JP2009082005A - Method for producing cell sheet with suppressed shrinkage - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique easily peeling a cell sheet from a cell culture carrier in producing the cell sheet, and also avoiding the cell sheet shrinkage. <P>SOLUTION: The technique comprises using a cell culture carrier comprising a substrate having a surface 45° or smaller in static water contact angle and an organic thin film laminated peelably on the surface of the substrate and having cell adhesiveness, and a shape-retaining sheet having sufficient mechanical strength and cell adhesiveness. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention can be used in fields related to cell tissue-based pharmaceuticals, for example, therapeutic fields such as regenerative medicine, drug discovery fields, diagnostic fields, examination fields, etc., a cell sheet with suppressed shrinkage and retained dimensions, a method for producing the same, The present invention relates to a cell culture carrier and a kit for producing the cell sheet.

  The technology for producing cell sheets is an important technology in the field of tissue engineering, but there are several problems.

  First, there is a problem that it is difficult to peel off a cell sheet cultured on a cell culture substrate from the substrate. In addition, even if the cell sheet can be peeled from the substrate, the cell sheet shrinks after peeling, and there is a problem that it becomes difficult to handle the cell sheet thereafter.

  For example, Patent Document 1 discloses a technique in which a temperature-responsive polymer is disposed on the surface of a cell culture substrate in order to facilitate cell sheet peeling, and the cell sheet is peeled off by changing the temperature after cell sheet culture. Yes. However, this technique is not applicable to all cell types, and some cell types are difficult to detach. Moreover, the problem that the cell sheet after peeling contracts has not been solved. Another problem is that it is difficult to make a thick cell sheet with this technique.

  Patent Document 2 discloses a technique for coating the surface of a support with fibrin glue for the purpose of facilitating peeling of a cell sheet. Patent Document 2 describes that fibrin glue is decomposed and lost by an enzyme contained in cells, and the cells remain suspended while being bound in the form of a sheet, so that the cell sheet can be easily peeled off. However, in Patent Document 2, it is difficult to say that sufficient peelability is achieved because the cell sheet is peeled using a scraper. Moreover, it is considered that some cell types cannot sufficiently degrade fibrin glue. Further, the problem that the cell sheet shrinks after peeling has not been solved.

  In addition to this, a technique for facilitating peeling of the cell sheet by controlling the adhesive force between the cell sheet and the culture substrate is disclosed (see, for example, Patent Documents 3 to 5). However, neither technique is applicable to all cell types. Moreover, the problem that the cell sheet contracts cannot be solved by these techniques.

  As a more difficult technique, there is a technique for multilayering cell sheets. In the field of tissue engineering, techniques for regenerating thin tissues such as skin and tissues with a small proportion of cells such as cartilage have already been put into practical use. There are also many clinical trials for injecting cells useful for treatment into the affected area. However, tissue engineering techniques have not yet been established for tissues with a large proportion of cells (as is the case with many tissues). In particular, in order to artificially manufacture a tissue body having high oxygen demand (a tissue body in which blood vessels are stretched) or a tissue body composed of parenchymal cells such as the heart and liver, the thickness of the cell sheet is set to 200 μm or more. However, there is a problem that when a normal cell sheet is laminated more than this thickness, oxygen does not reach the internal cells and necroses.

  So far, for the purpose of supplying oxygen to cells, many techniques have been studied in which cells are seeded on a porous scaffold (carrier) made of, for example, a bioabsorbable material to form a cell tissue body. However, these techniques have problems in that it is difficult to uniformly distribute cells to the inside of the scaffold, and the transplanted tissue is easily fibrillated.

  In addition, there is a problem that when the cell sheets are multilayered, contraction is more likely to occur compared to a single-layer cell sheet.

  Furthermore, when a plurality of cell sheets are stacked, if the distance between adjacent cell sheets can be shortened, network formation and interaction between the cell layers can be facilitated, and a regenerated tissue closer to the actual living tissue can be produced. It will be possible.

  Patent Documents 6 and 7 disclose a technique for culturing cells on a hydrogel thin film that has been vitrified to enhance its shape maintenance power. The hydrogel thin film such as collagen described in Patent Documents 6 and 7 can suppress shrinkage of the cell sheet cultured on the thin film by providing a support around the collagen. Further, since the cell sheet can be dynamically detached from the substrate, the cell sheet can be recovered with minimal invasiveness to the cells without lowering the temperature as in Patent Document 1 or requiring a digestive enzyme as in Patent Document 5. However, when the cell sheet is formed by culturing cells on the thin film with the hydrogel thin film described in Patent Documents 6 and 7 placed on the base material, the cell sheet is interposed between the hydrogel thin film. When the hydrogel thin film and the base material are strongly bonded, it is difficult to peel and collect the hydrogel thin film in a state in which the cell sheet is formed from the base material. There was a problem. In order to solve this problem, the purpose of suppressing the adhesion of the cell sheet to the substrate surface and the purpose of increasing the strength of the hydrogel thin film in order to prevent the cell sheet from being broken when the cell sheet is peeled off. It was necessary to thicken the gel thin film. However, a multilayered cell sheet obtained by laminating a plurality of cell sheets formed on a thick hydrogel thin film is not suitable as a regenerative tissue because the distance between adjacent cell sheets is long. .

International Publication WO2002 / 008387 Pamphlet International Publication WO2005 / 028638 Pamphlet JP 2006-346292 A JP 2006-94799 A JP 2005-261292 A International Publication WO2005 / 014774 Pamphlet JP-A-8-228768

  As described above, in the production of a cell sheet, a satisfactory technique that can easily peel the cell sheet from the cell culture carrier and avoids the contraction of the cell sheet has not yet been established. In addition, a technique suitable for multilayering cell sheets has not been provided yet.

  The present invention aims to solve these problems.

The present invention includes the following inventions.
(1) A cell comprising a substrate having a surface with a static water contact angle of 45 ° or less, and an organic thin film having cell adhesion, which is detachably laminated on the surface of the substrate. Culture carrier.
(2) The cell culture carrier according to (1), wherein the dry weight per unit area of the organic thin film is less than 5 μg / cm ² .
(3) The cell culture carrier according to (1), wherein the thickness of the organic thin film when dried is 1 μm or less.
(4) The cell culture carrier according to any one of (1) to (3), wherein the substrate surface is cell adhesion inhibitory.
(5) The cell culture carrier according to any one of (1) to (4), wherein the organic thin film is composed of a biological material.
(6) The cell culture carrier according to any one of (1) to (4), wherein the organic thin film is composed of an artificially synthesized biological material mimicking material.
(7) The cell culture carrier according to any one of (1) to (4), wherein the organic thin film is composed of a mixture of artificially synthesized biological material mimicking material and biological material.
(8) The cell culture carrier according to any one of (1) to (7), wherein the organic thin film is patterned.
(9) A sheet-like cell layer and a cell-adhesive organic thin film layer on a shape-retaining sheet having sufficient strength to suppress contraction of the sheet-like cell layer Is a method for producing a contraction-inhibiting cell sheet in which at least one layer is alternately laminated on the organic thin film of the cell culture carrier according to any one of (1) to (8) The cell layer formed in a sheet shape is laminated and bonded so that the stacking direction of the shape-retaining sheet is in contact with the cell layer, and the cell layer and the organic thin film are alternately formed on the surface of the shape-retaining sheet. The method according to claim 1, wherein after the laminated body is obtained by laminating and bonding, a laminating step of peeling the laminated body from the base material of the cell culture carrier is repeated one or more times.
(10) A sheet-like cell layer produced on the shape-retaining sheet, which is produced by the method according to (9) and has sufficient strength to suppress contraction of the sheet-like cell layer and is cell-adhesive And a contraction-inhibiting cell sheet in which at least one layer of an organic thin film having cell adhesiveness is alternately laminated.
(11) The cell culture carrier according to any one of (1) to (8), a shape-retaining sheet having sufficient strength to suppress contraction of the sheet-like cell layer, and cell adhesiveness A kit for producing a contraction-inhibiting cell sheet.

  According to the present invention, there is provided a cell sheet that can be easily detached from a cell culture carrier and in which shrinkage after peeling is suppressed. The peeling of the cell sheet according to the present invention does not use the change in the binding property between the cells and the substrate, and can be applied regardless of the cell type. If the organic thin film is appropriately patterned in the cell culture carrier of the present invention, the distribution of cells can be easily controlled, and a thick tissue can be formed. In addition, the sheet-like cell layer and the cell adhesion are obtained on the shape-retaining sheet having sufficient strength to suppress contraction of the sheet-like cell layer obtained by the present invention and having cell adhesion. The multi-layered shrinkage-suppressing cell sheet, in which a plurality of organic thin film layers are alternately laminated, is considered to easily form a network between adjacent cell layers because the organic thin film is very thin. Klein-like cell-layer interactions can also be expected.

(Structure of cell culture carrier)
The structure of the cell culture carrier of the present invention will be described with reference to FIG. FIG. 1 is a sectional view of a cell culture carrier 1 of the present invention. The cell culture carrier 1 includes an organic thin film 2 having cell adhesion and a substrate 4 having a surface 3 having a static water contact angle of 45 ° or less. The organic thin film 2 is detachably installed on the surface 5 of the substrate 6.

(How to use cell culture carrier)
An example of how to use the cell culture carrier 1 will be described with reference to FIG.
First, as shown in FIG. 2A, desired cells are cultured on the organic thin film 2 to form a sheet-like cell layer 5. Next, as shown in FIG. 2 (b), the shape-retaining sheet 6 having sufficient strength to suppress the contraction of the sheet-like cell layer and being cell-adhesive is formed on the cell layer 5. The holding sheet 6 is laminated so that the stacking direction is in contact with the cell layer 5, and incubated to adhere the cell layer 5 to the shape holding sheet 6, and the cell layer 5 and the organic thin film 2 are attached to the shape holding sheet 6. The laminated body 7 formed by alternately laminating and bonding is formed. Next, as shown in FIG. 2C, the laminate 7 is peeled from the base material 4. The laminate 7 thus obtained is a contraction-inhibiting cell sheet in which a sheet-like cell layer 5 and an organic thin film layer 2 are alternately laminated on a shape-retaining sheet 6.

  In order to make a cell layer multi-layered, the lamination process shown in FIGS. 2A to 2C is repeated twice or more. In the n-th lamination step (n is an integer of 2 or more), the laminate obtained in the (n-1) -th lamination step is used instead of the shape-retaining sheet in the first lamination step. That is, in the n-th (n is an integer of 2 or more) laminating step, the cell layer and the organic thin film obtained in the n-1 th laminating step are alternately laminated on the surface of the shape-retaining sheet. The adhered n-1th laminate is in contact with the cell layer formed in the form of a sheet on the organic thin film of the cell culture carrier of the present invention so that the lamination direction of the n-1th laminate is in contact with the cell layer. After stacking and adhering to obtain an n-th laminated body in which n layers of cell layers and organic thin films are alternately laminated and adhered to the surface of the shape-retaining sheet, the n-th laminated body is used as a base material for the cell culture carrier. It is the process of peeling from.

  Specifically, the laminated body 7 is brought into contact with the cell layer 5 ′ formed in a sheet shape on the organic thin film 2 ′ of the further cell culture carrier 1 ′, and the lamination direction of the laminated body 7 is in contact with the cell layer 5 ′. After further laminating a cell layer 5 ′ and an organic thin film layer 2 ′ on the laminate 7 by laminating and incubating them (FIGS. 2 (d) to 2 (e)), Two laminated body 7 'is peeled from base material 4' (FIG.2 (f)). The thus obtained second laminate 7 ′ is a contraction-suppressing cell in which sheet-like cell layers 5 and 5 ′ and organic thin film layers 2 and 2 ′ are alternately laminated on the shape-retaining sheet 6. It is a sheet.

  Further, the second laminated body 7 ′ is placed on the cell layer 5 ″ formed in a sheet shape on the organic thin film 2 ″ of the further cell culture carrier 1 ″, and the stacking direction of the second laminated body 7 ′ is the cell layer. After laminating to contact 5 ″, incubating and adhering, a further cell layer 5 ″ and organic thin film layer 2 ″ are laminated on the second laminate 7 ′ (FIG. 2 (g ) To (h)), the obtained third laminate 7 ″ is peeled from the substrate 4 ″ (FIG. 2 (i)).

  Shrinkage formed by alternately stacking n cell layers and n organic thin film layers on the shape-retaining sheet 6 by repeating the stacking process shown in FIGS. 2A to 2C n times. A suppressor cell sheet is formed. Although the example which repeated the said process 3 times was shown in FIG. 2, the repetition frequency is not specifically limited.

  The organic thin film may be patterned. As an example, FIG. 3 shows a cell culture carrier 11 in which a patterned organic thin film 12 is detachably arranged on a surface 13 of a base material. The organic thin film 12 is patterned so as to have two openings, and the surface 13 of the substrate is exposed. Since the surface 13 of the base material has a static water contact angle of 45 ° or less and is hydrophilic, the cells do not adhere and a cell layer (not shown) is formed along the pattern of the organic thin film 12. The cell sheet of the present invention produced using the cell culture carrier 11 having the organic thin film 12 patterned in this way has a hole at a desired position. Since the multilayered cell sheet of the present invention obtained by laminating a plurality of such cell sheets has a void at a desired position, oxygen or the like can be supplied to the inside of the thick cell sheet, It is expected that problems such as necrosis can be solved.

Next, the configuration of the present invention will be described in detail.
(Organic thin film)
Although an organic thin film will not be specifically limited if it has cell adhesiveness, Preferably it consists of biological material. The organic thin film is preferably biocompatible, particularly biodegradable. Specific materials include various collagens, peptide hydrogels, laminin, chondronectin, glycosaminoglycan, hyaluronic acid, proteoglycan, extracellular matrix component proteins other than those described above, and basement membrane reconstituted from mouse EHS tumor extract Examples thereof include gels of components (trade name: Matrigel), polymer compounds such as gelatin, agarose, oligonucleic acid, and polynucleic acid. The sheet prepared by rehydrating after vitrifying the hydrated gel, which is called “hydrogel thin film” in Patent Documents 6 and 7, is also used as the organic thin film of the present invention. it can. When a gelatin thin film is used as the organic thin film, it is preferable to use a gelatin thin film crosslinked by ultraviolet irradiation.

  A frame-like support for holding the dimensions of the organic thin film may be fixed to the peripheral portion of the organic thin film. However, in the present invention, such a support may not exist on the peripheral portion of the organic thin film. preferable.

  In consideration of the possibility that the therapeutic use of animal-derived biological materials will be restricted in the future, artificially synthesized biological material imitation materials are also materials that constitute organic thin films. As preferred. Examples thereof include gels made of polymer compounds such as various artificial peptides and derivatives thereof, artificial oligopeptides and derivatives thereof, artificial polypeptides and derivatives thereof, and artificial polysaccharides and derivatives thereof.

  Furthermore, an artificially synthesized biomimetic material mimic material and a biomaterial-derived mixture are also preferable as a material constituting the organic thin film. For example, it is preferable to use a mixture of an artificial peptide that is a biomaterial-mimetic material and gelatin that is a biomaterial.

The thickness of the organic thin film is such that the dry weight per unit area is, for example, 100 μg / cm ² or less, preferably 40 μg / cm ² or less, more preferably less than 5 μg / cm ² . Further, the lower limit of the thickness of the organic thin film is not particularly limited, but a thickness in which the dry weight per unit area is 2.5 μg / cm ² or more is preferable.

  The dry thickness of the organic thin film is also preferably 1 μm or less. Further, the lower limit of the thickness of the organic thin film is not particularly limited, but is preferably 100 nm or more, for example. Here, the numerical value of the thickness of the organic thin film is a numerical value obtained by measuring the average thickness using a stylus type surface shape measuring instrument (Dektak FPD-650, Japan Vacuum Technology).

  According to the method of the present invention, such a very thin layer of organic thin film can be used. For this reason, it is considered that the contraction-suppressing cell sheet obtained by the method of the present invention is likely to form a network between cell layers, and a paracrine-like cell layer interaction can also be expected. The shrinkage-suppressing cell sheet obtained in the present invention is easy to handle even with a thin organic thin film that can be expected to have such advantageous effects because the shape-retaining sheet with sufficient strength suppresses dimensional changes due to shrinkage. It is. Further, the base material having a surface with a static water contact angle of 45 ° or less used in the present invention has a sufficiently weak adhesive force with an organic thin film (however, it does not peel off naturally under normal cell culture conditions). Therefore, even a very thin organic thin film can be recovered without being damaged at the time of peeling from the substrate surface.

  The organic thin film may be patterned into a desired shape. As a patterning method, after preparing an organic thin film, the organic thin film is etched only in a specific region with a dissolving agent, a liquid organic material is directly applied to the substrate in a pattern by an inkjet or the like, and dried. Then, there are methods such as preparing an organic thin film and removing the resist, and applying a liquid organic material to the substrate through a mask having a hole and drying.

(Base material)
The substrate has a surface with a static water contact angle of 45 ° or less, and the organic thin film is detachably laminated on the surface. A substrate surface having such a static water contact angle generally tends to have cell adhesion inhibition properties. If the surface of the substrate is cell-adhesive, when the organic thin film is made very thin as described above, the cultured cells adhere to the substrate surface via the organic thin film, or the cells adhere to the organic thin film. Although it is decomposed by an enzyme and adheres to the surface of the base material, it is difficult to peel the formed shrinkage-suppressing cell sheet from the base material, but there is no such problem if the base material surface is cell adhesion inhibitory. Even when the organic thin film is very thin as described above, the shrinkage-inhibiting cell sheet can be easily peeled off from the substrate. Such a cell adhesion-inhibiting surface can be obtained by forming a film of an organic compound having a carbon-oxygen bond on the surface of the substrate.

  The base material is preferably a material capable of forming a film of an organic compound having a carbon-oxygen bond on the surface thereof. Specifically, inorganic materials such as metals, glass, ceramics, silicon, elastomers, plastics (for example, polyester resins, polyethylene resins, polypropylene resins, ABS resins, nylons, acrylic resins, fluororesins, polycarbonate resins, polyurethane resins, methyl resins) Organic materials represented by pentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride resin). The shape is not limited, and examples thereof include a flat shape such as a flat plate, a flat membrane, a film, and a porous membrane, and a three-dimensional shape such as a cylinder, a stamp, a multiwell plate, and a microchannel. When using a film, the thickness in particular is not restrict | limited, However, Usually, it is 0.1-1000 micrometers, Preferably it is 1-500 micrometers, More preferably, it is 10-200 micrometers.

  The cell adhesion-inhibiting surface can be formed by a hydrophilic film formed of an organic compound having a carbon-oxygen bond and having a static water contact angle of 45 ° or less.

  In the present invention, the carbon-oxygen bond means a bond formed between carbon and oxygen, and is not limited to a single bond but may be a double bond. Examples of the carbon-oxygen bond include a C—O bond, a C (═O) —O bond, and a C═O bond.

  Examples of the main raw material for the hydrophilic film include hydrophilic organic compounds such as water-soluble polymers, water-soluble oligomers, water-soluble organic compounds, surface active substances, and amphiphilic substances. These are physically or chemically cross-linked with each other and physically or chemically bonded to the substrate to form a hydrophilic film.

  Specific water-soluble polymer materials include polyalkylene glycol and derivatives thereof, polyacrylic acid and derivatives thereof, polymethacrylic acid and derivatives thereof, polyacrylamide and derivatives thereof, polyvinyl alcohol and derivatives thereof, and zwitterionic polymers. , Polysaccharides, and the like. Examples of the molecular shape include a straight chain, a branched one, and a dendrimer. More specifically, polyethylene glycol, a copolymer of polyethylene glycol and polypropylene glycol, such as Pluronic F108, Pluronic F127, poly (N-isopropylacrylamide), poly (N-vinyl-2-pyrrolidone), poly (2- Hydroxyethyl methacrylate), poly (methacryloyloxyethylphosphorylcholine), copolymers of methacryloyloxyethylphosphorylcholine and acrylic monomers, dextran, and heparin, but are not limited thereto.

  Specific water-soluble oligomer materials and water-soluble low molecular weight compounds include alkylene glycol oligomers and derivatives thereof, acrylic acid oligomers and derivatives thereof, methacrylic acid oligomers and derivatives thereof, acrylamide oligomers and derivatives thereof, saponified vinyl acetate oligomers and Derivatives thereof, oligomers composed of zwitterionic monomers and derivatives thereof, acrylic acid and derivatives thereof, methacrylic acid and derivatives thereof, acrylamide and derivatives thereof, zwitterionic compounds, water-soluble silane coupling agents, water-soluble thiol compounds, etc. be able to. More specifically, ethylene glycol oligomer, (N-isopropylacrylamide) oligomer, methacryloyloxyethylphosphorylcholine oligomer, low molecular weight dextran, low molecular weight heparin, oligoethylene glycol thiol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene Glycols, 2- [methoxy (polyethyleneoxy) -propyltrimethoxysilane, and triethylene glycol-terminated-thiols, but are not limited to these.

  The average thickness of the hydrophilic film is preferably 0.8 nm to 500 μm, more preferably 0.8 nm to 100 μm, more preferably 1 nm to 10 μm, and most preferably 1.5 nm to 1 μm. If the average thickness is 0.8 nm or more, it is preferable because protein adsorption and cell adhesion are not easily affected by the region not covered with the hydrophilic film on the substrate surface. Moreover, if the average thickness is 500 μm or less, coating is relatively easy.

  The hydrophilic film can be formed on the substrate surface by directly adsorbing the hydrophilic organic compound on the substrate, coating the hydrophilic organic compound directly on the substrate, or coating the hydrophilic organic compound on the substrate. A method of performing a crosslinking treatment later, a method of forming a hydrophilic film in a multi-stage manner in order to increase the adhesion to the substrate, a base layer is formed on the substrate in order to increase the adhesion to the substrate, Examples thereof include a method of coating a hydrophilic organic compound, a method of forming a polymerization initiation point on the substrate surface, and then polymerizing a hydrophilic polymer brush.

  Among the above film forming methods, particularly preferable methods include a method of forming a hydrophilic film in a multistage manner, and a base layer is formed on the base material in order to improve adhesion to the base material, and then hydrophilic organic Mention may be made of a method of coating a compound. This is because using these methods makes it easy to enhance the adhesion of the hydrophilic organic compound to the substrate. In this specification, the term “bonding layer” is used. The binding layer means a layer existing between the outermost hydrophilic film layer and the substrate in the case of forming a hydrophilic organic compound film in a multistage manner, and a base layer is provided on the substrate surface. When a hydrophilic film layer is formed on a base layer, the base layer is meant. The binding layer is preferably a layer containing a material having a binding portion (linker). Examples of the combination of the linker and the functional group at the end of the material to be bonded to the linker include epoxy group and hydroxyl group, phthalic anhydride and hydroxyl group, carboxyl group and N-hydroxysuccinimide, carboxyl group and carbodiimide, amino group and glutaraldehyde, etc. Is mentioned. In each combination, any of them may be a linker. In these methods, before coating with a hydrophilic material, a bonding layer is formed of a material having a linker on a substrate. The density of the material in the tie layer is an important factor that defines the bond strength. The density can be easily evaluated using the contact angle of water on the surface of the bonding layer as an index. For example, taking a silane coupling agent having an epoxy group at the end (epoxysilane) as an example, the water contact angle of the substrate surface to which epoxysilane is added is typically 45 ° or more, preferably 47 ° or more. Then, a substrate having sufficient cell adhesion inhibitory property can be made by adding an ethylene glycol-based material or the like in the presence of an acid catalyst.

(Shape retention sheet)
The shape-retaining cell sheet for producing the shrinkage-suppressing cell sheet has cell adhesiveness, and depending on the contraction force of the cell sheet when the cell sheet adheres to one surface of the shape-retaining sheet. There is no particular limitation as long as it has strength that does not deform (in the present invention, it is called “strength sufficient to suppress shrinkage of the cell sheet”). The shape-maintaining sheet is preferably biocompatible, particularly biodegradable. For example, polylactic acid, poly (lactic acid-butyric acid) copolymer, polybutyric acid, polyglycolic acid, collagen and its crosslinked body, gelatin and its crosslinked body, gel of biodegradable polymer material such as artificial polypeptide and its crosslinked body A sheet having a sufficient strength to suppress shrinkage of the cell sheet (typically, the dry weight per unit area of the polymer material constituting the gel is 100 μg / cm ^{2 to 1} mg / cm ^2, preferably the sheet is ^{200μg / cm 2 ~500μg / cm 2} , or a thickness after drying of the sheet) and the like is 0.5 mm to 5 mm. The sheet prepared by rehydrating after vitrifying the hydrated gel, which is called “hydrogel thin film” in Patent Documents 6 and 7, is a cell sheet as described above. As long as it has sufficient strength to suppress the shrinkage of the sheet, it can be used as a shape-retaining sheet in the present invention. When the shape-retaining sheet is laminated and adhered to the cell layer formed in the form of a sheet on the organic thin film of the cell culture carrier of the present invention, the shape-retaining sheet usually has a cell culture medium such as a cell culture solution. Absorbs liquid that does not adversely affect

(Method for producing cell culture carrier)
The method for producing the cell culture carrier of the present invention is not particularly limited. For example, the organic thin film forming step such as gelation is completed in a state where the solution for forming the organic thin film is applied on the surface of the base material. Can be manufactured.

(cell)
The cells cultured using the cell culture carrier of the present invention may be floating cells such as blood cells and lymphoid cells, and may be adhesive cells, but the present invention is suitable for cells having adhesiveness. used. Examples of such cells include hepatocytes, Kupffer cells, endothelial cells such as vascular endothelial cells and corneal endothelial cells, fibroblasts, osteoblasts, osteoclasts, periodontal ligament-derived cells, Epidermal cells such as epidermal keratinocytes, tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, epithelial cells such as corneal epithelial cells, breast cells, pericytes, muscle cells such as smooth muscle cells and cardiomyocytes, kidneys Examples include cells, pancreatic islets of Langerhans, nerve cells such as peripheral nerve cells and optic nerve cells, chondrocytes, and bone cells. These cells may be primary cells collected directly from tissues or organs, or may be passaged from one generation to another. Furthermore, these cells are undifferentiated embryonic stem cells, pluripotent stem cells such as pluripotent mesenchymal stem cells, unipotent stem cells such as vascular endothelial progenitor cells that have unipotency, and differentiation is completed. Any of the cells may be used. In addition, the cells may be cultivated as a single species, or two or more types of cells may be co-cultured.

(culture)
By culturing the cell culture carrier seeded with these cells in a culture solution, a sheet-like cell layer can be formed. Any culture medium can be used without particular limitation as long as it is a cell culture medium usually used in the art. For example, depending on the type of cells used, MEM medium, BME medium, DME medium, αMEM medium, IMDM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, RPMI1640 medium, etc. A basal medium as described in “Tissue culture technology third edition”, page 581, edited by the Japanese Society for Tissue Culture can be used. Furthermore, serum (such as fetal bovine serum), various growth factors, antibiotics, amino acids and the like may be added to the basal medium. A commercially available serum-free medium such as Gibco serum-free medium (Invitrogen) can also be used. Considering the clinical application of the finally obtained cell tissue, it is preferable to use a medium that does not contain animal-derived components.

  EXAMPLES The present invention will be specifically described below based on examples, but the contents of the present invention are not limited to the contents of the examples.

Substrate preparation and cell sheet collection
1-1. Preparation of a substrate having a static water contact angle of 45 ° or less and cell adhesion inhibition 39.0 g of toluene and 0.8 g of TSL8350 (manufactured by GE Toshiba Silicone) were mixed, and 450 μl of triethylamine was added while stirring. After stirring at room temperature for several minutes, the whole amount was transferred to a glass dish. A 10 cm square glass substrate that had been cleaned with UV was immersed therein and allowed to stand at room temperature for 16 hours. Thereafter, the glass substrate was washed with ethanol and water and dried by nitrogen blowing. Next, 25 μl of concentrated sulfuric acid was added dropwise while stirring 50 g of tetraethylene glycol (TEG). After stirring for several minutes, the whole amount was transferred to a glass dish. The above substrate was immersed therein and reacted at 80 ° C. for 20 minutes. After the reaction, the substrate was thoroughly washed with water and dried by nitrogen blowing. Thereby, the membrane | film | coat containing TEG was formed in the glass substrate surface. The surface static water contact angle was approximately 30 °. This substrate was cut into a size of 25 mm × 15 mm and used as a base material having a static water contact angle of 45 ° or less and cell adhesion inhibition. After autoclaving the substrate, add an appropriate amount of 5% fetal bovine serum-containing MEM medium, seeding 2.0 × 10 ⁵ bovine aortic vascular endothelial cells per substrate, and incubating in an incubator (37 ° C., 5% CO _When cultured for 24 hours in ₂ ), no cells adhered to the surface of the substrate.

1-2. Preparation of collagen thin film and thickness measurement Collagen solution was prepared to a final concentration of 2.4 mg / ml on ice using a collagen gel culture kit (Cellmatrix IA, Nitta Gelatin). 4 μl of this was added onto the base material prepared in 1-1 and gelled at 37 ° C. for 1 hour, and then dried in a clean bench for 3 hours to prepare a collagen thin film (collagen content per base unit area: 2 .6 μg / cm ² ). It was 100 nm as a result of measuring the average thickness of a collagen thin film with the stylus type surface shape measuring device (Dektak FPD-650, Japan Vacuum Technology). The collagen thin film thus prepared was adhered to the substrate even after PBS was added and hydrated.

1-3. Appropriate amount of 5% fetal bovine serum-containing MEM medium was added to the collagen thin film produced in cell sheet recovery 1-2. Here, 2.0 × 10 ⁵ bovine aortic vascular endothelial cells were seeded per substrate. When cultured for 24 hours in the incubator, the cells were confluent due to adhesion and proliferation of the collagen thin film. A thick collagen gel with a thickness of about 1 mm prepared on top of the confluent cells is layered, and the medium is sucked so that the cells are slightly immersed in the medium. The cells were cultured for 4 hours to adhere the collagen gel. Thereafter, the cell sheet was desorbed and recovered from the substrate by generating a water flow in the medium with a pipette or the like and applying a physical force to the cells. Moreover, the cell did not adhere at all to the base material after peeling the cell sheet. As a result of observing the thus prepared cell sheet with a phase contrast microscope (IX71, Olympus) (FIG. 4), the cell sheet did not cause cell contraction and remained as it was.

  The collagen gel used above was prepared by preparing a collagen solution to a final concentration of 2.4 mg / ml on ice using a collagen gel culture kit (Cellmatrix IA, Nitta Gelatin). This amount was gelled at 37 ° C. for 1 hour.

Lamination of cell sheets An appropriate amount of MEM medium containing 5% fetal bovine serum was added to the collagen thin film prepared in Example 1-2. Here, 2.0 × 10 ⁵ bovine aortic vascular endothelial cells were seeded per substrate. When cultured for 24 hours in an incubator, the cells were confluent due to adhesion and proliferation on the collagen thin film. The cell sheet prepared in Example 1 is overlaid on the confluent cells, the medium is sucked to such an extent that the cell sheet is slightly immersed in the medium, and the cell sheet is brought into contact with the confluent cells due to surface tension, and is stacked for 4 hours. Cells confluent with the cell sheet were allowed to adhere. Thereafter, the multilayer cell sheet could be detached and recovered from the substrate by generating a water flow in the medium with a pipette or the like and applying physical force to the cells. In a similar manner, this multilayer cell sheet was stacked on the above confluent cells and removed from the substrate, and the number of stacks was increased, so that four layers could be stacked.

Cell sheet collection by organic thin film other than collagen Base material prepared by 1-1 with 40 μl of a solution prepared by mixing 10 mg / ml peptide hydrogel before gelation and 20 mg / ml gelatin solution in a ratio of 10: 1 In addition to the above, it was dried in a clean bench for 3 hours to produce an organic thin film. When bovine aortic vascular endothelial cells were seeded on this organic thin film in the same manner as in Example 1-3, the cells became confluent due to adhesion and proliferation on the organic thin film. Thereafter, the cell sheet was desorbed and recovered from the substrate by generating a water flow in the medium with a pipette or the like and applying a physical force to the cells. Moreover, the cell did not adhere at all to the base material after peeling the cell sheet. As a result of observing the thus prepared cell sheet with a phase-contrast microscope (IX71, Olympus) (FIG. 5), the cell sheet did not cause cell contraction and remained as it was.

FIG. 1 shows a cross-sectional view of the cell culture carrier of the present invention. FIG. 2 shows the procedure of the method for producing the contraction-inhibiting cell sheet of the present invention. It is a figure following FIG. 2A which shows the procedure of the manufacturing method of the contraction suppression cell sheet | seat of this invention. FIG. 3 is a plan view of an embodiment of the cell culture carrier of the present invention in which an organic thin film is patterned. FIG. 4 shows the observation results of Example 1-3. FIG. 5 shows the observation results of Example 3.

Explanation of symbols

1, 1 ', 1'', 11 ... cell culture carrier 2, 2', 2 ", 12 ... organic thin film 3, 13 ... surface 4 having a static water contact angle of 45 ° or less 4 ′, 4 ″... Base material 5, 5 ′, 5 ″... Cell layer 6... Shape-retaining sheet 7, 7 ′, 7 ″. Sheet)

Claims (11)

  A cell culture carrier comprising: a base material having a surface with a static water contact angle of 45 ° or less; and an organic thin film having cell adhesion, which is detachably laminated on the surface of the base material. The cell culture carrier according to claim 1, wherein the dry weight per unit area of the organic thin film is less than 5 µg / cm ² .   The cell culture carrier according to claim 1, wherein the thickness of the organic thin film when dried is 1 µm or less.   The cell culture carrier according to any one of claims 1 to 3, wherein the substrate surface is cell adhesion inhibitory.   The cell culture carrier according to any one of claims 1 to 4, wherein the organic thin film is composed of a biological material.   The cell culture carrier according to any one of claims 1 to 4, wherein the organic thin film is composed of an artificially synthesized biological material-mimetic material.   The cell culture carrier according to any one of claims 1 to 4, wherein the organic thin film is composed of a mixture of artificially synthesized biological material mimicking material and biological material.   The cell culture carrier according to any one of claims 1 to 7, wherein the organic thin film is patterned. A sheet-like cell layer and an organic thin film layer having cell adhesion are at least provided on a shape-retaining sheet having sufficient strength to suppress contraction of the sheet-like cell layer and cell adhesion. A method for producing a contraction-inhibiting cell sheet, in which each layer is alternately laminated,
The said shape maintenance sheet | seat is a cell layer formed in the sheet form on the organic thin film of the cell culture carrier of any one of Claims 1-8, The lamination direction of the said shape maintenance sheet | seat is said cell layer. After laminating and adhering so as to be in contact with each other to obtain a laminate in which cell layers and organic thin films are alternately laminated and adhered to the surface of the shape-retaining sheet, the laminate is peeled from the substrate of the cell culture carrier The above-described method is characterized in that the laminating step is repeated one or more times.   A sheet-like cell layer and a cell on the shape-retaining sheet, which is produced by the method according to claim 9 and has sufficient strength to suppress contraction of the sheet-like cell layer and is cell adhesive. A shrinkage-suppressing cell sheet in which at least one layer of an organic thin film having adhesive properties is alternately laminated.   The cell culture carrier according to any one of claims 1 to 8, and at least a shape-retaining sheet having sufficient strength to suppress contraction of the sheet-like cell layer and being cell-adhesive, A kit for producing a contraction-inhibiting cell sheet.

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