JP7297282B2 - COMPOSITION, CELL CULTURE SUBSTRATE, AND METHOD FOR PRODUCING CELL CULTURE SUBSTRATE - Google Patents

Description

The present invention relates to compositions, cell culture substratums, and methods for producing cell culture substratums.

In recent years, three-dimensional cell culture models in vitro are known to be useful for various biological or medical assays, including drug screening for cancer chemotherapy. Substrate development is underway.
In Non-Patent Document 1, as a technique for controlling the morphology of cultured cells using a fluid cell culture substrate, a cell culture substrate composed of macromonomers formed from ε-caprolactone and DL-lactic acid and having a polymerizable group is disclosed. material is described.

K. UTO et al. , ACS Biomaterials Science and Engineering, 2016, 2, PP. 446-453

According to the fluid cell culture substratum described in Patent Document 1, it was possible to control the adhesion behavior of certain cells to the cell culture substratum, and as a result, obtain spheroids. However, as a result of further investigation by the present inventors, it was found that many adhesion cells cannot spread on the above-mentioned base material, making it difficult to culture them in forms other than spheroids. In other words, the present inventors have found that it is difficult to control the adhesion behavior of cells of the adhesive system to obtain desired culture forms, including forms other than spheroids.

In view of the above problems, the present invention provides a method for forming a cell culture substrate capable of forming a cell culture substrate that can easily control adhesion behavior and culture in a desired form even when culturing various cells. The object is to provide a composition of
Another object of the present invention is to provide a cell culture substrate, a method for producing a cell culture substrate, and a method for measuring cell traction force.

As a result of intensive studies aimed at achieving the above object, the inventors of the present invention have found that the above object can be achieved with the following configuration.

[1] having a repeating unit A and a repeating unit B, wherein the repeating unit A and the repeating unit B are not the same,
The repeating unit A is a repeating unit represented by formula 1 described later, and the repeating unit B is composed of a repeating unit represented by formula b1 described later and a repeating unit represented by formula b2 described later. A composition for forming a cell culture substrate containing an aliphatic polyester, which is at least one repeating unit selected from the group, and a photoradical generator.
[2] The composition according to [1], wherein the molar ratio of the content of the repeating unit A to the content of the repeating unit B in the aliphatic polyester is 60/40 or less.
[3] The composition according to [1] or [2], wherein the cell culture substrate is for measuring cell traction force.
[4] A cell culture substrate obtained by irradiating the composition according to any one of [1] to [3] with light.
[5] A method for producing a cell culture substratum, comprising irradiating the composition according to any one of [1] to [4] with light to obtain a cell culture substratum.
[6] A method for measuring the traction force of cells, comprising a step A of obtaining a cell culture substrate by irradiating the composition according to any one of [1] to [3] with light, and using the cell culture substrate A method for measuring the traction force of a cell, comprising a step B of culturing the cells to be measured using the above method to generate wrinkles on the cell culture substrate, and a step C of measuring the cycle of the wrinkles.

According to the present invention, even when culturing various cells, the composition for forming a cell culture substrate can easily control the adhesion behavior and can form a cell culture substrate that can be cultured in a desired form. can provide things. Moreover, according to the present invention, a cell culture substratum, a method for producing a cell culture substratum, and a method for measuring cell traction force can also be provided.

NIH3T3 cells are cultured using the composition before light irradiation and the cell culture substrate obtained by irradiating the composition with light for a predetermined time (1, 5, 15, 30, and 60 minutes), and after culturing It is a fluorescence microscope image after 24 hours. NIH3T3 cells are cultured using the composition before light irradiation and the cell culture substrate obtained by irradiating the composition with light for a predetermined time (1, 5, 15, 30, and 60 minutes), and after culturing It is a fluorescence microscope image after 24 hours. The third row also shows the results of culturing MCF-7. It is an optical microscope image of MCF-7 cultured using each cell culture substrate. FIG. 4 is force-distance curves for each cell culture substrate obtained using a composition containing 4b100. FIG. It is the elastic modulus of each cell culture substrate obtained using a composition containing 4b100. Figure 10 is a force-distance curve for each cell culture substrate obtained using a composition containing 4b500. It is the elastic modulus of each cell culture substrate obtained using a composition containing 4b500. It is a graph which put together the relationship between light irradiation time and an elastic modulus. Fig. 4 is a relaxation curve of each cell culture substrate obtained using a composition containing 4b100. Fig. 4 is a relaxation curve of each cell culture substrate obtained using a composition containing 4b500.

The present invention will be described in detail below.
Although the description of the constituent elements described below may be made based on representative embodiments of the present invention, the present invention is not limited to such embodiments.
In this specification, a numerical range represented by "-" means a range including the numerical values before and after "-" as lower and upper limits.

[Composition]
The composition according to the embodiment of the present invention comprises a repeating unit A represented by formula 1 (hereinafter also simply referred to as “unit A”) and a repeating unit represented by formula b1 (hereinafter “unit b1”). ), and at least one repeating unit B (hereinafter simply referred to as “unit B”) selected from the group consisting of repeating units represented by formula b2 (hereinafter also referred to as “unit b2”). ), and a composition for forming a cell culture substrate containing an aliphatic polyester having and a photoradical generator.

[Aliphatic polyester]
Compositions according to embodiments of the present invention contain an aliphatic polyester having A units and B units. The arrangement order of the units A and B in the aliphatic polyester is not particularly limited, and may be random, alternating, or block.

Unit A is a repeating unit represented by formula 1 below. In Formula 1, R ₁ represents a linear alkylene group. The alkylene group represented by R ₁ is not particularly limited, but a linear alkylene group having 1 to 20 carbon atoms is preferred .

The content of the unit A in the aliphatic polyester is not particularly limited, but in terms of obtaining a composition having a more excellent effect of the present invention, when the total repeating unit of the aliphatic polyester is 100 mol%, 90 mol % or less is preferable, 70 mol % or less is more preferable, and 60 mol % or less is even more preferable.
Although the lower limit is not particularly limited, 0.1 mol % or more is generally preferable.
When the content of the unit A is 60 mol% or less, it is easier to control the fluidity of the substrate obtained using the composition. This is because when the content of the unit A is 60 mol% or less, the composition becomes more fluid, and the fluidity of the obtained substrate can be controlled in a wider range by adjusting the light irradiation time. It is speculated that

Unit B is at least one selected from the group consisting of unit b1 represented by formula b1 below and unit b2 represented by formula b2 below.
In formula b1, _R2 represents a branched alkylene group. The alkylene group represented by R ₂ is not particularly limited, but a branched alkylene group having 2 to 20 carbon atoms is preferred .
In formula b2, _R3 represents an alkyl group . The alkyl group for R ₃ is not particularly limited, but may be a linear alkyl group having 1 to 20 carbon atoms or a branched alkyl group having 2 to 20 carbon atoms.

The content of the unit B in the aliphatic polyester is not particularly limited, but in terms of obtaining a composition having a more excellent effect of the present invention, when the total repeating unit of the aliphatic polyester is 100 mol%, 10 mol % or more is preferable, 30 mol % or more is more preferable, and 40 mol % or more is still more preferable.
The upper limit is not particularly limited, but generally 99.9 mol % or less is preferable.
When the content of the unit B is 40 mol% or more, it is easier to control the fluidity of the substrate obtained using the composition. This is because when the content of the unit B is 40 mol% or more, the composition becomes more fluid, and the fluidity of the resulting substrate can be controlled in a wider range by adjusting the light irradiation time. It is speculated that

The aliphatic polyester may have either unit b1 or unit b2, or both. When the aliphatic polyester has units b1 and units b2, the total content thereof is preferably within the above range.

The aliphatic polyester is not particularly limited as long as it has unit A and unit B, and may have other units and partial structures.
Among them, the aliphatic polyester preferably has a partial structure based on a polyhydric alcohol in order to obtain an aliphatic polyester having more excellent effects of the present invention.

Examples of polyhydric alcohols include, but are not limited to, trimethylolethane, ditrimethylolethane, trimethylolpropane, ditrimethylolpropane, pentaerythritol, dipentaerythritol, tripentaerythritol, glycerin, diglycerol, and trimethylolmelamine. be done.

When the aliphatic polyester has a partial structure based on the polyhydric alcohol, the structure of the aliphatic polyester is not particularly limited, but the polymer chains having the unit A and / or the unit B are based on the polyhydric alcohol A form in which they are linked by partial structures to form a branched structure is preferred.

The content molar ratio (A/B) of the content of the unit A to the content of the unit B in the aliphatic polyester is not particularly limited, but is preferably 60/40 or less.
When A/B is 60/40 or less, the loss modulus of the obtained cell culture substrate tends to exceed the storage modulus under conditions of 37°C (normal cell culture temperature) regardless of the light irradiation conditions. , the resulting cell culture substratum has a stronger property as a viscous liquid, making it easier to adjust the fluidity of the cell culture substratum to suit various cells with different stress fields.
Although the lower limit of A/B is not particularly limited, 1/100 or more is generally preferable.
Although the method for preparing A/B is not particularly limited, for example, a method of adjusting the charging ratio of monomers in the synthesis of aliphatic polyester can be mentioned.

The method for synthesizing the aliphatic polyester is not particularly limited, but in that it can be produced more easily and the content of the units A and B is easier to control, it is obtained by ring-opening polymerization of a cyclic compound. It is preferable that the

The cyclic compound is not particularly limited, and known cyclic compounds can be used. Among them, β-propiolactone, ε-caprolactone, γ-butyrolactone, δ-valerolactone, 15-pentadecanolide, and 16-hexadecanolide have substituents on the lactone ring, such as β-propiolactone, ε-caprolactone, γ-butyrolactone, 15-pentadecanolide, and 16-hexadecanolide. γ-valerolactone, γ-octanolactone, β-methyl-δ-valerolactone, δ-stearolactone, γ-octanoic lactone, 2-methyl-ε-caprolactone, 4- Lactone compounds having a substituent on the lactone ring such as methyl-ε-caprolactone, γ-octanolactone, γ-palmitolactone and α-methyl-γ-butyrolactone; lactide compounds such as glycolide and lactide; is mentioned.

[Photoradical generator]
A composition according to an embodiment of the present invention contains a photoradical generator. The photo-radical generator generates radicals upon irradiation with light, and has the effect of intramolecularly or intermolecularly cross-linking the aliphatic polyester, and the fluidity of the obtained cell culture substrate can be adjusted by light irradiation conditions. and

The photo-radical generator is not particularly limited as long as it generates radicals by light irradiation, and known photo-radical generators can be used without particular limitations.
Examples of photoradical generators include, but are not limited to, alkylphenone-based, acylphosphine oxide-based, oxime ester-based, and α-hydroxyketone-based agents.

Examples of alkylphenone radical generators include 2-benzyl-2-dimethylamino-1-(4-morpholinophenyl)-1-butanone, 2-(dimethylamino)-2-[(4-methylphenyl)methyl ]-[4-(4-morpholinyl)phenyl]-1-butanone, 2-methyl-1-[4-(methylthio)phenyl]-2-morpholinopropan-1-one, benzophenone, methylbenzophenone, o-benzoyl benzoin acid, benzoyl ethyl ether, 2,2-diethoxyacetophenone, 2,4-diethylthioxanthone, diphenyl-(2,4,6-trimethylbenzoyl)phosphine oxide, ethyl-(2,4,6-trimethylbenzoyl)phenylphosphine phosphate, 4,4′-bis(diethylamino)benzophenone, 1-hydroxy-cyclohexyl-phenylketone, 2,2-dimethoxy-1,2-diphenylethan-1-one, and 1-[4-(2-hydroxy ethoxy)-phenyl]-2-hydroxy-2-methyl-1-propan-1-one and the like.

Examples of acylphosphine oxide photoradical generators include 2,4,6-trimethylbenzoyl-diphenyl-phosphine oxide and bis(2,4,6-trimethylbenzoyl)-phenylphosphine oxide.

Examples of oxime ester photoradical generators include 1,2-octanedione, 1-[4-(phenylthio)-, 2-(O-benzoyloxime)], ethanone, 1-[9-ethyl-6- (2-methylbenzoyl)-9H-carbazol-3-yl]-, 1-(O-acetyloxime) and the like.

Examples of α-hydroxyketone photoradical generators include benzoin, benzoin methyl ether, benzoin butyl ether, 1-hydroxy-cyclohexyl-phenyl-ketone, 1-phenyl-2-hydroxy-2-methylpropan-1-one, 1-(4-i-propylphenyl)-2-hydroxy-2-methylpropan-1-one, 4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone, and 1-hydroxy cyclohexyl phenyl ketone and the like.

Among them, alkylphenone-based photoradical generators are preferred, and benzophenone and derivatives thereof are more preferred, in that a composition having more excellent effects of the present invention can be obtained.

Although the content of the photo-radical generator in the composition is not particularly limited, the total weight of the aliphatic polyester in the composition is generally 100 parts by weight in order to obtain a composition having more excellent effects of the present invention. , preferably 0.005 to 50 parts by mass, more preferably 0.01 to 10 parts by mass.
In addition, the composition may contain one type of photo-radical generator alone, or may contain two or more types. When the composition contains two or more photoradical generators, the total content is preferably within the above numerical range.

[Other ingredients]
As long as the composition according to the present embodiment contains an aliphatic polyester and a photoradical generator, it may contain other components within the range in which the effects of the present invention are exhibited. Other components include, for example, solvents.
The solvent is not particularly limited as long as it has an affinity for both the aliphatic polyester and the photoradical generator and dissolves both at least partially, and known solvents can be used.

Examples of solvents that can be used include hydrocarbon-based solvents, ketone-based solvents, ester-based solvents, ether-based solvents, halogen-based solvents, amide-based solvents, and mixtures thereof.
Examples of hydrocarbon solvents include hexane, cyclohexane, benzene, toluene, xylene, heptane, and decane.
Ketone solvents include, for example, acetone, methyl ethyl ketone, diethyl ketone, cyclohexanone, and isophorone.
Examples of ester solvents include ethyl acetate, methyl acetate, ethyl succinate, methyl carbonate, ethyl benzoate, and diethylene glycol diacetate.
Examples of ether solvents include diethyl ether, dibutyl ether, tetrahydrofuran, dioxane, diethylene glycol dimethyl ether, triethylene glycol diethyl ether, and diphenyl ether.
Halogen solvents include, for example, dichloromethane, chloroform, tetrachloromethane, dichloroethane, 1,1',2,2'-tetrachloroethane, chlorobenzene and dichlorobenzene.
Amide solvents include formamide, N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone and the like.

The content of the solvent in the composition is not particularly limited, but the solid content of the composition is preferably adjusted to 1 to 60% by mass, more preferably 10 to 55% by mass. more preferred.

[Method for producing cell culture substrate]
A method for producing a cell culture substratum according to an embodiment of the present invention is a method for producing a cell culture substratum, comprising irradiating the already-described composition with light to obtain a cell culture substratum.
The method of irradiating the composition with light is not particularly limited, but typically includes a method of coating the composition on a support to form a composition layer and irradiating the composition layer with light.

The support is not particularly limited, and known supports can be used. The material component of the support may be an organic substance, an inorganic substance, a mixture thereof, or a composite such as a laminate. Examples of organic materials include resin substrates. Examples of inorganic materials include glass substrates and metal substrates.

The method for forming a composition layer by coating the composition on the support is not particularly limited, and known methods can be used. Examples of coating methods include spin coating, spray coating, and dip coating.

After forming the composition layer on the support, a step of drying the composition layer may be included. The drying method is not particularly limited, and includes a method of heating the composition layer. The heating temperature is not particularly limited, and is preferably 10 to 70°C, more preferably 25 to 60°C. Alternatively, the composition layer may be maintained under reduced pressure.

Although the method of light irradiation is not particularly limited, it is preferably carried out in an atmosphere containing less water and less oxygen. When light is irradiated in an atmosphere containing less water and oxygen, the unintended molecular weight reduction reaction that is presumed to occur with the cross-linking reaction is less likely to occur, and the fluidity of the resulting cell culture substrate is more controlled. It's easy to do.
From the above point of view, the light irradiation is preferably carried out in an inert gas atmosphere, and more preferably carried out in an argon gas atmosphere.

Although the conditions for light irradiation are not particularly limited, it is preferable to irradiate with ultraviolet rays, and the wavelength is not particularly limited. is preferred, ultraviolet rays of 200 to 400 nm are more preferred, and light in the wavelength range of 320 to 400 nm is even more preferred.
The irradiation time can be arbitrarily selected according to the fluidity of the desired cell culture substratum. Although not particularly limited, for example, the irradiation may be performed for about 0.01 to 60 minutes.

Cells that can be cultured using this cell culture substrate are not particularly limited, but stem cells such as ES cells (embryonic stem cells), iPS cells (induced pluripotent stem cells), and mesenchymal stem cells; and animal cells such as mesenchymal somatic cells; tumor cells; and the like. Cells of insects, plants, bacteria, etc. can also be cultured.
These cells may also be primary cells taken directly from tissues and organs, or they may be passaged for several generations.
Also, a single type of cells may be cultured, or two or more types of cells may be co-cultured.

[Method for measuring cell traction force]
The method for measuring the traction force of cells according to the present embodiment comprises a step A of obtaining a cell culture substrate by irradiating the composition with light, and culturing the cells to be measured using the cell culture substrate, and culturing the cells. A method for measuring the traction force of cells, comprising a step B of generating wrinkles on a substrate and a step C of measuring the period of the wrinkles.
In the method for measuring the traction force of cells according to the present embodiment, a cell culture substrate obtained by irradiating the above composition with light is used. By using the cell culture substrate, culture in a desired form can be easily performed by adjusting the time and/or intensity of light irradiation according to the type of cells and the strength of the traction force.
Therefore, by controlling the fluidity of the cell culture substrate so that wrinkles, which will be described later, can be clearly observed, there is an advantage that the cell traction force can be measured more easily.
Each step will be described in detail below.

[Step A]
Step A is a step of irradiating the composition with light to obtain a cell culture substratum.
The method of irradiating the composition with light is not particularly limited, but the light irradiation conditions are such that when the cells to be measured are cultured, they have fluidity to the extent that wrinkles caused by the traction force of the cells can be observed under predetermined conditions. is preferably set.

[Step B]
Step B is a step of culturing cells to be measured using the cell culture substratum to form wrinkles on the cell culture substratum. The culture method is not particularly limited, and known methods can be applied. Since the substrate obtained using the above composition has an appropriate fluidity suitable for the cells to be cultured, periodic wrinkles are formed on the cell culture substrate due to the traction force of the cells extending on the cell culture substrate. can be generated.

Although the traction force of cells is not particularly limited, it is often 1 to 100 nN, and it is difficult to generate wrinkles when using a general cell culture substrate. Moreover, when the cell culture substratum described in Non-Patent Document 1 is used, it is difficult to generate wrinkles because many cells cannot be stretched.
On the other hand, in this step, since the cell culture substrate is used, the elastic modulus and fluidity of the cell culture substrate can be controlled according to the cells to be measured. A cell culture substrate having appropriate hardness can be prepared, and desired wrinkles can be generated.

[Step C]
Step C is a step of measuring the period of wrinkles. The cycle of wrinkles reflects the traction force of cells, and if these are measured, the traction force of cells can be calculated by a conventional method. In addition, in this step, in addition to the wrinkle cycle, the amplitude, length, and the like may be measured, and the cell traction force may be calculated using these.

An optical microscope, a confocal laser microscope, an atomic force microscope, a scanning electron microscope, or the like can be used to measure the period of wrinkles in this step. Among them, an optical microscope is preferred, and a phase-contrast microscope is more preferred, from the viewpoint of easier measurement.

The present invention will be described in more detail based on examples below. The materials, amounts used, proportions, treatment details, treatment procedures, etc. shown in the following examples can be changed as appropriate without departing from the gist of the present invention. Therefore, the scope of the present invention should not be construed to be limited by the examples shown below.

[Synthesis of Aliphatic Polyester]
Ring-opening copolymerization of ε-caprolactone (CL) and D,L-lactide (DLLA) under a nitrogen stream using pentaerythritol (corresponding to a polyhydric alcohol) as an initiator and tin octylate as a catalyst. A four-branched aliphatic polyester “P(CL-co-DLLA)” was synthesized by (scheme below). The content molar ratio of the content of ε-caprolactone to the content of D,L-lactide in the aliphatic polyester was 60/40.

The "4b100" formed above is 4-branched, meaning that m+n is 100 in each branch. Also, "4b500" (m+n is 500) was synthesized by the same method.

[Preparation of composition]
The obtained aliphatic polyester and benzophenone were dissolved in toluene. At this time, the content of the aliphatic polyester is 10 to 100 parts by weight with respect to 100 parts by weight of toluene, and the content of benzophenone is 0.005 to 5 parts by weight with respect to 100 parts by weight of toluene. bottom.

[Creation of cell culture substrate]
Using the above composition, a film was formed on a glass substrate by spin coating under the following conditions to obtain a cell culture substrate (uncrosslinked). Then, it was irradiated with light for a predetermined time in an argon gas atmosphere to prepare cell culture substrates with different fluidities.

Spin coating conditions: 2000 rpm, 120 sec, room temperature
Light irradiation conditions: (cut <320 nm or less with copper sulfate, irradiation for 0-60 min)

[Cell culture test]
Using the cell culture substrate obtained above, cell experiments using various cells (mouse fibroblasts (NIH3T3), human mammary adenocarcinoma cells (MCF-7), canine kidney tubular epithelial cells (MDCK), etc.) did
More specifically, the samples were sterilized by ultraviolet irradiation and ethylene oxide gas (EGO) sterilization, protein coating that promotes adhesion such as fibronectin was applied as necessary, and seeded at a predetermined cell density.
The morphology of cell adhesion to the substrate was observed over time using a phase-contrast/fluorescence microscope. The results are shown in FIGS. 1 and 2. FIG.

FIG. 1 shows the composition before light irradiation (described as “0 min”) and the cell culture obtained by light irradiation of the composition for a predetermined time (1, 5, 15, 30, and 60 minutes). It is a fluorescence microscope image of NIH3T3 cells cultured using the base material and 24 hours after the culture.
Note that "In air" in the upper row indicates that the light irradiation was performed in the air, and "Under Ar" in the lower row indicates that the light irradiation was performed in an argon gas atmosphere. .
From the above results, it was found that the composition according to the embodiment of the present invention can easily control the cell adhesion morphology simply by changing the light irradiation time.

In addition to each photograph of FIG. 1, FIG. 2 also shows the results of culturing MCF-7 on the third row. From the above results, it was found that not only fibroblasts but also epithelial cells can control adhesion morphology.

FIG. 3 shows a glass substrate (indicated as "Glass" in the figure);
"4b100"only;
"4b500"only;
A composition "4b100+BP" containing 4b100 and benzophenone was used without light irradiation (indicated as "0 min" in the figure);
A composition "4b100+BP" containing 4b500 and benzophenone was used without light irradiation (indicated as "0 min" in the figure);
Using each of the cultured MCF-7, and,
Cell culture substrate obtained by irradiating "4b100+BP" with light for 10 to 60 minutes;
Cell culture substrate obtained by irradiating "4b500+BP" with light for 10 to 60 minutes;
An optical microscope image of MCF-7 cultured using each of the above is shown.

From the results shown in FIG. 3, it was found that by changing the degree of polymerization of the aliphatic polyester (the difference between 4b500 and 4b100), the range in which the fluidity can be adjusted can be controlled.
It was also found that when the above cell culture substratum is used, periodic wrinkles are formed on the surface of the cell culture substratum due to the traction force of the cells when MCF-7 adheres. In addition, the shape, length, etc. of wrinkles formed differ depending on the light irradiation time, and it is presumed that wrinkles optimal for observation are formed even when the cell type is changed.

[Evaluation of fluidity of cell culture substrate]
Aliphatic polyesters 4b100 and 4b500 prepared above were prepared, the aliphatic polyester and benzophenone were dissolved in toluene, and each composition for forming a cell culture substrate was prepared. At this time, the content of the aliphatic polyester is 50 (4b100) parts by weight, 30 (4b500) parts by weight with respect to 100 parts by weight of toluene, and the content of benzophenone is 2 (4b100) parts by weight with respect to 100 parts by weight of toluene. Parts by weight, 1.2 (4b500) parts by weight.
Also, as a comparative example, a composition similar to the above was prepared except that it did not contain benzophenone.

<Elastic modulus>
The elastic modulus of the cell culture substrate was measured using an atomic force microscope. The measurement sample was a cell culture substrate obtained by irradiating each of the above compositions with ultraviolet rays for a predetermined period of time.
In the measurement, the force applied to the cantilever is measured while the cantilever is brought close to the cell culture substrate at a predetermined speed (5 μm / s, 42 μm / s for the comparative example), and the force-distance curve ( force-distance curves) and elastic modulus.
The force-distance curve of each cell culture substrate obtained using the composition containing 4b100 is shown in FIG. 4, and the elastic modulus is shown in FIG. The force-distance curve of each cell culture substrate obtained using the composition containing 4b500 is shown in FIG. 6, and the elastic modulus is shown in FIG.

In FIGS. 4-7, "-BP" indicates the measured values for the substrates formed from the comparative compositions that did not contain benzophenone. Further, "0 min" means that no ultraviolet irradiation was performed, and "10 min" to "60 min" mean cell culture substrates that were irradiated with ultraviolet light for 10 to 60 minutes, respectively.

From the above results, it was found that a cell culture substratum can be obtained by light irradiation using a predetermined composition, and that the elastic modulus of the cell culture substratum can be arbitrarily controlled by the time of light irradiation. . Specifically, it was shown that as the light irradiation time increases, the elastic modulus of the obtained cell culture substrate increases.

FIG. 8 shows a graph summarizing the relationship between light irradiation time and elastic modulus. From the results of FIG. 8, it was shown that the elastic modulus of the cell culture substrate can be arbitrarily controlled by the light irradiation time, and that the adjustable elastic modulus range can be adjusted by the degree of polymerization of the aliphatic polyester.

<Liquidity>
The fluidity of the cell culture substratum was measured using an atomic force microscope using the same composition as used in the elastic modulus measurement. The measurement was performed by pressing the cantilever into the obtained cell culture substrate at a predetermined speed (5 μm/s, 42 μm/s for the comparative example) and observing the attenuation behavior of the force.
The force relaxation curve of each cell culture substrate obtained using the composition containing 4b100 is shown in FIG. 9, and the force relaxation curve of each cell culture substrate obtained using the composition containing 4b500 is shown in FIG. It was shown to.
From the above results, it was found that the fluidity of the obtained cell culture substrate can be arbitrarily controlled by light irradiation time. It was also found that the range of adjustable fluidity can be arbitrarily controlled by changing the degree of polymerization of the aliphatic polyester (copolymer).

Claims (4)

A composition for forming a cell culture substrate,
an aliphatic polyester having only repeating units A and repeating units B as repeating units;
and an alkylphenone-based photoradical generator,
In the aliphatic polyester,
The repeating unit A and the repeating unit B are not the same,
The repeating unit A is a repeating unit represented by formula 1,
The composition, wherein the repeating unit B is at least one repeating unit selected from the group consisting of repeating units represented by formula b1 and repeating units represented by formula b2.

In formula 1, R ₁ represents a linear alkylene group, in formula b1, R ₂ represents a branched alkylene group, in formula b2, R ₃ represents an alkyl group, and two R ₃ may be the same or different. 2. The composition according to claim 1, wherein the content molar ratio of the repeating unit A content to the repeating unit B content in the aliphatic polyester is 60/40 or less. A cell culture substrate obtained by irradiating the composition according to claim 1 or 2 with light. A method for producing a cell culture substratum, comprising irradiating the composition according to claim 1 or 2 with light to obtain a cell culture substratum.

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