JP2017077240A - Adhesive cell culturing substrate, and cell culture vessel and cell culture method using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an adhesive cell culturing substrate which does not require nano imprinting of an uneven structure and can be formed, even after gamma-ray sterilization, with spheroid adhering to a base material surface.SOLUTION: An adhesive cell culturing substrate has albumin adsorption on a cell culture surface of more than 830 ng/cm.SELECTED DRAWING: Figure 4

Description

  The present invention relates to an adhesive cell culture substrate, a cell culture container having the adhesive cell culture substrate, and a cell culture method using the adhesive cell culture substrate.

  Cells that form organs such as the liver, pancreas, skin, and blood vessels are three-dimensionally forming a network and expressing their functions in vivo.

  Therefore, in research on regenerative medicine that regenerates the functions of these organs, when cells of these organs are cultured, a culture in which cells can form a three-dimensional network (that is, three-dimensional culture) is required. It is done. A spheroid is a tissue in which cells form a three-dimensional network. However, when cells are cultured on the surface of a general resin-made cell culture substrate, the cells spread in a plane and proliferate (two-dimensional culture), and a three-dimensional network is not formed.

  As a technique for performing three-dimensional culture, a method of culturing in a floating state in a container in which a resin layer having low cell adhesion is formed (Patent Document 1), or a method of using a substrate on which a concavo-convex structure is nanoimprinted (Patent) Document 2) is known.

JP 2008-061609 A JP 2014-210404 A

  When culture is performed using a substrate having low cell adhesiveness as described in Patent Document 1, the motility of the cells increases, and thus the adhesiveness between cells increases. This makes it difficult to control the size of the formed spheroids. Moreover, since the size of spheroids increases in suspension culture, there is a risk that cells existing in the center of the spheroids are necrotized. In addition, when culturing cells, it is essential to periodically change the medium, but in the case of a substrate with low cell adhesion as described in Patent Document 1, since spheroids are formed in a floating state, When removing the medium at the time of medium exchange, spheroids are removed together with the medium, and long-term culture is difficult.

  Although a spheroid formation is promoted by using a substrate imprinted with a concavo-convex structure as described in Patent Document 2 (that is, a substrate having a nano concavo-convex structure), the substrate having such a fine structure Is expensive due to imprint processing, and it is necessary to remove air bubbles accumulated in the recesses during use, which complicates the operation. In addition, in a substrate imprinted with a concavo-convex structure (that is, a substrate formed with a nano concavo-convex structure), since spheroids are weakly attached to the substrate, spheroids easily associate with each other, and it is difficult to control the size of the spheroids There are cases. Furthermore, since the spheroids are easily separated from the base material, there is a problem that the spheroids are easily lost when the medium is changed.

  Instruments used for cell culture need to be sterilized in advance before being used for culture. Known sterilization treatments for cell culture instruments include autoclave sterilization, ethylene oxide gas sterilization, and gamma ray sterilization. However, autoclave sterilization is not suitable for sterilization of instruments with low heat resistance such as thermoplastic resin, and ethylene oxide gas is used for regenerative medical purposes from the viewpoint of safety due to residual gas absorbed by instruments. Concerns remain for use in sterilizing cell culture instruments. On the other hand, gamma ray sterilization can be applied to sterilization of instruments having low heat and pressure resistance, and has an advantage that there is no problem of residual gas such as ethylene oxide gas. Regarding the sterilization means as described above, the present inventors have found that there is a problem that conventional cell culture substrates are not suitable for gamma ray sterilization. That is, it has been found that when a conventional cell culture substrate is sterilized with gamma rays and used for cell culture, three-dimensional network formation by cells is remarkably reduced.

  Therefore, the present invention has been made in view of the above circumstances, and does not require an imprint of a concavo-convex structure (that is, formation of a nano concavo-convex structure), and even after gamma sterilization, a spheroid is used as a base material. An object of the present invention is to provide an adhesive cell culture substrate that can be formed on a surface.

  The present inventors have intensively studied to solve the above problems. As a result, it has been found that the above problems can be solved by an adhesive cell culture substrate having an albumin adsorption amount on a cell culture surface of a predetermined value or more, and the present invention has been completed.

  According to the present invention, it is possible to form the spheroids in a state where they are attached to the surface of the substrate without requiring imprinting of the concavo-convex structure (that is, formation of the nano concavo-convex structure) and after gamma ray sterilization. An adhesive cell culture substrate that can be produced can be provided. Since the spheroids are attached to the substrate surface, it is possible to avoid the spheroids from meeting and becoming larger, and it is also possible to avoid losing the spheroids during medium exchange.

FIG. 1 is a diagram schematically showing a method for measuring the albumin adsorption amount. FIG. 2 schematically shows the cell culture container of the present invention. FIG. 3 is a photomicrograph of rat primary hepatocytes (5th day of culture) sterilized by gamma sterilization of 6FDA / TPEQ film (albumin adsorption amount = 830 ng / cm ² ) baked at 300 ° C. FIG. 4 is a photomicrograph of rat primary hepatocytes (5th culture day) sterilized by gamma sterilization of 6FDA / TPEQ film (albumin adsorption amount = 1009 ng / cm ² ) baked at 340 ° C.

<Base material for adhesive cell culture>
One embodiment of the present invention is an adhesive cell culture substrate in which the amount of albumin adsorbed on the cell culture surface exceeds 830 ng / cm ² . By using such an adherent cell culture substrate, spheroids can be formed on the substrate surface without requiring a nano-imprint with a concavo-convex structure and after gamma sterilization. . The mechanism is still unclear, but is presumed as follows.

  When the adhesive cell culture substrate is sterilized by gamma irradiation, a reforming reaction of the material for forming the substrate such as a resin occurs on the surface of the substrate, resulting in a chemical bond on the substrate. The presence of such chemical bonds on the substrate is considered to improve the adhesion of cells to the substrate. For this reason, it is thought that spheroid formation becomes difficult because the cells spread and spread easily in a plane and become two-dimensional culture.

Albumin is a protein that is abundant in plasma, but is known to be adsorbed to various materials such as resin materials by hydrophobic interaction or electrostatic interaction. In general, it is known that the adhesiveness of cells to a substrate becomes strong when the surface of the substrate has an appropriate hydrophobicity and is difficult to adhere to a hydrophilic surface. Therefore, in the adhesive cell culture substrate having an albumin adsorption amount of a predetermined value or more, when the cultured cells are seeded on the substrate, the adhesion of the cells to the substrate surface is not excessively strong, and spheroid formation is not caused. It is speculated that it will be possible. On the other hand, when the amount of albumin adsorbed on the cell culture surface is 830 ng / cm ² or less, floating spheroids are formed because cells exhibit two-dimensional growth or cells cannot adhere to the substrate. . This is because the substrate with an albumin adsorption amount of 830 ng / cm ² or less on the cell culture surface is (1) the hydrophobicity of the cell culture surface is in an appropriate range for cell adhesion, and the cell adhesion to the substrate is strong. As a result, cells expand in a plane and proliferate, or (2) components such as proteins (such as collagen and elastin) that constitute the extracellular matrix secreted by cells, and base materials for cell adhesion proteins such as integrins This is probably due to the low adhesion to the cells and the cells floating. Note that the above mechanism is speculation and does not limit the technical scope of the present invention.

  Embodiments of the present invention will be described below. In addition, this invention is not limited only to the following embodiment.

  In this specification, “X to Y” indicating a range means “X or more and Y or less”. Unless otherwise specified, measurement of operation and physical properties is performed under conditions of room temperature (20 to 25 ° C.) / Relative humidity 40 to 50% RH.

<Base material for adhesive cell culture>
The adhesive cell culture substrate (hereinafter simply referred to as “substrate”) according to the present invention is characterized in that the amount of adsorbed albumin measured by the method described later exceeds 830 ng / cm ² . Thereby, it is possible to form the spheroid in a state of being attached on the surface of the substrate without requiring nano-imprinting of the concavo-convex structure and after gamma ray sterilization. In the present specification, when simply described as “albumin adsorption amount”, it refers to a value measured using an adhesive cell culture substrate not irradiated with gamma rays. Although the albumin adsorption amount tends to decrease after gamma irradiation, if the albumin adsorption amount before gamma irradiation is in the above range, a high albumin adsorption amount is achieved even after gamma irradiation. Therefore, spheroid formation is promoted.

(Measurement method of albumin adsorption)
Hereinafter, a method for measuring the albumin adsorption amount will be described with reference to FIG. 1 as appropriate. FIG. 1 shows a step of adsorbing albumin to an adhesive cell culture substrate in a method for measuring albumin adsorption. As shown in FIG. 1, an adhesive cell culture substrate 1b, a 300 μl albumin solution 2 and an adhesive cell culture substrate 1a are arranged on a pedestal 3 in this order. At this time, the adhesive cell culture substrates 1a and 1b are arranged so that the cell culture surface is in contact with the albumin solution 2. The pedestal 3 is housed in the petri dish 4 to ensure vibration stability. The pedestal 3 housed in the petri dish 4, the adhesive cell culture substrate 1 b, the albumin solution 2, and the adhesive cell culture substrate 1 a disposed on the pedestal 3 are collectively referred to as a unit 10. The albumin solution 2 is prepared by dissolving bovine serum albumin powder in Dulbecco's PBS (−) immediately before use so that the final concentration is 20 μg / mL. Adhesive cell culture substrates 1a and 1b are each circular with a diameter of 14 mm. The unit 10 is allowed to stand in a 5 vol% CO ₂ incubator at 37 ° C. containing a vat containing water and humidified, and the albumin solution is recovered after 7 hours from the storage. If the subsequent albumin concentration measurement is not performed immediately after collection, the collected solution is stored at −20 ° C. The albumin concentration in the collected albumin solution is measured by the ELISA method. The ELISA method can be performed using a commercially available sandwich ELISA kit. In the present invention, the ELISA kit for albumin measurement described in the examples (Bethyl Laboratories) or an equivalent thereof is used. . Initial concentration (20 [mu] g / mL) and the area of the difference and the film of the albumin concentration after 7 hours (3.08cm ² in one per side 1.54 cm ² × 2 sheets) per unit area of the substrate albumin The adsorption amount (ng / cm ² ) is calculated.

The amount of adsorbed albumin measured by the above method on the adhesive cell culture substrate not irradiated with gamma rays is not particularly limited as long as it exceeds 830 ng / cm ² , but is preferably 840 ng / cm ² or more, more preferably 900 ng / cm ² or more, further preferably 950ng / cm ² or more, particularly preferably 1000 ng / cm ² or more. Thereby, since a cell adheres to a base material moderately without floating, it becomes possible to form a spheroid in the state where it adhered on the surface of a base material even after gamma ray irradiation. The upper limit of the albumin adsorption amount is not particularly limited, from the viewpoint of spheroid formation is better, preferably 1200 ng / cm ² or less, more preferably 1150ng / cm ² or less, more preferably 1100 ng / cm ² Or less, particularly preferably 1050 ng / cm ² or less.

  The albumin adsorption amount of the substrate can be set to a desired numerical value by selecting an appropriate material. Although not particularly limited, more specifically, the firing temperature at the time of production of the resin forming the base material, or a functional group that exhibits water repellency in the molecular structure constituting the resin (for example, trifluoromethyl). The amount of albumin adsorption can be controlled by adjusting the ratio of fluorine-containing groups such as groups. For example, the amount of albumin adsorbed on the substrate can be increased by increasing the firing temperature during resin production. Although this detailed mechanism is unknown, it is assumed that, for example, in the case of a resin such as polyimide, it is related to the improvement of the imidization rate by the improvement of the firing temperature. Further, by increasing the proportion of fluorine atoms per unit structure of the resin, albumin becomes difficult to access the resin, possibly due to improved water repellency, so that the amount of albumin adsorbed on the substrate can be reduced. In the present invention, the control means is not limited to the above as long as the albumin adsorption amount of the base material is in a desired range.

In the present invention, any substrate for adhesive cell culture may be used as long as the amount of albumin adsorbed on the cell culture surface in the non-gamma irradiation state exceeds 830 ng / cm ² . Although the amount of albumin adsorbed on the cell culture surface is reduced by gamma irradiation, the adherent cell culture substrate has an albumin adsorbed amount of 800 ng / cm ² or more after gamma irradiation on spheroid-forming materials. It is preferable from the viewpoint. The above-mentioned “adsorbed amount of albumin on the cell culture surface after gamma irradiation” is a value measured by the above-described method for measuring the amount of adsorbed albumin after gamma irradiation treatment (8 kGy) of the substrate for cell culture. is there. The amount of albumin adsorbed on the cell culture surface after gamma irradiation is more preferably 810 ng / cm ² or more, further preferably 820 ng / cm ² or more, particularly preferably 830 ng / cm ² or more, and most preferably 900 ng / cm ² or more. It is. The upper limit of the amount of albumin adsorbed after gamma irradiation is not particularly limited, but is preferably 1200 ng / cm ² or less, more preferably 1100 ng / cm ² or less from the viewpoint of preventing cells from growing two-dimensionally. , still more preferably 1050 ng / cm ² or less, particularly preferably 1000 ng / cm ² or less.

  The cell culture surface of the adhesive cell culture substrate preferably has a static water contact angle of 75 ° or more and a falling angle of 15 ° or more. When the adhesive cell culture substrate satisfies such conditions, spheroid formation on the adhesive cell culture substrate is further promoted. From the viewpoint of spheroid formation, the static water contact angle is more preferably more than 80 °, more preferably more than 81 °, and the upper limit of the static water contact angle is, for example, less than 150 °, preferably less than 120 °, More preferably, it is 100 ° or less, more preferably less than 90 °. From the viewpoint of spheroid formation, the falling angle is preferably as high as possible in the order of 18 ° or more, 20 ° or more, 22 ° or more, 24 ° or more. The upper limit value of the sliding angle is, for example, less than 80 °, preferably less than 70 °, more preferably less than 60 °, and still more preferably less than 50 °. In addition, said static water contact angle and sliding angle are the values measured by the method as described in an Example.

The material of the substrate for adhesive cell culture can be exemplified by a resin or the like, but preferably contains a resin from the viewpoint of easy control of the albumin adsorption amount. Such a resin is not particularly limited as long as it is highly biocompatible and can be used as an adhesive cell culture substrate, and the amount of albumin adsorbed on the cell culture surface exceeds 830 ng / cm ² . Although not particularly limited, for example, the resin contained in the adhesive cell culture substrate is exemplified by fluorine resin, polyimide resin (for example, fluorine-containing polyimide resin), polysulfone, polyethersulfone, polydimethylsiloxane, and the like, and blends thereof. From the viewpoint that the albumin adsorption amount can be easily controlled by the firing temperature, a polyimide resin is preferably used. That is, in one preferred embodiment of the present invention, the adhesive cell culture substrate contains a polyimide resin. As a polyimide resin, the polyimide resin containing the structural unit shown by the following formula | equation (I) can be illustrated. Further, from the viewpoint of good spheroid formation, improved heat resistance, and easier control of the firing temperature, a resin having a fluorine atom in the molecule is preferred, and a fluorinated polyimide (fluorinated polyimide resin) is more preferred. preferable. The polyimide resin used in the present invention is typically obtained by imidizing a polyamic acid obtained by polymerizing at least one acid dianhydride and one or more diamines. The polyimide resin may contain polyamic acid as part of its chemical structure. As a method for producing the polyimide resin, for example, a two-step synthesis method can be used. The two-stage synthesis method of polyimide resin is a method of synthesizing polyamic acid as a precursor and converting the polyamic acid into polyimide acid. The polyamic acid as the precursor may be a polyamic acid derivative. Examples of the polyamic acid derivatives include polyamic acid salts, polyamic acid alkyl esters, polyamic acid amides, polyamic acid derivatives from bismethylidene pyromellitide, polyamic acid silyl esters, and polyamic acid isoimides. Polyimides include pyromellitic dianhydride, biphenyltetracarboxylic dianhydride, benzophenone tetracarboxylic dianhydride and other acid anhydrides, and diamines such as oxydiamine, paraphenylenediamine, metaphenylenediamine, and benzophenonediamine. The polyimide which becomes can be illustrated. Examples of the resin having a fluorine atom include 4,4′-hexafluoroisopropylidenediphthalic anhydride (6FDA) / 1,4-bis (aminophenoxy) benzene (TPEQ) copolymer, 6FDA / 4,4. '-Oxydiphthalic anhydride (ODPA) / TPEQ copolymer, 4,4'-(4-4'-isopropylidenediphenoxy) diphthalic acid (BPADA) / 2,2-bis [4- (4-aminophenoxy) ) Phenyl] hexafluoropropane (HFBAPP) and other fluorine-containing polyimide resins containing structural units represented by the following formula (I); ethylene-tetrafluoroethylene copolymer and the like.

In the above formula (I), X ⁰ represents an oxygen atom, a sulfur atom, or a divalent organic group; Y represents a divalent organic group; Z ¹ , Z ² , Z ³ , Z ⁴ , Z ⁵ and Z ⁶ each independently represent a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom or an iodine atom, and p is 0 or 1. In the polyimide resin, the chemical structure represented by the formula (I) may be different for each structural unit of the resin, or may be the same. It is preferable that at least one of X ⁰ , Y, Z ¹ , Z ² , Z ³ , Z ⁴ , Z ⁵ , and Z ⁶ contains one or more fluorine atoms.

In the above formula (I), when p = 0, X ⁰ may not be present (in other words, the left and right benzene rings may be directly bonded), but when p = 1 the left and right benzene ring linked via X ^0.

The divalent organic group represented by X ^0, specifically, an alkylene group, an arylene group, arylene group, Arirenchio group and the like. Among these, an alkylene group, arylene group, Arirenchio group Are more preferable, and an alkylene group and an aryleneoxy group are more preferable, and these may be substituted with a fluorine atom. Carbon number of the said alkylene group is 1-12, for example, Preferably it is 1-6.

The alkylene group substituted with a fluorine atom is an example of X ^0, for _{example, -C (CF 3) 2 -} , - C (CF 3) 2 -C (CF 3) 2 - and the like can be exemplified . Among the above-described alkylene groups that are examples of X ⁰ , —C (CF ₃ ) ₂ — is preferable.

The arylene group is an example of X ^0, for example, it can be exemplified by the following.

The arylene group is an example of X ^0, for example, it can be exemplified by the following.

The Arirenchio group examples of X ^0, for example, can be exemplified by the following.

From the standpoint that spheroids can be satisfactorily formed on a substrate after irradiation with gamma rays, even if the cell has a decreased proliferation activity, the divalent organic group represented by X ⁰ is the above b-2 to b- Is preferably selected from the group consisting of 10 and c-2 to c-10, more preferably selected from the group consisting of b-7 to b-9 and c-7 to c-9. A structure represented by 8 is more preferable.

The above-mentioned arylene group, aryleneoxy group and arylenethio group which are examples of X ⁰ are each independently a halogen atom (for example, a fluorine atom, a chlorine atom, a bromine atom or an iodine atom, preferably a fluorine atom or a chlorine atom) An atom, more preferably a fluorine atom.), A group selected from the group consisting of a methyl group and a trifluoromethyl group. There may be a plurality of these substituents, in which case the types of the substituents may be the same or different. Suitable substituents substituted on the arylene group, aryleneoxy group and arylenethio group are a fluorine atom and / or a trifluoromethyl group, and preferably a fluorine atom. The arylene group, aryleneoxy group and arylenethio group are preferably substituted with at least one fluorine atom when Y does not contain a fluorine atom.

  In the above formula (I), the divalent organic group represented by Y is not particularly limited, and examples thereof include a divalent organic group having an aromatic ring. Specifically, a group consisting of one benzene ring or a group having a structure in which two or more benzene rings are bonded via a carbon atom (that is, a single bond or an alkylene group), an oxygen atom, a sulfur atom or directly. Can be mentioned. Specifically, the following groups can be exemplified.

The divalent organic group having an aromatic ring described above as an example of Y is a halogen atom (for example, a fluorine atom, a chlorine atom, a bromine atom or an iodine atom, preferably a fluorine atom or a chlorine atom, if it can be substituted). , More preferably a fluorine atom), and may be substituted with a group selected from the group consisting of a methyl group and a trifluoromethyl group. There may be a plurality of these substituents, in which case the types of the substituents may be the same or different. A preferred substituent substituted with a divalent organic group having an aromatic ring is preferably a fluorine atom and / or a trifluoromethyl group, and more preferably, particularly when X ⁰ does not contain a fluorine atom. Is a fluorine atom.

  From the viewpoint of spheroid formation, Y is a structure selected from the group consisting of d-3, d-9, e-1 to e-4, f-6, and f-7 in the above formula (I). It is preferably a structure of e-1, e-3 or e-4, more preferably a structure of e-4.

In the above formula (I), Z ¹ , Z ² , Z ³ , Z ⁴ , Z ⁵ , and Z ⁶ may be the same or different, and each independently represents a hydrogen atom or a fluorine atom. , A chlorine atom, a bromine atom or an iodine atom, and at least one of X ⁰ and Y does not contain a fluorine atom, at least one of Z ¹ , Z ² , Z ³ , Z ⁴ , Z ⁵ and Z ⁶ One is preferably a fluorine atom.

From the viewpoint of spheroid formation, in a preferred embodiment of the present invention, in the above formula (I), the divalent organic group represented by X ⁰ is —C (CF ₃ ) ₂ —, the above b- _{2 to} b. -10 and c-2 to c-10; and Y is selected from the group consisting of d-3, d-9, e-1 to e-4, f-6, and f-7. Selected. In a more preferred embodiment of the present invention, in the above formula (I), the divalent organic group represented by X ⁰ is selected from the group consisting of b-7 to b-9 and c-7 to c-9. And Y is selected from the group consisting of e-1, e-3 and e-4.

  The polyimide resin composed of the structural unit represented by the above formula (I) can be obtained by a method of baking polyamic acid obtained by polymerization of acid dianhydride and diamine. Below, the synthesis | combination process of 6FDA / TPEQ copolymer is shown as one specific example. The imidation ratio of the “polyimide resin composed of the structural unit represented by the formula (I)” may not be 100%. That is, the polyimide resin composed of the structural unit represented by the formula (I) may be composed only of the structural unit represented by the above formula (I), but within the range where the objective effect of the present invention is not impaired. In addition, a structural unit in which the cyclic imide structure remains as amic acid without dehydration and ring closure may be included in part.

  The polyamic acid synthesis reaction is preferably performed in an organic solvent. The organic solvent used in the polyamic acid synthesis reaction is not particularly limited as long as the reaction between the raw material acid dianhydride and the diamine can proceed efficiently and is inert to these raw materials. . For example, N-methylpyrrolidone (NMP), N, N-dimethylacetamide, N, N-dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, sulfolane, methyl isobutyl ketone, acetonitrile, benzonitrile, nitrobenzene, nitromethane, acetone, methyl ethyl ketone, isobutyl ketone And polar solvents such as methanol; nonpolar solvents such as toluene and xylene. Among these, it is preferable to use a polar solvent. These organic solvents may be used alone or as a mixture of two or more.

When a polyimide resin composed of the structural unit represented by the above formula (I) is used as a material for forming the adhesive cell culture substrate, in order to increase the amount of albumin adsorbed on the cell culture surface, What is necessary is just to make baking temperature high. Although the detailed mechanism by which the amount of adsorbed albumin is improved by increasing the firing temperature is unknown, it is presumed to be related to the imidization rate, and the imidation rate of polyamic acid is increased by high-temperature firing, and the polyimide resin It is presumed that the charge of the resin is changed by reducing the ratio of secondary amine derived from the diamine as the raw material. Although the firing temperature varies depending on the molecular structure of the resin, it cannot be specified unconditionally. For example, when firing a 6FDA / TPEQ copolymer, a BPADA / TPEQ copolymer, or a BPADA / HFBAPP copolymer, 340 ° C. or higher. It is. By baking at such a high temperature zone, the adhesive cell culture substrate has an albumin adsorption amount exceeding 830 ng / cm ² on the cell culture surface, and even after gamma sterilization, the nano uneven structure Spheroids can be formed on the substrate surface without the need for By using a resin having a fluorine atom in the molecule (for example, a fluorine-containing polyimide resin containing a structural unit represented by the formula (I)), it becomes easy to control the amount of adsorbed albumin during baking at a high temperature as described above. . The upper limit of the firing temperature is not particularly limited, but is, for example, less than 500 ° C., preferably 450 ° C. or less. The firing time is, for example, 0.2 to 10 hours.

  The weight average molecular weight of the resin as the material for forming the adhesive cell culture substrate is, for example, 5,000 to 2,000,000, preferably 8,000 to 1,000,000, and more preferably. 20,000 to 500,000. In the present specification, the weight average molecular weight of the resin is a value measured by the following method. When the weight average molecular weight is in the above range, the spheroid-forming property after gamma irradiation becomes better.

(Measurement of weight average molecular weight)
Device: HCL-8220GPC manufactured by Tosoh Corporation
Column: TSKgel Super AWM-H
Eluent (LiBr · H ₂ O, phosphoric acid-containing NMP): 0.01 mol / L
Measurement method: A 0.5% by weight solution is prepared with an eluent, and the molecular weight is calculated based on a calibration curve prepared with polystyrene.

The adhesive cell culture substrate according to the present invention only needs to have an albumin adsorption amount of more than 830 ng / cm ² on the cell culture surface, and includes only other main components such as the above resin. It may be a thing. For example, the adhesive cell culture substrate has a resin layer made of a resin having an albumin adsorption amount exceeding 830 ng / cm ² on a support member such as a resin having a low albumin adsorption amount (eg, polystyrene), inorganic glass or metal. The structure which has in a cell culture surface may be sufficient. Alternatively, the adhesive cell culture substrate may further contain additive components such as a plasticizer and an antioxidant. The thickness of the substrate is not particularly limited and can be set arbitrarily, but is, for example, 0.1 μm to 10 mm, preferably 1 μm to 1 mm.

  The adhesive cell culture substrate according to the present invention can be used for spheroid formation without being subjected to a nano uneven structure forming process, but does not exclude those having a nano uneven structure. The formation processing of the nano uneven structure with respect to the adhesive cell culture substrate can be performed, for example, by the method described in JP-A No. 2014-210404.

<Cell culture container, cell culture method>
In one embodiment of the present invention, a cell culture container having the above-mentioned adhesive cell culture substrate is provided. The cell culture vessel of the present invention may be configured by combining the cell culture substrate of the present invention and other members, or the cell culture substrate of the present invention and other members are integrated. It may be comprised, and may be comprised only by the base material for cell cultures of this invention. FIG. 2 illustrates one embodiment of the cell culture container according to the present invention. The cell culture vessel may be composed of the adhesive cell culture substrate 1 as shown in FIG. 2 (A), or the adhesive cell culture substrate 1 as shown in FIGS. 2 (B) and (C). It may consist of the support member 20. In FIG. 2, the inner shape and the outer shape when the cell culture container is viewed in plan from the opened side can be arbitrary shapes such as a circle and a polygon (square, triangle, etc.), respectively. Examples of the material constituting the support member 20 include inorganic glass; carbon; metal such as silicon; polyolefin resin such as polyethylene, polypropylene and cyclic olefin; polyester resin such as polyethylene terephthalate (PET); acrylic such as polymethyl methacrylate. Resin; epoxy resin; polyvinyl chloride, polyvinylidene chloride, polystyrene resin, polyvinyl acetate, ABS (acrylonitrile-butadiene-styrene) resin, polycarbonate resin, vinyl ether, polyacetal, polyphenylene ether (PPE), polyaryl ether, polyphenylenesulfur Examples include fido (PPS), polyether ether ketone (PEEK), polyaryl ether ketone, phenol resin, polyether nitrile (PEN), and the like.

  The cell culture vessel of the present invention may be any shape as long as it includes the cell culture substrate of the present invention. For example, it can be in the form of various containers such as plates for culture such as single or multiwell plates, petri dishes, dishes, flasks, bags and the like. The cell culture container of the present invention may also be in the form of a cell culture container in a culture apparatus such as a mass culture apparatus or a perfusion culture apparatus.

  In one embodiment of the present invention, a cell culture method including the step of culturing adhesive cells on the above-mentioned adhesive cell culture substrate is provided. The above-mentioned adhesive cell culture substrate can form spheroids by culturing adhesive cells on the surface thereof without forming a nano uneven structure. Moreover, since the above-mentioned adhesive cell culture substrate can form spheroids even after sterilization with gamma rays, it can be used in a cell culture instrument combined with a member having low heat and pressure resistance. Further, since gamma ray sterilization is possible, there is an advantage that there is no problem of residual gas such as ethylene oxide gas. In a further embodiment of the present invention, there is provided a cell culturing method in which the adherent cells are three-dimensionally cultured in the culturing step.

  The type of cells cultured by the cell culture method of the present invention is not particularly limited, and normal cells, cancer cells, and fused cells such as hybridomas can be used, and cells that have been subjected to artificial treatment such as gene transfer. May be. Although it is not particularly limited, for example, three-dimensional culture of artificial pluripotent stem cells (Induced pluripotent stem cells: iPS cells), embryonic stem cells (Embryonic stem cells: ES cells), mesenchymal stem cells, etc. is generally performed. Adipose cells, hepatocytes, kidney cells, pancreatic cells, mammary cells, endothelial cells, epithelial cells, smooth muscle cells, myoblasts, cardiomyocytes, nerve cells, including the required cells and various progenitor cells and stem cells, Glial cells, dendritic cells, chondrocytes, osteoblasts, osteoclasts, bone cells, fibroblasts, various blood cells, retinal cells, cornea-derived cells, gonad-derived cells, various line cells, other mesenchymal precursors And cells such as cells and various cancer cells. The biological species from which these cells are derived is not particularly limited, and various cells derived from humans and non-human animals can be used. Examples of the biological species from which the cells are derived include, for example, primates such as humans, rhesus monkeys, green monkeys, cynomolgus monkeys, chimpanzees, tamarins and marmosets, rodents such as mice, rats, hamsters and guinea pigs, dogs, cats, rabbits, pigs, Examples include cows, goats, sheep, horses, chickens, quails, minks, pineapples, and zebrafish.

  What is necessary is just to select the culture medium used for a cell culture suitably according to a cell. The type of medium is not particularly limited. For example, any cell culture basic medium, differentiation medium, primary culture medium, or the like can be used. Specifically, Eagle small essential medium (EMEM), Dulbecco's modified Eagle medium (DMEM), α-MEM, Glasgow MEM (GMEM), IMDM, RPMI1640, Ham F-12, MCDB medium, Williams medium E, Hepatocyte two medium And a mixed medium thereof, but are not limited to these, and any medium containing components necessary for growth and differentiation of cells can be used. Furthermore, a medium supplemented with serum, various growth factors, differentiation-inducing factors, antibiotics, hormones, amino acids, sugars, salts and the like may be used. The culture temperature is not particularly limited, but is usually about 25 to 40 ° C.

  Examples of tissues formed by three-dimensional culture include spheroids and three-dimensional cell aggregates. Spheroids or three-dimensional cell aggregates are spheroids or three-dimensional cell aggregates formed of a single cell such as a hepatocyte, and various fibroblasts, vascular endothelial cells, etc., and two or more different types such as hepatocytes. It may be a spheroid mixed with cell types or a three-dimensional cell aggregate. Examples of cells that can be used include the various cells described above. In one embodiment, there is provided the above-mentioned adhesive cell culture substrate, wherein the spheroid is adhered to the cell culture surface. In another embodiment, a cell culture container having an adhesive cell culture substrate having a spheroid adhered to the cell culture surface is provided.

  The effects of the present invention will be described using the following examples and comparative examples. However, the technical scope of the present invention is not limited only to the following examples. Unless otherwise specified, the following operations were performed at room temperature (25 ° C.).

<Comparative Example 1>
1. Preparation of Polyamic Acid Resin Composition 2.976 g (10.2 mmol) of 1,4-bis (aminophenoxy) benzene (TPEQ), 4,4′-hexafluoroisopropylidenediphthalic anhydride in a 100 ml three-necked flask (6FDA) 4.524 g (10.2 mmol) and N, N-dimethylacetamide 42.5 g were charged. By stirring at room temperature for 5 days under a nitrogen atmosphere, a fluorine-containing polyamic acid resin composition (solid content concentration: 15.0% by mass) was obtained. The weight average molecular weight of the polyamic acid was 180,000. The weight average molecular weight of the polyamic acid and the weight average molecular weight of the fluorinated polyimide resin after firing are substantially the same.

2. Preparation of fluorinated polyimide film The obtained fluorinated polyamic acid resin composition was formed into a film so that the thickness of the fluorinated polyimide film after firing was 30 μm on a glass substrate using a die coater, Firing was performed at 300 ° C. for 1 hour in a nitrogen atmosphere. Thereafter, the film was peeled from the glass to obtain a fluorine-containing polyimide film 1.

<Example 1>
Except having changed the calcination temperature into 340 degreeC, operation similar to the comparative example 1 was performed and the fluorine-containing polyimide film 2 was obtained.

<Example 2>
1. Preparation of polyamic acid resin composition In a 100 ml three-necked flask, 3.842 g (7.38 mmol) of 1,4-bis (aminophenoxy) benzene (TPEQ), 4,4 ′-(4-4′-isopropylidene) Phenoxy) diphthalic acid (BPADA) 2.158 g (7.38 mmol) and N-methylpyrrolidone 34 g were charged. The polyamic acid resin composition (solid content concentration 15.0 mass%) was obtained by stirring at room temperature for 5 days under nitrogen atmosphere. The weight average molecular weight of the polyamic acid was 120,000. In addition, the weight average molecular weight of a polyamic acid and the weight average molecular weight of the polyimide resin after baking are substantially the same.

2. Preparation of polyimide film The obtained polyamic acid resin composition was formed into a film shape so that the thickness of the polyimide film after firing was 30 μm on a glass substrate using a die coater, and the film was formed at 340 ° C. for 1 hour. Firing was performed in a nitrogen atmosphere. Thereafter, the film was peeled from the glass to obtain a polyimide film 3. The structural unit of the BPADA / TPEQ copolymer is shown below.

<Example 3>
1. Preparation of Polyamic Acid Resin Composition 3.006 g (5.77 mmol) of 2,2-bis [4- (4-aminophenoxy) phenyl] hexafluoropropane (HFBAPP), 4,4′- in a 100 ml three-necked flask 2.994 g (5.77 mmol) of (4-4′-isopropylidenediphenoxy) diphthalic acid (BPADA) and 34 g of N-methylpyrrolidone were charged. By stirring at room temperature for 5 days under a nitrogen atmosphere, a fluorine-containing polyamic acid resin composition (solid content concentration: 15.0% by mass) was obtained. The weight average molecular weight of the polyamic acid was 100,000. The weight average molecular weight of the polyamic acid and the weight average molecular weight of the fluorinated polyimide resin after firing are substantially the same.

2. Preparation of fluorinated polyimide film The obtained fluorinated polyamic acid resin composition was formed into a film so that the thickness of the fluorinated polyimide film after firing was 30 μm on a glass substrate using a die coater, Firing was performed at 340 ° C. for 1 hour in a nitrogen atmosphere. Thereafter, the film was peeled off from the glass to obtain a fluorine-containing polyimide film 4. The structural unit represented by the formula (I) of the BPADA / HFBAPP copolymer is shown below.

<Measurement of static water contact angle>
Apparatus: Automatic contact angle meter (manufactured by Kyowa Interface Science: DM-500)
Measuring method: The adhesion angle of the droplet immediately after dropping 2 μl of water on the film was measured (measurement temperature: 25 ° C.).

<Measurement of sliding angle>
Apparatus: Automatic contact angle meter (manufactured by Kyowa Interface Science: DM-500)
Measurement method: After 25 μl of water was dropped on the film, the base material was continuously tilted, and the angle when the water flowed down was defined as the falling angle. (Measurement temperature: 25 ° C.).

<Measurement of albumin adsorption amount>
Bovine serum albumin (Sigma; A8022-10G) was dissolved in Dulbecco's PBS (-) (Wako; 041-20211) to an albumin concentration of 20 μg / mL to obtain an albumin solution. One piece of the above film (circular with a diameter of 14 mm) was set on a pedestal on a petri dish, and 300 μL of the albumin solution was mounted on the film.

Put another film of the same type (circular with a diameter of 14 mm) on the albumin solution, sandwich the albumin solution between the two films, set the area where the film and the albumin solution contact each other, and set the unit Obtained. This was left still in a 5 volume% CO ₂ incubator at 37 ° C. containing a vat containing water and humidified. After 7 hours, the unit was taken out and the albumin solution was recovered. The collected solution was stored at −20 ° C. until measurement.

The albumin concentration in the collected albumin solution was measured. The concentration was measured using Albamin, Bovine, ELISA Quantitation kit (Bethyl Laboratories), and the measurement protocol was in accordance with the attached manual. From the initial concentration (20 [mu] g / mL) area and the difference in the film between the albumin concentration after 7 hours (one per side 1.54cm ² × 3.08cm ² with two sheets), albumin adsorption amount per unit area ( μg / cm ² ) was calculated.

  The measurement results are shown in Table 2. In Table 2, the measurement of albumin adsorption was performed using a film before and after gamma ray sterilization described later, and the contact angle and the falling angle were measured using a film before gamma ray sterilization.

<Acquisition of rat primary hepatocytes>
Specific virtual pathogen free Wistar rats, male, 9 weeks old, weighing 200 g were purchased from Japan SLC. Rat primary hepatocytes were obtained by referring to the method described in Chapter 10 of the Cultured Cell Experiment Handbook (Yodosha), “Hepatocytes”. Specifically, Wistar rats were laparotomized under isoflurane anesthesia, a catheter was inserted into the portal vein, and a preperfusion solution having the composition described below was injected. The chest cavity was then opened and the inferior vena cava entering the right atrium was incised to release blood. After confirming that blood removal from the liver was sufficiently performed, the perfusion was stopped, and the perfusion solution was replaced with a collagenase solution having the composition described below. After confirming that the intercellular tissue was digested by collagenase, the perfusion was stopped. After the liver was cut off and transferred to a glass petri dish, chilled EMEM High Glucose medium (Wako) was added, and the cells were dispersed by pipetting. Next, undigested tissue was removed with a 150 mm filter. The cell suspension was centrifuged at 50G for 1 minute several times to remove non-parenchymal cells. The survival rate of the obtained hepatocytes was measured by the trypan blue exclusion method, and hepatocytes with a survival rate of 85% or more were used as rat primary hepatocytes in the culture test.

<Three-dimensional culture evaluation using rat primary hepatocytes>
In the cell culture test, fluorine-containing polyimide films 1 and 2 (circular with a diameter of 14 mm) prepared as described above were used after sterilization with gamma rays (conditions for gamma sterilization: 8 kGy).

Rat primary hepatocytes obtained by the above-described method are suspended in a serum medium having the following composition, and rat primary hepatocytes of 6.25 × 10 ⁵ cells / mL so as to be 1.33 × 10 ⁴ cells / cm ^2. 0.4 mL of the suspension was seeded on a film subjected to gamma ray sterilization treatment and cultured under conditions of 37 ° C. and 5% CO ₂ . The medium was exchanged 4 hours after seeding, after removing the entire medium on the first day, the third day, and the fifth day of culture, and then adding 0.4 mL of serum medium. On the fifth day of the culture, spheroid formation on each film and the presence or absence of adherent cells were confirmed. As a result, no spheroids were formed on the film 1, and many cells that adhered and extended were observed (FIG. 3). On the other hand, almost no adherent cells were observed on the film 2, and it was confirmed that spheroids were formed on the surface of the substrate (FIG. 4).

(Composition of serum medium)
William's E medium (Wako Pure Chemical), 10% (w / v) FBS (Wako Pure Chemical), 8.6 nM insulin, 255 nM dexamethasone, 50 ng / mL EGF, 5KIU / mL aprotinin, antibiotics (penicillin (100 units / mL) / streptomycin (100 μg / mL) / amphotericin B (0.25 μg / mL)).

<Three-dimensional culture evaluation using rat frozen hepatocytes>
A culture test was performed using frozen hepatocytes having a reduced cell proliferative activity compared to primary hepatocytes.

  In the cell culture test, the polyimide film 3 and the fluorine-containing polyimide film 4 (circular with a diameter of 14 mm) prepared as described above were used after sterilization with gamma rays.

  Rat frozen hepatocytes and hepatocyte how medium were purchased from Oriental Yeast Co., Ltd. and used for experiments. Further, before starting the experiment, 50 μL of 0.4 (w / v)% trypan blue solution (manufactured by Wako Pure Chemical Industries, Ltd.) was added to 200 μL of the serum medium having the above composition to prepare a staining solution.

  A vial containing frozen rat hepatocytes was taken out of liquid nitrogen and placed in a 37 ° C. water bath to thaw the cell suspension. After wiping the vial with 70% (v / v) ethanol, it is transferred into a safety cabinet, and the whole cell suspension in the vial is added to 45 mL of Hepatocyte how medium previously warmed to 37 ° C., at room temperature, 86 × g. For 6 minutes. After discarding the supernatant, 10 mL of the serum medium was added to prepare a cell suspension. 50 μL of the cell suspension and 50 μL of the staining solution were mixed, the number of viable cells and viability were measured using a hemocytometer, and a culture test was performed using rat frozen hepatocytes having a viability of 85% or more.

6.25 × 10 ⁵ cells / mL rat frozen hepatocytes obtained by suspending the rat frozen hepatocytes obtained by the above-described method in a serum medium having the above composition to obtain 1.33 × 10 ⁴ cells / cm ^2. 0.4 mL of the suspension was seeded on a film subjected to gamma ray sterilization treatment, and cultured under conditions of 37 ° C. and 5% by volume CO ₂ . The medium was exchanged 4 hours after seeding, after removing the entire medium on the first day, the third day, and the fifth day of culture, and then adding 0.4 mL of serum medium. On day 5 of culture, spheroid formation on the film and the presence or absence of two-dimensional adherent cells were confirmed.

  In addition, the adsorbed amount of albumin not irradiated with gamma rays for the BPADA / TPEQ copolymer in Example 2 and the BPADA / HFBAPP copolymer in Example 3 was higher than that after gamma rays.

1,1a, 1b Adhesive cell culture substrate (film),
2 albumin solution,
3 pedestals,
4 Petri dish,
10 units,
20 support member,
100, 101, 102 Cell culture vessel.

Claims (7)

An adhesive cell culture substrate, wherein the amount of albumin adsorbed on the cell culture surface exceeds 830 ng / cm ² .   The substrate for adhesive cell culture according to claim 1, wherein the static water contact angle of the cell culture surface is 75 ° or more and the falling angle is 15 ° or more.   The adhesive cell culture substrate according to claim 1 or 2, comprising a polyimide resin.   The adhesive cell culture substrate according to claim 3, wherein the polyimide resin is a fluorine-containing polyimide resin.   The cell culture container which has the base material for adhesive cell cultures of any one of Claims 1-4.   The cell culture method including the process of culture | cultivating an adhesive cell on the base material for adhesive cell cultures of any one of Claims 1-4.   The cell culture method according to claim 6, wherein, in the culturing step, the adherent cells are three-dimensionally cultured.