JP2006288217A - Cell culture substrate and cell culture method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a flexible and tough cell culture substrate and a cell culture substrate that does not damage cells, is not mixed with a substrate in separating and recovering cultured cells and rapidly recovers cultured cells and to provide a cell culture method for easily recovering cultured cells. <P>SOLUTION: The cell culture substrate comprises a dried polymer hydrogel that is constituted of a polymer of a water-soluble organic monomer and a water-swellable clay mineral and has a three-dimensional network structure and reversibly changes from hydrophilicity to hydrophobicity with an external environmental change. The cell culture method comprises using the cell culture substrate, culturing cells at a temperature at which the cell culture substrate exhibits hydrophobicity, lowering the temperature of the cell culture substrate to a temperature at which the cell culture substrate exhibits hydrophilicity so as to separate the cultured cells from the cell culture substrate. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a cell culture substrate comprising a dried organic-inorganic composite polymer hydrogel having a three-dimensional network structure composed of a polymer of a water-soluble organic monomer and a water-swellable clay mineral.

  Conventionally, plastic (eg, polystyrene) or glass containers have been used as cell culture substrates for animal tissues and the like. These containers are subjected to surface treatment such as plasma treatment or coating of silicon, cell adhesion factor or the like in order to effectively perform cell culture. Therefore, when these cell culture vessels are used as a culture substrate, the cultured and proliferated cells adhere to the surface of the surface-treated vessel, and in order to isolate and recover the cells, a protein such as trypsin is used. It was necessary to peel from the container surface using a hydrolase or chemical. The operation of detaching cells with such enzymes and chemicals is not only complicated, but there is a risk that impurities such as bacteria and DNA or RNA may be mixed. In addition, not only the binding part of the cell and the base material is cut, but also the binding between the cells is cut, so that the cell can not be taken out in a proliferating shape (eg, a sheet shape) There was a problem that the properties of the cells changed. Also, when cultivating multiple types of cells, it is necessary to exchange a plurality of culture solutions, etc., but since the container cannot be replaced, various chemicals etc. remain inside the container. There were many problems from the viewpoint of the use of cultured cells, such as the possibility of contamination of cultured cells.

  In recent years, using a substrate with a thin coating of a temperature-responsive polymer on the surface of a cell culture container, the cell is kept in a hydrophobic state at the cell culture temperature to adhere cells, and after the culture, the polymer is treated at a low temperature. A technique has been reported in which the adhesiveness between cells and polymers is lowered by making them hydrophilic, 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 and Non-Patent Document 1). 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 in the meantime, the cell is also exposed to a low temperature state. there were. Furthermore, when the substrate is used, the cultured cells are used in the next experiment (for example, when transplanted into the body of an animal or when co-cultured with other cells), it becomes a sheet. The cells need to be peeled off from the polymer-coated container and moved without a support, and the peeled cell sheet with very low strength must be grabbed and moved to the next use position, etc. It had the problem that it was easy to get damaged or the operability was very bad. Therefore, after culturing the cells, it can be completely separated from the base material in a short time without being contaminated or damaged, and the cell sheet can be taken out at an arbitrary place, moved to the next step, and used. It was requested to do.

  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 Masayuki Yamato, Mitsuo Okano “The Frontiers of Nanobiotechnology”, Chapter 6 340-P. 347, CMC Publishing (published in 2003)

  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 cell culture substrate used in the present invention comprises a dried polymer hydrogel having a three-dimensional network structure composed of a water-soluble organic monomer polymer and a water-swellable clay mineral, and the three-dimensional network. The polymer hydrogel having a structure is characterized in that its state reversibly changes its hydrophilicity and hydrophobicity due to a change in the external environment. When the dried product of the polymer hydrogel absorbs water and the medium in a liquid medium, when the surface is hydrophobic, the cells can be suitably cultured and grown on the surface. Since the adhesion to the cells can be reduced under conditions that show the property, the cells can be easily and quickly peeled and recovered without causing damage to the cultured and grown cells and exfoliation of the base material. . Moreover, since it is a flexible and tough material, it can implement | achieve using a cell in the form of the sheet | seat of various shapes on the surface, and using it for the next experiment in the state which maintained the shape.

  That is, the present invention comprises a dried product 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 the polymer hydrogel becomes hydrophilic as the external environment changes. Cell culture substrate characterized by reversibly changing the property and hydrophobicity, a cell culture method for culturing cells on the cell culture substrate, and culturing cells on the cell culture substrate, Provided is a cell separation method for separating cells cultured from the cell culture substrate by setting the substrate to a temperature exhibiting hydrophilicity.

  The cell culture substrate of the present invention is a dried product 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 the polymer hydrogel Since the hydrophilicity and hydrophobicity change reversibly as the external environment changes, the surface smoothness and hydrophobicity of the polymer hydrogel can be controlled to a state suitable for cell adhesion and extension. Excellent culture performance. The cell culture substrate of the present invention can obtain a surface state particularly suitable for cell culture by drying the polymer hydrogel. In addition, by immersing the dried product in a cell culture medium, it is possible to obtain a moisture content suitable for cell culture. From these results, excellent culture characteristics are exhibited for various types of cells. I can do it. In addition, when the dried product obtained by drying the polymer hydrogel of the present invention is swollen again, it has excellent flexibility and toughness. The cells cultured stably can be transferred while being held. 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. Moreover, the dried product of the polymer hydrogel of the present invention is less contaminated than the polymer hydrogel containing a solvent, and can be stably stored for a long period before use as a culture substrate.

  A cell culture substrate made of a dried polymer hydrogel whose hydrophilicity and hydrophobicity reversibly change depending on the external environment shows excellent adhesion to cells under hydrophobic conditions. It can be grown, and under hydrophilic conditions, it can reduce adhesion to cells, so cells can be detached without the use of protein hydrolases such as trypsin or chemicals. In addition, the cells can be easily collected without causing the substrate to be peeled and mixed. 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 dried 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 for the cell culture substrate of the present invention is not limited as long as it has a property of being dissolved in water and interacts 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 coordination bond, a covalent bond and the like with a mineral 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 needs to be able to form a polymer hydrogel having a stable shape by forming a three-dimensional network structure with a water-swellable clay mineral, such as an acrylic compound or a vinyl compound. Can be used. In particular, since the cultured cells can be easily separated from the obtained cell culture substrate, it is effective that the water-soluble or water-absorbing hydrophilicity and hydrophobicity reversibly change in accordance with changes in the external environment. is there. 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.

  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 dried polymer hydrogel are sufficiently exhibited, the preparation is easy, and the obtained cell culture substrate exhibits higher strength.

  The cell culture substrate of the present invention comprises a polymer hydrogel having a three-dimensional network structure, wherein the polymer of the water-soluble organic monomer and the water-swellable clay mineral are a polymer before drying. The hydrogel contains in its structure an aqueous solution containing water or a salt as a solvent.

  The amount of the solvent contained in the polymer hydrogel is set according to the purpose and is not generally defined. However, in order for the polymer hydrogel to exhibit the mechanical properties described below, the solid content in the polymer hydrogel is preferably set. A solvent having a mass ratio of 0.1 to 50 is more preferably 0.1 to 10. The amount of these solvents can be arbitrarily controlled by changing the amount of the solvent during polymerization or by reversibly changing the state of the polymer hydrogel from hydrophilic to hydrophobic after polymerization.

  The cell culture substrate used in the present invention is obtained by drying a polymer hydrogel having a three-dimensional network structure of a water-soluble organic monomer polymer and a water-swellable clay mineral. Sometimes it is used in a state immersed in a liquid medium. At that time, the cell culture substrate is a polymer hydrogel swollen by impregnating the medium, but the moisture content is different from the moisture content of the polymer hydrogel before drying. The water content can be arbitrarily set by changing the ratio of the polymer of water-soluble organic monomer constituting the polymer hydrogel and the clay mineral, and reversibly changing the state of the polymer hydrogel from hydrophilic to hydrophobic. However, by impregnating the medium with the polymer hydrogel at a temperature higher than the lower critical solution temperature of the polymer hydrogel, the moisture content is suitable for cell culture. The thing whose mass ratio of the solvent with respect to solid content in hydrogel is 0.01-5 is used, More preferably, the thing of 0.01-1 is used. Within such a mass ratio range, the surface state of the polymer hydrogel is suitable for cell culture due to its smoothness and water content, and exhibits excellent culture characteristics for various types of cells. I can do it.

  The dried product of the polymer hydrogel used for the cell culture substrate of the present invention is a dried product of the polymer hydrogel having a three-dimensional network structure in which the polymer of the water-swellable clay mineral and the water-soluble organic monomer is used. Therefore, after culturing cells, the cells can be maintained in shape without being destroyed when the cells are peeled from the polymer hydrogel. Further, the dried polymer hydrogel used for the cell culture substrate of the present invention can move without destroying the sheet of cultured cells when it is necessary to move the cells to the next experimental position after culturing. The dried product of the polymer hydrogel can realize a tensile elastic modulus of 1 kPa or more, a tensile strength of 20 kPa or more, and a elongation at break of 50% or more in a state where the moisture content is 90% before drying, and has these characteristics. Those 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. . A polymer hydrogel having such mechanical properties can obtain a surface state suitable for cell culture by making it dry. Furthermore, when used as a cell culture substrate, the dried product of the polymer hydrogel is immersed in a liquid culture medium for cell culture so that it is rehydrated and becomes a polymer hydrogel having a water content suitable for cell culture. Under the condition of a moisture content of 90%, the polymer hydrogel having such mechanical properties retains excellent mechanical properties even in a hydrophobic state when cell culture is performed after immersing in a medium after drying. After cell culture, it exhibits excellent shape stability, handleability, mobility, etc. regardless of shape.

  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. Moreover, what re-swelled the dry thing of this polymer hydrogel with the culture medium for cell culture similarly has hydrophilic property and hydrophobicity according to external environmental conditions. For this reason, the cell culture substrate made of the dried polymer hydrogel can cultivate the cells suitably, and the cultured cells can be easily and quickly detached without causing the destruction of the cultured cells and the contamination of the substrate. It can be recovered.

  When culturing cells, it is necessary to sterilize the cell culture substrate, and the sterilization step can be performed by irradiation with radiation, steam sterilization, gas sterilization, etc. on the dried polymer hydrogel. It is preferably performed by irradiation with radiation such as gamma rays. As a result, the cell culture substrate can be sterilized without greatly reducing the properties of the polymer hydrogel and without leaving a substance that adversely affects the cells.

  The critical temperature at which the hydrophilicity and hydrophobicity of the dried polymer hydrogel re-swelled with the cell culture medium can change the hydrophilicity and hydrophobicity suitably, so that the critical temperature is 0 to 50 ° C. It is preferable that the temperature is within a range. Cell culturing is performed under conditions above the critical temperature, and the cells can be detached by changing the temperature below the critical temperature after culturing. 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 dried polymer hydrogel used for the cell culture substrate of the present invention can be used alone or coated on a support having a smooth or uneven surface such as metal, ceramic, or plastic. In addition, since it has high mechanical properties, it can be formed into various shapes, and can be used in sheet form, fiber form, hollow fiber form, and spherical form.

  The cells that can be cultured using the cell culture substrate of the present invention are not particularly limited as long as they are human and animal tissue cells. For example, vascular cells, fibroblasts, muscle cells, nerve cells, Examples include chondrocytes, osteoblasts, hepatocytes, pancreatic cells, corneal cells and the like. Of these, vascular endothelial cells, dermal fibroblasts, hepatocytes, hepatoma cells, chondrocytes and the like are preferably used. In particular, in the present invention, it can be suitably used for culturing skin fibroblasts, vascular endothelial cells, chondrocytes and the like.

  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 is dried to obtain a dried product, which can be used as the cell culture substrate of the present invention. The obtained dried polymer hydrogel 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 suitably the temperature at the time of the said polymerization reaction according to the kind of water-soluble organic monomer to be used, a polymerization catalyst, an initiator, etc., for example, it can set to the range of 0 to 100 degreeC. In addition, when the polymerization temperature is set to a temperature lower than or equal to the LCST of the polymer hydrogel (or more than UCST), the polymer hydrogel after polymerization becomes hydrophilic and the resulting dried product becomes hydrophilic. By impregnating and maintaining at a temperature of LCST or higher (or UCST or lower), the hydrophilic state can be changed to a hydrophobic state, which is 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 the obtained dried product is also hydrophobic and cell culture can be performed as it is. I can do it. That is, it is possible to obtain a dried product of a polymer hydrogel used as a cell culture substrate by using either polymerization method. 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.

  As a method for drying the polymer hydrogel obtained as described above, a known method for drying a sample is generally used. For example, by leaving it in a dry atmosphere for 3 days, a dried product of the target polymer hydrogel is obtained. Is obtained. The dried product of the polymer hydrogel of the present invention can exhibit the characteristics described above not only in a completely dry state but also in a state of absorbing moisture in the air.

  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.

  In preparing the polymer hydrogel, a known and commonly used organic crosslinking agent may be used in the 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.

  In the preparation of the polymer hydrogel, an additive containing a polymer compound or a low-molecular compound in addition to the polymer of the water-soluble organic monomer is included as long as the effects of the present invention are not impaired. For example, cell adhesion factors such as collagen and hyaluronic acid, cell growth factors, hydroxyapatite particles and the like can be added.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, the scope of the present invention is not limited only to these Examples.

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. N, N′-methylenebisacrylamide (Wako Pure Chemical Industries, Ltd .: hereinafter abbreviated as BIS) was used as the organic crosslinking agent. 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 constant temperature room at 20 ° C., 57.06 g of ultrapure water and a Teflon (registered trademark) stirrer were placed in a flat bottom glass container purged with nitrogen, and 2.88 g of Laponite XLG was added while stirring, A solution was prepared. 6.78 g of NIPA was added thereto, and dissolved by stirring 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 two 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. 20 hours after the start of polymerization, a substantially colorless and transparent uniform sheet-like hydrogel (A) was obtained in a polystyrene container.

  The sheet-like hydrogel (A) is slightly swollen in water at 20 ° C. and adjusted to a moisture content of 90%, then cut into a size of 1 cm × 5 cm, and a tensile test apparatus (stock) It was mounted on Shimadzu Corporation "table type universal testing machine AGS-H"), and a tensile test was conducted at a distance between grades of 30 mm and a tensile speed of 100 mm / min. % And the elastic modulus was 13.3 kPa. After carefully removing the water adhering to the surface of the hydrogel (A), the contact angle with water at 20 ° C. and 50 ° C. of the surface of the hydrogel (A) was determined using a contact angle measuring device (“CA-” manufactured by Kyowa Interface Science Co., Ltd.). X200 "). The contact angle of water at each temperature was 30 ° at 20 ° C. and 59 ° at 50 ° C. holding state. From this, it was confirmed that the obtained hydrogel (A) exhibits both hydrophilic and hydrophobic characteristics depending on temperature conditions.

  On the other hand, the sheet-like hydrogel (A) is immersed in 2 L of ultrapure water at 20 ° C. for 2 days to swell the hydrogel and then taken out, and then immersed in 1 L of 50 ° C. ultrapure water for 2 days. It was taken out after shrinking. After repeating the purification operation by washing three times, the ends of the purified sheet-like hydrogel (A) are fixed with clips so as not to deform, and are dried in a clean bench for 3 days, so that it is almost transparent. A dried hydrogel (A) was obtained. The obtained dried hydrogel (A) was sealed in a gas barrier plastic bag and irradiated with gamma rays (radiation source: Cobalt 60, Radier Kogyo Co., Ltd.). The irradiation dose was 25 kGy. No particular deformation or discoloration was observed in the dried hydrogel (A) after irradiation. The obtained dried hydrogel (A) after irradiation with gamma rays was cut into a size of 8 cm in diameter to obtain a cell culture substrate (A) comprising the dried 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. 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.

The cells were cultured using the cell culture dish containing the cell culture substrate (A) thus obtained. 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. Two dishes containing the cell culture substrate (A) were prepared and seeded simultaneously under the same conditions. One week after seeding, one dish containing the cell culture substrate (A) was 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 adhered on the culture substrate (A) and sufficiently proliferated. The cell culture substrate (A) was removed from the dish containing the other cell culture substrate (A), and the fetal bovine serum that had been kept at 20 ° C. It was transferred to a tissue culture dish containing a minimum essential eagle medium containing 1%. 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). At this time, the cell culture substrate (A) was not damaged at all, and no adherent was observed on the separated cells. The extracted cells were treated with trypsin-EDTA to separate each cell into individual states, and then subjected to trypan blue staining to measure the number of viable cells. It was confirmed that the number of cells, which was ⁶ , increased to 1.2 × 10 ⁸ after culture.

  Moreover, when the dried hydrogel (A) obtained by drying the hydrogel (A) was immersed in the medium used in the above experiment and kept in a 37 ° C. incubator for 24 hours, the water content was 30%. It was confirmed to return to the hydrogel.

(Example 2)
Cells were cultured using the cell culture dish containing the cell culture substrate (A) obtained in Example 1 above. Normal human skin fibroblasts (Dainippon Pharmaceutical Co., Ltd.) were used as the cells to be cultured. Culturing was performed in a 37 ° C. incubator containing 5% carbon dioxide using CS-C medium (Dainippon Pharmaceutical Co., Ltd.). Two dishes containing the cell culture substrate (A) were prepared and seeded simultaneously under the same conditions. One week after seeding, one dish containing the cultured cell culture substrate (A) was 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 the cells adhered on the cell culture substrate (A) and proliferated sufficiently. Remove the whole cell culture substrate (A) from the dish in which the other cell culture substrate (A) was cultured, and remove the CS-C medium kept at 20 ° C. in advance. It was transferred to a tissue culture dish containing. 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 to separate each cell into individual states and then subjected to trypan blue staining to count the number of viable cells. At the start of culture, 2.5 × It was confirmed that the number of cells, which was 10 ⁶ , increased to 9.9 × 10 ⁷ after culturing.

  Moreover, when the dried hydrogel (A) obtained by drying the hydrogel (A) was immersed in the medium used in the above experiment and kept in a 37 ° C. incubator for 24 hours, the water content was 30%. It was confirmed to return to the hydrogel.

(Example 3)
A substantially colorless and transparent, uniform sheet-like hydrogel (B) was synthesized in the same manner as in Example 1 except that the amount of Laponite XLG to be added was 1.44 g. Using the obtained hydrogel (B), the contact angle with water at 20 ° C. and 50 ° C. was measured in the same manner as in Example 1. As a result, 30 ° at 20 ° C. and 60 ° at 50 ° C. holding state, It was confirmed that the obtained cell culture substrate (B) exhibits both hydrophilic and hydrophobic characteristics depending on the temperature conditions.
In addition, a dried hydrogel (B) was prepared in the same manner as in Example 1 using the obtained hydrogel (B). The dried hydrogel (B) obtained was irradiated with gamma rays in the same manner as in Example 1. No particular deformation or discoloration was observed in the dried hydrogel (B) after irradiation. The obtained dried hydrogel (B) after irradiation with gamma rays was cut into a size of 8 cm in diameter to obtain a cell culture substrate (B) comprising the dried hydrogel (B).
Using the cell culture substrate (B) thus obtained, HepG2 cells were cultured in the same manner as in Example 1, with the cells, medium, and culture conditions. One week after cell seeding, the surface was observed with an optical microscope in the same manner as in Example 1. As a result, it was confirmed that the cells adhered to the cell culture substrate (B) and proliferated sufficiently. It was. In the same manner as in Example 1, after another cell culture substrate (B) was used for cell culture, the cell culture substrate (B) was removed from the dish containing the cell culture substrate (B). The cultured cells were taken out and transferred to a tissue culture dish containing a minimum essential eagle medium containing 10% of fetal bovine serum previously maintained at 20 ° C. The cells were allowed to stand at 20 ° C. for 10 minutes after being capped, 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). At this time, the cell culture substrate (B) was not damaged at all, and no adherent was observed on the separated cells. When the number of viable cells was measured by the same method as in Example 1, it was confirmed that the number of cells that was 2.0 × 10 ⁶ at the start of the culture increased to 4.9 × 10 ⁷ after the culture. It was.

  Moreover, when the dried hydrogel (B) obtained by drying hydrogel (B) was immersed in the culture medium used in the above experiment and kept in a 37 ° C. incubator for 24 hours, the water content was 28%. It was confirmed to return to the hydrogel.

(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 1. 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 taken out. 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)
The organic cross-linked hydrogel was polymerized in the same manner as in Example 1 except that no clay mineral was used and that the NIPA monomer was added and then 5 mol% of the organic cross-linking 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 (E) whitened at 20 ° C. was obtained. The obtained sheet-like hydrogel (E) was purified in the same manner as in Example 1, and then transferred to a cell culture dish. However, the hydrogel sheet (E) was very brittle and was purified and transferred. Was difficult. Moreover, the contact angle of this hydrogel sheet (E) was 49 degrees in a 50 degreeC holding state.

Next, cell culture was performed in the same manner as in Example 2 using the cell culture dish containing the hydrogel sheet (E). One week after the start of culture, a portion of the hydrogel sheet in the dish was cut out and stained with trypan blue, and no cell growth on the hydrogel sheet was confirmed. In addition, an attempt was made to remove the hydrogel sheet from the dish, but the hydrogel sheet was destroyed and could not be removed.

Claims (13)

It is composed of a dried polymer hydrogel having a three-dimensional network structure composed of a water-soluble organic monomer polymer and a water-swellable clay mineral, and the polymer hydrogel becomes hydrophilic and hydrophobic as the external environment changes. A cell culture substrate characterized by reversibly changing sex. 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 cell culture substrate according to claim 1 or 2, wherein the polymer of the water-soluble organic monomer has a lower critical solution temperature. 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. 4. The cell culture substrate according to 3. 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 3, which is at least one selected from the group consisting of N-acryloylpyrrolidine. 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. The cell culture medium according to any one of claims 1 to 6, 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 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. A cell culture substrate according to any one of claims 1 to 8, wherein the cell culture substrate is irradiated with radiation and sterilized, and then cell culture is performed in a state in which a medium solution is absorbed. Cell culture method. The cell culture method according to claim 9, wherein the amount of the medium solution to be absorbed is a mass in the range of 0.01 to 5 with respect to the mass of the cell culture substrate. The cell culture method according to claim 9 or 10, wherein a temperature at which the medium solution is absorbed is higher than a lower critical solution temperature. The cell culture method according to any one of claims 9 to 11, wherein the cell culture is a culture at a temperature at which the cell culture substrate exhibits hydrophobicity. After culturing the cells by the method according to claim 9 to 12, the temperature of the cell culture substrate is lowered, and the cells cultured by setting the temperature of the cell culture substrate to be hydrophilic are removed from the cell culture substrate. A method for separating cultured cells to be separated.