JP2005110604A - Cell culture substrate and cell culture method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a flexible and tough cell culture substrate hardly causing breakage of a cell and contamination of the substrate when separating and collecting the cultured cells to enable the cultured cells to be rapidly collected; and to provide a cell culture method by which the collection of the cultured cells is easily carried out. <P>SOLUTION: The flexible and tough cell culture substrate is achieved by a polymer of a water-soluble organic monomer, and a polymer hydrogel having a three-dimensional structure constituted of a water-swellable clay mineral. The cell culture substrate comprising the polymer hydrogel allows the cells to be suitably cultured and proliferated under a condition exhibiting hydrophobicity, and the adhesion to the cells is reduced under a condition exhibiting hydrophilicity by using the polymer hydrogel exhibiting hydrophilicity and hydrophobicity by various kinds of the setting of conditions exemplified by a temperature condition to enable the cultured cells to be easily and rapidly peeled and collected without causing the breakage of the cultured and proliferated cells, and the contamination of the peeled substrate. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a cell culture substrate composed of a polymer hydrogel having a three-dimensional network structure composed of a polymer of a water-soluble organic monomer and a water-swellable clay mineral, and a cell culture substrate composed of the polymer hydrogel. The present invention relates to a cell culture method and a cell culture separation method used.

  Conventionally, plastic or glass containers have been used as cell culture substrates for animal tissues and the like. In order to control the adhesion of cultured cells, these containers are subjected to a surface treatment such as plasma treatment or coating of silicon or cell adhesion factor. When these cell culture vessels are used as the culture substrate, cells cultured and grown on the cell culture vessel are adhered to the surface of the vessel that has been surface-treated, and protein hydrolases such as trypsin and chemicals are used. In this case, since the cells are removed from the surface of the container and collected, impurities such as germs, DNA, or RNA may be mixed when the cells are detached with an enzyme or a chemical. In addition, not only the binding part of the cell and the substrate is cut, but also the bond between the cells is cut, and not only can the cells not be recovered in a proliferating shape, but also the property of the cell changes. there were. That is, when cells were collected, they fell apart and it was difficult to take out the cells. Moreover, the process of detaching cells with enzymes or chemicals was complicated. In addition, when performing multiple types of cell culture, it is necessary to change the type of culture solution, etc., but since the container cannot be replaced, various chemicals and the like remain inside the container, and these are the cells. There was a risk of contamination in the culture.

  In addition, using a base material coated with a temperature-responsive polymer on the surface of the cell culture vessel, the polymer is maintained in a hydrophobic state at the cell culture temperature to adhere the cells, and after the culture, the polymer is treated at a low temperature to remove the polymer. By making it hydrophilic, the adhesion between the cells and the polymer is reduced, and the cells are peeled off from the base material in a sheet form without using hydrolase or chemicals. (For example, refer to Patent Documents 1 and 2). However, the performance of the base material varies greatly depending on the degree of cross-linking of the polymer. When the cross-linking is insufficient, the polymer is partially detached from the base material together with the cells when the cells are peeled off. It was difficult to separate them. In addition, when the crosslinking is sufficient, the response speed of the temperature responsiveness becomes very poor, and there is a problem that it takes a long time to make the polymer hydrophilic, and during that time, there is a problem that the cells are also exposed to a low temperature state. . Further, when the base material is used, when the cultured cells are used for the next experiment, for example, when transplanted into the body of an animal, or when co-cultured with other cells, the sheet-like cells The cell sheet itself must be peeled off from the polymer-coated container, and only the cell sheet needs to be moved, forcibly grabbing the peeled, very weak cell sheet Therefore, the cell sheet has to be moved to the next experimental position, and the cell sheet is easily damaged, and the operability is very poor. From these things, after culturing the cells, it was necessary to completely separate them without contamination or damage, and to take out the cell sheet at an arbitrary place in a short time, and to move to the next step for use. .

  On the other hand, a polymer hydrogel having a three-dimensional network formed by combining a water-soluble organic polymer and a layered clay mineral is disclosed (see Patent Document 3). The polymer compound has characteristics such as excellent water absorption and extremely high extensibility, and is a useful material in various fields, but its usefulness as a cell culture substrate has not been known.

Japanese Patent Publication No. 6-104061 JP-A-5-192138 JP 2002-53629 A

  The problem to be solved by the present invention is a flexible and tough cell culture substrate, as well as a cell that can quickly recover cultured cells without cell breakage or substrate contamination when the cultured cells are separated and recovered It is an object of the present invention to provide a culture substrate and a cell culture method that allows easy collection of cultured cells.

  The polymer hydrogel having a three-dimensional network structure composed of a polymer of a water-soluble organic monomer and a water-swellable clay mineral used in the present invention can suitably culture cells on its surface, Since it is a tough material, it can be realized that cells are cultured in the form of a sheet on its surface and used for the next experiment while maintaining its shape. Furthermore, by using a polymer hydrogel exhibiting hydrophilicity and hydrophobicity by setting various conditions including temperature conditions, the cell culture substrate made of the polymer hydrogel can be suitably used under conditions exhibiting hydrophobicity. Can be cultured and grown, and under conditions that show hydrophilicity, adhesion to cells can be reduced, so there is no damage to the cultured or grown cells or exfoliation of the substrate. It can be peeled and collected easily and quickly.

  That is, the present invention relates to a cell culture substrate comprising a polymer hydrogel having a three-dimensional network structure composed of a polymer of a water-soluble organic monomer and a water-swellable clay mineral, and culturing cells on the cell culture substrate. And a cell separation method for separating cells cultured from the cell culture substrate by culturing cells on the cell culture substrate and then setting the substrate to a temperature that exhibits hydrophilicity. .

  The cell culture substrate of the present invention comprises a polymer hydrogel having a three-dimensional network structure composed of a polymer of a water-soluble organic monomer and a water-swellable clay mineral, and thus has excellent flexibility and toughness. Therefore, even when the cultured cells are transferred together with the substrate, the stably cultured cells can be transferred while maintaining the shape. Further, when co-culture is performed after the initial cell culture, the culture can be performed again without contamination by the culture medium or chemicals.

  A cell culture substrate made of a polymer hydrogel whose hydrophilicity and hydrophobicity reversibly change depending on the external environment shows excellent adhesion to cells under hydrophobic conditions. In addition, under hydrophilic conditions, adhesion to cells can be reduced, and cells can be detached without the use of protein hydrolases such as trypsin or chemicals. The cells can be easily recovered without causing material peeling and mixing. Furthermore, since the change from hydrophobic to hydrophilic or from hydrophilic to hydrophobic is rapid, there is little influence on cells when changing the external environment including temperature.

  The cell culture substrate of the present invention comprises a polymer hydrogel having a three-dimensional network structure composed of a polymer of a water-soluble organic monomer and a water-swellable clay mineral.

  The water-soluble organic monomer used in the polymer hydrogel of the present invention may be any one that has a property of being dissolved in water and interacting with a water-swellable clay mineral that can be uniformly dispersed in water. Those having a functional group capable of forming a hydrogen bond, an ionic bond, a coordinate bond, a covalent bond, and the like are preferable. Specific examples of water-soluble organic monomers having these functional groups include water-soluble organic monomers having an amide group, amino group, ester group, hydroxyl group, tetramethylammonium group, silanol group, epoxy group, and the like. Of these, water-soluble organic monomers having an amide group are preferred. In addition, the water referred to in the present invention includes a mixed solvent with an organic solvent miscible with water, the main component of which is water.

  The polymer of the water-soluble organic monomer in the present invention is not limited as long as it can form a three-dimensional network structure with a water-swellable clay mineral to form a polymer hydrogel having a stable shape, such as an acrylic compound or a vinyl compound. Etc. can be used. Among them, since cultured cells can be easily separated from the obtained cell culture substrate, it is effective to have both water-solubility or hydrophilicity to absorb water and hydrophobicity. Particularly preferred are those in which the hydrophilicity and hydrophobicity of the polymer in an aqueous solution vary with temperature, pH, solute concentration, and solvent composition. Specifically, for example, in the case of temperature, a polymer having a lower critical solution temperature (hereinafter abbreviated as LCST) that becomes hydrophobic at a critical temperature (Tc) or higher, or a hydrophilic polymer at Tc or higher. A polymer having an upper critical solution temperature (hereinafter abbreviated as UCST) is more preferably used. In the case of the solute concentration, for example, a polymer that becomes hydrophobic when the concentration of sodium chloride in the solvent is a certain concentration or higher and hydrophilic when the concentration is less than a certain concentration is preferably used. Further, in the case of a solvent composition, for example, at a certain temperature, a polymer that becomes hydrophobic when the methanol concentration with respect to water in the solvent is a certain concentration or more and becomes hydrophilic when the concentration is below a certain concentration is also preferably used.

  Examples of water-soluble organic monomers that give such polymers are preferably N-substituted acrylamide derivatives, N, N-disubstituted acrylamide derivatives, N-substituted methacrylamide derivatives, N, N-disubstituted methacrylamide derivatives, and the like. Specifically, N-isopropylacrylamide, N-isopropylmethacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N -Ethoxyethyl acrylamide, N-ethoxyethyl methacrylamide, N-tetrahydrofurfuryl acrylamide, N-tetrahydrofurfuryl methacrylamide, N-ethyl acrylamide, N-ethyl-N-methyl acrylamide, N, N-di Chill acrylamide, N- methyl -N-n-propyl acrylamide, N- methyl -N- isopropylacrylamide, N- acryloyl Lupi Peri Dinh, N- acryloylpyrrolidine Din and the like.

  Examples of the polymer of the organic monomer include poly (N-isopropylacrylamide), poly (Nn-propylacrylamide), poly (N-cyclopropylmethacrylamide), poly (N-isopropylmethacrylamide), poly ( Nn-propylmethacrylamide), poly (N-ethoxyethylacrylamide), poly (N-ethoxyethylmethacrylamide), poly (N-tetrahydrofurfurylacrylamide), poly (N-tetrafurfurylmethacrylamide), poly Examples thereof include (N-ethylacrylamide), poly (N, N-diethylacrylamide), poly (N-acryloylpiperidine), and poly (N-acryloylpyrrolidine).

  As the polymer of the water-soluble organic monomer, in addition to the polymer from the single water-soluble organic monomer as described above, a copolymer obtained by polymerizing a plurality of different water-soluble organic monomers selected from these is used. It is also effective. A polymer composed of the above water-soluble organic monomer is preferred, but a copolymer of the above water-soluble organic monomer and other water-soluble organic monomer or organic solvent-soluble organic monomer is also hydrophilic and hydrophobic. Any material that exhibits both sexes can be used. Specific examples of the organic monomer used for copolymerization include acrylamides such as N-alkylacrylamide, N, N-dialkylacrylamide, and acrylamide, or N-alkylmethacrylamide, N, N-dialkylmethacrylamide, and methacrylamide. And other methacrylamides. More preferably, N-alkylacrylamide or N, N-dialkylacrylamide is used. As the alkyl group, those having 1 to 4 carbon atoms are preferably selected. In addition, acryloylmorpholine, N, N-dimethylaminopropylacrylamide, N-acryloylmethyl homopiperadin, N-acryloylmethylpiperazine, and the like can also be used.

  The clay mineral used for the cell culture substrate of the present invention is required to have a property of swelling between layers in water or an aqueous solution. More preferably, at least a part can be peeled and dispersed in layers in water, more preferably in water to a thickness of 1 to 10 layers, particularly preferably in water to a thickness of 1 to 3 layers. It is a layered clay mineral that can be dispersed uniformly. For example, water-swellable smectite or water-swellable mica can be used. Specifically, water-swellable hectorite containing sodium as an interlayer ion, water-swellable montmorillonite, water-swellable saponite, water-swellable synthetic mica Is mentioned.

  Since the polymer hydrogel used for the cell culture substrate of the present invention has a three-dimensional network structure of the polymer of the water-swellable clay mineral and the water-soluble organic monomer, the cell culture substrate comprising the polymer hydrogel is After culturing cells, the cells can be maintained in shape without being destroyed when the cells are peeled from the polymer hydrogel. The polymer hydrogel used for the cell culture substrate of the present invention can move without destroying the cultured cell sheet when it is necessary to move the cell to the next experimental position after culturing. The polymer hydrogel can realize a tensile elastic modulus of 1 kPa or more, a tensile strength of 20 kPa or more, and an elongation at break of 50% or more, and those having these characteristics can be preferably used. Further, it is more preferable if the tensile elastic modulus is 5 kPa or more, the tensile strength is 50 kPa or more, and the elongation at break is 50% or more, and more preferable if the tensile elastic modulus is 10 kPa or more, the tensile strength is 80 kPa or more, and the elongation at break is 100% or more. .

  The polymer hydrogel has hydrophilicity and hydrophobicity depending on external environmental conditions due to the polymer of the water-soluble organic monomer forming a three-dimensional network structure. For this reason, the cell culture substrate made of the polymer hydrogel can cultivate the cells suitably, and the cultured cells can be easily and quickly detached and recovered without causing destruction of the cultured cells or exfoliation of the substrate. Can do.

  Since the critical temperature at which the hydrophilicity and hydrophobicity of the polymer hydrogel changes can suitably culture and detach cells, the critical temperature is preferably in the temperature range of about 0 to 50 ° C. Since the critical temperature is affected by UCST and LCST of the polymer of the monomer used, a material having UCST and LCST within the temperature range is selected, and the type of water-swellable clay mineral to be used, This can be realized by appropriately adjusting the ratio of the water-soluble swellable clay mineral and the like.

  The polymer hydrogel used for the cell culture substrate of the present invention can be used alone or coated on a support having a smooth surface or an uneven surface such as metal, ceramic or plastic. Moreover, it can be formed in various shapes, and can be used in a sheet shape, a fiber shape, a hollow fiber shape, or a spherical shape.

  The polymer hydrogel used for the cell culture substrate of the present invention contains water, an organic solvent miscible with water, or an aqueous solution containing a salt in the structure. It is also possible to replace the whole with an organic solvent that is miscible with water after the preparation of the polymer hydrogel. Examples of the organic solvent miscible with water include methanol, ethanol, propanol, acetone, methyl ethyl ketone, tetrahydrofuran, dimethylacetamide, dimethylformamide, dimethyl sulfoxide, and a mixed solvent thereof.

  As a method for producing the cell culture substrate of the present invention, for example, after preparing a uniform dispersion containing a water-soluble organic monomer, a water-swellable clay mineral, and water, the water-soluble organic monomer is added in the presence of the water-swellable clay mineral. A polymer hydrogel is prepared by polymerization. Here, the three-dimensional network of the water-soluble organic monomer polymer and the clay mineral is formed by the clay mineral exfoliated in the form of a layer acting as a crosslinking agent. The obtained polymer hydrogel can be used as it is as a cell culture substrate, but it can be more preferably used as a cell culture substrate by adjusting the coating on the support or the shape as necessary.

  The polymerization reaction of the water-soluble organic monomer in the presence of the water-swelling clay mineral is carried out by, for example, a method of polymerizing using a peroxide as a polymerization initiator, or a radical polymerization using a conventional method such as heating or radiation irradiation. It can be made. The radical polymerization initiator and the catalyst can be appropriately selected from conventional radical polymerization initiators and catalysts, and preferably have a dispersibility in water and are uniformly contained in the entire system. Can do. Particularly preferred is a radical polymerization initiator having a strong interaction with the layered clay mineral. As the polymerization initiator, water-soluble peroxides such as potassium peroxodisulfate and ammonium peroxodisulfate, water-soluble azo compounds and the like can be preferably used. Specifically, VA-044 manufactured by Wako Pure Chemical Industries, Ltd. V-50, V-501 and the like can be preferably used. In addition, a water-soluble radical initiator having a polyethylene oxide chain can also be used. As the catalyst, tertiary amine compounds such as N, N, N ′, N′-tetramethylethylenediamine and β-dimethylaminopropionitrile can be suitably used.

  What is necessary is just to select the temperature at the time of the said polymerization reaction suitably according to the kind etc. of the water-soluble organic monomer to be used, a polymerization catalyst, and an initiator, for example, it can set to the range of 0 to 100 degreeC. In addition, when the polymerization temperature is set to a temperature of LCST or lower (or UCST or higher) of the polymer hydrogel, the polymer hydrogel after polymerization becomes hydrophilic, but is maintained at a temperature of LCST or higher (or UCST or lower). By this, it can change from a hydrophilic state to a hydrophobic state, and it becomes a state suitable for cell culture. In addition, when polymerization is performed at a temperature of LCST or higher (or UCST or lower), the polymer hydrogel is obtained in a hydrophobic state, so that cell culture can be performed as it is. That is, regardless of which polymerization method is used, it is possible to obtain a polymer gel used as a cell culture substrate. However, when polymerization is performed at a temperature of LCST or lower (or USCT or higher), shrinkage occurs when the polymer hydrogel obtained by polymerization is changed to a hydrophobic state, but at a temperature of LCST or higher (or UCST or lower). When polymerization is performed, the polymer hydrogel obtained by polymerization is kept in a hydrophobic state, which is preferable because cell culture can be performed without shrinking as it is. The above is the other external environment (solvent composition such as salt added to control the pH of the solution, hydrophilicity or hydrophobicity during polymerization, solute concentration of additives, etc., organic solvent miscible with water) It is possible to carry out the change as long as it does not interfere with the polymerization of the organic monomer.

  The polymerization time varies depending on the polymerization conditions such as the catalyst, the initiator, the polymerization temperature, and the amount (thickness) of the polymerization solution, and cannot be defined unconditionally.

  In the production of the cell culture substrate of the present invention, cell culture substrates having various sizes and shapes can be prepared by changing the shape of the polymerization vessel at the time of polymerization or by cutting the gel after polymerization. . For example, it is possible to prepare a cell culture substrate having an arbitrary shape such as a fibrous shape, a rod shape, a flat plate shape, a cylindrical shape, a hollow shape, a spiral shape, or a spherical shape. It is also possible to produce the obtained cell culture substrate in the form of fine particles by a method in which a conventional surfactant is allowed to coexist during the polymerization reaction. The cell culture substrate of the present invention is preferably used by being laminated on a non-hydrophilic support such as a plastic or glass petri dish generally used for cell culture. Such a laminated member may be polymerized at the upper part of the support and used for cell culture as it is, or after polymerization in another container, it may be filled on the surface of the substrate and used for cell culture.

  The ratio of the water-soluble organic monomer and the clay mineral constituting the cell culture substrate of the present invention may be appropriately selected depending on the type of the water-soluble organic monomer and clay mineral to be used. In view of excellent mechanical properties and uniformity, the mass ratio of the clay mineral to the water-soluble organic monomer polymer is preferably 0.01 to 10, more preferably 0.03 to 2, particularly preferably 0.1. ~ 1. When the mass ratio is in this range, the characteristics of the polymer hydrogel are sufficiently exhibited, the preparation is easy, and the obtained cell culture substrate exhibits higher strength.

  The amount of the solvent contained in the polymer hydrogel used for the cell culture substrate in the present invention is set according to the purpose and is not unconditionally defined. However, in order for the polymer hydrogel to exhibit the above mechanical properties, it is preferable. Are those having a mass ratio of the solvent to the solid content in the polymer hydrogel in a hydrophobic state of 0.1 to 50, more preferably 0.1 to 10. The amount of these solvents can be arbitrarily controlled by the amount of the solvent at the time of polymerization or the holding state when the polymer hydrogel is changed to a hydrophobic state after the polymerization.

  In the polymer hydrogel used for the cell culture substrate of the present invention, a known and commonly used organic crosslinking agent may be used during polymerization for the purpose of improving the properties. The concentration of the organic crosslinking agent to be used is not particularly limited and can be selected according to the purpose. Examples of the organic crosslinking agent that can be used include conventionally known N, N′-methylenebisacrylamide, N, N′-propylenebisacrylamide, di (acrylamidomethyl) ether, 1,2-diacrylamide ethylene glycol, 1,3- Bifunctional compounds such as diacryloyl ethylene urea, ethylene diacrylate, N, N′-diallyl tartaramide, N, N′-bisacrylyl cystamine, and trifunctional compounds such as triallyl cyanurate and triallyl isocyanurate Can be exemplified.

  For the cell culture substrate of the present invention, the obtained polymer hydrogel material can be dried by a conventional method to remove a part or all of the solvent to obtain a dried product. Such a dried product can reversibly regenerate a polymer hydrogel material as a cell culture substrate by re-adding water or a solvent such as an organic solvent miscible with water.

  In addition, the polymer hydrogel used for the cell culture substrate of the present invention contains a polymer compound or a low molecular compound in addition to the polymer of the water-soluble organic monomer as an additive within a range not impairing the effects of the present invention. Included. For example, cell adhesion factors such as collagen and hyaluronic acid, cell growth factors, hydroxyapatite particles and the like can be added.

(Production Example 1)
For the clay mineral, water-swellable synthetic hectorite (“Laponite XLG” manufactured by Rockwood Ltd.) having a composition of [Mg _5.34 Li _0.66 Si ₈ O ₂₀ (OH) ₄ ] Na ⁺ _0.66 is vacuumed. Used after drying. The organic monomer was used after purifying N-isopropylacrylamide (manufactured by Kojin Co., Ltd .: hereinafter abbreviated as NIPA) by a known method to remove the polymerization inhibitor. The polymerization initiator was dissolved in ultrapure water obtained by deoxidizing potassium peroxodisulfate (manufactured by Kanto Chemical Co., Inc .: hereinafter abbreviated as KPS) at a rate of KPS / water = 0.40 / 20 (g / g). And used as an aqueous solution. As the catalyst, N, N, N ′, N′-tetramethylethylenediamine (manufactured by Wako Pure Chemical Industries, Ltd .: hereinafter abbreviated as TEMED) was used. The ultrapure water was used after thoroughly bubbling high-purity nitrogen through a particulate removal filter in advance to remove the oxygen contained therein.

  In a thermostatic chamber at 20 ° C., 57.06 g of ultrapure water and a Teflon (registered trademark) stirrer were placed in a flat bottom glass container whose interior was purged with nitrogen, and 1.44 g of Laponite XLG was added while stirring. A solution was prepared. To this, 6.78 g of NIPA was added and stirred in a nitrogen atmosphere to obtain a colorless transparent solution. Next, 3 g of KPS aqueous solution and 48 μl of TEMED were added to the colorless transparent solution with stirring. This solution was placed in a nitrogen atmosphere in advance and transferred to three polystyrene containers (9 cm × 15 cm) with lids, from which oxygen in the container had been removed. Then, the polymerization was carried out by standing in a constant temperature water bath at 20 ° C. for 20 hours. The operations from preparation of the solution to polymerization were all performed in a clean bench, and further performed in a nitrogen atmosphere in which oxygen was blocked. After 20 hours from the start of polymerization, a colorless and transparent uniform sheet-like hydrogel (A) was obtained in a polystyrene container.

This sheet-like hydrogel (A) is cut into a size of 1 cm × 5 cm, and it is applied to a tensile tester (“desktop universal testing machine AGS-H” manufactured by Shimadzu Corporation) so as not to slip at the chuck portion. As a result of carrying out a tensile test with the distance between the scores = 30 mm and the tensile speed = 100 mm / min, the tensile strength at break was 85 kPa, the elongation at break was 1200%, and the elastic modulus was 11.2 kPa.
Next, the sheet-like hydrogel (A) is immersed in 2 L of ultrapure water at 20 ° C. for 2 days to swell the hydrogel, then taken out, and then immersed in 1 L of 50 ° C. ultrapure water for 12 hours. The hydrogel was contracted and then removed. After the purification operation by washing was repeated three times, the purified sheet-like hydrogel (A) was cut into a size of 8 cm in diameter to obtain a cell culture substrate (A) comprising the hydrogel (A). It was transferred into a dish for cell culture (“Falcon 3003” manufactured by Becton Dickinson Labware), covered, and allowed to stand at 37 ° C. At this time, since the cell culture substrate (A) was hydrophobized, it was completely whitened. In addition, all operations from the purification to the transfer of the cell culture substrate (A) into the cell culture dish were performed in a clean bench.

  After carefully removing the water adhering to the surface of the cell culture substrate (A) obtained by these operations, the contact angle of water at 20 ° C. and 50 ° C. on the surface of the cell culture substrate (A) is determined as the contact angle. It measured using the measuring apparatus (Kyowa Interface Science Co., Ltd. product "CA-X200"). The contact angle of water at each temperature is 25 ° at 20 ° C. and 62 ° when kept at 50 ° C. The obtained cell culture substrate (A) exhibits both hydrophilic and hydrophobic characteristics depending on the temperature conditions. It was confirmed.

(Example 1)
Using the cell culture substrate (A) -containing cell culture dish obtained in Preparation Example 1, cells were cultured. The cells to be cultured were human hepatic epithelial cell-derived cancer cell HepG2 cell line (Dainippon Pharmaceutical Co., Ltd.). The culture is performed using a minimum essential eagle medium (manufactured by SIGMA) containing 10% fetal bovine serum (manufactured by ICN) (containing pyruvic acid (manufactured by ICN) and non-essential amino acids (manufactured by ICN) as additives). This was carried out in a 37 ° C. incubator containing 5% carbon dioxide. One week after seeding, a part of the end of the cell culture substrate (A) in the dish was cut out and left in a constant temperature bath at 20 ° C. for 5 minutes, and then the surface was observed with an optical microscope. It was confirmed that the cells adhered on the cell culture substrate (A) and proliferated sufficiently. A minimum essential containing 10% of fetal bovine serum taken out of the cultured cell culture substrate (A) -containing dish together with the cells cultured with the cell culture substrate (A) and kept at 20 ° C. in advance. -Transferred to a tissue culture dish containing Eagle's medium. The cells were allowed to stand at 20 ° C. for 10 minutes after being covered, and then the cells grown on the cell culture substrate (A) were picked with forceps to separate the cells from the cell culture substrate (A) in a sheet form. At this time, the cell culture substrate (A) was not damaged at all, and no adhesion was observed on the sheet-like cells. The extracted sheet-like cells were treated with trypsin-EDTA, and each cell was separated into individual states, followed by trypan blue staining to count the number of viable cells. At the start of culture, 2.5 × 10 ⁶ It was confirmed that the number of cells increased to 3.1 × 10 ⁷ after culturing.

(Production Example 2)
A sheet-like hydrogel (B) was obtained in the same manner as in Production Example 1 except that the polymerization was performed in a constant temperature water bath at 50 ° C. When the tensile test of the sheet-like hydrogel (B) was conducted in the same manner as in Reference Example 1, the tensile break strength was 71 kPa, the break elongation was 300%, and the elastic modulus was 11.2 kPa. This sheet-like hydrogel (B) was purified in the same manner as in Production Example 1, and then cut into a size of 8 cm in diameter to obtain a cell culture substrate (B). The contact angle that can be placed on the surface of the obtained cell culture substrate (B) was 55 ° in a state maintained at 50 ° C.

(Example 2)
Using the cell culture substrate (B) -containing cell culture dish obtained in Production Example 2, HepG2 cells were cultured in the same manner as in Example 1. One week after seeding the cells, a portion of the end of the hydrogel sheet in the dish was cut out and left in a constant temperature bath at 20 ° C. for 5 minutes, and the surface was observed with an optical microscope. It was confirmed that it was adhered on the culture substrate (B) and sufficiently proliferated. A minimum of 10% of fetal bovine serum that has been taken out of the cultured cell culture substrate (B) -containing dish together with the cultured cell culture substrate (B) and kept at 20 ° C. It was transferred to a tissue culture dish containing an essential eagle medium. The cells were allowed to stand at 20 ° C. for 10 minutes after being covered, and then the cells grown on the cell culture substrate (B) were picked with forceps to separate the cells from the cell culture substrate (B) in a sheet form. At this time, the cell culture substrate (B) was not damaged at all, and no deposits were observed on the sheet-like cells. When the number of viable cells was measured for the extracted sheet-like cells by the same method as in Example 2, the number of cells that was 2.5 × 10 ⁶ at the start of the culture was 3.3 × 10 ⁷ after the culture. It was confirmed that the number increased.

(Production Example 3)
In a thermostatic chamber at 20 ° C., 57.06 g of ultrapure water and a Teflon (registered trademark) stirrer were placed in a flat bottom glass container whose interior was purged with nitrogen, and 1.44 g of Laponite XLG was added while stirring. A solution was prepared. To this was added 6.78 g of NIPA and 1.0 g of a 3.0 mg / ml solution of pig skin-derived pepsin-solubilized collagen (Nitta Gelatin Co., Ltd., “Type I”), and stirred in a nitrogen atmosphere to give a colorless transparent solution Got. Next, 3.0 g of KPS aqueous solution and 48 μl of TEMED were added to the colorless transparent solution with stirring. In the same manner as in Reference Example 1, this solution was allowed to stand in a constant temperature water bath at 20 ° C. for 20 hours for polymerization. The operations from preparation of the solution to polymerization were all performed in a clean bench, and further performed in a nitrogen atmosphere in which oxygen was blocked. After 20 hours from the start of polymerization, a colorless and transparent uniform sheet-like hydrogel (C) was formed in the polystyrene container, and was carefully taken out from the polystyrene container. Using this sheet-like hydrogel (C), a tensile test was conducted in the same manner as in Example 1. As a result, the tensile strength at break was 81 kPa, the elongation at break was 1080%, and the elastic modulus was 8.8 kPa.

  This sheet-like hydrogel (C) was purified three times in the same manner as in Production Example 1, and then cut into a size of 8 cm in diameter to obtain a cell culture substrate (C). The obtained cell culture substrate (C) was transferred to a cell culture dish in the same manner as in Example 1, and maintained at 37 ° C. The contact angle of water on the surface of the cell culture substrate (C) was 23 ° at 20 ° C. and 49 ° at 50 ° C. holding state.

(Example 3)
Using the cell culture substrate (C) -containing cell culture dish obtained in Reference Example 3, HepG2 cells were cultured in the same manner as in Example 1. One week after seeding the cells, a part of the end of the cell culture substrate (C) in this dish was cut off and left in a constant temperature bath at 20 ° C. for 5 minutes, and then the surface was observed with an optical microscope. However, it was confirmed that the cells adhered to the cell culture substrate (C) and proliferated sufficiently. A minimum of 10% of fetal bovine serum that has been taken out of the cultured cell culture substrate (C) -containing dish together with the cultured cell culture substrate (C) and previously maintained at 20 ° C. It was transferred to a tissue culture dish containing an essential eagle medium. The cells were allowed to stand at 20 ° C. for 10 minutes after being covered, and then the cells grown on the cell culture substrate (C) were picked with forceps to separate the cells from the cell culture substrate (C) in a sheet form. At this time, the cell culture substrate (C) was not damaged at all, and no deposits were observed on the sheet-like cells. When the number of viable cells was measured for the extracted sheet-like cells by the same method as in Example 1, the number of cells that was 2.0 × 10 ⁶ at the start of the culture was 3.9 × 10 ⁷ after the culture. It was confirmed that the number increased.

(Comparative Example 1)
Cell culture was performed using the cell culture dish “Falcon 3003” without any surface treatment. Cells, medium and culture conditions were the same as in Example 2. One week after the start of the culture, the dish surface was observed with an optical microscope, and it was confirmed that the cells adhered and proliferated. The cultured dish was placed in a constant temperature bath at 20 ° C., allowed to stand for 10 minutes, and then the cells on the dish were tried to be removed. In addition, when cultured cells were separated using trypsin by a known method, the cells were separated into individual cells, and it was impossible to take out the cells in a sheet form.

(Comparative Example 2)
N-isopropylacrylamide was dissolved in isopropyl alcohol to prepare a 50 wt% solution. 0.2 ml of this solution was added to the surface of the cell culture dish “Falcon 3003” and irradiated with an electron beam to form a poly (N-isopropylacrylamide) film on the dish surface. The irradiation amount was 30 Mrad. After irradiation, the dish surface was washed with ultrapure water. Using this dish, cell culture similar to that in Example 1 was performed, and it was confirmed that the cells adhered and proliferated on the surface of the dish. The dish was allowed to stand for 10 minutes in a constant temperature bath at 20 ° C., and then an attempt was made to take out the cells. However, separation of the dish surface from the cells was insufficient, and it was impossible to detach the cells in a sheet form.

(Comparative Example 3)
The organic crosslinked hydrogel was polymerized in the same manner as in Preparation Example 1 except that no clay mineral was used and that the NIPA monomer was added and then 5 mol% of the organic crosslinking agent was added. As the organic crosslinking agent, N, N′-methylenebisacrylamide (manufactured by Wako Pure Chemical Industries, Ltd.) was used as it was. As a result, a sheet-like hydrogel (D) whitened at 20 ° C. was obtained. The obtained sheet-like hydrogel (D) was purified in the same manner as in Preparation Example 1 and then transferred to a cell culture dish. However, the hydrogel sheet (D) was very brittle and was purified and transferred. Was difficult. Moreover, the contact angle of this hydrogel sheet (D) was 49 degrees in a 50 degreeC holding state.

Next, cell culture was performed in the same manner as in Example 1 using this hydrogel sheet (D) -containing cell culture dish. One week after the start of the culture, a part of the end of the hydrogel sheet in the dish was cut out and stained with trypan blue, and it was confirmed that cells were growing on the hydrogel sheet. Attempts were made to remove the cultured cells from the hydrogel sheet-containing dish in which this culture was performed, but the hydrogel sheet was destroyed during the separation of the cells and the hydrogel sheet, and the cells could not be detached at all.

Claims (11)

A cell culture substrate comprising a polymer hydrogel having a three-dimensional network structure composed of a polymer of a water-soluble organic monomer and a water-swellable clay mineral. The cell culture substrate according to claim 1, wherein the polymer hydrogel is a polymer hydrogel in which hydrophilicity and hydrophobicity are reversibly changed in accordance with a change in external environment. The cell culture substrate according to claim 1, wherein the polymer hydrogel is a polymer hydrogel in which hydrophilicity and hydrophobicity reversibly change at a certain temperature as a boundary. The polymer hydrogel is a polymer hydrogel having a tensile elastic modulus of 1 kPa or more, a tensile strength of 20 kPa or more, and a breaking elongation of 50% or more under the condition of a moisture content of 90%. The cell culture substrate described. The cell culture medium according to any one of claims 1 to 4, wherein the polymer hydrogel is a polymer hydrogel obtained by polymerizing a water-soluble organic monomer in the presence of a water-swellable clay mineral uniformly dispersed in water. Wood. The water-soluble organic monomer is at least one selected from the group consisting of N-substituted acrylamide derivatives, N, N-disubstituted acrylamide derivatives, N-substituted methacrylamide derivatives, and N, N-disubstituted methacrylamide derivatives. The cell culture substrate according to 1 to 5. The water-soluble organic monomer is N-isopropyl (meth) acrylamide, Nn-propyl (meth) acrylamide, N-cyclopropyl (meth) acrylamide, N-ethoxyethyl (meth) acrylamide, N-tetrahydrofurfuryl (meth) ) Acrylamide, N-ethylacrylamide, N-ethyl-N-methylacrylamide, N, N-diethylacrylamide, N-methyl-Nn-propylacrylamide, N-methyl-N-isopropylacrylamide, N-acryloylpiperidine The cell culture substrate according to claim 1, which is at least one selected from the group consisting of N-acryloylpyrrolidine. The cell culture substrate according to any one of claims 1 to 7, wherein the polymer of the water-soluble organic monomer has a lower critical solution temperature. The cell culture substrate according to any one of claims 1 to 8, wherein the polymer of the water-soluble organic monomer is a polymer obtained by polymerization under a condition exhibiting hydrophobicity. The water-swellable clay mineral is at least one selected from the group consisting of water-swellable hectorite, water-swellable montmorillonite, water-swellable saponite, and water-swellable synthetic mica. A cell culture substrate according to any one of the above. Using the cell culture substrate according to any one of claims 1 to 12, after culturing cells at a temperature at which the cell culture substrate exhibits hydrophobicity, the temperature of the cell culture substrate is lowered, A cell culture method for separating cells cultured from a temperature at which the cell culture substrate exhibits hydrophilicity from the cell culture substrate.