JP6989116B2 - Cell sheet manufacturing method and cell culture support - Google Patents

Description

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

A transplantation method using a cell sheet has been developed for the purpose of regenerating damaged living tissue. Most of the cell sheets used for transplantation are prepared using attachment-dependent cells among animal cells including human cells. In order to prepare a cell sheet, it is necessary to attach animal cells on the surface of a substrate, incubate them in the form of a sheet, and exfoliate them while maintaining their morphology. For example, Patent Document 1 states that skin cells are below or below the upper limit critical lysis temperature on a cell culture support whose substrate surface is coated with a polymer having an upper or lower critical lysis temperature of 0 ° C to 80 ° C. Described is a skin cell culture method in which cultured skin cells are exfoliated by culturing at a critical dissolution temperature or higher and then lowering the upper critical dissolution temperature or higher or the lower critical dissolution temperature or lower.

Japanese Unexamined Patent Publication No. 05-192138

When a cell sheet was prepared using the cell culture support described in Patent Document 1, the cell sheet may not be exfoliated or suffered chilling injury depending on the cell type. Therefore, an object of the present invention is to provide a cell culture support which has good cell proliferation and can peel off a cell sheet without relying on low temperature treatment, and a method for producing a cell sheet using the same.

The first aspect is on a cell culture support having a polymer having a structural unit derived from (meth) acrylate having an alkyl group having 14 to 22 carbon atoms and a structural unit derived from (meth) acrylic acid on the surface. Includes culturing cells at temperature to form a cell sheet on a cell culture support and holding the cell culture support at a second temperature higher than the first temperature to recover the cell sheet. This is a method for producing a cell sheet.
The second embodiment comprises a substrate and a polymer comprising a constituent unit derived from a (meth) acrylate having an alkyl group having 14 to 22 carbon atoms and a constituent unit derived from (meth) acrylic acid arranged on the substrate. A cell culture support comprising.
A third aspect is a composition comprising a polymer containing a structural unit derived from a (meth) acrylate having an alkyl group having 14 to 22 carbon atoms and a structural unit derived from (meth) acrylic acid, and a liquid medium.
The fourth aspect is based on a composition containing a polymer containing a structural unit derived from (meth) acrylate having an alkyl group having 14 to 22 carbon atoms and a structural unit derived from (meth) acrylic acid, and a liquid medium. A method for producing a cell culture support, which comprises applying the above.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a cell culture support which has good cell proliferation and can peel off a cell sheet without relying on low temperature treatment, and a method for producing a cell sheet using the same.

It is a figure which shows the surface tension of the cell culture dish to which a polymer was attached. It is a figure which shows the state of cell proliferation after 48 hours of culture. It is a figure which shows the state of cell proliferation after 72 hours of culture. It is a figure which shows the peeling state of a cell sheet. It is a figure which shows the surface tension of the glass cell culture dish to which a polymer was attached. It is a figure which shows the state of cell proliferation after 48 hours of culture. It is a figure which shows the state of cell proliferation after 72 hours of culture. It is a figure which shows the peeling state of a cell sheet. It is a figure which shows the surface tension of the cell culture dish to which a polymer was attached. It is a figure which shows the peeling state of a cell sheet. This is an experimental schedule for evaluating the cell proliferation ability of the cell sheet. It is an evaluation result of the cell proliferation ability of a cell sheet.

In the present specification, the content of each component in the composition is the total amount of the plurality of substances present in the composition when a plurality of substances corresponding to each component are present in the composition, unless otherwise specified. Means. (Meta) acrylic acid means at least one of acrylic acid and methacrylic acid, and (meth) acrylate means at least one of acrylic acid ester and methacrylic acid ester.

<Method of manufacturing cell sheet>
A method for producing a cell sheet is a cell culture support having a polymer having a structural unit derived from (meth) acrylate having an alkyl group having 14 to 22 carbon atoms and a structural unit derived from (meth) acrylic acid on the surface. To cultivate cells at the first temperature to form a cell sheet on the cell culture support, to hold the cell culture support at a second temperature higher than the first temperature, and to recover the cell sheet. including.

By culturing cells on a cell culture support having a polymer having a specific composition on the surface, the cells adhere to the cell culture support and show good proliferative properties to obtain a uniform cell sheet. By holding the cell culture support having the cell sheet formed on the surface at a temperature higher than the culture temperature, the surface state of the cell culture support changes and the cell sheet peels off. This gives a cell sheet exfoliated from the cell culture support without chilling injury.

(Cell culture support)
The cell culture support used in the method for producing a cell sheet is a structural unit derived from a base material and a (meth) acrylate having an alkyl group having 14 to 22 carbon atoms arranged on the surface of the base material, and (meth). It comprises a polymer containing a structural unit derived from acrylic acid. By having a specific configuration, the polymer has adhesiveness to the base material and can impart appropriate hydrophilicity to the surface of the base material. Further, the polymer has a peculiar temperature-sensitive property that it exhibits adherence to cultured cells at the first temperature, which is the culture temperature, and loses adherence to cultured cells at a second temperature higher than the culture temperature.

The material of the base material used for the cell culture support can be appropriately selected from the materials of the base material usually used depending on the purpose and the like. Examples of the material of the base material include polystyrene, polyethylene, a polyvinyl resin such as polypropylene, and glass. Further, the shape, size, etc. of the base material are not particularly limited as long as cell culture is possible, and may be appropriately selected according to the shape, size, etc. of the target cell sheet. The base material can be appropriately selected from commercially available base materials for cell culture according to the purpose and the like.

The polymer placed on the surface of the substrate contains a first structural unit derived from a (meth) acrylate having an alkyl group having 14 to 22 carbon atoms and a second structural unit derived from (meth) acrylic acid. .. The number of carbon atoms of the alkyl group contained in the first structural unit is preferably 16 to 22 because of the adhesion of the polymer to the substrate. Specific examples of the monomer forming the first structural unit include linear alkyls such as tetradecyl (meth) acrylate, hexadecyl (meth) acrylate, stearyl (meth) acrylate, eicosanyl (meth) acrylate, and behenyl (meth) acrylate. (Meta) acrylates having a group can be mentioned.

The molar ratio of the first structural unit to the second structural unit (first structural unit: second structural unit) contained in the polymer is, for example, 2: 8 to 5: 5, and 2: 8 to 4: 6. preferable. The polymer may be a random copolymer or a block copolymer, but is preferably a block copolymer.

The polymer may further contain the first structural unit and other structural units other than the second structural unit to the extent that the effect of the present invention is not impaired. The monomer constituting the other structural unit is not particularly limited as long as it can be copolymerized with (meth) acrylate and (meth) acrylic acid having an alkyl group having 14 to 22 carbon atoms, and for example, styrene, butyl acrylate and the like. Examples thereof include (meth) acrylates having an alkyl group having 12 or less carbon atoms.

The molecular weight of the polymer is, for example, 3,500 to 200,000 as a weight average molecular weight (Mw), preferably 6,000 to 70,000. Further, the degree of polydispersity (Mw / Mn) is, for example, 1.05 to 15, preferably 1.2 to 2. The weight average molecular weight and the degree of polydispersity can be determined in terms of polystyrene using GPC.

When the polymer is a block copolymer, the weight average molecular weight of the first structural unit portion is, for example, 500 or more, 1,000 or more, 2,000 or more, 3,000 or more, 4,000 or more, or 5,000 or more. It is 100,000 or less, 50,000 or less, 10,000 or less, 8,000 or less, or 7,000 or less. The weight average molecular weight of the second structural unit portion is, for example, 500 or more, 1,000 or more, or 2,000 or more, and 100,000 or less, 20,000 or less, or 15,000 or less. The degree of polymerization of the first structural unit portion is, for example, 2 or more, 5 or more or 10 or more, and 800 or less, 500 or less, 100 or less or 50 or less. The degree of polymerization of the second structural unit portion is, for example, 5 or more, 10 or more or 30 or more, and 800 or less, 500 or less or 100 or less.

The polymer can be produced by a commonly used production method. For example, when the polymer is a block copolymer, it can be produced by various living polymerization methods (radicals, anions, cations). Examples of the living radical polymerization method include an NPM method, an ATRP method, a RAFT method, and the like. Specifically, for example, the production method described in International Publication No. 2012/098750 can be referred to.
Moreover, as an initiator used for the polymerization of a polymer, for example, BlockBuilder MA (manufactured by Arkema) and the like can be mentioned.

The amount of polymer to be disposed on the surface of the substrate, for example, 30 [mu] g / cm ² or more 160 [mu] g / cm ² or less, 50 [mu] g / cm ² or more 80 [mu] g / cm ² or less. Further, the polymer may be used alone or in combination of two or more polymers having different configurations.

(Cell culture)
The cell sheet is formed by seeding desired cells on a cell culture support and culturing at the first temperature (culture temperature). The cells to be cultured on the cell culture support are not particularly limited as long as they are animal cells, and examples thereof include cells immediately after being collected from a living body and established cell lines. Examples of the origin of animal cells include mammals including humans. The cells may be somatic cells or stem cells.

A commonly used medium can be used for culturing the cells. The medium used for culturing may be any medium generally used for culturing animal cells, and for example, various basal cultures containing no serum such as RPMI medium, Dalveco modified eagle medium (DMEM), MEM medium, and F12 medium. A liquid (standard culture medium) can be mentioned. Serum for promoting cell proliferation is added to this medium, or as a substitute for serum, for example, cell growth factors such as FGF, EGF and PDGF, known serum components such as transferrin and the like are added. May be good. The concentration when adding serum can be appropriately changed depending on the culture state at that time, and can be, for example, from 5% by volume to 10% by volume. The medium may be supplemented with various vitamins, antibiotics such as streptomycin, differentiation-inducing substances and the like.

The density of cells to be seeded on the cell culture support may be appropriately selected depending on the cell type and the like as long as a cell sheet can be formed. For example, it can be ^{from 3 × 10 4} to 6 × 10 ⁴ cells / cm ^{2 per culture area.}

The cells are cultured at the first temperature and can be appropriately selected depending on the cells. For example, it can be 35 to 37 ° C. Cell culture may be performed in an incubator with a 5% CO _{2 concentration.} Cell sheets derived from various cells may be formed by culturing under normal culture conditions for about 3 to 5 days until a confluent state is reached. The formation of the cell sheet can be confirmed by microscopic observation.

(Recovery of cell sheet)
The recovery of the cell sheet formed on the cell culture support is performed by holding the cell culture support at a second temperature (exfoliation temperature) higher than the first temperature. By holding the cell culture support on which the cell sheet is formed at the second temperature, the cell sheet can be peeled off from the cell culture support and the cell sheet can be recovered. In the peeling of the cell sheet, the cell culture support may be shaken if necessary, or a part of the outer edge of the cell sheet may be physically peeled and then the cell culture support may be shaken.

The second temperature may be higher than the first temperature and may be a temperature that does not damage the cells. The second temperature is preferably a temperature at which the adhesion of the polymer to the substrate is maintained. The second temperature can be, for example, 38 ° C to 45 ° C, preferably 38 ° C to 40 ° C. The time for retaining the second temperature in the recovery of the cell sheet can be, for example, 10 to 180 minutes.

In the method for producing a cell sheet, the cultured cells can be collected in the sheet state without extremely lowering the temperature, and the cells are not chilling injury. In addition, the recovered cell sheet has high uniformity, and agglomeration due to shrinkage of the cell sheet is suppressed. Furthermore, good cell proliferation is maintained in the cells constituting the cell sheet.

<Composition>
The composition according to the present embodiment includes a polymer containing a structural unit derived from (meth) acrylate having an alkyl group having 14 to 22 carbon atoms and a structural unit derived from (meth) acrylic acid, and a liquid medium. The composition is applied to the surface treatment of a substrate used for cell culture, and exhibits a unique temperature-sensitive property that exhibits adhesion to cultured cells at a culture temperature and loses adhesion to cells at a temperature higher than the culture temperature. A polymer layer having the above can be formed on the surface of the substrate. That is, the composition is used as a coating agent for a cell culture substrate.

Details of the polymer contained in the composition are as described above. The composition may contain one kind of polymer alone, or may contain two or more kinds of polymers having different compositions in combination.

The liquid medium can be appropriately selected from commonly used liquid media as long as it can dissolve at least a part of the polymer. The liquid medium is preferably soluble in the polymer at the desired concentration and is preferably inert to the applied substrate. The liquid medium can contain at least one of a water-soluble organic solvent such as, for example, dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), ethanol and the like. Among them, the liquid medium preferably contains DMSO because the surface coating of the cell culture substrate can be easily and quickly performed without the need for special means and no special sterilization operation is required. Further, the liquid medium may contain water or may be a mixture of a water-soluble organic solvent and water.

The content of the polymer in the composition can be, for example, 1 (w / v)% or less, preferably 0.025 (w / v)% or more and 0.05 (w / v)% or less.

The composition can be prepared, for example, by dissolving or dispersing the polymer in a liquid medium to a desired concentration. The composition can impart hydrophilicity to the surface of the substrate by forming a polymer layer on the surface of the substrate to which it is applied. The substrate on which the polymer layer is formed can be used as a cell culture support in a method for producing a cell sheet.

<Manufacturing method of cell culture support>
The cell culture support can be produced by applying the composition to a substrate for cell culture and adhering a polymer to the surface of the substrate. The method for applying the composition may be appropriately selected from commonly used methods such as dropping, coating, spraying, and dipping. The amount of the composition applied may be appropriately selected so that the amount of the polymer adhering to the surface of the base material is within a desired range in consideration of the area of the base material and the concentration of the polymer in the composition.

After applying the composition to the surface of the substrate, at least a part of the liquid medium contained in the composition may be removed. This allows, for example, the polymer to adhere to the surface of the substrate. The liquid medium can be removed, for example, by volatilizing the liquid medium under reduced pressure or normal pressure. When the liquid medium contains a water-soluble organic solvent, the water-soluble organic solvent may be removed by adding an aqueous medium such as physiological saline and then removing the supernatant. The coating time from applying the composition to the surface of the substrate to removing the liquid medium is, for example, 30 minutes or more, preferably 60 minutes or more. Also, for example, it is 180 minutes or less. The temperature at which the polymer is attached to the surface of the substrate is, for example, 20 ° C. or higher and 50 ° C. or lower, preferably about room temperature (25 ° C.).

The method for producing a cell culture support is, for example, a simple method of adding a composition as a surface modifier to a substrate for cell culture, and therefore, it can be easily applied to any substrate such as a commercially available product. Can be done. In addition, since the same surface characteristics can be easily imparted to the surface of any substrate, a cell sheet of uniform quality can be stably produced by using the obtained cell culture support. ..

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to these Examples.

(Manufacturing Example 1)
Stearyl acrylate (STA; manufactured by Tokyo Chemical Industry Co., Ltd.) 6.6 g (20 mmol) was dissolved in 6.6 g of butyl acetate, and BlockBuilder MA No. 33 (manufactured by Arkema) was added, and the mixture was polymerized at a polymerization temperature of about 105 ° C., a stirring speed of 75 rpm, and a nitrogen atmosphere for 4.5 hours. Next, a solution prepared by dissolving 3.415 g (47 mmol) of acrylic acid (AA; manufactured by Tokyo Chemical Industry Co., Ltd.) in 3.42 g of butyl acetate was added, and the mixture was polymerized at 105 ° C. for 27 hours. The obtained polymerization solution was purified by reprecipitation to obtain a block copolymer. The charging ratio of STA, which is the first structural unit, and AA, which is the second structural unit, used in the production of the block copolymer was STA: AA = 3: 7.
When the weight average molecular weight (Mw) of the obtained block copolymer was determined in terms of polystyrene using GPC, the weight average molecular weight of the first structural unit portion was about 4,519, and the weight average of the second structural unit portion was obtained. The molecular weight was about 3,371. The weight average molecular weight of the first structural unit portion was determined by sampling the reaction solution before adding acrylic acid. The composition of the weight average molecular weight in the obtained polymer was STA: AA = 5000: 3000.

(Example 1)
The polymer obtained in Production Example 1 was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 0.05% (w / v) to prepare a composition for cell culture dish coating.
1.5 mL of the obtained composition was added dropwise to a 35 mm untreated dish (IWAKI # 1000-035) made of polystyrene (PS) to spread it over the entire culture surface, and then 2 ml of PBS was added and rinsed. A cell culture support with a polymer attached to the surface was prepared.

In order to evaluate the surface condition of the obtained cell culture support, 100 μL of PBS was added dropwise at room temperature, and the condition of the droplet was observed. At the same time, the surface condition of the dish (a) treated only with PBS and the dish (b) treated only with DMSO and rinsed with PBS was evaluated by dropping PBS in the same manner. The results are shown in FIG.

From FIG. 1, it can be seen that the cell culture support (c) to which the polymer is attached has a lower surface tension and is hydrophilic as compared with the PBS-treated dish (a) and the DMSO-treated dish (b). When PBS was added to the cell culture support to which the polymer was attached, the mixture was allowed to stand at 37 ° C. for 72 hours, heated at 45 ° C. for 30 minutes, and then the surface condition was evaluated in the same manner, the surface tension remained low. Met. From this, it was found that the polymer layer was not peeled off by the heat treatment at 45 ° C. for 30 minutes.

(Example 2)
A cell sheet was prepared using the cell culture support prepared in Example 1. DMEM medium (10% FCS added) was added to the cell culture support, and a human tongue cancer-derived squamous cell carcinoma cell line (NA) was ^{inoculated at 3 × 10 5} cells / dish. Cell culture was performed for 72 hours in an incubator at 37 ° C. and a 5% CO _{2 concentration to form a cell sheet.}
Then, it was shaken at 45 ° C. for 30 to 60 minutes.

(Comparative Example 1)
Example 2 except that a 35 mm untreated dish made of polystyrene (PS) (# 1000-035 manufactured by IWAKI) treated with DMSO only was used instead of the cell culture support prepared in Example 1. Cell culture was performed in the same manner as above.

(Comparative Example 2)
Same as Example 2 except that a polystyrene (PS) 35 mm TC-treated dish (FALCON, # 353001) treated with DMSO alone was used instead of the cell culture support prepared in Example 1. Cell culture was performed.
Then, it was shaken at 45 ° C. for 30 to 60 minutes.

The results of observing the state of the cultured cells in Example 2, Comparative Examples 1 and 2 using a microscope (× 10) are shown in FIGS. 2 and 3. FIG. 2 shows the proliferative state of cells after 48 hours, and FIG. 3 shows the state of cell proliferation after 72 hours.
Further, FIG. 4 shows the states of Example 2 and Comparative Example 2 after shaking at 45 ° C. for 30 minutes.

From FIG. 2, when Example 2 and Comparative Example 2 are compared, it can be seen that the cells proliferate more uniformly in Example 2. Further, from FIG. 3, it can be seen that in Example 2 and Comparative Example 2, the cells proliferate confluently and form a sheet.
From FIG. 4, it can be seen that the cultured cells were exfoliated in the form of a sheet in Example 2, whereas the exfoliation of the cultured cells was not observed in Comparative Example 2.

(Production Example 2) Production of FITC Labeled Polymer 5 g of stearyl acrylate (STA; manufactured by Tokyo Chemical Industry Co., Ltd.) was dissolved in 5 g of butyl acetate, and BlockBuilder MA No. 33 (manufactured by Arkema) 0.381 g was added, and the mixture was polymerized at a polymerization temperature of about 110 ° C. and in a nitrogen atmosphere for 5 hours. Next, a solution prepared by dissolving 200 mg of fluorescein isothiocyanate (FITC) and 4 g of acrylic acid (AA; manufactured by Tokyo Chemical Industry Co., Ltd.) in 4 g of butyl acetate was added, and the mixture was polymerized at 110 ° C. for 29 hours. The obtained polymer solution was purified by reprecipitation to obtain a FITC-labeled polymer.

(Production Example 3) Production of anthracene-labeled polymer 100 mg of the block copolymer obtained in Production Example 1 and 107 mg of tetramethylammonium hydrochloride / pentahydrate were added to 15 mL of N, N-dimethylformamide. 160 mg of 9-chloromethylanthracene was added thereto, and the mixture was stirred at 70 ° C. for 6 hours. The obtained pale yellow solution was added dropwise to ethanol to precipitate the polymer. The precipitate was washed with ethanol and vacuum dried to give a white anthracene labeled polymer.

(Example 3)
Example 1 and Example 1 except that the FITC-labeled polymer obtained in Production Example 2 or the anthracene-labeled polymer obtained in Production Example 3 was used as the labeled polymer in place of the polymer obtained in Production Example 1. Cell culture supports were prepared in the same manner, and using these, cell culture was carried out in the same manner as in Example 2 to form a cell sheet. Then, it was shaken at 45 ° C. for 60 minutes.

The detached cell sheets were transferred into PBS of different cell culture dishes, and the fluorescence of the label was observed for the transferred cell sheets and the cell culture support after the cell sheets were detached. For the FITC-labeled polymer, an excitation wavelength of 470 nm and a detection wavelength of 525 nm were used, and for the anthracene-labeled polymer, an excitation wavelength of 360 nm and a detection wavelength of 447 nm were used.

As a result of fluorescence observation, almost no fluorescence was detected in the peeled cell sheet, but clear fluorescence was detected in the cell culture support after peeling the cell sheet regardless of which labeled polymer was used. ..
From the above, it can be seen that even if the cell sheet is peeled off at 45 ° C. after the cell sheet is formed, the polymer does not peel off from the substrate.

(Example 4)
The polymer obtained in Production Example 1 was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 0.05% (w / v) to prepare a composition for cell culture dish coating.
1.5 mL of the obtained composition was added dropwise to a glass 35 mm untreated dish (Matsunami Glass Ind. Co., Ltd., # D111300) to spread over the entire culture surface, and then 2 ml of PBS was added and rinsed. , A cell culture support in which a polymer was attached to the glass surface was prepared.

In order to evaluate the surface condition of the obtained cell culture support, 100 μL of PBS was added dropwise at room temperature, and the condition of the droplet was observed. At the same time, the surface condition of the dish (a) treated with DMSO alone and rinsed with PBS was evaluated by dropping PBS in the same manner. The results are shown in FIG.
From FIG. 5, it can be seen that the cell culture support (b) to which the polymer is attached has a lower surface tension and is hydrophilic as compared with the DMSO-treated dish (a).

(Example 5)
Cell culture was performed in the same manner as in Example 2 except that the cell culture support prepared in Example 4 was used instead of the cell culture support prepared in Example 1, to prepare a cell sheet. ..
Then, it was shaken at 45 ° C. for 120 minutes.

The results of observing the state of the cultured cells in Example 5 using a microscope (× 10) are shown in FIGS. 6 and 7, respectively. FIG. 6 shows the cell proliferation state after 48 hours, and FIG. 7 shows the cell proliferation state after 72 hours of Comparative Example 3. Further, FIG. 8 shows the state of Example 5 after shaking at 45 ° C. for 120 minutes.

From FIGS. 6 and 7, it can be seen that in Example 5, the cells proliferated uniformly, and after 72 hours, the cells proliferated confluently to form a sheet. From FIG. 8, it can be seen that in Example 5, the cultured cells were exfoliated in the form of a sheet.

(Manufacturing Example 4)
Production Example 1 except that the conditions are changed so that the weight average molecular weight of the first structural unit portion (STA) and the weight average molecular weight of the second structural unit portion (AA) are STA: AA = 5000: 1000. The polymer was produced in the same manner.

(Manufacturing Example 5)
Production Example 1 except that the conditions are changed so that the weight average molecular weight of the first structural unit portion (STA) and the weight average molecular weight of the second structural unit portion (AA) are STA: AA = 5000: 12000. The polymer was produced in the same manner.

(Manufacturing Example 6)
Production Example 1 except that the conditions are changed so that the weight average molecular weight of the first structural unit portion (STA) and the weight average molecular weight of the second structural unit portion (AA) are STA: AA = 10000: 10000. The polymer was produced in the same manner.

(Manufacturing Example 7)
Hexadecyl acrylate (HDA) is used instead of stearyl acrylate, and the weight average molecular weight of the first structural unit portion (HDA) and the weight average molecular weight of the second structural unit portion (AA) are HDA: AA = 8000: 8000. A polymer was produced in the same manner as in Production Example 1 except that the conditions were changed so as to be.

(Example 6)
A cell culture support was prepared under coating conditions at room temperature for 2 hours in the same manner as in Example 1 except that the polymers of Production Examples 4 and 5 were used instead of the polymer of Production Example 1. In order to evaluate the surface condition of the obtained cell culture support, 100 μL of PBS was added dropwise at room temperature, and the condition of the droplet was observed. At the same time, the surface condition of the dish (Noncoat) treated with DMSO only and rinsed with PBS was evaluated by dropping PBS in the same manner. The results are shown in FIG.

It can be seen that the cell culture support to which the polymer is attached has a lower surface tension than the non-coat dish.

(Example 7)
Using the cell culture supports obtained in Examples 1 and 6, cell culture was performed in the same manner as in Example 2. Further, the second temperature was 43 ° C. or 45 ° C., and the shaking time was 30 minutes, 60 minutes or 120 minutes, and the detachability of the cell sheet from the cell culture support was evaluated. The results are shown in FIG. 10 and Table 1. Note that FIG. 10 shows a state after shaking at 43 ° C. for 60 minutes. The evaluation in Table 1 was performed by visual observation, and the evaluation criteria were as follows.
Evaluation Criteria A: Completely peeled off.
B: Partially peeled off, but incomplete.
C: No peeling.

From FIG. 10 and Table 1, it can be seen that the larger the weight average molecular weight of the second structural unit portion, the better the peelability.

(Example 8)
Using the polymers obtained in Production Examples 1, 6 and 7, cell culture supports were obtained under coating conditions at room temperature for 2 hours in the same manner as in Example 1. Using the obtained cell culture support, cells were cultured in the same manner as in Example 2 except that the culture time was set to 5 days, and then permeated at 45 ° C. for 120 minutes to peel the cell sheet from the cell culture support. did. The exfoliated cell sheet was pipette-operated to disperse the cells into a single cell. After cell counting, seeds were seeded on a 96-well culture plate (FALCON, # 353072) at 1 × 10 ⁴ cell / well, and cell culture was carried out under the same conditions for 3 days. At that time, every 24 hours, using Cell Counting Kit-8 (CCK-8; manufactured by Dojin Kagaku Co., Ltd.), WST-8 assay was performed by a method according to the instruction manual to evaluate the number of viable cells. The experimental schedule is shown in FIG. 11A and the WST-8assay results are shown in FIG. 11B. The results of the cultured cells obtained in the same manner except that the cell culture dish of Non Coat not coated with the polymer was used are shown.

From FIG. 11B, the cells constituting the detached cell sheet had good cell proliferation ability and could be re-engrafted.

Claims (7)

Cells are cultured at first temperature on a cell culture support having a polymer having a structural unit derived from (meth) acrylate having an alkyl group having 14 to 22 carbon atoms and a structural unit derived from (meth) acrylic acid on the surface. To form a cell sheet on the cell culture support,
A method for producing a cell sheet, which comprises holding the cell culture support at a second temperature higher than the first temperature and recovering the cell sheet. Claim 1 in which the content ratio of the structural unit derived from the (meth) acrylate having an alkyl group having 14 to 22 carbon atoms to the structural unit derived from (meth) acrylic acid is 2: 8 to 5: 5. The manufacturing method described in. The production method according to claim 1 or 2, wherein the polymer is a block copolymer of (meth) acrylate having an alkyl group having 14 to 22 carbon atoms and (meth) acrylic acid. Cell culture comprising a substrate and a polymer comprising a constituent unit derived from a (meth) acrylate having an alkyl group having 14 to 22 carbon atoms and a constituent unit derived from (meth) acrylic acid arranged on the substrate. Support. A coating agent for a cell culture substrate, which comprises a polymer containing a structural unit derived from (meth) acrylate having an alkyl group having 14 to 22 carbon atoms and a structural unit derived from (meth) acrylic acid, and a liquid medium. The coating agent according to claim 5, wherein the liquid medium contains dimethyl sulfoxide. A method for producing a cell culture support, which comprises applying the coating agent according to claim 5 or 6 onto a substrate.

Images