linear polymer polysaccharide containing four sugar molecules (glucose with 1-3 bonds, glucuronic acid with 1-4 bonds, glucose with 1-4 bonds, and rhamnose with 1-4 bonds) as constituent units. It is a polysaccharide represented by the following formula (I), wherein R1 and R2 are each a hydrogen atom, and n is an integer of 2 or greater. R1 may contain a glyceryl group, and R2 may contain an acetyl group. The content of the acetyl and glyceryl groups is preferably no more than 10%, more preferably no more than 1%.

The structures of the present invention can take various forms depending on the specific compound. In the case of deacylated gellan gum, when mixed with a liquid culture medium, it absorbs metal ions (e.g., calcium ions) in the medium, forming an indeterminate structure with the metal ions, and suspending cells and/or tissues. The viscosity of the medium composition of the present invention prepared from deacylated gellan gum is no more than 8 mPa·s, preferably no more than 4 mPa·s, and more preferably no more than 2 mPa·s, making it easier to recover cells or tissues.

The specific compound in the present invention can be obtained by chemical synthesis. When the compound is a naturally occurring substance, it is preferably obtained from various plants, various animals, various microorganisms containing the compound through extraction, separation and purification by conventional techniques. For extraction, water and supercritical gas can be used to effectively extract the compound. For example, as a production method of gellan gum, microorganisms are cultivated and produced in a fermentation medium, and the sticky substance (mucosal substance) produced outside the fungus is recovered by conventional purification methods, and after steps such as drying and grinding, it is powdered. When it is deacylated gellan gum, alkali treatment is applied when the sticky substance is recovered, and the glyceryl and acetyl groups bonded to the 1-3 bonded glucose residues are deacylated and recovered. Examples of purification methods include liquid-liquid extraction, fraction precipitation, crystallization, various ion exchange chromatography, gel filtration chromatography using Sephadex LH-20 or the like, adsorption chromatography using activated carbon, silica gel or the like, adsorption and desorption treatment of active substances by thin layer chromatography, high performance liquid chromatography using reverse phase columns or the like, and impurities can be removed and the compound can be purified by using these methods alone or in combination in any order, or by repeated use. Examples of microorganisms that produce gellan gum include, but are not limited to, Sphingomonas elodea and microorganisms obtained by genetically modifying Sphingomonas elodea.

When it is deacylated gellan gum, commercially available products such as “KELCOGEL (registered trademark of CP Kelco) CG-LA” manufactured by SANSHO Co., Ltd. and “KELCOGEL (registered trademark of CP Kelco)” manufactured by San-Ei Gen F.F.I., Inc. can be used. As a natural type of gellan gum, “KELCOGEL (registered trademark of CP Kelco)” manufactured by San-Ei Gen F.F.I., Inc. can be used.

The concentration of a specific compound in the culture medium depends on the type of specific compound and can be appropriately determined within a range such that the specific compound can form the above-mentioned structure in the liquid culture medium and can evenly suspend (preferably suspend and allow to stand) cells and/or tissues without substantially increasing the viscosity of the liquid. It is generally 0.0005%-1.0% (weight/volume), preferably 0.001%-0.4% (weight/volume), more preferably 0.005%-0.1% (weight/volume), and still more preferably 0.005%-0.05% (weight/volume). For example, in the case of deacylated gellan gum, it is added to the culture medium at 0.001%-1.0% (weight/volume), preferably 0.003%-0.5% (weight/volume), more preferably 0.005%-0.1% (weight/volume), more preferably 0.01%-0.05% (weight/volume), and most preferably 0.01%-0.03% (weight/volume). In the case of xanthan gum, it is added to the culture medium at 0.001%-5.0% (weight/volume), preferably 0.01%-1.0% (weight/volume), more preferably 0.05%-0.5% (weight/volume), and most preferably 0.1%-0.2% (weight/volume). In the case of a mixture of kappa-carrageenan and locust bean gum, it is added to the culture medium at 0.001%-5.0% (weight/volume), preferably 0.005%-1.0% (weight/volume), more preferably 0.01%-0.1% (weight/volume), and most preferably 0.03%-0.05% (weight/volume). In the case of natural type gellan gum, it is added to the culture medium at 0.05%-1.0% (weight/volume), preferably 0.05%-0.1% (weight/volume).

When multiple (preferably two) of the above-mentioned polysaccharides are used in combination, the concentration of the polysaccharides can form the above-mentioned structure in the liquid culture medium and can evenly suspend (preferably suspend and allow to stand) cells and/or tissues without substantially increasing the viscosity of the liquid. For example, when using a combination of DAG or a salt thereof and a polysaccharide other than DAG and a salt thereof, the concentration of DAG or a salt thereof is, for example, 0.005-0.02% (weight/volume), preferably 0.01-0.02% (weight/volume), and the concentration of the polysaccharide other than DAG and a salt thereof is, for example, 0.005-0.4% (weight/volume), preferably 0.1-0.4% (weight/volume). Specific examples of combinations of concentration ranges include the following:

DAG or its salt: 0.005 - 0.02% (preferably 0.01 - 0.02%) (weight/volume)

Polysaccharides other than DAG

Xanthan gum: 0.1 - 0.4% (weight/volume)

Sodium alginate: 0.1 - 0.4% (weight/volume)

Locust bean gum: 0.1 - 0.4% (weight/volume)

Methylcellulose: 0.1 - 0.4% (w/v) (preferably 0.2 - 0.4% (w/v))

Carrageenan: 0.05 - 0.1% (weight/volume)

Dimethicone: 0.05 - 0.1% (weight/volume).

The concentration can be calculated using the following formula.

Concentration (%) = weight of specific compound (g) / volume of medium composition (ml) × 100.

The above-mentioned compounds can also be further converted into different derivatives by chemical synthesis methods, and the derivatives obtained therefrom can also be effectively used in the present invention. Specifically, in the case of deacylated gellan gum, derivatives of the compound represented by formula (I), wherein the hydroxyl groups of R1 and/or R2 are replaced by C1-3 alkoxy groups, C1-3 alkylsulfonyl groups, monosaccharide residues such as glucose, fructose, etc., oligosaccharide residues such as sucrose, lactose, etc., or amino acid residues such as glycine, arginine, etc., can also be used in the present invention. In addition, the compound can also be cross-linked using a cross-linking agent such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

The specific compound or its salt to be used in the present invention can exist in any crystalline form, and this depends on production conditions, and can exist as any hydrate.Such crystalline forms, hydrates and mixtures thereof are also included in the scope of the present invention.In addition, they can exist as solvates containing organic solvents such as acetone, ethanol, tetrahydrofuran etc.Such forms are all included in the scope of the present invention.

The specific compounds to be used in the present invention may exist as tautomers, geometric isomers or tautomers, or mixtures of geometric isomers, or mixtures thereof, formed by endo- or exo-cyclic isomerization. When a compound of the present invention has an asymmetric center, whether or not the compound is formed by isomerization, it may exist as a resolved optical isomer or as a mixture containing the same in any proportion.

The culture medium composition of the present invention may contain metal ions, such as divalent metal ions (calcium ions, magnesium ions, zinc ions, ferrous ions, copper ions, etc.), preferably calcium ions. Two or more metal ions may be used in combination, for example, calcium ions and magnesium ions, calcium ions and zinc ions, calcium ions and ferrous ions, and calcium ions and copper ions. Those skilled in the art can determine the appropriate combination. In one embodiment, the culture medium composition contains metal ions, causing the polymer compound to aggregate and form a three-dimensional network (for example, a polysaccharide to form a microgel via the metal ions), thereby forming the structure of the present invention. The concentration of the metal ions can be appropriately determined within a range such that the specific compound can form the aforementioned structure in the liquid culture medium and can uniformly suspend (preferably, allow the suspension to remain stationary) cells and/or tissues without substantially increasing the viscosity of the liquid culture medium. The salt concentration is, but is not limited to, 0.1 mM to 300 mM, preferably 0.5 mM to 100 mM. The metal ions can be mixed with the culture medium, or a separate salt solution can be prepared and added to the culture medium. The culture medium composition of the present invention may contain the following: extracellular matrix, adhesion molecules, etc.

The present invention also includes a culture method for proliferating cells or tissues by using the medium composition, a method for recovering the obtained cells or tissues by, for example, filtration, centrifugation, or magnetic separation, and a method for producing spheres by using the medium composition.

When cells and/or tissues are cultured in vitro, the structure composed of the specific compound to be used in the present invention exhibits the effect of suspending the cells and/or tissues in a liquid containing the structure of the specific compound (preferably the effect of suspending and allowing the suspension to stand). Through the suspension effect, when compared to monolayer culture, a greater amount of cells and/or tissues per given volume can be cultured. When conventional floating culture methods are accompanied by rotation or shaking operations, the proliferation rate and recovery rate of cells and/or tissues can become lower, or the function of the cells may be impaired due to the shear forces acting on the cells and/or tissues. Using the culture medium composition of the present invention, which contains the structure of the specific compound, cells and/or tissues can be evenly dispersed without the need for operations such as shaking, and the target cells and/or tissues can be easily obtained in large quantities without losing cell function. In addition, when cells and/or tissues are suspended and cultured in conventional culture medium containing a gel matrix, observation and recovery of cells and/or tissues are sometimes difficult, and their functions are sometimes impaired during the recovery process. However, using the culture medium composition containing the structure of the specific compound of the present invention, cells and/or tissues can be suspended and cultured, observed, without damaging their functions, and can be recovered. In addition, conventional culture media containing gel base materials sometimes exhibit high viscosity, making it difficult to replace the culture medium. However, since the culture medium composition containing the structure of the specific compound of the present invention has low viscosity, it can be easily replaced with a pipette, pump, or the like.

The human-derived cells and/or tissues cultured by the methods of the present invention can be transplanted for therapeutic purposes in patients suffering from a disease or condition. In this case, the target disease, the type of condition, the pretreatment method, and the cell transplantation method are appropriately selected by one of ordinary skill in the art. The engraftment of the transplanted cells in the recipient, the recovery from the disease or condition, the presence or absence of transplant-related side effects, and the therapeutic effect are appropriately examined and determined using conventional methods for transplantation therapy.

Furthermore, because cells and/or tissues effectively proliferate using the methods of the present invention, the culture medium compositions containing the specific compounds and structures of the present invention can be used as reagents for cell research. For example, when elucidating factors that control the differentiation and proliferation of cells and tissues, cells are co-cultured with the target factor and analyzed for changes in cell number and type, cell surface differentiation markers, and expressed genes. In this case, using the culture medium compositions of the present invention can effectively expand the number of target cells analyzed and efficiently recover them. When elucidating the target factor, those skilled in the art can appropriately select culture conditions, culture equipment, culture medium type, type of compound of the present invention, specific compound content, type of additives, additive content, culture period, culture temperature, and the like within the ranges described herein. Cells that proliferate or emerge during culture can be observed using a standard microscope in the art. In this case, the cultured cells can be stained with specific antibodies. Genes whose expression is altered by the target factor can be detected by extracting DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) from the cultured cells and analyzing them using Southern blotting, Northern blotting, RT-PCR, and the like. In addition, cell surface differentiation markers are detected by ELISA and flow cytometry using specific antibodies, and the effects of target factors on differentiation and proliferation can be observed.

When cells and/or tissues are cultured by the culture method of the present invention, culture tools commonly used for cell culture, such as culture dishes, flasks, plastic bags, Teflon (registered trademark) bags, dishes, schale, tissue culture dishes, multidishes, microplates, microwell plates, multiplates, multiwell plates, cell culture slides, cell culture bottles, spinner flasks, tubes, trays, culture bags, roller bottles, etc., can be used for culture. While the materials of these culture media are not particularly limited, examples include glass, plastics such as polyvinyl chloride, cellulose polymers such as ethyl cellulose and acetyl cellulose, polystyrene, polymethacrylate, polycarbonate, polysulfone, polyurethane, polyester, polyamide, polystyrene, polypropylene, polyethylene, polybutadiene, poly(ethylene-vinyl acetate) copolymers, poly(butadiene-styrene) copolymers, poly(butadiene-acrylonitrile) copolymers, poly(ethylene-ethyl acrylate) copolymers, poly(ethylene-methacrylate) copolymers, polychloroprene, styrene resins, chlorosulfonated polyethylene, ethylene vinyl acetate, and acrylic block copolymers. These plastics are not only excellent in permeability to oxygen, carbon dioxide, and the like, but also in industrial molding processability, can withstand various sterilization treatments, and are preferably transparent materials to allow observation of the interior of the culture media. The sterilization method used is not particularly limited, and examples include radiation sterilization, ethylene oxide gas sterilization, and autoclave sterilization. In addition, these plastics can be subjected to a variety of surface treatments (e.g., plasma treatment, corona treatment, etc.). In addition, these culture tools can be pre-coated with extracellular matrix, cell adhesion molecules, etc. Examples of coating materials include collagen types I to XIX, gelatin, fibronectin, vitronectin, laminins-1 to 12, nitogen, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, various elastins, various proteoglycans, various cadherins, desmocolin, desmoglein, various integrins, E-selectin, P-selectin, L-selectin, immunoglobulins, hyaluronic acid, superfamily, Matrigel, poly-D-lysine, poly-L-lysine, chitin, chitosan, agarose, alginate gel, hydrogel, and cleavage fragments thereof. These coating materials having amino acid sequences artificially altered by genetic recombination techniques can also be used. Coating materials used to inhibit the adhesion of cells and/or tissues to culture tools can also be used. Examples of coating materials include, but are not limited to, silicon, poly(2-hydroxymethyl methacrylate), poly(2-methoxymethylacrylate), poly(2-methacryloyloxyethylphosphorylcholine), poly-N-isopropylacrylamide, mebiol gel (registered trademark), and the like.

Cells and/or tissues can also be cultured by automatically performing cell inoculation, culture medium replacement, cell image acquisition, and the recovery of cultured cells, controlling pH, temperature, oxygen concentration, etc. simultaneously in a closed environment under mechanical control and using a bioreactor and an automatic incubator capable of high-density culture. As methods for using such devices to supply new culture medium and add required substances to cells and/or tissues during culture, batch feeding culture, continuous culture, and perfusion culture are available, and all of these methods can be used for the culture method of the present invention. Culture containers for bioreactors and automatic incubators include open culture containers (e.g., culture containers with lids) that are easy to open-close and have a large contact area with the outside world, and closed culture containers (e.g., cartridge type culture containers) that are difficult to close at the end and have a small contact area with the outside world. Both culture containers can be used for the culture method of the present invention.

When cells and/or tissues are cultured using the specific compounds of the present invention, a culture medium composition can be prepared by mixing a culture medium for culturing cells and/or tissues with the specific compound. Classification of such compositions by culture medium includes natural culture medium, semi-synthetic culture medium, and synthetic culture medium. Classification by form includes semi-solid culture medium, liquid culture medium, powder culture medium (hereinafter sometimes referred to as powder culture medium), and the like. When the cells and/or tissues are derived from animals, any culture medium used for culturing animal cells can be used. Examples of the culture medium include Dulbecco's modified Eagle's medium (DMEM), hamF12 medium (Ham's Nutrient Mixture F12), DMEM/F12 medium, McCoy's 5A medium, Eagle MEM medium (Eagle's Minimum Essential Medium; EMEM), αMEM medium (α-modified Eagle's Minimum Essential Medium; αMEM), MEM medium (Minimal Essential Medium), RPMI1640 medium, Iscove's modified Dulbecco's medium (IMDM), MCDB131 medium, William's medium E, IPL41 medium, Fischer's medium, StemPro34 (manufactured by Invitrogen), X-VIVO 10 (manufactured by Cambrex Corporation), X-VIVO 15 (manufactured by Cambrex Corporation), HPGM (manufactured by Cambrex Corporation), StemSpan H3000 (manufactured by STEMCELL Technologies), StemSpan SFEM (manufactured by STEMCELL Technologies), Technologies), Stemline II (manufactured by Sigma Aldrich), QBSF-60 (manufactured by Qualitybiological), StemPro hESC SFM (manufactured by Invitrogen), Essential8 (registered trademark) medium (manufactured by Gibco), mTeSR1 or 2 medium (manufactured by STEMCELL Technologies), ReproFF or ReproFF2 (manufactured by ReproCELL), PSGro hESC/iPSC medium (manufactured by System Biosciences), NutriStem (registered trademark) medium (manufactured by Biological Industries), CSTI-7 medium (manufactured by Cell Science & Technology Institute, Inc.), MesenPRO RS medium (manufactured by Gibco), MF-Medium (registered trademark) mesenchymal stem cell proliferation medium (manufactured by TOYOBO CO., LTD.), Sf-900II (manufactured by Invitrogen), Opti-Pro (manufactured by Invitrogen), etc.

The culture medium to be used for culturing cancer cells can be the above-mentioned culture medium added with cell adhesion factors, and its examples include Matrigel, collagen gel, gelatin, poly-L-lysine, poly-D-lysine, laminin and fibronectin. Two or more of these cell adhesion factors can be added in combination. In addition, the culture medium to be used for culturing cancer cell spheres can be further mixed with thickeners such as guar gum, tamarind gum, propylene glycol alginate, locust bean gum, gum arabic, tara gum, tamarind gum, methylcellulose, etc.

Examples of the culture medium to be used for culturing hepatocytes include, in addition to the above-mentioned culture media, HepatoZYME-SFM (manufactured by Life Technologies), HCM (registered trademark)-Hepatocyte Medium Bullet Kit (registered trademark, manufactured by Lonza), HBM (registered trademark)-Hepatocyte Basal Medium (manufactured by Lonza), HMM (registered trademark)-Hepatocyte Maintenance Medium (manufactured by Lonza), Modified Lanford's Medium (manufactured by NISSUI PHARMACEUTICAL CO., LTD.), ISOM's Medium, Hepatocyte Proliferation Medium (manufactured by Takara Bio Inc.), Hepatocyte Maintenance Medium (manufactured by Takara Bio Inc.), Hepatocyte Basal Medium (manufactured by Takara Bio Inc.), Active Maintenance Super Medium (manufactured by In Vitro ADMET Laboratories), etc. These culture media may contain cell adhesion factors, and examples thereof include Matrigel, collagen gel, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin. Two or more of these cell adhesion factors can also be added in combination. In addition, the culture medium to be used for culturing cancer cell spheres or hepatocyte spheres can be further mixed with a thickener such as guar gum, tamarind gum, propylene glycol alginate, locust bean gum, gum arabic, tara gum, tamarind gum, methylcellulose, etc.

When the cells and/or tissues are derived from plants, examples of culture media include those obtained by adding auxins and (if necessary) plant growth control substances (plant hormones) such as cytokines at appropriate concentrations to basal media commonly used for culturing plant tissues, such as Murashige Skoog (MS) medium, Linsmaier Skoog (LS) medium, White's medium, Gamborg's B5 medium, niche medium, HeLa medium, Morel's medium, etc., or modified media in which these medium components are modified to optimal concentrations (e.g., to half the concentration of ammonia nitrogen, etc.). If necessary, these media may be further supplemented with casein-degrading enzymes, corn steep liquor, vitamins, etc. Examples of auxins include, but are not limited to, 3-indoleacetic acid (IAA), 3-indolebutyric acid (IBA), 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), etc. For example, auxins can be added to the culture medium at a concentration of about 0.1 to about 10 ppm. Examples of cytokines include, but are not limited to, kinetin, benzyl adenine (BA), zeatin, etc. For example, cytokines can be added to the culture medium at a concentration of about 0.1 to about 10 ppm.

Those skilled in the art can freely add sodium, potassium, calcium, magnesium, phosphorus, chlorine, various amino acids, various vitamins, antibiotics, serum, fatty acids, sugars, etc. to the above-mentioned culture medium according to the purpose. For culturing cells and/or tissues of animal origin, those skilled in the art can also add one or more other chemical components and biogenic substances according to the purpose.

Examples of components to be added to cells and/or tissues for animal origin include fetal bovine serum, human serum, horse serum, insulin, transferrin, lactoferrin, cholesterol, ethanolamine, sodium selenite, monothioglycerol, 2-mercaptoethanol, bovine serum albumin, sodium pyruvate, polyethylene glycol, various vitamins, various amino acids, agar, agarose, collagen, methylcellulose, various cytokines, various hormones, various growth factors, various extracellular matrices, various cell adhesion molecules, and the like. Examples of cytokines to be added to the culture medium include, but are not limited to, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-14 (IL-14), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), interferon-α (IFN-α), interferon-β (IFN-β), interferon-γ (IFN-γ), granulocyte colony-stimulating factor (G-CSF), monocyte colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), flk2/flt3 ligand (FL), leukemia cell inhibitory factor (LIF), oncostatin M (OM), erythropoietin (EPO), thrombopoietin (TPO), etc.

Examples of hormones to be added to the culture medium include, but are not limited to, melatonin, serotonin, thyroxine, triiodothyronine, epinephrine, norepinephrine, dopamine, anti-mullerian hormone, adiponectin, adrenocorticotropic hormone, angiotensinogen and angiotensin, antidiuretic hormone, atrial natriuretic peptide, calcitonin, cholecystokinin, corticotropin-releasing hormone, erythropoietin, follicle-stimulating hormone, gastrin, ghrelin, glucagon, gonadotropin-releasing hormone, growth hormone-releasing hormone, human chorionic gonadotropin, human placental lactogen, growth hormone, inhibin, Insulin, insulin-like growth factor, leptin, luteinizing hormone, melanocyte-stimulating hormone, oxytocin, parathyroid hormone, prolactin, gastrin-releasing peptide, somatostatin, thrombopoietin, thyroid-stimulating hormone, thyrotropin-releasing hormone, cortisol, androstenedione, testosterone, dehydroepiandrosterone, androstenedione, dihydrotestosterone, estradiol, estrone, estriol, progesterone, calcitriol, calcifediol, prostaglandins, leukotrienes, prostaglandins, thromboxanes, prolactin-releasing hormone, lipotropin, brain natriuretic peptide, neuropeptide Y, histamine, endothelin, pancreatic polypeptide, rennet, and enkephalin.

Examples of growth factors to be added to the culture medium include, but are not limited to, transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), macrophage inflammatory protein 1 alpha (MIP-1α), epidermal cell growth factor (EGF), fibroblast growth factor-1, 2, 3, 4, 5, 6, 7, 8 or 9 (FGF-1, 2, 3, 4, 5, 6, 7, 8, 9), nerve growth factor (NGF), hepatocyte growth factor (HGF), leukemia inhibitory factor (LIF), protease ligand I, protease ligand II, platelet-derived growth factor (PDGF), cholinergic vasoactive differentiation factor (CDF), chemokines, Notch ligands (Delta1, etc.), Wnt proteins, angiopoietin-like proteins 2, 3, 5 or 7 (Angpt2, 3, 5, 7), insulin-like growth factor (IGF), insulin-like growth factor binding protein-1 (IGFBP), pleiotrophin, etc.

In addition, these cytokines and growth factors with amino acid sequences artificially altered by genetic recombination techniques can also be added. Examples include IL-6/soluble IL-6 receptor complex, Hyper IL-6 (a fusion protein of IL-6 and soluble IL-6 receptor), and the like.

Examples of various extracellular matrices and various cell adhesion molecules include collagen types I to XIX, fibronectin, vitronectin, laminins-1 to 12, nitogen, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, various elastins, various proteoglycans, various cadherins, desmocolin, desmoglein, various integrins, E-selectin, P-selectin, L-selectin, the immunoglobulin superfamily, Matrigel, poly-D-lysine, poly-L-lysine, chitin, chitosan, agarose, hyaluronic acid, alginate gel, various hydrogels, cleavage fragments thereof, and the like.

Examples of antibiotics to be added to the culture medium include sulfonamides and preparations, penicillin, phenoxyethyl penicillin, methicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, nafcillin, ampicillin, penicillin, amoxicillin, cyclohexylcillin, carbenicillin, ticarcillin, piperacillin, azlocillin, mezlocillin, mecillinam, andinocillin, cephalosporins and their derivatives, oxolinic acid, amlofloxacin, temafloxacin, nalidixic acid, piromidic acid, ciprofloxacin, cinoxacin, norfloxacin, mefloxacin, rosaxacin, ofloxacin, enoxacin, pipemidic acid, sulbactam, clavulanic acid, β-bromopenisillanic acid, β-chloropenisillanic acid, 6-acetylmethylene-penisillanic acid, acid), cefuroxime, sultampicillin, adinoshirin, and sulbactam formaldehyde hudrate ester, tazobactam, aztreonam, sulfazethin, isosulfazethin, norcardicin, m-carboxyphenyl, phenylacetamidophosphonic acid methyl, chlortetracycline, oxytetracycline, tetracycline, demeclocycline, doxycycline, metacycline, and minocycline.

When a specific compound of the present invention is added to the above-mentioned culture medium, when used, the specific compound is dissolved or dispersed in a suitable solvent (this serves as a culture medium additive). Subsequently, the culture medium additive can be added to the culture medium so that the concentration of the specific compound in the culture medium is such that, as detailed above, the cells and/or tissues can be uniformly suspended (preferably suspended and allowed to stand) without substantially increasing the viscosity of the liquid, for example, 0.0005%-1.0% (weight/volume), preferably 0.001%-0.4% (weight/volume), more preferably 0.005%-0.1% (weight/volume), and even more preferably 0.005%-0.05% (weight/volume). For example, in the case of deacylated gellan gum, it is added to the culture medium at 0.001%-1.0% (weight/volume), preferably 0.003%-0.5% (weight/volume), more preferably 0.005%-0.1% (weight/volume), and most preferably 0.01%-0.03% (weight/volume). On the other hand, in the case of deacylated gellan gum, it is added to the culture medium at 0.0005%-1.0% (weight/volume), preferably 0.001%-0.5% (weight/volume), more preferably 0.003%-0.1% (weight/volume), and most preferably 0.005%-0.03% (weight/volume). In the case of xanthan gum, it is added to the culture medium at 0.001%-5.0% (weight/volume), preferably 0.01%-1.0% (weight/volume), more preferably 0.05%-0.5% (weight/volume), and most preferably 0.1%-0.2% (weight/volume). In the case of a mixture of kappa-carrageenan and locust bean gum, it is added to the culture medium at 0.001%-5.0% (weight/volume), preferably 0.005%-1.0% (weight/volume), more preferably 0.01%-0.1%, and most preferably 0.03%-0.05% (weight/volume). In the case of a mixture of deacylated gellan gum and dimethicone, it is added to the culture medium at 0.001%-1.0% (weight/volume), most preferably 0.005%-0.01% (weight/volume). In the case of a mixture of deacylated gellan gum and methylcellulose, it is added to the culture medium at 0.001%-1.0% (weight/volume), most preferably 0.005%-0.2% (weight/volume). In the case of a mixture of deacylated gellan gum and locust bean gum, it is added to the culture medium at 0.001%-1.0% (weight/volume), most preferably 0.01%-0.1% (weight/volume). In the case of a mixture of deacylated gellan gum and sodium alginate, it is added to the culture medium at 0.001%-1.0% (weight/volume), most preferably 0.01%-0.1% (weight/volume). In the case of a mixture of deacylated gellan gum and xanthan gum, it is added to the culture medium at 0.001%-1.0% (weight/volume), most preferably 0.01%-0.1% (weight/volume). A mixture of deacylated gellan gum and kappa-carrageenan is added to the culture medium at 0.001%-1.0% (weight/volume), most preferably 0.01%-0.1% (weight/volume). The concentration can be calculated by the following formula.

Concentration (%) = weight of specific compound (g) / volume of medium composition (ml) × 100.

Examples of suitable solvents for culture medium additives include, but are not limited to, aqueous solvents such as water, dimethyl sulfoxide (DMSO), various alcohols (e.g., methanol, ethanol, butanol, propanol, glycerol, propylene glycol, butylene glycol, etc.), and the like. In this case, the concentration of the specific compound is 0.001%-5.0% (weight/volume), preferably 0.01%-1.0% (weight/volume), and more preferably 0.1%-0.6% (weight/volume). It is also possible to further add additives to enhance the effect of the specific compound or to reduce the concentration when used. Examples of such additives include one or more guar gum, tamarind gum, propylene glycol alginate, locust bean gum, gum arabic, tara gum, tamarind gum, methylcellulose, carboxymethylcellulose, agarose, tamarind seed gum, and polysaccharides such as pullulan. It is also possible to immobilize the specific compound on the surface of the carrier or to carry the specific compound within the carrier during culture. The specific compound can have any shape during supply or storage. The specific compound can be in the form of a solid such as a tablet, pill, capsule, granule, or a liquid such as a solution or suspension obtained by dissolving in a suitable solvent using a solubilizer, or can be bonded to a substrate or a single substance. Examples of additives used for formulation include preservatives such as parabens, excipients such as lactose, glucose, sucrose, mannitol, etc., lubricants such as magnesium stearate, talc, etc., adhesives such as polyvinyl alcohol, hydroxypropyl cellulose, gelatin, etc., surfactants such as fatty acid esters, etc., plasticizers such as glycerol, etc., etc. These additives are not limited to those mentioned above and can be freely selected as long as they are available to those of ordinary skill in the art. The specific compound of the present invention can be sterilized when necessary. There is no specific limitation on the sterilization method, and for example, radiation sterilization, ethylene oxide gas sterilization, high pressure sterilization, filtration sterilization, etc. can be mentioned. When filter sterilization (hereinafter sometimes referred to as filtration sterilization) is performed, the material of the filter portion is not particularly limited, and examples thereof include glass fiber, nylon, PES (polyethersulfone), hydrophilic PVDF (polyvinylidene fluoride), cellulose mixed esters, cellulose acetate, polytetrafluoroethylene, etc. Although the pore size in the filter is not particularly limited, it is preferably 0.1 μm to 10 μm, more preferably 0.1 μm to 1 μm, and most preferably 0.1 μm to 0.5 μm. When the specific compound is in a solid state or a solution state, sterilization treatment can be applied.

The medium composition of the present invention can be obtained by adding a solution or dispersion of the specific compound prepared as above to a liquid medium to form the above structure in the liquid medium. Since the medium generally contains a sufficient concentration of metal ions to ionically assemble the polymer compound or form a three-dimensional network of the polymer compound, the medium composition of the present invention can be obtained by simply adding the solution or dispersion of the specific compound of the present invention to the liquid medium. Alternatively, the medium can be added to a medium additive (a solution or dispersion of the specific compound). In addition, the medium composition of the present invention can also be prepared by mixing the specific compound and the medium components in an aqueous solvent (e.g., water, including ion-exchanged water, ultrapure water, etc.). Examples of the mixing embodiment include, but are not limited to, (1) mixing a liquid medium and a medium additive (solution), (2) mixing a liquid medium and the above polymer compound (solid such as powder, etc.), (3) mixing a medium additive (solution) and a powder medium, (4) mixing a powder medium and the above polymer compound (solid such as powder, etc.) with an aqueous solvent, and so on. In order to prevent the specific compound from being unevenly distributed in the medium composition of the present invention, embodiment (1) or (4), or (1) or (3) is preferred.

When dissolving a specific compound in a solvent (e.g., an aqueous solvent such as water or a liquid culture medium) or dissolving a specific compound and a powdered culture medium in a solvent, the mixture is preferably heated to facilitate dissolution. The heating temperature is, for example, 80°C to 130°C, preferably 100°C to 125°C (e.g., 121°C), at which temperature heat sterilization is performed.

After heating, the resulting solution of the specific compound is cooled to room temperature. The above structure composed of the specific compound can be formed by adding the above metal ions to a solution (e.g., by adding the solution to a culture medium). Alternatively, the above structure composed of the specific compound can also be formed by heating (e.g., 80°C - 130°C, preferably 100°C - 125°C (e.g., 121°C)) the specific compound while dissolved in a solvent containing the above metal ions (e.g., an aqueous solvent such as water and a liquid culture medium), and then cooling the resulting solution to room temperature.

An example of a method for preparing the medium composition of the present invention is shown below, which should not be construed as limiting. A specific compound is added to ion-exchanged water or ultrapure water. The mixture is then heated and stirred at a temperature at which the specific compound can dissolve (e.g., not less than 60°C, not less than 80°C, not less than 90°C) to allow the compound to dissolve until it becomes transparent.

After dissolution, the mixture is allowed to cool with stirring and sterilized (for example, autoclave at 121°C for 20 min). After cooling to room temperature, the sterilized aqueous solution is added to a given culture medium to be used for static culture while stirring (for example, a homogenizer, etc.) to evenly mix the solution and the culture medium. There is no specific limitation on the method for mixing the aqueous solution with the culture medium, and they can be mixed manually, such as by pipetting, or by using instruments such as a magnetic stirrer, a mechanical stirrer, a homogenizer, and a homogenizer. In addition, the culture medium composition of the present invention can be filtered through a filter after mixing. The pore size of the filter to be used for the filtration treatment is 5 μm-100 μm, preferably 5 μm-70 μm, and more preferably 10 μm-70 μm.

Alternatively, the powder culture and the above-mentioned polymer compound (solid such as powder etc.) are mixed with an aqueous solvent, and the mixture is heated at the above-mentioned temperature to prepare the culture composition of the present invention.

For example, when preparing deacylated gellan gum, deacylated gellan gum is added to ion-exchanged water or ultrapure water to 0.1%-1% (weight/volume), preferably 0.2%-0.5% (weight/volume), more preferably 0.3%-0.4% (weight/volume). In addition, on the other hand, when preparing deacylated gellan gum, deacylated gellan gum is added to ion-exchanged water or ultrapure water to 0.1%-1% (weight/volume), preferably 0.2%-0.8% (weight/volume), more preferably 0.3%-0.6% (weight/volume).

Subsequently, the deacylated gellan gum is dissolved by heating and stirring at any temperature (as long as it dissolves) until it becomes transparent. The temperature may be no less than 60°C, preferably no less than 80°C, and more preferably no less than 90°C (e.g., 80-130°C). After dissolution, the mixture is allowed to cool with stirring and then sterilized by autoclaving, for example, at 121°C for 20 minutes. After cooling to room temperature, the aqueous solution is added to, for example, DMEM/F-12 culture medium while stirring using a homomixer or the like to the desired final concentration (e.g., a 0.3% aqueous solution: culture medium ratio of 1:19 for a final concentration of 0.015%), and the mixture is uniformly mixed.

The method for mixing the aqueous solution and the culture medium is not particularly limited, and can be mixed manually, such as by pipetting, or by using an instrument such as a magnetic stirrer, a mechanical stirrer, a homogenizer, and a homogenizer. In addition, the culture medium composition of the present invention can be filtered through a filter after mixing. The pore size of the filter to be used for the filtration process is 5 μm to 100 μm, preferably 5 μm to 70 μm, and more preferably 10 μm to 70 μm.

Furthermore, after preparing the medium composition of the present invention, the structure can be precipitated by centrifugation.

Those skilled in the art can freely select the form and state of cells and/or tissues to be cultured by the method of the present invention. Preferred specific examples include, but are not limited to, a state in which cells and/or tissues are individually dispersed in a culture medium composition, a state in which cells and/or tissues are attached to the surface of a carrier, a state in which cells and/or tissues are embedded in a carrier, a state in which multiple cells gather and form cell aggregates (spheres), or a state in which two or more cells gather and form cell aggregates (spheres), etc. More preferred are states in which cells and/or tissues are attached to the surface of a carrier, a state in which cells and/or tissues are embedded in a carrier, a state in which multiple cells gather and form cell aggregates (spheres), and a state in which two or more cells gather and form cell aggregates (spheres). Further preferred are states in which cells and/or tissues are attached to the surface of a carrier, a state in which multiple cells gather and form cell aggregates (spheres), and a state in which two or more cells gather and form cell aggregates (spheres). Among these states, the state in which cell aggregates (spheres) are formed can be mentioned as the most preferred state to be cultured by the culture method of the present invention, since cell-cell interactions and cell structures close to those in the in vivo environment are reconstructed, long-term culture can be performed while maintaining cell function, and cell recovery is relatively easy.

As carriers that support cells and/or tissues on the surface, there can be mentioned microcarriers and glass beads, ceramic beads, polystyrene beads, dextran beads, etc. composed of various polymers. As examples of polymers, there can be used vinyl resins, polyurethane resins, epoxy resins, polystyrene, polymethyl methacrylate, polyesters, polyamides, polyimides, silicone resins, phenol resins, melamine resins, urea resins, aniline resins, ionomer resins, polycarbonates, collagen, dextran, gelatin, cellulose, alginate, mixtures thereof, etc. The carrier can be coated with a compound that enhances cell adhesion or substance release from cells. Examples of such coating materials include poly(monostearylglycerol succinate), poly-D,L-lactide-co-glycolide, sodium hyaluronate, n-isopropylacrylamide, collagen types I to XIX, fibronectin, vitronectin, laminins 1 to 12, nitogen, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, various elastins, various proteoglycans, various cadherins, desmocolin, desmoglein, various integrins, E-selectin, P-selectin, L-selectin, immunoglobulin superfamily, Matrigel, poly-D-lysine, poly-L-lysine, chitin, chitosan, agarose, alginate gel, various hydrogels, and further, cleavage fragments thereof. Two or more coating materials may be combined. In addition, one or more polysaccharides such as guar gum, tamarind gum, locust bean gum, gum arabic, tara gum, tamarind gum, methylcellulose, carboxymethyl cellulose, agarose, tamarind gum, pullulan (pullulan) etc. can also be mixed with the culture medium of the carrier to be used for culturing on the surface supporting cells and/or tissues.Carrier can also contain magnetic material, for example, ferrite.The diameter of carrier is tens of microns to hundreds of microns, more preferably 100 μm-200 μm, and its specific gravity is preferably close to 1, more preferably 0.9-1.2, especially preferably about 1.0. Examples of carriers include, but are not limited to, Cytodex 1 (registered trademark), Cytodex 3 (registered trademark), Cytoline 1 (registered trademark), Cytoline 2 (registered trademark), Cytopore 1 (registered trademark), Cytopore 2 (registered trademark), (above, GE Healthcare Life Sciences), Biosilon (registered trademark) (NUNC), Cultispher-G (registered trademark), Cultispher-S (registered trademark) (above, Thermo SCIENTIFIC), HILLEXCT (registered trademark), ProNectin F-COATED (registered trademark), and HILLEX II (registered trademark) (Solo Hill Engineering), GEM (registered trademark) (Global Eukaryotic Microcarrier), etc. The carrier can be sterilized when necessary. Sterilization methods are not specifically limited, and for example, radiation sterilization, ethylene oxide gas sterilization, high pressure sterilization, dry heat sterilization, etc. can be mentioned. The method for culturing animal cells using a carrier is not particularly limited, and a culture method using a general flow layer culture vessel or a filling layer culture vessel, etc., can be used. Here, the carrier supporting cells and/or tissues on the surface and the medium composition containing the structure of the specific compound of the present invention allow for uniform dispersion even without shaking, etc. As a result, the target cells and/or tissues can be cultured without loss of cell function.

The cells and/or tissues cultured by this method can be collected by centrifugation and filtration, and the cells and/or tissues are supported by the carrier after culture. In this case, the centrifugation and filtration can be performed after adding the liquid culture medium used. For example, without limitation, the gravitational acceleration (G) of the centrifugation is 50G to 1000G, more preferably 100G to 500G, and the pore size of the filter used for the filtration treatment is 10 μm-100 μm. In addition, the cultured carrier can be recovered by magnetic force by encapsulating a magnetic material such as ferrite in the carrier. The cells and/or tissues cultured by this method can be collected by releasing the carrier using various chelating agents, heat treatment or enzymes.

When cells and/or tissues are embedded in a carrier, materials consisting of various polymers can be selected as carriers. Examples of such polymers include collagen, gelatin, alginate, chitosan, agarose, polyglycolic acid, polylactic acid, fibrin adhesives, polylactic acid-polyglycolic acid copolymers, proteoglycans, glycosaminoglycans, sponges such as polyurethane foam, DSEA-3D (registered trademark), poly-N-substituted acrylamide derivatives, poly-N-substituted methacrylamide derivatives, and copolymers thereof, polyvinyl methyl ether, polypropylene oxide, polyethylene oxide, temperature-sensitive polymers such as partially acetified polyvinyl alcohol, polyacrylamide, polyvinyl alcohol, methylcellulose, cellulose nitrate, cellulose butyrate, polyethylene oxide, and hydrogels such as poly(2-hydroxyethyl methacrylate)/polycaprolactone. In addition, carriers for embedding cells can be prepared using two or more of these polymers. In addition, the carrier can have physiologically active substances in addition to these polymers. As examples of physiologically active substances, cell growth factors, differentiation-inducing factors, cell adhesion factors, antibodies, enzymes, cytokines, hormones, lectins, extracellular matrices and the like can be mentioned, and most of these can also be included. Examples of cell adhesion factors include poly (monostearylglycerol succinate), poly-D,L-lactide-co-glycolide, sodium hyaluronate, n-isopropylacrylamide, collagen types I to XIX, gelatin, fibronectin, vitronectin, laminins 1 to 12, nitogen, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, various elastins, various proteoglycans, various cadherins, desmocolin, desmoglein, various integrins, E-selectin, P-selectin, L-selectin, immunoglobulin superfamily, Matrigel, poly-D-lysine, poly-L-lysine, chitin, chitosan, agarose, alginate gel, various hydrogels, and further, cleavage fragments thereof, etc. In this case, two or more cell adhesion factors may be combined. In addition, one or more thickeners such as guar gum, tamarind gum, propylene glycol alginate, locust bean gum, gum arabic, tara gum, tamarind gum, methylcellulose, carboxymethylcellulose, agarose, tamarind gum, pullulan, etc. can also be mixed with the culture medium for culturing the carrier for embedding cells and/or tissues.

The method for embedding cells and/or tissues in these carriers is not particularly limited, and for example, a method comprising aspirating a mixture of cells and the aforementioned polymer with a syringe and adding it dropwise to a culture medium from an approximately 25G to 19G injection needle, or adding it dropwise to a culture medium using a micropipette, etc. can be used.

The size of the bead-like carrier formed here is determined by the shape of the tip of the tool used to add the mixture of cells and the aforementioned polymer dropwise, and is preferably tens of microns to hundreds of microns, more preferably 100 μm-2000 μm. The number of cells that can be cultured on the bead-like carrier is not specifically limited and can be freely selected according to the size of the beads. For example, five million cells can be embedded in a bead-like carrier with a diameter of about 2000 μm. Cells can be individually dispersed in the carrier or multiple cells can be gathered to form cell aggregates. Here, the carrier in which cells and/or tissues are embedded and the culture medium composition containing the structure of the specific compound of the present invention allow for uniform dispersion, even without operations such as shaking. As a result, the target cells and/or tissues can be cultured without losing cell function. The cells and/or tissues cultured by this method can be collected by centrifugation and filtration, and the cells and/or tissues are embedded in the carrier after cultivation. In this case, the centrifugation and filtration can be performed after adding the liquid culture medium used. For example, without limitation, the gravitational acceleration (G) of the centrifugation is 50 G to 1000 G, more preferably 100 G to 500 G, and the pore size of the filter used for the filtration treatment is 10 μm to 100 μm. By this method, cells and/or tissues cultured in the culture medium can be collected by disintegrating the carriers using various chelating agents, heat, enzymes, etc., thereby dispersing them.

The method for forming cell aggregates (balls) is not specifically limited, and can be suitably selected by those of ordinary skill in the art. Examples thereof include methods using a container with a cell non-adhesive surface, hanging drop method, rotary culture method, three-dimensional scaffold method, centrifugation, methods using coagulation (coagulation) by an electric field or magnetic field, etc. For example, using a method with a container with a cell non-adhesive surface, the target cells are cultured in a culture vessel such as a culture dish (schale) applied with a surface treatment to suppress cell adhesion, thus forming a ball. Using such cell non-adhesive culture vessels, first collect the target cells, prepare their cell suspensions and spread them in a culture vessel for cultivation. When cultivation continues for about 1 week, the cells spontaneously form a ball. As the cell non-adhesive surface used herein, the surface of a commonly used culture vessel such as a culture dish can be used, which is coated with a substance that suppresses cell adhesion, etc. Examples of such substances include agarose, agar, polyHEMA (copolymer of poly(2-hydroxyethyl methacrylate)-2-methacryloyloxyethyl phosphorylcholine and other monomers (e.g., butyl methacrylate, etc.), poly(2-methoxymethyl acrylate), poly-N-isopropylacrylamide, mebiol gel (registered trademark), etc. When cytotoxicity is not present, the substance is not limited thereto.

As a method for forming cell aggregates (spheres), the method described in NATURE BIOTECHNOLOGY, VOL. 28, NO. 4, APRIL 2010, 361-366, NATURE PROTOCOLS, VOL. 6, NO. 5, 2011, 689-700, NATURE PROTOCOLS, VOL. 6, NO. 5, 2011, 572-579, StemCell Research, 7, 2011, 97-111, Stem Cell Rev and Rep, 6, 2010, 248-259 can also be used.

In addition, the culture medium for sphere formation may also contain components that promote sphere formation or maintenance. Examples of components having such effects include dimethyl sulfoxide, superoxide dismutase, ceruloplasmin, catalase, peroxidase, L-ascorbic acid, L-ascorbyl phosphate, tocopherol, flavonoids, uric acid, bilirubin, selenium-containing compounds, transferrin, unsaturated fatty acids, albumin, theophylline, forskolin, glucagon, dibutyryl cAMP, etc. As selenium-containing compounds, ROCK inhibitors such as sodium selenite, sodium selenate, dimethyl selenium, hydrogen selenide, selenomethionine, seleno-methylselenocysteine, alanine butyramine selenoether, selenocysteine, selenohomocysteine, adenosine-5'-triphosphate, seleno-adenosylselenomethionine, Y27632, fasudil (HA1077), H-1152, and Wf-536 can be mentioned. In order to obtain a target cell aggregate of uniform size, a plurality of concave surfaces (concaves) having the same diameter as the target cell aggregate can also be introduced into a cell non-adhesive culture vessel to be used. When these concave surfaces are in contact with each other or within the diameter range of the target cell aggregate, cells are laid on the concave surfaces. The laid cells will not form cell aggregates between the concave surfaces, but will definitely form cell aggregates with a size corresponding to their volume in the concave surfaces, thereby providing a cell aggregate group with uniform size. As the shape of the concave surface in this case, it is preferably hemispherical or conical.

Alternatively, spheres can be formed based on supports that exhibit cell adhesion. Examples of such supports include collagen, polyrotaxanes, polylactic acid (PLA), polylactic-co-glycolic acid (PLGA) copolymers, hydrogels, and the like.

Spheres can also be formed by co-culturing with feeder cells. Any adhesive cells can be used as feeder cells to promote sphere formation. Preferably, feeder cells for various cells are desirable. While not limiting, for example, when forming spheres with cells derived from liver or cartilage, COS-1 cells and vascular endothelial cells are preferred examples of feeder cells.

Spheres can also be formed using a culture composition containing a structure of a specific compound of the present invention. In this case, the specific compound can be added to the culture medium for sphere formation so that the concentration of the specific compound, as detailed above, is such that cells and/or tissues can be uniformly suspended (preferably suspended and allowed to stand) without substantially increasing the viscosity of the liquid, for example, 0.0005%-1.0% (weight/volume), preferably 0.001%-0.3% (weight/volume), more preferably 0.005%-0.1% (weight/volume), and even more preferably 0.01%-0.05% (weight/volume). On the other hand, the specific compound can be added to the culture medium for sphere formation so that the concentration of the specific compound is as described in detail above, at which cells and/or tissues can be uniformly suspended (preferably suspended and allowed to stand) without substantially increasing the viscosity of the liquid, for example, 0.0005%-1.0% (weight/volume), preferably 0.001%-0.3% (weight/volume), more preferably 0.003%-0.1% (weight/volume), and further preferably 0.005%-0.05% (weight/volume).

The spheres are prepared by uniformly dispersing the target cells in a culture medium containing a structure of a specific compound and allowing them to be cultured by standing for 3 to 10 days. The prepared spheres can be recovered by centrifugation and filtration. For example, without limitation, the gravitational acceleration (G) of the centrifugation is 50G to 1000G, more preferably 100G to 500G, and the pore size of the filter used for the filtration is 10 μm-100 μm. In addition, using magnetic microparticles coated with antibodies that specifically bind to the target cells on the surface, the cultured spheres can be recovered by magnetic force. Examples of such magnetic microparticles include Dynabeads (manufactured by Veritas Ltd.), MACS microbeads (manufactured by Miltenyi Biotec), BioMag (manufactured by TechnoChemicals Corporation), etc.

The size of the sphere varies depending on the cell type and culture period and is not particularly limited. When it has a spherical or ellipsoidal shape, its diameter is 20 μm to 1000 μm, preferably 40 μm to 500 μm, more preferably 50 μm to 300 μm, and most preferably 80 μm to 200 μm.

By continuing static culture, such spheres can maintain their proliferation capacity for at least 10 days, preferably at least 13 days, and more preferably at least 30 days. During static culture, by regularly performing mechanical division, or single cell processing and aggregation, the proliferation capacity can be maintained virtually indefinitely.

There is no particular limitation on the culture container to be used for culturing spheres, as long as it generally allows animal cell culture. For example, flasks, dishes, culture dishes (schale), tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, cell culture slides, cell culture bottles, spinner flasks (spinner flasks), culture dishes, tubes, trays, culture bags, roller bottles, EZ SPHERE (manufactured by ASAHI GLASS CO., LTD.), Sumilon cell tight plates (manufactured by SUMITOMOBAKELITE), etc. can be mentioned.

When evaluating a large number of anticancer drugs, drug candidate compounds, or pharmaceutical products, microplates, microwell plates, multiplates, and multi-well plates are preferably used among these culture containers. Although the shape of the well bottom of these plates is not particularly limited, flat bottoms, U-shaped bottoms, and V-shaped bottoms can be used, and U-shaped bottoms are preferably used. Although the material of these culture tools is not particularly limited, for example, glass, plastics such as polyvinyl chloride, cellulose polymers, polystyrene, polymethacrylate, polycarbonate, polysulfone, polyurethane, polyester, polyamide, polystyrene, polypropylene, etc. can be mentioned.

The culture medium to be used for static culture of the spheres may contain cell adhesion factors, examples of which include Matrigel, collagen gel, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin. Two or more of these cell adhesion factors may also be added in combination. In addition, the culture medium to be used for culturing the spheres may be mixed with thickeners such as guar gum, tamarind gum, propylene glycol alginate, locust bean gum, gum arabic, tara gum, tamarind gum, methylcellulose, carboxymethylcellulose, agarose, tamarind gum, pullulan, and the like.

Use of a medium composition comprising the structure of the specific compound of the present invention can provide a uniform dispersion in the culture medium even without shaking, etc. As a result, target cells and/or tissues can be cultured as spheres without loss of cell function.

The spheres statically cultured using this method can be collected by centrifugation and filtration after culture. In this case, the centrifugation and filtration can be performed after adding the used liquid culture medium. For example, without limitation, the gravitational acceleration (G) of the centrifugation is 50 G to 1000 G, more preferably 100 G to 500 G, and the pore size of the filter used for the filtration is 10 μm to 100 μm. In addition, the cultured spheres can be recovered magnetically using magnetic microparticles coated with antibodies that specifically bind to the target cells. Examples of such magnetic microparticles include Dynabeads (manufactured by Veritas Ltd.), MACSmicrobeads (manufactured by Miltenyi Biotec), and BioMag (manufactured by Techno Chemicals Corporation). The recovered spheres can be further broken down and dispersed into single cells by treatment with various chelating agents, heat, filters, enzymes, etc. Cell recovery and exchange of the culture medium composition can also be achieved by centrifugation, filtration, or recovery using magnetic conduction bioreactors and automated incubators that can conduct under mechanical control and in a closed environment.

As a method for static culture of plant-derived cells and/or tissues, callus (which is an undifferentiated plant cell aggregate) can be cultivated. Callus can be induced by methods known to each plant species to be used. For example, when necessary, with 70% ethanol, 1% sodium hypochlorite solution etc., the surface sterilization of a part of the tissue (for example, root, stem, leaf part, seed, growing point, embryo, pollen etc.) of the plant body that is differentiated, will have a tissue part (for example, the root part of about 1-about 5 mm ² ) cut out (when necessary) with a knife etc., the tissue part is placed on a pre-sterilized callus induction medium with a clean bench etc. by aseptic operation, and aseptically cultured under suitable conditions. The callus induced here can be liquid cultured for a large number of proliferations, or can also be maintained as a seed strain (seed strain) by being passed down through generations on a subculture medium. Subculture can be carried out using any liquid culture medium and solid culture medium.

The amount of plant cell aggregates inoculated when static culture is started using the culture medium composition of the present invention varies depending on the proliferation rate of the target cells, the culture method (batch culture, fed-batch culture, continuous culture, etc.), the culture cycle, etc. For example, when a plant cell aggregate such as callus is to be cultured, it is inoculated into the culture medium composition of the present invention so that the wet weight of the cell aggregate relative to the culture medium composition of the present invention is 4-8 (weight/volume (w/v))%, preferably 5-7 (w/v)%. The specific size of the plant cell aggregate during the culture process is 1 mm-40 mm, preferably 3 mm-20 mm, and more preferably 5 mm-15 mm. As used herein, "specific size" refers to the diameter when, for example, the plant cell aggregate has a spherical shape, the long diameter when it has an ellipsoidal shape, and the maximum length possible when it has other shapes.

The temperature when culturing cells and/or tissues is generally 25-39°C for animal cells, preferably 33-39°C. The _CO₂ concentration in the culture atmosphere is generally 4-10% by volume, and preferably 4-6% by volume. The culture period is generally 3-35 days, which can be freely set according to the culture objectives. The culture temperature for plant cells is generally 20-30°C, and when light is required, they can be cultured under illumination conditions of 2000-8000 lux.

Although the culture period is generally 3 to 70 days, it can be freely set according to the culture purpose.

When cell and/or tissue are cultivated by method of the present invention, the cell and/or tissue prepared separately are added in culture medium composition of the present invention, and are mixed to generate uniform dispersion.In this case, mixing method has no specific restriction, and for example, use the manual mixing of suction etc., use instrument such as agitator, vortex mixer, microparticle culture plate mixer, shaking device etc. to mix and can be mentioned.After mixing, culture medium can be kept standing, or culture medium can be rotated, shaken or stirred as needed.Number of rotations and frequency can be suitably set according to the target of those of ordinary skill in the art.When culture medium composition needs to be exchanged during the static culture cycle, by centrifugal or filtration treatment, cell and/or tissue are separated from culture medium composition, and new culture medium composition can be added in cell and/or tissue.Or, cell and/or tissue are suitably concentrated by centrifugal or filtration treatment, and new culture medium composition can be added in concentrated liquid.

For example, without limitation, the gravitational acceleration (G) of the centrifugation is 50 G to 1000 G, more preferably 100 G to 500 G, and the pore size of the filter used for the filtration treatment is 10 μm-100 μm. In addition, using magnetic particles coated with antibodies that specifically bind to target cells on the surface, cultured cells and/or tissues can be separated by magnetic force. Examples of such magnetic particles include Dynabeads (manufactured by Veritas Ltd.), MACS microbeads (manufactured by Miltenyi Biotec), BioMag (manufactured by Techno Chemicals Corporation), magnetic microspheres (manufactured by Polysciences Inc.), etc. The exchange of the culture medium composition can also be carried out by using a bioreactor and an automatic incubator that can conduct (conducting) under mechanical control and in a closed environment.

Since cancer cells can be effectively proliferated by the method of the present invention, the culture medium composition containing the specific compound of the present invention can be used to evaluate the anticancer drug for cancer cells. For example, when explaining the anticancer drug that inhibits the proliferation of cancer cells, cancer cells are co-cultured with the anticancer drug, and the number and type of cells and the changes of cell surface differentiation markers and expressed genes are analyzed. In this case, using the culture medium composition of the present invention, the number of target cells to be analyzed can be effectively expanded, and effectively recovered. In the present invention, in particular, the culture medium additive for cancer cells containing deacylated gellan gum or its salt and the culture medium composition for cancer cells containing this additive can be used to evaluate cancer cell proliferation or anticancer activity, etc. In this case, the concentration of deacylated gellan gum or its salt is as mentioned above.

Although cancer cells proliferate even by using diterpenoid, deacylated gellan gum is more preferable since the proliferative effect on cancer cells is particularly excellent and it can be used at a low concentration (preferred concentration mentioned above), which in turn prevents the occurrence of bubbles in the culture medium and facilitates the recovery of cancer cells.

More preferred screening methods for anticancer drugs include, for example, methods comprising (a) culturing cancer cells in the medium composition of the present invention in the presence and absence of a test substance, and (b) analyzing changes in cancer cell proliferation. This method may further include selecting a substance that inhibits cancer cell proliferation compared to that in the absence of the test substance and/or recovering the cancer cells. A change indicates an increase or decrease in cancer cell proliferation. The analysis can be performed using the above-described method, but is not limited thereto.

When evaluating the activity of an anticancer drug, those skilled in the art can appropriately determine the culture conditions, culture tools, culture equipment, type of culture medium, type of specific compound, content of specific compound, type of additive, content of additive, culture period, culture temperature, type of anticancer drug, content of anticancer drug, etc. within the range described in this specification. Cells that proliferate or emerge during culture can be observed using a standard microscope in the art. When measuring cell number, colony formation assays, crystal violet assays, thymidine uptake assays, trypan blue staining, ATP (adenosine triphosphate) measurement, 3-(4,5-dimethylthio-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining, WST-1 (registered trademark) staining, WST-8 (registered trademark) staining, flow cytometry, methods using an automated cell counter, etc. can be used. Of these, WST-8 (registered trademark) staining is most preferably used. When evaluating cytotoxicity, lactate dehydrogenase (LDH) activity measurement, CytoTox-ONE (registered trademark) assay, etc. can be used. Alternatively, cultured cells can be stained with specific antibodies and cell surface differentiation markers detected by ELISA or flow cytometry, allowing observation of the effects of anticancer drugs on proliferation and apoptosis. Furthermore, genes that show differential expression due to cancer resistance can be identified by extracting DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) from cultured cells and analyzing them using Southern blotting, Northern blotting, RT-PCR, and other methods.

Because the method of the present invention maintains the survival and function of hepatocytes, the culture medium composition containing the specific compound of the present invention can be used to evaluate the various effects of drugs or drug candidate substances on hepatocytes. For example, when the toxic effects of drug candidate substances are elucidated, hepatocytes are co-cultured with the evaluation target test substance, and the number and type of cells and cell surface differentiation markers and expressed gene changes are analyzed. In this case, using the culture medium composition of the present invention, the survival and function of the target hepatocytes can be maintained and analyzed, and the hepatocytes can be effectively recovered.

As a method for screening candidate pharmaceutical substances that act on hepatocytes, there can be mentioned a method comprising: (a) a step of culturing hepatocytes in the medium composition of the present invention in the presence and absence of a test substance, and (b) a step of analyzing changes in the physiological functions of the hepatocytes.

As methods for evaluating the efficacy or toxicity of candidate drug substances acting on hepatocytes, methods comprising: (a) culturing hepatocytes in the medium composition of the present invention in the presence and absence of a test substance, and (b) analyzing changes in the physiological functions of the hepatocytes. These methods may further include selecting a substance that inhibits or increases the physiological functions of the hepatocytes compared to those in the absence of the test substance, and/or recovering the hepatocytes. Changes refer to increases or decreases in the physiological functions of the hepatocytes (e.g., hepatocyte proliferation, cytochrome P450 enzyme activity, etc.). Increases in the physiological functions of the hepatocytes can be evaluated as indicating low efficacy or toxicity, while decreases in the physiological functions of the hepatocytes can be evaluated as indicating high efficacy or toxicity, and so on.

When evaluating the activity of a drug candidate substance, a person of ordinary skill in the art can appropriately select culture conditions, culture tools, culture equipment, type of culture medium, type of specific compound, content of specific compound, type of additive, content of additive, culture period, culture temperature, type and content of the drug or drug candidate substance, etc. from the ranges described in this specification. Cells maintained or emerging through culture can be observed using a standard microscope in the art. When measuring cell number, colony formation assays, crystal violet assays, thymidine uptake assays, trypan blue staining, ATP (adenosine triphosphate) measurement, 3-(4,5-dimethylthiocarbazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining, WST-1 (registered trademark) staining, WST-8 (registered trademark) staining, flow cytometry, methods using an automated cell counting instrument, etc. can be used. Among them, the WST-8 (registered trademark) staining method is most preferably used. When evaluating cytotoxicity, lactate dehydrogenase (LDH) activity measurement methods, CytoTox-ONE (registered trademark) assays, etc. can be used. Alternatively, cultured cells can be stained with specific antibodies and cell surface differentiation markers detected by ELISA (enzyme-linked immunosorbent assay) or flow cytometry to observe the effects of drugs or drug candidates on proliferation and apoptosis. Furthermore, genes whose expression is differentially altered by drugs or drug candidates can be discovered by extracting DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) from cultured cells and performing Southern blotting, Northern blotting, RT-PCR, and other assays. Furthermore, proteins whose expression is differentially altered by drugs or drug candidates can be detected by ELISA, Western blotting, flow cytometry, and other assays. Furthermore, cytochrome P450 enzyme activity can be measured by measuring the enzyme's ability to convert substrate structures using methods such as radioisotope analysis, high-performance liquid chromatography, luminescence, and colorimetric analysis.

Example

The present invention is explained in more detail below by way of Analysis Examples and Experimental Examples specifically describing the medium composition of the present invention as examples; however, the present invention is not limited thereto.

(Analytical Example 1: Viscosity Measurement and Cell Suspension Test of a Culture Medium Containing Deacylated Gellan Gum)

Preparation of culture medium containing deacylated gellan gum and viscosity measurement

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in pure water to 0.4% (w/v) and dissolved by heating and stirring at 90°C. The aqueous solution was allowed to cool to room temperature while stirring and sterilized in an autoclave at 121°C for 20 min. A 2-fold concentration of DMEM/F-12 culture medium (manufactured by Aldrich, 50 mL) and sterilized water (47.5 mL) were placed in a 300 mL high beaker and stirred at room temperature (3000 rpm) by a homomixer. An aqueous deacylated gellan gum solution (2.5 mL) was added and the mixture was stirred for 1 min to prepare a deacylated gellan gum culture medium composition with a final concentration of 0.01%. Culture medium compositions with aqueous deacylated gellan gum solutions having final concentrations of 0.02, 0.03, and 0.05% (w/v) were similarly prepared. The viscosity of the medium composition was measured using an E-type viscometer (manufactured by TokiSangyo Co., Ltd., Viscometer TVE-22L, standard rotor 1°34'×R24) at 37°C and 100 rpm for 5 minutes.

Cell suspension test in culture medium containing deacylated gellan gum

Human cervical cancer cell line HeLa (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) was suspended at 250,000 cells/mL in EMEM medium containing 10% (v/v) fetal bovine serum (manufactured by WAKO). This suspension (10 mL) was plated on an EZ SPHERE (manufactured by ASAHI GLASS CO., LTD.) and cultured in a CO ₂ incubator (5% CO ₂ ) for 3 days. The resulting suspension (10 mL) of spheres (100-200 μm in diameter) was centrifuged (200 g, 5 min) to allow the spheres to precipitate, and the supernatant was removed to produce a sphere suspension (1.0 mL). Next, 1.0 mL of the culture medium containing deacylated gellan gum prepared above was placed in a 1.5 mL Eppendorf tube, and the HeLa cell sphere suspension (10 μL) was further added. The cell aggregates were dispersed by tapping, incubated at 37°C, and the cell dispersion state was visually observed after 1 hour.

(Comparative Example) Preparation of a culture medium containing methylcellulose and collagen

Preparation of culture medium containing methylcellulose

DMEM/F-12 culture medium (manufactured by Aldrich, 100 mL) was placed in a 200 mL pear-shaped flask, and methylcellulose (M0387, manufactured by Aldrich, 0.1 g) was added. The mixture was stirred while cooling on an ice bath to dissolve the methylcellulose. Using this solution, culture medium compositions containing an aqueous methylcellulose solution added at a final concentration of 0.1, 0.3, 0.6, or 1.0% (w/v) were prepared.

Preparation of collagen-containing culture medium

A 10-fold concentration of DMEM/F-12 medium (manufactured by Aldrich, 1 mL), a reconstitution buffer (manufactured by Nitta Gelatin Inc., 1 mL), and purified water (1.5 mL) were added to 0.3% Type I-A cell matrix (manufactured by Nitta Gelatin Inc., 6.5 mL), and the mixture was stirred on ice to produce a medium containing 0.2% collagen. Similarly, medium compositions containing collagen at a final concentration of 0.01, 0.05, 0.1, or 0.2% (w/v) were prepared.

The medium composition prepared above was subjected to a HeLa cell spheroid suspension test and viscosity measurement in the same manner as the medium containing deacylated gellan gum. Due to the measuring range of the instrument, the viscosity of 1.0% (w/v) methylcellulose was measured at 50 rpm.

[Experimental Example]

Although the usefulness of the medium composition of the present invention in cell culture is specifically explained in the following experimental examples, the present invention is not limited thereto. The _CO2 concentration (%) in the _CO2 incubator is shown by the volume % of _CO2 in the atmosphere. PBS represents phosphate-buffered saline (manufactured by Sigma Aldrich Japan), and FBS represents fetal bovine serum (manufactured by Biological Industries). In addition, (w/v) represents weight/volume.

(Experimental Example 1: Cell proliferation test by dispersed single cells)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to 0.3% (w/v) and dissolved by heating and stirring at 90°C. The aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum to IMDM medium (manufactured by Gibco) containing 10% (v/v) fetal bovine serum and 10 ng/mL thrombopoietin (manufactured by WAKO) at a final concentration of 0.015% (w/v). Next, the human leukemia cell line UT7/TPO was placed in the medium composition supplemented with the above-mentioned deacylated gellan gum to 20,000 cells/mL and distributed into a 6-well flat-bottom microplate (manufactured by Corning Incorporated) at 5 mL/well. Similarly, human cervical cancer cell line HeLa was plated at 20,000 cells/mL onto a medium composition obtained by adding 0.015% (w/v) deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) to EMEM medium containing 10% (v/v) fetal bovine serum (manufactured by WAKO), and the composition was dispensed into a 6-well flat-bottom microplate (manufactured by Corning Incorporated) at 5 mL/well. The cell suspension was cultured while being kept still in a _CO2 incubator (5% _CO2 ) for 3 days. Subsequently, a portion of the medium was recovered, the same amount of trypan blue staining solution (manufactured by Invitrogen Corporation) was added, and the number of viable cells was measured using a hemocytometer (manufactured by ERMA INC.).

The results showed that UT7/TPO cells and HeLa cells could be uniformly cultured in a suspended state using the medium composition of the present invention and efficiently proliferated in the medium composition. The cell counts of UT7/TPO cells and HeLa cells after 3 days of static suspension culture are shown in Table 4.

[Table 4]

UT7/TPO cells HeLa cells Cell number (×10000/mL) 38 40

(Experimental Example 2: Cell Proliferation Test of Spheres Derived from Cultured Cell Lines)

Human liver cancer cell line HepG2 (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) was suspended at 250,000 cells/mL in DMEM medium containing 10% (v/v) fetal bovine serum (manufactured by WAKO), and the suspension (10 mL) was plated on EZ SPHERE (manufactured by ASAHI GLASS CO., LTD.) and cultured in a CO ₂ incubator (5% CO ₂ ) for 7 days. Similarly, human cervical cancer cell line HeLa (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) was suspended at 250,000 cells/mL in EMEM medium containing 10% (v/v) fetal bovine serum (manufactured by WAKO), and the suspension (10 mL) was plated on EZ SPHERE (manufactured by ASAHI GLASS CO., LTD.) and cultured in a CO ₂ incubator (5% CO ₂ ) for 7 days. A suspension (2.5 mL) of spheres (100-200 μm in diameter) obtained for each cell line was centrifuged (200 G, 5 min) to allow the spheres to precipitate, and the supernatant was removed. Next, the above-mentioned culture medium (10 mL) was added to the spheres (approximately 800 spheres) to suspend them, and the suspension was transferred to a flat-bottom tube (manufactured by BM Equipment Co., Ltd.). Similarly, a culture medium composition obtained by adding 0.015% (w/v) deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) to the above-mentioned culture medium was used to produce a sphere suspension, which was then transferred to a flat-bottom tube (manufactured by BM Equipment Co., Ltd.). A medium composition supplemented with 0.015% (w/v) deacylated gellan gum was prepared by first suspending deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) in ultrapure water (Milli-Q water) to 0.3% (w/v), dissolving it by stirring with heating at 90°C, sterilizing the aqueous solution in an autoclave at 121°C for 20 min, and adding the solution at a 1/20 dilution to a DMEM medium containing 10% (v/v) fetal bovine serum.

After the sphere suspension was incubated at 37°C in a _CO2 incubator (5% _CO2 ) for 3 days, twice the volume of culture medium was added. The mixture was centrifuged (200g, 5 min) to allow the spheres to settle, and the supernatant was removed. At this point, a portion of the spheres was removed and their shape was observed using an optical microscope (manufactured by OLMPUS, CK30-F100). The recovered spheres were then washed once with PBS (10 mL), 1 mL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added, and the mixture was incubated at 37°C for 5 min. The above culture medium (9 mL) was added, and the cells were collected by centrifugation (200g, 5 min). The same amount of trypan blue staining solution (manufactured by Invitrogen Corporation) was added to a portion of the obtained cell suspension (2 mL), and the number of live and dead cells was measured using a hemocytometer (manufactured by ERMA INC.).

The results demonstrated that spheres of HepG2 and HeLa cells could be cultured in a suspended state using the medium composition of the present invention, and that the cells proliferated efficiently in the medium composition. Furthermore, the medium composition of the present invention demonstrated a lower proportion of dead cells during cell proliferation compared to existing culture media, and exhibited an excellent cell proliferation-promoting effect. Spheres cultured in existing culture media settled to the bottom of the culture vessel. Furthermore, the shape of the cultured spheres was observed using an optical microscope. The medium composition of the present invention showed no sphere binding, whereas sphere binding was observed in existing culture media.

The relative numbers of HepG2 cells and HeLa cells are shown in Table 5, where the number of cells cultured in a medium without deacylated gellan gum is 1. Furthermore, the relative ratio of dead cells is shown in Table 6, where the ratio of dead cells (number of dead cells/number of live cells) cultured in a medium without deacylated gellan gum is 1. The suspended states of spheres of HepG2 cells and HeLa cells cultured in the medium composition of the present invention are shown in Figures 1 and 2, respectively. Furthermore, the shape of the cultured HeLa cell spheres is shown in Figure 3.

[Table 5]

Deacylated gellan gum HepG2 cells HeLa cells does not exist Relative cell number 1.0 1.0 exist Relative cell number 1.7 1.5

[Table 6]

Deacylated gellan gum HepG2 cells HeLa cells does not exist Relative mortality rate 1.0 1.0 exist Relative mortality rate 0.5 0.5

(Experimental Example 3: Cell Proliferation Test of Human Pluripotent Stem Cells in Adhesion Culture)

Human pluripotent stem cells (hPSCs) are generally proliferated and maintained on feeder cells or culture dishes coated with Matrigel under flat plane culture conditions that allow adhesion. To evaluate the toxicity of deacylated gellan gum to hPSCs, deacylated gellan gum was added to mTeSR medium (manufactured by STEM CELL Technologies) under flat culture conditions using Matrigel (manufactured by Becton, Dickinson and Company) at a concentration of 0.000%-0.020% (w/v), and the effect on hPSC proliferation was examined. In this case, the Kyoto University 253G1 strain was cultured as human iPS cells and the Kyoto University KhES-1 strain was cultured as a human ES cell line. By first suspending deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) in ultrapure water (Milli-Q water) to 0.3% (w/v), dissolving it by stirring under heating at 90°C, sterilizing the aqueous solution in an autoclave at 121°C for 20 min, and adding the solution to the mTeSR medium at a given concentration, a medium composition containing deacylated gellan gum at the above concentration was prepared. As a result, by using a medium containing deacylated gellan gum, the same level of cell number as that of a general mTeSR medium could be obtained for both human iPS cells and human ES cells, and no toxicity of deacylated gellan gum was observed. The results are shown in Figure 4. The cell number after culture as shown in FIG4 is a relative value of the cell number obtained by plating hPSCs on a culture dish coated with Matrigel and culturing the cells in mTeSR medium containing deacylated gellan gum for 5 days until the cell number in mTeSR medium without deacylated gellan gum became 1.

(Experimental Example 4: Precipitation Inhibition Test of Deacylated Gellan Gum in the Culture of Human Pluripotent Stem Cell Spheroids)

hPSCs form spheres on low-adhesion culture dishes such as petri dishes, etc. For example, spheres can be formed by any method described in NATURE BIOTECHNOLOGY, VOL. 28, NO. 4, APRIL 2010, 361-366, NATURE PROTOCOLS, VOL. 6, NO. 5, 2011, 689-700, NATURE PROTOCOLS, VOL. 6, NO. 5, 2011, 572-579, Stem Cell Research, 7, 2011, 97-111, Stem Cell Rev and Rep, 6, 2010, 248-259. hPSCs (Kyoto University 253G1 strain or Kyoto University KhES-1 strain) maintained on feeder cells (mouse fetal fibroblasts) were recovered, the feeder cells were removed by natural sedimentation, and the hPSCs were resuspended in mTeSR medium supplemented with the Rho kinase inhibitor Y-27632 (10 μM). Next, hPSC colonies of a given size were seeded on petri dishes (manufactured by BD Falcon) and cultured in a CO ₂ incubator (5% CO ₂ ) at 37°C to form spheres. The medium was exchanged with mTeSR medium without Y-27632 on days 1 and 3 after passage, and the cells were passaged every 5 days in mTeSR medium containing Y-27632. The hPSC spheres thus prepared (day 4 of culture) were suspended in a medium composition obtained by adding deacylated gellan gum to mTeSR medium (prepared in a manner similar to that in Experimental Example 3) to a concentration of 0.000%-0.020% (w/v) and transferred to a cuvette. The cuvette was left to stand overnight in a _CO2 incubator (5% _CO2 ) at 37°C, and the effect of deacylated gellan gum on inhibiting sphere precipitation was examined. The results are shown in Figure 5. As shown in Figure 5, the addition of deacylated gellan gum allowed the spheres to be maintained in a three-dimensional suspended state in the culture medium over the entire concentration range. On the other hand, in the existing culture medium without deacylated gellan gum, the spheres were found to settle to the bottom surface of the culture vessel and could not remain suspended. Furthermore, the effect of deacylated gellan gum was shared by both human iPS cells and human ES cells. The above results show that deacylated gellan gum can maintain hPSC spheres in a suspended state.

(Experimental Example 5: Cell Proliferation Test in Culture of Human Pluripotent Stem Cell Spheres)

We examined whether hPSCs can be cultured in a three-dimensional suspended state in tubes. hPSC spheres (600-800/3 mL) prepared and passaged in a manner similar to Experimental Example 4 were plated on mTeSR medium containing 0.000%, 0.015%, or 0.020% (w/v) deacylated gellan gum in 5 mL polystyrene tubes (manufactured by BD Falcon) so that each tube had the same number of spheres and cultured in a CO ₂ incubator (5% CO ₂ ) at 37°C for 5 days. On the first and third days after passage, the medium was exchanged by adding 3 volumes of DMEM/F-12 medium (manufactured by Sigma Ltd.), the spheres were pelleted by centrifugation (100G, 3 min), and new medium was added to the spheres. At the 5th day, add equal amount of DEME/F-12 culture medium (manufactured by Sigma Ltd.), reclaim all balls by centrifugation (100G, 3 min) and dissociate them into single cells with trypsin-EDTA solution (manufactured by Invitrogen), and measure cell number by NucleoCounter (manufactured by chemometec).The result is, in the culture medium without deacylated gellan gum, ball is precipitated on the bottom of pipe to form large cell aggregates, and does not show proliferation.However, in the culture medium containing 0.015% or 0.020% (w/v), the size of the ball with three-dimensional suspension state increases, and as confirmed by the cell number of about 10 times obtained at the 5th day for the cell number of 1 laid, find cell proliferation.The results are shown in Figure 6.Figure 6 relatively shows the cell number that was 1 to the cell number of 1 at the 5th day. With human ES cells, it is practical to obtain 3,000,000 cells per 3 mL of culture in a polystyrene tube on day 5 (corresponding to approximately 1,000,000,000 cells in 1000 mL of culture medium).

(Experimental Example 6: Undifferentiation Maintenance Confirmation Test in Culture of Human Pluripotent Stem Cell Spheres)

The hPSC sphere cells were suspended and statically cultured in mTeSR culture medium containing 0.015% or 0.020% (w/v) deacylated gellan gum, and the maintenance of their undifferentiated characteristics was checked by flow cytometry analysis. In a polystyrene tube, human ES cells (KhES-1) were passaged 3 times and human iPS cells (253G1) were passaged 4 times in a sphere state. Cells were recovered and stained with SSEA4 antibody (#MAB4304, manufactured by Millipore) and TRA-1-60 (#MAB4360, manufactured by Millipore) antibody (which is a surface marker showing the undifferentiated characteristics of hPSC), and the positive rate of cell staining with antibodies was evaluated using FACSCantoII (manufactured by Becton, Dickinson and Company). The results are shown in Figure 7. As shown in Figure 7, in A: human iPS cells (253G1) and B: human ES cells (KhES-1), no less than 90% of cells suspended and statically cultured in an additive medium containing deacylated gellan gum expressed pluripotent stem cell markers, such as cells maintained on Matrigel. As a negative control, staining with only a secondary antibody was performed. From the above, it is clarified that in both human iPS cells and human ES cells, hPSC spheres suspended and statically cultured in an additive medium containing deacylated gellan gum maintain undifferentiated properties.

(Experimental Example 7: Characterization Analysis of Sphere-Cultured Human Pluripotent Stem Cells-1)

Spheres of human iPS cells (253G1) or human ES cells (KhES-1) were subcultured a total of nine times using mTeSR medium (manufactured by STEM Cell Technologies) containing 0.020% (w/v) deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) prepared similarly to Experimental Example 3, and spheres of each cell type on passage day 1 were plated on mouse fetal fibroblasts. The next day, the spheres were treated with 300 μg/mL thymidine (manufactured by Sigma Aldrich) overnight. Subsequently, the spheres were treated with 100 ng/mL colchicine (manufactured by Nacalai Tesque), dissociated into single cells using trypsin-EDTA, and subjected to hypotonic treatment with 0.075 M KCl. The cells were then fixed with Carnoy's fixative (methanol:acetic acid = 3:1).

The karyotype of fixed cells was analyzed using the Q-banding method (experiments commissioned by Chromosome Science Lab. Ltd.). The results demonstrated that both human iPS cells and human ES cells cultured in suspension or static culture in the medium composition of the present invention retained normal karyotypes. The results are shown in Figure 8.

(Experimental Example 8: Characterization Analysis of Sphere-Cultured Human Pluripotent Stem Cells-2)

Spheres of human iPS cells (253G1) were subcultured 21-22 times every 5 days using mTeSR medium (manufactured by STEM Cell Technologies) containing 0.020% (w/v) deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) prepared similarly to Experimental Example 3. The cultured spheres were fixed with 4% (w/v) paraformaldehyde (manufactured by Nacalai Tesque) using a method similar to Experimental Example 4. These spheres were immersed in PBS containing 20% (w/v) sucrose and embedded in an embedding medium (O.C.T compound, manufactured by Japanese Cherry Finetek Japan Co., Ltd.) for freezing. 12 μm thick sections were prepared in a cryostat (manufactured by Thermo Scientific) and stained with antibodies against NANOG (#4903, manufactured by Cell Signaling), OCT3/4 (#sc-5279, manufactured by Santa Cruz), and SSEA4 (#MAB4304, manufactured by Millipore) to show undifferentiation of hPSCs. The results showed that cells suspended and statically cultured in a culture medium composition containing deacylated gellan gum expressed undifferentiated markers of pluripotent stem cells. As described above, it was shown that human iPS cell spheres suspended and statically cultured for less than 100 days in a culture medium composition containing deacylated gellan gum maintained undifferentiated characteristics. The results are shown in FIG9 .

(Experimental Example 9: Cell Proliferation Test by Cultivating Cell Lines Attached to Microcarriers)

The microcarrier Cytodex (registered trademark) 1 (manufactured by GE Healthcare Life Sciences) was suspended in PBS at 0.02 g/mL and the suspension was left overnight. The supernatant was discarded and the microcarrier was washed twice with fresh PBS. Subsequently, it was resuspended in PBS at 0.02 g/mL and sterilized in an autoclave at 121°C for 20 min. Next, the microcarrier was washed twice with 70% ethanol and three times with PBS, and suspended in DMEM medium containing 10% (v/v) fetal bovine serum (manufactured by WAKO) at 0.02 g/mL. Using this microcarrier suspension, a DMEM culture medium (containing 10% (v/v) fetal bovine serum, 20 mL) containing 120 mg of Cytodex (registered trademark) 1 and 4,000,000 HepG2 cells was prepared. The cell suspension was then cultured in a beaker pre-treated with a silicone coating agent (manufactured by AGC TECHNO GLASS Co., Ltd.) while stirring at 37°C (100 rpm) for 6 hours. At this point, microscopic observation confirmed that the HepG2 cells were attached to the microcarriers. Subsequently, the microcarriers to which the cells had adhered were washed twice with DMEM culture medium containing 10% (v/v) fetal bovine serum and suspended in the same culture medium (3 mL).

The above microcarrier suspension (300 μL) was added to each of a DMEM medium (20 mL) containing 10% (v/v) fetal bovine serum and a medium composition obtained by adding 0.015% (w/v) deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) to the medium, and the mixture was cultured at 37°C for 3 days. In the case of a medium without deacylated gellan gum, the mixture was cultured while stirring with a stirrer (100 rpm). After culture, the attachment state of the cells on the microcarriers was confirmed using a microscope, and the microcarriers were precipitated by centrifugation (200G, 5 min). The microcarriers were washed with PBS (10 mL), 1 mL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added, and the mixture was incubated at 37°C for 5 min. In addition, add DMEM culture medium (9 mL) containing 10% (v/v) fetal bovine serum, remove microcarrier by Cell Strainer (manufactured by BD Falcon, mesh size 70 μm). Cells are recovered from the filtrate obtained by centrifugation (200G, 5min). Cells are suspended in culture medium (500 μL), and in a portion thereof, the same amount of trypan blue staining solution (manufactured by Invitrogen Corporation) is added, and the number of living cells is measured by hemocytometer (manufactured by ERMA INC.). As a result, the culture medium without deacylated gellan gum contains 123,000 cells, while the culture medium containing deacylated gellan gum contains 1,320,000 cells. As mentioned above, it is confirmed that the culture medium composition containing the structure of the specific mixture of the present invention is excellent in cell proliferation promotion compared to existing culture medium, even when using microcarrier culture cells. The attachment state of HepG2 cells after 3 days of culture using the microcarrier composition containing the structure of the specific compound of the present invention is shown in Figure 10.

(Experimental Example 10: Cell Suspension Test Using Cell Line-Derived Spheres)

Xanthan gum (KELTROL CG, manufactured by SANSHO Co., Ltd.) is suspended in ultrapure water (Milli-Q water) to a concentration of 1% (w/v), and dissolved by heated stirring at 90°C. Using the aqueous solution, DMEM/F-12 culture medium compositions with a final concentration of 0.1, 0.15 or 0.2% (w/v) xanthan gum are prepared. In addition, an aqueous solution containing 0.2% (w/v) κ-carrageenan (GENUGEL WR-80-J, manufactured by SANSHO Co., Ltd.) and 0.2% (w/v) locust bean gum (GENUGUM RL-200-J, manufactured by SANSHO Co., Ltd.) is prepared by heating at 90°C. Using this aqueous solution, a DMEM/F-12 medium (manufactured by Sigma Ltd.) composition containing 0.03, 0.04, or 0.05% (w/v) of κ-carrageenan and locust bean gum was prepared.

In the same manner as in Experimental Example 2, HeLa cell spheres were formed, and several dozen spheres were added to each of the culture media prepared above (1 mL). The mixture was left to stand at 37°C for 1 hour, and the suspended state of the spheres was visually observed. The results confirmed that HeLa cell spheres remained suspended in all of the above-mentioned culture medium compositions. Furthermore, it was confirmed that adding an equal amount of culture medium to the cell suspension and centrifuging it (300-400g, 5 minutes) resulted in the precipitation and recovery of the HeLa cell spheres. The suspended state of HeLa cell spheres cultured in the culture medium compositions of the present invention is shown in Figure 11. Furthermore, the viscosity measured in a manner similar to that in Analytical Example 1 is shown in Tables 7 and 8.

(Experimental Example 11: Cell Suspension Test Using Filter-Filtered Culture Medium Composition)

A DMEM/F-12 medium composition containing 0.015% deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was prepared in a manner similar to that in Experimental Example 2. This medium composition (1 mL) was then filtered through a 70 μm filter and a 40 μm filter (manufactured by BD Falcon), a 30 μm filter and a 20 μm filter (manufactured by AS ONE Corporation), a 10 μm filter (manufactured by Partec), and a 5 μm filter, a 1.2 μm filter, a 0.45 μm filter, and a 0.2 μm filter (manufactured by Sartorius Stedim Japan). Several dozen HepG2 spheres prepared in a manner similar to that in Experimental Example 2 were added to the filtrate and allowed to stand at 37°C for 1 hour, and the suspended state of the sphere cells was visually observed. The results showed that HepG2 cell spheres remained suspended in a culture medium composition that had been filtered through a filter with a diameter of 10 μm or greater, but precipitated in a culture medium composition that had been filtered through a filter with a diameter of less than 5 μm. Furthermore, it was confirmed that HepG2 cell spheres in suspension were precipitated and recovered by centrifugation at 300G for 5 minutes at room temperature or by adding an equal amount of culture medium and centrifuging at 200G for 5 minutes at room temperature.

(Experimental Example 12: Ball Formation Test)

In the same manner as in Experimental Example 2, a composition containing 0.01% deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) and 10% (v/v) fetal bovine serum in EMEM culture medium (manufactured by WAKO) was prepared. Next, HeLa cells were added to a concentration of 1000 cells/mL and distributed into a 24-well plate (manufactured by Corning Incorporated). The plate was suspended and cultured for 9 days at 37°C, and the formation of spheres was confirmed using a microscope. In addition, the spheres were precipitated by centrifugation (300 G, 5 min) and washed once with PBS (5 mL). 100 μL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added, and the mixture was incubated at 37°C for 5 min. Here, EMEM culture medium (100 μL) containing 10% (v/v) fetal bovine serum was added to the obtained cell suspension (100 μL), and a trypan blue staining solution (manufactured by Invitrogen Corporation) was added to a subset of the cell suspension in the same amount, and the number of viable cells was measured by a hemocytometer (manufactured by ERMA INC.). As a result, it was confirmed that the number of HeLa cells increased to 170,000 cells/mL. The spheres of HeLa cells formed in the culture medium composition of the present invention are shown in Figure 12.

(Experimental Example 13: Optical Microscope Observation of Structure)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in pure water to 0.4% (w/v) and dissolved by heating and stirring at 90°C. DMEM/F-12 culture medium (manufactured by Aldrich, 95 mL) was placed in a 300 mL high beaker at twice the concentration, and an aqueous deacylated gellan gum solution (5 mL) was added while stirring with a magnetic stirrer at room temperature. The mixture was stirred for 5 minutes to generate a culture medium composition containing deacylated gellan gum at a final concentration of 0.02%. In addition, the culture medium composition was stirred for 5 minutes by a homomixer (3000 rpm). The prepared culture medium composition was observed using an optical microscope (KEYENCE Corporation, BIOREVO BZ-9000). The observed structure is shown in Figure 13.

(Experimental Example 14: Preparation by mixing and heating powdered culture medium and DAG)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd., 20 mg) and DMEM/F-12 culture medium (manufactured by Life Technologies, 1.58 g) are placed in a 200 mL conical flask, and pure water (100 mL) is poured into it. The mixture is sterilized at 121 ° C for 20 minutes in an autoclave to prepare the DMEM/F-12 culture medium composition with 0.02% decarboxylation gellan gum concentration. Dextran beads Cytodex 1 (size 200 μm, manufactured by GE Healthcare Life Sciences) are added to the culture medium prepared, and the dispersion state of pearl is determined by visual observation. For evaluation, suspended state is ○, and partial precipitation/dispersion state is Δ, and precipitation state is ×. The results are shown in Table 9.

[Table 9]

Deacylated gellan gum concentration% (w/v) state Cytodex1 dispersion 0.05 liquid 0.02 liquid 0.01 liquid

(Experimental Example 15: Preparation of a culture medium composition containing polysaccharides)

Xanthan gum (KELTROL CG, manufactured by SANSHO Co., Ltd.) was suspended in pure water to a concentration of 0.5% (w/v) and dissolved by stirring with heating at 90° C. Similarly, 0.5% (w/v) aqueous solutions of sodium alginate (Duck alginic acid NSPM, manufactured by FOOD CHEMIFA Co., Ltd.), locust bean gum (GENUGUM RL-200-J, manufactured by SANSHO Co., Ltd.), κ-carrageenan (GENUGEL WR-80-J, manufactured by SANSHO Co., Ltd.), and diterpenoid gum (KELCO CRETED G-F, manufactured by SANSHO Co., Ltd.) were prepared.

This aqueous solution was mixed with a 0.2 or 0.1% (w/v) deacylated gellan gum solution and a 10-fold concentration of DMEM/F-12 medium. The mixture was heated at 80°C for 30 minutes, allowed to cool to room temperature, and a 7.5% aqueous sodium bicarbonate solution was added to prepare a DMEM/F-12 medium composition containing deacylated gellan gum at final concentrations of 0.01% and 0.02% (w/v) and other polysaccharides at final concentrations of 0.1%, 0.2%, 0.3%, and 0.4% (w/v). Furthermore, a deacylated gellan gum-containing medium was prepared as described above, and methylcellulose powder (cP400, manufactured by WAKO) was added. The mixture was stirred in an ice bath to dissolve methylcellulose to prepare a DMEM/F-12 medium composition containing deacylated gellan gum at final concentrations of 0.01 and 0.02% (w/v) and other methylcellulose at final concentrations of 0.1, 0.2, 0.3, and 0.4% (w/v).

Polystyrene beads (500-600 μm in size, manufactured by Polysciences Inc.) were added to the culture medium prepared above, and the dispersion of the beads was confirmed by visual observation. The evaluation was performed with a ◯ rating for a suspended state, a Δ rating for a partially precipitated/dispersed state, and a × rating for a precipitated state. The results are shown in Table 10.

(Experimental Example 16: Viscosity Measurement of Polysaccharide-Containing Medium Composition)

DMEM/F-12 culture media containing deacylated gellan gum and other polysaccharides at final concentrations of 0.005% and 0.01% (w/v) were prepared using a method similar to that used for the polysaccharide mixture of Experimental Example 15. The final concentrations of the polysaccharides were set to 0.1% (w/v) for xanthan gum, sodium alginate, and locust bean gum, 0.2% (w/v) for methylcellulose, and 0.05% (w/v) for kappa-carrageenan and dimethicone. The properties of each culture medium composition and the viscosity measured using a method similar to that used in Analytical Example 1 are shown in Tables 11-16.

[Table 11]

Xanthan gum concentration% (w/v) Deacylated gellan gum concentration% (w/v) state Viscosity (mPa･s) 0.1 0.005 liquid 4.36 0.1 0.010 liquid 4.59

[Table 12]

Sodium alginate concentration% (w/v) Deacylated gellan gum concentration% (w/v) state Viscosity (mPa･s) 0.1 0.005 liquid 1.53 0.1 0.010 liquid 1.75

[Table 13]

Locust bean gum concentration% (w/v) Deacylated gellan gum concentration% (w/v) state Viscosity (mPa･s) 0.1 0.005 liquid 1.92 0.1 0.010 liquid 2.36

[Table 14]

Methylcellulose concentration% (w/v) Deacylated gellan gum concentration% (w/v) state Viscosity (mPa･s) 0.2 0.005 liquid 3.36 0.2 0.010 liquid 3.81

[Table 15]

κ-carrageenan concentration% (w/v) Deacylated gellan gum concentration% (w/v) state Viscosity (mPa･s) 0.05 0.005 liquid 1.04 0.05 0.010 liquid 1.28

[Table 16]

Dietary gum concentration% (w/v) Deacylated gellan gum concentration% (w/v) state Viscosity (mPa･s) 0.1 0.005 liquid 2.76 0.1 0.010 liquid 3.04

(Experimental Example 17: Preparation of a culture medium composition having a changed divalent metal ion concentration)

Using DMEM/F-12 (D9785, manufactured by Aldrich) without calcium chloride, magnesium sulfate, and magnesium chloride, a DMEM/F-12 medium composition containing 0.02% (w/v) deacylated gellan gum was prepared in the same manner as in Experimental Example 14. DMEM/F-12 medium compositions were prepared with calcium chloride or magnesium sulfate and magnesium chloride added so that the final concentrations were set to the specified amounts of the DMEM/F-12 medium. Considering the specified composition of the DMEM/F-12 medium, the final concentrations were set to 0.116 g/L for calcium chloride, 0.049 g/L for magnesium sulfate, and 0.061 g/L for magnesium chloride.

To the prepared medium composition, dextran beads Cytodex 1 (manufactured by GE Healthcare Life Sciences) were added, and the dispersion state of the beads was determined by visual observation 2 days later. The evaluation was performed with a ○ for a suspended state, a Δ for a partially precipitated/dispersed state, and a × for a precipitated state. The results are shown in Table 17.

(Experimental Example 18: Preparation of a culture medium composition to which divalent metal ions are subsequently added)

A salt solution was prepared by dissolving a 0.1% (w/v) deacylated gellan gum solution, a 5-fold concentration of DMEM/F-12 medium (containing no calcium chloride, magnesium sulfate, or magnesium chloride, D9785, manufactured by Aldrich), calcium chloride (1167 mg), magnesium sulfate (489 mg), and magnesium chloride (287 mg) in pure water (300 mL). The aqueous deacylated gellan gum solution and pure water were placed in a 200 mL beaker and stirred at 200 rpm using an anchor-type stirring blade. Solution A, a mixture of the medium solution and water, was added, and the mixture was stirred for 10 minutes. Subsequently, the salt solution was added, and a 7.5% aqueous sodium bicarbonate solution (1.6 mL) was further added to prepare a DMEM/F-12 medium composition containing deacylated gellan gum at a final concentration of 0.02%. The amounts of each solution mixed are shown in the table. Four hours after preparation, the six medium compositions were evaluated for dispersion on polystyrene beads and Cytodex 1. The results are shown in Tables 18 and 19.

(Experimental Example 19: Preparation of various culture medium compositions)

A 0.1% (w/v) deacylated gellan gum solution and a high-concentration culture medium solution were prepared. As high-concentration culture medium solutions, MEM (M0268, manufactured by Aldrich) with a 10-fold concentration, RPMI-1640 (R6504, manufactured by Aldrich) with a 10-fold concentration, and DMEM (autoclaved corresponding culture medium, manufactured by Nissui) with a 5-fold concentration were prepared. The 0.1% (w/v) deacylated gellan gum solution, various high-concentration culture media, and purified water for concentration adjustment were mixed, and the mixture was heated at 80°C for 30 minutes. The mixture was allowed to cool to room temperature, and a 7.5% aqueous sodium bicarbonate solution was added to prepare culture medium compositions containing deacylated gellan gum at final concentrations of 0.01, 0.02, and 0.03% (w/v).

The polystyrene beads and dextran beads Cytodex1 of the nine prepared medium compositions were evaluated for their suspension and dispersion states, with suspension being rated as ○, partial precipitation/dispersion as Δ, and precipitation as ×. The results are shown in Tables 20 and 21.

(Experimental Example 20: Measurement of Particle Size Distribution of a Culture Medium Composition Containing Deacylated Gellan Gum)

According to Analytical Example 1, a DEME/F-12 medium composition containing 0.038% (w/v) deacylated gellan gum was prepared. The medium was prepared by stirring at 3000 rpm and 6000 rpm for 1 minute using a homomixer. The particle size distribution of the medium composition was measured using a Beckman Instruments Coulter, Inc. Multisizer 4 (an accurate particle size distribution measuring instrument based on the Coulter principle) and the median particle size (d50) of the volume-standardized particle size distribution was determined. The results are shown in Table 22.

[Table 22]

Homomixer revolutions for culture medium preparation d50 (μm) 3000 rpm 1.709 6000 rpm 1.499

(Experimental Example 21: Phosphorylation of Deacylated Gellan Gum)

Deacylated gellan gum (1 g) and pure water (40 mL) were measured in a 100 mL glass test tube, and the mixture was heated at 100°C for 30 minutes to prepare a suspension. An aqueous phosphoric acid solution (85%, 1 g) was added to this suspension, and the mixture was heated under reflux for 5 hours. Subsequently, it was allowed to cool to room temperature while stirring for 12 hours, and the resulting white suspension was poured into 99% ethanol (500 mL). The resulting flocculent white solid was collected by filtration and dried to produce a light brown solid (0.4 g), which is the phosphorylated substance of the deacylated gellan gum. The introduction of the phosphate groups was verified by Fourier transform infrared spectroscopy (manufactured by SHIMADZUCORPORATION, IR-Prestage 21) (1700 cm-1; P-OH, 1296 cm-1, 1265 cm-1; P=O). The light brown solid was decomposed by a microwave heating digestion instrument (ETHOS TC, manufactured by Milestone General), and the phosphorus atom content was measured by an inductively coupled plasma optical emission spectrometer (ICP-OES) (SPS 5520, manufactured by SIINanoTechnology). The result was 3.5 wt% (n=2).

(Experimental Example 22: Preparation of a culture medium composition containing phosphorylated decarboxylated gellan gum)

An optional amount of phosphorylated decarboxylated gellan gum (30 mg) and DMEM/F-12 medium (manufactured by Life Technologies, 1.56 g) were placed in a 200 mL Erlenmeyer flask, and purified water (100 mL) was added. The mixture was sterilized in an autoclave at 121°C for 20 minutes to prepare a DMEM/F-12 medium composition with a decarboxylated gellan gum concentration of 0.03%. Dextran Cytodex 1 beads (manufactured by GE Healthcare Life Sciences) were added to the prepared medium, and the dispersion of the beads was determined by visual observation. The beads were found to be dispersed at a phosphorylated deacylated gellan gum concentration of 0.03% (w/v).

(Experimental Example 23: Preparation of a culture medium composition containing decarboxylated gellan gum)

The aqueous deacylated gellan gum solution and the culture medium solution were mixed at the rates shown in the table below to prepare a DMEM/F-12 culture medium composition having a deacylated gellan gum concentration of 0.02%, and the dispersion state of polystyrene beads (size 500-600 μm, manufactured by Polysciences Inc.) was evaluated. The results are shown in Tables 23 and 24. After being maintained for 1 day or longer, the styrene beads were dispersed under all conditions.

[Table 23]

Deacylated gellan gum/purified water DMEM/F12 powder culture medium/pure water Placement time 20 mg / 10 mL 1.56 g / 90 mL 5 min 20 mg / 20 mL 1.56 g / 80 mL 5 min 20 mg / 30 mL 1.56 g / 70 mL 5 min 20 mg / 40 mL 1.56 g / 60 mL 6 hours 20 mg / 50 mL 1.56 g / 50 mL 6 hours 20 mg / 60 mL 1.56 g / 40 mL 6 hours 20 mg / 70 mL 1.56 g / 30 mL 6 hours 20 mg / 80 mL 1.56 g / 20 mL 1 day 20 mg / 90 mL 1.56 g / 10 mL 1 day

Add "DMEM/F12 powdered medium/pure water" to "deacylated gellan gum/pure water".

[Table 24]

Deacylated gellan gum/purified water DMEM/F12 powder culture medium/pure water Placement time 20 mg / 10 mL 1.56 g / 90 mL 5 min 20 mg / 20 mL 1.56 g / 80 mL 5 min 20 mg / 30 mL 1.56 g / 70 mL 1 hour 20 mg / 40 mL 1.56 g / 60 mL 6 hours 20 mg / 50 mL 1.56 g / 50 mL 6 hours 20 mg / 60 mL 1.56 g / 40 mL 6 hours 20 mg / 70 mL 1.56 g / 30 mL 1 day 20 mg / 80 mL 1.56 g / 20 mL 1 day 20 mg / 90 mL 1.56 g / 10 mL 1 day

Add "deacylated gellan gum/pure water" to "DMEM/F12 powder medium/pure water".

(Experimental Example 24: Preparation of culture medium composition using a filter)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to a final concentration of 0.02 or 0.04% (w/v) and dissolved by heating at 90°C for 30 minutes or at 121°C for 20 minutes. Furthermore, the aqueous solution (100 mL) was filtered with a polyethersulfone membrane filter having a pore size of 0.22 μm (manufactured by Corning Incorporated). Next, the filtrate was mixed with a 2- to 4-fold concentration of DMEM/F-12 medium (manufactured by Sigma Aldrich), and the mixture was shaken for 1 hour using a gentle mixer (SI-24, manufactured by TAITEC Co., Ltd.) to prepare a medium composition containing deacylated gellan gum at a final concentration of 0.01 or 0.015% (w/v) (for example, 25 mL of each 0.02% (w/v) aqueous deacylated gellan gum solution was mixed with a 2-fold concentration of DMEM/F-12 medium to prepare a 0.01% (w/v) deacylated gellan gum medium composition (50 mL)). By a method similar to that in Experimental Example 2, spheres of HepG2 cells were formed, and several dozen spheres were added to the culture medium (1 mL) prepared above, placed at 37°C, and the suspended state of the sphere cells was visually observed after 1 hour and overnight. As a result, it was confirmed that the spheres of HepG2 cells remained suspended in all the above-mentioned culture medium compositions. In addition, add twice the culture medium mentioned, and the cell suspension is centrifuged (500G, 5 min).Determine the ball precipitation of HepG2 cells, and cell can be recovered in all culture medium compositions.After one night, the dispersion state of ball is determined and evaluated by visual observation, and wherein suspended and dispersed state is ○, and partial precipitation/dispersion state is Δ, and precipitation state is ×.Evaluation result is shown in Table 25.

(Experimental Example 25: Cell proliferation test of spheres derived from cultured cell lines)

Human embryonic kidney cell line HEK293 (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) was suspended at 250,000 cells/mL in EMEM medium containing 10% (v/v) fetal bovine serum (manufactured by WAKO), and this suspension (10 mL) was plated on an EZ SPHERE (manufactured by ASAHI GLASS CO., LTD.) and cultured in a CO ₂ incubator (5% CO ₂ ) for 2 days. The suspension (10 mL) of HEK293 cell spheres (diameter 100-200 μm) obtained here was centrifuged (200G, 5 min) to allow the spheres to precipitate, the supernatant was removed, and the spheres were suspended in 1 mL. Next, culture medium (10 mL) was added to the sphere suspension (200 μL, cell number approximately 200,000) to suspend them and the suspension was transferred to a flat-bottom tube (manufactured by BM Equipment Co., Ltd.). Similarly, using a medium composition obtained by adding 0.015% (w/v) deacylated gellan gum (KELCOGELCG-LA, manufactured by SANSHO Co., Ltd.) to the above-mentioned medium, a sphere suspension was produced and transferred to a flat-bottom tube (manufactured by BM Equipment Co., Ltd.). A medium composition supplemented with 0.015% (w/v) deacylated gellan gum was prepared by first suspending deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) in ultrapure water (Milli-Q water) to 0.3% (w/v), dissolving it by heating with stirring at 90°C, sterilizing the aqueous solution in an autoclave at 121°C for 20 min, and adding the solution at a dilution of 1/20 to an EMEM medium containing 10% (v/v) fetal bovine serum.

After the above-mentioned sphere suspension was statically cultured at 37°C in a _CO2 incubator (5% _CO2 ) for 5 days, two times the volume of culture medium was added. The mixture was centrifuged (500G, 5 min) to allow the spheres to precipitate, and the supernatant was removed. Next, the recovered spheres were washed once with PBS (10 mL), 1 mL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added, and the mixture was incubated at 37°C for 5 min. The above-mentioned culture medium (9 mL) was added, and the cells were collected by centrifugation (200G, 5 min). The same amount of trypan blue staining solution (manufactured by Invitrogen Corporation) was added to a portion of the obtained cell suspension (2 mL), and the number of live and dead cells was measured by a hemocytometer (manufactured by ERMA INC.). As a control, a culture medium composition without deacylated gellan gum was produced, and a similar experiment was performed.

The results demonstrated that HEK293 cell spheres can be cultured in a suspended state using the medium composition of the present invention, and that the cells proliferate efficiently in the medium composition. Furthermore, the medium composition of the present invention demonstrated a lower proportion of dead cells during cell proliferation compared to a medium composition lacking deacylated gellan gum, and exhibited an excellent cell proliferation-promoting effect. Spheres cultured in the existing medium settled to the bottom of the culture vessel.

The relative number of HEK293 cells is shown in Table 26, where the number of cells cultured in a medium without deacylated gellan gum is 1. In addition, the relative ratio of dead cells is shown in Table 27, where the ratio of dead cells (number of dead cells/number of live cells) cultured in a medium without deacylated gellan gum is 1.

[Table 26]

Deacylated gellan gum HEK293 cells does not exist Relative cell number 1.0 exist Relative cell number 1.6

[Table 27]

Deacylated gellan gum HEK293 cells does not exist Relative dead cell ratio 1.0 exist Relative dead cell ratio 0.3

(Experimental Example 26: Cell proliferation test by culturing insect cells)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to 0.3% (w/v) and dissolved by heating with stirring at 90°C. The aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, a culture medium composition was prepared by adding deacylated gellan gum to Sf-900 (registered trademark) III SFM culture medium (manufactured by Gibco) at a final concentration of 0.015% (w/v). Immediately thereafter, Sf9 cells (manufactured by Gibco) derived from Spodoptera frugiperda were inoculated into the above-mentioned culture medium composition added with deacylated gellan gum at 100,000 cells/mL and distributed to the wells of a 24-well flat-bottom microplate (manufactured by Corning Incorporated) at 1 mL/well. The cell suspension was cultured by keeping it still in an incubator at 25°C for 5 days. Subsequently, a portion of the culture medium was recovered, the same amount of trypan blue dye solution (manufactured by Invitrogen Corporation) was added, and the number of viable cells was measured using a hemocytometer (manufactured by ERMA INC.). As a control, a culture medium composition without deacylated gellan gum was produced and a similar experiment was performed.

The results demonstrated that the medium composition of the present invention can be used to uniformly culture Sf9 cells in a suspended state and proliferate in the medium composition. Furthermore, the medium composition of the present invention demonstrated superior cell proliferation-promoting effects compared to a medium composition lacking deacylated gellan gum. Table 28 shows the cell count of Sf9 cells after 5 days of static suspension culture.

[Table 28]

Deacylated gellan gum Sf9 cell number (×10000) does not exist 33.5 exist 47.4

(Experimental Example 27: Cell proliferation test by culturing CD34-positive cells)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to 0.3% (w/v) and dissolved by heating with stirring at 90°C. The aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum at a final concentration of 0.015% (w/v), 20 ng/mL thrombopoietin (manufactured by WAKO), and 100 ng/mL stem cell factor (SCF, manufactured by WAKO) to StemSpan SFEM medium (manufactured by StemCell Technologies). Next, the CD34 positive cells (manufactured by Lonza) derived from human umbilical cord blood were seeded into the above-mentioned culture medium composition added with deacylated gellan gum to 10,000 cells/mL and distributed to the wells of a 24-well flat-bottom microplate (manufactured by Corning Incorporated) with 1 mL/well. The cell suspension was placed in a _CO2 incubator (5% _CO2 ) at 37°C for 7 days. Subsequently, a portion of the culture medium was recovered, the same amount of trypan blue staining solution (manufactured by Invitrogen Corporation) was added, and the number of living cells was measured by a hemocytometer (manufactured by ERMA INC.). 3 times the volume of culture medium was added to the culture medium and the mixture was centrifuged (500 G, 5 min) to allow all cells to be precipitated. As a control, a culture medium composition without deacylated gellan gum was produced, and a similar experiment was performed.

The results confirmed that the medium composition of the present invention can be used to uniformly culture CD34-positive cells in a suspended state and proliferate in the medium composition. Furthermore, the medium composition of the present invention was confirmed to exhibit a cell proliferation-promoting effect at a level equivalent to or higher than that of an existing medium lacking deacylated gellan gum. Furthermore, it was confirmed that centrifugation leads to cell precipitation and allows for cell recovery. The relative number of cells proliferated from CD34-positive cells after 7 days of suspended static culture, when the number of cells cultured in a medium lacking deacylated gellan gum is 1, is shown in Table 29.

[Table 29]

Deacylated gellan gum Relative cell number does not exist 1.0 exist 1.2

(Experimental Example 28: Ball Formation Test)

In the same manner as in Experimental Example 2, a composition containing 0.015% deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) and 10% (v/v) fetal bovine serum in DMEM medium (manufactured by WAKO) was prepared. Next, HepG2 cells were added to a cell concentration of 15,000 cells/mL and distributed to a 24-well plate (manufactured by Corning Incorporated) with 1 mL. The plate was suspended and cultured for 7 days at 37°C, and the formation of spheres was confirmed under a microscope. In addition, the sphere cells were precipitated by centrifugation (400 G, 5 min) and washed once with PBS (5 mL). 100 μL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added, and the mixture was incubated at 37°C for 5 min. Here, DMEM culture medium (100 μL) containing 10% (v/v) fetal bovine serum was added to the obtained cell suspension (100 μL), and trypan blue staining solution (manufactured by Invitrogen Corporation) was added in the same amount to a subset of the cell suspension, and the number of viable cells was measured by a hemocytometer (manufactured by ERMA INC.). As a result, it was confirmed that HepG2 cells formed spheres in the culture medium composition of the present invention and increased to 80,800 cells/mL. The spheres of HepG2 cells formed in the culture medium composition of the present invention are shown in Figure 14.

(Experimental Example 29: Cell suspension test using cell line-derived spheres)

Diet gum (KELKO-CRETE DG, manufactured by SANSHO Co., Ltd.) is suspended in ultrapure water (Milli-Q water) to a concentration of 0.3% (w/v) and dissolved by heating with stirring at 90°C. Using this aqueous solution, a DMEM/F-12 culture medium composition with a final concentration of 0.1% (w/v) diet gum is prepared. In addition, an aqueous solution containing 0.5% (w/v) natural type gellan gum (KELCO glue HT, manufactured by San-Ei Gen F.F.I., Inc.) is prepared by heating at 90°C. Using this aqueous solution, a DMEM/F-12 culture medium (manufactured by Sigma Ltd.) containing 0.05 or 0.1% (w/v) natural type gellan gum is prepared.

In the same manner as in Experimental Example 2, HeLa cell spheres were generated, and several dozen spheres were added to each of the culture media (1 mL) prepared above. The mixture was kept still at 37°C for 1 hour, and the suspended state of the spheres was visually observed. The results confirmed that HeLa cell spheres remained suspended in all of the above-mentioned culture medium compositions. Furthermore, centrifugation (200G, 5 minutes) of a cell suspension containing 0.1% (w/v) diterpenoids confirmed that the HeLa cell spheres were precipitated and recovered.

(Experimental Example 30: Cell Suspension Test Using Magnetic Beads Having Cell Adhesion Ability-1)

A suspension of GEM (registered trademark, Global Eukaryotic Microcarrier, manufactured by GL Sciences Inc.) coated with laminin or fibronectin was dispensed into a 1.5 mL volume microtube (manufactured by Eppendorf) at 500 μL, and the GEM was accumulated from the GEM suspension using a magnetic stand (TA4899N12, manufactured by TAMAGAWA SEIKI CO., LTD.) and the solvent was removed. Furthermore, the GEM was washed twice with a DMEM culture medium (manufactured by WAKO, 500 μL) containing 10% (v/v) fetal bovine serum and suspended in the same culture medium (500 μL). The suspension was dispensed into a Sumilon cell tight plate 24F (manufactured by SUMITOMO BAKELITE), a low cell adhesion plate, at 50 μL/well. Next, separately prepared HepG2 cells were added at 250,000 cells/mL, and the final volume was adjusted to 500 μL/well using the same medium. The cell suspension was manually stirred, and the plate was placed in a _CO2 incubator (5% _CO2 ) overnight. After confirming cell adhesion on the GEM using a microscope, the cell suspension was transferred to a 1.5 mL microtube (manufactured by Eppendorf), the GEM with adhered cells was accumulated using the above-mentioned magnetic rack, and the supernatant was removed.

A DMEM medium (manufactured by WAKO) composition containing 0.015% deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) and 10% (v/v) fetal bovine serum was prepared in a similar manner to Experimental Example 2. 1 mL of each of this medium composition or the above medium without deacylated gellan gum was added to the GEM (laminin- or fibronectin-coated) to which HepG2 cells had adhered, as prepared above, and the cells were suspended and transferred to a Sumilon Cell Compact Plate 24F. The plate was then placed in a _CO2 incubator (5% _CO2 ) for 6 days, and the cell culture medium was transferred to a 1.5 mL microtube (manufactured by Eppendorf). The cell-adherent GEM was accumulated by gentle pipetting on the magnetic stand, and the supernatant was removed. The GEM was washed once with PBS (1 mL), 200 μL of a trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added, and the mixture was incubated at 37°C for 10 minutes. To the 200 μL cell suspension obtained here, 800 μL of a DMEM medium containing 10% (v/v) fetal bovine serum was added, the same amount of a trypan blue staining solution (manufactured by Invitrogen Corporation) was added to a portion of the cell suspension, and the number of viable cells was measured using a hemocytometer (manufactured by ERMA INC.).

The results demonstrated that using the medium composition of the present invention, GEMs to which HepG2 cells adhered could be cultured in a suspended state and effectively proliferated in the medium composition. Furthermore, the medium composition of the present invention was shown to exhibit a cell proliferation-promoting effect superior to that of existing culture media lacking deacylated gellan gum. Furthermore, it was demonstrated that GEMs to which HepG2 cells adhered could be collected from the medium composition of the present invention using magnetic force, and further, that HepG2 cells could be recovered from the GEMs.

The cell counts of HepG2 cells after culturing on GEM in a medium containing or without deacylated gellan gum for 6 days are shown in Table 30. In addition, the suspended state of laminin-coated GEM with attached HepG2 cells when cultured in the medium composition of the present invention is shown in FIG14 .

(Experimental Example 31: Cell Suspension Test-2 Using Magnetic Beads Having Cell Adhesion Ability)

In the same manner as in Experimental Example 30, fibronectin-coated GEM (registered trademark, Global Eukaryotic Microcarrier, manufactured by GL Sciences Inc.) was suspended in MF-Medium (registered trademark) mesenchymal stem cell proliferation culture medium (manufactured by TOYOBO CO., LTD.). The suspension was distributed to Sumilon cell tight plates 24F (manufactured by SUMITOMO BAKELITE) at 50 μL/well, which are low-adhesion plates for cells. Next, separately prepared human bone marrow-derived mesenchymal stem cells (manufactured by Cell Applications) were added at 250,000 cells/mL, and in the same manner as in Experimental Example 30, the plate was allowed to stand overnight in a _CO incubator (5% _CO ) to prepare GEM adhered with mesenchymal stem cells.

A MF-Medium (registered trademark) mesenchymal stem cell proliferation medium (manufactured by TOYOBO CO., LTD.) composition containing 0.015% deacylated gellan gum (KELCOGELCG-LA, manufactured by SANSHO Co., Ltd.) was prepared using a method similar to that used in Experimental Example 2. 1 mL of each of this medium composition or the above-mentioned medium without deacylated gellan gum was added to the mesenchymal stem cell-adherent GEM (fibronectin-coated) prepared above, suspended, and transferred to a Sumilon Cell Compact Plate 24F. The plate was then placed in a _CO2 incubator (5% _CO2 ) for 4 days, and the cell culture medium was transferred to a 1.5 mL microtube (manufactured by Eppendorf). The cell-adherent GEM was accumulated by gentle pipetting on the magnetic stand, and the supernatant was removed. The GEM was washed once with PBS (1 mL), 200 μL of a trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added, and the mixture was incubated at 37°C for 10 minutes. To the 200 μL cell suspension obtained here, 800 μL of a DMEM medium containing 10% (v/v) fetal bovine serum was added, the same amount of a trypan blue staining solution (manufactured by Invitrogen Corporation) was added to a portion of the cell suspension, and the number of viable cells was measured using a hemocytometer (manufactured by ERMA INC.).

The results demonstrated that using the medium composition of the present invention, GEMs with attached mesenchymal stem cells can be cultured in a suspended state and effectively proliferate in the medium composition. Furthermore, the medium composition of the present invention demonstrated a cell proliferation-promoting effect superior to that of existing culture media lacking deacylated gellan gum. Furthermore, it was demonstrated that GEMs with attached mesenchymal stem cells can be collected from the medium composition of the present invention using magnetic force, and further, that mesenchymal stem cells can be recovered from the GEMs.

The cell numbers of mesenchymal stem cells after culturing on GEM in a medium containing deacylated gellan gum or without deacylated gellan gum for 4 days are shown in Table 31.

[Table 31]

Deacylated gellan gum Mesenchymal stem cell count (×10000/mL) does not exist 11.3 exist 20.9

(Experimental Example 32: Cell suspension test using alginate beads)

The following test is carried out according to the method for the three-dimensional culture kit of alginate manufactured by PG Research. The HepG2 cells prepared separately are added to a sodium alginate solution (manufactured by PG research, 2.5 mL) with 400,000 cells/mL, and human recombinant laminin 511 (manufactured by Veritas Ltd.) is further added to prepare a cell suspension with 5 μg/mL. The cell suspension is recovered with a 5 mL syringe (manufactured by TERUMOCORPORATION) with a gavage needle, and a 22G injection needle (manufactured by TERUMO CORPORATION) is installed to the syringe. Immediately thereafter, the cell suspension is added to each hole of a 24-well flat-bottom microplate (manufactured by PG research) with 10 drops of each aqueous calcium chloride solution (manufactured by PG research). The mixture is kept at room temperature for 10 min, confirming the formation of alginate beads, removing the calcium chloride solution, adding PBS (2 mL), and the mixture is left at room temperature for 15 min. Furthermore, PBS was removed, and DMEM medium (manufactured by WAKO, 2 mL) containing 10% (v/v) fetal bovine serum was added, and the mixture was allowed to stand at room temperature for 15 minutes. The medium was removed, and a DMEM medium (manufactured by WAKO) composition containing 0.03% deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) and 10% (v/v) fetal bovine serum (prepared by a method similar to that in Experimental Example 2) or the above medium without deacylated gellan gum was added to each well at 1 mL, and the mixture was statically cultured in a _CO2 incubator (5% _CO2 ) for 8 days. The medium was exchanged on the 4th day of culture.

The cultured alginate beads were transferred to a 1.5 mL micro tube (manufactured by Eppendorf) using a 1 mL pipette tip, sodium citrate solution (1 mL, manufactured by PG research) was added to each tube, and the mixture was stirred at room temperature for 15 minutes to dissolve the alginate beads. Next, the cells were pelleted by centrifugation at 300G for 3 minutes, and the supernatant was removed. 200 μL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added to the cells, and the mixture was incubated at 37°C for 5 minutes. To the obtained cell suspension (200 μL), 800 μL of DMEM medium containing 10% (v/v) fetal bovine serum was added, and to a portion of the cell suspension, the same amount of trypan blue staining solution (manufactured by Invitrogen Corporation) was added, and the number of viable cells was measured using a hemocytometer (manufactured by ERMA INC.).

The results demonstrated that using the medium composition of the present invention, alginate beads encapsulating HepG2 cells could be cultured in a suspended state and effectively proliferated in the medium composition. Furthermore, the medium composition of the present invention demonstrated a cell proliferation-promoting effect superior to that of existing culture media lacking deacylated gellan gum.

The cell numbers of HepG2 cells when cultured for 8 days in alginate beads in a medium containing or without deacylated gellan gum are shown in Table 32. In addition, when alginate beads encapsulating HepG2 cells were cultured in the medium composition of the present invention, the suspended state is shown in FIG16 .

[Table 32]

Deacylated gellan gum HepG2 cell count (×10000/mL) does not exist 34.9 exist 51.8

(Experimental Example 33: Cell Suspension Test Using Collagen Gel Capsules)

A: Tissue Culture Collagen Cell Matrix (registered trademark) Type IA (cell matrix, manufactured by Nitta Gelatin Inc.), B: 10-fold concentrated DMEM/F-12 medium (manufactured by Aldrich), and C: Reconstitution buffer (prepared by adding sodium bicarbonate (2.2 g) and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)) (4.77 g) to a 0.05N sodium hydroxide solution (100 mL) and sterilizing the mixture by filtration) were mixed at a ratio of A:B:C = 8:1:1 while cooling on ice. Furthermore, human recombinant laminin 511 (manufactured by Veritas Ltd.) was added at 5 μg/mL to prepare a collagen mixed solution (500 μL). To the mixed solution, HepG2 cells prepared separately at 200,000 cells/mL were added, and the total amount was recovered using a 1.5 mL syringe (manufactured by TERUMO CORPORATION) with a 25G injection needle (manufactured by TERUMO CORPORATION). Next, the cell suspension was added dropwise using the syringe to a flat-bottomed tube (manufactured by BM Equipment Co., Ltd.) containing 10% (v/v) fetal bovine serum in a DMEM medium (manufactured by WAKO) (10 mL) pre-incubated at 37°C. The mixture was incubated in a 37°C water bath for 10 minutes, and the formation of an indefinite collagen gel capsule with a diameter of approximately 2 mm was confirmed. Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was added to a final concentration of 0.04% by a method similar to that in Experimental Example 2, and the capsule was suspended by gentle stirring. Next, the tube was statically cultured in a CO ₂ incubator (5% CO ₂ ) for 5 days.

PBS (25 mL) was added to the culture medium containing the collagen gel capsules, and the collagen gel capsules were precipitated by centrifugation at 400G for 5 minutes, and the supernatant was removed. PBS (25 mL) was added again, and the mixture was centrifuged, and the supernatant was removed to a remaining amount of 5 mL. To this solution, 1% (w/v) collagenase L (manufactured by Nitta Gelatin Inc., 20 μL) was added, and the mixture was shaken at 37°C for 2 hours. After confirming that the collagen gel had dissolved, PBS (10 mL) was added, and the cells were precipitated by centrifugation at 400G for 5 minutes, and the supernatant was removed. 1 mL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added to the cells, and the mixture was incubated at 37°C for 5 minutes. To the resulting cell suspension, 4 mM DMEM medium containing 10% (v/v) fetal bovine serum was added, and the cells were precipitated by centrifugation at 400G for 5 minutes, and the supernatant was removed. The obtained cells were suspended in 2 mL of the same medium as above, and the same amount of trypan blue staining solution (manufactured by Invitrogen Corporation) was added to a portion thereof, and the number of living cells was measured by a hemacytometer (manufactured by ERMA INC.).

The results demonstrated that the medium composition of the present invention enables the culture of collagen gel capsules encapsulating HepG2 cells in a suspended state, and that the cells effectively proliferate in the medium composition. Furthermore, the medium composition of the present invention demonstrated a cell proliferation-promoting effect superior to that of existing culture media lacking deacylated gellan gum.

The cell counts of HepG2 cells when cultured for 5 days in collagen gel capsules in a medium containing or without deacylated gellan gum are shown in Table 33. Furthermore, when collagen gel capsules encapsulating HepG2 cells were cultured in the medium composition of the present invention, the suspended state is shown in FIG17 .

[Table 33]

Deacylated gellan gum HepG2 cell count (×10000/mL) does not exist 62.4 exist 106.0

(Experimental Example 34: Ball Recovery Test Using Filter)

By a method similar to that in Experimental Example 2, a DMEM medium (manufactured by WAKO) composition containing 0.015% deacylated gellan gum (KELCOGELCG-LA, manufactured by SANSHO Co., Ltd.) and 10% (v/v) fetal bovine serum was prepared. In addition, as a control, the same medium without deacylated gellan gum was prepared. HepG2 cell spheres were formed by a method similar to that in Experimental Example 2, and 86,000 cells were added to the culture medium (1 mL) prepared as above, and the mixture was allowed to stand at 37°C for 1 hour, and the sphere cell suspension was visually observed. In addition, the cell suspension was added to Cell Strainers (manufactured by Becton, Dickinson and Company) with a mesh size of 40 μm to capture the spheres on the filter. Subsequently, PBS (10 mL) was drained from the back of the filter to recover the spheres in a 15 mL tube, and the spheres were precipitated by centrifugation at 300G for 5 minutes. The supernatant was recovered, 500 μL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added to the spheres, and the mixture was incubated at 37°C for 5 min. DMEM medium (1 mL) containing 10% (v/v) fetal bovine serum was added to the obtained cell suspension, and the same amount of trypan blue staining solution (manufactured by Invitrogen Corporation) was added to a portion thereof, and the number of viable cells was measured using a hemocytometer (manufactured by ERMA INC.). The results showed that the HepG2 cell spheres remained suspended in the above-mentioned medium composition. In addition, it was confirmed that the cells of the HepG2 cell spheres could be recovered at a recovery rate equivalent to that of the medium without deacylated gellan gum by treating the sphere suspension containing 0.015% deacylated gellan gum through a filter. The relative number of cells recovered from the medium containing deacylated gellan gum is shown in Table 34, where the number of HepG2 cells recovered using the filter and the medium without deacylated gellan gum is 1.

[Table 34]

Deacylated gellan gum Relative HepG2 cell number does not exist 1.0 exist 1.1

(Experimental Example 35: Cell Suspension Test Using a Combination Reagent of Multiple Polysaccharides)

A DMEM/F-12 medium composition containing xanthan gum (KELTROL CG, manufactured by SANSHO Co., Ltd.), sodium alginate (Duck alginic acid NSPM, manufactured by FOOD CHEMIFA Co., Ltd.), locust bean gum (GENUGUM RL-200-J, manufactured by SANSHO Co., Ltd.), methylcellulose (cP400, manufactured by WAKO), κ-carrageenan (GENUGEL WR-80-J, manufactured by SANSHO Co., Ltd.), pectin (GENU pectin LM-102AS, manufactured by SANSHO Co., Ltd.) or a combination of diterpenoid (KELCO CRETE DG-F, manufactured by SANSHO Co., Ltd.) and deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was prepared by a method similar to that in Experimental Example 15. In the same manner as in Experimental Example 2, HepG2 cell spheres were generated, and several dozen spheres were added to each of the culture media (1 mL) prepared above. The mixture was left to stand at 37°C for 1 hour or overnight, and the suspended state of the spheres was visually observed. The results confirmed that HepG2 cell spheres remained suspended in all of the above-mentioned medium compositions. Furthermore, adding a double amount of medium and centrifuging the cell suspension (500g, 5 minutes) resulted in the sedimentation and recovery of HepG2 cell spheres in all of the medium compositions. The dispersion state of the spheres after overnight observation was determined by visual observation, with suspended and dispersed states indicated as ○, partially precipitated/dispersed states indicated as Δ, and precipitated states indicated as ×. The evaluation results are shown in Tables 35 and 36. In the tables, - indicates not performed.

Comparison of dispersibility of beads and cells-1

The dispersion states of dextran beads Cytodex (registered trademark) 1 (manufactured by GE Healthcare Life Sciences) and HeLa cell spheroids were compared between the culture medium containing deacylated gellan gum (Comparative Example) prepared above and the culture medium containing methylcellulose. The results are shown in the table. Since the dispersion states of Cytodex 1 and HeLa cell spheroids correlate well, Cytodex 1 can be used as a spheroid model.

Comparison of dispersibility of beads and cells-2

The dispersion states of polystyrene beads (500-600 μm, manufactured by Polysciences Inc.) and HepG2 cell spheroids were compared between the polysaccharide prepared in Experimental Example 15 and a culture medium containing deacylated gellan gum. Suspended and dispersed states were evaluated as ○, partially precipitated/dispersed states as Δ, and precipitated states as ×. The results are shown in the table. Since the dispersion states of the polystyrene beads and HepG2 cell spheroids correlated well, the polystyrene beads can be used as a spheroid model.

(Experimental Example 36: Floating Culture Test of Rice-Derived Plant Callus)

Fifty fully mature rice Nipponbare seeds (purchased from Koto Agricultural Cooperatives) selected with a saline solution were transferred to a 50 mL polystyrene tube (manufactured by BD Falcon), washed with sterile water (50 mL), and stirred in 70% ethanol water (30 mL) for 1 minute. The ethanol water was removed, Kitchen Haiter (manufactured by Kao Corporation, 30 mL) was added, and the mixture was stirred for 1 hour. The Kitchen Haiter was removed and washed four times with sterile water (50 mL). The sterilized seeds were cultured on Murashige Skoog basal medium (M9274, manufactured by Sigma Aldrich) containing 2 μg/mL 2,4-dichlorophenoxyacetic acid (manufactured by Sigma Aldrich) and agar at 1.5 mL/well in a 24-well flat-bottom microplate (manufactured by Corning Incorporated). They were cultured at 30°C, 16 hr dark/8 hr dark conditions for 3 weeks, and cream-colored calli (1-2 mm) grown on the seed blastocysts were harvested.

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) is suspended in ultrapure water (Milli-Q water) to 0.3% (w/v), and dissolved by heated and stirred at 90 ℃.The aqueous solution was sterilized 20 minutes at 121 ℃ in an autoclave.Use this solution, by adding deacylated gellan gum at a final concentration of 0.03% (w/v) in the Murashige Skoog basal medium (M9274, manufactured by Sigma Aldrich) containing 2 μ g/mL 2,4-dichlorophenoxyacetic acid (manufactured by Sigma Aldrich) to prepare culture medium compositions.15 calli prepared above are added in this culture medium compositions with 10 mL/ flat-bottomed tubes (manufactured by BM Equipment Co., Ltd.), and shaken at 25 ℃ for 7 days. As a result, it was confirmed that the rice-derived callus can be cultured in a suspended state using the medium composition of the present invention, and the callus is maintained in the medium composition. When the rice-derived callus is cultured in the medium composition of the present invention, the suspended state is shown in FIG18 .

(Experimental Example 37: Cell proliferation test by dispersing HeLa cells)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to 0.3% (w/v) and dissolved by heating and stirring at 90°C. This aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum to a final concentration of 0.015% (w/v) or 0.030% (w/v) to DMEM medium containing 10% (v/v) fetal bovine serum (manufactured by WAKO). Next, the human cervical cancer cell line HeLa (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) was seeded at 50,000 cells/mL into the above-mentioned medium composition supplemented with deacylated gellan gum and distributed to 200 μL/well of a 96-well flat-bottom ultra-low adhesion surface microplate (manufactured by Corning Incorporated, #3474). As a negative control, HeLa cells were suspended in the above-mentioned medium without deacylated gellan gum and the suspension was dispersed. The plate was then kept stationary in a _CO2 incubator (37°C, 5% _CO2 ) for 8 days for culture. After 3 and 8 days of culture, WST-8 solution (manufactured by DOJINDO Laboratories, 20 μL) was added to the medium, and the mixture was incubated at 37°C for 100 minutes. The absorbance was measured at 450 nm using an absorbance spectrometer (manufactured by Molecular Devices, SPECTRA MAX 190), and the number of viable cells was measured by subtracting the absorbance of the culture medium alone. The cell-containing culture medium after 8 days of culture was stirred with a pipette, and the resulting stirred solution (20 μL) was mixed with 0.4% Trypan Blue Stain (manufactured by Invitrogen, 20 μL), and the cell density was measured under a microscope.

The results demonstrated that HeLa cells could be cultured in a uniformly dispersed state without forming excessively large cell aggregates using the medium composition of the present invention, and that they proliferated efficiently in the medium composition. Microscopic observation of HeLa cell aggregates after 8 days of culture is shown in Figure 19 . Furthermore, the absorbance at 450 nm (corresponding to the number of HeLa cells) after 3 and 8 days of static culture is shown in Table 40 . The number of HeLa cells after 8 days of culture is shown in Table 41 .

[Table 41]

Experimental group Negative control 3.7 Deacylated gellan gum 0.015% 8.9 Deacylated gellan gum 0.030% 11.2

(Experimental Example 38: Cell proliferation test by dispersing A549 cells and HCT116 cells)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to 0.3% (w/v) and dissolved by heating and stirring at 90°C. The aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum to a final concentration of 0.015% (w/v) in DMEM medium or McCoy's 5a medium (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) containing 10% (v/v) fetal bovine serum (manufactured by WAKO). Next, human lung cancer cell line A549 (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) or human colorectal cancer cell line HCT116 (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) were seeded at 50,000 cells/mL into the above-mentioned medium composition supplemented with deacylated gellan gum and dispensed at 200 μL/well into the wells of a 96-well flat-bottom ultra-low adhesion surface microplate (manufactured by Corning Incorporated, #3474). As a negative control, A549 and HCT116 cells were suspended in the above-mentioned medium without deacylated gellan gum and the suspension was dispersed. The plate was then kept stationary in a _CO2 incubator (37°C, 5% _CO2 ) for 7 days for culture. After culturing for 3, 5, and 7 days, a WST-8 solution (manufactured by DOJINDO Laboratories, 20 μL) was added to the culture medium, and the mixture was incubated for 100 min at 37° C. The absorbance was measured at 450 nm using an absorbance spectrometer (manufactured by Molecular Devices, SPECTRA MAX 190), and the number of viable cells was measured by subtracting the absorbance of the culture medium alone.

The results demonstrated that the medium composition of the present invention enables A549 and HCT116 cells to be cultured in a uniformly dispersed state without forming excessively large cell aggregates, and that they proliferate efficiently in the medium composition. Microscopic observation of A549 and HCT116 cell aggregates after 5 days of culture is shown in FIG20 . Furthermore, the absorbance at 450 nm (corresponding to the number of A549 cells) after 3, 5, and 7 days of static culture (corresponding to the number of HCT116 cells) is shown in Table 42, and the absorbance at 450 nm (corresponding to the number of HCT116 cells) is shown in Table 43.

(Experimental Example 39: Cell proliferation test using a low-adhesion surface plate with a U-shaped bottom)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to 0.3% (w/v) and dissolved by heating and stirring at 90°C. This aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum to a final concentration of 0.015% (w/v) to DMEM medium containing 10% (v/v) fetal bovine serum (manufactured by WAKO). Next, the human cervical cancer cell line HeLa (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) was seeded at 50,000 cells/mL into the above-mentioned medium composition supplemented with deacylated gellan gum and dispensed at 200 μL/well into the wells of a 96-well U-bottom low-adhesion surface microplate (manufactured by SUMITOMO BAKELITE, #MS-9096U). As a negative control, HeLa cells were suspended in the above-mentioned medium without deacylated gellan gum and the suspension was dispersed. The plate was then kept stationary in a _CO2 incubator (37°C, 5% _CO2 ) for 7 days for culture. A WST-8 solution (manufactured by DOJINDO Laboratories, 20 μL) was added to the culture medium after 2, 5, and 7 days of culture, and the mixture was incubated at 37°C for 100 minutes. The absorbance was measured at 450 nm by an absorbance spectrometer (manufactured by Molecular Devices, SPECTRA MAX 190), and the number of viable cells was measured by subtracting the absorbance of the culture medium alone.

The results confirmed that efficient proliferation was achieved even in other low-adhesion plates using the medium composition of the present invention. The absorbance at 450 nm (corresponding to the number of HeLa cells) after 2, 5, and 7 days of static culture is shown in Table 44.

(Experimental Example 40: Cell proliferation test using a low-adhesion surface plate of another company)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to 0.3% (w/v) and dissolved by heating and stirring at 90°C. This aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum to a DMEM medium containing 10% (v/v) fetal bovine serum (manufactured by WAKO) at a final concentration of 0.005% and 0.030% (w/v). Next, the human cervical cancer cell line HeLa (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) was inoculated at 50,000 cells/mL into the above-mentioned medium composition supplemented with deacylated gellan gum and dispensed at 200 μL/well into the wells of a 96-well flat-bottomed low-adhesion surface microplate (manufactured by IWAKI, #Ez-BindShut). As a negative control, HeLa cells were suspended in the above-mentioned medium without deacylated gellan gum and the suspension was dispersed. Next, the plate was kept stationary in a _CO2 incubator (37°C, 5% _CO2 ) for 7 days for culture. WST-8 solution (manufactured by DOJINDO Laboratories, 20 μL) was added to the culture medium after 3 days of culture, and the mixture was incubated at 37°C for 100 min. The absorbance was measured at 450 nm by an absorbance spectrometer (manufactured by Molecular Devices, SPECTRA MAX 190), and the number of viable cells was measured by subtracting the absorbance of the culture medium alone.

The results confirmed that efficient proliferation was achieved even in other low-adhesion plates using the medium composition of the present invention. The absorbance at 450 nm (corresponding to the number of HeLa cells) after 3 days of static culture is shown in Table 45.

(Experimental Example 41: Comparative Cell Proliferation Test Using Happy Cell ASM Medium)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to 0.3% (w/v) and dissolved by heating and stirring at 90°C. The aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum to a final concentration of 0.015% (w/v) to DMEM medium containing 10% (v/v) fetal bovine serum (manufactured by WAKO). HappyCell ASM medium (manufactured by biocroi) and DMEM medium (manufactured by WAKO) were adjusted to a given concentration in advance (mixed at a 1:1 ratio). Next, the human cervical cancer cell line HeLa (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) or the human lung cancer cell line A549 (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) were seeded at 50,000 cells/mL into the above-mentioned medium composition or Happy Cell ASM medium composition supplemented with deacylated gellan gum, and dispensed at 200 μL/well into the wells of a 96-well flat-bottom ultra-low adhesion surface microplate (manufactured by Corning Incorporated, #3474). As a negative control, HeLa and A549 cells were suspended in the above-mentioned medium without deacylated gellan gum and the suspension was dispersed. The plates were then kept stationary in a _CO2 incubator (37°C, 5% _CO2 ) for 5 days for culture. To the culture medium after culturing for 3 and 5 days, a WST-8 solution (manufactured by DOJINDO Laboratories, 20 μL) was added, and the mixture was incubated at 37° C. for 100 min. The absorbance was measured at 450 nm by an absorbance spectrometer (manufactured by Molecular Devices, SPECTRA MAX 190), and the number of viable cells was measured by subtracting the absorbance of the culture medium alone.

The results demonstrated that cells proliferated more efficiently using the medium composition of the present invention compared to Happy Cell ASM. The absorbance at 450 nm (corresponding to the number of HeLa cells) after 3 and 5 days of static culture is shown in Table 46, and the absorbance at 450 nm (corresponding to the number of A549 cells) is shown in Table 47.

(Experimental Example 42: Cell proliferation test using other polysaccharides)

KELCO CRETE DG-F (manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to 1.5% (w/v) and dissolved by heating and stirring at 90°C. The aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding KELCO CRETE DG-F to a DMEM medium containing 10% (v/v) fetal bovine serum (manufactured by NISSUI PHARMACEUTICAL CO., LTD.) at a final concentration of 0.2% and 0.3% (w/v). Next, the human lung cancer cell line A549 (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) was inoculated into the above-mentioned medium composition supplemented with dimethicone at 50,000 cells/mL and dispensed into the wells of a 96-well flat-bottom ultra-low adhesion surface microplate (manufactured by Corning Incorporated, #3474) at 200 μL/well. As a negative control, A549 cells were suspended in the above-mentioned medium without dimethicone and the suspension was dispersed. Next, the plate was kept stationary in a _CO2 incubator (37°C, 5% _CO2 ) for 3 days for culture. WST-8 solution (manufactured by DOJINDO Laboratories, 20 μL) was added to the culture medium after 3 days of culture, and the mixture was incubated at 37°C for 100 min. The absorbance was measured at 450 nm by an absorbance spectrometer (manufactured by Molecular Devices, SPECTRA MAX 190), and the number of viable cells was measured by subtracting the absorbance of the culture medium alone.

The results confirmed the effective proliferation of the medium composition of the present invention containing other polysaccharides. The absorbance at 450 nm (corresponding to the number of A549 cells) after 3 days of static culture is shown in Table 48.

(Experimental Example 43: Cell Proliferation Test Using Various Anticancer Drugs)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to 0.3% (w/v) and dissolved by heating and stirring at 90°C. The aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum to a final concentration of 0.030% (w/v) and various anticancer drugs to a DMEM medium containing 10% (v/v) fetal bovine serum (manufactured by WAKO) at final concentrations of 0.001, 0.01, 0.1, and 1 μM. The anticancer drugs used were doxorubicin (manufactured by WAKO), paclitaxel (manufactured by WAKO), or mitomycin C (manufactured by WAKO). Next, human cervical cancer cell line HeLa (manufactured by DS PHARMABIOMEDICAL CO., LTD.) was seeded at 50,000 cells/mL into the above-mentioned medium composition supplemented with deacylated gellan gum, and 200 μL/well was dispensed into the wells of a 96-well flat-bottom ultra-low adhesion surface microplate (manufactured by Corning Incorporated, #3474).

As no addition, HeLa cells are suspended in the above-mentioned culture medium containing only the deacylated gellan gum with a final concentration of 0.030% (w/v) and the suspension is dispersed. Immediately thereafter, the plate is kept stationary for 7 days in a _CO incubator (37°C, 5% CO ₂ ) and cultured. After cultivation for 3, 5, and 7 days, WST-8 solution (manufactured by DOJINDO Laboratories, 20 μL) is added to the culture medium, and the mixture is incubated for 100 min at 37°C. Absorbance is measured at 450 nm by an absorbance spectrometer (manufactured by Molecular Devices, SPECTRA MAX 190), and the number of living cells is measured by the absorbance measurement of the culture medium alone. The culture medium containing cells after 5 days of cultivation is stirred with a pipette and the solution (20 μL) after the stirring obtained is mixed with trypan blue stain 0.4% (manufactured by Invitrogen, 20 μL), and cell density is measured under a microscope.

The results demonstrate that the medium composition of the present invention can be used to effectively evaluate anticancer drugs using a cell proliferation assay. Furthermore, the absorbance at 450 nm (corresponding to the number of HeLa cells) after 3, 5, and 7 days of static culture is shown in Table 49. The cell density of HeLa cells after 5 days is shown in Table 50.

[Table 50]

Experimental group No additives 4.8 Doxorubicin 0.001 μM 5.1 Doxorubicin 0.01 μM 2.6 Doxorubicin 0.1 μM 1.7 Doxorubicin 1 μM 0.6 Paclitaxel 0.001 μM 5.3 Paclitaxel 0.01 μM 1.6 Paclitaxel 0.1 μM 0.9 Paclitaxel 1 μM 0.2 Mitomycin C 0.01 μM 1.0 Mitomycin C 0.1 μM 0.4 Mitomycin C 1 μM 0.1

(Experimental Example 44: Maintenance and Functional Testing of Human Primary Hepatocytes)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to 0.3% (w/v) and dissolved by heating with stirring at 90°C. The aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum to HBM culture medium (manufactured by Lonza Japan) supplemented with additives (HCM single Quots (registered trademark), BSA-free fatty acids, EGF, ascorbic acid, transferrin, insulin, GA-1000, hydrocortisone 21 hemisuccinate; Lonza Japan) at a final concentration of 0.015% or 0.030% (w/v). Next, frozen primary human hepatocytes (manufactured by Xenotech) were seeded at 250,000 cells/mL into the above-mentioned medium composition supplemented with deacylated gellan gum and dispensed at 200 μL/well into the wells of a 96-well U-bottom ultra-low adhesion surface microplate (manufactured by SUMITOMO BAKELITE, PrimeSurface, #MS-9096U). As a negative control, primary human hepatocytes were suspended in the above-mentioned medium without deacylated gellan gum and the suspension was dispersed. The plate was then incubated in a _CO2 incubator (37°C, 5% _CO2 ) for 3 days.

1. Measure the number of viable cells

To the culture medium after culturing for 4 hours, 8 hours, and 1 day, a WST-8 solution (manufactured by DOJINDO Laboratories, 20 μL) was added, and the mixture was incubated for 100 minutes at 37° C. The number of viable cells was measured by measuring absorbance at 450 nm using an absorbance spectrometer (manufactured by Molecular Devices, SPECTRA MAX 190).

2. Analysis of Albumin Secretion

After culturing for 3 days, the culture medium containing the hepatocytes was recovered, and the culture supernatant was recovered by centrifugation (400 g, 3 min). The concentration of human albumin in the culture medium was measured using an Albumin ELISA Quantitation Kit (manufactured by Bethyl Laboratories).

3. mRNA expression analysis by real-time PCR

The culture medium containing hepatocytes after 8 hours of recovery cultivation, and by centrifugal (400g, 3 min) reclaiming cell.Use RNeasy Mini test kit (manufactured by QIAGEN) to extract total RNA from cell.Use total RNA and PrimeScript (registered trademark) RT Master Mix (manufactured by Takara Bio Inc.), use GeneAmp PCR System 9700 (manufactured by Applied Biosystems) to carry out reverse transcription reaction to synthesize cDNA.By dispersing and diluting 1/10 with sterile water, obtain as each cDNA sample for PCR reaction.In addition, as the sample to be used for calibration curve, use the cDNA dispersed and mixed, and be arranged in the quantitative range of 1/3 to 1/243 dilution with 3 times of common ratios.Use every kind of cDNA sample, calibration sample, Premix Ex Taq (registered trademark) (manufactured by Takara Bio Inc.) and multiple Taqman probes (manufactured by Applied Biosystems) and 7500 Real-time PCR System (manufactured by Applied Biosystems) to carry out PCR reaction. Specificity was calculated using GAPDH mRNA as an endogenous control, and the expression of each mRNA was corrected with the value of GAPDH (glyceraldehyde 3-phosphate dehydrogenase), with the negative control as 100%.

Each probe used (manufactured by Applied Biosystems) is shown below.

GAPDH:HS99999905

Albumin: HS99999922

Cyp3A4:HS00604506

Cyp2C9:HS02383631

PXR (Pregnane X receptor): HS01114267

ApoA1 (Apolipoprotein A1):HS00163641.

The results demonstrated that the medium composition of the present invention maintains human primary hepatocytes in a dispersed state and inhibits the decrease in viable cell count through protection. Furthermore, the medium composition demonstrated higher albumin production and higher expression of mRNA panels related to pharmacokinetics compared to the negative control. The absorbance at 450 nm (corresponding to the number of human primary hepatocytes) after 4 hours, 8 hours, and 1 day of static culture is shown in Table 51. The albumin values of the culture supernatant after 3 days of suspension static culture are shown in Table 52. Furthermore, the expression values of each mRNA based on the negative control after 8 hours of static culture, which was taken as 100%, are shown in Table 53. The cellular status of human primary hepatocytes after 4 hours of culture is shown in Figure 21.

[Table 52]

Experimental group Albumin (ng/mL) Negative control 452 Deacylated gellan gum 0.015% 614 Deacylated gellan gum 0.030% 685

[Table 53]

Negative control Deacylated gellan gum 0.015% Deacylated gellan gum 0.030% albumin 100 124 135 Cyp3A4 100 101 113 Cyp2C9 100 106 125 PXR 100 117 202 ApoA1 100 95 153

(Experimental Example 45: Maintenance and Functional Testing of Cynomolgus Monkey Primary Hepatocytes)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to 0.3% (w/v) and dissolved by heating with stirring at 90°C. The aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum to HBM culture medium (manufactured by Lonza Japan) supplemented with additives (HCM single Quots (registered trademark), BSA-free fatty acids, EGF, ascorbic acid, transferrin, insulin, GA-1000, hydrocortisone 21 hemisuccinate; Lonza Japan) at a final concentration of 0.015% or 0.030% (w/v). Next, frozen cynomolgus monkey primary hepatocytes (manufactured by Ina Research) were seeded at 250,000 cells/mL into the above-mentioned medium composition supplemented with deacylated gellan gum and dispensed at 200 μL/well into the wells of a 96-well U-bottom ultra-low adhesion surface microplate (manufactured by SUMITOMO BAKELITE, PrimeSurface, #MS-9096U). As a negative control, cynomolgus monkey primary hepatocytes were suspended in the above-mentioned medium without deacylated gellan gum and the suspension was dispersed. The plate was then kept stationary in a _CO2 incubator (37°C, 5% _CO2 ) for 3 days for culture.

1. Measure the number of viable cells

To the culture medium after culturing for 4 hours, 8 hours, 1 day, and 3 days, a WST-8 solution (manufactured by DOJINDO Laboratories, 20 μL) was added, and the mixture was incubated at 37° C. for 100 minutes. The number of viable cells was measured by measuring absorbance at 450 nm using an absorbance spectrometer (manufactured by Molecular Devices, SPECTRA MAX 190).

2. Analysis of Albumin Secretion

After culturing for 3 days, the culture medium containing the hepatocytes was recovered, and the culture supernatant was recovered by centrifugation (400 g, 3 min). The concentration of human albumin in the culture medium was measured using an Albumin ELISA Quantitation Kit (manufactured by Bethyl Laboratories).

3. mRNA Expression Analysis by Real-time PCR

The culture medium containing hepatocytes after 1,2,3 days of recovery cultivation, and by centrifugal (400g, 3 min) reclaiming cells.Use RNeasy Mini test kit (manufactured by QIAGEN) to extract total RNA from cells.Use total RNA and PrimeScript (registered trademark) RT Master Mix (manufactured by Takara Bio Inc.), use GeneAmp PCR System 9700 (manufactured by Applied Biosystems) to carry out reverse transcription reaction to synthesize cDNA.By dispersing and diluting 1/10 with sterile water, obtain as each cDNA sample for PCR reaction.In addition, as the sample to be used for calibration curve, use the cDNA dispersed and mixed, and be arranged in the quantitative range of 1/3 to 1/243 dilution with 3 times of common ratios.Use every kind of cDNA sample, calibration sample, Premix Ex Taq (registered trademark) (manufactured by Takara Bio Inc.) and multiple Taqman probes (manufactured by Applied Biosystems) and 7500 Real-time PCR System (manufactured by Applied Biosystems) to carry out PCR reaction. Specificity was calculated using GAPDH mRNA as an endogenous control, and the expression of each mRNA was corrected by the value of GAPDH.

Each probe used (manufactured by Applied Biosystems) is shown below.

GAPDH:Rh02621745

Albumin: Rh02789672

ApoA1 (Apolipoprotein A1):Rh02794272.

The results demonstrated that the medium composition of the present invention inhibits the decrease in viable cell count by protecting cynomolgus monkey primary hepatocytes. Furthermore, hepatocytes cultured in the medium composition demonstrated higher albumin production and higher expression of albumin and ApoA1 mRNAs compared to the negative control. The absorbance at 450 nm (corresponding to the number of cynomolgus monkey primary hepatocytes) after 4 hours, 8 hours, 1 day, and 3 days of static culture is shown in Table 54. The albumin levels in the culture supernatant after 3 days of static culture are shown in Table 55. Furthermore, the albumin mRNA expression levels after 2 and 3 days of static culture, based on the negative control as 100%, are shown in Table 56, and the ApoA1 mRNA expression levels are shown in Table 57. The cell status of cynomolgus monkey primary hepatocytes after 4 hours of culture is shown in Figure 22.

[Table 55]

Experimental group Albumin (ng/mL) Negative control 420 Deacylated gellan gum 0.015% 485 Deacylated gellan gum 0.030% 499

[Table 56]

albumin Negative control Deacylated gellan gum 0.015% Deacylated gellan gum 0.030% Day 2 100 124 111 Day 3 100 132 153

[Table 57]

ApoA1 Negative control Deacylated gellan gum 0.015% Deacylated gellan gum 0.030% Day 2 100 139 221 Day 3 100 118 105

(Experimental Example 46: Maintenance and Functional Testing of Hepatocytes in Collagen-Coated Microplates)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to 0.3% (w/v) and dissolved by heating with stirring at 90°C. The aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum to HBM culture medium (manufactured by Lonza Japan) supplemented with additives (HCM single Quots (registered trademark), BSA-free fatty acids, EGF, ascorbic acid, transferrin, insulin, GA-1000, hydrocortisone 21 hemisuccinate; Lonza Japan) at a final concentration of 0.015% or 0.030% (w/v). Next, frozen cynomolgus monkey primary hepatocytes (manufactured by Ina Research) were seeded at 100,000 cells/mL into the above-mentioned medium composition supplemented with deacylated gellan gum and dispensed at 200 μL/well into the wells of a 96-well collagen-coated microplate (manufactured by IWAKI, 4860-010). As a negative control, cynomolgus monkey primary hepatocytes were suspended in the above-mentioned medium without deacylated gellan gum and the suspension was dispersed. The plate was then incubated in a _CO2 incubator (37°C, 5% _CO2 ) for 3 days.

1. Measure the number of viable cells

To the culture medium after 1 day of culture, a WST-8 solution (manufactured by DOJINDO Laboratories, 20 μL) was added, and the mixture was incubated for 100 min at 37° C. The number of viable cells was measured by measuring absorbance at 450 nm using an absorbance spectrometer (manufactured by Molecular Devices, SPECTRA MAX 190).

2. Analysis of the amount of albumin to be secreted

After culturing for 3 days, the culture medium containing the hepatocytes was recovered, and the culture supernatant was recovered by centrifugation (400 g, 3 min). The concentration of human albumin in the culture medium was measured using an Albumin ELISA Quantitation Kit (manufactured by Bethyl Laboratories).

The results demonstrated that the medium composition of the present invention protected primary hepatocytes, even when using collagen-coated plates, by suppressing a decrease in the number of viable cells. Furthermore, the medium composition demonstrated a higher albumin production capacity than the negative control. The absorbance at 450 nm (corresponding to the number of cynomolgus monkey primary hepatocytes) after one day of static culture is shown in Table 58. Furthermore, the albumin values of the culture supernatant after three days of static culture are shown in Table 59.

[Table 59]

Experimental group Albumin (ng/mL) Negative control 97 Deacylated gellan gum 0.015% 161 Deacylated gellan gum 0.030% 157

(Experimental Example 47: Comparative Test Using Happy Cell ASM Medium)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to 0.3% (w/v) and dissolved by heating and stirring at 90°C. The aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, HBM medium (manufactured by Lonza Japan) supplemented with additives (HCM single Quots (registered trademark), BSA-fatty acid-free, EGF, ascorbic acid, transferrin, insulin, GA-1000, hydrocortisone 21 hemisuccinate; Lonza Japan) was mixed with DMEM medium (manufactured by WAKO) in a 1:1 ratio and deacylated gellan gum was added to a final concentration of 0.015% (w/v) to prepare a medium composition. HappyCell ASM medium (manufactured by biocroi) and DMEM medium were prepared in advance to a given concentration (mixed in a 1:1 ratio). Next, frozen primary human hepatocytes (manufactured by Xenotech) were seeded at 250,000 cells/mL into the above-mentioned medium composition supplemented with deacylated gellan gum or the Happy Cell ASM medium composition, and dispensed at 200 μL/well into the wells of a 96-well U-bottom ultra-low adhesion surface microplate (manufactured by SUMITOMO BAKELITE). As a negative control, primary human hepatocytes were suspended in the above-mentioned medium without deacylated gellan gum and the suspension was dispersed. The plates were then kept stationary in a _CO2 incubator (37°C, 5% _CO2 ) for 6 days for culture.

1. Measure the number of viable cells

To the culture medium after culturing for 2 hours, 4 hours, 8 hours, 1 day, 4 days, and 6 days, a WST-8 solution (manufactured by DOJINDO Laboratories, 20 μL) was added, and the mixture was incubated at 37° C. for 100 minutes. The number of viable cells was measured by measuring the absorbance at 450 nm using an absorbance spectrometer (manufactured by Molecular Devices, SPECTRA MAX 190).

The results demonstrated that the culture medium composition of the present invention is superior to Happy Cell ASM in its ability to protect primary hepatocytes and inhibit a decrease in viable cell count. Table 60 shows the absorbance at 450 nm (corresponding to the number of human primary hepatocytes) after static culture for 2 hours, 4 hours, 8 hours, 1 day, 4 days, and 6 days.

(Experimental Example 48: Toxicity test of compound on hepatocytes)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to 0.3% (w/v) and dissolved by heating and stirring at 90°C. The aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Separately, HepG2 cells were mixed with DMEM medium (manufactured by WAKO) at a density of 100,000 cells/mL, and the cell suspension was dispensed into the wells of a 96-well flat-bottom ultra-low adhesion surface microplate (manufactured by Corning Incorporated, #3474) at 100 μL/well. Troglitazone (manufactured by WAKO, #71750) was added to the aqueous deacylated gellan gum solution at various concentrations, and this solution (10 μL) was added to the cell suspension (100 μL). The above treatments prepared cell suspensions with 0.18% (v/v) DMSO, 20.0, 40.0, 60.0, and 100 μmol/L troglitazone, and 0.015% (w/v) deacylated gellan gum. The plates were then incubated in a _CO2 incubator (37°C, 5% _CO2 ) for 1 day.

1. Measure the number of viable cells

The culture medium (50 μL) after 1 day of culture was dispensed into a 96-well titer plate (manufactured by Corning Incorporated), Cell Titer-Glo (registered trademark) reagent (manufactured by Promega, 50 μL) was added to the culture medium, and the mixture was incubated at room temperature for 10 min. The luminescence intensity was measured by a multiplate reader (manufactured by Molecular Devices, FlexStation3) to determine the number of viable cells.

2. Lactate Dehydrogenase (LDH) Activity Measurement

DMEM culture medium (manufactured by WAKO, 100 μL) was added to the culture medium (100 μL) after culturing for 1 day, and the plate was centrifuged at 440G for 15 min. The supernatant (100 μL) was dispersed into a 96-well titration plate (manufactured by Corning Incorporated), the reaction mixture (100 μL) in a cytotoxicity detection kit (manufactured by Roche Applied Science) was added, and the mixture was placed in the dark at room temperature for 30 minutes. Then, according to the protocol of the above-mentioned kit, the absorbance at 490 nm was measured by an absorbance spectrometer (manufactured by Molecular Devices, SPECTRA MAX 190), thereby measuring the ratio of disordered cells, that is, cell disorder rate (%).

As a result, it was confirmed that troglitazone has cytotoxicity to hepatocytes using the medium composition of the present invention. The relative cell number and cell disorder ratio (%) are shown in Table 61 when the no-addition condition after 1 day of culture is 1.

(Experimental Example 49: Cell proliferation test using A549 by ATP quantification method)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to 0.3% (w/v) and dissolved by heating and stirring at 90°C. This aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum to a final concentration of 0.005% (w/v), 0.015% (w/v), or 0.030% (w/v) to a DMEM medium containing 10% (v/v) fetal bovine serum (manufactured by WAKO). Next, the human lung cancer cell line A549 (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) was seeded at 100,000 cells/mL into the above-mentioned medium composition supplemented with deacylated gellan gum and dispensed at 100 μL/well into the wells of a 96-well flat-bottom ultra-low adhesion surface microplate (manufactured by Corning Incorporated, #3474). As a negative control, A549 cells were suspended in the above-mentioned medium without deacylated gellan gum and the suspension was dispersed. The plate was then incubated in a _CO2 incubator (37°C, 5% _CO2 ) for 5 days. ATP reagent (100 μL) (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, manufactured by Promega) was added to the culture medium after 1, 3, and 5 days of culture to generate a suspension, which was left at room temperature for about 10 minutes and the luminescence intensity (RLU value) was measured by FlexStation3 (manufactured by Molecular Devices), and the number of viable cells was measured by subtracting the luminescence value of the culture medium alone. For WST-8 measurement, WST-8 solution (manufactured by DOJINDO Laboratories, 10 μL) was added to the cells after 3 days of culture, and the mixture was incubated at 37°C for 100 minutes. The absorbance was measured at 450 nm by an absorbance spectrometer (manufactured by Molecular Devices, SPECTRA MAX 190), and the number of viable cells was measured by subtracting the absorbance of the culture medium alone.

As a result, it was confirmed by the ATP measurement method that A549 cells proliferated efficiently when using the medium composition of the present invention. The RLU values (ATP measurement, luminescence intensity) after 1, 3, and 5 days of static culture are shown in Table 62. The absorbance (WST-8) at 450 nm and the RLU value (ATP measurement, luminescence intensity) after 3 days of static culture are shown in Table 63.

[Table 63]

Experimental group WST-8 ATP Negative control 0.617 11183 Deacylated gellan gum 0.005% 0.906 17623 Deacylated gellan gum 0.015% 1.149 21021 Deacylated gellan gum 0.030% 1.239 20492

(Experimental Example 50: Comparison of Monolayer Culture Methods in Cell Proliferation Tests Using Anticancer Drugs)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to 0.3% (w/v) and dissolved by heating and stirring at 90°C. The aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum to a DMEM medium containing 10% (v/v) fetal bovine serum (manufactured by WAKO) at a final concentration of 0.015% (w/v), and a medium composition without deacylated gellan gum was prepared. Next, the human cervical cancer cell line HeLa (manufactured by DS PHARMABIOMEDICAL CO., LTD.) was seeded at 37,000 cells/mL in the above-mentioned medium composition supplemented with deacylated gellan gum, and 135 μL/well was dispensed into the wells of a 96-well flat-bottom ultra-low adhesion surface microplate (manufactured by Corning Incorporated, #3474). In the monolayer culture method, the human cervical cancer cell line HeLa was seeded at 37,000 cells/mL in the above-mentioned medium composition without deacylated gellan gum, and 135 μL/well was dispensed into the wells of a 96-well flat-bottom microplate (manufactured by Corning Incorporated, #3585). Each plate was maintained in a _CO2 incubator (37°C, 5% _CO2 ) for culture. On day 1 of culture, a medium composition containing 10-fold concentrations of various anticancer drugs to form a final concentration of 0.001-1 μM and a final concentration of 0.015% (w/v) deacylated gellan gum (deacylated gellan gum supplemented group) and a medium composition containing 10-fold concentrations of various anticancer drugs alone (monolayer culture group) were added at 15 μL each, and the cells were cultured continuously for 3 days. The anticancer drugs used were doxorubicin (manufactured by WAKO), paclitaxel (manufactured by WAKO), or mitomycin C (manufactured by WAKO). On day 4, ATP reagent (150 μL) (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, manufactured by Promega) was added to the culture medium to generate a suspension, which was left at room temperature for approximately 10 minutes and the luminescence intensity (RLU value) was measured by FlexStation3 (manufactured by Molecular Devices). The number of viable cells was measured by subtracting the luminescence value of the culture medium alone. For WST-8 measurement, a WST-8 solution (manufactured by DOJINDO Laboratories, 15 μL) was added, and the mixture was incubated for 100 min at 37° C. The absorbance was measured at 450 nm using an absorbance spectrometer (manufactured by Molecular Devices, SPECTRA MAX 190), and the number of viable cells was measured by subtracting the absorbance of the culture medium alone.

As a result, it was found that the cell proliferation test method using the medium composition of the present invention strongly demonstrated the efficacy of mitomycin C compared to the monolayer culture method. The % control values of the RLU values (ATP measurement, luminescence intensity) on the fourth day of static culture are shown in Table 64. The % control values of the absorbance at 450 nm (WST-8 measurement) on the fourth day of static culture are shown in Table 65.

(Experimental Example 51: Comparison of cell proliferation assay using an apoptosis-inducing agent with a monolayer culture method)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to 0.3% (w/v) and dissolved by heating and stirring at 90°C. The aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum to a DMEM medium containing 10% (v/v) fetal bovine serum (manufactured by WAKO) at a final concentration of 0.015% (w/v), and a medium composition without deacylated gellan gum was prepared. Next, the human cervical cancer cell line HeLa (manufactured by DS PHARMABIOMEDICAL CO., LTD.) was seeded at 37,000 cells/mL in the above-mentioned medium composition supplemented with deacylated gellan gum, and 135 μL/well was dispensed into the wells of a 96-well flat-bottom ultra-low adhesion surface microplate (manufactured by Corning Incorporated, #3474). In the monolayer culture method, the human cervical cancer cell line HeLa was seeded at 37,000 cells/mL in the above-mentioned medium composition without deacylated gellan gum, and 135 μL/well was dispensed into the wells of a 96-well flat-bottom microplate (manufactured by Corning Incorporated, #3585). Each plate was maintained in a _CO2 incubator (37°C, 5% _CO2 ) for culture. On day 1 of culture, 15 μL of each of the following media compositions were added: a 10-fold concentration of various apoptosis-inducing agents to a final concentration of 0.2-10 μM and a final concentration of 0.015% (w/v) deacylated gellan gum (deacylated gellan gum supplementation group); and a 10-fold concentration of various apoptosis-inducing agents alone (monolayer culture group). The cells were cultured for three consecutive days. The apoptosis-inducing agents used were the Apoptosis Inducer set (Merck Millipore, APT800: actinomycin D, camptothecin, cycloheximide, dexamethasone, etoposide). On day 4, ATP reagent (150 μL) (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, manufactured by Promega) was added to the culture medium to produce a suspension, which was left at room temperature for approximately 10 minutes. The luminescence intensity (RLU value) was measured using a FlexStation3 (manufactured by Molecular Devices), and the number of viable cells was measured by subtracting the luminescence value of the culture medium alone. For WST-8 measurement, a WST-8 solution (manufactured by DOJINDO Laboratories, 15 μL) was added, and the mixture was incubated at 37°C for 100 minutes. The absorbance was measured at 450 nm using an absorbance spectrometer (manufactured by Molecular Devices, SPECTRA MAX 190), and the number of viable cells was measured by subtracting the absorbance of the culture medium alone.

As a result, it was found that the cell proliferation test method using the medium composition of the present invention strongly demonstrated the efficacy of camptothecin and etoposide compared to the monolayer culture method. The % control values of the RLU values (ATP measurement, luminescence intensity) on the fourth day of static culture are shown in Table 66. The % control values of the absorbance at 450 nm (WST-8 measurement) on the fourth day of static culture are shown in Table 67.

(Experimental Example 52: Comparison of HeLa cell proliferation assay using trametinib and MK-2206 with a monolayer culture method)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to 0.3% (w/v) and dissolved by heating and stirring at 90°C. The aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum to a DMEM medium containing 10% (v/v) fetal bovine serum (manufactured by WAKO) at a final concentration of 0.015% (w/v), and a medium composition without deacylated gellan gum was prepared. Next, the human cervical cancer cell line HeLa (manufactured by DS PHARMABIOMEDICAL CO., LTD.) was seeded at 37,000 cells/mL into the above-mentioned medium composition supplemented with deacylated gellan gum, and 135 μL/well was dispensed into the wells of a 96-well flat-bottom ultra-low adhesion surface microplate (manufactured by Corning Incorporated, #3474). In a monolayer culture method, the human cervical cancer cell line HeLa was seeded at 7,400 cells/mL into the above-mentioned medium composition without deacylated gellan gum, and 135 μL/well was dispensed into the wells of a 96-well flat-bottom microplate (manufactured by Corning Incorporated, #3585). Each plate was maintained in a _CO2 incubator (37°C, 5% _CO2 ) for culture. On the first day of cultivation, a medium composition (deacylated gellan gum addition group) of deacylated gellan gum with a final concentration of 0.001-30 μM and a final concentration of 0.015% (w/v) and a medium composition (monolayer culture group) containing only 10 times the concentration of a variety of anticancer drugs were added with 15 μL each, and the cells were cultured continuously for 5 days. The anticancer drugs used were trametinib (manufactured by Santa Cruz, a MEK inhibitor) and MK-2206 (manufactured by Santa Cruz, an Akt inhibitor). On the 6th day, ATP reagent (150 μL) (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, manufactured by Promega) was added to the culture medium to generate a suspension, which was placed at room temperature for about 10 min and measured for luminescence intensity (RLU value) by FlexStation3 (manufactured by Molecular Devices), and the number of viable cells was measured by subtracting the luminescence value of the culture medium alone.

As a result, it was found that the cell proliferation test method using the medium composition of the present invention strongly demonstrated the efficacy of MK-2206 and trametinib compared to the monolayer culture method. The RLU values (ATP measurement, luminescence intensity) on the fourth day of static culture as a percentage of the control value are shown in Table 68.

(Experimental Example 53: Comparison of A549 Cell Proliferation Assay Using Trametinib and MK-2206 with a Monolayer Culture Method)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to 0.3% (w/v) and dissolved by heating and stirring at 90°C. The aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum to a DMEM medium containing 10% (v/v) fetal bovine serum (manufactured by WAKO) at a final concentration of 0.015% (w/v), and a medium composition without deacylated gellan gum was prepared. Next, human lung cancer cell line A549 (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) was seeded at 14,800 cells/mL into the above-mentioned medium composition supplemented with deacylated gellan gum, and 135 μL/well was dispensed into the wells of a 96-well flat-bottom ultra-low adhesion surface microplate (manufactured by Corning Incorporated, #3474). In the monolayer culture method, human lung cancer cell line A549 was seeded at 14,800 cells/mL into the above-mentioned medium composition without deacylated gellan gum, and 135 μL/well was dispensed into the wells of a 96-well flat-bottom microplate (manufactured by Corning Incorporated, #3585). Each plate was maintained in a _CO2 incubator (37°, 5% _CO2 ) for culture. On the first day of culture, a medium composition (deacylated gellan gum addition group) of deacylated gellan gum with a final concentration of 0.001-30 μM and a final concentration of 0.015% (w/v) and a medium composition (monolayer culture group) containing only 10 times the concentration of a variety of anticancer drugs were added with 15 μL each, and the cells were cultured continuously for 5 days. The anticancer drugs used were trametinib (manufactured by Santa Cruz, a MEK inhibitor) and MK-2206 (manufactured by Santa Cruz, an Akt inhibitor). On the 6th day, ATP reagent (150 μL) (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, manufactured by Promega) was added to the culture medium to generate a suspension, which was placed at room temperature for about 10 min and measured for luminescence intensity (RLU value) by FlexStation3 (manufactured by Molecular Devices), and the number of viable cells was measured by subtracting the luminescence value of the culture medium alone.

As a result, it was found that the cell proliferation test method using the medium composition of the present invention strongly demonstrated the efficacy of MK-2206 compared to the monolayer culture method. The % control value of the RLU value (ATP measurement, luminescence intensity) on the 4th day of static culture is shown in Table 69.

(Experimental Example 54: Comparison with the Monolayer Culture Method in the Proliferation of HeLa Cells Stimulated with Human HB-EGF)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to 0.3% (w/v) and dissolved by heating and stirring at 90°C. The aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum to a DMEM medium containing 10% (v/v) fetal bovine serum (manufactured by WAKO) at a final concentration of 0.015% (w/v), and a medium composition without deacylated gellan gum was prepared. Next, the human cervical cancer cell line HeLa (manufactured by DS PHARMABIOMEDICAL CO., LTD.) was seeded at 37,000 cells/mL into the above-mentioned medium composition supplemented with deacylated gellan gum, and 135 μL/well was dispensed into the wells of a 96-well flat-bottom ultra-low adhesion surface microplate (manufactured by Corning Incorporated, #3474). In the monolayer culture method, the human cervical cancer cell line HeLa was seeded at 37,000 cells/mL into the above-mentioned medium composition without deacylated gellan gum, and 135 μL/well was dispensed into the wells of a 96-well flat-bottom microplate (manufactured by Corning Incorporated, #3585). Each plate was maintained in a _CO2 incubator (37°C, 5% _CO2 ) for culture. On day 1 of culture, 15 μL of each of a medium composition containing 10-fold concentration of human HB-EGF (heparin-binding EGF-like growth factor, manufactured by PEPROTECH) to a final concentration of 10, 30, and 100 ng/ml and a final concentration of 0.015% (w/v) deacylated gellan gum (deacylated gellan gum supplemented group) and a medium composition containing 10-fold concentration of human HB-EGF alone (monolayer culture group) were added, and the cells were cultured continuously for 7 days. On days 6 and 8, ATP reagent (150 μL) (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, manufactured by Promega) was added to the culture medium to produce a suspension, which was left at room temperature for approximately 10 minutes. The luminescence intensity (RLU value) was measured using a FlexStation3 (manufactured by Molecular Devices), and the number of viable cells was measured by subtracting the luminescence value of the culture medium alone.

The results showed that the HeLa cell proliferation test method using the medium composition of the present invention strongly demonstrated the cell proliferation-promoting effect of human HB-EGF compared to the monolayer culture method. The RLU values (ATP measurement, luminescence intensity) on day 6 of static culture as compared to the control values are shown in Table 70. The RLU values (ATP measurement, luminescence intensity) on day 8 of static culture as compared to the control values are shown in Table 71.

(Experimental Example 55: Comparison with the Monolayer Culture Method in the Proliferation of A549 Cells Stimulated with Human HB-EGF)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to 0.3% (w/v) and dissolved by heating and stirring at 90°C. The aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum to a DMEM medium containing 10% (v/v) fetal bovine serum (manufactured by WAKO) at a final concentration of 0.015% (w/v), and a medium composition without deacylated gellan gum was prepared. Next, human lung cancer cell line A549 (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) was seeded into the above-mentioned medium composition supplemented with deacylated gellan gum to a density of 14,800 cells/mL and dispensed at 135 μL/well into the wells of a 96-well flat-bottom ultra-low adhesion surface microplate (manufactured by Corning Incorporated, #3474). In a monolayer culture method, human lung cancer cell line A549 was seeded into the above-mentioned medium composition without deacylated gellan gum at a density of 14,800 cells/mL and dispensed at 135 μL/well into the wells of a 96-well flat-bottom microplate (manufactured by Corning Incorporated, #3585). Each plate was maintained in a _CO2 incubator (37°, 5% _CO2 ) for culture. On day 1 of culture, a medium composition containing 10-fold concentration of human HB-EGF (manufactured by PEPROTECH) to form final concentrations of 10, 30, and 100 ng/ml and a final concentration of 0.015% (w/v) deacylated gellan gum (deacylated gellan gum supplemented group) and a medium composition containing only 10-fold concentration of human HB-EGF (monolayer culture group) were added at 15 μL each, and the cells were cultured continuously for 7 days. On days 6 and 8, ATP reagent (150 μL) (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, manufactured by Promega) was added to the culture medium to generate a suspension, which was left at room temperature for approximately 10 minutes, and the luminescence intensity (RLU value) was measured using a FlexStation3 (manufactured by Molecular Devices), and the number of viable cells was measured by subtracting the luminescence value of the culture medium alone.

As a result, it was found that the A549 cell proliferation test method using the medium composition of the present invention strongly demonstrated the cell proliferation-promoting effect of human HB-EGF compared to the monolayer culture method. The RLU values (ATP measurement, luminescence intensity) on the 6th day of static culture as compared to the control values are shown in Table 72. The RLU values (ATP measurement, luminescence intensity) on the 8th day of static culture as compared to the control values are shown in Table 73.

(Experimental Example 56: Comparison with the Monolayer Culture Method in the Proliferation of A431 Cells Stimulated with Human HB-EGF)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) to 0.3% (w/v) and dissolved by heating and stirring at 90°C. The aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum to an EMEM medium (manufactured by DSPHARMA BIOMEDICAL CO., LTD.) containing 10% (v/v) fetal bovine serum at a final concentration of 0.015% (w/v), and a medium composition without deacylated gellan gum was prepared. Next, the human squamous cell carcinoma cell line A431 (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) was seeded at 37,000 cells/mL into the above-mentioned medium composition supplemented with deacylated gellan gum, and 135 μL/well was dispensed into the wells of a 96-well flat-bottom ultra-low adhesion surface microplate (manufactured by Corning Incorporated, #3474). In the monolayer culture method, the human squamous cell carcinoma cell line A431 was seeded at 37,000 cells/mL into the above-mentioned medium composition without deacylated gellan gum, and 135 μL/well was dispensed into the wells of a 96-well flat-bottom microplate (manufactured by Corning Incorporated, #3585). Each plate was maintained in a _CO2 incubator (37°, 5% _CO2 ) for culture. On the first day of culture, a medium composition containing 10-fold concentration of human HB-EGF (manufactured by PEPROTECH) to form a final concentration of 10, 30, and 100 ng/ml and a final concentration of 0.015% (w/v) deacylated gellan gum (deacylated gellan gum supplemented group) and a medium composition containing only 10-fold concentration of human HB-EGF (monolayer culture group) were added at 15 μL each, and the cells were cultured continuously for 7 days. On the 6th and 8th days, ATP reagent (150 μL) (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, manufactured by Promega) was added to the culture medium to generate a suspension, which was left at room temperature for about 10 minutes, and the luminescence intensity (RLU value) was measured by FlexStation3 (manufactured by Molecular Devices), and the number of viable cells was measured by subtracting the luminescence value of the culture medium alone.

The results showed that the A431 cell proliferation test method using the medium composition of the present invention strongly demonstrated the cell proliferation-promoting effect of human HB-EGF compared to the monolayer culture method. The RLU values (ATP measurement, luminescence intensity) on day 6 of static culture as compared to the control values are shown in Table 74. The RLU values (ATP measurement, luminescence intensity) on day 8 of static culture as compared to the control values are shown in Table 75.

(Experimental Example 57: Comparison with the Monolayer Culture Method in the Proliferation Activity of SKOV3 Cells Stimulated by Human HB-EGF)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended to 0.3% (w/v) in ultrapure water (Milli-Q water) and dissolved by stirring while heating at 90°C. The aqueous solution was sterilized at 121°C for 20 minutes in an autoclave. Using this solution, a medium composition was prepared by adding deacylated gellan gum to a final concentration of 0.015% (w/v) to McCoy's 5a medium (manufactured by DSPHARMA BIOMEDICAL CO., LTD.) containing 15% (v/v) fetal bovine serum, and a medium composition without deacylated gellan gum was prepared. Next, the human ovarian cancer cell line SKOV3 (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) was seeded at 37,000 cells/mL in the above-mentioned medium composition supplemented with deacylated gellan gum, and 135 μL/well was dispensed into a 96-well flat-bottom ultra-low adhesion surface microplate (manufactured by Corning Incorporated, #3474). In a monolayer culture method, the human ovarian cancer cell line SKOV3 was seeded at 37,000 cells/mL in the above-mentioned medium composition without deacylated gellan gum, and 135 μL/well was dispensed into the wells of a 96-well flat-bottom microplate (manufactured by Corning Incorporated, #3585). Each plate was cultured while maintaining a static state in a _CO2 incubator (37°C, 5% _CO2 ). On the 1st day of cultivation, 15 μ L of 10-fold concentrations of human HB-EGF (manufactured by PEPROTECH) were added to produce a culture medium composition (deacylated gellan gum addition group) of deacylated gellan gum at a final concentration of 10, 30, and 100 ng/ml and a final concentration of 0.015% (w/v), and a culture medium composition (monolayer culture group) containing only 10-fold concentrations of human HB-EGF, and the cells were cultured continuously for 8 days. On the 6th and 9th days, ATP reagent (150 μ L) (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, manufactured by Promega) was added to the culture medium to produce a suspension, which was placed at room temperature for approximately 10 minutes, and luminous intensity (RLU value) was measured by FlexStation3 (manufactured by Molecular Devices), and the number of viable cells was measured by subtracting the luminescent value of the culture medium alone.

The results showed that the SKOV3 cell proliferation test method using the medium composition of the present invention strongly demonstrated the cell proliferation-promoting effect of human HB-EGF compared to the monolayer culture method. Table 76 shows the RLU values (ATP measurement, luminescence intensity) as a percentage of the control value on day 6 of static culture. Table 77 shows the RLU values (ATP measurement, luminescence intensity) as a percentage of the control value on day 9 of static culture.

(Experimental Example 58: Comparison with the Monolayer Culture Method in VEGF mRNA Expression in HeLa Cells Stimulated by Human HB-EGF)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended to 0.3% (w/v) in ultrapure water (Milli-Q water) and dissolved by stirring while heating at 90°C. The aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum to a final concentration of 0.015% (w/v) to a DMEM medium (manufactured by WAKO) containing 10% (v/v) fetal bovine serum, and a medium composition without deacylated gellan gum was prepared. Next, the human cervical cancer cell line HeLa (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) was seeded at 37,000 cells/mL into the above-mentioned medium composition supplemented with deacylated gellan gum, and the culture was dispensed at 135 μL/well into a 96-well flat-bottom ultra-low adhesion surface microplate (manufactured by Corning Incorporated, #3474). In the monolayer culture method, the human cervical cancer cell line HeLa was seeded at 37,000 cells/mL into the above-mentioned medium composition without deacylated gellan gum, and the culture was dispensed at 135 μL/well into the wells of a 96-well flat-bottom microplate (manufactured by Corning Incorporated, #3585). Each plate was cultured while remaining stationary in a _CO2 incubator (37°C, 5% _CO2 ). On the 1st day of cultivation, 15 μ L of people HB-EGF (manufactured by PEPROTECH) containing 10 times of concentration were added separately to produce the culture medium composition (deacylated gellan gum addition group) of the deacylated gellan gum of 10,30 and 100 ng/ml final concentration and final concentration 0.015% (w/v), and the culture medium composition (monolayer culture group) of people HB-EGF only containing 10 times of concentration, and cells were continuously cultured for 6 days. The culture medium containing cancer cells was reclaimed on the 7th day, and the cells were reclaimed by centrifugation (400g, 3 minutes). Total RNA was extracted from cells using RNeasy Mini test kit (manufactured by QIAGEN). Total RNA and PrimeScript (registered trademark) RT Master Mix (manufactured by Takara Bio Inc.) were used, reverse transcription reaction was carried out using GeneAmp PCRSystem 9700 (manufactured by Applied Biosystems), and cDNA was synthesized. Each cDNA sample for PCR reaction was diluted to 1/10 with sterile water. In addition, the sample for calibration curve is to distribute and mix in the quantitative range of 1/3-1/243 dilution with 3 times of common ratios, and the cDNA of adjustment. Use each cDNA sample, correction sample, Premix Ex Taq (registered trademark) (manufactured by Takara Bio Inc.) and multiple Taqman probes (manufactured by Applied Biosystems), and 7500 real-time PCR systems (manufactured by Applied Biosystems) to carry out PCR reaction. Use the mRNA of GAPDH (3-glyceraldehyde-3-phosphate dehydrogenase) as endogenous control, utilize the value of GAPDH to correct the expression of VEGF (vascular endothelial growth factor) mRNA, and use negative control as 100% to calculate specificity. Shown hereinafter are each probes used (manufactured by Applied Biosystems).

GAPDH: HS99999905

VEGF: HS00173626.

As a result, it was found that HeLa cells cultured using the medium composition of the present invention exhibited a stronger effect of human HB-EGF on promoting VEGF mRNA expression than cells cultured using the monolayer culture method. Table 78 shows VEGF mRNA expression values on day 7 of static culture, when the negative control is 100%.

(Experimental Example 59: Effect of Gefitinib on Cell Proliferation of A549 Cells Stimulated by Human HB-EGF)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended to 0.3% (w/v) in ultrapure water (Milli-Q water) and dissolved by stirring while heating at 90°C. The aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum to a final concentration of 0.015% (w/v) to a DMEM medium (manufactured by WAKO) containing 10% (v/v) fetal bovine serum, and a medium composition without deacylated gellan gum was prepared. Next, human lung cancer cell line A549 (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) was seeded at 14,800 cells/mL into the above-mentioned medium composition supplemented with deacylated gellan gum and dispensed at 135 μL/well into the wells of a 96-well flat-bottom ultra-low adhesion surface microplate (manufactured by Corning Incorporated, #3474). Each plate was cultured while maintaining a static state in a _CO2 incubator (37°C, 5% _CO2 ). On the 1st day of cultivation, for each anticancer drug preparation final concentration 0.1-30 μ m and for people HB-EGF preparation final concentration 0 ng/ml or 100 ng/ml, separately add 15 μ L and contain the culture medium composition (deacylated gellan gum adds group) of the deacylated gellan gum of each anticancer drug, people HB-EGF (manufactured by PEPROTECH) and final concentration 0.015% (w/v) of 10 times of concentration, and by cell continuous culture 5 days.Used anticancer drug is gefitinib (manufactured by Santa Cruz, EGF receptor inhibitor).At the 6th day, in culture medium, add ATP reagent (150 μ L) (CellTiter-Glo (registered trademark) Luminescent Cell ViabilityAssay, manufactured by Promega), to produce suspension, it was at room temperature stood about 10 minutes, and by FlexStation3 (manufactured by Molecular Devices) measurement luminous intensity (RLU value), and by the luminous value of subtracting independent culture medium, measure the quantity of living cells.

The result is that, according to the A549 cell proliferation test method using the culture medium composition of the present invention and human HB-EGF, the culture condition with the addition of HB-EGF shows a stronger inhibitory effect of gefitinib. In Table 79, the % control value of the RLU value (ATP measurement, luminous intensity) on the 6th day of static culture is shown.

(Experimental Example 60: Effects of Gefitinib and Erlotinib on the Growth and Proliferation of A431 Cells Stimulated by Human HB-EGF)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended to 0.3% (w/v) in ultrapure water (Milli-Q water) and dissolved by stirring while heating at 90°C. The aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum to an EMEM medium (manufactured by DSPHARMA BIOMEDICAL CO., LTD.) containing 10% (v/v) fetal bovine serum at a final concentration of 0.015% (w/v), and a medium composition without deacylated gellan gum was prepared. Next, the human squamous cell carcinoma cell line A431 (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) was seeded at 37,000 cells/mL in the above-mentioned medium composition supplemented with deacylated gellan gum and dispensed at 135 μL/well into the wells of a 96-well flat-bottom ultra-low adhesion surface microplate (manufactured by Corning Incorporated, #3474). Each plate was cultured by maintaining it in a _CO2 incubator (37°C, 5% _CO2 ). On the first day of culture, each anticancer drug was prepared at a final concentration of 0.1-30 μM and human HB-EGF was prepared at a final concentration of 0 ng/ml or 100 ng/ml, and 15 μL of a medium composition containing 10-fold concentrations of each anticancer drug, human HB-EGF (manufactured by PEPROTECH), and deacylated gellan gum at a final concentration of 0.015% (w/v) was added to each (deacylated gellan gum supplemented group), and the cells were cultured continuously for 7 days. The anticancer drugs used were gefitinib (manufactured by Santa Cruz, an EGF receptor inhibitor) and erlotinib (manufactured by Santa Cruz, an EGF receptor inhibitor). On days 4, 6, and 8, an ATP reagent (150 μL) (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, manufactured by Promega) was added to the culture medium to produce a suspension, which was allowed to stand at room temperature for about 10 minutes, and the luminescence intensity (RLU value) was measured by FlexStation3 (manufactured by Molecular Devices), and the number of viable cells was measured by subtracting the luminescence value of the culture medium alone.

The result is that, by the culture method of combining culture medium composition of the present invention and people HB-EGF, A431 cell proliferation is observed under low adhesion culture condition.In addition, by the A431 cell proliferation test method of combining culture medium composition of the present invention and people HB-EGF, the inhibitory effect of gefitinib and erlotinib on the cell proliferation induced by HB-EGF can be evaluated. For people HB-EGF growth promotion activity, in table 80, the RLU values (ATP measurement, luminous intensity) of the 4th day, the 6th day, the 8th day of static culture are shown. In addition, for the effect of each anticancer drug on people HB-EGF proliferation promotion activity, in table 81, the % control value of the RLU values (ATP measurement, luminous intensity) of the 4th day, the 8th day are shown.

(Experimental Example 61: Cell proliferation test by dispersing MCF-7 cells)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended to 0.3% (w/v) in ultrapure water (Milli-Q water) and dissolved by stirring while heating at 90°C. The aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum to an EMEM medium (manufactured by DSPHARMA BIOMEDICAL CO., LTD.) containing 10% (v/v) fetal bovine serum at a final concentration of 0.005% (w/v) or 0.015% (w/v). Next, the human breast cancer cell line MCF-7 (manufactured by DS PHARMABIOMEDICAL CO., LTD.) was seeded at 50,000 cells/mL into the above-mentioned medium composition supplemented with deacylated gellan gum and dispensed into the wells of a 96-well flat-bottom ultra-low adhesion surface microplate (manufactured by Corning Incorporated, #3474) at 100 μL/well. As a negative control, MCF-7 cells were suspended in the above-mentioned medium without deacylated gellan gum and the suspension was dispensed. Next, the plate was cultured for 5 days by keeping it stationary in a _CO2 incubator (37°C, 5% _CO2 ). After culturing for 2 days and 5 days, ATP reagent (100 μL) (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, manufactured by Promega) was added to the culture medium to produce a suspension, which was left at room temperature for about 10 minutes and measured for luminescence intensity (RLU value) by FlexStation3 (manufactured by Molecular Devices), and the number of viable cells was measured by subtracting the luminescence value of the culture medium alone. For WST-8 measurement, after culturing for 2 days and 5 days, WST-8 solution (manufactured by DOJINDO Laboratories, 10 μL) was added to the cells, and the mixture was incubated at 37°C for 100 minutes, and the absorbance at 450 nm was measured by an absorbance spectrometer (manufactured by Molecular Devices, SPECTRA MAX190), and the number of viable cells was measured by subtracting the absorbance of the culture medium alone.

The result is to confirm that, using the culture medium composition of the present invention, according to the ATP measurement method and the WST-8 measurement method, MCF-7 cells effectively proliferate. In Table 82, the RLU value (ATP measurement, luminous intensity) after static culture 2,5 days is shown. In Table 83, the absorbance of 450 nm (WST-8) after static culture 2,5 days is shown. In Figure 23, the microscopic observation results of the aggregates of MCF-7 cells after 5 days of cultivation are shown.

(Experimental Example 62: Cell proliferation test when A375 cells and MNNG/HOS cells are dispersed)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended to 0.3% (w/v) in ultrapure water (Milli-Q water) and dissolved by stirring while heating at 90°C. The aqueous solution was sterilized in an autoclave at 121°C for 20 minutes. Using this solution, a medium composition was prepared by adding deacylated gellan gum to a final concentration of 0.005% (w/v) or 0.015% (w/v) to a DMEM medium or EMEM medium (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) containing 10% (v/v) fetal bovine serum (manufactured by WAKO). Next, each of the human melanoma cell line A375 (manufactured by ATCC) and the human osteosarcoma cancer cell line MNNG/HOS (manufactured by ATCC) was seeded at 50,000 cells/mL into the above-mentioned medium composition supplemented with deacylated gellan gum and dispensed at 100 μL/well into the wells of a 96-well flat-bottom ultra-low adhesion surface microplate (manufactured by Corning Incorporated, #3474). As a negative control, A375 cells and MNNG/HOS cells were suspended in the above-mentioned medium without deacylated gellan gum and the suspension was dispensed. The plates were then cultured for 4 days by keeping them stationary in a _CO2 incubator (37°C, 5% _CO2 ). After 4 days of culture, ATP reagent (100 μL) (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, manufactured by Promega) was added to the culture medium to produce a suspension, which was left at room temperature for about 10 minutes and measured for luminescence intensity (RLU value) by FlexStation3 (manufactured by Molecular Devices), and the number of viable cells was measured by subtracting the luminescence value of the culture medium alone. After 4 days of culture, WST-8 solution (manufactured by DOJINDO Laboratories, 10 μL) was added to the culture medium, and the mixture was incubated at 37°C for 100 minutes, and the absorbance at 450 nm was measured by an absorbance spectrometer (manufactured by Molecular Devices, SPECTRA MAX 190), and the number of viable cells was measured by subtracting the absorbance of the culture medium alone.

The results confirmed that using the medium composition of the present invention, A375 cells and MNNG/HOS cells can be cultured in a uniformly dispersed state without forming cell aggregates of excessive size, and effectively proliferate in the medium composition. Figure 24 shows the microscopic observation results of aggregates of A375 cells and MNNG/HOS cells after 4 days of culture. In addition, in A375 cells, the absorbance (WST-8) and RLU values (ATP measurement, luminescence intensity) at 450 nm after 4 days of static culture are shown in Table 84. In MNNG/HOS cells, the absorbance (WST-8) and RLU values (ATP measurement, luminescence intensity) at 450 nm after 4 days of static culture are shown in Table 85.

[Table 84]

Experimental group WST-8 ATP Negative control 0.738 55193 Deacylated gellan gum 0.005% 2.088 98739 Deacylated gellan gum 0.015% 3.336 115365

[Table 85]

Experimental group WST-8 ATP Negative control 0.294 41529 Deacylated gellan gum 0.005% 0.843 66913 Deacylated gellan gum 0.015% 2.197 102199

(Experimental Example 63: Cell proliferation test by dispersing MIAPaCa-2 cells)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended to 0.3% (w/v) in ultrapure water (Milli-Q water) and dissolved by stirring under heating at 90°C. The aqueous solution was sterilized at 121°C for 20 minutes in an autoclave. Using this solution, a culture medium composition was prepared by adding deacylated gellan gum to a DMEM culture medium containing 10% (v/v) fetal bovine serum (manufactured by WAKO) at a final concentration of 0.005% (w/v) or 0.015% (w/v). Subsequently, the human pancreatic cancer cell line MIAPaCa-2 (manufactured by ATCC) was inoculated at 50,000 cells/mL into the above-mentioned culture medium composition supplemented with deacylated gellan gum and dispensed into the wells of a 96-well flat-bottom ultra-low adhesion surface microplate (manufactured by Corning Incorporated, #3474) at 100 μL/well. As a negative control, MIAPaCa-2 cells were suspended in the above-mentioned culture medium without deacylated gellan gum and the suspension was distributed. Immediately thereafter, the plates were cultured by keeping them stationary for 6 days in a _CO incubator (37°C, 5% _CO ). After 6 days of cultivation, ATP reagent (100 μL) (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, manufactured by Promega) was added to the culture medium to produce a suspension, which was placed at room temperature for approximately 10 minutes and measured for luminous intensity (RLU value) by FlexStation3 (manufactured by Molecular Devices), and the number of viable cells was measured by subtracting the luminescent value of the culture medium alone. After culturing for 6 days, a WST-8 solution (manufactured by DOJINDO Laboratories, 10 μL) was added to the culture medium, and the mixture was incubated at 37° C. for 100 minutes, the absorbance at 450 nm was measured by an absorbance spectrometer (manufactured by Molecular Devices, SPECTRA MAX 190), and the number of viable cells was measured by subtracting the absorbance of the culture medium alone.

The results confirmed that using the culture medium composition of the present invention, MIAPaCa-2 cells can be cultured in a uniformly dispersed state without forming cell aggregates of excessive size, and effectively proliferate in the culture medium composition. Figure 25 shows the microscopic observation results of aggregates of MIAPaCa-2 cells after 6 days of culture. In addition, Table 86 shows the absorbance (WST-8) and RLU value (ATP measurement, luminescence intensity) at 450 nm of MIAPaCa-2 cells after 4 days of static culture.

[Table 86]

Experimental group WST-8 ATP Negative control 2.030 52674 Deacylated gellan gum 0.005% 3.102 86650 Deacylated gellan gum 0.015% 3.621 85412

(Experimental Example 64: Concentration and Dilution of a Culture Medium Containing Deacylated Gellan Gum)

The DMEM medium (manufactured by WAKO) containing 0.015% (w / v) deacylated gellan gum (KELCOGELCG-LA, manufactured by SANSHO Co., Ltd.) prepared in the same manner as in Experimental Example 2 was dispensed into a 15 mL centrifuge tube (manufactured by VIOLAMO) in 10 mL portions, and the deacylated gellan gum was precipitated by centrifugation (700G, 5 min) in a cantilever spinner LC-200 (manufactured by TOMY SEIKO Co., Ltd.). The supernatant (8 mL) was removed by aspirator to concentrate the deacylated gellan gum-containing medium. Furthermore, a DMEM medium (manufactured by WAKO) without deacylated gellan gum was added to the concentrated medium and mixed by pipetting to produce a medium having an optional concentration ratio.

On the other hand, human liver cancer cell HepG2 (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) is suspended in the DMEM culture medium containing 10% (v/v) fetal bovine serum (manufactured by WAKO) with 500000 cells/mL, this suspension (10 mL) is inoculated in EZ SPHERE (manufactured by ASAHI GLASS CO., LTD.), and by cell at 37 ℃, _CO incubator (5% _CO ) incubator, cultivate 7 days.Herein, the suspension (10 mL) of the ball (diameter 100 – 200 μm) obtained is centrifuged (200G, 5 minutes), to allow precipitation, and remove supernatant, to prepare ball suspension (1.0 mL).To being prepared into the culture medium of above-mentioned optional concentration, add this ball suspension of 100 μ L, and disperse ball and incubate at 37 ℃ by aspiration, and the dispersion state of this ball of visual observation after 1 hour.In table 87, shown result.

As shown in Table 87, after being prepared as a medium composition, deacylated gellan gum can be concentrated and diluted to an optional concentration. It was confirmed that the medium composition concentrated and diluted in this manner had a spherical suspension effect.

[Table 87]

Concentration rate of deacylated gellan gum (multiple) 0.33 0.66 1.00 1.67 2.50 5.00 The state of the ball (settled or suspended) Precipitated Suspended Suspended Suspended Suspended Suspended

(Experimental Example 65: Production of DMEM/Ham's F12 Medium Containing Deacylated Gellan Gum)

Deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd., 120 mg) was suspended in pure water (72 mL) and dissolved by stirring and heating at 90 ° C. Pure water was added thereto to prepare 0.017% (w/v) solution (720 mL) of deacylated gellan gum, and the solution was sterilized using a sterilizing filter (pore size 0.22 μm). On the other hand, pure water corresponding to 1/10 of the amount recommended for preparing the culture medium was added to the culture medium containing the mixed powder culture medium of equal amounts of DMEM/Ham's F12 (Life Technologies Corporation) and sodium bicarbonate, to prepare 80 mL of 10 times the concentration of the aqueous solution, and the solution was sterilized using a sterilizing filter (pore size 0.22 μm). This was stirred and mixed under 25 ° C sterilization conditions to prepare the target culture medium (800 mL) with 0.015% (w/v) deacylated gellan gum concentration.

Industrial Applicability

The culture medium composition of the present invention exhibits excellent effects in suspending cells and/or tissues and is extremely useful for large-scale cultivation of cells and/or tissues derived from animals and plants while maintaining their function. Furthermore, cells and/or tissues cultured using the methods of the present invention are extremely useful in evaluating the efficacy and toxicity of chemical substances and pharmaceuticals, large-scale production of useful substances such as enzymes, cell growth factors, and antibodies, and in the field of regenerative medicine for replenishing organs, tissues, and cells lost due to disease and deficiency.

This application is based on patent application No. 2012-164227 (filing date: July 24, 2012), 2012-263801 (filing date: November 30, 2012), and 2013-017836 (filing date: January 31, 2013) filed in Japan, the contents of which are incorporated herein in their entirety.

Claims (27)

1. A liquid culture medium composition capable of culturing suspended cells or tissues, the composition having a viscosity of not more than 8 mPa·s at 37°C, and comprising a polysaccharide and a liquid culture medium, the polysaccharide being deacylated gellan gum or a salt thereof, the concentration of the deacylated gellan gum or a salt thereof in the liquid culture medium composition being 0.005-0.05% by weight/volume. 2. The liquid culture medium composition of claim 1, further comprising polysaccharides other than deacylated gellan gum or its salts. 3. The liquid culture medium composition of claim 1 or 2, wherein the liquid culture medium comprises metal ions. 4. The liquid culture medium composition of claim 3, wherein the metal ion is a calcium ion. 5. The liquid culture medium composition of claim 3, wherein the polysaccharide is assembled or forms a three-dimensional network by the metal ions. 6. The liquid culture medium composition of claim 4, wherein the polysaccharide is assembled or formed into a three-dimensional network by the metal ions. 7. A method for producing a liquid culture medium composition capable of culturing suspended cells or tissues, comprising mixing a polysaccharide and a liquid culture medium, wherein the polysaccharide is deacylated gellan gum or a salt thereof, and the concentration of the deacylated gellan gum or a salt thereof in the liquid culture medium composition is 0.005-0.05% by weight/volume. 8. The production method of claim 7, wherein the composition has a viscosity of not more than 8 mPa·s at 37°C. 9. The production method of claim 7, wherein the liquid culture medium composition further comprises polysaccharides other than deacylated gellan gum or its salts. 10. The production method of claim 8, wherein the liquid culture medium composition further comprises polysaccharides other than deacylated gellan gum or its salts. 11. A method of production according to any one of claims 7-10, wherein the liquid culture medium composition comprises metal ions. 12. The production method of claim 11, wherein the metal ion is a calcium ion. 13. A production method according to any one of claims 7-10, comprising mixing a polysaccharide dissolved or dispersed in a solvent with a liquid culture medium. 14. The production method of claim 13, wherein the polysaccharide dissolved or dispersed in the solvent is in a sterilized state. 15. The production method of claim 14, wherein sterilization is carried out by autoclaving. 16. The production method of claim 14, wherein sterilization is performed by filtration sterilization. 17. The production method of claim 16, wherein the filtration sterilization comprises passing through a 0.1-0.5 μm filter. 18. Use of the liquid culture medium composition of any one of claims 1-6 for culturing cells or tissues, wherein the cells are not embryonic stem cells or germ cells. 19. A cell or tissue culture comprising a liquid culture medium composition of any one of claims 1-6 and cells or tissues, wherein the cells are not embryonic stem cells or germ cells. 20. A method for culturing cells or tissues, comprising culturing cells or tissues in a liquid culture medium composition of any one of claims 1-6, wherein the cells are not embryonic stem cells or germ cells. 21. The culture method of claim 20, wherein the cells are selected from pluripotent stem cells, cancer cells, and hepatocytes. 22. A method for recovering cells or tissues, comprising isolating cells or tissues from a culture of claim 19. 23. The recycling method of claim 22, wherein the separation is performed by filtration, centrifugation or magnetic separation. 24. A method for producing spheres, comprising culturing adhesive cells in a liquid culture medium composition of any one of claims 1-6, said cells being neither embryonic stem cells nor germ cells. 25. The cell or tissue culture of claim 19, wherein the cells are adhesive cells or non-adhesive cells. 26. The cell or tissue culture of claim 25, wherein the aforementioned adhesive cells are spheres. 27. The cell or tissue culture of claim 26, wherein the aforementioned adhesive cells are selected from pluripotent stem cells, cancer cells, and hepatocytes.