JP2008237088A - Base medium and method for cell culture - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a base medium for cell culture, which is composed of a polymer composite having uniformly and finely dispersed clay mineral in an organic polymer in wide range content rate of clay mineral and having flexible and also tough excellent dynamic physical properties, further capable of recovering cultured cells quickly, without breaking the cells or mixing the base medium on separating and recovering the cultured cells, and to provide a method easily recovering the cultured cells. <P>SOLUTION: This base medium for the cell culture uses the polymer composite (C) having finely dispersed a water swellable clay mineral (B) in a polymer (A) using a water soluble (meth)acrylic acid ester (a) as a monomer. By using the base medium, culturing the cells under a temperature condition that the base medium for cell culture shows bio-adhesion, and then lowering the temperature of the base medium for cell culture so as to make the temperature of the base medium for cell culture show non-bio-adhesion, and it is possible to separate the cultured cells from the base medium for cell culture, without developing the breakage of the cells or mixing of the base medium. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a cell culture substrate comprising a polymer complex in which a water-swellable clay mineral is finely dispersed in an organic polymer, and a cell culture method using the cell culture substrate comprising the polymer complex, The present invention relates to a method for separating cultured cells.

  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. Moreover, when cells were collected, they fell apart, and it was difficult to take them out in sheet form. 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, as a polymer composite material obtained by compounding an organic polymer and an inorganic material, a material in which an organic polymer is filled with talc, calcium carbonate, etc. in addition to glass fiber and carbon fiber has been known for a long time. . In recent years, dispersion of nanometer-scale inorganic components in organic polymers and compounding them has resulted in improved mechanical properties and improved thermal properties, attracting attention as organic / inorganic nanocomposite materials. .

  In recent years, the present inventors have obtained a copolymer of a water-soluble (meth) acrylic acid ester, or at least one of water-soluble (meth) acrylamide and N-substituted (meth) acrylamide and a water-soluble (meth) acrylic acid ester. Developed a polymer composite formed by forming a three-dimensional network of a water-swellable clay mineral with an organic polymer composed of the above copolymer and stretched compared to a polymer or copolymer that does not contain a clay mineral. It has been reported that mechanical properties such as strength and strength are remarkably improved (see Patent Document 3), and although it is a useful material in various fields, a technique relating to a cell culture substrate is not disclosed.

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 cell culture comprising a polymer complex having a wide range of clay mineral content, wherein clay minerals are uniformly and finely dispersed in an organic polymer, and have flexible and tough excellent mechanical properties. Cell culture substrate capable of collecting rapidly cultured cells without cell breakage or substrate contamination when separating and collecting cultured cells and further cultured cells, and cell culture method for easy collection of cultured cells Is to provide

  As a result of various studies, the present inventors have found that a polymer composite in which a water-swellable clay mineral is finely dispersed in a polymer using a water-soluble (meth) acrylic ester can solve the above problems. The present invention was completed.

  That is, the present invention relates to a polymer composite in which a water-swellable clay mineral (B) is finely dispersed in a polymer (A) using a water-soluble (meth) acrylic acid ester (a) as a monomer ( A cell culture substrate characterized by using C) is provided.

  Furthermore, the present invention uses the above cell culture substrate, and after culturing cells under temperature conditions where the cell culture substrate exhibits cell adhesion, the temperature of the cell culture substrate is lowered, and the cell culture Provided is a cell culture method characterized in that the cultured cells are separated from the cell culture substrate by setting the temperature at which the substrate exhibits cell non-adhesiveness.

  Since the cell culture substrate of the present invention is a polymer mainly using a water-soluble (meth) acrylic acid ester, it is possible to finely disperse a water-swellable clay mineral in a wide content ratio range in the polymer. it can. And, compared to a polymer or copolymer that does not contain clay minerals, cells can be cultured and propagated on its surface, and if the conditions for holding the polymer complex after culturing are changed The adhesion to the cells can be reduced, and the cells can be easily and quickly peeled and recovered without causing damage to the cultured and proliferated cells or peeling and mixing of the base material. Incidentally, the cell adhesion was not observed on the surface of the polymer of (meth) acrylic acid ester containing no clay mineral or its crosslinked product, as shown in the comparative example. Moreover, since the cell culture substrate of the present invention is a flexible and tough material, it can be used in a state in which cells are cultured in various shapes on the surface and the shape is maintained.

  The cell culture substrate of the present invention can contain a water-swellable clay mineral finely and uniformly at a nanometer level and in a wide concentration range, and has good stretchability and dynamics such as excellent strength and elastic modulus. Since it is composed of a polymer composite having physical properties, the smoothness and hydrophobicity of the surface of the polymer composite can be controlled to be in a state suitable for cell adhesion and extension, so that it has excellent culture performance. In addition, the cell culture substrate of the present invention can obtain a moisture content suitable for cell culture by immersing it in a cell culture medium. From these results, excellent culture for various types of cells. The characteristics can be shown. In addition, cell adhesion and non-adhesion of the polymer complex can be reversibly changed with changes in the external environment. In addition, when the polymer composite of the present invention is swollen, it has excellent flexibility and toughness, so that the cultured cells can be stably cultured while maintaining the shape even when the cultured cells are transferred together with the substrate. Can be transported. 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. In addition, the polymer composite of the present invention can be stably stored for a long period of time before use as a culture substrate.

  A cell culture substrate composed of a polymer complex in which cell adhesion and cell non-adhesiveness reversibly change depending on the external environment can cultivate and proliferate cells appropriately under cell adhesion conditions. Under non-cell-adhesive conditions, cells can be detached without the use of protein hydrolases such as trypsin or chemicals, so that cells can be easily recovered without causing cell damage or substrate contamination. Is possible. 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 is a polymer obtained from a water-soluble (meth) acrylic acid ester, or at least one of (meth) acrylamide and N-substituted (meth) acrylamide and a water-soluble (meth) acrylic acid ester. It consists of a polymer composite (C) in which a water-swellable clay mineral (B) is finely dispersed in an organic polymer (A) consisting of a copolymer of the above, and has excellent mechanical properties such as stretchability and flexibility. Show.

  The water-soluble (meth) acrylic acid ester, (meth) acrylamide, and N-substituted (meth) acrylamide used in the present invention are preferably soluble in water or a mixed solvent of water and an organic solvent. On the other hand, the organic polymer (A) obtained by polymerizing or copolymerizing these has more hydrophobicity, does not dissolve in water, does not swell due to excessive water absorption, and is stable in water. Those present are preferred. That is, the polymer composite (C) constituting the cell culture substrate of the present invention is synthesized mainly from a water-soluble monomer and a water-swellable clay mineral, but the obtained polymer composite preferably has water-swellability. It is a low and highly hydrophobic material. However, in order to change the balance between hydrophilicity and hydrophobicity of the polymer composite or to strengthen the interaction with other components, hydrophilic groups, ionic groups and / or hydrophobic groups may be added as necessary. It is also possible to introduce it into a polymer or copolymer.

  The water-soluble (meth) acrylic acid ester (a) is substantially soluble in water and can finely disperse the water-swellable clay mineral (B) at the time of polymerization. For example, methoxyethyl acrylate (MEA), ethoxy Examples include ethyl acrylate, methoxyethyl methacrylate, ethoxyethyl methacrylate and the like. The polymer obtained from the water-soluble (meth) acrylic acid ester (a) of the present invention includes a single-monomer polymer or a multi-monomer copolymer selected from these (meth) acrylic acid esters.

  (Meth) acrylamide and N-substituted (meth) acrylamide are (meth) acrylamide and alkyl (meth) acrylamide having 1 or more carbon atoms in the alkyl group, specifically, N-methylacrylamide and N-ethyl. Acrylamide, N-cyclopropylacrylamide, N-isopropylacrylamide, N, N-dimethylacrylamide, N-methyl-N-ethylacrylamide, N-methyl-N-isopropylacrylamide, N-methyl-Nn-propylacrylamide, N , N-diethylacrylamide, N-ethyl-N-isopropylacrylamide, N-ethyl-Nn-propylacrylamide, N-acryloylpyrrolidine, N-acryloylpiperidine, N-acryloylmorpholine, N-acryloyl Methyl homopiperazine, N- acryloyl methyl piperazine or N- methyl methacrylamide.

  When the organic polymer (A) comprises a copolymer of (meth) acrylamide and at least one of N-substituted (meth) acrylamide (b) and a water-soluble (meth) acrylate (a), The molar ratio ((b) / (a)) of the polymer (a) and (b) is to increase the hardness and elastic modulus while maintaining the hydrophobic property of the resulting polymer composite at room temperature. The molar ratio is preferably 1 or less, and in particular, in order to have high flexibility at room temperature and to reduce the water absorption rate in water, the molar ratio is preferably 0.5 or less, more preferably 0.25 or less. is there.

  The glass transition temperature of the organic polymer (A) is not necessarily limited, and a wide range is used. From the viewpoint of workability, stretchability at room temperature, and stretchability, the organic polymer (A) has a glass transition temperature. Is preferably 100 ° C. or lower, more preferably 30 ° C. or lower, particularly preferably 0 ° C. or lower.

  As the water-swellable clay mineral (B), a swellable clay mineral that can be peeled in layers is used, preferably a clay mineral that can swell and uniformly disperse in water or a mixed solution of water and an organic solvent, particularly preferably. An inorganic clay mineral that can be uniformly dispersed in water at a molecular level (single layer) or a level close thereto is used. More specifically, water-swellable hectorite containing sodium as an interlayer ion, water-swellable montmorillonite, water-swellable saponite, water-swellable synthetic mica and the like can be mentioned.

  As the initiator and catalyst to be used, known radical polymerization initiators and catalysts can be appropriately selected and used. Preferably, those having water dispersibility and uniformly contained in the entire system are preferably used. Specifically, as a polymerization initiator, a water-soluble peroxide such as potassium peroxodisulfate or ammonium peroxodisulfate, a water-soluble azo compound such as VA-044, V-50, V-501 (all of which are Wako Pure Chemical Industries, Ltd.) In addition to Yaku Kogyo Co., Ltd., a mixture of iron ion (II) and hydrogen peroxide is exemplified.

  As the catalyst, tertiary amine compounds such as N, N, N ′, N′-tetramethylethylenediamine are preferably used. However, a catalyst is not necessarily used. The polymerization temperature is 0 to 80 ° C., and the polymerization time is several tens of seconds to several tens of hours.

  In the present invention, a small amount of a polyfunctional organic crosslinking agent may be used in combination as the organic crosslinking agent, and representatively N, N'-methylenebisacrylamide is exemplified.

  The polymer composite (C) constituting the cell culture substrate of the present invention has a structure in which the water-swellable clay mineral (B) is finely dispersed in the organic polymer (A).

  The finely dispersed state of the water-swellable clay mineral (B) varies depending on the concentration of the water-swellable clay mineral (B) and the synthesis conditions and stretching conditions of the polymer composite (C). When the water-swellable clay mineral (B) layered or separated within 10 layers is dispersed alone in the polymer composite (C), the water-swellable clay mineral (B) and the organic polymer (A ) In a state where the complex is formed and dispersed at the molecular level, and further the state in which the water-swellable clay mineral (B) in these states forms a three-dimensional network in the polymer complex (C), etc. A single or mixed state is used. Further, those in which these water-swellable clay minerals (B) are oriented by stretching are also used.

  As long as effective fine dispersion can be achieved, the interaction between the organic polymer (A) and the water-swellable clay mineral (B) is one of ionic bond, hydrogen bond, hydrophobic bond, coordinate bond, and covalent bond. There may be one or more. The composite still contains a considerable amount of water immediately after synthesis. For example, the water content (mass% with respect to the solid content of the polymer composite, hereinafter the same) is often 200 to 800% by mass. Are often weak. Such a composite is dried immediately after synthesis, and the water content is preferably 100% by mass or less, more preferably 50% by mass or less, and most preferably 20% by mass or less. The interaction between the molecule (A) and the water-swellable clay mineral (B) is greatly enhanced, and the characteristics of cell adhesion and excellent mechanical properties are expressed. The polymer composite obtained by drying once turns white even if it is again put into water, but it is uniform and does not swell greatly, and the strength is not greatly reduced.

  Further, for the purpose of imparting functionality to the polymer composite obtained by using other polymerizable organic molecules together with the polymerization component constituting the organic polymer (A) within a range not impeding the effects of the present invention. Various organic or inorganic functional molecules and particles may be added. In particular, it is preferable to add a high molecular compound or a low molecular compound that improves cell adhesion and cell proliferation in a range that does not impair the effects of the present invention. For example, cell adhesion such as collagen and hyaluronic acid is used. Factors, cell growth factors, hydroxyapatite particles and the like can be added.

  In the present invention, it is essential to finely disperse the water-swellable clay mineral (B) in the polymer composite (C), and the water-swellable clay mineral alone is used without using any ordinary organic crosslinking agent. It is possible to develop mechanical properties and transparency. Particularly preferably, it is prepared without using an organic crosslinking agent, but it may be preferable to use an organic crosslinking agent in combination depending on the selection of monomers and reaction conditions and for cell adhesion and physical property control. When clay minerals are not used, that is, when only a linear polymer is used or only an organic cross-linking agent is used, the resulting composite has no cell adhesiveness and low mechanical properties such as stretchability and strength. . When an organic cross-linking agent is used in combination with a water-swellable clay mineral, a polymer composite in which the elastic modulus and stretchability are controlled while the cell adhesion is maintained can be obtained. Specifically, by using an organic crosslinking agent in combination, it is possible to control to reduce stretchability and increase elastic modulus. The amount of the organic crosslinking agent used in combination is 2 mol% or less, more preferably 1 mol% or less, and particularly preferably 0.5 mol% or less.

  In the polymer complex (C) constituting the cell culture substrate of the present invention, the mass ratio of the water-swellable clay mineral (B) to the organic polymer (A) is preferably 0.003 to 3, more preferably Preferably it is 0.005-2, Most preferably, it is 0.01-1. If the mass ratio is less than 0.003, the mechanical properties tend to be insufficient as a polymer composite, and if it exceeds 3, uniform fine dispersion of the water-swellable clay mineral tends to be difficult.

  The polymer composite (C) that constitutes the cell culture substrate of the present invention has a water-swelling that is uniform and transparent in the dry matter, regardless of the content of the water-swellable clay mineral, and lowers the transparency. Micron-sized heterogeneous aggregation of the clay mineral is not observed. Finally, the content of the water-swellable clay mineral is measured by thermal mass spectrometry (TGA), and the fine dispersibility is measured by observation with a transmission electron microscope (TEM). In the polymer composite, it was confirmed by TGA that the total amount of the water-swellable clay mineral used was contained in the polymer composite, and a layered clay mineral layer having a thickness of 1 to several nanometers was uniformly dispersed. This is confirmed by TEM.

  The polymer composite constituting the cell culture substrate of the present invention exhibits excellent mechanical properties, particularly high stretchability and stretchability, even when no commonly used organic crosslinking agent is added. It is estimated that molecules and finely dispersed water-swellable clay minerals interact to form a three-dimensional network. On the other hand, a linear polymer that does not contain a water-swellable clay mineral and an organic cross-linking agent added thereto to form a three-dimensional network are extremely low compared to the polymer composite as shown in the comparative example. It shows only mechanical properties. This suggests that the polymer complex constituting the cell culture substrate of the present invention forms an unprecedented effective complex structure. Many of the highly hydrophobic organic polymers (A) are present as free chains without being constrained by a cross-linking agent, and thus have excellent mechanical properties but are hydrophilic, water-swellable clay minerals (B ) To have hydrophobicity suitable for cell adhesion, and excellent cell adhesion is realized.

  The formation of the above structure is not only fine dispersion of water-swellable clay minerals by transmission electron microscope and X-ray diffraction measurement, but also the following excellent stretchability and high breaking strength, dynamic viscoelasticity measurement and differential scanning calorimetry It was also confirmed by measuring the glass transition temperature close to the free chain of the organic polymer between water-swellable clay mineral layers by analysis (DSC) or the like.

  The polymer composite constituting the cell culture substrate of the present invention exhibits excellent mechanical properties. For example, the elongation is about several hundred% to 3000% even when the water content is substantially zero. It is also possible to obtain a polymer composite having a large elongation at break of 3000% or more. In addition, the polymer composite of the present invention exhibits a very high breaking strength as compared with organic crosslinked polymers and linear polymers that do not contain clay minerals. Specifically, the tensile strength of the polymer composite is 500 kPa or more, the tensile breaking elongation is 200% or more, and the elastic modulus at a tensile elongation of 100% is 50 kPa or more. In addition, a stretched product of the polymer composite can be obtained by stretching the obtained polymer composite to 100% or more, preferably 100% to 3000%. The obtained polymer composite stretched product has excellent stretchability (100% to 1500%) despite being after being stretched, and has particularly high flexibility and excellent stretchability. It is the characteristic to have. Specifically, the tensile strength of the stretched polymer composite is 1000 kPa or more, the tensile elongation at break is 200% or more, and the elastic modulus at 100% tensile is 100 kPa or more. The obtained polymer composite stretched product also has excellent cell adhesion, and the cell is stretched or contracted in a stretched state or within a range that does not interfere with cell adhesion and extension. Culture is possible.

  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.

  The polymer complex constituting the cell culture substrate of the present invention becomes white by absorbing water when immersed in water. However, even when immersed in a liquid medium used for cultured cells that can be used in the present invention, the polymer complex is almost hydrated. It can maintain a transparent state. For this reason, even if the medium is immersed, the mechanical properties hardly change from the dry state, and excellent stretchability and the like can be maintained.

  In addition, when the polymer complex constituting the cell culture substrate of the present invention is used as a cell culture substrate, it must be polymerized and purified after drying, and the method is not particularly limited. Purification can be performed by washing with water or an organic solvent capable of retaining the structure of the complex. For example, a purified polymer composite can be obtained by immersing the polymer composite in water or an organic solvent in a clean bench, purifying by appropriately replacing water or the organic solvent, and drying again. .

  The polymer complex (C) constituting the cell culture substrate of the present invention has a property of changing from cell adhesiveness to cell nonadhesiveness after cell culture according to external environmental conditions. For this reason, the cell culture substrate made of the polymer complex can cultivate the cells suitably, and the cultured cells can be easily and quickly removed without causing destruction of the cultured cells or exfoliation of the substrate. It is possible to peel and collect.

  In order to change the cell adhesiveness and cell non-adhesiveness of the polymer complex according to external environmental conditions, it is convenient to change the external temperature, and it is most convenient because it has less burden on the cells. After the cell culture is performed in a temperature range of 33 ° C. to 40 ° C. where the polymer complex has cell adhesion, the polymer complex exhibits cell non-adhesiveness in a temperature range of about 0 ° C. to 25 ° C. By holding, the cultured cells can be quickly detached and recovered.

  It is presumed that the moisture content of the polymer composite (C) and the fine structure on the outermost surface change as the mechanism by which the cell adhesiveness and non-adhesiveness change due to temperature change. When the temperature for holding the polymer complex is in the range of 33 ° C. to 40 ° C., the water content of the polymer complex is less than 10% with respect to the weight in the dry state, and hydrophobicity having excellent cell adhesion However, when the holding temperature is about 0 ° C. to 25 ° C., the water content slightly increases, becomes 10% or more with respect to the weight in the dry state, becomes hydrophilic and becomes non-cell-adhesive. The change in the water content varies depending on the amount of clay mineral contained in the polymer composite and the temperature to be retained, but the water content is preferably 20% or more with respect to the dry weight, and 30% or more. More preferably.

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

EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, the scope of the present invention is not limited only to these Examples.
(Production Example 1)
The water-swellable clay mineral includes a water-swellable synthetic hectorite having a composition of [Mg _5.34 Li _0.66 Si ₈ O ₂₀ (OH) ₄ ] Na ⁺ _0.66 (“Laponite XLG” manufactured by Rockwood Ltd.) ) Was used after vacuum drying. As the acrylic ester, 2-methoxyethyl acrylate (manufactured by Wako Pure Chemical Industries, Ltd .: hereinafter abbreviated as MEA) was purified by a known method and used after removing the polymerization inhibitor. As a polymerization initiator, potassium peroxodisulfate (manufactured by Kanto Chemical Co., Inc .: hereinafter abbreviated as KPS) was dissolved in deoxygenated ultrapure water 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 used for the polymerization was used after the contained oxygen was removed.

  Into a flat bottom glass container purged with nitrogen, 57.06 g of ultrapure water was added, and 0.96 g of Laponite XLG was added with stirring to prepare a colorless and transparent solution. To this, 8.58 g of MEA was added 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 transferred to two polystyrene containers (9 cm × 15 cm) with lids that had been allowed to stand in a nitrogen atmosphere in advance so as not to come into contact with oxygen, and then sealed and placed in a constant temperature water bath at 20 ° C. And then allowed to stand 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. Twenty hours after the start of polymerization, a white and uniform sheet-like water-containing polymer composite (C′-1) was released in water in a polystyrene container.

  In the obtained water-containing polymer composite (C′-1), non-uniform aggregation due to water-swellable clay mineral was not observed, and it was a uniform white solid (water content 372 mass%). A transparent polymer composite (C-1) was obtained by vacuum drying until the mass became constant at 100 ° C. The polymer polymerization yield calculated from the dry mass was 99.5% by mass. This polymer complex (C-1) was put in a sterilized polystyrene container, and 1 L of water for injection (manufactured by Otsuka Pharmaceutical) was added and sealed. The water for injection was changed 3 times every 24 hours to obtain a purified water-containing polymer complex. This water-containing polymer composite was again vacuum-dried to obtain a purified transparent polymer composite (C-1). These purification steps were all performed in a clean bench. The polymer composite (C-1) thus obtained was subjected to thermal mass analysis up to 600 ° C. (TG-DTA-220 manufactured by Seiko Denshi Co., Ltd .: air flow, temperature increase: 10 ° C./min), and clay mineral The content rate was determined. The clay mineral content (inorganic clay / polymer composite) was 9.5% by mass, and the calculated values from the polymerization solution composition almost coincided. Further, in the measurement of Fourier transform infrared absorption spectrum (FT-IR) by KBr method, characteristic peaks of poly (2-methoxyethyl acrylate) (PMEA) and water-swellable clay mineral were confirmed.

  After embedding the dried polymer composite (C-1) in an epoxy resin, an ultrathin section having a thickness of 50 μm was prepared and observed with a transmission electron microscope (using JEM-200CX manufactured by JEOL Ltd.) However, it was observed that the layered clay mineral having a thickness of 1 to several nm was finely and uniformly dispersed. Further, when X-ray diffraction measurement (using RX-7 manufactured by Rigaku Corporation: CuKα ray) of the dried polymer composite (C-1) was performed, no large peak was observed on the low angle side. From the above results, the solid obtained in this example is a polymer composite composed of a water-swellable clay mineral and PMEA, the clay mineral content is 9.5%, and the water-swellable clay mineral. Was found to be uniformly finely dispersed.

As a result of measuring the dynamic viscoelasticity of the dried polymer composite (C-1) using DMA-200 manufactured by Seiko Denshi Kogyo Co., Ltd. at a measurement frequency of 1 Hz and a temperature increase of 2 ° C./minute, the polymer composite ( C-1) exhibited a glass transition temperature (Tg) at about -34 ° C. Further, the elastic modulus was high at a temperature lower than Tg, and the elastic modulus of the rubber region was stable over a wide temperature range at a temperature higher than Tg. On the other hand, in the differential scanning calorimetry (DSC) measurement (using Perkin Elmer DSC-7), a glass transition temperature was observed at −34 ° C., and this temperature was a linear polymer dry prepared without using clay minerals. It was equivalent to the glass transition temperature of the product.
The dried polymer composite (C-1) is cut into a size of 1 cm × 5 cm, and a tensile test apparatus (“Tabletop Universal Testing Machine AGS-H” manufactured by Shimadzu Corporation) without slipping at the chuck portion. ), And a tensile test was performed at a distance between ratings = 20 mm and a tensile speed = 100 mm / min. As a result, the initial elastic modulus was 27 MPa, the breaking elongation was 1850%, and the breaking strength was 2.6 MPa. The residual strain of the polymer composite after the tensile test was about 100%, indicating rubbery elasticity and toughness. Due to such excellent mechanical properties, rubber-like stretchability without using any organic crosslinking agent, fine dispersion of water-swellable clay mineral, glass transition temperature of polymer, etc., this polymer composite In the body (C-1), it was concluded that the water-swellable clay mineral was finely dispersed in the organic polymer from the fine dispersion of the organic polymer and the water-swellable clay mineral, the glass transition temperature of the polymer, etc. It was.

Example 1
The polymer composite (C-1) obtained in Production Example 1 was cut to a size of 8 cm in diameter to obtain a cell culture substrate (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. All these operations 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 9.7 × 10 ⁷ after culturing. In addition, two separately prepared cell culture substrates (A-1 and A-2) are placed in a cell culture dish and allowed to stand at 37 ° C., and then kept at 37 ° C. in advance. The above medium was added and kept at 37 ° C. for 1 week. Thereafter, the cell culture substrate (A-1) was removed from the cell culture dish, and the cell culture substrate (A-2) was allowed to stand at 20 ° C. for 10 minutes and then removed. When the water content was determined by drying each cell culture substrate, the cell culture substrate (A-1) taken out at 37 ° C. was 8% of the dry weight. The cell culture substrate (A-2) taken out at 20 ° C. was 25%, and it was confirmed that the water content was increased by lowering the standing temperature.

(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. The CS-C medium that has been kept at 20 ° C. is taken out from the dish in which the other cell culture substrate (A) has been cultivated and the cell culture substrate (A) is cultured together. It was transferred to a tissue culture dish containing it. 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 deposits were 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 measure 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 7.2 × 10 ⁷ after culturing. Moreover, when the change of the water content was calculated | required similarly to Example 1, what was 8% at 37 degreeC was confirmed that it increased with 28% at 20 degreeC.

(Production Example 2)
Instead of using 8.58 g of MEA, 5.5 g of MEA (4.2 × 10 ⁻² mol) and N-isopropylacrylamide (NIPA: manufactured by Kojin Co., Ltd.) 1.18 g (1.0 × 10 ⁻² mol) A water-containing polymer composite (C′-2) was prepared in the same manner as in Production Example 1 except that the mixture of was used. The obtained water-containing polymer composite was a polymer composite composed of a uniform water-swellable clay mineral and copolymer, and non-uniform aggregation of the water-swellable clay mineral was not observed. The obtained water-containing polymer composite (C′-2) was vacuum dried at 100 ° C. in the same manner as in Production Example 1, and then further purified to obtain a transparent dried polymer composite (C-2). Obtained. When measured in the same manner as in Production Example 1, the clay mineral content in the polymer composite was 12.2%. Further, when a tensile test was performed on the dried polymer composite (C-2) in the same manner as in the preparation example, the elastic modulus was 4.8 MPa, the breaking strength was 4.5 MPa, the breaking elongation was 700% toughness, and there was flexibility. It was a polymer composite.

(Example 3 and Example 4)
The polymer composite (C-2) obtained in Production Example 2 was cut to a size of 8 cm in diameter to obtain a cell culture substrate (B). It was transferred into a dish for cell culture (“Falcon 3003” manufactured by Becton Dickinson Labware), covered, and allowed to stand at 37 ° C. All these operations were performed in a clean bench.

In the same manner as in Example 1 and Example 2, cells were cultured using the cell culture substrate (B). Human hepatoma HepG2 cell line (Example 3) and normal human skin fibroblasts (Example 4) were used as cells. When the surface of the cell culture substrate (B) cultured after 1 week after seeding each cell was observed with an optical microscope, the cells adhered to the cell culture substrate (B) and proliferated sufficiently. Was confirmed. When cells were detached from another cell culture substrate (B) that had been cultured in the same manner as in Example 1 and Example 2, either of Example 3 or Example 4 was used. Were able to separate the cells from the cell culture substrate (B). In addition, when the number of cells after culture was measured by the same method as in Example 1, it was 2.0 × 10 ⁶ cells (Example 3) and 2.5 × 10 ⁶ cells (Example 4) at the start of culture. It was confirmed that the number of cells increased to 1.0 × 10 ⁸ cells (Example 3) and 8.9 × 10 ⁷ cells (Example 4), respectively. Moreover, when the change of the moisture content was calculated | required similarly to Example 1, what was 10% at 37 degreeC in both cases of Example 3 and Example 4 increased to 60% at 20 degreeC. confirmed.

(Production Example 3)
A hydrous polymer composite (C′-3) was prepared in the same manner as in Production Example 1 except that 0.084 g of N, N′-methylenebisacrylamide (manufactured by Wako Pure Chemical Industries, Ltd.) was added after adding Laponite XLG. ) The obtained water-containing polymer composite (C′-3) was a uniform solid, and nonuniform aggregation such as a water-swellable clay mineral was not observed. The obtained water-containing polymer composite (C′-3) was vacuum-dried at 100 ° C. in the same manner as in Production Example 1, and then further purified to obtain a transparent and dry polymer composite (C-3). Got. A tensile test of this dried polymer composite (C-3) was conducted in the same manner as in Preparation Example 1. As a result, a high elastic modulus and elongation at break of elastic modulus of 3.8 MPa, breaking strength of 1.8 MPa, and breaking elongation of 400% were obtained. showed that.

(Example 5)
The polymer composite (C-3) obtained in Production Example 3 was cut into a size of 8 cm in diameter to obtain a cell culture substrate (C). It was transferred into a dish for cell culture (“Falcon 3003” manufactured by Becton Dickinson Labware), covered, and allowed to stand at 37 ° C. All these operations were performed in a clean bench.

In the same manner as in Example 2, normal human fibroblasts were cultured using the cell culture substrate (C). One week after seeding the cells, the surface of the cultured cell culture substrate (C) was observed with an optical microscope. The cells adhered to the cell culture substrate (C) and proliferated sufficiently. It was confirmed that it was. When cells were detached from another cell culture substrate (C) that had been cultured in the same manner as in Example 2, the cultured cells were separated from the cell culture substrate (D). I was able to. Further, when the number of cells after culture was measured by the same method as in Example 1, it was confirmed that the number of cells which was 2.5 × 10 ⁶ at the start of the culture increased to 9.2 × 10 ^7. It was done. Moreover, when the change of the water content was calculated | required similarly to Example 1, what was 8% at 37 degreeC was confirmed that it increased with 28% at 20 degreeC.

(Production Example 4)
The dried polymer composite (C-1) obtained in Preparation Example 1 is uniaxially stretched up to 30 times the original length in the same manner as the tensile test described in Preparation Example 1, and polymer composite A stretched body was prepared. The residual strain at the time of preparing the polymer composite stretched product was 110%.

(Example 6)
The polymer composite stretched product obtained in Production Example 4 was cut into a size of 8 cm in diameter to obtain a cell culture substrate (D). It was transferred into a dish for cell culture (“Falcon 3003” manufactured by Becton Dickinson Labware), covered, and allowed to stand at 37 ° C. All these operations were performed in a clean bench.

In the same manner as in Example 2, normal human fibroblasts were cultured using the cell culture substrate (D). One week after seeding the cells, the surface of the cultured cell culture substrate (D) was observed with an optical microscope. The cells adhered to the cell culture substrate (D) and proliferated sufficiently. It was confirmed that it was. When the cells were detached from the other cell culture substrate (D) that had been cultured in the same manner as in Example 2, the cultured cells were separated from the cell culture substrate (D). I was able to. Moreover, when the number of each cell after culture | cultivation was measured by the method similar to Example 1, it confirmed that the cell number which was 2.5 * 10 < ⁶ > at the time of culture | cultivation increased to 8.0 * 10 < ⁷ >. It was done. Moreover, when the change of the water content was calculated | required similarly to Example 1, what was 8% at 37 degreeC was confirmed that it increased with 29% at 20 degreeC.

(Comparative Example 1)
Polymerization was carried out at 20 ° C. for 20 hours in the same manner as in Production Example 1 except that no water-swellable clay mineral was used, and a cloudy solid polymer was obtained. This polymer was soft but very fragile, and had strong adhesion to a polystyrene container, and when it was attempted to be removed from the polystyrene container, it was difficult to peel off, and it was immediately destroyed upon peeling. Further, the polymer was further finely disrupted and purified in the same manner as in Example 1, then transferred to a cell culture dish and cultured, and the cells did not adhere to the polymer at all. Could not be cultured.

(Comparative Example 2)
20 ° C. at 20 ° C. in the same manner as in Preparation Example 1 except that no water-swelling clay mineral is used, MEA is added, and N, N′-methylenebisacrylamide is added as an organic crosslinking agent at 3 mol% of MEA. Polymerization with time was performed to obtain an organic crosslinked product of PMEA. This organic cross-linked product of PMEA is a cloudy and very brittle gel, and has strong adhesion to a polystyrene container, and when it was attempted to be removed from the polystyrene container, it was difficult to peel off, and it was destroyed immediately after peeling. . Furthermore, after refine | purifying by the method similar to Example 1 using the finely destroyed organic cross-linked product of PMEA, it was transferred to a dish for cell culture, and the cells were cultured. The cells were classified into the organic cross-linked product of PMEA. The cells did not adhere at all, and the cells could not be cultured.

Claims (8)

A polymer composite (C) in which a water-swellable clay mineral (B) is finely dispersed in a polymer (A) using a water-soluble (meth) acrylic acid ester (a) as a monomer is used. A cell culture substrate characterized by the above. The cell according to claim 1, wherein the polymer (A) is a polymer using one or more (b) selected from (meth) acrylamide and N-substituted (meth) acrylamide as a monomer. Culture substrate. The polymer (A) is a molar ratio of the water-soluble (meth) acrylic acid ester (a) and one or more (b) selected from the (meth) acrylamide and N-substituted (meth) acrylamide. The cell culture substrate according to claim 2, which is a polymer reacted so that ((b) / (a)) is 1 or less. The mass ratio ((B) / (A)) of the water-swellable clay mineral (B) and the polymer (A) in the polymer composite (C) is 0.003 to 3. The cell culture substrate according to any one of 2 and 3. The water-soluble (meth) acrylic acid ester (a) is at least one monomer selected from methoxyethyl acrylate, ethoxyethyl acrylate, methoxyethyl methacrylate and ethoxyethyl methacrylate. The cell culture substrate according to any one of the above. The cell culture substrate according to any one of claims 1, 2, 3, 4 and 5, wherein a stretched product of the polymer composite (C) is used. The cell culture substrate according to any one of claims 1, 2, 3, 4, 5 and 6, wherein the polymer complex (C) reversibly changes cell adhesion and cell non-adhesiveness due to a temperature change. . The cell culture substrate according to any one of claims 1 to 7, wherein the cell culture substrate is cultured under temperature conditions where the cell culture substrate exhibits cell adhesion, and then the temperature of the cell culture substrate is lowered. A cell culture method comprising separating the cultured cells from the cell culture substrate by setting the cell culture substrate to a temperature at which the cell culture substrate exhibits cell non-adhesiveness.