JP5657744B2 - Method for producing cell culture support - Google Patents

Description

  The present invention relates to a method for producing a cell culture support.

  In the production method in which a temperature-responsive polymer is already grafted onto a substrate surface using radiation, the present inventors uniformly coat the substrate with a monomer to be covalently bonded to the surface to obtain a homogeneous and stable graft surface. Therefore, it has been invented that an oligomer or a prepolymer is preferably mixed into a coating composition containing a monomer before polymerization, and a patent application has been filed (Japanese Patent Application No. 2007-066700).

  Conventionally, cell culture supports prepared in a production method in which a temperature-responsive polymer is grafted onto a substrate surface using radiation tend to have a slightly weaker cell adhesion than polystyrene cell culture supports usually used for cell culture. is there. For this reason, even in the case of adherent cells, the number of adherent cells is small compared to a polystyrene support, which is a normal cell culture support, and therefore, cell growth is delayed or unnecessary for growing desired cells. In some cases, the number of cells increases, differentiation into different cells proceeds, and it is inconvenient to obtain a desired cell sheet depending on the cell type. For this reason, there are cases where the substrate surface is precoated with gelatin, collagen, fibronectin or the like, and cell culture is carried out by changing the constitution of the untreated support, cell adhesion and surface adhesion protein. Some cells with strong adhesiveness are difficult to peel off even below the lower critical lysis temperature of the polymer, so try to apply physical stimuli such as pipetting or tapping, or use temperature treatment with diluted trypsin or EDTA. Even when used in combination, a cell sheet may not be obtained.

  When the cell sheet is used as a tissue for regenerative medicine, the medium used for the culture contains autoserum or is serum-free. Furthermore, ES cells, mesenchymal cells, iPS cells, precursor cells, and the like for the purpose of differentiation / induction of various cells are often cultured using a serum-free medium. In cell culture using such a serum-free medium, cell support adhesion tends to be weakened, and complicated adhesion condition setting, complicated operation, sacrifice of reproducibility, etc. are caused by the above-mentioned natural product-derived precoat. It was.

  For this reason, the cell adhesion of the cell culture support is required to be in an appropriate state for adhering / proliferating desired cells by pre-coating on the support, and in the production method in which the temperature-responsive polymer is grafted to the substrate surface. However, a method capable of controlling the strength of adhesiveness and peelability to replace the precoat is desired.

  In addition, in the Japanese Patent Application No. 2007-111757, the present inventors used various plastic materials for temperature-responsive grafts that were only implemented using silane-treated glass, such as polystyrene and some glass slides. Although it shows temperature responsiveness when the base substrate is different, the cell adhesion force and cell detachment force change, and a cell sheet can be produced in a short period of time. Although responsive cell adhesion / detachment was observed, it was difficult to obtain a cell sheet. Among them, a production method for grafting an optimal temperature-responsive polymer onto a base material surface on a material at an appropriate position was required.

  The present invention provides a cell culture support in which at least one stimulus-responsive polymer selected from the group consisting of a temperature-responsive polymer, a pH-responsive polymer, and an ion-responsive polymer is immobilized on a surface via a covalent bond. It is an object of the present invention to provide a technique for optimizing cell adhesion, cell sheet peelability, or single cell peelability according to the type of substrate.

  In the production of a cell culture support in which a stimulus-responsive polymer is immobilized on a surface through a covalent bond, the present inventor is formed by polymerizing the monomer capable of forming the stimulus-responsive polymer and the monomer. By selecting the amount of the monomer in the coating composition containing an oligomer or prepolymer and an organic solvent according to the type of substrate, cell adhesion, cell sheet release or single cell release We found that sex could be optimized. That is, the present invention includes the following inventions.

(1) A method for producing a cell culture support in which at least one stimulus-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 for coating comprising a monomer that can be polymerized by radiation irradiation to form the stimulus-responsive polymer, an oligomer or prepolymer obtained by polymerizing the monomer, and an organic solvent is irradiated with radiation. An application step of applying the stimulus-responsive polymer to a substrate having a surface including a material capable of being introduced via a covalent bond to form a coating film on the surface of the substrate; and applying radiation to the coating film Irradiating the surface of the substrate with a radiation irradiation step for causing the formation reaction of the stimuli-responsive polymer, and a drying step for drying the coating film. The blending amount of the mer is a blending amount determined so that at least one of the cell adhesion of the cell culture support to be produced, the peelability of the cell sheet, and the peelability of a single cell has a predetermined degree, Method.
(2) The base material is polyethylene terephthalate whose surface is subjected to adhesion treatment, polyethylene terephthalate whose surface is coated with urethane acrylate, or polycarbonate whose surface is coated with urethane acrylate, and blending of the monomer in the coating composition The method of (1), wherein the amount is from 3 to 20% by weight.
(3) The base material is a polycarbonate whose surface is plasma-treated, or a blend or polymer alloy of polycarbonate and ABS resin which has been surface-treated with plasma treatment, and the blending amount of the monomer in the coating composition is 2-8. The method according to (1), wherein the method is by weight.
(4) The base material is a porous polycarbonate whose surface has been plasma-treated, or a blend or polymer alloy of a porous polycarbonate and ABS resin whose surface has been plasma-treated, in the coating composition. The method of (1), wherein the amount of the monomer is 1 to 4% by weight.
(5) The ratio of the amount of the monomer and the amount of the oligomer or prepolymer in the coating composition is such that the cell adhesion of the cell culture support to be produced, the peelability of the cell sheet, and the single cell The method according to any one of (1) to (4), wherein the ratio is determined so that at least one of the peelability is a predetermined level.
(6) A cell culture support produced by the method according to any one of (1) to (5).
(7) A process for producing a cell culture support in which at least one stimulus-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 for coating comprising a monomer that can be polymerized by radiation irradiation to form the stimulus-responsive polymer, an oligomer or prepolymer obtained by polymerizing the monomer, and an organic solvent is irradiated with radiation. An application step of applying the stimulus-responsive polymer to a substrate having a surface including a material capable of being introduced via a covalent bond to form a coating film on the surface of the substrate; and applying radiation to the coating film In the manufacturing process, including a radiation irradiation process for promoting the formation reaction of the stimulus-responsive polymer on the surface of the substrate and a drying process for drying the coating film, That cell adhesiveness of the cell culture support, a method of modulating at least one of the peeling of the release property and a single cell of the cell sheet,
The method including adjusting the compounding quantity of the said monomer in the said composition for application | coating according to the kind of said base material.
(8) The method according to (7), comprising adjusting the ratio of the amount of the monomer and the amount of the oligomer or prepolymer in the coating composition according to the type of the substrate.

  According to one aspect of the present invention, when a stimulus-responsive polymer is grafted to the surface of a substrate in the prior art, the cell adhesiveness is low, and it is difficult to form a cell sheet. Cell culture supports capable of stably mass-producing and producing cell sheets equivalent to those using polystyrene as a base material even for high-base materials or substrates with high porosity (low specific gravity) be able to.

  According to another aspect of the present invention, a substrate surface suitable for culturing cells with weak cell adhesion to the substrate or cell types with weak intercellular adhesion and difficult to produce confluent on the substrate is formed. be able to.

  According to yet another aspect of the present invention, the substrate surface is suitable for culturing a cell type that has strong cell adhesion and is difficult to peel off even if the adhesion between the extracellular matrix and the substrate is weakened by low-temperature treatment. Can be formed.

FIG. 1a shows the formulation of the coating solution used in the examples. FIG. 1b shows the formulation of the coating solution used in the examples.

(Stimulus responsive polymer)
The present invention relates to a cell in which at least one stimulus-responsive polymer selected from the group consisting of a temperature-responsive polymer, a pH-responsive polymer, and an ion-responsive polymer is immobilized on a surface (that is, grafted) by a covalent bond. The present invention relates to a method for producing a culture support.
The stimulus-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, which is not preferable. 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, hydroxypropyl cellulose, block copolymers of polypolypropylene oxide and polyethylene oxide, and polyalkylenes 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.

(Base material)
The base material to which the coating composition is applied is not particularly limited as long as the surface thereof includes a material into which the stimulus-responsive polymer can be introduced through covalent bonding by irradiation with radiation. Only the surface may contain a material that can be covalently bonded to the responsive polymer by irradiation, or the entire substrate may contain such a material. Examples of the material for the base material include glasses, plastics, ceramics, metals, and the like that are usually used for cell culture, but are not particularly limited as long as the material can be used for cell culture. An arbitrary layer may be provided on the surface of the substrate or the intermediate layer as long as the object of the present invention is not hindered, and an arbitrary treatment may be performed. For example, the support surface can be hydrophilized using a treatment technique such as ozone treatment, plasma treatment, or sputtering.

  Materials that constitute the substrate and can themselves form a covalent bond with the responsive polymer include polystyrene, low-density polyethylene, medium-density polyethylene, high-density polyethylene, polyurethane, urethane acrylate, and polymethyl methacrylate. Examples thereof include acrylic resins such as polyamide (nylon), polycarbonate, natural rubber having a conjugated bond, synthetic rubber having a conjugated bond, and silicon rubber containing polysilicon. The substrate may be composed of a blend polymer or polymer alloy containing two or more of these materials.

  Examples of the base material surface-treated so as to be covalently bonded to the responsive polymer include polyethylene terephthalate whose surface is easily bonded, synthetic resin whose surface is corona-treated or plasma-treated, and acrylic resin such as urethane acrylate. And synthetic resins coated with the above. The substrate may be composed of a blend polymer or polymer alloy containing two or more of these materials. Examples of the synthetic resin include nylon, low density polyethylene, medium density polyethylene, polypropylene or polyethylene terephthalate, polycarbonate, and polystyrene. The synthetic resin may be composed of a blend polymer or polymer alloy containing two or more of these materials.

  Examples of the shape of the substrate include a dish shape and a film shape. When using a film-shaped base material, after forming a graft polymer layer on the film-shaped base material surface, it can be 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.

(Coating composition)
In the method of the present invention, a coating composition comprising a monomer that can be polymerized by irradiation with radiation to form the polymer, an oligomer or prepolymer obtained by polymerizing the monomer, and an organic solvent is used. Since this coating composition contains an oligomer or a prepolymer, it is difficult to crystallize 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 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, these are collectively referred to as “oligomer or prepolymer”, but the oligomer and the prepolymer are not particularly distinguished, and may be simply referred to as a polymer.

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 viscosity of the coating composition is preferably 5 × 10 ⁻³ Pa · s to 10 Pa · s.

(Method of optimizing the function of the cell culture support by adjusting the composition of the coating composition)
Highly hydrophilic substrates, highly wettable substrates, and substrates with high porosity (low specific gravity) have low cell adhesion when stimuli-responsive molecules are introduced onto the cell surface by grafting, There exists a tendency for the peelability of a cell sheet and a single cell to be inferior. On the other hand, a substrate with high hydrophobicity, a substrate with low wettability, and a substrate with low porosity (high specific gravity) have cell adhesion when a stimulus-responsive molecule is introduced onto the cell surface by grafting. It tends to be high and the cell sheet and single cell peelability tend to be excellent. The feature of the present invention is that the cell of the cell culture support obtained by adjusting the blending amount of the monomer in the coating composition according to the properties of the substrate such as hydrophilicity / hydrophobicity, wettability, porosity, etc. At least one of adhesiveness, cell sheet detachability, and single cell detachability is set to a predetermined level. The ratio of the amount of the monomer and the amount of the oligomer or prepolymer in the coating composition is determined according to at least one of cell adhesion, cell sheet detachability and single cell detachability of the obtained cell culture support. Is more preferably set to a predetermined level.

  The hydrophilicity / hydrophobicity of the substrate can be evaluated by the water contact angle of the surface. For example, water contact angles of typical materials that can be used for the substrate are as follows.

Also, the water contact angle of polyethylene terephthalate subjected to easy adhesion treatment used in the examples is 78 °, the water contact angle of polycarbonate treated with plasma is 72 °, and the porous polycarbonate treated with plasma (pore size 0. 4 μm, pore density 1.0 × 10E8 / cm ² ) is 65 °.

  According to the experimental results shown in the examples, the substrate having a small water contact angle (high hydrophilicity) has a relatively small amount of monomer in the coating composition, so that cell adhesion, cell sheet At least one of detachability and single cell detachability can be optimized. On the other hand, for substrates with a large water contact angle (low hydrophilicity), cell adhesion, cell sheet detachment and single cell detachment can be achieved by using a relatively large amount of monomer in the coating composition. At least one of the sexes can be optimized.

  Specifically, when the base material is polyethylene terephthalate whose surface is bonded, polyethylene terephthalate whose surface is coated with urethane acrylate, and polycarbonate whose surface is coated with urethane acrylate, the coating composition is for coating It is preferable to contain 3 to 20% by weight of monomer with respect to the total amount of the composition. At this time, it is preferable that the cell culture support produced has a dry thickness of 0.05 to 10 μm of the graft polymer layer immobilized on the surface thereof. In this case, the weight ratio of the monomer blended in the coating composition to the oligomer or prepolymer is preferably 0.6 to 6.7.

  When the substrate is a polycarbonate whose surface is plasma-treated, a blend of a surface-treated plasma-treated polycarbonate and an ABS resin, or a polymer alloy, the coating composition has a monomer content of 2 to 2 with respect to the total amount of the coating composition. It is preferable to contain 8% by weight. At this time, it is preferable that the cell culture support produced has a dry thickness of 0.05 to 10 μm of the graft polymer layer immobilized on the surface thereof. In this case, the weight ratio of the monomer blended in the coating composition to the oligomer or prepolymer is preferably 0.4 to 2.0.

  When the base material is a porous polycarbonate whose surface is plasma-treated, a porous polycarbonate whose surface is plasma-treated and a blend or polymer alloy of ABS resin, the coating composition is the total amount of the coating composition. The content of the monomer is preferably 1 to 4% by weight. At this time, it is preferable that the cell culture support produced has a dry thickness of 0.05 to 10 μm of the graft polymer layer immobilized on the surface thereof. In this case, the weight ratio of the monomer blended in the coating composition to the oligomer or prepolymer is preferably 0.2 to 0.8.

(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 necessary coating amount for the graft polymer to exhibit a 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 in this example 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)
The method of the present invention includes a radiation irradiation step of irradiating the coating film with radiation to advance a formation reaction (that is, grafting) of the stimulus-responsive polymer on the surface of the substrate. The formation reaction (grafting) referred to here was not only a phenomenon in which the polymer chain stretches after the free monomer was bonded to the substrate surface, but also was formed in situ from the monomer by polymerization by radiation irradiation. It includes a phenomenon in which a free polymer binds to the substrate surface.

Examples of the radiation used include α rays, β rays, γ rays, electron beams, ultraviolet rays, and the like. In the synthesis for producing 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.

(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 polymer layer of the cell culture support formed through the above-described steps includes not only polymer molecules immobilized by covalent bonding on the substrate surface but also free polymer molecules, monomers or Oligomer molecules exist. Therefore, it is preferable to further include a washing step for washing in order to remove these free polymers or monomer or oligomer molecules.
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 washing | 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. In the cell culture support of the present invention, at least one of cell adhesion, cell sheet detachability, and single cell detachability is optimized in a desired range. In addition, the cell culture support produced by the method of the invention has a more uniform graft polymer layer compared to the cell culture support produced by the conventional method using a raw material monomer / solvent mixture as a coating composition. Features.

(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.

Production of cell culture support (Test 1)
As Examples 1 to 3, an isopropyl acrylamide monomer (manufactured by Kojin Co., Ltd.) and polyisopropyl acrylamide (manufactured by Aldrich, 565311) were dissolved in isopropyl alcohol so as to have the weight% shown in FIGS. (Coating composition). In FIGS. 1a and b,% by weight is% by weight relative to the total amount of the coating solution. In the following examples, they are referred to as coating solutions S, A, B, C, D, E, F, and G.

This coating solution was applied to the following three types of substrates.
Base material 1: Easy adhesion polyethylene terephthalate (PET), water contact angle: 78
Base material 2: Polycarbonate (PC) sheet (200μm thickness, manufactured by Teijin Limited), water contact angle: 72 (after plasma treatment)
Base material 3: Cyclopore membrane sheet (20 μm thick, manufactured by Whatman Corp., material polycarbonate, hole diameter 0.4 μm, hole density 1.0 x 10E8 / cm ² ), contact angle: 65 (after plasma treatment)

The coating solution was applied to the equipment 1 to 3 with a 4th wire bar with a coating thickness of about 5 g / m ² , and the dry surface quality was confirmed by drying with a dryer. The coated surface of the coated substrate was irradiated with an electron beam at a dose of 24 Mrad with a low voltage electron beam irradiation device (EC 250/15/180 L manufactured by Iwasaki Electric Co., Ltd.).
The coated film prepared under each condition was washed and dried, and the one cut into a dish size was fixed to the dish with an adhesive and EOG sterilized to obtain a cell culture support.
In Examples 1 to 3, a cell culture support was prepared using three kinds of coating liquid S (monomer 40%, polymer 2%), B, and E on the easy-adhesion PET of the substrate 1. As Comparative Example 1, a commercially available polystyrene cell culture dish (Tissue Cell Poly Styrene (TCPS) manufactured by Becton Deckton) was used.

The evaluation method was performed by the following three types. The cells used for the evaluation were bovine carotid artery normal vascular endothelial cells (Human Science Research Resource Bank JCRB0099 commonly known as HH). The culture solution used was Eagle Dulbecco's modified medium supplemented with 10% bovine serum.
Evaluation 1: Evaluation of cell adhesion
6 × 10 ⁴ HH cell suspensions passaged and detached by trypsin treatment in 35 mm cell culture dish were seeded on the support (dish) of Examples 1 to 3 and Comparative Example 1, and the medium was changed 6 hours after seeding. At the same time, the supernatant culture was cultured in another cell culture dish for 18 hours, and non-adherent cells were observed.
Evaluation 2: Evaluation of peelability of single cell
35mm cell culture Ditsushiyu implement 10 ⁴ HH cell suspension were passaged, detached by trypsinization to Examples 1-3, were seeded in support of Comparative Example 1 (dish), low temperature incubator (20 ° C. After 24 hours 5% CO ₂ ) was observed for detachment of adherent cells before migration (0 minutes), 10 minutes, 30 minutes, 60 minutes and 120 minutes after migration.
Evaluation 3: Evaluation of cell sheet peelability
6 × 10 ⁴ HH cell suspensions passaged and detached by trypsin treatment on 35 mm cell culture dish were seeded on the support (dish) of Examples 1 to 3 and Comparative Example 1, and the cells were cultured 6 days later. Proliferation / monolayer HH cells are moved to a low temperature incubator (20 ° C, 5% CO ₂ ), and after 20 minutes, the bottom of the dish is cut into the monolayer cells about 3-5mm, and the round cell sheet is completely detached. Was observed and timed. The measurement was performed twice, and the time required for peeling was measured for each.

  The evaluation results are shown in Table 2. The 6-hour adhesion of HH cells to a temperature-responsive cell culture support based on easy-adhesion PET was observed with a slight cell adhesion residue (all less than 5%) compared to the comparative cell culture dish. The difference was not so great as to cause problems in adhesion, growth, passage, and cell sheet detachment of the target cells. When the cell adhesion residue was compared between supports having different compositions of the coating solution, a tendency toward a slight increase was observed with a high monomer ratio.

  Comparative Example 1 in which a temperature-responsive polymer is not grafted on the substrate surface when seeding a small number of cells per support area of evaluation 2 and performing cell detachment by a low temperature treatment from a state where there is not much cell-cell adhesion In the cell culture dish (TCPS), the cells remained adhered after 2 hours. However, in the grafted easy-adhesion PET, it was observed that the cells detached from the support over time and floated in the culture solution. When the 1.5 mm square field was compared between the supports having different composition of the coating solution at each observation elapsed time, it was observed that the cell ratio disappeared completely from the field of view with a low monomer ratio.

  HH cells were allowed to adhere and proliferate for 6 days after seeding on the support to obtain a confluent state. This support (dish) was observed to be cooled and peeled off by the method of Evaluation 3. In the comparative cell culture dish in which the temperature-responsive polymer was not grafted on the surface of the base material, the cell sheet was not peeled at all other than the cut portion even after 1 day. Cell sheets were obtained in all of the supports in which the temperature-responsive polymer was grafted to the easy-adhesion PET substrate. It was shown that the support prepared with the coating liquid B has a shorter peeling time than the support prepared with the coating liquids S and E. Thus, there existed optimum conditions for the monomer / polymer ratio of the coating solution for processing an easily-adhesive PET substrate into a cell culture support.

  In Examples 4 to 8, cells were applied to 200 μm-thick polycarbonate sheet (base material 2) by applying coating solutions A to E to coating, drying, electron beam irradiation, washing, drying, and dish as in Examples 1-3. A culture support was obtained. This support was tested for cell adhesion and detachment by HH cell seeding in Evaluation Methods 1 to 3. The results are shown in Table 3.

The substrate 2 before processing was subjected to plasma treatment. The plasma treatment was performed by filling with oxygen at about 60 to 200 mm Torr under reduced pressure and plasma discharge at 400 W for 3 minutes.
When HH cells were seeded by the methods of Evaluations 1 and 3, the number of cells remaining after 6 hours of support on the support surface was higher as the monomer ratio of the coating solution was higher, and support using coating solutions A and B was used. The body did not get enough adhesion and proliferation in 6 days and did not become confluent. CE obtained a confluent state with sufficient growth. In the low temperature treatment of evaluation 3, cell sheets were formed in a short time with all of C to E. However, it was observed that the adhesion between cells was weak at C and was damaged when the sheet was peeled off.
In the single cell exfoliation of Evaluation 2, the cells in the visual field were exfoliated within 60 minutes on all the supports and floated in the culture solution.

  In Examples 9 to 13, coating liquids C to G are applied to a coating, drying, electron beam irradiation, washing, drying, and dish in the same manner as in Examples 1 to 3 on a 20 μm-thick cyclopore membrane sheet (base material 3). A cell culture support was obtained. This support was tested for cell adhesion and detachment by HH cell seeding in Evaluation Methods 1 to 3. The results are shown in Table 4.

Plasma treatment was performed on the base material 3 before processing. The plasma treatment was performed by filling with oxygen at about 60 to 200 mm Torr under reduced pressure and plasma discharge at 400 W for 3 minutes.
When HH cells were seeded by the methods of Evaluations 1 and 3, the number of cells remaining after 6 hours of adhesion to the support surface was higher as the monomer ratio of the coating solution was higher, and support using coating solutions C and D was used. The body did not get enough adhesion and proliferation in 6 days and did not become confluent. EG obtained a confluent state with sufficient growth. In the low temperature treatment of evaluation 3, cell sheets were formed in a short time with all of C to E. However, it was observed that the adhesion between cells was weak at C and was damaged when the sheet was peeled off.
In the single cell exfoliation of Evaluation 2, the cells in the visual field were exfoliated within 60 minutes on all the supports and floated in the culture solution.

Claims (13)

A method for producing a cell culture support in which a stimulus-responsive polymer that is a temperature-responsive polymer is immobilized on a surface by covalent bonding,
A coating composition comprising a monomer that can be polymerized by radiation irradiation to form the stimulus-responsive polymer, an oligomer or prepolymer obtained by polymerizing the monomer, and an organic solvent, An application step in which a polymer is applied to a substrate with a surface containing a material that can be introduced via a covalent bond to form a coating on the surface of the substrate;
Radiation irradiation step of irradiating the coating film with radiation to advance the formation reaction of the stimulus-responsive polymer on the substrate surface;
A drying step of drying the coating film,
The blending amount of the monomer in the coating composition is such that at least one of the cell adhesion of the cell culture support to be produced, the peelability of the cell sheet, and the peelability of a single cell is a predetermined level. It is a blending amount determined based on the water contact angle of the substrate,
The molecular weight of the oligomer or prepolymer rather greater than 3,300,
The method, wherein the monomer is isopropylacrylamide monomer and the oligomer or prepolymer is polyisopropylacrylamide . Performing the irradiation step after the drying step, the method according to claim 1. The base material is polyethylene terephthalate whose surface is bonded, polyethylene terephthalate whose surface is coated with urethane acrylate, or polycarbonate whose surface is coated with urethane acrylate, and the amount of the monomer in the coating composition is 3 3. A process according to claim 1 or 2 which is -20% by weight. The method of claim 3 , wherein the weight ratio of the monomer to the oligomer or prepolymer is 0.6 to 6.7. The base material is a polycarbonate whose surface is plasma-treated, or a blend or polymer alloy of a polycarbonate and ABS resin which has been surface-treated with plasma treatment, and the amount of the monomer in the coating composition is 2 to 8% by weight. The method according to claim 1 or 2 , wherein: The method according to claim 5 , wherein a weight ratio of the monomer to the oligomer or prepolymer is 0.4 to 2.0. The base material is a porous polycarbonate whose surface is plasma-treated, or a blend or polymer alloy of a porous polycarbonate whose surface is plasma-treated and an ABS resin, and the blending amount of the monomer in the coating composition The method according to claim 1 or 2 , wherein is 1 to 4% by weight. The method according to claim 7 , wherein a weight ratio of the monomer to the oligomer or prepolymer is 0.2 to 0.8. The ratio of the blending amount of the monomer and the blending amount of the oligomer or prepolymer in the coating composition is such that the cell adhesion of the cell culture support to be produced, the peelability of the cell sheet, and the peelability of a single cell. The method according to any one of claims 1 to 8 , wherein the ratio is determined based on a water contact angle of the substrate so that at least one of the two has a predetermined degree. Cell culture support manufactured by the method according to any one of claims 1-9. A process for producing a cell culture support in which a stimulus-responsive polymer that is a temperature-responsive polymer is immobilized on a surface by covalent bonding, and a monomer that can be polymerized by irradiation to form the stimulus-responsive polymer And a coating composition containing an oligomer or prepolymer obtained by polymerizing the monomer and an organic solvent, and a surface containing a material into which the stimulus-responsive polymer can be introduced through a covalent bond by irradiation with radiation. Applying to the base material to form a coating film on the surface of the base material, and irradiating the coating film with radiation to advance the formation reaction of the stimulus-responsive polymer on the base material surface In the manufacturing process including a radiation irradiation process and a drying process for drying the coating film, at least one of cell adhesion, cell sheet detachability, and single cell detachability of the produced cell culture support. There is provided a method of modulating,
Adjusting the blending amount of the monomer in the coating composition according to the water contact angle of the substrate;
The molecular weight of the oligomer or prepolymer rather greater than 3,300,
The method, wherein the monomer is isopropylacrylamide monomer and the oligomer or prepolymer is polyisopropylacrylamide . The method according to claim 11 , wherein the radiation irradiation step is performed after the drying step. The method according to claim 11 or 12 , comprising adjusting a ratio of the amount of the monomer and the amount of the oligomer or prepolymer in the coating composition according to a water contact angle of the substrate. .

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