JP2007215519A - Carrier for cell culture - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a carrier for cell culture, hardly curled in a dried state and having improved wettability; to provide a technique for stably and easily superposing a cell; to provide a technique by which the cells at the upper layer and the lower layer are sufficiently stuck when cell layers are superposed; and to provide a transparent carrier for the cell culture, enabling the state of the cells under culture to be observed directly, and allowing the cultured cells to be separated in a short time. <P>SOLUTION: The carrier for cell culture having a multilayered structure including a water-containing polymer gel layer has a hydrophilic layer on the surface at the opposite side of the cell culture surface side of the water-containing polymer gel layer. The hydrophilic layer contains a natural polymer, a synthetic polymer or a mixed polymer thereof, and the water-containing polymer gel is chitosan. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a cell culture technique, specifically, a cell culture carrier, a cell culture method using the cell culture carrier, a cell culture obtained by the culture method, and a cell layer using the cell culture And a stratified cell layer obtained by the stratification method.

  Hydrophilic polymer gels such as chitosan and gelatin have a structure similar to that of living organisms, and have the property of expanding and contracting due to external conditions such as temperature, acidity and alkalinity, so they can be applied to artificial organs and tissues such as artificial muscles. In addition to application to the medical field of controlling the release amount by encapsulating a drug inside, attempts have been made to use it as a scaffold for cell growth when cells are cultured as gels containing various cytokines.

  Among the hydrophilic polymer gels, poly N-isopropylacrylamide (hereinafter referred to as “PNIPAM”), which is a temperature-responsive polymer, swells and is liquid at low temperatures, but undergoes a phase transition near 34 ° C. and rapidly shrinks. Gels. Conventionally, in stratification of cultured cells, cells cultured on gelled PNIPAM under a temperature condition of 37 ° C are stacked on another cell layer together with PNIPAM, and then lowered to 34 ° C or lower to liquefy PNIPAM. And the method of directly superimposing cells was taken (Patent Document 1: Japanese Patent Publication No. 6-1004061; Non-Patent Document 1: Tatsuya Shimizu, Mitsuo Okano, Bioscience and Bioindustry, 58 (12), 851 (2000); Non-Patent Document 2: Masayuki Yamato, Mitsuo Okano, Rin, 56 (1), 53 (2001); Non-Patent Document 3: Masayuki Yamato, Mitsuo Okano, Materials Integration, 13 (2), 58 (2000); Non-Patent Literature 4: Masayuki Yamato, Ai Kushida, Mitsuo Okano, Protein, Nucleic Acid, Enzyme, 45 (10), 72 (2000); Non-Patent Literature 5: Masayuki Yamato, Shihiro Hirose, Hari Masami Moto, Mitsuo Okano, Protein / Nucleic Acid / Enzyme, 45 (13), 162 (2000)).

  When cell culture is performed on PNIPAM, cells usually grow in a single layer. At this time, an extracellular matrix such as collagen (hereinafter referred to as “ECM”) is formed between adjacent cells. And in order for a cell to proliferate, it is necessary to adhere to ECM. However, the upper part of the cell and the cell and the PNIPAM that is the basal layer are not adhered to other cells, and an ECM necessary for cell adhesion is not formed.

  Therefore, the cell layers of monolayers cultured on PNIPAM were overlaid, and the cell layer was solubilized and removed under conditions of 34 ° C or lower, and the cells were stacked so that the cells were in direct contact with each other, or stacked on top The cells did not have enough scaffold to grow, so stable growth could not be desired. In addition, since liquefied PNIPAM acts as a cytotoxin and inhibits the normal growth of cells, it was an extremely unsuitable and unstable technique for cell stratification.

  In order to solve the problems of the above methods, cell culture in a medium in which extracellular matrix components are layered on various gels and gelled, and a medium that can easily remove unnecessary gels after layering are used. The study on the establishment of the culture system was continued. In Japanese Patent No. 3261456 (Patent Document 2), a cell comprising a porous membrane and an alginate gel layer and an extracellular matrix component gel layer or an extracellular matrix component sponge layer formed on the porous membrane. Culture carriers have been proposed. However, with this cell culture carrier, the extracellular matrix layer is thick, and it was impossible to store the cell culture carrier in a dry film state. In addition, there is a problem that the stratified cells cannot be stably obtained because it interferes with the stratification operation of cells. Furthermore, since the porous membrane described in the patent publication is not transparent and the growth of living cells during cell culture cannot be observed directly or visually with a microscope, the cell culture placed on a petri dish Although the state of cells on the cell carrier was estimated to be equivalent from the state of cell growth on the petri dish around the carrier, the cell growth state on the petri dish around the cell culture carrier However, it does not always match the cell state of the cell, and there is a problem that the cell growth is insufficient or overconfluent. Therefore, in order to reliably grasp the cell growth state, a cell culture carrier having transparency like a petri dish used in normal cell culture has been desired.

  Further, in the method described in the patent publication, cultured cells are detached from the cell culture support using an EDTA aqueous solution for dissolving the alginate gel layer, but the cells are damaged by the invasion of the aqueous EDTA solution at this time. was there. Therefore, there has been a demand for a cell culture carrier that can minimize damage to the cells when the cultured cells are detached from the cell culture carrier.

  Japanese Patent Laid-Open No. 2005-10537 (Patent Document 3) describes a cell culture carrier and a cell culture method in which damage to cells is minimized, but the cell culture carrier is curled in a dry state. When installed in a cell culture container, handling may be poor. In addition, wettability may be poor, and there is a concern that cell adhesion and growth may be hindered by repelling the medium.

Japanese Patent Publication No.6-104061 Japanese Patent No. 3261456 JP 2005-10537 Tatsuya Shimizu, Mitsuo Okano, Bioscience and Bioindustry, 58 (12), 851 (2000) Masayuki Yamato, Mitsuo Okano, Outside, 56 (1), 53 (2001) Masayuki Yamato, Mitsuo Okano, Materials Integration, 13 (2), 58 (2000) Masayuki Yamato, Ai Kushida, Mitsuo Okano, Protein / Nucleic Acid / Enzyme, 45 (10), 72 (2000) Masayuki Yamato, Shihiro Hirose, Masami Harimoto, Mitsuo Okano, Protein / Nucleic Acid / Enzyme, 45 (13), 162 (2000)

  An object of the present invention is to provide a cell culture carrier that does not curl in a dry state and has improved wettability. Furthermore, the present invention provides a technique for stably and easily overlaying cells, and a technique capable of sufficiently adhering the cells in the upper layer and the cells in the lower layer when the cell layers are overlaid, The aim is to open the way to cell stratification technology that has been considered difficult under in vitro conditions except for some tissues such as skin. Specifically, an object of the present invention is to provide a transparent cell culture carrier capable of directly observing the state of cells in culture and capable of peeling the cultured cell layer in a short time. .

  The present inventors have intensively studied to solve the above problems, and in a cell culture carrier having a multilayer structure including a polymer hydrous gel layer, the polymer hydrogel layer has a surface opposite to the cell culture surface side. It has been found that by providing a hydrophilic layer, a cell culture carrier that does not curl in a dry state and has improved wettability can be provided. The present invention has been completed based on these findings.

  That is, according to the present invention, it is a cell culture carrier having a multilayer structure including a polymer hydrogel layer, and has a hydrophilic layer on the surface opposite to the cell culture surface side of the polymer hydrogel layer. Characteristic cell culture carriers are provided. According to a preferred embodiment of the present invention, there is provided a cell culture carrier in which the hydrophilic layer in the above cell culture carrier contains a natural polymer, a synthetic polymer or a mixed polymer thereof.

  According to a preferred embodiment of the present invention, there is provided a cell culture carrier in which the polymer-containing gel is chitosan in the above cell culture carrier. According to a further preferred aspect of the present invention, in any one of the above cell culture carriers, the cell culture carrier further comprises a gel layer containing an anionic polysaccharide and a polyvalent metal ion, and the gel layer is a polymer hydrous gel layer. A cell culture carrier adjacent to the cell; the cell culture carrier in which the gel containing the anionic polysaccharide and the polyvalent metal ion is a calcium alginate gel; Cell culture carrier that is an adhesive gel layer; cell culture carrier in which cell adhesive gel contains gelatin and / or collagen; cell culture carrier in which cell adhesive gel is cell adhesive collagen gel; cell adhesive gel The cell culture carrier wherein is modified with a cell adhesion factor; the cell culture carrier wherein the cell adhesion factor is fibronectin, laminin, or vitronectin; In fine any of the above carrier for cell culture, the cell culture carrier is an electron beam, gamma rays, cell culture carrier which is sterile by irradiation either or more of ultraviolet light is provided.

  From another aspect of the present invention, there is provided a cell culture method including a step of culturing cells using any one of the above cell culture carriers, and further, cells cultured using any of the above cell culture carriers A cell culture method comprising a step of obtaining a cell sheet by solubilization treatment of a gel layer containing an anionic polysaccharide and polyvalent metal ions in a culture; the solubilization treatment comprises a chelating agent and the polyvalent metal cation is 2.6 mM. The cell culture method performed using the following medium; and a cell sheet obtained by any one of the above cell culture methods are provided.

  According to still another aspect of the present invention, there is provided a step of further culturing a cell culture obtained by a cell culturing method including a step of culturing cells using any of the above cell culture carriers on another cell culture carrier. A cell transfer method comprising: a cell culture or cell sheet obtained by solubilizing a gel layer containing an anionic polysaccharide and a polyvalent metal ion in a cell culture obtained by the cell transfer method; The cell culture or cell sheet using a medium containing a polyvalent metal cation of 2.6 mM or less; and a step of culturing the cell culture obtained by the cell transfer method on other cultured cells A cell stratification method; a cell stratification method comprising a step of culturing a cell culture cultured using any one of the above cell culture carriers on other cultured cells; and a cell stratification method obtained by the cell stratification method. Anionic polysaccharide and stratified cell culture or stratified cell sheet obtained by solubilization treatment of the gel layer containing the polyvalent metal ions are provided in a cell culture.

  The present invention provides a cell culture carrier that does not curl in a dry state and has improved wettability. In addition, according to the present invention, there is provided a cell culture carrier capable of directly observing the living state of cells during cell culture and easily layering cell layers.

  The cell culture carrier means a cell culture carrier having a multilayer structure including a polymer hydrated gel layer and having a hydrophilic layer on the surface opposite to the cell culture surface side of the polymer hydrated gel layer. . The shape of the cell culture carrier of the present invention is not particularly limited, but is preferably a sheet shape.

  The cell culture carrier of the present invention is characterized by having a hydrophilic layer on the surface opposite to the cell culture surface side of the polymer hydrous gel layer. In the present specification, the layer may be referred to as a film.

  In the present invention, the polymer hydrogel refers to a water-absorbing polymer that does not dissolve particularly in water but has water in the polymer and has a two-dimensional or three-dimensional support structure throughout the system. In the present invention, the term “not soluble in water” means that the substantial solubility in water at a neutral pH at room temperature is 0.5 g / 100 g or less, preferably 0.1 g / 100 g or less, further substantially Most preferably, it does not dissolve. Here, room temperature refers to a range of 20 ° C to 30 ° C, preferably 25 ° C. Moreover, neutral pH means the range of pH5-9, Preferably it is the range of pH6-8. In the present invention, a polymer hydrogel layer is used that allows a chelating agent to reach the other surface from one surface of the layer by diffusing a substance such as a chelating agent into the layer. In the present invention, as the polymer hydrogel layer, a layer that does not allow an anionic polysaccharide such as an alginate gel and a polyvalent metal ion to reach from one surface of the layer to the other surface is used. The polymer hydrogel layer used in the present invention is not particularly limited as long as it is such a layer, and may be a synthetic polymer, a natural polymer, or a biopolymer. Examples of the polymer hydrogel include cross-linked acrylamide gel, cross-linked acrylic acid gel, agar, cross-linked gelatin, cross-linked dextran, chitosan, silica gel and the like, and chitosan is preferably used.

  In the cell culture carrier of the present invention, the “polymer hydrous gel layer” is preferably a support. Here, the support in the cell culture carrier means a layer that is a base for producing a cell culture carrier having a multilayer structure.

  In the present invention, the polymer hydrous gel layer has a thickness of 0.01 μm or more and 20 μm or less. Preferably they are 0.1 micrometer or more and 10 micrometers or less, More preferably, they are 0.5 micrometer or more and 5 micrometers or less. If the polymer hydrogel layer is too thin, a sufficient film cannot be formed, causing problems such as tearing, tearing, and holes. If the polymer hydrogel layer is too thick, the medium component and the chelating agent component during stripping operation The problem is that the diffusion is slow and the cells are adversely affected.

  The polymer water-containing gel layer in the cell culture carrier of the present invention can be prepared using various generally known methods for preparing gel membranes. Examples include a method of casting a polymer solution (casting method), a method of applying with a bar coater (bar coating method), and a method of applying with a gap coater (gap coating method). A gap coat coating method is preferred.

  The polymer hydrogel used in the present invention may be one having a viscosity in a solution state defined later in the present specification of 100 mPa · s or more and 50000 mPa · s or less, but is 300 mPa · s or more and 30000 mPa · s. It is preferably s or less, more preferably 600 mPa · s or more and 20000 mPa · s or less.

  The viscosity of the polymer solution prepared under the same conditions is an index of the molecular weight of the polymer, and the larger the value of the viscosity, the higher the molecular weight. The viscosity in the solution state of the gel defined in this specification is a solution before gelation obtained by adding 12 g of the polymer to 1000 g of a 1% by mass acetic acid aqueous solution at 25 ° C. with a B-type viscometer. This is the viscosity when When the viscosity is low, that is, when the molecular weight is small, it is suggested that the strength of the produced polymer hydrogel layer is weak. In addition, since the solution is low in viscosity, the solution begins to flow and the film thickness becomes non-uniform, or only the outermost surface of the solution is dried to cause a skinning phenomenon, or the solvent is dried. It takes time. On the other hand, when the viscosity is too high, that is, when the molecular weight is too high, the produced polymer hydrogel film has strength, but is not castable when the film is formed, or the film thickness becomes non-uniform, or the coating film There is a problem that it cannot be formed. By using a polymer hydrogel having a viscosity in the above range, it is possible to obtain a polymer hydrogel layer having high strength and a certain thickness as described above.

  In the present invention, hydrophilicity provided on the surface opposite to the cell culture surface side of the polymer hydrous gel layer (that is, the surface opposite to the outermost surface of the cell culture surface across the polymer hydrogel layer). The layer is a layer composed of a hydrophilic polymer. The hydrophilic polymer is a water-absorbing polymer that does not dissolve in water but has water in the polymer and has a two-dimensional or three-dimensional support structure throughout the entire system. This refers to a functional polymer. In the present invention, the term “not soluble in water” means that the substantial solubility in water at a neutral pH at room temperature is 0.5 g / 100 g or less, preferably 0.1 g / 100 g or less, further substantially Most preferably, it does not dissolve. Here, room temperature refers to a range of 20 ° C to 30 ° C, preferably 25 ° C. Moreover, neutral pH means the range of pH5-9, Preferably it is the range of pH6-8.

In the present invention, the hydrophilic polymer can be made to reach the chelating agent from one side of the layer to the other side by diffusing a substance such as a chelating agent into the layer when the gel layer is formed. Is used. In the present invention, when a hydrophilic polymer is used as a gel layer, a gel containing an anionic polysaccharide such as an alginate gel and a polyvalent metal ion is allowed to reach from one surface of the layer to the other surface. Use something that cannot. The hydrophilic polymer used in the present invention is not particularly limited as long as the above conditions are satisfied, and may be a natural polymer (natural polymer, biopolymer), a synthetic polymer, or a mixed polymer thereof. Examples of hydrophilic polymers include cross-linked acrylamide gel, cross-linked acrylic acid gel, agar, cross-linked gelatin, cross-linked dextran, cross-linked agarose, chitosan, silica gel, etc., but cross-linked acrylamide gel, cross-linked gelatin, cross-linked dextran should be used. Is preferred.
In the present invention, the hydrophilic polymer used in the hydrophilic layer provided on the surface opposite to the cell culture surface side of the polymer hydrous gel layer may be used alone, or a plurality of hydrophilic polymers may be mixed. May be used.

  The hydrophilic layer provided on the surface opposite to the cell culture surface side of the water-containing polymer gel layer of the present invention can contain an additive as necessary. The additive is not limited as long as it does not prevent formation of a hydrophilic layer or / and cell culture. For example, a hardening agent, a thickener, a surfactant, an electrostatic removing agent, a pH adjusting agent, a swelling agent. And humidity control agents. These additives may be inorganic or organic, and may be low molecular compounds, high molecular compounds or oligomers.

The thickness of the hydrophilic layer on the side opposite to the cell culture surface side of the polymer hydrogel layer in the present invention is preferably 0.01 μm or more and 50 μm or less, more preferably 0.1 μm or more and 25 μm or less, Preferably they are 0.5 micrometer or more and 15 micrometers or less. If the layer is too thin, sufficient water content cannot be obtained and curling cannot be improved. If the layer is too thick, curling is improved, but the permeation of the medium components is slow and the cells are adversely affected or peeled off. There is a problem that the diffusion of the chelating agent component during the operation is slow and the peelability is deteriorated.
In the present specification, the layer thickness indicates a value measured in a sufficiently dry state unless otherwise specified. In this specification, the thickness of this layer is sometimes referred to as “dry film thickness”. The thickness of the layer can be measured using an electron microscope cross-sectional image, a micrometer film thickness meter, an ellipsometer, an angle variable XPS, a light interference film thickness meter, etc., preferably a micrometer film thickness meter, an electron It can be performed using a microscope cross-sectional image and a light interference film thickness meter.

  In the cell culture carrier of the present invention, the hydrophilic layer on the surface opposite to the cell culture surface side of the polymer water-containing gel layer is formed on the polymer water-containing gel on various generally known methods for preparing gel films. Can be used. Examples include a method of casting a polymer solution (casting method), a method of applying with a bar coater (bar coating method), and a method of applying with a gap coater (gap coating method). A gap coat coating method is preferred.

  The hydrophilic polymer forming the hydrophilic layer on the surface opposite to the cell culture surface side of the polymer hydrogel layer used in the present invention has a viscosity in a solution state of 10 mPa · s or more and 50000 mPa · s. Although the following can be used, it is preferably 30 mPa · s or more and 30000 mPa · s or less, more preferably 60 mPa · s or more and 20000 mPa · s or less.

  As a preferred embodiment of the present invention, a polymer water-containing gel layer is adjacent to a gel layer containing an anionic polysaccharide and a polyvalent metal ion. Examples of the anionic polysaccharide include alginic acid, dextran sulfate, carboxymethylcellulose, carboxymethyldextran, and hyaluronic acid. Alginic acid is preferably used.

  Alginic acid exists naturally as a cell wall-constituting polysaccharide or intercellular packing material of brown algae, and these can be collected as raw materials. Specific examples of the raw material brown algae include the genus Dhabilliidae (for example, D. potatorum), the genus Asbophila (for example, A. nodosum), the genus Ascomophilum (for example, Macombu, Nagacombu), Examples include brown algae of the genus Alameda (for example, arame), the order of the genus Coleaceae (for example, Kajime, Urome), and the genus Lessoniaceae (for example, L. flavikans). Commercially available alginic acid can also be used. The G / M ratio of alginic acid is not particularly limited, but the larger the G / M ratio, the greater the gel-forming ability, so the larger G / M ratio is preferred, specifically 0.1 to 1. Preferably, it is 0.2-0.5.

  “Alginic acid gel” means a gel formed by forming a chelate structure between a carboxylic acid group in a molecule of alginic acid and a polyvalent metal ion, and “alginate gel layer” means a layered alginate gel. To do. Alginic acid is a block copolymer composed of glucuronic acid (G) and mannuronic acid (M). It is thought that polyvalent metal cations enter the pocket structure of M block to form an egg box and gel. Yes. Specific examples of polyvalent metal cations that can cause gelation of alginic acid include barium (Ba), lead (Pb), copper (Cu), strontium (Sr), cadmium (Cd), calcium (Ca), zinc (Zn ), Nickel (Ni), cobalt (Co), manganese (Mn), iron (Fe), magnesium (Mg) and other metal ions can be exemplified, and among these, calcium ions, magnesium ions, barium ions are particularly preferable. And strontium ions. The “alginate gel” may be a polyion complex gel of an organic polymer compound having alginic acid and a cation residue. Examples of the organic polymer compound having a cation residue mentioned here include compounds having a plurality of amino groups such as polylysine, chitosan, gelatin and collagen.

  The gelation of alginic acid can be performed according to a conventional method. The gelation of alginic acid can be performed using, for example, ion exchange. For example, when calcium ions are added to a sodium alginate aqueous solution, ion exchange occurs rapidly, and a calcium alginate gel is obtained. More specifically, a 0.2-5% sodium alginate aqueous solution is applied on the polymer hydrous gel layer, and then immersed in a 0.01-1.0 M calcium chloride aqueous solution to impregnate calcium chloride, and at 20-30 ° C. for 3 minutes to A calcium alginate gel layer is obtained by leaving it to stand for 3 hours. Thus, when gelatinization of alginic acid is performed using the polymer hydrogel, a cell culture carrier comprising a polymer hydrogel layer and an alginate gel layer formed on the gel layer can be obtained.

  The thickness of the gel layer containing the anionic polysaccharide and the polyvalent metal ion in the cell culture carrier of the present invention is preferably 0.01 μm or more and 50 μm or less, preferably 0.1 μm or more and 10 μm or less, and 0.5 μm or more and 8 μm or less. More preferably. If the solid content of the alginate gel layer is too small, a sufficient film-like layer cannot be formed and holes are formed, and if it is too much, curling or cracking occurs in the dried membrane, deformation in the culture process or alginate gel dissolution process. This causes problems such as poor dissolution.

  The cultured cell layer formed on the cell culture carrier of the present invention can be detached as a cell sheet by solubilizing a gel layer containing an anionic polysaccharide and a polyvalent metal ion. The cell sheet includes a cultured cell layer and preferably a cell adhesion layer described later. The solubilization treatment of the gel layer containing the anionic polysaccharide and the polyvalent metal ion can be carried out by removing the cation component constituting the gel layer containing the anionic polysaccharide and the polyvalent metal ion. In the case of metal ions, the cell culture carrier on which the cultured cell layer is formed is soaked in 1) a medium to which ions that form a complex with a polyvalent metal cation such as phosphoric acid or a sparingly soluble salt are added. Immerse in a medium to which an aqueous chelating agent solution is added 3) Immerse in a medium with reduced polyvalent metal ions or 4) Conceal the polyvalent metal ions in the culture medium of the cells with a chelating agent it can. Usually, there are many phosphate ions in a cell culture medium. Therefore, the solubilization treatment of the gel layer containing the anionic polysaccharide and the polyvalent metal ion is such that the polyvalent metal cation concentration is lower than the concentration of the polyvalent metal cation in the minimum medium usually used for cell culture, and a chelating agent is used. It is preferable to carry out using the culture medium containing. Specifically, the concentration is preferably 2.6 mM or less, more preferably 3 μM or less, still more preferably 0.5 μM or less, and most preferably substantially 0. The concentration of the chelating agent is preferably 2.3 mM or more and 26000 mM or less, more preferably 2.3 mM or more and 2600 mM or less. By using a medium having a reduced polyvalent metal cation concentration as described above, it is possible to solubilize a gel layer containing an anionic polysaccharide and a polyvalent metal ion with reduced invasion of a chelating agent into cells.

  Examples of the chelating agent used in the present invention include ethylenediamine diorthydroxyphenylacetic acid, diaminopropanetetraacetic acid, nitrilotriacetic acid, hydroxyethylethylenediaminetriacetic acid, dihydroxyethylglycine, ethylenediaminediacetic acid, ethylenediaminedipropionic acid, and iminodiacetic acid. , Diethylenetriaminepentaacetic acid, hydroxyethyliminodiacetic acid, 1,3-diaminopropanoltetraacetic acid, triethylenetetraminehexaacetic acid, transcyclohexanediaminetetraacetic acid, ethylenediaminetetraacetic acid (edta), glycol etherdiaminetetraacetic acid, O, O'- Bis (2-aminoethyl) ethylene glycol-N, N, N ', N'-tetraacetic acid (egta), ethylenediaminetetrakismethylenephosphonic acid, diethylenetriaminepentamethylenephosphonic acid, nitrilotrimethylenediamine Sphonic acid, 1-hydroxyethylidene-1,1-diphosphonic acid, 1,1-diphosphonoethane-2-carboxylic acid, 2-phosphonobutane-1,2,4-tricarboxylic acid, 1-hydroxy-1-phosphonopropane-1 , 3,3-tricarboxylic acid, catechol-3,5-disulfonic acid, sodium pyrophosphate, sodium tetrapolyphosphate, sodium hexametaphosphate, 1-hydroxypropylidene-1,1-diphosphonic acid, 1-aminoethylidene-1, Examples thereof include 1-diphosphonic acid and salts thereof. Among these, preferred are edta, egta, ethylenediaminetetraphosphonic acid, and 1-hydroxyethylidene-1,1-diphosphonic acid.

  Further, in the medium for solubilizing the gel layer containing the anionic polysaccharide and the polyvalent metal ion, the concentration of the cationic amino acid is lower than the concentration of the cationic amino acid in the minimum medium usually used for cell culture. The concentration is preferred. Specifically, the concentration is preferably 1.0 mM or less, more preferably 2 μM or less, still more preferably 0.5 μM or less, and most preferably substantially 0. The cationic amino acid component refers to L-Lysin (Lys), L-Arginine (Arg), L-Histidine (His), L-Cystine (Cys), and salts thereof.

  What is necessary is just to implement the solubilization process of the gel layer containing an anionic polysaccharide and a polyvalent metal ion by immersing once in the above-mentioned culture medium for solubilization processes. In the case of multiple times, the solubilizing medium used may be the same or different.

  During the solubilization treatment of the gel layer containing the anionic polysaccharide and the polyvalent metal ion using the chelating agent, that is, when the cell culture carrier on which the cultured cell layer is formed is immersed in the solubilization treatment medium, The chelating agent is preferably soaked from the hydrophilic layer side of the surface opposite to the cell culture surface side of the polymer hydrogel layer. As a result, the hydrophilic layer on the opposite side to the cell culture surface side of the polymer hydrogel and the polymer hydrogel layer is easily separated from the gel layer containing the anionic polysaccharide and the polyvalent metal ion. The cell sheet containing the cultured cell layer can be easily peeled from the hydrophilic layer on the surface opposite to the cell culture surface side of the polymer hydrogel and the polymer hydrogel layer. It is not necessary to completely remove the gel layer containing the anionic polysaccharide and the polyvalent metal ion by the solubilization treatment of the gel layer containing the anionic polysaccharide and the polyvalent metal ion. Although a gel layer containing a polyvalent metal ion may remain, it is preferable to remove the gel layer containing an anionic polysaccharide and a polyvalent metal ion as much as possible.

  Another preferred embodiment of the present invention includes a cell culture carrier whose cell culture surface side outermost surface is a cell adhesive gel layer. “Cell-adhesive gel layer” means a layered cell-adhesive hydrogel, and any natural or synthetic compound may be used as long as it is non-cytotoxic and adheres to cells under normal culture conditions. Preferably, it is a layered extracellular matrix component gel. The extracellular matrix is generally defined as "a complex body of biopolymers, which are stable biological structures that exist outside the cells in animal tissues and are synthesized and secreted and accumulated outside the cells." (Biochemical Dictionary (Third Edition) p.570, Tokyo Chemical Doujin Co., Ltd.), the role of materially supporting cells and the role of regulating cell activity (ie, transmitting extracellular information to cells) It plays a role in changing its activity). “Extracellular matrix component” means a component of the extracellular matrix, and specific examples thereof include collagen, elastin, proteoglycan, glucosaminoglycan (hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, Keratan sulfate, etc.), fibronectin, laminin, vitronectin, gelatin, etc., among which collagen, atelocollagen, matrigel (gel made of type IV collagen, laminin, heparan sulfate), hyaluronic acid, and gelatin are particularly preferable. It can be illustrated. The extracellular matrix component can be obtained according to a conventional method. Commercially available extracellular matrix components may also be used. Gelation of the cell adhesion component can be performed according to a conventional method. These extracellular matrices may be used alone, may be used as a mixture of two or more, or may be used as a laminate of two or more. For example, when the cell adhesion component is collagen, a collagen gel can be obtained by incubating a 0.3 to 0.5% aqueous collagen solution at 37 ° C. for 10 to 20 minutes. When the extracellular matrix component is gelled, a gelling agent may be used as necessary.

  The thickness of the cell adhesive gel layer in the present invention is 0.005 μm or more and 5.0 μm or less, preferably 0.005 μm or more and 1.0 μm or less, and more preferably 0.005 μm or more and 0.5 μm or less. When the cell-adhesive gel layer of the present invention is thick, cracks are generated in the layer when it is dried, and cell transfer becomes extremely difficult.

  The cell adhesion gel layer of the cell culture carrier of the present invention may be further surface modified with a cell adhesion factor. The cell adhesion factor is not particularly limited and generally known ones can be used, and examples thereof include fibronectin, laminin, vitronectin and the like.

  When forming a cell adhesive gel layer on a gel layer containing an anionic polysaccharide and polyvalent metal ions, the gel layer containing an anionic polysaccharide and polyvalent metal ions and the cell adhesive component gel layer are separated. However, it is preferable to add the cell-adhesive component-containing aqueous solution to the gel layer containing the anionic polysaccharide and the polyvalent metal ion, and then gel the aqueous solution. Since the cell-adhesive gel layer does not have sufficient physical strength for desorption, the cell-adhesive gel layer is peeled off from the container (for example, dish, petri dish, etc.) on which the cell-adhesive gel layer is formed. Because it is difficult. The ultra-thin cell adhesive gel layer is prepared by immersing a gel layer containing an anionic polysaccharide and a polyvalent metal ion in a re-adhesive adhesive component solution (immersion method) or applying (application method). Alternatively, it can be easily obtained by casting (casting method), but any of these methods may be used for preparing the cell culture carrier of the present invention. Of these, the casting method is preferably used. For example, when a collagen gel layer is formed on an alginate gel, a commercially available 0.3-0.5% collagen aqueous solution is diluted to an appropriate concentration as necessary, and this solution is cast on the calcium alginate gel prepared by the above method. By drying, a collagen gel layer formed on an alginate gel is obtained.

  The cultured cell layer formed on the cell culture carrier of the present invention can be peeled off as a cell sheet by solubilizing the gel layer containing anionic polysaccharide and polyvalent metal ion as described above. The cell culture carrier of the present invention may be provided with a physical reinforcing jig on the surface of the hydrophilic layer opposite to the cell adhesive gel layer in order to improve the operability at that time. The material of the physical reinforcing jig is not particularly limited as long as it does not affect the cells, but metals (for example, iron, stainless steel, titanium, gold, etc.), plastics (for example, polystyrene, polycarbonate, polyethylene, Polypropylene, acrylic, etc.) and inorganic materials such as ceramics, and stainless steel, titanium and plastics are preferred.

  The physical reinforcing jig may have any shape as long as it can improve the handleability of the cell culture carrier of the present invention, but is preferably plate-shaped and has a thickness of 0.1 μm or more and 10 mm or less. It is preferably 1 μm or more and 1 mm or less, more preferably 10 μm or more and 200 μm or less.

  The shape of the physical reinforcing jig is not particularly limited as long as there is a portion where the cell culture carrier of the present invention can be seen for cell observation, but a circle, a polygon (triangle, square, hexagon, etc.), or a combination thereof (Sector type etc.) etc. are mentioned as an example. Among them, a shape close to a circle is preferable. In addition, the cell culture carrier of the present invention can be seen for cell observation with one or more portions. In order to easily distinguish the surface to which the reinforcing jig is bonded, the shape is preferably asymmetric.

  The physical reinforcing jig may be adhered to the polymer gel film by any method as long as it does not affect the cell culture. For example, after a polymer gel film is prepared, a commercially available adhesive (for example, Aron Alpha, Bond, etc.) is used for adhesion, or when creating a polymer gel film, the reinforcing jig is not dried. You may make it adhere by putting it in.

  Depending on the material of the physical reinforcing jig, the edge of the auxiliary jig may be sharp. In this case, not only the cell culture carrier is torn by the sharp edges of the auxiliary jig, but also there is a concern that it may be dangerous for the handling operator. Therefore, it is preferable to eliminate sharp edges. As a method for eliminating sharp edges, any method may be used as long as cell culture is not hindered, but examples include physical polishing (for example, polishing with a file) and chemical processing (for example, chemical etching). It is done. In the present invention, when the physical reinforcing jig is made of stainless steel, it is preferable to perform chemical treatment such as chemical etching.

  In order to improve the operability when peeling the cultured cell layer formed on the cell culture carrier of the present invention as a cell sheet, in the cell culture carrier having a cell adhesive gel layer on the cell culture surface side outermost surface, An unmodified portion of the cell adhesive gel layer, that is, a portion where the cell adhesive gel layer is not formed may be provided on a part of the outermost surface of the cell culture surface side. The unmodified portion of the cell adhesive gel layer becomes a polymer hydrous gel layer or a gel layer containing an anionic polysaccharide and a polyvalent metal ion. By providing the cell-adhesive gel layer unmodified portion, it is possible to improve the handleability when peeling the polymer hydrous gel layer and the cell-adhesive gel layer. That is, since the polymer hydrogel layer can be removed without touching the cell adhesive gel layer by grasping the polymer hydrogel layer of the unmodified portion of the cell adhesive gel layer with tweezers or the like, there is little adverse effect on the cells. The unmodified portion of the cell adhesive gel layer is preferably a corner portion of the polymer-containing gel layer in the cell culture carrier of the present invention.

  Any method may be used as a method for providing the unmodified portion of the cell adhesive gel layer as long as cell culture is not hindered. For example, a generally well-known masking method may be mentioned. That is, the portion of the polymer hydrous gel layer that is not modified with the cell adhesive gel layer is covered in advance with another material, the polymer hydrous gel layer is modified with the cell adhesive gel component, and then the covered other material is removed. This is a method of providing an unmodified portion of the cell adhesive gel layer.

  The material of the other material covered to provide the unmodified portion of the cell adhesive gel layer may be any material as long as it does not interfere with cell culture. For example, silicon rubber, commercially available masking tape or cello tape (registered trademark) , Plastics (for example, polystyrene, polycarbonate, polyethylene, polypropylene, acrylic, etc.), metals (for example, iron, stainless steel, titanium, gold, etc.) and the like. Silicon rubber and commercially available masking tape are preferred.

  The shape of the unmodified portion of the cell adhesive gel layer is not particularly limited as long as cell culture is not hindered, but may be a circle, a polygon (triangle, square, hexagon), or a combination thereof (fan shape, etc.). A triangular shape and a sector shape are preferable. The size of the unmodified portion of the cell adhesive gel layer is not particularly limited as long as cell culture is not hindered, but is equivalent to a circle and has a diameter of 0.1 mm to 10 mm, preferably 1 mm to 5 mm.

  Specific examples of cells that can be cultured using the cell culture carrier of the present invention include fibroblasts, vascular endothelial cells, chondrocytes, hepatocytes, small intestinal epithelial cells, epidermal keratinocytes, osteoblasts, bone marrow mesenchyme A cell etc. can be illustrated and a fibroblast can be illustrated as a preferable thing. When culturing cells, a culture solution (eg, D-MEM medium, MEM medium, HamF12 medium, HamF10 medium) having a cell concentration of 1 to 15,000 cells / ml is usually added onto the cell adhesive gel layer. Cell culture conditions can be appropriately selected according to the cells to be cultured. When culturing cells on a cell adhesive gel layer, it is usually performed until a confluent monolayer cell layer is formed on the cell adhesive gel layer.

  Specifically, the cell culture using the cell culture carrier of the present invention can be performed as follows. Place the cell culture carrier inside a petri dish, etc., add an appropriate culture medium (for example, D-MEM medium, MEM medium, HamF12 medium, HamF10 medium) in the petri dish, soak the medium for 5 minutes and replace the medium. After being repeated for 12 to 24 hours, the culture solution is infiltrated into the cell culture carrier. Discard the culture medium in the petri dish, seed the cells on the cell adhesion gel layer of the cell culture carrier, and add an appropriate culture medium (for example, D-MEM medium, MEM medium, HamF12 medium, HamF10 medium) to the petri dish. . After standing at 37 ° C. for 1 to 2 hours and holding (adhering) the cell adhesive gel layer, the culture is continued at 37 ° C. When culturing, the culture solution may be exchanged as necessary. Usually, the culture medium is changed every 0.5 to 2 days of culture.

  The cell culture cultured using the cell culture carrier of the present invention includes the cell culture carrier of the present invention and a cell layer held on the cell culture carrier. The “cell layer held on the cell culture carrier” is preferably a cell layer formed on a cell adhesive gel layer.

  Since the cell sheet obtained by solubilizing the gel layer containing the anionic polysaccharide and the polyvalent metal ion contains the cell layer, it can be used for layering and transferring the cell layer. When the cell layers are layered, the cell culture cultured using the cell culture carrier of the present invention is overlaid with or without applying a load on the cells cultured in advance so that the cell culture surface faces each other. Further, after further culturing, the gel layer containing the anionic polysaccharide and the polyvalent metal ion may be solubilized, or the cell sheet obtained by solubilizing the gel layer containing the anionic polysaccharide and the polyvalent metal ion The cell sheets obtained by solubilizing the gel layer containing the anionic polysaccharide and the polyvalent metal ion may be layered separately. In addition, a cell sheet or cell culture containing a cell layer that has been layered by the above-described method or the like may be layered on a cell layer that has been separately prepared. The cell layer prepared separately may be a cell layer of a cell culture cultured using the cell culture carrier of the present invention, a cell layer of a cell culture cultured using another cell culture carrier, It may be a cell sheet. The cell type of the cell layer to be stratified may be the same or different. The number of cell layers to be stratified is not particularly limited, but is usually 1 to 10, preferably 1 to 5, and more preferably 1 to 3.

  When transferring the cell layer, the cell culture cultured on another cell culture substrate using the cell culture carrier of the present invention is placed so that the cell culture surface is on the other cell culture substrate side. The gel layer containing the anionic polysaccharide and the polyvalent metal ion may be solubilized after being placed in a state where the load is applied or not applied, and further cultured, and the anionic polysaccharide and the polyvalent metal ion are contained. The cell sheet obtained by solubilizing the gel layer may be transferred to another medium. The cell culture to be transferred may be a stratified cell culture.

  Preferable stratification and transcription methods include anionic polysaccharides and polyvalent metals after culturing a cell culture cultured using the cell culture carrier of the present invention on cells previously cultured or on another cell culture substrate. The method of solubilizing the gel layer containing ion is mentioned.

  As a method for culturing a cell in a state where a load is applied, any method may be used as long as the load is sufficiently applied so that unevenness does not occur in the cell or the substrate to which the cell is transferred. Here, when cells are sealed when a load is applied, they suffocate, so at least one cell culture substrate on the transfer side or the receiving side is a water-permeable gel, a hydrophilic polymer gel, or a combination thereof. It is preferable that it is made of. In addition, in order to transfer without unevenness, it is necessary to apply a load in a state where the cell surface is sufficiently covered. However, since uniform contact prevents oxygen diffusion, nonwoven fabric (nylon, polyester, stainless steel, etc.) It is preferable to apply the load without hindering the diffusion of oxygen through the like.

The load of the cell culture method under load is preferably 0.1 g / cm ² or more and 50 g / cm ² or less, more preferably 0.5 g / cm ² or more and 10 g / cm ² or less. The time for culturing the cell under load is not limited as long as sufficient cell transfer can be realized, but it is preferably 4 hours to 72 hours, more preferably 6 hours to 48 hours. In the present invention, it is preferable to culture without applying a load.

  When preparing the cell culture carrier of the present invention, a preparation solution containing carbodiimides may be used for the purpose of improving adhesion. Carbodiimides and N-hydroxysuccinimide may be added to any layer preparation solution, but impregnation in a gel layer preparation solution containing an anionic polysaccharide and polyvalent metal ions or a polymer hydrogel in advance. Or it is preferable to immerse in the liquid co-dissolved with calcium chloride after coating the gel layer containing anionic polysaccharide and polyvalent metal ion. The carbodiimide is preferably water-soluble, such as 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride. When the carbodiimide is used, the concentration is preferably 0.01 mg / l or more and 200 g / l or less. At this time, N-hydroxysuccinimide may be used as a catalyst, and the concentration is preferably 1% by mass or more and 50% by mass or less with respect to the carbodiimide. In the present invention, it is preferable not to use carbodiimide unless particularly required.

  The cell culture carrier of the present invention may be sterilized by any method, but sterilization by radiation such as electron beam, γ-ray, X-ray, ultraviolet ray is preferably used, and electron beam, γ-ray, ultraviolet ray is more preferably used. Electron beam sterilization is particularly preferred. The irradiation dose for electron beam sterilization of the present invention is preferably 0.1 kGy or more and 65 kGy or less, particularly preferably 1 kGy or more and 40 kGy or less. Sterilization by applying high heat such as chemical sterilization such as EOG sterilization or high-pressure steam gas sterilization is not preferable because it degrades the cell adhesive layer and the gel layer containing anionic polysaccharide and polyvalent metal ions. The cell culture carrier thus sterilized can be stored at room temperature for a long time under aseptic conditions. The sterilization method of the present invention may be carried out singly or in combination of a plurality of types, and the same type of sterilization method may be used repeatedly.

  If, for example, a vascular endothelial cell layer or a hepatocyte layer is used as the cell layer to be layered, a three-dimensional tissue structure of the liver can be constructed. This three-dimensional tissue structure can be applied to in vitro drug permeability tests, and can also be applied to animal experiment alternative models and transplanted organs. The layered cell layer can be cultured under culture conditions according to the type of cells constituting the cell layer. In the culture, for example, a medium such as D-MEM medium, MEM medium, HamF12 medium, HamF10 medium or the like can be used.

  The present invention will be specifically described by the following examples, but the present invention is not limited to the examples.

[Example 1] Production of cell culture carrier A 20 g of inert gelatin (made by Nitta Gelatin) was gradually added to 1500 g of distilled water, and then dissolved by stirring at 40 ° C for 2 hours. Immediately after adding 15 ml of 4% N, N-ethylenebis (vinylsulfone acetamide) aqueous solution, the dry film thickness is 1.5 μm with an applicator on a polyethylene terephthalate film (length 20 cm, width 18 cm, film thickness 195 μm). And dried at 30 ° C. overnight.

  On this gelatin film, 6 g of Daichitosan 100D (manufactured by Dainichi Seika Kogyo Co., Ltd.) is gradually added to 500 g of a 1% by weight acetic acid aqueous solution and dissolved by stirring for 7 hours at room temperature. This aqueous solution filtered through the filter FG-30 was applied with an applicator so as to have a dry film thickness of 4 μm, and dried at 37 ° C. overnight.

  The dried membrane was immersed in a methanol solution of 1.9 mass% sodium hydroxide for 30 minutes, and then immersed in a PBS (Dulbecco's Phosphate buffered Saline) solution for 30 minutes. Thereafter, it was immersed in distilled water for 30 minutes to obtain a crosslinked gelatin / chitosan gel film. The obtained membrane was dried at room temperature overnight and then stored in a plastic bag.

  On this crosslinked gelatin / chitosan film, 2% by mass of Kimika Argin B-1 aqueous solution (manufactured by Kimika Co., Ltd.) was further applied so as to have a wet coating thickness of 300 μm. This coated product was immersed in a 25% methanol aqueous solution containing 0.3 M calcium chloride for 30 minutes and then washed with running water for 30 minutes to obtain a crosslinked gelatin / chitosan / calcium alginate laminated gel film.

A frame made of silicon rubber and aluminum metal (inner diameter length 12 cm, width 7 cm, thickness 5 mm) was placed on the undried crosslinked gelatin / chitosan / calcium alginate laminated gel film obtained above. Cellmatrix IP (manufactured by Nitta Gelatin Co., Ltd.) 1 ml of Ham's F-12 medium 10-fold concentrated in 8 ml of Cellmatrix IP (manufactured by Nitta Gelatin Co., Ltd.) was added to the mixture which was mixed with a stirrer for 3 minutes under ice-cooling. _A solution prepared by adding 1 ml of ₃ g of 2.2 g or 4.7 g dissolved in 100 ml of 0.05 N NaOH aqueous solution and mixing without foaming was cast into the above frame in an amount of 6.5 ml. This was gelled at 37 ° C. for 3 hours, washed with distilled water and dried to obtain a crosslinked gelatin / chitosan / calcium alginate laminate / collagen layer modified film.

On the crosslinked gelatin / chitosan / calcium alginate laminate / collagen layer modified membrane obtained above, fibronectin (derived from Itoham's bovine plasma) was dissolved to a concentration of 1 mg / 1 ml. Cast to cm ² and dried at room temperature. The completed cell culture carrier was placed in a plastic bag and stored at room temperature. In this way, cell culture carrier A was prepared.

  A similar cell culture carrier could be prepared by using crosslinked polyacrylamide or crosslinked dextran instead of crosslinked gelatin.

  As comparative samples, samples prepared in the same manner as sample A were prepared as B and C, respectively, except that the sample was prepared without a cross-linked gelatin layer, and polymethyl methacrylate was applied and prepared using a solvent instead of cross-linked gelatin.

[Example 2] Evaluation of cell culture carrier The handleability of the cell culture carrier obtained in Example 1 was evaluated by the following method.
(1) Evaluation of curl The cell culture carrier was cut into strips having a width of 2 cm and a length of 5 cm, and the state of the sample after being left on a flat surface at 25 ° C. and 50% for 1 hour was evaluated according to the following criteria.

A: The strip is flat without curling.
○: Some curls are seen on the strip, and there is a part that floats 3mm above the plane.
Δ: Some curls are seen on the strip, and there is a part that rises 3 mm or more from the plane.
X: The curling of the strip is intense, and the portion that rises from the plane is 5 mm or more.
◎ and ○ are acceptable.

(2) Evaluation of the hydrophilicity / hydrophobicity of the sample The cell culture carrier was cut into a strip of 2 cm width and 10 cm length, and the contact angle between the cell adhesive surface and the opposite surface after standing at 25 ° C. and 50% for 1 hour. Measured and evaluated according to the following criteria. The results are shown in Table 1.

A contact angle is 30 to 60 degrees.
○: The contact angle is 60 to 70 degrees.
Δ: The contact angle is 70 to 90 degrees.
X: The contact angle is 90 degrees or more.
◎ and ○ are acceptable.

[Example 3] Sterilization of cell culture carrier The crosslinked gelatin / chitosan / calcium alginate laminate / collagen layer modified membrane obtained above was sterilized by UV sterilization for 1, 2, 3 hours, electron beam sterilization 20, 40, 60, 80, 100 When 6 types of sterilization of kGy were performed, no bacteria were found in any of them. At this time, 8000 cells / m ² of bacteria were confirmed from the sample that was not sterilized.

[Example 4] Cell culture using cell culture carrier (1) Cell culture Polystyrene cell culture petri dish and polystyrene with the cell culture carrier obtained in Example 1 placed with the collagen layer facing upward For the cell culture petri dish (comparative example), UV sterilization or electron beam sterilization was performed, and after adding the medium and immersing it for 5 minutes and then changing the medium three times, the medium was infiltrated into the cell culture carrier. It was. As the medium, Eagle's minimum medium (10% fetal bovine serum) was used.

BAE (bovine aortic vascular endothelial cells) cells cultured in advance were collected by trypsin treatment, and the cell concentration was adjusted to 35000 cell / ml. After discarding the medium in the petri dish, this cell solution was seeded in the petri dish so that the cell number was 7000 cell / cm ² and the medium was added. These were cultured at 37 ° C. for 3 days using a CO ₂ incubator.

(2) Results Among the cell culture carriers used, the polystyrene cell culture petri dish having the cell culture carrier of the present invention on the bottom has transparency, so that the growth state of the cells during cell culture can be observed in detail. There was no problem in cell adhesion and toxicity, and it was almost the same state as the sample of the petri dish for cell culture made of polystyrene alone.

  Similar results were obtained even when the cell type was changed to HEPG2 (human liver cancer-derived cells), CHL (Chinese hamster lung-derived cells) or CV-1 (African green monkey kidney-derived cells).

[Example 5] Separation and transfer of cell layer A sample cultured as in Example 4 using the cell culture carrier obtained in Example 1 was immersed in the following solubilization medium at 37 ° C for 20 minutes. The peeling state of the cell adhesive layer from the cell culture carrier was observed. Subsequently, the peeled cell adhesive layer sheet was placed on a polystyrene cell culture petri dish and a medium was added thereto, followed by culturing at 37 ° C. for 3 days using a CO ₂ incubator and observing with an optical microscope. . The evaluation of the peeling situation and the cell viability was performed according to the following criteria. The results are shown in Table 2.

<Solubilization medium>
(a) Eagle's minimum medium
(b) What added 200 mol% of 1-hydroxyethylidene-1,1-diphosphonic acid to the minimum Eagle medium with respect to the total number of calcium ions and magnesium ions present in the minimum Eagle medium

<Evaluation Criteria for Peeling Status> “A” and “B” are acceptable ranges.
(Double-circle): It peels so that a cell adhesion layer may float naturally from a polymer hydrous gel layer.
○: The cell adhesion layer can be easily detached from the polymer hydrous gel layer by pulling it with tweezers.
(Triangle | delta): Although it can peel from a polymer hydrous gel layer by pulling a cell adhesion layer with tweezers, there exists a part which does not peel.
X: Even if the cell adhesion layer is pulled with tweezers, it does not peel from the polymer hydrogel.

<Evaluation Criteria of Cell Viability> ◎ and ○ are acceptable.
A: More than 90% of cells are alive.
○: More than 70% of cells are alive.
Δ: More than 40% of cells are alive.
X: Living cells are 40% or less.

  As can be seen from the results in Table 2, the cell culture carrier of the present invention showed good exfoliation properties and high cell viability.

[Example 6] Multilayer culture of cell layer
The following cells were cultured in the same manner as in Example 4 in a petri dish for cell culture made of polystyrene that had been subjected to UV sterilization for 3 hours.
・ CHL (Chinese hamster lung-derived cells)
・ BRL (Buffalo Rat Liver 3A, ATCC No.: CRL 1442)
・ BAE (bovine aortic vascular endothelial cells)
・ Rat primary hepatocytes

  On each of the three types of cells cultured on the petri dish, the cells were transferred in the same manner as in Example 5 using the stripping solution (b) of Example 5 to obtain stratified cells. After culturing the stratified cells in the medium for 7 days or 14 days, staining with trypan blue and confirming the situation with an optical microscope, it was found that any stratified cells were well cultured in the region of the present invention. all right.

Claims (22)

A cell culture carrier having a multilayer structure including a polymer hydrous gel layer, wherein the cell culture carrier has a hydrophilic layer on a surface opposite to the cell culture surface side of the polymer hydrogel layer. The cell culture carrier according to claim 1, wherein the hydrophilic layer contains a natural polymer, a synthetic polymer or a mixed polymer thereof. The cell culture carrier according to claim 1 or 2, wherein the polymer hydrogel is chitosan. The cell culture carrier according to any one of claims 1 to 3, wherein the cell culture carrier further includes a gel layer containing an anionic polysaccharide and a polyvalent metal ion, and the gel layer is a polymer hydrogel layer. Adjacent cell culture carrier. The cell culture carrier according to claim 4, wherein the gel layer containing an anionic polysaccharide and a polyvalent metal ion is an alginate gel layer. The cell culture carrier according to claim 4, wherein the gel containing an anionic polysaccharide and a polyvalent metal ion is a calcium alginate gel. The cell culture carrier according to any one of claims 1 to 6, wherein the cell culture surface side outermost surface is a cell adhesive gel layer. The cell culture carrier according to claim 7, wherein the cell adhesive gel is a gel containing gelatin and / or collagen. The cell culture carrier according to claim 7, wherein the cell adhesive gel is a cell adhesive collagen gel. The cell culture carrier according to any one of claims 7 to 9, wherein the cell adhesive gel is modified with a cell adhesion factor. The cell culture carrier according to claim 10, wherein the cell adhesion factor is fibronectin, laminin, or vitronectin. The cell culture carrier according to any one of claims 1 to 11, wherein the cell culture carrier is sterilized by irradiation with one or a plurality of electron beams, γ rays, and ultraviolet rays. A method for culturing cells, comprising a step of culturing cells using the cell culture carrier according to any one of claims 1 to 12. A step of obtaining a cell sheet by solubilizing a gel layer containing an anionic polysaccharide and a polyvalent metal ion in a cell culture cultured using the cell culture carrier according to any one of claims 4 to 12. A cell culture method comprising. The cell culture method according to claim 14, wherein the solubilization treatment is performed using a medium containing a chelating agent and having a polyvalent metal cation of 2.6 mM or less. 16. A cell sheet obtained by the cell culture method according to claim 14 or 15. A step of further culturing a cell culture obtained by a cell culturing method including a step of culturing cells using the cell culture carrier according to any one of claims 4 to 12 on another cell culture carrier. Cell transcription method. A cell culture or a cell sheet obtained by solubilizing a gel layer containing an anionic polysaccharide and a polyvalent metal ion in a cell culture obtained by the cell transfer method according to claim 17. The cell culture or cell sheet according to claim 18, wherein the solubilization treatment is performed using a medium containing a chelating agent and having a polyvalent metal cation of 2.6 mM or less. A cell stratification method comprising a step of culturing a cell culture obtained by the cell transcription method according to claim 17 on another cultured cell. A cell stratification method comprising a step of culturing a cell culture cultured using the cell culture carrier according to any one of claims 4 to 12 on another cultured cell. A stratified cell culture or a stratified cell sheet obtained by solubilizing a gel layer containing an anionic polysaccharide and a polyvalent metal ion in a cell culture obtained by the cell stratification method according to claim 21.