JP5080848B2 - Cell culture support and production method thereof - Google Patents

Description

  The present invention relates to a cell culture support for stably mass-producing production of cell sheets.

  The cell sheet is a sheet-like cell aggregate in which cells are connected to each other in at least a single layer by intercellular bonding. Cell sheets are used in regenerative medicine. The cell sheet can be obtained by culturing cells on a support such as a petri dish, but the cell sheet formed on the support is firmly bonded to the support surface via an adhesion molecule or the like. It is not easy to quickly peel the cell sheet from the culture support without breaking the bonds between the cells.

  Thus, methods for efficiently peeling a cell sheet from a cell culture support have been studied so far. The peeling method can be roughly divided into two. The first method is a method of weakening the bond between the support and the cells using an enzyme reaction. The second method is a method using a support with weak cell adhesion or a support with varying cell adhesion.

  In the first method, specifically, an enzyme is used to intercellular adhesion molecules such as protease (proteolytic enzyme) and collagenase (collagen degrading enzyme) (tight bond, adhesive bond, desmosome bond, gap bond, hemidesmosome). It is a method of degrading the protein constituting the binding), the collagen-binding tissue surrounding the periphery of the culture, and the extracellular matrix (Extracellular Matrix: ECM) formed between the cells and the support. This method weakens not only cell-support surface binding but also cell-cell binding. This method has long been used in the field of cell culture. Since the binding substance decomposed by this method is a substance produced in cells, tissues, and organs to be cultured, the binding substance decomposed under a certain condition and period can be regenerated even after detachment.

  However, there is a problem that it takes time to regenerate the binding substance. In addition, this method is not desirable as a method for producing a cell sheet used for regenerative medicine because the cell sheet is damaged in many ways.

  Then, the 2nd method using the support body with weak cell adhesive force and the support body with which cell adhesive force changes is newly developed.

  Examples of the support having a weak cell adhesion include those disclosed in Patent Document 1 and Patent Document 2. These documents disclose a technique in which ultra-fine pillars called nanopillars are set on the surface of a support and culture is performed thereon. According to this technique, the support and the culture material are adhered to each other only in a very small area, and the recovery and peeling are easy and the damage is small.

  However, as described in Non-Patent Documents 1 and 2, the cell adhesion and the behavior of the adhered cells are different between the case of adhering to a flat surface and the case of adhering to an uneven surface, and cell adhesion and extension are slow on the nanopillar. And there is a problem that a temporary foot is generated from the cell surface. In addition, when the concave portion has a width of 20 μm or more, there is a problem that cells infiltrate.

  As a support that changes cell adhesion, there is a support in which a cell growth surface is coated with a temperature-responsive polymer (Patent Document 3). For the purpose of changing the cell adhesive force, it is most preferable to use a temperature-responsive polymer, but other than that, a pH-responsive polymer or an ion-responsive polymer can also be used. Patent Documents 1 and 2 mention that a temperature-responsive polymer is combined with culture using a nanopillar. Patent Document 4 also mentions the use of a temperature-responsive polymer in cell culture.

  In Patent Document 3, as a method for producing a temperature-responsive polymer layer, a monomer polymerization reaction and at least one end of the temperature-responsive polymer are covalently bonded to a molecule constituting the substrate by electron beam irradiation to form a substrate surface. A graft polymerization method in which a temperature responsive polymer is immobilized (grafted) is described.

  On the other hand, in Patent Document 15, when culturing epithelial cells on a porous membrane for cell culture insert, both the upper layer portion and the lower layer portion are filled with a medium with the porous membrane as a boundary, and the cells are cultured. In the meantime, a method for stratifying and culturing epithelial cells in which a medium is supplied to cells from the lower layer through a porous membrane is disclosed. Patent Document 15 discloses disposing a temperature-responsive polymer on the porous film. The technique of insert culture disclosed in Patent Document 15 is a method for culturing cells on a permeable porous membrane aiming at in vitro cell culture that is physiologically similar to the in vivo environment. Similarly, it is a method that allows components contained in a liquid medium to diffuse to both the luminal side and the basement membrane side of cells. By using the technique of insert culture, three-dimensional culture, co-culture of two types of cells, cell migration / invasion assay using passage of cells through porous membrane, drug using passage of drug through porous membrane Permeation assays and the like are possible. Cell sheets with cell culture inserts that are released from feeder cells and cell growth factors placed on the bottom surface are layered more than cells that are cultured without a feeder or filled with a medium containing cell growth factors directly on a petri dish. In addition, the vertical orientation can be adjusted. For example, corneal epithelial cells are layered on the basal plane downward, and mucosal cells are oriented with microvilli upward.

  In Patent Document 17, the myocardium cultured on a base material provided with a temperature-responsive polymer for myocardial treatment using a cell sheet is peeled off as a cell sheet to improve the function of the heart as an implantable cardiomyocyte sheet. It is shown to suppress the deformation of.

  It is shown in Non-Patent Document 6 that the cardiomyocyte sheet produced here self-pulsates, the two-layered cell sheet adheres without stitching with the above-mentioned ECM, and the pulsation synchronizes in a short period of time. .

  However, the self-pulsating cell sheet produced here has a random pulsation direction and can be used for myocardial therapy to align with the orientation of the transplanted heart by transplanting to a pulsatile-oriented heart, or even a single cardiomyocyte sheet alone from the outside It is known that the orientation is aligned in the direction of stretching / shrinking by applying force and repeating stretching / shrinking in the culture solution.

  However, the ECM of the sheet disappears (digests) when the cell sheet detached from the base material is held in the culture solution until the cardiomyocyte sheet is stretched and contracted. There was a problem that it was difficult to place.

  By the way, the present applicant has many technologies for anti-fogging films and decorative plywood in the field of printed coating technology using electron beam materials. The present applicants have found that a coating film obtained by adding a polymerizable oligomer or prepolymer to printing, coating, and adhesion curing by electron beam irradiation has excellent cosmetic coating film properties. (Patent Document 5)

JP 2004-170935 A JP 2005-168494 A Japanese Patent Publication No. 6-104061 JP-A-5-192130 Japanese Patent No. 2856862 Japanese Patent No. 31660 Japanese Patent No. 3491717 JP-A-9-12651 JP-A-10-248557 Japanese Patent Laid-Open No. 11-34943 JP 2001-329183 A JP 2002-18270 A Japanese Patent Laid-Open No. 5-244938 International Publication WO01 / 068799 Pamphlet JP 2005-130838 A JP 2006-320304 A JP 2003-306434 A M. J. Dalby et al., Biomaterials 25 (2004) 5415-5422 C. C. Berry et al., Biomaterials 25 (2004) 5781-5788 W. D. Snyder et al., J. Am. Chem. Soc. 97, 4999 (1967) L. F. Fieser et al., “Reagents for Organic Synthesis”, Willey, New York, N.Y., 1967 O. H. Kwon, J. Biomed. Mater. Res., (2000) Apr .; 50 (1): 82-9 Y. Haraguchi et al., Biomaterials 27 (2006) 4765-4774

  The technique of grafting an environmentally responsive polymer such as a temperature responsive polymer on the substrate surface of a cell culture support has many advantages. However, there is still room for improvement with respect to the material constituting the substrate.

  Various irradiation conditions when the temperature-responsive polymer is grafted onto the substrate surface using radiation are determined so that the polymer to be grafted exists uniformly on the substrate. However, even when the polymer is successfully immobilized on the surface of the base material and the hydrophilicity / hydrophobicity change due to the polymer occurs, the cell sheet peeling may be difficult to proceed depending on the material constituting the base material.

  Regarding the material constituting the base material, in Patent Documents 3 and 4, the material of the support to be coated is not only a substance usually used for cell culture, such as glass, modified glass, polystyrene, polymethyl methacrylate, In general, materials that can be given a form, such as polymer compounds other than those described above, ceramics, metals, etc., can be used, and the shape is not limited to petri dishes, but plates, fibers, (porous) particles Moreover, it is described that the shape of a container (flask or the like) generally used for cell culture or the like may be given. However, actually, other than the polystyrene used for petri dishes as a general-purpose substrate, the surface of the glass slide or the glass surface treated with a silane coupling agent or the porous polyethylene terephthalate described in Non-Patent Document 5 (PET ) There are only research reports on films and presentations at academic conferences.

  Although it has been found that a temperature-responsive graft polymer surface can be formed by radiation on the glass surface, the temperature-responsive graft polymer surface formed in this way has a problem that it does not show significant cell sheet detachment compared to that of a polystyrene petri dish surface. It was. In addition, when a temperature-responsive polymer is grafted on the surface of a substrate treated with a silane coupling agent, significant cell sheet detachment due to temperature responsiveness is obtained compared to the case where a temperature-responsive polymer is grafted on a polystyrene surface. It is known that cell adhesion to the surface of the substrate is deteriorated, and a good cell sheet cannot be formed.

  In addition, when Non-Patent Document 5 was traced using a PET microporous film, it was confirmed that temperature responsiveness was not exhibited depending on manufacturing conditions. In order to produce a temperature-responsive cell sheet support for an effective cell culture insert, a technology that binds and functions a temperature-responsive polymer to the surface of a microporous film is indispensable. Is not provided.

  As a preliminary experiment of the present invention, when a cell sheet was cultured using a general-purpose plastic PET base material on which a temperature-responsive graft polymer was formed, the substrate had a cell sheet peeling function. Not confirmed. Further, even when polycarbonate was used as a base material, both the hydrophilicity / hydrophobicity change and the cell sheet peeling were weaker than when polystyrene was used as the base material.

  Patent Document 17 describes "implantable mammalian cardiomyocyte sheet" and "cardiomyocyte sheet used for myocardial regeneration therapy". These techniques have been observed in small animals such as rats. However, in "transplantation" and "regenerative treatment" in large animals such as humans, cows, and pigs, the cardiomyocyte sheet layered in two layers as in the example has a contraction-relaxation function that pumps blood throughout the body. There were a number of problems in the construction of myocardial tissue.

  One is the need for multi-layered cardiomyocyte sheets with 5 layers or 6 layers or more with sufficient strength and contraction / relaxation function for large-animal "transplantation" and "regenerative treatment". The cardiomyocyte sheet is necrotic due to lack of oxygen at the center. The inventors of Patent Document 17 and the presenters of Non-Patent Document 6 have transplanted three layers of cell sheets to prevent necrosis due to the stratification of cell sheets, and after building a capillary network, the three layers of cell sheets This is achieved by laminating.

  The other is culturing confluently using a culture dish coated with poly-N-isopropylacrylamide, a temperature-responsive polymer, and the pulsatile orientation of the sheet-like cardiomyocyte tissue to be peeled after low-temperature treatment. The cardiomyocyte tissue to be “transplanted” or “regenerative treatment” needs to be the same, but the cardiomyocyte sheet to be exfoliated has no pulsatile orientation. In order to adjust the expansion / contraction direction of the cell sheet, it is transplanted near a muscle tissue or the like having a predetermined expansion / contraction direction as used for necrosis prevention, or the expansion / contraction movement is passively trained from outside in a cell culture solution.

To solve both of these problems, the prepared cardiomyocyte sheet is transplanted into a mammal for a certain period of time or muscle training is performed in a cell culture solution. At this time, the extracellular material formed on the exfoliated surface of the sheet-like cardiomyocyte tissue to be exfoliated after low-temperature treatment after culturing confluently using a culture dish coated with poly-N-isopropylacrylamide, which is a temperature-responsive polymer The matrix (ECM) contributes to the rapid adhesion between cells and the engraftment to the host myocardium, as well as electrical coupling (gap coupling). This ECM disappears when transplanted or directly in contact with the culture medium. Therefore, it is necessary to provide temperature responsiveness to the surface of the implantable material that is flexible and stretchable and has high gas permeability.
It is an object of the present invention to provide a cell culture support in which the above problems are eliminated.

This application includes the following inventions.
(1) A cell culture support comprising a substrate on which at least one environmentally responsive polymer selected from the group consisting of a temperature responsive polymer, a pH responsive polymer and an ion responsive polymer is immobilized on the surface by a covalent bond A composition comprising a monomer that can be polymerized by irradiation to form the environmentally responsive polymer and an organic solvent, and a surface that can be covalently bonded to the environmentally responsive polymer by irradiation. An application step of applying to a substrate and forming a coating film on the surface of the substrate; and a polymerization reaction for irradiating the coating film with radiation to form the environmentally responsive polymer; and the environmentally responsive polymer; A polyethylene comprising a radiation irradiation step for causing a binding reaction to bond to a substrate surface and a drying step for drying the coating film, wherein the substrate has a surface subjected to easy adhesion treatment Synthetic resin whose surface is corona-treated or plasma-treated, synthetic resin whose surface is coated with acrylic resin, natural rubber with conjugated bond, synthetic rubber with conjugated bond, silicon rubber containing polysilicon, and fine Said method comprising at least one material selected from the group consisting of porous polycarbonates.

(2) The method according to (1), wherein the synthetic resin is at least one selected from the group consisting of nylon, low density polyethylene, medium density polyethylene, polypropylene, polyethylene terephthalate, polycarbonate, and polystyrene.

(3) The synthetic resin whose surface is corona-treated or plasma-treated is selected from the group consisting of microporous nylon, polyethylene, polypropylene, polyethylene terephthalate, and polycarbonate whose surface is corona-treated or plasma-treated. The method according to (1), which is at least one kind.

(4) At least one environmental responsiveness selected from the group consisting of a temperature-responsive polymer, a pH-responsive polymer, and an ion-responsive polymer, produced by the method according to any one of (1) to (3) Polyethylene terephthalate with polymer surface immobilized by covalent bond, surface with easy adhesion treatment, synthetic resin with surface treated with corona or plasma, synthetic resin with surface coated with acrylic resin, conjugated bond A cell culture support comprising a substrate comprising at least one material selected from the group consisting of natural rubber, synthetic rubber having a conjugated bond, silicon rubber containing polysilicon, and microporous polycarbonate.

(5) The cell culture support according to (4), wherein the thickness of the environmentally responsive polymer layer immobilized on the surface of the substrate is 0.001 to 10 μm when dried.
(6) A method for producing a cell sheet, comprising a step of culturing cells on the cell culture support according to (4) or (5).
(7) A cell sheet produced by the method according to (6).

  According to the method for producing a cell culture support according to the present invention, since the environment-responsive polymer can be grafted directly on the surface of the substrate without using a silane coupling agent, the cell adhesion to the substrate at the time of culture is uniform. A uniform confluent state is achieved and a less damaged cell sheet is obtained.

  According to the method for producing a cell culture support according to the present invention, an environmentally responsive polymer can be effectively grafted onto a microporous synthetic resin film without impairing the medium (medium) permeability of the film. The cell culture support thus produced can be used for insert culture.

  According to the method for producing a cell culture support according to the present invention, an environment-responsive polymer can be grafted onto the surface of rubber or plastic that can be transplanted or stretched for cell orientation.

  When the cell culture support produced by the method according to the present invention includes a layer of a temperature-responsive graft polymer on its surface, the wettability of the surface changes according to the temperature, and reversibly good cell adhesion / Shows peelability. In addition, ECM is maintained in vivo and ex vivo (for example, in a culture solution) even after the cell sheet is peeled off due to low temperature. For this reason, the detached cell sheets can be joined within 30 minutes without performing suturing including gap bonding. In this way, a cell sheet that retains many of the various cell binding functions can be obtained.

  Further, according to the method of the present invention, since the environmentally responsive polymer can be grafted on the surface of the implantable material that is flexible and stretchable and has high gas permeability, the ECM of the cell sheet is in vivo. The problem of disappearing in more than half a day can be avoided if the cell is directly transplanted or touched with a culture solution.

  The cell sheet produced using the cell culture support according to the present invention is suitable for use in regenerative medicine and the like because the surface adhesion factor is not impaired.

(Base material)
The substrate to which the coating composition is applied in the present invention is covalently bonded to at least one environmentally responsive polymer selected from the group consisting of a temperature responsive polymer, a pH responsive polymer, and an ion responsive polymer by irradiation. A base material having a surface that can be made of polyethylene terephthalate whose surface is easily bonded, a synthetic resin whose surface is corona-treated or plasma-treated, a synthetic resin whose surface is coated with an acrylic resin, and having a conjugate bond It includes at least one material selected from the group consisting of natural rubber, synthetic rubber having a coconjugated bond, silicon rubber containing polysilicon, and microporous polycarbonate. The base material is provided with at least one surface (cell adhesion surface) on which cells can be cultured to form a cell sheet, and at least the cell adhesion surface may be formed of the material. Of course, the whole base material may be comprised with the said material. Examples of the shape of the substrate include a dish shape and a film shape. In the case of using a film-shaped substrate, an environment-responsive polymer graft polymer layer can be formed on the surface of the film-shaped substrate and then processed into a shape suitable for cell culture (for example, a dish shape). In processing, a member made of another material can be used in combination with the base material as necessary. In the case of using a dish-shaped substrate, it is sufficient that at least the inner bottom portion of the dish serving as the cell adhesion surface is covered with the graft polymer layer.

  As a material for the base material, polyethylene terephthalate whose surface is subjected to easy adhesion treatment can be suitably used. Polyethylene terephthalate is suitable as a cell support material because it is excellent in properties such as transparency, dimensional stability, mechanical properties, electrical properties, and chemical resistance. As the “polyethylene terephthalate whose surface is easily bonded” that can be used in the present invention, an easy-adhesive layer is provided on polyethylene terephthalate with an easy-adhesive agent such as polyester, acrylate ester, polyurethane, polyethyleneimine, and silane coupling agent. Can be mentioned. In order to impart easy adhesion to the polyethylene terephthalate film, an easy adhesion imparting paint is applied by an in-line coating method or an offline coating method. Examples of the easy-adhesion imparting coating material include a combination of the easy-adhesive and a melamine resin as a crosslinking agent component. The in-line coating method is a method in which paint is applied during the film formation process, and the off-line coating method is a method in which a biaxially stretched polyethylene terephthalate film obtained in film formation is applied to a coater, and the paint is applied and dried. It is a method to do. Considering the cost, the in-line coating method is preferable. Examples of paint application methods include roll coating, gravure coating, micro gravure coating, reverse coating, reverse gravure coating, bar coating, roll brushing, air knife coating, curtain coating, and die coating. Any coating method may be applied singly or in combination as appropriate.

As a material for the base material, a synthetic resin having a corona-treated or plasma-treated surface can also be suitably used. The synthetic resin that can be used in this case, for example nylon, low density polyethylene (density refers to polyethylene of less than 910 kg / m ³ or more 930 kg / m ^3), medium density polyethylene (density 930 kg / m ³ or more 942Kg / m less than ³ May be polypropylene, polyethylene terephthalate, polycarbonate, polystyrene, or a blend polymer or polymer alloy containing two or more of these materials.

  The base material is nylon, polyethylene, polypropylene, polyethylene terephthalate, or polycarbonate, or a microporous material composed of a blend polymer or polymer alloy containing two or more of these materials, and its surface is corona-treated. Or what was plasma-processed can also be used conveniently. However, microporous polycarbonate whose surface is not corona-treated or plasma-treated can also be used suitably. These microporous materials can effectively covalently bond an environmentally responsive polymer without impairing the permeability of the medium (medium). In this case, the substrate is preferably in the form of a film. A film-like microporous substrate is suitable for use in insert culture.

  The pore size and pore density of the microporous material are appropriately selected from a range suitable for cell culture inserts and the like. When a porous membrane is used in a cell migration / invasion assay that utilizes the passage of cells through pores, a pore size that is large enough for cells to pass through is required. In addition, when the porous membrane is used for three-dimensional culture, co-culture of two types of cells, drug permeation assay, etc., a pore size of φ20 μ or less that does not allow cells to migrate / infiltrate is preferable. When the cell sheet made of is peeled off using an environmentally responsive polymer, the thickness is preferably 20 μm or less without migration / invasion, and preferably 3 μm or less in order to smoothly peel the cell sheet without being caught by the pores. Moreover, since the material and thickness of the porous membrane and the density of the pores affect the transit time of the medium, those suitable for the intended cell culture insert are preferable.

  As a material for the substrate, a synthetic resin whose surface is coated with an acrylic resin can also be suitably used. By using such a synthetic resin, coating of the coating agent is improved, and the graft ratio of graft polymerization by radiation irradiation is improved. In this case, as the synthetic resin, those similar to the synthetic resin whose surface is subjected to corona treatment or plasma treatment can be suitably used. As the acrylic resin, urethane acrylate is preferable.

  As the material for the base material, natural rubber having a conjugated bond, synthetic rubber having a conjugated bond, or silicon rubber containing polysilicon can also be suitably used. These may be appropriately surface-treated. On such materials, radical dissociation and proton release of conjugated bonds by irradiation are easy and graft polymerization is easy. The natural rubber having a conjugated bond is preferably a substance mainly composed of cis-polyisoprene [(C5H8) n] contained in the sap of rubber tree. As the synthetic rubber having a conjugated bond, polybutadiene, butadiene / acrylonitrile, chloroprene, ethylene propylene and the like are preferable. Silicon rubber containing polysilicon has the following basic units:

を含むものが好ましい。

The thing containing is preferable.

(Environmentally responsive polymer)
The present invention relates to a substrate in which at least one environmentally responsive polymer selected from the group consisting of a temperature responsive polymer, a pH responsive polymer, and an ion responsive polymer is immobilized (i.e., grafted) to the surface by a covalent bond. The present invention relates to a method for producing a cell culture support.

  The environmentally responsive polymer is particularly preferably a temperature responsive polymer, but is not limited thereto.

  The temperature-responsive polymer that can be suitably used in the present invention is hydrophobic at the cell culture temperature (usually about 37 ° C.) and hydrophilic at the temperature at the time of recovering the cultured cell sheet. The temperature at which the temperature-responsive polymer changes from hydrophobic to hydrophilic (critical solution temperature in water (T)) is not particularly limited, but from the viewpoint of ease of recovery of the cell sheet after culture, The temperature is preferably lower than the cell culture temperature. By including such a temperature-responsive polymer component, since cell scaffolds (cell adhesion surfaces) are sufficiently secured during cell culture, cell culture can be performed efficiently. On the other hand, at the time of collecting the cell sheet after culturing, the hydrophobic part is changed to hydrophilic, and the cultured cell sheet is separated from the cell culture substrate, thereby making it easier to collect the cell sheet. be able to. In particular, a temperature-responsive polymer that exhibits hydrophilicity at a temperature lower than a predetermined critical dissolution temperature and exhibits hydrophobicity at a temperature equal to or higher than the same temperature is preferable. The critical solution temperature in such a temperature-responsive polymer is particularly called the lower critical solution temperature.

  Specifically, the temperature-responsive polymer that can be suitably used in the present invention is preferably a polymer having a lower critical solution temperature T of 0 to 80 ° C, preferably 0 to 50 ° C. If T exceeds 80 ° C., the cells may die, which is not preferable. If T is lower than 0 ° C., the cell growth rate is generally extremely reduced or the cells are killed. Such suitable polymers include acrylic or methacrylic polymers. Suitable polymers are also described, for example, in US Pat. Specific suitable polymers include, for example, poly-N-isopropylacrylamide (T = 32 ° C.), poly-Nn-propyl acrylamide (T = 21 ° C.), poly-Nn-propyl methacrylamide (T = 32 ° C.), poly-N-ethoxyethyl acrylamide (T = about 35 ° C.), poly-N-tetrahydrofurfuryl acrylamide (T = about 28 ° C.), poly-N-tetrahydrofurfuryl methacrylamide (T = about 35 ° C.) ), And poly-N, N-diethylacrylamide (T = 32 ° C.). Examples of other polymers include poly-N-ethylacrylamide, poly-N-isopropylmethacrylamide, poly-N-cyclopropylacrylamide, poly-N-cyclopropylmethacrylamide, poly-N-acryloylpyrrolidine, poly-N- Polyalkylene oxide block copolymers represented by alkyl-substituted cellulose derivatives such as acryloyl piperidine, polymethyl vinyl ether, methyl cellulose, ethyl cellulose, and hydroxypropyl cellulose, block copolymers of polypolypropylene oxide and polyethylene oxide, and polyalkylene An oxide block copolymer is mentioned.

  Examples of the monomer for forming these polymers include monomers having a monomer homopolymer having T = 0 to 80 ° C. and capable of being polymerized by irradiation. Examples of the monomer include (meth) acrylamide compounds, N- (or N, N-di) alkyl-substituted (meth) acrylamide derivatives, (meth) acrylamide derivatives having a cyclic group, and vinyl ether derivatives. More than seeds may be used. When a single monomer is used alone, the polymer formed on the substrate is a homopolymer, and when multiple monomers are used together, the polymer formed on the substrate is a copolymer. These forms are also encompassed by the present invention. In addition, when it is necessary to adjust T depending on the type of proliferating cell, when it is necessary to enhance the interaction between the coating substance and the cell culture support, and the balance between the hydrophilicity and hydrophobicity of the cell support is adjusted. If necessary, other monomers other than those described above may be further added for copolymerization. Further, a graft or block copolymer of the above-mentioned polymer used in the present invention and another polymer, or a mixture of the polymer of the present invention and another polymer may be used. Moreover, it is also possible to crosslink within a range where the original properties of the polymer are not impaired.

  As the pH responsive polymer and the ion responsive polymer, those suitable for the cell sheet to be prepared can be appropriately selected.

(Coating composition)
In the method of the present invention, a coating composition containing a monomer that can be polymerized by irradiation with radiation to form the environmentally responsive polymer and an organic solvent is used. In the coating composition, it is preferable to further mix and use an oligomer or prepolymer obtained by polymerizing the monomer. By using the coating composition containing an oligomer or a prepolymer, crystallization is suppressed even when the amount of the organic solvent is small. For this reason, when this composition for application | coating is apply | coated to the base-material surface and superposition | polymerization is advanced by radiation irradiation, a uniform environmentally responsive polymer layer can be formed over the whole surface of a base-material surface.

  The radiation polysynthetic monomer is as described above. The coating composition contains one or more monomers.

  The size of the oligomer or prepolymer contained in the coating composition is not particularly limited as long as it is a dimer or larger, and preferably has a molecular weight of more than about 3,300 (typically 28 molecular polymers). More than 700 are more preferable. The upper limit is not particularly limited, and may be a molecular weight of 1 million or more. In the present invention, the term “prepolymer” refers to a polymer before irradiation.

  The organic solvent is not particularly limited as long as it can dissolve the monomer, oligomer or prepolymer, but those having a boiling point of 120 ° C. or less, particularly 60 to 110 ° C. under normal pressure are preferred. Preferable examples of the solvent include methanol, ethanol, n (or i) -propanol, 2 (or n) -butanol, and water, and one or more of them may be used. Other solvents such as 1-pentanol, 2-ethyl-1-butanol, 2-butoxyethanol, and ethylene (or diethylene) glycol or monoethyl ether thereof may be used. As other additives, acids such as sulfuric acid, Mole salt and the like may be added to the above solution.

The content of the monomer in the coating composition is preferably 5 to 70% by weight.
When the coating composition contains an oligomer or prepolymer, the content of the oligomer or prepolymer in the composition is preferably 0.1 to 20% by weight. The weight ratio of the monomer and the oligomer or prepolymer contained in the composition is preferably 500: 1 to 1:20.
The viscosity of the coating composition is preferably 5 × 10 ⁻³ Pa · s to 10 Pa · s.

(Coating process)
The method of this invention includes the application | coating process which apply | coats the said composition for application | coating to the surface of the said base material, and forms a coating film on the surface.

The coating amount of the coating film formed in this step may be 50 mg / m ² or more, which is a coating amount necessary for the environment-responsive graft polymer to exhibit its function (for example, temperature responsiveness). The upper limit of the coating amount is not particularly limited, but is preferably less than 40 g / m ^{2 and} more preferably 10 g / m ² or less. When the coating amount is 40 g / m ² or more, the thickness is increased and the coating thickness is not stable, the thickness is increased and the radiation penetration / irradiation amount is not stable, and in the film derived from the irradiation energy It has been confirmed by the present inventors that unevenness occurs in the coating amount of the graft polymer by convection. In order to shorten the washing time for washing the ungrafted free polymer, the coating amount is desirably 10 g / m ² or less.

  As a method for applying the coating composition to a substrate on a small area, any known method may be used, and examples thereof include a coating method using a spin coater, a bar coater and the like, and a spray coating method. As a coating method for a large area, a blade coating method, a gravure coating method, a rod coating method, a knife coding method, a reverse roll coating method, an offset gravure coating method and the like can be used.

  In the solid formation, a known coating method such as a gravure coating method, a roll coating method, a slot coating method, a kiss coating method, a spray coating method, or a fountain coating method can be used. In the pattern formation of the pattern layer, a known printing method such as a gravure printing method, a screen printing method, or an offset printing method can be used. As a method of applying the coating composition to the substrate, a continuous coating method or printing method can also be used. Specifically, the continuous coating method or printing method includes hot melt coating, hot lacquer coating, gravure direct coating, gravure reverse coating, die coating, micro gravure coating, slide coating, slit reverse coating, curtain coating, knife coating, and air coating. Application methods such as roll coating can be used, but these are merely examples, and those skilled in the art can use those that can be applied for a while.

(Radiation irradiation process)
In the method of the present invention, the coating film is irradiated with radiation to cause a polymerization reaction for forming the environmentally responsive polymer and a bonding reaction (that is, grafting) for bonding the environmentally responsive polymer and the substrate surface. Including a radiation irradiation step. The bonding reaction (grafting) here is not only a phenomenon in which a free polymer formed in situ from a monomer, oligomer or prepolymer by polymerization by radiation irradiation binds to the surface of the substrate, but also a free monomer is bonded to the substrate. A phenomenon in which the polymer chain extends from the monomer as a starting point after bonding to the surface, a phenomenon in which a free prepolymer or oligomer derived from the coating composition is bonded to the substrate surface, or a polymer or oligomer bonded to the substrate surface Including the phenomenon that the polymer chain extends from the base point.

  Examples of the radiation used include α rays, β rays, γ rays, electron beams, ultraviolet rays, and the like. In order to produce a desired graft polymer, γ rays and electron beams are energy efficient, and electron beams are particularly preferable from the viewpoint of productivity. With respect to ultraviolet rays, it can be used by combining an appropriate polymerization initiator and an anchor agent with a substrate.

  The range of radiation dose is preferably 5 Mrad to 50 Mrad for electron beams, and preferably 0.5 Mrad to 5 Mrad for γ rays.

  When a coating composition containing an oligomer or prepolymer that has undergone polymerization to some extent is used as the coating composition, the formation of an environmentally responsive polymer by a polymerization reaction, the surface of the substrate, and the environment by only one irradiation. It is possible to complete the grafting reaction with the responsive polymer.

(Drying process)
The method of the present invention includes a drying step of drying the coating film to remove an organic solvent derived from the coating composition.

  Since the coating film formed in the coating process does not form crystals due to the effect of the residual solvent amount, the coating film before drying may be irradiated with radiation and then dried. After that, radiation may be applied. However, if radiation is applied to a wet coating before drying, it may be affected by environmental changes, foreign matter, coating thickness fluctuations, etc., so radiation should be applied after the coating is dried. Is preferred.

  Although it does not specifically limit as a drying method, Typically, a dry air drying method, a hot air (warm air) drying method, a (far) infrared drying method etc. are mentioned.

(Washing process)
The environmentally responsive polymer layer of the cell culture support formed through the above-described steps includes not only polymer molecules immobilized by covalent bonds on the substrate surface, but also free polymer molecules that are not immobilized. Unreacted monomer molecules are present. In addition, when the coating composition contains an oligomer or prepolymer molecule, unreacted oligomer molecule or prepolymer molecule may be present. Therefore, it is preferable to further include a washing step for washing in order to remove these free polymers or unreacted substances.

  Although it does not specifically limit as a washing | cleaning method, Typically, immersion washing | cleaning, swing washing | cleaning, shower washing | cleaning, spray washing | cleaning, ultrasonic cleaning, etc. are mentioned. The cleaning liquid typically includes various water-based, alcohol-based, hydrocarbon-based, chlorine-based, acid / alkali cleaning liquids. The combination of the washing method and the washing solution may be appropriately selected according to the cell culture support to be washed.

(Cell culture support produced by the method of the present invention)
The present invention also relates to a cell culture support produced by the method of the present invention. The cell culture support according to the present invention comprises polyethylene terephthalate having an adhesive surface treated with a covalent bond, and a synthetic resin having a corona or plasma treated surface, and an acrylic surface. Including at least one material selected from the group consisting of a synthetic resin coated with a resin, a natural rubber having a conjugated bond, a synthetic rubber having a conjugated bond, a silicon rubber containing polysilicon, and a microporous polycarbonate A substrate is provided.

  The cell culture support of the present invention preferably has a dry thickness of the graft polymer layer immobilized on its surface of 0.001 to 10 μm.

The coating amount of the various environmental responsive polymers in cell culture support surface is preferably 5~80μg / cm ^2, more preferably 6~40μg / cm ^2. When the coating amount of the environmentally responsive polymer exceeds 80 μg / cm ² , the cells do not adhere on the surface of the cell culture support, and conversely, when the coating amount is less than 5 μg / cm ² , the cells are cultured in a monolayer and are in a tissue state. In addition, it becomes difficult to peel and collect the cultured cells from the support. Such an environmentally responsive polymer coating amount can be analyzed, for example, by Fourier transform infrared spectrometer total reflection method (FT-IR-ATR method), analysis by coating or non-coating dyeing or dyeing of fluorescent material, and contact angle measurement. It is possible to obtain surface analysis by means of, for example, alone or in combination.

(Method for creating cell culture sheet)
Using the cell culture support of the present invention, various cells, such as epithelial cells and endothelial cells constituting each tissue and organ in the living body, skeletal muscle cells exhibiting contractility, smooth muscle cells, cardiomyocytes, nervous system Constituent neurons, glial cells, fibroblasts, liver parenchymal cells related to metabolism in the living body, non-hepatic parenchymal cells and fat cells, stem cells existing in various tissues as cells having differentiation potential, bone marrow cells, ES cells A cell sheet can be produced from the above. The cell sheet thus prepared is suitable for use in regenerative medicine and the like because the adhesion factor on the surface is not impaired and the portion in contact with the cell culture surface has a uniform quality. In addition, the cell sheet can be applied to detection devices such as biosensors.

(Test 1)
Two types of temperature-responsive polymer prepolymers were tested.
Polyisopropylacrylamide (Aldrich product number 535311) having a molecular weight of 20000 to 25000 commercially available from Aldrich was used as prepolymer 1 in the following experiments.

  Polyisopropylacrylamide obtained by redox synthesis by the following procedure was used as prepolymer 2 in the following experiments. Into a 500 mL separable flask, 17.8 g of N-isopropylacrylamide and 150 mL of pure water were added, and dissolved and dispersed under stirring. Under nitrogen gas flow, 0.24 g of ammonium persulfate and 0.30 mL of N, N, N ′, N′-tetramethylethylenediamine were added at room temperature to initiate polymerization. After the polymerization, the gel was taken out by heating and dried in an electric dryer at 100 ° C. to obtain Prepolymer 2. The dried gel was pulverized and subjected to GPC analysis with an NMP solvent. As a result, the prepolymer 1, a polyisopropylacrylamide product number P3241 (molecular weight 5700) manufactured by Polymer Source, and a polyisopropylacrylamide product number P7142 (molecular weight 258000) manufactured by Polymer Source In comparison, the molecular weight was about 35 to 400,000.

  1 kg of a solution in which prepolymer 1 was dissolved in isopropyl alcohol so that the prepolymer 1 was 0.5 wt% and the isopropylacrylamide monomer was 40 wt% was prepared. This solution was designated as Composition 1 for coating.

  1 kg of a solution dissolved in isopropyl alcohol was prepared so that the prepolymer 1 was 2 wt% and the isopropylacrylamide monomer was 40 wt%. This solution was designated as Composition 2 for coating.

  1 kg of a solution dissolved in isopropyl alcohol was prepared so that the prepolymer 1 was 5 wt% and the isopropylacrylamide monomer was 40 wt%. This solution was designated as “Coating composition 3”.

  1 kg of a solution in which prepolymer 2 was dissolved in isopropyl alcohol so that the prepolymer 2 was 0.5 wt% and the isopropylacrylamide monomer was 40 wt% was prepared. This solution was designated as Composition 4 for coating.

  1 kg of a solution in which prepolymer 2 is 2 wt% and isopropyl acrylamide monomer is 40 wt% is dissolved in isopropyl alcohol. This solution was designated as Composition 5 for coating.

  1 kg of a solution dissolved in isopropyl alcohol was prepared so that the prepolymer 2 was 5 wt% and the isopropylacrylamide monomer was 40 wt%. This solution was designated as composition 6 for coating.

Each of the coating compositions 1 to 6 is 9 mg / m ² with a solid gravure direct roll of 48 lines on a continuous base film of easy-adhesive polyethylene terephthalate (PET) coated with an adhesive and protected with a release film. The coating amount was 5 m / min, dried with a continuous 1 m hot air dryer at 45 ° C., and irradiated with an electron beam in an environment having an oxygen concentration of 50 ppm or less with a continuous electron beam irradiation unit. The irradiation amount was adjusted to 10, 12, 16, 20, 24 Mrad by changing the electron flow rate of the electron beam. After the electron beam irradiation, the coated film was washed with ion exchange water at 5 ° C. to remove residual monomers and polymers not bonded to the coated film surface. It was dried in a clean bench, further sterilized with ethylene oxide gas (EOG), and further sufficiently deaerated to obtain a coated film. The smoothness of the coated surface of this product was examined by examining the surface for unevenness under an optical microscope. The coated surface (cell culture surface) of the coated film produced using the coating compositions 1 to 6 had a smooth and transparent surface.

Each of the coating films produced using the coating compositions 1 to 6 was cut into a dish size and fixed to the inner bottom surface of the dish with an adhesive so that the coated surface was upward, and EOG sterilized. Using the cell culture support thus obtained, bovine aortic vascular endothelial cells were cultured. That is, the cells were cultured at 37 ° C. in 5% carbon dioxide using Dulbecco's modified Eagle medium (DMEM) containing 10% fetal calf serum (FCS) on the cell culture support. After confirming that the cells have fully proliferated, the culture solution is transferred together with the dish into a chamber at 20 ° C. and 5% CO ₂ , and left for 30 minutes to detach the attached / proliferated cells, and the detachment rate of proliferating cells is recovered. Was determined according to the following equation. Proliferation cell exfoliation recovery rate (%) = 100 × total number of exfoliated cells / total number of proliferated cells. At that time, in order to measure the total number of cells peeled and collected and the total number of proliferated cells, the cells must be in individual states. Therefore, the total number of cells separated and collected was measured by cooling the cells to 20 ° C. and allowing them to stand, and then treating the collected cell mass with trypsin-EDTA so that the cells were in individual states. The total number of cells grown is the total number of cells peeled and collected by the above method, and the total number of cells that have been peeled into individual states by trypsin-EDTA treatment after cooling to 20 ° C. Obtained by adding together. In addition, a cell sheet-like lump that has been cultured in a chamber at 37 ° C. for 5 days and is entirely confined except for the surroundings of the dish is cut with a scalpel into the sub-confluent cell layer surrounding the dish, The whole dish was transferred into a chamber at 20 ° C. and 5% CO ₂ , and detachment of the cell sheet was observed.

  In all examples of cell sheet preparation using the cell culture support prepared using the coating compositions 1 to 6, all cells exhibit a high proliferation cell detachment recovery rate exceeding 90%, and the entire cell sheet is exfoliated cleanly and without distortion. I was able to. Regarding the composition having a polymer content of 2 wt% (that is, the coating compositions 2 and 5), a cell culture support was prepared from a coated film formed at the end of the 40-minute coating after coating with 200 m. There was no difference in cell recovery and cell sheet peeling performance from the cell culture support from the initial coating film, and it was good.

(Test 2)
In this test example, a sample in a state where an electron beam was not irradiated when a continuous coating film using the coating composition 1, 2 or 3 in Test 1 was prepared. This film was irradiated with gamma rays from cobalt 60 at room temperature and 1.5 Mrad for 2 hours. This was washed and dried in the same manner as described in Test 1, and the cut and pasted dish was EOG sterilized to obtain a cell culture support. The prepared cell culture supports were all smooth and transparent on the surface, and the cell recovery and cell sheet peeling were good.

(Test 3)
As Examples 1 to 14, on each base film shown in Table 1, a solution in which prepolymer 1 described in Test 1 was dissolved in isopropyl alcohol so that the prepolymer 1 was 1 wt% and the isopropylacrylamide monomer was 40 wt% (Coating Composition 7) After coating with a wire bar (counter 4), drying the dryer, the coated surface was irradiated with an electron beam and washed and dried in the same procedure as in Test 1. The amount of electron beam irradiation was 10 Mrad, 15 Mrad, and 25 Mrad. The coated film after washing and drying was cut into a circle, and the coated back surface and the inner bottom surface of the Petri dish were bonded with a double-sided tape. This was subjected to EOG sterilization to obtain a cell culture support. Using the obtained cell culture support, bovine aortic vascular endothelial cells were cultured in the same procedure as in Test 1 to prepare a cell sheet. The cell recovery rate and cell sheet peeling were observed in the same procedure as in Test 1.

The self-prepared film of Example 2 was obtained by coating the film of Example 1 with the composition shown in Table 2 on a wire bar (count 4) and irradiating it with ultraviolet light using a high-pressure mercury lamp at 170 mJ / cm ² (365 nm). It was obtained by curing the coated surface. This cured coated surface was used in the same manner as other films. The polystyrene film of Comparative Example 1 was used after removing and drying the antifogging agent with ethanol in advance.

  The films of Examples 3 to 5 were those having a corona-treated surface for printing, and the corona-treated surface was coated with the coating composition and irradiated with an electron beam. The films of Examples 6 to 14 were subjected to plasma treatment, and the obtained plasma treated surface was applied with a coating composition and irradiated with an electron beam. The plasma treatment was performed by filling with oxygen at a reduced pressure of about 60 to 150 mm Torr and performing plasma discharge at 400 W for 3 minutes. For the synthetic rubbers of Examples 12 to 14, in addition to the coating composition 7 with wire bar coating (A), the prepolymer 1 described in Test 1 was 5 wt%, and the isopropyl acrylamide monomer was 10 wt%. A wire bar-coated liquid (coating composition 8) dissolved in alcohol (B) was prepared. In Example 15, the microporous polycarbonate of Example 10 was similarly processed and evaluated without plasma treatment. Comparative Example 2 was obtained by processing and evaluating the microporous polyethylene of Example 8 in the same manner without plasma treatment.

  The results of the measurement of the cell recovery rate and the observation of the cell sheet peeling are as shown in Table 3. With polyimide, neither cell recovery nor cell sheet peeling was observed, and with polyester, both the recovery rate and cell sheet peeling were weaker as functional supports than polystyrene. When easy-adhesive PET, urethane acrylate, nylon, or low density polyethylene (LDPE) was used, the cell culture support produced with a lower radiation dose showed better function than when polystyrene was used.

  All of the microporous films treated with plasma showed good functions from the cell culture support prepared with a lower radiation dose than polystyrene with respect to the observation of cell recovery and cell sheet peeling. The microporous polycarbonate showed a good function without plasma treatment.

  With respect to the microporous polycarbonate film having a pore size of 5 μm in Example 11, the cell sheet is peeled from the same confluent state, but some cells fit into the pore, and the cell is caught in the pore, and the cell sheet peels slowly. The condition was observed. Depending on the state of catching, even if it was peeled off, the cell sheet remained damaged, and not all peeled off. Example 9 also had a large pore size (about 5 μm at the maximum), and partial peeling was observed under all radiation conditions, but the whole was not peeled off as a single good cell sheet.

  Regarding the polysilicon, Example 14A shows that the other cell culture support prepared under the same conditions became confluent when the entire culture except for the periphery of the dish became confluent when cultured in a chamber at 37 ° C. for 5 days. It took 8 days for culture. Example 14B became confluent after 5 days of culture.

  The polystyrene film had some distortion due to the substrate becoming 70 ° C. by irradiation with 25 Mrad. PET and polyimide also had a substrate temperature of 70 ° C., but the substrate had heat resistance and no distortion was observed. Nylon, polyethylene, polypropylene, polybutadiene, and polysilicon did not raise the substrate temperature, and no distortion was observed. Regarding poram (microporous polyethylene), some distortion occurred due to heat generation of the contained calcium carbonate fine particles.

(Test 4)
In this test, the permeability of the culture solution of the substrate obtained by bonding the temperature-responsive polymer to the surface of the microporous film (Examples 6 to 11) subjected to the plasma treatment used in Test 3 was examined.

  The coating composition 7 described in Test 3 was applied to the base film of Example 6, and the film irradiated once with an electron beam irradiation amount of 15 Mrad was washed and dried to obtain a coated film. For comparison, an uncoated base film was also prepared. These films were set in a suction glass holder having a filter diameter of 47 mm. When this suction glass holder is placed in a 1 L suction bottle connected to an aspirator and filtered through distilled water at 20 ° C. and 40 ° C. alternately, the base film not coated as shown in Table 4 is used. Both hot water and cold water passed, and the coated film surface-modified with the temperature-responsive polymer passed hot water and hardly allowed cold water to pass. The temperature dependence of the permeation amount for water was reversible, and reproducibility was observed in the permeation amount switching by switching between cold water and hot water.

  It was confirmed that all the plasma-treated microporous films had the ability to permeate the culture solution at 37 ° C. as in Table 4.

Claims (8)

Method for producing cell culture support comprising a substrate on which at least one environmentally responsive polymer selected from the group consisting of a temperature responsive polymer, a pH responsive polymer and an ion responsive polymer is immobilized on the surface by a covalent bond Because
A roll-shaped film-shaped substrate having a surface that can be covalently bonded to the environmentally responsive polymer by radiation irradiation is prepared, and a monomer and an organic solvent that can be polymerized by irradiation to form the environmentally responsive polymer. A coating step of continuously applying the composition to the substrate to form a coating film on the surface of the substrate;
A radiation irradiation step of continuously irradiating the coating film with radiation to advance a polymerization reaction for forming the environmentally responsive polymer and a binding reaction for binding the environmentally responsive polymer and the substrate surface;
A drying step of drying the coating film,
The base material is a polyethylene terephthalate whose surface is easily adhered, a synthetic resin whose surface is corona-treated or plasma-treated, a synthetic resin whose surface is coated with an acrylic resin, a natural rubber having a conjugated bond, and a conjugated bond. The method as described above, comprising at least one material selected from the group consisting of synthetic rubber, silicon rubber containing polysilicon, and microporous polycarbonate.   The method according to claim 1, further comprising a substrate processing step of cutting the substrate into a shape suitable for cell culture after the drying step. A method for producing a cell culture support according to claim 1 or 2,
The composition used in the coating step further contains an oligomer or a prepolymer,
The said irradiation process WHEREIN: The said reaction which advances the superposition | polymerization reaction which forms the said environmentally responsive polymer by one continuous irradiation, and the coupling | bonding reaction which combines the said environmentally responsive polymer and a substrate surface.   The method according to any one of claims 1 to 3, wherein the synthetic resin is at least one selected from the group consisting of nylon, low density polyethylene, medium density polyethylene, polypropylene, polyethylene terephthalate, polycarbonate, and polystyrene.   The synthetic resin whose surface is corona-treated or plasma-treated is at least one selected from the group consisting of microporous nylon, polyethylene, polypropylene, polyethylene terephthalate, and polycarbonate whose surface is corona-treated or plasma-treated. The method according to any one of claims 1 to 3, wherein   6. At least one environmentally responsive polymer selected from the group consisting of a temperature-responsive polymer, a pH-responsive polymer, and an ion-responsive polymer produced by the method according to claim 1 is covalently bonded. Polyethylene terephthalate that has been surface-adhered and fixed on the surface, synthetic resin whose surface is corona-treated or plasma-treated, synthetic resin whose surface is coated with an acrylic resin, natural rubber with conjugate bonds, conjugated A cell culture support comprising a substrate comprising at least one material selected from the group consisting of a synthetic rubber having a bond, a silicone rubber containing polysilicon, and a microporous polycarbonate.   The cell culture support according to claim 6, wherein a thickness of the layer of the environmentally responsive polymer immobilized on the surface of the base material is 0.001 to 10 μm.   A method for producing a cell sheet, comprising a step of culturing cells on the cell culture support according to claim 6 or 7.