JP2020018174A - Culture substrate of pluripotent stem cell, and method for producing pluripotent stem cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a culture substrate capable of efficient cell culture of pluripotent stem cells and cell detachment in a short time or with a weak external stimulus, and a production method using the same. SOLUTION: The surface of a base material 1 has a layer made of a temperature-responsive polymer whose response temperature to water is in the range of 0 ° C to 50 ° C, and the average roughness H / layer of the layer at a temperature equal to or higher than the response temperature. A culture substrate 10 for pluripotent stem cells, characterized in that the ratio of thickness D is 0.5 or more and 1 or less. In the culture substrate, the ratio of the average roughness / layer thickness of the layer due to the temperature-responsive polymer at a temperature above the response temperature and the average roughness / layer thickness of the layer due to the temperature-responsive polymer at a temperature below the response temperature. The difference in the ratios of may be 0.1 or more and 1 or less. [Selection diagram] Fig. 1

Description

  The present invention relates to a culture substrate that enables efficient cell culture of pluripotent stem cells and enables cell detachment in a short time or with a weak external stimulus, and a production method using the same.

  Cell culture is used to understand biochemical phenomena and to produce useful substances. In recent years, the discovery of pluripotent stem cells such as ES cells and iPS cells and advances in culture technology have led to the development of cells such as regenerative medicine. A great deal of attention has been paid to treatments using.

  Many cells have adhesive properties, and are known to adhere to biological macromolecules such as collagen, fibronectin, and laminin and to proliferate and differentiate in the body. Similarly, in cell culture, many cells having adhesiveness need to adhere to some kind of base material during culture. Conventionally, surface-treated glass or polymer has been used as a substrate. For example, there is a substrate obtained by performing gamma irradiation or silicone coating on polystyrene. Further, a support having a surface coated with a biopolymer such as collagen or fibronectin is also used.

  Proliferating cells generally need to be subcultured on another substrate after culturing on the substrate, and in many cases, proteolytic enzymes are used. Proteolytic enzymes are responsible for degrading proteins on cell surfaces and breaking bonds between cells and substrates and breaking bonds between cells. On the other hand, it is known that proteolytic enzymes have a large effect on cell viability, and a technique of separating cells from a substrate without using proteolytic enzymes is important as a method that does not damage cells. Similarly, in regenerative medicine, it is required that cells or organized cells be separated from a substrate and returned to the body without damaging the cells cultured outside the body and without breaking the bonds between the cells. Therefore, there is a need for a method of separating the substrate from the substrate without using a protease.

  In order to solve the above problem, Patent Document 1 discloses a culture substrate in which the surface of a substrate is coated with a temperature-responsive polymer. According to such a substrate, the adhesive force on the substrate surface is weakened by the sol transition of the temperature-responsive polymer due to the temperature drop of the surrounding environment, so that the cells can be separated and the cells can be separated and collected without causing damage. it can. However, when the type of the cell changes, the characteristics of the base material necessary for culturing the cell and exfoliating it well change. In particular, pluripotent stem cells are different from ordinary cells in that they form colonies and proliferate, so that the characteristics of the required base material are greatly different. Patent Document 1 does not disclose the characteristics of a substrate suitable for pluripotent stem cells, and has a problem that the method cannot be applied to culture of pluripotent stem cells. In addition, Patent Document 1 requires a high-cost electron beam irradiation, and has a problem that it is not suitable for a substrate used for culturing a large amount of cells.

Japanese Patent Application Laid-Open No. H2-211865

  An object of the present invention is to provide a culture substrate that enables efficient cell culture of pluripotent stem cells and enables cell detachment with a short time or weak external stimulus, and a production method using the same. It is in.

  The inventors of the present invention have conducted intensive studies in view of the above points, and as a result, the above problem has been solved by using a culture substrate having a layer made of a temperature-responsive polymer having an average roughness / layer thickness ratio of 0.5 or more and 1 or less. Can be solved, and the present invention has been completed.

  That is, according to the present invention, the base material surface has a layer made of a temperature-responsive polymer having a response temperature to water in the range of 0 ° C to 50 ° C, and the average roughness of the layer at a temperature equal to or higher than the response temperature / Provided is a culture substrate for pluripotent stem cells, wherein the ratio of the layer thickness is 0.5 or more and 1 or less.

  According to the present invention, it is possible to provide a culture substrate that enables efficient cell culture of pluripotent stem cells, and that enables cell detachment with a short time or weak external stimulus, and a production method using the same. it can.

Schematic diagram showing the culture substrate of the present invention Example of LCST measurement result by measuring transmittance of aqueous solution Example of measurement result of response temperature by water contact angle measurement Atomic force microscope image of the culture substrate of Example 1 Atomic force microscope image of the culture substrate of Example 2 Atomic force microscope image of the culture substrate of Example 3 Atomic force microscope image of the culture substrate of Comparative Example 2 Atomic force microscope image of the culture substrate of Comparative Example 3

  Hereinafter, embodiments for carrying out the present invention (hereinafter, simply referred to as “the present embodiment”) will be described in detail. The following embodiment is an exemplification for describing the present invention, and is not intended to limit the present invention to the following contents. The present invention can be appropriately modified and implemented within the spirit of the invention.

  As used herein, the term “temperature-responsive polymer” refers to a polymer whose degree of hydrophilicity / hydrophobicity changes with a change in temperature. In addition, when the temperature of a polymer is raised from low to high in the presence of water molecules, the degree of hydrophilicity / hydrophobicity of the polymer changes to more hydrophobic at a certain temperature, and the water solubility is increased. There is a temperature-responsive polymer that changes from water-insoluble to water-insoluble, and this boundary temperature is referred to as “Lower Critical Solution Temperature (LCST)”. In general, when a temperature-responsive polymer dissolves in water at a temperature lower than LCST, it becomes insoluble in water at a temperature higher than LCST. When the temperature-responsive polymer is water-insoluble, it has no LCST, but has a response temperature at which the degree of hydrophilicity / hydrophobicity changes with temperature change.

  In addition, in the present specification, the “substance derived from a living body” is a substance existing in the body of an organism, but may be a natural product, or may be a substance artificially synthesized by genetic recombination technology or the like. Alternatively, a substance chemically synthesized based on the biological substance may be used. Although there is no particular limitation on biological substances, for example, nucleic acids, proteins, and polysaccharides, which are the basic materials constituting living organisms, and nucleotides, nucleosides, amino acids, various sugars, and lipids, vitamins, and hormones, which are constituents thereof, are used. is there.

  Further, in the present specification, “pluripotent stem cell adhesion” means that the pluripotent stem cells adhere to the culture substrate at a temperature at which the pluripotent stem cells are cultured.

  In addition, in the present specification, “a block segment has water-insolubility” indicates that at least a part of a homopolymer composed of only monomer units constituting the block segment is insoluble in water.

As used herein, “pluripotent stem cell proliferation” refers to the ease of proliferation of pluripotent stem cells at the culture temperature, and “proliferative” means that the pluripotent stem cells are cultured at the culture temperature. It adheres to the material and shows that it can proliferate. Furthermore, high proliferation indicates that the cells proliferate into more pluripotent stem cells when compared in the same culture period.
Further, in the present specification, "pluripotent stem cell detachability" refers to the ease of detachment of the pluripotent stem cells grown on the culture substrate from the culture substrate, and "having detachability" 3 shows that pluripotent stem cells grown on the culture substrate can be detached from the culture substrate by external stimuli. Further, “high detachability” indicates that the pluripotent stem cells detach from the culture substrate by a weak external stimulus such as short-time cooling or weak stress. Further, “having a cooling releasability” indicates that the releasability is increased and the releasability is enhanced by cooling the culture substrate as compared with the case where the culture substrate is not cooled. Here, in the present specification, the term "external stimulus" refers to a mechanical stimulus such as ultrasonic waves, vibration, convection, light, electricity, an electromagnetic stimulus such as magnetism, and a thermodynamic stimulus such as heating or cooling. Excludes those caused by biological reactions such as enzyme reactions.

  The culture substrate of the present invention has a layer made of a temperature-responsive polymer having a response temperature to water in the range of 0 ° C to 50 ° C on the surface of the substrate. By having a layer made of a temperature-responsive polymer, the culture substrate of the present invention has the ability to remove pluripotent stem cells by cooling. When there is no layer made of the temperature-responsive polymer, the pluripotent stem cells do not have a cooling exfoliation property. Here, in the present invention, the term “culture substrate” refers to the entire article (for example, a portion indicated by reference numeral 10 in FIG. 1) on which pluripotent stem cells are cultured, and is composed of a substrate and a temperature-responsive polymer. Including layers. In the present invention, the term “substrate” refers to a base substrate (for example, a portion indicated by reference numeral 1 in FIG. 1) covered with a layer made of a temperature-responsive polymer.

  In the present invention, the temperature-responsive polymer has a response temperature in the range of 0 ° C to 50 ° C. When the response temperature is in the range of 0 ° C. to 50 ° C., the culture substrate of the present invention has pluripotent stem cell proliferation near body temperature (37 ° C.) and cools and exfoliates in a temperature range that does not damage cells. Has the property. When it does not have a response temperature, it does not have cooling peelability. The response temperature is in the range of 10 ° C. to 40 ° C., since it is suitable for imparting pluripotent stem cell proliferation near body temperature and for imparting cooling exfoliation in a temperature range that does not damage cells. Is preferably in the range of 15 ° C to 35 ° C, more preferably 15 ° C to 30 ° C, and most preferably 15 ° C to 25 ° C. At a temperature equal to or higher than the response temperature, since the temperature-responsive polymer has hydrophobicity, the protein is easily adsorbed, and it becomes possible to adhere and culture the cells using the adsorbed protein as a scaffold. On the other hand, when the temperature is lowered, the exfoliation of cells is promoted by changing to a hydrophilic property. If the response temperature is less than 0 ° C., it is difficult to impart cooling and exfoliating properties in a temperature range that does not damage cells, and if it exceeds 50 ° C., it does not have pluripotent stem cell proliferation near body temperature and cell culture Becomes difficult. In addition, when a medium at room temperature is used during medium exchange in the culturing operation, the response temperature is in the range of 0 ° C to 30 ° C because it is suitable for suppressing cell detachment during medium exchange. The temperature is more preferably in the range of 5 ° C to 25 ° C, particularly preferably 5 ° C to 20 ° C, most preferably 10 ° C to 20 ° C.

  In the present invention, the ratio of the average surface roughness / layer thickness of the layer made of the temperature-responsive polymer is 0.5 or more. When the ratio of the average surface roughness / layer thickness of the layer made of the temperature-responsive polymer is 0.5 or more, the culture substrate of the present invention has pluripotent stem cell proliferation and cooling exfoliation. If the average roughness / layer thickness ratio is less than 0.5, it does not have pluripotent stem cell proliferation or does not have cold exfoliation. Since it is suitable for enhancing the cooling releasability of pluripotent stem cells, the ratio of the average surface roughness / layer thickness is more preferably 0.6 or more, particularly preferably 0.7 or more, and 0. 9 or more is most preferable. Here, in the present invention, the “average roughness” of the surface of the layer made of the temperature-responsive polymer refers to the average height of the contour curve obtained according to JIS B0601: 2013 (for example, the length indicated by the symbol H in FIG. 1). ) Is shown, an atomic force microscope image of a representative surface of the culture substrate is measured, and the roughness is calculated by obtaining an average height of 1 μm as a reference length in 10 randomly selected roughness curves. be able to. In the present invention, the “layer thickness” of the layer made of the temperature-responsive polymer refers to the surface from the interface between the substrate and the layer made of the temperature-responsive polymer to the peak of the surface structure of the layer made of the temperature-responsive polymer. It indicates the length in the outward direction (for example, the length indicated by symbol D in FIG. 1), and in the range where the layer thickness exceeds 10 nm, the cross-sectional image is obtained by transmission electron microscopy using an ultrathin section of a culture substrate prepared by a microtome. The distance can be calculated by measuring with a microscope, measuring the distances of ten randomly selected points, and averaging the distances. When the layer thickness is in the range of 10 nm or less, it can be measured using an ellipsometer.

  Here, the ratio of the average roughness / layer thickness of the layer made of the temperature-responsive polymer is determined by immersing the culture substrate in the response temperature-responsive temperature water for 24 hours or more, and drying the sample rapidly by blowing air or the like. Can be measured by the method described above.

  In the present invention, the ratio of the average roughness / layer thickness of the layer by the temperature-responsive polymer at a temperature equal to or higher than the response temperature and the ratio of the average roughness / layer thickness of the layer by the temperature-responsive polymer at a temperature lower than the response temperature Is preferably 0.1 or more and 1 or less. When the ratio of the average roughness / layer thickness of the layer made of the temperature-responsive polymer at a temperature equal to or higher than the response temperature and a temperature lower than the response temperature is different, it is possible to enhance the releasability of the pluripotent stem cells upon cooling. Since it is suitable for enhancing the cooling exfoliation property of pluripotent stem cells, the difference in the ratio of the average roughness / layer thickness of the layer by the temperature-responsive polymer at a temperature equal to or higher than the response temperature and a temperature lower than the response temperature is zero. 0.3 or more, more preferably 0.5 or more, and most preferably 0.7 or more.

  In the present invention, it is preferable that the average length (for example, the length indicated by the symbol L in FIG. 1) of the roughness curve element of the layer made of the temperature-responsive polymer is 1 μm or less. When the average length of the roughness curve element of the layer made of the temperature-responsive polymer is 1 μm or less, the pluripotent stem cells can be uniformly cultured, and the cooling exfoliation property can be enhanced. The pluripotent stem cells are uniformly cultured, and, because they are suitable for enhancing the cooling detachment, the average length is more preferably 0.8 μm or less, particularly preferably 0.6 μm or less. , 0.4 μm or less. Here, in the present invention, the “average length of a roughness curve element” of a layer made of a temperature-responsive polymer indicates the average length of a contour curve determined according to JIS B0601: 2013. It can be calculated by measuring an atomic force microscope image of a representative surface and calculating an average length with 1 μm as a reference length in ten randomly selected roughness curves.

  In the present invention, a layer made of a temperature-responsive polymer preferably has a layer thickness of 5 to 1000 nm, more preferably 5 to 200 nm, because it is suitable for enhancing pluripotent stem cell proliferation and detachability. Particularly preferred is 20 to 100 nm, most preferably 35 to 80 nm.

  In the present invention, the surface structure of the layer made of the temperature-responsive polymer is preferably derived from the phase-separated structure of the temperature-responsive polymer. Because the surface structure of the layer made of the temperature-responsive polymer is derived from the phase-separated structure of the temperature-responsive polymer, the repetition of the surface structure is more complicated than when the surface structure is artificially patterned. The absence of seams is suitable for uniformly culturing pluripotent stem cells in all regions of the culture substrate, and is also suitable for increasing the productivity of the culture substrate. The method for forming the surface structure derived from the phase separation of the temperature-responsive polymer is not particularly limited. For example, a method of performing phase separation when applying the temperature-responsive polymer to a substrate, For example, a method of applying a temperature-responsive polymer to be used.

  In the present invention, the shape of the surface structure of the layer made of the temperature-responsive polymer is not particularly limited, and any of a line shape, a wrinkle structure, a dot shape, a sea-island shape, a hole structure, and the like can be used. Since it is suitable for uniformly culturing pluripotent stem cells over the entire surface of the culture substrate, a wrinkled structure, a sea-island-like structure, or a hole structure is preferable.

  In the present invention, the laminin adsorption rate in the following test is preferably 10% or more.

A solution obtained by adding 2 to 2.5 μL of a laminin 511-E8 fragment solution at a concentration of 0.5 mg / mL to 1 mL of phosphate buffered saline was added in an amount of 0.2 mL / cm per unit area of the culture substrate. ² Laminin adsorption rate calculated from the following formula when dropped on a substrate and allowed to stand at 37 ° C. for 24 hours.

When the laminin adsorption rate is 10% or more, multipotent stem cell proliferation can be enhanced. Further, since it is suitable for enhancing the pluripotent stem cell proliferation, the laminin adsorption rate is more preferably 20% or more, particularly preferably 30% or more, and most preferably 40% or more. preferable.

  In the present invention, the type of the temperature-responsive polymer is not particularly limited, but a block copolymer coated on the substrate, a copolymer immobilized on the substrate via a reactive group such as an azide group, or the like. Coalescence can be suitably used.

In the present invention, the temperature-responsive polymer is preferably a block copolymer containing the following block segments (A) and (B).
(A) A temperature-responsive polymer block segment having a response temperature in the range of 0 ° C to 80 ° C.
(B) A water-insoluble block segment.

  When the block copolymer contains the block segment (A), the culture substrate of the present invention can easily impart pluripotent stem cell proliferation near body temperature, and can be cooled and peeled to a temperature range that does not damage cells. Easy to impart properties.

  Here, in the present invention, the expression “LCST of the block segment (A)” indicates an LCST of a homopolymer composed of only the repeating units constituting the block segment (A). The LCST of the block segment (A) is a temperature at which the homopolymer is insolubilized in water. For example, in an aqueous solution in which the homopolymer is dissolved at 0.6 wt%, while heating the aqueous solution at a rate of 1 ° C./min, It can be determined by measuring the transmittance of light having a wavelength of 500 nm. From the low temperature until the LCST is reached, the transmittance is substantially constant, but the transmittance becomes sharply reduced due to cloudiness near the LCST, and thereafter, a curve is obtained in which the transmittance is substantially constant again (for example, FIG. 2). ). In this curve, the LCST can be obtained by calculating the temperature at which the transmittance at a temperature lower than the LCST and the transmittance of the average value of the transmittance at the temperature equal to or higher than the LCST (middle point method). The temperature range to be measured includes a temperature range of 5 ° C. or higher where the transmittance is substantially constant at a temperature lower than the LCST, and further includes a temperature range of 5 ° C. or higher where the transmittance is constant at a temperature of the LCST or higher.

  When the block copolymer of the present invention has a water-insoluble block segment, the response temperature is measured by determining the temperature at which the degree of hydrophilicity / hydrophobicity is insoluble in water but changes with temperature. be able to. For example, the degree of hydrophilicity / hydrophobicity of the block copolymer is measured by immersing the substrate coated with the block copolymer in water and measuring the contact angle of bubbles in water. Next, by changing the temperature of water, the temperature of the block copolymer is changed, and after waiting for the temperature to stabilize, the contact angles at various temperatures are obtained by measuring the contact angles of the bubbles again. . Below the response temperature, the contact angle of the bubbles is generally constant at a large value (the contact angle with water is small), but the contact angle of the bubbles is small at the boundary of the response temperature (the contact angle with water is large) Above the response temperature, a substantially constant curve is obtained (for example, FIG. 3). In this curve, the response temperature can be obtained by calculating the temperature that is the contact angle of the average value of the contact angle at a temperature lower than the response temperature and the contact angle at a temperature higher than the response temperature (middle point method). The temperature range to be measured includes a temperature range of 10 ° C. or higher where the contact angle is substantially constant at a temperature lower than the response temperature, and further includes a temperature range of 10 ° C. or higher where the contact angle is substantially constant at a temperature higher than the response temperature. It is.

  As a typical method for controlling the response temperature of a temperature-responsive polymer, there is a method of adjusting the property of a monomer to be copolymerized and the copolymerization rate. In the case where a hydrophilic monomer is copolymerized with a temperature-responsive polymer, the response temperature shifts to a higher temperature as the composition increases. On the other hand, in the case where a hydrophobic monomer is copolymerized with a temperature-responsive polymer, the response temperature shifts to a lower temperature side as the composition increases. From such a viewpoint, the preferred LCST of the block segment (A) varies depending on the nature of the monomer of the block segment (B) to be copolymerized. From the viewpoint of suitability, the LCST of the block segment (A) is preferably 10 to 75 ° C, more preferably 10 to 60 ° C, and particularly preferably 20 to 50 ° C.

  The monomer unit constituting the block segment (A) is not particularly limited, and examples thereof include (meth) acrylamide compounds such as acrylamide and methacrylamide; N, N-diethylacrylamide, N-ethylacrylamide, and Nn -Propylacrylamide, Nn-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N N-alkyl-substituted (meth) acrylamide derivatives such as -tetrahydrofurfurylacrylamide and N-tetrahydrofurfuryl methacrylamide; N, N-dimethyl (meth) acrylamide, N, N-ethyl N, N-dialkyl-substituted (meth) acrylamide derivatives such as tylacrylamide and N, N-diethylacrylamide; 1- (1-oxo-2-propenyl) -pyrrolidine, 1- (1-oxo-2-propenyl) -piperidine , 4- (1-oxo-2-propenyl) -morpholine, 1- (1-oxo-2-methyl-2-propenyl) -pyrrolidine, 1- (1-oxo-2-methyl-2-propenyl) -piperidine And (meth) acrylamide derivatives having a cyclic group such as 4- (1-oxo-2-methyl-2-propenyl) -morpholine; vinyl ethers such as methyl vinyl ether; and proline derivatives such as N-proline methyl ester acrylamide. It is preferable to set the response temperature of the block copolymer to 0 to 50 ° C. N-diethylacrylamide, Nn-propylacrylamide, N-isopropylacrylamide, Nn-propylmethacrylamide, N-ethoxyethylacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfurylmethacrylamide is preferred, and N- N-propylacrylamide and N-isopropylacrylamide are more preferable, and N-isopropylacrylamide is particularly preferable. Further, when a medium at room temperature is used for medium exchange in the culturing operation, N-isopropylacrylamide and N-proline are preferable because the response temperature of the block copolymer is suitable to be lower than room temperature. Methyl ester acrylamide is preferred.

  In the present invention, when the block copolymer contains the block segment (B), it is easy to suppress the incorporation of a temperature-responsive polymer into the culture solution, which is preferable. The monomer unit constituting the block segment (B) is not particularly limited, and for example, 2-hydroxyphenyl acrylate, 2-hydroxyphenyl methacrylate, 3-hydroxyphenyl acrylate, 3-hydroxyphenyl methacrylate, 4-hydroxy Phenyl acrylate, 4-hydroxyphenyl methacrylate, N- (2-hydroxyphenyl) acrylamide, N- (2-hydroxyphenyl) methacrylamide, N- (3-hydroxyphenyl) acrylamide, N- (3-hydroxyphenyl) methacrylamide , N- (4-hydroxyphenyl) acrylamide, N- (4-hydroxyphenyl) methacrylamide, styrene, n-butyl acrylate, n-butyl methacrylate, isobutyl acrylate , Isobutyl methacrylate, t-butyl acrylate, t-butyl methacrylate, n-hexyl acrylate, n-hexyl methacrylate, n-octyl acrylate, n-octyl methacrylate, n-decyl acrylate, n-decyl methacrylate, n-dodecyl acrylate, n Hydrophobic monomer units such as -dodecyl methacrylate, n-tetradecyl acrylate, n-tetradecyl methacrylate; 4-azidophenyl acrylate, 4-azidophenyl methacrylate, 2-((4-azidobenzoyl) oxy) ethyl acrylate And a monomer unit having a reactive group such as 2-((4-azidobenzoyl) oxy) ethyl methacrylate.

  In the present invention, the block segment (B) may also contain a repeating unit for controlling the response temperature of the block copolymer. Examples of the repeating unit for controlling the response temperature of the block copolymer include a hydrophilic or hydrophobic component, and are not particularly limited. Examples thereof include 2-dimethylaminoethyl acrylate, 2-dimethylaminoethyl methacrylate, Those having an amino group such as -diethylaminoethyl acrylate, 2-diethylaminoethyl methacrylate, N- [3- (dimethylamino) propyl] acrylamide; N- (3-sulfopropyl) -N-methacryloyloxyethyl-N, N Those having betaines such as -dimethylammonium betaine, N-methacryloyloxyethyl-N, N-dimethylammonium-α-N-methylcarboxybetaine; hydroxyethyl acrylate, hydroxyethyl methacrylate, N- (2-hydroxyethyl) amine Lilamide, polyethylene glycol monoacrylate, polyethylene glycol monomethacrylate, polypropylene glycol monoacrylate, polypropylene glycol monomethacrylate, methoxypolyethylene glycol monoacrylate, methoxypolyethylene glycol monomethacrylate, diethylene glycol monomethyl ether acrylate, diethylene glycol monomethyl ether methacrylate, diethylene glycol monoethyl ether acrylate, Diethylene glycol monoethyl ether methacrylate, 2-methoxyethyl acrylate, 2-methoxyethyl methacrylate, 2-ethoxyethyl acrylate, 2-ethoxyethyl methacrylate, 3-butoxyethyl acrylate, 3-butoxy Ethyl methacrylate, 3-butoxyethyl acrylamide, furfuryl acrylate, furfuryl methacrylate, tetrahydrofurfuryl acrylate, tetrahydrofurfuryl methacrylate, etc. having a polyethylene glycol group or a methoxyethyl group; methoxymethyl acrylate, methoxymethyl methacrylate, 2-ethoxy Those having an acrylate group such as methyl acrylate, 2-ethoxymethyl methacrylate, 3-butoxymethyl acrylate, 3-butoxymethyl methacrylate, 3-butoxymethyl acrylamide; 2-methacryloyloxyethyl phosphorylcholine, 2-acryloyloxyethyl phosphorylcholine, 3- (Meth) acryloyloxypropyl phosphorylcholine, 4- (meth) acryloyl Xyloxybutyl phosphorylcholine, 6- (meth) acryloyloxyhexyl phosphorylcholine, 10- (meth) acryloyloxydecyl phosphorylcholine, ω- (meth) acryloyl (poly) oxyethylene phosphorylcholine, 2-acrylamidoethyl phosphorylcholine, 3-acrylamidopropylphosphorylcholine, Examples thereof include those having a phosphorylcholine group such as -acrylamidobutylphosphorylcholine, 6-acrylamidohexylphosphorylcholine, 10-acrylamidodecylphosphorylcholine, and ω- (meth) acrylamide (poly) oxyethylenephosphorylcholine.

  Although the structure of the block copolymer containing the block segments (A) and (B) in the present invention is not particularly limited, a diblock copolymer containing at least the block segments (A) and (B) is used. It is preferred that they are united. Further, the block segment (A) and the block segment (B) may be directly bonded, or may be bonded via a spacer. When two or more types of repeating units constitute the block segment (B), their arrangement may be a block arrangement, a random arrangement, or an alternating arrangement.

  In the present invention, the ratio of the constitutional unit of the block segment (A) in the block copolymer containing the block segments (A) and (B) is preferably from 40 to from the viewpoint of enhancing the cooling releasability of the pluripotent stem cells. It is 99 wt%, more preferably 60-99 wt%, particularly preferably 80-99 wt%, and most preferably more than 90 wt%. On the other hand, from the viewpoint of increasing the water insolubility of the block copolymer, the structural unit ratio of the block segment (B) is preferably 1 to 50 wt%, preferably 1 to 25 wt%, more preferably 3 to 15 wt%. %, Most preferably 5 to 15 wt%.

  The molecular weight of the temperature-responsive polymer in the present invention is not particularly limited, but is preferably 1,000 to 1,000,000 in number average molecular weight, and 2000 to 500,000 in order to enhance the strength of the block copolymer. Is more preferable, it is particularly preferably 5,000 to 300,000, and most preferably 10,000 to 200,000.

  The temperature-responsive polymer in the present invention may contain a chain transfer agent, a polymerization initiator, a polymerization inhibitor, and the like, if necessary. The chain transfer agent is not particularly limited and generally used ones can be suitably used. For example, dithiobenzoate, trithiocarbonate, 4-cyano-4-[(dodecylsulfonylthiocarbonyl) sulfonyl] pentane Neuac Acid, 2-cyanopropan-2-yl N-methyl-N- (pyridin-4-yl) carbamodithioate, methylmethyl 2-propionate (4-pyridinyl) carbamodithioate can be exemplified. . The polymerization initiator is not particularly limited, and generally used ones can be suitably used. Examples thereof include azobisisobutyronitrile, 1,1′-azobis (cyclohexanecarbonitrile), and di-tert. -Butyl peroxide, tert-butyl hydroperoxide, hydrogen peroxide, potassium peroxodisulfate, benzoyl peroxide, triethylborane, diethylzinc and the like. Further, the polymerization inhibitor is not particularly limited and generally used ones can be suitably used. Examples thereof include hydroquinone, p-methoxyphenol, triphenylferdazyl, 2,2,6,6-tetramethylpiperidyl. Nyl-1-oxyl, 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxyl and the like can be mentioned.

  The method for synthesizing the temperature-responsive polymer in the present invention is not particularly limited, and is disclosed in "Radical Polymerization Handbook", published by NTT Corporation, p. 161 to 225 (2010).

In the present invention, the type of the temperature-responsive polymer is not particularly limited, but does not cause variation in the coating, and is suitable for making the state of the pluripotent stem cells to be cultured uniform. It is preferably a copolymer or a mixture of two types of block copolymers, and more preferably one type of block copolymer. Here, in the present invention, the “type of temperature-responsive polymer” is regarded as the same type of block copolymer when all the blocks constituting the block copolymer are the same. Here, the term "block is the same" means that when the block is mainly composed of one type of monomer unit, the case where the most frequently contained monomer unit in the wt% ratio is the same, When the block is composed of two or more types of monomer units, the case where the upper two monomer units having a large wt% ratio are the same is shown. Since it is more preferable to make the state of the cultured pluripotent stem cells uniform, the polydispersity (weight average molecular weight M _w / number average molecular weight M _n ) of each block is preferably 1 to 20, It is more preferably from 1 to 10, particularly preferably from 1 to 5, and most preferably from 1 to 2.

  In the present invention, since the temperature-responsive polymer is suitable for suppressing contamination of the pluripotent stem cells, the temperature-responsive polymer has a number average molecular weight of 5,000 or less, %, More preferably 30% or less, particularly preferably 10% or less, most preferably 5% or less. The amount of the temperature-responsive polymer having a number-average molecular weight of 5000 or less contained in the temperature-responsive polymer is not particularly limited, but can be measured, for example, by gel permeation chromatography.

  In the present invention, the layer made of the temperature-responsive polymer may have a biological substance as needed. The biological substance is not particularly limited, and examples thereof include Matrigel, laminin, vitronectin, fibronectin, and collagen. These biological substances may be natural products, artificially synthesized by genetic recombination technology or the like, fragments cut with restriction enzymes or the like, or synthetic proteins based on these biological substances. Alternatively, it may be a synthetic peptide.

  In the present invention, as the Matrigel, for example, Matrigel (manufactured by Corning Incorporated) or Geltrex (manufactured by Thermo Fisher Scientific) can be preferably used as a commercial product because of its availability.

  Although the type of the laminin is not particularly limited, for example, laminin 511, laminin 521, or laminin 511, which has been reported to exhibit high activity against α6β1 integrin expressed on the surface of human iPS cells. E8 fragments can be used. The laminin may be a natural product, may be artificially synthesized by genetic recombination technology or the like, or may be a synthetic protein or a synthetic peptide based on the laminin. From the standpoint of availability, for example, iMatrix-511 (manufactured by Nippi Corporation) can be suitably used as a commercially available product.

  The vitronectin may be a natural product, may be artificially synthesized by a genetic recombination technique or the like, or may be a synthetic protein or a synthetic peptide based on the vitronectin. For ease of availability, commercially available products such as vitronectin, human plasma-derived (manufactured by Wako Pure Chemical Industries, Ltd.) and synthemax (manufactured by Corning Incorporated), and Vitronectin (VTN-N) (manufactured by Thermo Fisher Scientific) are preferably used. be able to.

  The fibronectin may be a natural product, may be artificially synthesized by a genetic recombination technique or the like, or may be a synthetic protein or a synthetic peptide based on the fibronectin. From the viewpoint of easy availability, for example, a fibronectin solution, a human plasma-derived product (manufactured by Wako Pure Chemical Industries, Ltd.), and Retronectin (manufactured by Takara Bio Inc.) can be suitably used as commercially available products.

  The type of the collagen is not particularly limited, and for example, type I collagen or type IV collagen can be used. The collagen may be a natural product, artificially synthesized by genetic recombination technology or the like, or a synthetic peptide based on the collagen. For ease of availability, commercially available products such as collagen I, human (manufactured by Corning Incorporated) and collagen IV, human (manufactured by Corning Incorporated) can be suitably used.

  In the present invention, the material of the substrate on which the layer made of the temperature-responsive polymer is coated is not particularly limited, but not only substances such as glass and polystyrene that are usually used for cell culture, but also generally can be given a form. For example, high molecular compounds such as polycarbonate, polyethylene terephthalate, polyvinylidene fluoride, polytetrafluoroethylene, polyethylene, polypropylene, and polymethyl methacrylate, ceramics, and metals can be used. From the viewpoint of easiness of the culturing operation, the material of the substrate preferably contains at least one of glass, polystyrene, polycarbonate, polyethylene terephthalate, polyvinylidene fluoride, polytetrafluoroethylene, polyethylene, and polypropylene, and glass, polystyrene, and polycarbonate. And more preferably at least one of polyethylene terephthalate, and polystyrene, polycarbonate and polyethylene terephthalate are particularly preferred because they are suitable for enhancing flexibility.

  The shape of the substrate is not particularly limited, and may be a planar shape such as a plate or a film, or may be a fiber, a porous particle, a porous membrane, or a hollow fiber. Further, a container (cell culture dish such as a Petri dish, a flask, a plate, or the like) generally used for cell culture or the like may be used. From the viewpoint of easiness of the culturing operation, a porous membrane having a planar shape such as a plate or a film or a flat membrane is preferable.

  As a method of forming a layer made of a temperature-responsive polymer on the surface of a substrate according to the present invention, (1) a method of coating a substrate with a temperature-responsive polymer by chemical bonding to form a layer, and (2) The method of coating and forming a layer by physical interaction can be performed alone or in combination. That is, (1) UV irradiation, electron beam irradiation, gamma ray irradiation, plasma treatment, corona treatment, or the like can be used as a method by chemical bonding. Furthermore, when the temperature-responsive polymer and the substrate have appropriate reactive functional groups, generally used organic reactions such as a radical reaction, an anion reaction and a cation reaction can be used. (2) As a method based on physical interaction, a matrix having good compatibility with a temperature-responsive polymer and good coatability as a medium, coating, brushing, dip coating, spin coating, bar coding, Various commonly known methods such as flow coating, spray coating, roll coating, air knife coating, blade coating, gravure coating, microgravure coating, and slot die coating can be used.

  In the present invention, the type of pluripotent stem cells is not particularly limited. For example, ES cells, iPS cells, and nuclear transplanted ES cells (ntES cells) can be used, and iPS cells are particularly preferable. The animal from which the pluripotent stem cells are derived is not particularly limited, but may be, for example, a mammal. Examples of mammals include rodents (mouse, rat, etc.) and primates (monkey, human, etc.), and may be experimental animals or companion animals. In the present invention, it is preferably derived from a primate, and particularly preferably derived from a human.

The present invention also relates to a method for culturing pluripotent stem cells using the culture substrate. The method is a method for producing pluripotent stem cells, which produces undifferentiated pluripotent stem cells through the following steps [1] to [3].
[1] A step of seeding the culture substrate with pluripotent stem cells.
[2] a step of culturing the pluripotent stem cells seeded on the culture substrate in a liquid at a temperature equal to or higher than the response temperature of the temperature-responsive polymer.
[3] a step of cooling the culture substrate to a temperature lower than the response temperature of the temperature-responsive polymer, and detaching the pluripotent stem cells cultured in the liquid from the substrate.

  Hereinafter, the steps [1] to [3] in the production method of the present invention will be described in detail.

  Step [1] in the method for producing pluripotent stem cells of the present invention is a step of using the culture substrate and seeding the culture substrate with pluripotent stem cells. In the present invention, “to inoculate cells” means that a medium in which cells are dispersed (hereinafter, referred to as “cell suspension”) is applied onto a culture substrate, or is injected into the culture substrate. Fig. 4 shows contact between a cell suspension and a culture substrate. By using a substrate having a layer made of a temperature-responsive polymer having a response temperature in the range of 0 ° C to 50 ° C, exfoliating the pluripotent stem cells from the culture substrate by a temperature change in the step [3] described below. Can be. When the culture substrate does not have a layer made of a temperature-responsive polymer, the pluripotent stem cells cannot be detached from the substrate in step [3] due to a temperature change. In addition, since the culture substrate has a biological material immobilized on a layer made of a temperature-responsive polymer, pluripotent stem cells can be cultured in the step [2] described below. If the culture substrate does not have a biological substance immobilized on the layer made of the temperature-responsive polymer, the pluripotent stem cells cannot be cultured in the step [2].

  In the steps [1] to [3], the culture is performed under conditions effective for maintaining the undifferentiated state of the pluripotent stem cells. Conditions that are effective for maintaining undifferentiation are not particularly limited.For example, the density of pluripotent stem cells at the start of culture is preferably set to the preferable range described as the cell density at the time of seeding. Performed in the presence of a suitable liquid medium. As a medium effective for maintaining undifferentiated pluripotent stem cells, for example, known as a factor for maintaining undifferentiated pluripotent stem cells, insulin, transferrin, selenium, ascorbic acid, A medium containing one or more of sodium bicarbonate, basic fibroblast growth factor, transforming growth factor β (TGFβ), CCL2, activin, and 2-mercaptomethanol can be suitably used. Insulin, transferrin, selenium, ascorbic acid, sodium bicarbonate, basic fibroblast growth factor, transforming growth factor β (TGFβ) are particularly suitable for maintaining the undifferentiation of pluripotent stem cells. It is more preferable to use a medium containing the medium, and it is most preferable to use a medium to which basic fibroblast growth factor has been added.

  The type of medium to which the basic fibroblast growth factor has been added is not particularly limited. For example, commercially available products include DMEM (manufactured by Sigma-Aldrich Co. LLC) and Ham's F12 (Sigma-Aldrich Co. LLC). D-MEM / Ham's F12 (manufactured by Sigma-Aldrich Co. LLC), Primate ES Cell Medium (manufactured by REPROCELL), StemFit AK02N (manufactured by Ajinomoto Co.), StemFit AK03 (manufactured by Ajinomoto Co.) )), MTeSR1 (manufactured by STEMCELL TECHNOLOGIES), TeSR-E8 (manufactured by STEMCELL TECHNOLOGIES), ReproNive (manufactured by REPROCELL), ReproXF (manufactured by RE) ROCELL), ReproFF (manufactured by REPROCELL), ReproFF2 (manufactured by REPROCELL), NutriStem (manufactured by Biologic Industries), iSTEM (manufactured by Takara Bio Inc.), GS2-M (manufactured by Takara Bio Inc.) Co., Ltd.) and hPSC Growth Medium DXF (manufactured by PromoCell). Primate ES Cell Medium (manufactured by REPROCELL), StemFit AK02N (manufactured by Ajinomoto Co.) or StemFit AK03 (manufactured by Ajinomoto Co.) is preferable because it is suitable for maintaining the undifferentiated state of pluripotent stem cells. , StemFit AK02N (manufactured by Ajinomoto Co.) or StemFit AK03 (manufactured by Ajinomoto Co.) is more preferable, and StemFit AK02N (manufactured by Ajinomoto Co.) is particularly preferable.

In the step [1], the method of seeding the pluripotent stem cells is not particularly limited. For example, the method can be performed by injecting a cell suspension into the base material. The cell density at the time of seeding is not particularly limited, but is preferably 1.0 × 10 ^{2 to} 1.0 × 10 ⁶ cells / cm ^{2 so} that the cells can be maintained and proliferated, 5.0 × 10 ^{2 to} 5.0 × 10 ⁵ cells / cm ² are more preferable, 1.0 × 10 ^{3 to} 2.0 × 10 ⁵ cells / cm ² are particularly preferable, and 1.2 × 10 ³ to 1 0.0 × 10 ⁵ cells / cm ² are most preferred.

Since the medium used in the step [1] is suitable for maintaining the survival of pluripotent stem cells, a Rho-binding kinase inhibitor is further added to the medium containing the basic fibroblast growth factor. It is preferable to use a prepared medium. In particular, when human pluripotent stem cells are used and the Rho-binding kinase inhibitor is added in a state where the cell density of human pluripotent stem cells is low, survival of human pluripotent stem cells can be maintained. May be effective. The Rho-linked kinase inhibitors, for example, (R) - (+) - trans-N- (4-pyridyl) -4- (1-aminoethyl) -cyclohexanecarboxamide · 2HCl · H 2 O ( Wako Pure Chemical Industries (Co. Y-27632), 1- (5-Isoquinolinesulfonyl) homopiperazine Hydrochloride (HA1077, manufactured by Wako Pure Chemical Industries, Ltd.) can be used. The concentration of the Rho-binding kinase inhibitor added to the medium is a range that is effective for maintaining the survival of human pluripotent stem cells and does not affect the undifferentiated state of human pluripotent stem cells, It is preferably 1 μM to 50 μM, more preferably 3 μM to 20 μM, still more preferably 5 μM to 15 μM, and most preferably 8 μM to 12 μM.

  Shortly after the start of the step [1], the pluripotent stem cells start to adhere to the culture substrate.

  In step [2] of the method for producing pluripotent stem cells of the present invention, the seeded pluripotent stem cells are cultured at a temperature equal to or higher than the response temperature of the temperature-responsive polymer. When the culture temperature is equal to or higher than the response temperature of the temperature-responsive polymer, pluripotent stem cells can be proliferated. If the culture temperature is lower than the response temperature of the temperature-responsive polymer, the pluripotent stem cells cannot grow. The culture temperature is preferably 30 to 42 ° C., more preferably 32 to 40 ° C., particularly preferably 36 to 38 ° C., and most preferably 37 ° C., since it is suitable for cell growth ability, physiological activity and function maintenance. It is.

  It is preferable to perform the first medium exchange 22 to 26 hours after the start of the step [2]. It is preferable to perform the second medium exchange 48 to 72 hours after that, and then perform the medium exchange every 24 to 48 hours. During this time, the pluripotent stem cells proliferate and form a cell mass called a colony. Culture is continued until the size of the colony becomes about 1 mm, and then the process proceeds to step [3].

  In step [3] of the method for producing pluripotent stem cells of the present invention, the culture substrate on which the pluripotent stem cells are cultured is cooled to a temperature lower than the response temperature of the temperature-responsive polymer, The sex stem cells are detached from the culture substrate. In order to exfoliate the pluripotent stem cells in a short time and to reduce damage due to cooling, the temperature at the time of cooling is preferably 0 to 30 ° C, more preferably 3 to 25 ° C, and still more preferably 5 to 25 ° C. 20 ° C. In order to reduce damage to cells, the cooling time is preferably 120 minutes or less, more preferably 60 minutes or less, particularly preferably 30 minutes or less, and most preferably 15 minutes or less.

  The method of cooling the culture substrate in the step [3] is not particularly limited. For example, a method of cooling the culture substrate in a cool box, a method of cooling the culture substrate on a cool plate, A method of replacing the medium with a cooled medium or a buffer solution and allowing the medium to stand for a predetermined time can be used.

  Further, in the step [3], a step of causing convection in the liquid containing the cultured pluripotent stem cells at the time of cooling in order to exfoliate the pluripotent stem cells in a short time and further reduce damage due to cooling. May be included. The method of generating convection is not particularly limited, but, for example, a method of mechanically generating forced convection inside a liquid by a method such as pipetting a culture solution, a pump, or using a stirring blade, Examples of the method include a method of applying physical vibration to the substrate and a method of generating natural convection such as Marangoni convection by giving a temperature difference.

Hereinafter, the present invention will be described in detail with reference to embodiments for carrying out the present invention. However, this is an exemplification for describing the present invention, and is not intended to limit the present invention to the following contents. Further, the present invention can be appropriately modified and implemented within the scope of the present invention. Unless otherwise specified, commercially available reagents were used.
<Polymer composition>
Nuclear magnetic resonance measurement apparatus (JEOL Ltd., trade name JNM-GSX400) Proton nuclear magnetic resonance spectroscopy ⁽¹ H-NMR) spectrum analysis using, or nuclear magnetic resonance measuring device (Bruker, trade name AVANCEIIIHD500) Was determined by carbon nuclear magnetic resonance spectroscopy ( ^13C -NMR) spectrum analysis using
<Molecular weight and molecular weight distribution of polymer>
The weight average molecular weight (Mw), number average molecular weight (Mn) and molecular weight distribution (Mw / Mn) were measured by gel permeation chromatography (GPC). The GPC apparatus uses HLC-8320GPC manufactured by Tosoh Corporation, the column uses two TSKgel Super AWM-H manufactured by Tosoh Corporation, the column temperature is set to 40 ° C., and the eluent contains 10 mM sodium trifluoroacetate. The measurement was performed using N, N-dimethylformamide containing 1,1,1,3,3,3-hexafluoro-2-isopropanol or 10 mM lithium bromide. The measurement sample was prepared at 1.0 mg / mL and measured. For the calibration curve of the molecular weight, polymethyl methacrylate of a known molecular weight (manufactured by Polymer Laboratories Ltd.) was used.
<Polymer layer thickness>
The layer thickness of the polymer coated on the substrate was measured by an atomic force microscope (SPM-9600 manufactured by Shimadzu Corporation). The cantilever was made of BL-AC40TS-C2 (manufactured by Olympus Corporation), and the surface of the polymer was scratched with tweezers, and the depth of the scratch was measured to measure the thickness of the polymer.
<Average roughness and average length of polymer surface structure>
The average roughness and average length of the surface structure of the polymer coated on the base material were determined in accordance with JIS B0601: 2013 on a surface roughness curve of 10 points measured by an AFM device (SPM-9600 manufactured by Shimadzu Corporation) It was calculated by averaging.
<Laminin adsorption rate>
A solution obtained by adding 2 to 2.5 μL of a laminin 511-E8 fragment solution at a concentration of 0.5 mg / mL to 1 mL of phosphate buffered saline was added in an amount of 0.2 mL / cm per unit area of the culture substrate. ^{(2) The} solution was dropped on a substrate, allowed to stand at 37 ° C. for 24 hours, and determined by the following formula.

Example 1
[Synthesis of block copolymer]
In a test tube, 0.40 g (0.1 mmol) of 4-cyano-4-[(dodecylsulfanylthiocarbonyl) sulfanyl] pentanoic acid, 7.11 g (50 mmol) of n-butyl methacrylate, azobis (isobutyronitrile) 33 mg (0.2 mmol) was added and dissolved in 50 mL of 1,4-dioxane. After deaeration by nitrogen bubbling for 30 minutes, the reaction was carried out at 70 ° C. for 24 hours. After completion of the reaction, the reaction solvent was distilled off under reduced pressure using a rotary evaporator, and the reaction solution was concentrated. The concentrated solution was poured into 250 mL of methanol, and the precipitated yellow oily substance was recovered and dried under reduced pressure to obtain an n-butyl methacrylate polymer.

0.9 g (0.3 mmol) of the n-butyl methacrylate polymer, 8.14 g (72 mmol) of N-isopropylacrylamide, and 5 mg (0.03 mmol) of azobisisobutyronitrile were added to a test tube, and 1,4- It was dissolved in 15 mL of dioxane. After deaeration by nitrogen bubbling for 30 minutes, the reaction was carried out at 65 ° C. for 17 hours. After completion of the reaction, the reaction solvent was diluted with acetone, poured into hexane (500 mL), and the precipitated solid was recovered and dried under reduced pressure. Further, it was dissolved again in acetone, poured into 500 mL of pure water, and the precipitated solid was recovered and dried under reduced pressure to obtain a diblock copolymer of N-isopropylacrylamide and n-butyl methacrylate.
[Preparation of base material coated with block copolymer]
The block copolymer was dissolved in ethanol to obtain a 0.6 wt% solution. 50 μL of this solution was dropped on a 3.5 cm-diameter dish (manufactured by Corning Incorporated, material: polystyrene), and spin-coated at 2000 rpm for 60 seconds. After drying at room temperature for 1 hour, a substrate coated with the block copolymer was prepared.
[Surface structure of substrate surface coated with block copolymer]
The substrate coated with the block copolymer was immersed in water at 37 ° C. for 24 hours, dried by blowing air, and observed with an atomic force microscope. Further, the substrate coated with the block copolymer was immersed in water at 25 ° C. for 24 hours, dried by blowing air, and similarly observed with an atomic force microscope. FIG. 4 shows the results. A surface structure is formed at a temperature equal to or higher than the response temperature, and the ratio of the surface roughness / layer thickness at a temperature equal to or higher than the response temperature is 0.81, and the surface roughness / layer thickness at a temperature equal to or higher than the response temperature and lower than the response temperature. Was 0.71 and the average length of the roughness curve elements was 0.24 μm.
[Measurement of Response Temperature of Block Copolymer] The bubble contact angle (θ) (°) of the substrate coated with the block copolymer at 37 ° C. and 10 ° C. in water was measured. The water contact angle (180-θ) (°) was calculated. θ was measured using a contact angle meter DM300 manufactured by Kyowa Interface Science Co., Ltd. to measure the contact angle of 3 μL of bubbles in water. Table 1 shows the contact angles with water at 37 ° C and 10 ° C. The contact angle with water at 10 ° C. is lower than the contact angle with water at 37 ° C., and the base material coated with the block copolymer exhibits temperature response, and the response temperature is in the range of 10 ° C. to 37 ° C. I found it. In addition, the bubble contact angle (θ) (°) was measured at 5 ° C. intervals in the range of 20 to 45 ° C., and the contact angle with water (180−θ) (°) at each temperature was determined. The temperature was calculated. The response temperature was 33 ° C.
[Laminin adsorption rate]
The laminin adsorption rate determined by the laminin adsorption test was 35%.
[Evaluation of cell culture and detachment]
A medium StemFitAK02N (manufactured by Ajinomoto Co., Inc.) was added at 0.2 mL / cm ² to the substrate coated with the block copolymer, and human iPS cell 201B7 strain was further added to 1300 cells / cm ² , iMatrix-511 solution ((trade name)). Was added at a concentration of 2.5 μL / mL. The cells were cultured at 37 ° C. in an environment with a CO ₂ concentration of 5%. Until 24 hours after the cell seeding, Y-27632 (manufactured by Wako Pure Chemical Industries, Ltd.) (concentration: 10 μM) was added to the medium.

  At 24 hours, 96 hours, and 144 hours after cell seeding, the state of the cells was observed with a phase-contrast microscope. In each case, cell adhesion and proliferation were confirmed, and the medium was replaced with a fresh medium. After the culture medium was exchanged 144 hours after the cell seeding, the substrate was cooled to 4 ° C., vibration was applied by tapping the side surface of the substrate, pipetting was performed, and observation was performed again with a phase contrast microscope. The cells were detached and collected.

Example 2
[Synthesis of block copolymer]
A block copolymer was synthesized in the same manner as in Example 1 [Synthesis of block copolymer].
[Preparation of base material coated with block copolymer]
The block copolymer was dissolved in ethanol to prepare a 0.9 wt% solution. 50 μL of this solution was dropped on a 3.5 cm-diameter dish (manufactured by Corning Incorporated, material: polystyrene), and spin-coated at 2000 rpm for 60 seconds. Dried at room temperature for 1 hour. Further, the substrate was immersed in pure water at room temperature for 24 hours and washed to prepare a substrate coated with a block copolymer.
[Surface structure of substrate surface coated with block copolymer]
The substrate coated with the block copolymer was immersed in water at 37 ° C. for 24 hours, dried by blowing air, and observed with an atomic force microscope. FIG. 5 shows the results. A surface structure is formed at a temperature equal to or higher than the response temperature, and a ratio of surface roughness / layer thickness at a temperature equal to or higher than the response temperature is 0.78, and a surface roughness / layer thickness at a temperature equal to or higher than the response temperature and lower than the response temperature is used. Was 0.65, and the average length of the roughness curve elements was 0.58 μm.
[Wettability of substrate surface coated with block copolymer]
The measurement was performed in the same manner as in Example 1 [Wettability of the substrate coated with the block copolymer], except that the substrate coated with the block copolymer was used.

The contact angle with water at 10 ° C. is lower than the contact angle with water at 37 ° C., and the base material coated with the block copolymer exhibits temperature response, and the response temperature is in the range of 10 ° C. to 37 ° C. I found it.
[Laminin adsorption rate]
The laminin adsorption rate determined by the laminin adsorption test was 22%.
[Evaluation of cell culture and detachment]
Culture was performed in Example 1 [Evaluation of cell culture and evaluation of exfoliation], except that the substrate coated with the block copolymer was used.

  At 24 hours, 96 hours, and 144 hours after cell seeding, the state of the cells was observed with a phase-contrast microscope. In each case, cell adhesion and proliferation were confirmed, and the medium was replaced with a fresh medium. After the culture medium was exchanged 144 hours after the cell seeding, the substrate was cooled to 4 ° C., vibration was applied by tapping the side surface of the substrate, pipetting was performed, and observation was performed again with a phase contrast microscope. The cells were detached and collected.

Example 3
[Synthesis of block copolymer]
A block copolymer was synthesized in the same manner as in Example 1 [Synthesis of block copolymer].
[Preparation of base material coated with block copolymer]
The block copolymer was dissolved in ethanol to obtain a 0.05 wt% solution. 190 μL of this solution was added dropwise to a 3.5 cm-diameter dish (manufactured by Corning Incorporated, material: polystyrene), and dried under reduced pressure at room temperature for 1 hour to prepare a substrate coated with a block copolymer.
[Surface structure of substrate surface coated with block copolymer]
The substrate coated with the block copolymer was immersed in water at 37 ° C. for 24 hours, dried by blowing air, and observed with an atomic force microscope. FIG. 6 shows the results. A surface structure is formed at a temperature equal to or higher than the response temperature, and the ratio of surface roughness / layer thickness at a temperature equal to or higher than the response temperature is 0.53, and a surface roughness / layer thickness at a temperature equal to or higher than the response temperature and lower than the response temperature. Was 0.41 and the average length of the roughness curve elements was 0.09 μm.
[Wettability of substrate surface coated with block copolymer]
The measurement was performed in the same manner as in Example 1 [Wettability of the substrate coated with the block copolymer], except that the substrate coated with the block copolymer was used.

The contact angle with water at 10 ° C. is lower than the contact angle with water at 37 ° C., and the base material coated with the block copolymer exhibits temperature response, and the response temperature is in the range of 10 ° C. to 37 ° C. I found it.
[Laminin adsorption rate]
The laminin adsorption rate determined by the laminin adsorption test was 38%.
[Evaluation of cell culture and detachment]
Culture was performed in Example 1 [Evaluation of cell culture and evaluation of exfoliation], except that the substrate coated with the block copolymer was used.

  At 24 hours, 96 hours, and 144 hours after cell seeding, the state of the cells was observed with a phase-contrast microscope. In each case, cell adhesion and proliferation were confirmed, and the medium was replaced with a fresh medium. After the culture medium was exchanged 144 hours after the cell seeding, the substrate was cooled to 4 ° C., vibration was applied by tapping the side surface of the substrate, pipetting was performed, and observation was performed again with a phase contrast microscope. The cells were detached and collected.

Comparative Example 1
The substrate was not coated with the block copolymer, a 3.5 cm diameter dish (manufactured by Corning Incorporated, material: polystyrene) was used as it was, and the others were evaluated in the same manner as in Example 1.
[Laminin adsorption rate]
The laminin adsorption rate determined by the laminin adsorption test was 58%.
[Evaluation of cell culture and detachment]
Culture was performed in the same manner as in Example 1 [Evaluation of cell culture and evaluation of detachment], except that the dish having a diameter of 3.5 cm was used as it was.

  24 hours, 96 hours, and 144 hours after the cell seeding, the state of the cells was observed with a phase contrast microscope. In each case, cell adhesion and proliferation were confirmed, and the medium was replaced with a fresh one. After the culture medium was exchanged 144 hours after the cell seeding, the substrate was cooled to 4 ° C., vibration was applied by tapping the side surface of the substrate, pipetting was performed, and observation was performed again with a phase contrast microscope. The cells adhered and proliferated sufficiently in culture up to 144 hours later, but were not detached by the cooling operation. In the absence of the block copolymer, cells could not be recovered due to the temperature drop.

Comparative Example 2
[Synthesis of block copolymer]
A block copolymer was synthesized in the same manner as in Example 1 [Synthesis of block copolymer].
[Preparation of base material coated with block copolymer]
The block copolymer was dissolved in ethanol to form a 2 wt% solution. 50 μL of this solution was dropped on a 3.5 cm-diameter dish (manufactured by Corning Incorporated, material: polystyrene), and spin-coated at 2000 rpm for 60 seconds. Dried at room temperature for 1 hour. Further, the substrate was immersed in pure water at room temperature for 24 hours and washed to prepare a substrate coated with a block copolymer.
[Surface structure of substrate surface coated with block copolymer]
The substrate coated with the block copolymer was immersed in water at 37 ° C. for 24 hours, dried by blowing air, and observed with an atomic force microscope. FIG. 7 shows the results. The surface structure hardly forms even at a temperature higher than the response temperature, and the ratio of the surface roughness / layer thickness at the temperature higher than the response temperature is 0.15, and the surface roughness at a temperature higher than the response temperature and a temperature lower than the response temperature is lower. The difference in the ratio of the thickness / layer thickness was 0.02, and the average length of the roughness curve elements was 0.15 μm.
[Laminin adsorption rate]
The laminin adsorption rate determined by the laminin adsorption test was 6%.
[Evaluation of cell culture and detachment]
Culture was carried out in the same manner as in Example 1 [Evaluation of cell culture and evaluation of peeling], except that a substrate coated with the copolymer was used.

  24 hours, 96 hours, and 144 hours after the cell seeding, the state of the cells was observed with a phase contrast microscope. The cells did not adhere and could not be cultured.

Comparative Example 3
[Synthesis of block copolymer]
A block copolymer was synthesized in the same manner as in Example 1 [Synthesis of block copolymer].
[Preparation of base material coated with block copolymer]
The block copolymer was dissolved in ethanol to obtain a 0.05 wt% solution. 50 μL of this solution was dropped on a 3.5 cm-diameter dish (manufactured by Corning Incorporated, material: polystyrene), and spin-coated at 2000 rpm for 60 seconds. Dried at room temperature for 1 hour. Further, the substrate was immersed in pure water at room temperature for 24 hours and washed to prepare a substrate coated with a block copolymer.
[Surface structure of substrate surface coated with block copolymer]
The substrate coated with the block copolymer was immersed in water at 37 ° C. for 24 hours, dried by blowing air, and observed with an atomic force microscope. FIG. 8 shows the results. Almost no surface structure is formed even at a temperature higher than the response temperature. The ratio of the surface roughness / layer thickness at the temperature higher than the response temperature is 0.41, and the surface roughness at the temperature higher than the response temperature and the temperature lower than the response temperature is lower. The difference in the ratio of the thickness / layer thickness was 0.08, and the average length of the roughness curve elements was 0.01 μm.
[Laminin adsorption rate]
The laminin adsorption rate determined by the laminin adsorption test was 42%.
[Evaluation of cell culture and detachment]
Culture was carried out in the same manner as in Example 1 [Evaluation of cell culture and evaluation of peeling], except that a substrate coated with the copolymer was used.

  24 hours, 96 hours, and 144 hours after the cell seeding, the state of the cells was observed with a phase contrast microscope. In each case, cell adhesion and proliferation were confirmed, and the medium was replaced with a fresh one. After the culture medium was exchanged 144 hours after the cell seeding, the substrate was cooled to 4 ° C., vibration was applied by tapping the side surface of the substrate, pipetting was performed, and observation was performed again with a phase contrast microscope. The cells adhered and proliferated sufficiently in culture up to 144 hours later, but were not detached by the cooling operation.

Reference Signs List 1 base material 2 block copolymer 10 culture base H average roughness L average length

Claims (6)

On the surface of the base material, there is a layer made of a temperature-responsive polymer having a response temperature to water in the range of 0 ° C. to 50 ° C., and the ratio of the average roughness / layer thickness of the layer at a temperature equal to or higher than the response temperature is 0. A culture substrate for pluripotent stem cells, wherein the number is 5 or more and 1 or less. The difference between the ratio of the average roughness / layer thickness of the layer by the temperature-responsive polymer at a temperature equal to or higher than the response temperature and the ratio of the average roughness / layer thickness of the layer by the temperature-responsive polymer at a temperature lower than the response temperature is as follows: The culture substrate for pluripotent stem cells according to claim 1, wherein the culture ratio is 0.1 or more and 1 or less. The culture substrate for pluripotent stem cells according to claim 1 or 2, wherein the average length of the roughness curve element of the layer made of the temperature-responsive polymer is 1 µm or less. The pluripotent stem cell according to any one of claims 1 to 3, wherein the surface structure of the layer made of the temperature-responsive polymer is derived from a phase-separated structure of the temperature-responsive polymer. Culture substrate. The culture substrate for pluripotent stem cells according to any one of claims 1 to 4, wherein the laminin adsorption rate in the following test is 10% or more.
A solution obtained by adding 2-2.5 μL of a laminin 511-E8 fragment solution having a concentration of 0.5 mg / mL to 1 mL of phosphate buffered saline was added in an amount of 0.2 mL / cm ² per unit area of the substrate. The laminin adsorption rate calculated from the following formula when dropped on a substrate and allowed to stand at 37 ° C. for 24 hours.

A method for producing pluripotent stem cells, comprising producing undifferentiated pluripotent stem cells through the following steps [1] to [3].
[1] A step of seeding the culture substrate according to any one of claims 1 to 5 with a pluripotent stem cell.
[2] a step of culturing the pluripotent stem cells seeded on the culture substrate in a liquid at a temperature equal to or higher than the response temperature of the temperature-responsive polymer.
[3] a step of cooling the culture substrate to a temperature lower than the response temperature of the temperature-responsive polymer, and detaching the pluripotent stem cells cultured in the liquid from the substrate.

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