JP2012231788A - Cultural base material of bone marrow-derived cell, culture method, and culture bone marrow-derived cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cultural base material of a bone marrow-derived cell which controls the adhesion between a surface of the cultural base material and the bone marrow-derived cell, and has high culture (proliferation) ability and high peelability by a low temperature treatment after culturing, a culture method of the bone marrow-derived cell, and the culture bone marrow-derived cell.SOLUTION: The cell culture base material for culturing the bone marrow-derived cell contains a polymer (A) of a methoxyethyl acrylate (a), and one or more inorganic materials (C) selected from a water-swellable clay mineral and silica.

Description

  The present invention is a cell culture substrate for culturing bone marrow-derived cells, which contains a water-soluble (meth) acrylic acid ester (a) polymer (A) and an inorganic material (C). The present invention relates to a material, a method for culturing bone marrow-derived cells, and cultured bone marrow-derived cells obtained thereby.

  Conventionally, plastic (for example, polystyrene) containers have been mainly 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. The culture properties of these cell culture containers vary depending on the cell type. For example, in the case of bone marrow-derived mesenchymal cells, the growth of the cells is very slow. Especially, when the serum is not put in the medium, the cells hardly grow. On the other hand, for cells that can proliferate, such as fibroblasts, cultured (proliferated) cells adhere to the surface of the container, and in order to isolate and recover the cells, protein hydrolases such as trypsin, chemicals, It was necessary to peel from the container surface using physical manipulation. The operation of peeling cells by such enzymes, chemicals, and physical operations not only cuts the binding part of the cell and the base material, but also cuts the bond between cells, so that the cell is proliferating. It could not be removed in the form (for example, in the form of a sheet) or the basal protein of the cell was damaged, which could cause unexpected cell property changes.

  In recent years, the surface of a cell culture vessel is coated with a polymer having a low critical solution temperature (LCST) such as poly N-substituted (meth) acrylamide, and the polymer is in a hydrophobic state at the cell culture temperature. The cells adhere to the polymer, and after culturing, the polymer is treated at a low temperature to make it hydrophilic, thereby reducing the adhesion between the cell and the polymer, and the cell can be used without hydrolase or chemicals. Techniques for peeling cells from a material into a sheet have been reported (see, for example, Patent Documents 1 and 2 and Non-Patent Document 1).

  However, when such a culture substrate is sterilized by radiation (for example, γ-rays), the temperature responsiveness of the polymer having LCST is greatly reduced, and the original cells are not easily detached. .

  On the other hand, an organic-inorganic composite particle (X) in which a polymer (P) of a monomer containing a (meth) acrylic acid ester monomer (a) and a water-swellable clay mineral (B) form a three-dimensional network. A cell culture substrate having a dry film of an organic-inorganic composite (X) formed by drying a dispersion of the above on its surface is disclosed (for example, see Patent Document 3).

  However, the above-mentioned conventional documents do not disclose specific means relating to a method for culturing and detaching bone marrow-derived cells.

JP 6-104061 JP-A-5-192138 Japanese Patent No. 4430124

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

  The problem to be solved by the present invention is to control the adhesion between the surface of the culture substrate and the bone marrow-derived cells, and to culture bone marrow-derived cells having high culture (proliferation) properties and high detachability by low-temperature treatment after the culture. To provide a base material, a method for culturing bone marrow-derived cells, and cultured bone marrow-derived cells.

  As a result of diligent research to solve the above-mentioned problems, the present inventors have found that a cell culture substrate containing a polymer (A) of a water-soluble (meth) acrylic acid ester (a) and an inorganic material (C), And a water-soluble (meth) acrylic acid ester (a) polymer (A), an N-substituted (meth) acrylamide polymer (B), and an inorganic material (C). Then, bone marrow-derived cells have excellent growth properties, and when using the culture substrate, bone marrow-derived cells can be cultured while maintaining low adhesion between the substrate surface and the cells, Furthermore, the inventors have found a method for culturing bone marrow-derived cells that can be easily detached from the substrate surface by low-temperature treatment after culturing, and have completed the present invention.

That is, the present invention is a cell culture substrate for culturing bone marrow-derived cells,
Bone marrow characterized by containing water-soluble (meth) acrylic acid ester (a) polymer (A) and one or more inorganic materials (C) selected from water-swellable clay minerals and silica A cell culture substrate for cell culture is provided.

  The present invention further provides the above-mentioned cell culture substrate for bone marrow-derived cell culture, which contains a polymer (B) of N-substituted (meth) acrylamide.

The present invention also provides a cell comprising a polymer (A) of a water-soluble (meth) acrylic acid ester (a), and one or more inorganic materials (C) selected from water-swellable clay minerals and silica. A method for culturing bone marrow-derived cells, comprising culturing bone marrow-derived cells on a culture substrate, and then peeling the cultured cells from the substrate,
Provided is a method for culturing bone marrow-derived cells, wherein the mass ratio ((C) / (A)) between the polymer (A) and the inorganic material (C) is in the range of 0.03 to 0.7.

  The present invention also provides a method for culturing bone marrow-derived cells, wherein the cell culture substrate further contains a polymer (B) of N-substituted (meth) acrylamide.

  In this method, it is preferable that the mass ratio ((B) / (A)) between the polymer (A) and the polymer (B) is in the range of 0.01 to 0.7.

  The present invention also provides bone marrow-derived cells produced by the culture method.

  The greatest feature of the cell culture substrate of the present invention is that the constituent part of the inorganic material (C) is responsible for the growth of bone marrow-derived cells, and the polymer (A) of the water-soluble (meth) acrylate (a) is a cell. Furthermore, the polymer (B) of N-substituted (meth) acrylamide is responsible for peeling of bone marrow-derived cells due to temperature change. Each part can be independently adjusted according to the state of proliferation and detachment of bone marrow-derived cells. For example, at the time of culture (37 ° C.), bone marrow-derived cells proliferate with high proliferation ability while maintaining weak adhesion to the culture surface, and after completion of the culture, the temperature is lowered to 30 ° C. or less (for example, room temperature). The polymer (B) part exhibits higher hydrophilicity, the adhesion between the culture surface and the cells is further weakened, and the cells can be easily detached. In addition, when the adhesion between the water-soluble (meth) acrylic acid ester (a) polymer (A) and the cells is sufficiently weak, the medium can be treated at a low temperature without blending the polymer (B). The adhesiveness between the two is further weakened, and bone marrow-derived cells can be easily detached and collected (see Examples 1 and 9). Bone marrow-derived cells as used herein refer to cells derived from bone marrow and immortalized bone marrow derived from humans or other animals.

  Another feature of the cell culture substrate of the present invention is that it has high proliferative ability to bone marrow-derived cells without using animal-derived serum during culture. ), The cultured cells can be easily detached from the surface of the substrate only by low-temperature treatment, and the cells can be recovered with high yield without damaging the cells.

  The polymer (A) interacts and bonds with the inorganic material (C) mainly by ionic bonds or hydrogen bonds. This bonding force is strong, and the polymer and the inorganic material (C) cannot be easily separated.

  The method for culturing bone marrow-derived cells of the present invention has a high proliferation ability while maintaining a weak adhesive force between the cells and the culture surface, and the cultured cells are treated without using a drug (trypsin or the like). Can be easily peeled and recovered from the surface of the culture substrate.

  In addition, the culture substrate used in the present culture method has a feature that radiation sterilization such as γ rays and electron beams is possible.

  By using the monomer (a), the initial adhesion of bone marrow-derived cells can be kept low, and a cell culture substrate having good cell growth and exfoliation properties can be obtained. In addition, the water-soluble (meth) acrylic acid ester (a) is produced when these cell culture substrates are laminated on the surface of a support such as a plastic substrate such as polystyrene, and the adhesion between them is strong. Has the feature that can be easily.

  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.

  In addition, as necessary, other copolymerization monomers, such as acrylic monomers having an anion group such as a sulfone group or a carboxyl group, quaternary ammonium groups, etc., to the extent that does not affect the culture properties and physical properties. An acrylic monomer having a cationic group, an acrylic monomer having a zwitterionic group having a quaternary ammonium group and a phosphate group, an acrylic monomer having an amino acid residue having a carboxyl group and an amino group, and an acrylic having a sugar residue -Based monomers, acrylic monomers having hydroxyl groups, amphiphilic acrylic monomers having a hydrophilic chain such as polyethylene glycol and a hydrophobic group such as nonylphenyl group, N-substituted (meth) acrylamide derivatives, N , N-disubstituted (meth) acrylamide derivatives, N, N′-methylenebi It can be used in combination, such as acrylamide.

  More specifically, the N-substituted (meth) acrylamide derivative is an alkyl (meth) acrylamide having an alkyl group having 1 or more carbon atoms, such as N-methylacrylamide, N-ethylacrylamide, 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-acryloylmethylhomopiperazine, N-acryloyl Etc. chill piperazine or N- methyl methacrylamide.

  The inorganic material (C) used in the present invention is one or more inorganic materials selected from water-swellable clay minerals and silica. Examples of water-swellable clay minerals include water-swellable clay minerals that can be peeled in layers, preferably clay minerals that can swell and uniformly disperse in water or a mixed solution of water and an organic solvent, particularly preferably water. An inorganic clay mineral that can be uniformly dispersed at a molecular level (single layer) or at a level close thereto is used. Specific examples include water-swellable hectorite containing sodium as an interlayer ion, water-swellable montmorillonite, water-swellable saponite, and water-swellable synthetic mica. You may mix and use these clay minerals.

Examples of the silica (SiO ₂ ) used in the present invention include colloidal silica, preferably colloidal silica that can be uniformly dispersed in an aqueous solution and has a particle size of 10 nm to 500 nm, and particularly preferably a colloidal particle having a particle size of 10 to 50 nm. Silica is used.

  In the cell culture substrate of the present invention, the mass ratio of the water-soluble (meth) acrylic acid ester (a) polymer (A) to the N-substituted (meth) acrylamide polymer (B) ((B) / ( A)) is preferably 0.01 to 0.7, more preferably 0.03 to 0.5, and particularly preferably 0.1 to 0.3. When the mass ratio ((B) / (A)) is within this range, it is preferable because it can have both good culturing properties and high peelability for bone marrow-derived cells.

  In the cell culture substrate of the present invention, the mass ratio ((C) / (A)) of the polymer (A) and the inorganic material (C) is preferably 0.03 to 0.7, 0.05 to 0.3 is more preferable, and 0.07 to 0.1 is particularly preferable. When the mass ratio ((C) / (A)) is within this range, it is preferable because it can have both good culturing properties and high peelability for bone marrow-derived cells.

Furthermore, the bone marrow-derived cells cultured on the cell culture substrate of the present invention can be allowed to stand by lowering the medium temperature to 30 ° C. or lower, and the cells can be naturally detached, or the medium temperature can be reduced to 30 ° C. or lower. After lowering, the cells can be easily detached by using the methods listed below.
(1) A method of detaching cells by gently shaking the medium in the culture vessel.
(2) A method of peeling by a pipetting operation in which a medium is sucked and taken out with a pipette.
(3) A method in which a glass rod, pipette tip, rubber spatula or the like is inserted between the cell sheet and the cell culture substrate, and the cell sheet is lifted off,
(4) There is a method of peeling while sucking with a straw-like device.

  Since bone marrow-derived cells produced by the culture method of the present invention do not use a proteolytic enzyme such as trypsin, the basal proteins of the cells are not damaged, are in a state closer to the cell morphology in vivo, and have high cell activity. It is considered that the post-transplant fixation and curability are high.

  In the method for producing a culture substrate of the present invention, a water-soluble (meth) acrylic acid ester (a) polymer (A) and an N-substituted (meth) acrylamide polymer (B) interact with an inorganic material (C). It will not specifically limit if it acts and can form an organic inorganic composite. For example, an aqueous medium (E) in which a water-soluble (meth) acrylic acid ester (a), the inorganic material (C), and a polymerization initiator (D) are mixed is applied to a support, and the water-soluble (meth) The manufacturing method which forms the thin layer of the composite_body | complex (X) of a polymer (A) and the said inorganic material (C) by polymerizing acrylic ester (a) is mentioned.

The aqueous medium (E) used in the production method can include the monomer (a), the inorganic material (C), and the like, and is not particularly limited as long as an organic-inorganic composite having good physical properties can be obtained by polymerization. For example, it may be water or an aqueous solution containing a solvent miscible with water and / or other compounds, and further contains antiseptics, antibacterial agents, antibiotics, coloring agents, fragrances, enzymes as necessary. , Proteins, collagen, saccharides, amino acids, peptides, DNAs, salts, water-soluble organic solvents, surfactants, polymer compounds, leveling agents, and the like.
As the polymerization initiator (D) used in the present invention, a known radical polymerization initiator can be appropriately selected and used. Preferably, those having water solubility or 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 Fe ²⁺ and hydrogen peroxide is exemplified.

  As the catalyst, tertiary amine compounds such as N, N, N ′, N′-tetramethylethylenediamine are preferably used. However, the catalyst is not necessarily used. The polymerization temperature is, for example, 0 ° C. to 100 ° C. according to the type of polymerization catalyst or initiator. The polymerization time can also be carried out for several tens of seconds to several tens of hours.

  On the other hand, the photopolymerization initiator is less susceptible to oxygen inhibition and has a high polymerization rate, and therefore is suitably used as the polymerization initiator (D). Specifically, acetophenones such as p-tert-butyltrichloroacetophenone, benzophenones such as 4,4′-bisdimethylaminobenzophenone, ketones such as 2-methylthioxanthone, benzoin ethers such as benzoin methyl ether, hydroxy Examples include α-hydroxy ketones such as cyclohexyl phenyl ketone, phenyl glyoxylates such as methyl benzoyl formate, and metallocenes.

As the light used in this step, electron beam, γ-ray, X-ray, ultraviolet ray, visible light, etc. can be used. Among them, the apparatus and handling are easy and the viewpoint of not causing crosslinking simultaneously with polymerization of the monomer (b). It is preferable to use ultraviolet rays. The intensity of the irradiated ultraviolet light is preferably 10 to 500 mW / cm ² and the irradiation time is generally about 0.1 to 200 seconds. In radical polymerization by normal heating, oxygen works as an inhibitor of polymerization. However, in the present invention, it is not always necessary to prepare a solution in an atmosphere in which oxygen is blocked and to perform polymerization by ultraviolet irradiation, and these are performed in an air atmosphere. It is possible. However, it may be desirable that the polymerization rate can be further increased by performing ultraviolet irradiation in an inert gas atmosphere.

Moreover, as a second production example of the culture substrate of the present invention, the concentration of the inorganic material (C) in the aqueous medium (E) falls within the range represented by the following formula (1) or formula (2). Thus, after the monomer (a), the inorganic material (C), and the polymerization initiator (D) are mixed in an aqueous medium (E), the monomer (a) is polymerized to polymer (A) and A first step of producing a dispersion (L) of a complex (X) with the inorganic material (C);
A method for producing a cell culture substrate comprising sequentially performing the second step of forming the thin layer of the complex (X) by applying the dispersion (L) to a substrate and then drying the dispersion. It is done.
Formula (1) When Ra <0.19
Concentration (% by mass) of inorganic material (C) <12.4Ra + 0.05
Formula (2) When Ra ≧ 0.19
Concentration (% by mass) of inorganic material (C) <0.87Ra + 2.17
(In the formula, the concentration (mass%) of the inorganic material (C) is a value obtained by dividing the mass of the inorganic material (C) by the total mass of the aqueous medium (E) and the inorganic material (C) and multiplying by 100, Ra Is a mass ratio ((C) / (A)) between the inorganic material (C) and the polymer (A).)

  When the concentration (mass%) of the inorganic material (C) with respect to the aqueous medium is within the range represented by the formula (1) or the formula (2), a good dispersion (L) of the composite (X) is obtained. The coating on the support is easy, and a smooth and uniform thin coating is obtained, which is preferable.

  The dispersion (L) produced by the production method of the present invention may be used as it is, or may be used after undergoing a purification step such as washing with water. Further, a leveling agent, a surfactant, a peptide, a protein, collagen, an amino acid, a polymer compound or the like may be added to the dispersion (L).

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

(Example 1) Example of culture equipment (not including polymer (B)) comprising polymer (A) and inorganic material (C).
[Preparation of reaction solution containing water-soluble (meth) acrylic acid ester (a), inorganic material (C), aqueous medium (E)]
A reaction solution (3.2 g of methoxyethyl acrylate, 0.2 g of a water-swellable clay mineral Laponite XLG (manufactured by Rockwood Additives Ltd.) as an inorganic material (C) and 100 g of water as an aqueous medium (E) are uniformly mixed. 1) was prepared.

[Preparation of a solution in which the polymerization initiator (D) is dissolved in the solvent (F)]
A solution (2) is prepared by uniformly mixing 9.8 g of methanol as a solvent (F) and 0.2 g of 1-hydroxycyclohexyl phenyl ketone “Irgacure 184” (manufactured by Ciba Geigy) as a polymerization initiator (D). did.

[Preparation of dispersion (L) of complex (X) (first step)]
250 μl of the solution (2) is added to the total amount of the reaction solution (1) and dispersed uniformly, and then irradiated with ultraviolet rays having an ultraviolet intensity of 40 mW / cm ² at 365 nm for 180 seconds, and a milky white complex (X) dispersion liquid (L1) was produced.

  Ra = 0.06 in this reaction system, concentration (mass%) of inorganic material (C) = 0.20 (%) <12.4Ra + 0.05 = 0.79

[Preparation of culture substrate (thin layer of complex (X)) (second step)]
A dispersion (L1) of the above complex (X) is placed in a polystyrene petri dish (IWAKI tissue culture dish 3000-035) having a diameter of 35 mm, and the dispersion is thinly applied to the surface of the petri dish at 3000 revolutions using a spin coater. After coating, the cell dish is dried for 10 minutes in a hot air dryer at 80 ° C., and then the petri dish is washed with sterilized water, and then the petri dish is dried at 40 ° C. for 5 hours in a sterile bag to obtain the cell culture substrate 1. It was.

[Culture and recovery of bone marrow-derived cells]
Mononuclear cells collected from GFP transgenic mouse bone marrow after sterilizing the obtained cell culture substrate 1 with an electron beam with an irradiation dose of 10 kGy (Japan Irradiation Service Co., Ltd.) and adding an appropriate amount of medium DMEM (containing 10% serum) The cells were seeded at 2.0 × 10 ⁶ spheres / Dish (3.38 cm ² ) and cultured in 5% carbon dioxide at 37 ° C. for 6 days. Next, remove the culture medium in the dish and the suspended mononuclear cells, add a cold culture medium that has been chilled in the refrigerator in advance, leave it for 10 minutes, and then pipette up and down about 10 times with a pipette. As a result, it was observed that most bone marrow-derived cells were detached from the surface of the culture substrate 1. Bone marrow-derived cells that were naturally detached were collected, and further, using D-PBS and 0.25% Trypsin-EDTA, the bone marrow-derived cells remaining in the dish were separated and collected, and the number of each collected cells was measured. The number of cells spontaneously detached and collected by the low-temperature treatment was 8.6 × 10 ⁴ , and the number of cells collected by the Trypsin treatment was 1.8 × 10 ⁴ . When the cell recovery rate by low-temperature treatment was determined by the following formula (5), the cell recovery rate was about 82%.
Formula (5) Cell recovery rate (%) = {number of cells recovered by low-temperature treatment / (number of cells recovered by low-temperature treatment + number of cells recovered by trypsin treatment)} × 100
In addition, the total number of bone marrow-derived cells recovered from the culture substrate 1 (10.4 × 10 ⁴ ) was obtained when an uncoated petri dish (IWAKI tissue culture 3000-035) was used (2.5 × 10 ^four) was about 4.2 times.

  In addition, it was confirmed that the bone marrow-derived cells collected by the natural detachment by the low-temperature treatment and the trypsin treatment each had a normal cell morphology under a microscope.

  From this example, the culture substrate (not including the polymer (B)) composed of the polymer (A) and the inorganic material (C) has a higher cell culture property than a normal tissue culture dish, It can be understood that the adhesion between the culture substrate surface and the cells is low, and the cells can be easily detached only by low-temperature treatment.

(Example 2) Example of culture substrate composed of polymers (A), (B) and inorganic material (C).
[Preparation of aqueous solution of polymer (B) of N-substituted (meth) acrylamide]
After mixing 1.7 g of N-isopropylacrylamide (manufactured by Kojin Co., Ltd.), 10 g of water, and 140 μl of the solution (2), the glass container containing the solution is cooled (about 10 ° C.) and ultraviolet light at 365 nm. After irradiating ultraviolet rays having an intensity of 40 mW / cm ² for 180 seconds to polymerize N-isopropylacrylamide, 5 g of water was further added to prepare an aqueous solution of polymer (B) (PNIPA2). The viscosity of this solution was measured using a DIGITAL VISCOMATE viscometer (MODEL VM-100A, manufactured by Yamaichi Electronics Co., Ltd.), and the viscosity was 368 mPa · s. The solution temperature at the time of measurement was 24.2 ° C.

Moreover, as a result of measuring with Shodex GPC System-21 apparatus (made by Showa Denko KK), the weight average molecular weight Mw of this poly N-isopropylacrylamide was 3.40 * 10 < ⁶ >. An N, N-dimethylformamide (DMF) solution containing 10 mmol / L LiBr was used as a solvent for the measurement. STANDARD SH-75 and SM-105 kit (manufactured by Showa Denko KK) were used as polystyrene standard substances used for the calculation of molecular weight.

[Preparation of reaction solution containing water-soluble (meth) acrylic acid ester (a), inorganic material (C), aqueous medium (E)]
A reaction solution (3.2 g of methoxyethyl acrylate, 0.2 g of a water-swellable clay mineral Laponite XLG (manufactured by Rockwood Additives Ltd.) as an inorganic material (C) and 100 g of water as an aqueous medium (E) are uniformly mixed. 2) was prepared.

[Preparation of dispersion (L) of complex (X) (first step)]
Disperse the milky white complex (X) by adding 250 μl of the above solution (2) to the total amount of the reaction solution (2) and uniformly dispersing it, and then irradiating it with ultraviolet light having an ultraviolet intensity of 365 m at 40 mW / cm ² for 180 seconds. A liquid (L2) was produced.

  Ra = 0.06 in this reaction system, concentration (mass%) of inorganic material (C) = 0.20 (%) <12.4Ra + 0.05 = 0.79

[Preparation of culture substrate (thin layer of complex (X)) (second step)]
7 g of an aqueous solution of N-isopropylacrylamide polymer (B) (PNIPA2) was added to the total amount of the dispersion (L2) and mixed uniformly, and then a polystyrene petri dish (IWAKI tissue culture 3000-035 with a diameter of 35 mm) was added. ), Apply the dispersion to the surface of the petri dish at 3000 rpm using a spin coater, dry it in a hot air drier at 80 ° C. for 10 minutes, and then wash the petri dish with sterilized water. The petri dish was dried in a bag at 40 ° C. for 5 hours to obtain a cell culture substrate 2.

[Culture and recovery of bone marrow-derived cells]
Mononuclear cells collected from the GFP transgenic mouse bone marrow after sterilizing the obtained cell culture substrate 2 with an electron beam with an irradiation dose of 10 kGy (Japan Irradiation Service Co., Ltd.) and then adding an appropriate amount of medium DMEM (containing 10% serum) The cells were seeded at 2.0 × 10 ⁶ spheres / Dish (3.38 cm ² ) and cultured in 5% carbon dioxide at 37 ° C. for 6 days. Next, remove the culture medium in the dish and the suspended mononuclear cells, add a cold culture medium that has been chilled in the refrigerator in advance, leave it for 10 minutes, and then pipette up and down about 10 times with a pipette. As a result, it was observed that most bone marrow-derived cells were detached from the surface of the culture substrate 2. Bone marrow-derived cells that were naturally detached were collected, and further, using D-PBS and 0.25% Trypsin-EDTA, the bone marrow-derived cells remaining in the dish were separated and collected, and the number of each collected cells was measured. The number of cells spontaneously detached and collected by low-temperature treatment was 12.0 × 10 ⁴ , and the number of cells collected by Trypsin treatment was 0.7 × 10 ⁴ . When the cell recovery rate by low-temperature treatment was determined by the following formula (5), the cell recovery rate was 94%.
Formula (5) Cell recovery rate (%) = {number of cells recovered by low-temperature treatment / (number of cells recovered by low-temperature treatment + number of cells recovered by trypsin treatment)} × 100
When the total number of bone marrow-derived cells recovered from the culture substrate 2 (12.7 × 10 ⁴ cells) was obtained using an uncoated petri dish (IWAKI tissue culture 3000-035) (Comparative Example 1) ( 2.5 times 10 ⁴ ).

  In addition, it was confirmed that the bone marrow-derived cells collected by the natural detachment by the low-temperature treatment and the trypsin treatment each had a normal cell morphology under a microscope.

  From this example, when the polymer (B) having further temperature responsiveness is blended with the polymer (A) and the inorganic material (C), it has good culturing property and at the same time, the cell recovery rate by low-temperature treatment. Can be understood to be even higher.

(Example 3)
[Preparation of reaction solution containing water-soluble (meth) acrylic acid ester (a), inorganic material (C), aqueous medium (E)]
3.2 g of methoxyethyl acrylate, 1 g of colloidal silica 20% by mass as inorganic material (C) (trade name Snowtex 20, manufactured by Nissan Chemical Industries, Ltd.) 1 g (SiO ₂ = 0.2 g), water as aqueous medium (E) 100 g was uniformly mixed to prepare a reaction solution (3).

[Preparation of dispersion (L) of complex (X) (first step)]
Disperse the milky white complex (X) by adding 250 μl of the solution (2) to the total amount of the reaction solution (3) and uniformly dispersing it, and then irradiating it with ultraviolet light having an ultraviolet intensity at 365 nm of 40 mW / cm ² for 180 seconds. A liquid (L3) was produced.

  Ra = 0.06 in this reaction system, concentration (mass%) of inorganic material (C) = 0.20 (%) <12.4Ra + 0.05 = 0.79

[Preparation of culture substrate (thin layer of complex (X)) (second step)]
1 g of an aqueous solution of N-isopropylacrylamide polymer (B) (PNIPA2) was added to the total amount of the dispersion (L3) and mixed uniformly, and then a polystyrene petri dish (IWAKI tissue culture 3000-035 with a diameter of 35 mm) was added. ), Apply the dispersion to the surface of the petri dish at 3000 rpm using a spin coater, dry it in a hot air drier at 80 ° C. for 10 minutes, and then wash the petri dish with sterilized water. The petri dish was dried in a bag at 40 ° C. for 5 hours to obtain a cell culture substrate 3.

[Culture and recovery of bone marrow-derived cells]
Mononuclear cells collected from GFP transgenic mouse bone marrow after sterilizing the obtained cell culture substrate 3 with an electron beam with an irradiation dose of 10 kGy (Japan Irradiation Service Co., Ltd.) and adding an appropriate amount of medium DMEM (containing 10% serum) The cells were seeded at 2.0 × 10 ⁶ spheres / Dish (9.8 cm ² ) and cultured in 5% carbon dioxide at 37 ° C. for 6 days. Next, remove the culture medium in the dish and the suspended mononuclear cells, add a cold culture medium that has been chilled in the refrigerator in advance, leave it for 10 minutes, and then pipette up and down about 10 times with a pipette. As a result, it was observed that most bone marrow-derived cells were detached from the surface of the culture substrate 3. Bone marrow-derived cells that were naturally detached were collected, and further, using D-PBS and 0.25% Trypsin-EDTA, the bone marrow-derived cells remaining in the dish were separated and collected, and the number of each collected cells was measured. The number of cells spontaneously detached and recovered by low-temperature treatment was 6.2 × 10 ⁴ , and the number of cells recovered by Trypsin treatment was 1.1 × 10 ⁴ . When the cell recovery rate by low-temperature treatment was determined by the following formula (5), the cell recovery rate was 85%.
Formula (5) Cell recovery rate (%) = {number of cells recovered by low-temperature treatment / (number of cells recovered by low-temperature treatment + number of cells recovered by trypsin treatment)} × 100
Further, the total number of bone marrow-derived cells recovered from the culture substrate 3 (7.3 × 10 ⁴ ) was obtained when using an uncoated petri dish (IWAKI tissue culture 3000-035) (2.5 × 10 6). About ⁴ times).

  In addition, it was confirmed that the bone marrow-derived cells collected by the natural detachment by the low-temperature treatment and the trypsin treatment each had a normal cell morphology under a microscope.

Further, the culture was performed for 18 days in the same manner. Next, remove the culture medium in the dish and the suspended mononuclear cells, add a cold culture medium that has been chilled in the refrigerator in advance, leave it for 10 minutes, and then pipette up and down about 10 times with a pipette. As a result, it was observed that most bone marrow-derived cells were detached from the surface of the culture substrate 3. Bone marrow-derived cells that were naturally detached were collected, and further, using D-PBS and 0.25% Trypsin-EDTA, the bone marrow-derived cells remaining in the dish were separated and collected, and the number of each collected cells was measured. The number of cells spontaneously detached and collected by low-temperature treatment was 7.0 × 10 ⁵ , and the number of cells collected by Trypsin treatment was 1.2 × 10 ⁴ . When the cell recovery rate by low-temperature treatment was determined by the above formula (5), the cell recovery rate was 85%.

In addition, the total number of bone marrow-derived cells recovered from the culture substrate 3 (7.0 × 10 ⁵ ) was obtained when an uncoated petri dish (IWAKI tissue culture 3000-035) was used (2.8 × 10 5). About ⁴ times).

  From this example, it can be understood that the culture substrate 3 in the case where silica is used as the inorganic material (C) has good culturing properties and a high recovery rate due to natural peeling.

(Example 4) Example of serum-free culture using the culture substrate 3 [Culture and recovery of bone marrow-derived cells]
The obtained cell culture substrate 3 was sterilized with an electron beam with an irradiation dose of 10 kGy (Japan Irradiation Service Co., Ltd.), and then used as a serum-free medium with appropriate amounts of STEMPRO LIPOMAX SUPPLEMENT (Life Technologies Japan A1085001) and GLUTAMAX I (Life Technologies Japan). 35050061) containing StemPro MSC SFM XenoFree (Life Technologies Japan A10675501) and seeding 7.0 × 10 ⁶ mononuclear cells collected from GFP transgenic mouse bone marrow with Dish (9.8 cm ² ), Culturing was performed at 37 ° C. in 5% carbon dioxide for 7 days. Next, remove the culture medium in the dish and the suspended mononuclear cells, add a cold culture medium that has been chilled in the refrigerator in advance, leave it for 10 minutes, and then pipette up and down about 10 times with a pipette. As a result, it was observed that most bone marrow-derived cells were detached from the surface of the culture substrate 3. Bone marrow-derived cells that were naturally detached were collected, and further, using D-PBS and 0.25% Trypsin-EDTA, the bone marrow-derived cells remaining in the dish were separated and collected, and the number of each collected cells was measured. The number of cells that were naturally detached and recovered by the low-temperature treatment was 3.9 × 10 ⁴ , and the number of cells that were recovered by the Trypsin treatment was 9 × 10 ³ . When the cell recovery rate by low-temperature treatment was determined by the following formula (5), the cell recovery rate was 81%.
Formula (5) Cell recovery rate (%) = {number of cells recovered by low-temperature treatment / (number of cells recovered by low-temperature treatment + number of cells recovered by trypsin treatment)} × 100
On the other hand, when cultured in a similar serum-free medium using an uncoated petri dish (IWAKI tissue culture 3000-035) (Comparative Example 2), bone marrow-derived cells were hardly cultured.

  In addition, it was confirmed that the bone marrow-derived cells collected by the natural detachment by the low-temperature treatment and the trypsin treatment each had a normal cell morphology under a microscope.

  From this example, it can be understood that the culture substrate 3 has good culturing properties and a high recovery rate due to spontaneous peeling even in completely serum-free culture.

(Example 5) The example which mix | blended the other high molecular compound.
[Preparation of reaction solution containing water-soluble (meth) acrylic acid ester (a), inorganic material (C), aqueous medium (E)]
A reaction solution (3.2 g of methoxyethyl acrylate, 0.4 g of water-swellable clay mineral Laponite XLG (manufactured by Rockwood Additives Ltd.) as an inorganic material (C) and 100 g of water as an aqueous medium (E) are uniformly mixed. 6) was prepared.

[Preparation of dispersion (L) of complex (X) (first step)]
Disperse the milky white complex (X) by adding 250 μl of the solution (2) to the total amount of the reaction solution (6) and uniformly dispersing it, and then irradiating it with ultraviolet light having an ultraviolet intensity at 365 nm of 40 mW / cm ² for 180 seconds. A liquid (L6) was produced.

  Ra = 0.13 of this reaction system, concentration (mass%) of inorganic material (C) = 0.40 (%) <12.4Ra + 0.05 = 1.66

[Preparation of culture substrate (thin layer of complex (X)) (second step)]
1 g of γ-polyglutamic acid (manufactured by Nippon Polyglu Co., Ltd.) was added to the total amount of the dispersion (L6) and mixed uniformly, and then placed in a polystyrene petri dish (IWAKI tissue culture 3000-035) having a diameter of 35 mm. The dispersion is thinly applied to the surface of the petri dish at 3000 revolutions using a spin coater, dried in a hot air drier at 80 ° C. for 10 minutes, then washed with sterile water, and then washed in a sterile bag. Was dried at 40 ° C. for 5 hours to obtain a cell culture substrate 6.

[Culture and recovery of bone marrow-derived cells]
Mononuclear cells collected from GFP transgenic mice after sterilizing the obtained cell culture substrate 6 with an electron beam with an irradiation dose of 10 kGy (Japan Irradiation Service Co., Ltd.) and adding an appropriate amount of medium DMEM (containing 10% serum) The cells were seeded at 2.0 × 10 ⁶ cells / Dish (9.8 cm ² ) and cultured in 5% carbon dioxide at 37 ° C. for 7 days. Next, remove the culture medium in the dish and the suspended mononuclear cells, add a cold culture medium that has been chilled in the refrigerator in advance, leave it for 10 minutes, and then pipette up and down about 10 times with a pipette. As a result, it was observed that most bone marrow-derived cells were detached from the surface of the culture substrate 6. Bone marrow-derived cells that were naturally detached were collected, and further, using D-PBS and 0.25% Trypsin-EDTA, the bone marrow-derived cells remaining in the dish were separated and collected, and the number of each collected cells was measured. The number of cells spontaneously detached and collected by the low temperature treatment was 6.4 × 10 ⁴ , and the number of cells collected by the Trypsin treatment was 3.2 × 10 ⁴ . When the cell recovery rate by low-temperature treatment was determined by the following formula (5), the cell recovery rate was 67%.
Formula (5) Cell recovery rate (%) = {number of cells recovered by low-temperature treatment / (number of cells recovered by low-temperature treatment + number of cells recovered by trypsin treatment)} × 100
In addition, when the total number of bone marrow-derived cells recovered from the culture substrate 6 (6.4 × 10 ⁴ cells) was obtained using an uncoated petri dish (IWAKI tissue culture 3000-035) (Comparative Example 1) ( 2.4 times 10 ⁴ ).

  In addition, it was confirmed that the bone marrow-derived cells collected by the natural detachment by the low-temperature treatment and the trypsin treatment each had a normal cell morphology under a microscope.

(Comparative Example 1)
Using a commercially available petri dish made of polystyrene having a diameter of 35 mm (IWAKI tissue culture 3000-035), mononuclear cells collected from GFP transgenic mouse bone marrow using a serum-containing medium were obtained at 37 ° C. in the same manner as in Example 1. After culturing for 7 days, cells were detached by the same low-temperature treatment, but the cells were hardly detached. Furthermore, using D-PBS and 0.25% Trypsin-EDTA, bone marrow-derived cells remaining in the dish were peeled and collected, and the number of cells was counted. The number of cells was 2.5 × 10 ⁴ .

  Compared to this comparative example, normal polystyrene tissue culture has low culture (proliferation) properties for bone marrow-derived cells, and the cultured cells adhere strongly to the dish surface, and can easily be used when no drug (trypsin) is used. It can be understood that it cannot be peeled off.

(Comparative example 2) Example of culture in a serum-free system using a commercially available dish Using a commercially available polystyrene dish having a diameter of 35 mm (IWAKI tissue culture 3000-035), an appropriate amount of STEMPRO LIPOMAX SUPPLEMENT (Life) Technologies Japan A1085001) and GLUTAMAX I (Life Technologies Japan 35050061) put an appropriate amount of StemPro MSC SFM XenoFree containing (Life Technologies Japan A1067501) to, GFP transgenic mouse mononuclear cells harvested from 7.0 × 10 ⁶ cells / Dish (9.8 cm ² ) was seeded and cultured in 5% carbon dioxide at 37 ° C. for 7 days. Next, except for the culture medium in the dish and the suspended mononuclear cells, a new culture medium was added and observed with a microscope. As a result, almost no bone marrow-derived cells were found.

  From this comparative example, it can be understood that bone marrow-derived cells can hardly adhere to the dish in a serum-free medium using an ordinary polystyrene tissue culture dish, and thus cannot be grown.

(Comparative example 3) Example of base material (not including inorganic material (C)) only of polymer (A) [Preparation of reaction solution containing water-soluble (meth) acrylic acid ester (a) and aqueous medium (E) ]
A reaction solution (3 ′) was prepared by uniformly mixing 3.2 g of methoxyethyl acrylate and 100 g of water as an aqueous medium (E).

  In the same manner as in Example 1, a substrate (3 ') was produced. In the same manner as in Example 1, mononuclear cells collected from GFP transgenic mouse bone marrow were seeded using a serum-containing medium and cultured for 6 days. However, when most cells adhering to the surface of the substrate (3 ′) were found. There wasn't.

  From this comparative example, it can be understood that the base material containing only the polymer (A) that does not contain the inorganic material (C) has too low adhesion to the cells, and the cells cannot grow.

(Reference Example) Example of inorganic material (C) exceeding the range of formulas (1) and (2) [Including water-soluble (meth) acrylic acid ester (a), inorganic material (C), aqueous medium (E) Preparation of reaction solution]
12.8 g of methoxyethyl acrylate, 1.6 g of water-swellable clay mineral Laponite XLG (manufactured by Rockwood Additives Ltd.) as inorganic material (C), and 100 g of water as aqueous medium (E) are uniformly mixed to form a reaction solution ( 4 ′) was prepared.

[Preparation of dispersion (L) of complex (X) (first step)]
250 μl of the solution (2) was added to the total amount of the reaction solution (4 ′) and dispersed uniformly, and then irradiated with ultraviolet rays having an ultraviolet intensity of 40 mW / cm ² at 365 nm for 180 seconds. Even when a large amount of water was added and stirred, the gel-like product was not finely dispersed and a dispersion was not obtained.

Ra = 0.125 of this reaction system, concentration (mass%) of inorganic material (C) = 1.6 (%) = 12.4Ra + 0.05 = 1.6
From this comparative example, when the amount of the inorganic material (C) used exceeds the range of the formulas (1) and (2), the whole reaction solution becomes a gel due to polymerization and cannot be applied to other supports, It can be understood that the material cannot be manufactured.

(Example 6)
A dispersion liquid (L1) having the same composition as in Example 1 was prepared, and 1 g of an aqueous solution (PNIPA2) of the N-isopropylacrylamide polymer (B) of Example 2 was added to the total amount of the dispersion liquid (L1). After mixing, a cell culture substrate 7 was prepared in the same manner as in Example 1.

[Culture and recovery of bone marrow-derived cells]
The cell culture substrate 7 obtained above was sterilized with an electron beam with an irradiation dose of 10 kGy (Japan Irradiation Service Co., Ltd.), and then cultured and collected mononuclear cells collected from GFP transgenic mouse bone marrow in the same manner as in Example 1. Went. As a result, the number of cells naturally detached and collected by the low temperature treatment was 5.7 × 10 ⁴ , and the number of cells collected by the trypsin treatment was 1.2 × 10 ⁴ . Cell recovery was about 83%.

In addition, the total number of bone marrow-derived cells recovered from the culture substrate 7 (6.9 × 10 ⁴ ) was obtained when an uncoated petri dish (IWAKI tissue culture 3000-035) was used (2.5 × 10 6). About ⁴ times).

(Example 7)
A dispersion (L1) having the same composition as in Example 1 was prepared, and 4 g of an aqueous solution (PNIPA2) of the N-isopropylacrylamide polymer (B) in Example 2 was added to the total amount of the dispersion (L1). After mixing, a cell culture substrate 8 was prepared in the same manner as in Example 1.

[Culture and recovery of bone marrow-derived cells]
The cell culture substrate 8 obtained above was sterilized with an electron beam with an irradiation dose of 10 kGy (Japan Irradiation Service Co., Ltd.), and then cultured and collected mononuclear cells collected from GFP transgenic mouse bone marrow in the same manner as in Example 1. Went. As a result, the number of cells spontaneously detached and collected by low-temperature treatment was 9.7 × 10 ⁴ , and the number of cells collected by trypsin treatment was 1.6 × 10 ⁴ . The cell recovery rate was about 86%.

In addition, the total number of bone marrow-derived cells recovered from the culture substrate 8 (11.3 × 10 ⁴ ) was obtained when an uncoated petri dish (IWAKI tissue culture 3000-035) was used (2.5 × 10 ⁴ ).

  In addition, it was confirmed that the bone marrow-derived cells collected by the natural detachment by the low-temperature treatment and the Trypsin treatment each had a normal cell morphology with a microscope.

  From the above Examples 6, 7, and 1, the composition component of the culture substrate contains N-isopropylacrylamide polymer (B), and the content (B / A) is increased, whereby low temperature treatment is performed. It can be understood that the cell recovery rate is greatly increased.

(Example 8) Example of culture and recovery of human bone marrow mononuclear cells The cell culture substrate 2 of Example 2 was sterilized with an electron beam with an irradiation dose of 10 kGy (Japan Irradiation Service Co., Ltd.), and then the GFP gene recombination of Example 1 Cells were cultured and collected in the same manner as in Example 1 except that human bone marrow-derived mononuclear cells CL2M-125A (Lonza Japan Co., Ltd.) were used instead of mononuclear cells collected from mouse bone marrow. As a result, the number of cells spontaneously detached and collected by the low-temperature treatment was 22.3 × 10 ^{4 and the} number of cells collected by the trypsin treatment was 0 (all the cells were naturally detached). Cell recovery was about 100%.

In addition, the total number of bone marrow-derived cells recovered from the culture substrate 2 (22.3 × 10 ⁴ ) was obtained using an uncoated petri dish (IWAKI tissue culture 3000-035) (8.33 × 10 6). About ⁴ times).

  From the above examples, it can be understood that good culturing properties and a high cell recovery rate are exhibited even for highly sensitive human-derived bone marrow mononuclear cells.

  In addition, after sterilizing the cell culture substrate 2 of Example 2 with an electron beam with an irradiation dose of 10 kGy (Japan Irradiation Service Co., Ltd.), human bone marrow-derived mononuclear cells CL2M-125A (Lonza Japan Co., Ltd.) are used. Cells were cultured and collected in the same manner as in Example 1. When the collected cell surface antigen was analyzed with a flow cytometer (Galios, Beckman Coulter, Inc.), CD73-positive cells were 99.2%, CD105-positive cells were 94.4%, and CD45-positive cells were 0.1 %Met.

  In addition, the cell culture substrate 2 of Example 2 was sterilized with an electron beam with an irradiation dose of 10 kGy (Japan Irradiation Service Co., Ltd.), and then Lonsa Japan Co., Ltd. was used except for using human bone marrow-derived mononuclear cells CL2M-125A. The cells were cultured in the same manner as in 1. The differentiation ability of cultured cells into bone cells, adipocytes, and chondrocytes was evaluated using R & D Systems / Mesenchymal Stem Cell, Human, Functional Identification Kit (SC006). Cells cultured in the induction medium for bone cells were positive for alizarin red staining. Cells cultured in the induction medium for adipocytes were positive by oil red o staining. Cells cultured in the induction medium for chondrocytes were positive by immunostaining using anti-aggrecan antibodies.

  From the above examples, it can be understood that cells containing mesenchymal stem cells can be efficiently cultured by culturing human bone marrow-derived mononuclear cells on the cell culture substrate 2 of Example 2.

(Example 9) Culture substrate comprising polymer (A) and inorganic material (C) (excluding polymer (B))
A dispersion (L3) having the same composition as in Example 3 was prepared, and a cell culture substrate 9 was prepared in the same manner as in Example 1.

[Culture and recovery of bone marrow-derived cells]
The cell culture substrate 9 obtained above was sterilized with an electron beam with an irradiation dose of 10 kGy (Japan Irradiation Service Co., Ltd.), and then cultured and collected mononuclear cells collected from GFP transgenic mouse bone marrow in the same manner as in Example 1. Went. As a result, the number of cells naturally detached and collected by low-temperature treatment was 2.0 × 10 ⁴ , and the number of cells collected by trypsin treatment was 1.4 × 10 ⁴ . Cell recovery was about 59%.

In addition, the total number of bone marrow-derived cells recovered from the culture substrate 9 (3.4 × 10 ⁴ ) was obtained when an uncoated petri dish (IWAKI tissue culture 3000-035) was used (0.6 × 10 6). About ⁴ times).

  In addition, it was confirmed that the bone marrow-derived cells collected by the natural detachment by the low-temperature treatment and the trypsin treatment each had a normal cell morphology under a microscope.

(Comparative Example 5) Example of B / A> 0.7 A dispersion (L3) having the same composition as in Example 3 was prepared, and the total amount of the dispersion (L3) was a polymer of N-isopropylacrylamide of Example 2. After adding 25 g of the aqueous solution (PNIPA2) of (B) (B / A = 0.78) and mixing uniformly, a substrate 5 ′ was produced in the same manner as in Example 3.

[Culture and recovery of bone marrow-derived cells]
After sterilizing the obtained base material 5 ′ with an electron beam with an irradiation dose of 10 kGy (Japan Irradiation Service Co., Ltd.), culture and recovery of mononuclear cells collected from GFP transgenic mouse bone marrow in the same manner as in Example 1. went. Although cultured for 6 days, few cells adhered to the surface of the substrate 5 ′.

From this comparative example, it can be understood that when the content (B / A) of the polymer (B) exceeds 0.7, the surface of the base material is not suitable for cell growth.

(Comparative example 6) Example of C / A <0.03 [Preparation of reaction solution containing water-soluble (meth) acrylic acid ester (a), inorganic material (C), aqueous medium (E)]
Methoxyethyl acrylate 3.2 g, 20 mass% colloidal silica aqueous solution (trade name Snowtex 20, manufactured by Nissan Chemical Industries, Ltd.) 0.4 g (SiO ₂ = 0.08 g) as inorganic material (C), aqueous medium (E) As a result, 100 g of water was uniformly mixed to prepare a reaction solution (6 ′).

Ra = 0.025 of this reaction system, concentration (mass%) of inorganic material (C) = 0.08 (%) <12.4Ra + 0.05 = 0.36
In the same manner as in Example 1, a substrate 6 ′ was produced. The C / A of the substrate was 0.025.

[Culture and recovery of bone marrow-derived cells]
The obtained substrate 6 ′ was sterilized with an electron beam with an irradiation dose of 10 kGy (Japan Irradiation Service Co., Ltd.) and then collected from GFP transgenic mouse bone marrow using a serum-containing medium in the same manner as in Example 1. Nucleocytes were seeded and cultured for 6 days, but few cells adhered to the surface of the substrate (6 ′).

(Comparative Example 7)
A light milky white complex (X) reaction solution in the same manner as in Example 1 except that the amount of the water-swellable clay mineral Laponite XLG as the inorganic material (C) was changed from 0.2 to 2.4 g. (7 ') was adjusted.

Ra = 0.75 of this reaction system, concentration (mass%) of inorganic material (C) = 2.34 (%) <0.87Ra + 2.17 = 2.82
Further, using the reaction solution (7 ′), a cell culture substrate 7 ′ was obtained in the same manner as in Example 1.

[Culture and recovery of bone marrow-derived cells]
The cell culture substrate 7 ′ obtained above was sterilized with an electron beam with an irradiation dose of 10 kGy (Japan Irradiation Service Co., Ltd.), and then the culture days were changed from 7 days to 14 days in the same manner as in Example 1. Mononuclear cells collected from GFP transgenic mouse bone marrow were cultured and collected. As a result, the number of cells that were naturally detached and collected by the low-temperature treatment was 0, and the number of cells collected by the trypsin treatment was 2.6 × 10 ⁴ . Cell recovery was about 0%.

When the total number of bone marrow-derived cells recovered from the culture substrate 7 ′ (2.6 × 10 ⁴ ) was cultured for 14 days using an uncoated petri dish (IWAKI tissue culture 3000-035) ( 0.5 × 10 ⁴ ) was about 5.2 times.

  In addition, it was confirmed that the bone marrow-derived cells collected by the natural detachment by the low-temperature treatment and the trypsin treatment each had a normal cell morphology under a microscope.

  From these comparative examples, it can be understood that when the content (C / A) of the inorganic material (C) is too small (<0.03), the adhesion to the cells is too low and the cells cannot grow.

  Similarly, even when C / A is in the range of 0.03 to 0.7, it can be understood that there are cases where culture cannot be performed when B / A> 0.7.

Note) TCPS: Uncoated Petri dish (IWAKI tissue culture 3000-035)

Note) TCPS: Uncoated Petri dish (IWAKI tissue culture 3000-035)

Claims (9)

Bone marrow characterized by containing water-soluble (meth) acrylic acid ester (a) polymer (A) and one or more inorganic materials (C) selected from water-swellable clay minerals and silica A cell culture substrate for cell culture,
A cell culture substrate for bone marrow-derived cell culture, wherein the mass ratio ((C) / (A)) between the polymer (A) and the inorganic material (C) is in the range of 0.03 to 0.7. One or more inorganic materials selected from water-soluble (meth) acrylic acid ester (a) polymer (A), N-substituted (meth) acrylamide polymer (B), and water-swellable clay mineral and silica A cell culture substrate for bone marrow-derived cell culture characterized by containing (C),
The mass ratio ((B) / (A)) between the polymer (A) and the polymer (B) is in the range of 0.01 to 0.7, and the polymer (A) and the inorganic material A cell culture substrate for bone marrow-derived cell culture having a mass ratio ((C) / (A)) to (C) in the range of 0.03 to 0.7. On a cell culture substrate containing a polymer (A) of a water-soluble (meth) acrylic acid ester (a) and one or more inorganic materials (C) selected from water-swellable clay minerals and silica A method for culturing bone marrow-derived cells, characterized by culturing bone marrow-derived cells and then peeling the cultured cells from the substrate,
A method for culturing bone marrow-derived cells, wherein the mass ratio ((C) / (A)) between the polymer (A) and the inorganic material (C) is in the range of 0.03 to 0.7. One or more inorganic materials selected from water-soluble (meth) acrylic acid ester (a) polymer (A), N-substituted (meth) acrylamide polymer (B), and water-swellable clay mineral and silica A method for culturing bone marrow-derived cells, comprising culturing bone marrow-derived cells on a cell culture substrate containing (C), and then peeling the cultured cells from the substrate,
The mass ratio ((B) / (A)) between the polymer (A) and the polymer (B) is in the range of 0.01 to 0.7, and the polymer (A) and the inorganic material A method for culturing bone marrow-derived cells wherein the mass ratio to (C) ((C) / (A)) is in the range of 0.03 to 0.7. The water-soluble (meth) acrylic acid ester (a) is at least one monomer selected from methoxyethyl acrylate, ethoxyethyl acrylate, methoxyethyl methacrylate and ethoxyethyl methacrylate. For culturing cells derived from bone marrow. The method for culturing bone marrow-derived cells according to any one of claims 3 to 5, wherein the bone marrow-derived cells are cultured without using animal-derived serum. The method for culturing bone marrow-derived cells according to any one of claims 3 to 5, wherein the cultured cells are detached from the substrate without using a proteolytic enzyme. The method for culturing bone marrow-derived cells according to any one of claims 3 to 7, wherein bone marrow-derived cells cultured on the cell culture substrate are detached from the substrate by low-temperature treatment at 25 ° C or lower and / or physical external stimulation. . Bone marrow-derived cells produced by the culture method according to any one of claims 3 to 8.