JP2019068761A - Cell culture support and cell culture plate - Google Patents

Abstract

To provide a cell culture support having a surface excellent in gas permeability and cell adhesiveness as well as being excellent for prolonged culture.SOLUTION: The present invention relates to a cell culture support having a silicone membrane which is surface-modified by a polymer layer comprising an amphipathic polymer and a cytophilic substance, the amphipathic polymer having a hydrophobic part and a hydrophilic part. The hydrophobic part has one or more structural units selected from a group consisting of a polyurethane-derived structural unit, a polyethylene-derived structural unit, a nylon-derived structural unit, and a silicone-derived structural unit. The hydrophilic part is a copolymer having one or more structural units selected from a group consisting of a polyamino acid-derived structural unit, a water soluble collagen-derived structural unit, gelatin-derived structural unit, cellulose-derived structural unit, a starch-derived structural unit, a polysulfonic acid-derived structural unit, and a polyethyleneglycol-derived structural unit.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a cell culture support and cell culture plate in which a cytophilic substance is surface-modified.

  In order to culture cells in an environment close to a living body, there is a culture system using a base material having gas permeability capable of supplying oxygen and discharging carbon dioxide. A culture system using a silicone membrane is known as a substrate used in such a culture system.

  For example, as a cell culture device capable of transporting and supplying oxygen to cells, Patent Document 1 discloses a cell culture device provided with a gas-permeable, liquid-impermeable membrane barrier such as a mixture of perfluorohydrocarbon and silicone. It is disclosed.

On the other hand, silicone membranes have the problem of poor adhesion to cells. Therefore, a method is known in which collagen is coated on a silicone membrane to adhere and culture cells.
For example, Patent Document 2 discloses a hepatocyte culture method in which hepatocytes embedded in extracellular matrix are disposed on a gas permeable membrane, and the hepatocytes are cultured while supplying oxygen from the gas permeable membrane side. ing. Specifically, a method of culturing while supplying oxygen is disclosed using a gas permeable membrane whose surface is coated with collagen. Then, as a method of coating collagen on a gas permeable membrane, a method of adsorbing collagen to the gas permeable membrane by oxygen plasma treatment and a method of covalently bonding using a chemically reactive functional group are described There is.

Japanese Patent Application Publication No. 2009-542230 International Publication No. 2011/024592

As described above, since the silicone film has poor adhesion to cells, it is necessary to improve cell adhesion by surface-modifying and modifying the silicone film.
However, in the method of coating collagen by oxygen plasma treatment as disclosed in Patent Document 2, the process is complicated, and it is difficult to plasma treat the surface of the silicone film uniformly, and adhesion There were problems such as the existence of low sex parts.
Further, the method of chemically bonding requires a very complicated synthesis such as introducing a chemically reactive functional group into the silicone film, and there is a problem that impurities and the like are easily mixed.

Under such circumstances, there is desired a cell culture system having a silicone membrane in which a collagen or the like is immobilized on the surface of the silicone membrane to realize a stable functional surface (a surface having adhesiveness).
An object of the present invention is to provide a support for cell culture which has a surface excellent in gas permeability and cell adhesion and is also excellent in long-term culture.

  As a result of intensive studies to solve the above problems, the present inventor has found that, by using a specific amphiphilic polymer, a cytophilic substance such as collagen stably adheres to a silicone membrane. Then, it was found that the following invention met the above object, and the present invention was made.

That is, the present invention relates to the following inventions.
<1> A silicone film surface-modified with a polymer layer containing an amphiphilic polymer and a cytophilic substance, the amphiphilic polymer having a hydrophobic part and a hydrophilic part, and the hydrophobic part being a polyurethane And at least one structural unit selected from the group consisting of a structural unit derived from polyethylene, a structural unit derived from polyethylene, a structural unit derived from nylon, and a structural unit derived from silicone, and the hydrophilic portion is a structural unit derived from polyamino acid At least one selected from the group consisting of a water-soluble collagen-derived structural unit, a gelatin-derived structural unit, a cellulose-derived structural unit, a starch-derived structural unit, a polysulfonic acid-derived structural unit and a polyethylene glycol-derived structural unit A support for cell culture which is a copolymer having a structural unit of
<2> In the amphiphilic polymer, the hydrophobic part is composed of a structural unit derived from polyurethane, and the hydrophilic part is composed of a structural unit derived from polyamino acid, a structural unit derived from polysulfonic acid, and a structural unit derived from polyethylene glycol The support for cell culture as described in <1> which is a copolymer which consists of one or more types of structural units selected from a group.
<3> The support for cell culture as described in <1> or <2> which consists of a silicone membrane surface-modified by the said polymer layer.
<4> The support for cell culture according to any one of <1> to <3>, wherein the cytophilic substance is distributed throughout the polymer layer.
<5> The support for cell culture according to any one of <1> to <4>, wherein the cytophilic substance is one or more selected from the group consisting of collagens, growth factors and mucopolysaccharides.
<6> The support for cell culture according to any one of <1> to <5>, wherein the support is a support to be used for long-term culture.
<7> A cell culture plate in which the support for cell culture according to any one of <1> to <6> is disposed on the bottom of a plate such that the polymer layer is a culture surface.

  According to the present invention, there is provided a support for cell culture which has a surface excellent in gas permeability and cell adhesion and is also excellent in long-term culture.

It is a growth curve when HepG2 is cultured using the cell culture plate of Example 1-1, 1-2, and Comparative Example 1-1. It is a growth curve when HepG2 is cultured using the cell culture plate of Example 1-3, 1-4, and Comparative Example 1-2.

  The embodiment of the present invention will be described in detail below, but the explanation of the constituent requirements described below is an example (representative example) of the embodiment of the present invention, and the present invention is not limited to the following unless its gist is changed. It is not limited to the content of. In addition, when using the expression "-" in this specification, it uses as an expression including the numerical value before and behind that.

[Support for cell culture]
The present invention comprises a silicone film surface-modified with a polymer layer containing an amphiphilic polymer and a cytophilic substance, wherein the amphiphilic polymer has a hydrophobic portion and a hydrophilic portion, and the hydrophobic portion comprises It has one or more structural units selected from the group consisting of structural units derived from polyurethane, structural units derived from polyethylene, structural units derived from nylon and structural units derived from silicone, and the hydrophilic part is a structure derived from polyamino acid Unit, structural unit derived from water-soluble collagen, structural unit derived from gelatin, structural unit derived from cellulose, structural unit derived from starch, structural unit derived from polysulfonic acid, and structural unit derived from polyethylene glycol The support for cell culture which is a copolymer having the above structural units (hereinafter, sometimes referred to as "support of the present invention"). To.

As mentioned above, silicone membranes have the property that it is difficult to adhere a cytophilic substance to the surface. On the other hand, by covering the surface of a silicone membrane with a polymer layer containing an amphiphilic polymer having a structure as in the present invention and a cytophilic substance, a support having a surface with excellent cell adhesion is provided. it can. In addition, when cells are cultured using the support of the present invention, the cultured cells are less likely to be detached from the support and can be cultured stably.
In addition, even if it coats the surface of a silicone membrane with a cytophilic substance using the copolymer which consists of polylactic acid and PEG as an amphiphilic polymer, the support body which has the surface excellent in adhesiveness can not be obtained. By using an amphiphilic polymer having a structure as in the present invention and a cytophilic substance, a surface with excellent adhesion can be obtained.

  In addition, the support of the present invention can supply oxygen to cells and discharge carbon dioxide through the silicone membrane, and when using the support of the present invention, it can be compared to a plastic plate that is not permeable to gas. A culture period of about 2 times or more is also possible. Therefore, the support of the present invention is suitable as a support for use in long-term culture.

  In addition, the support of the present invention is usually a surface on which spheroids (cell aggregates) are easily formed. For example, by culturing cells to form spheroids, cancer cells and the like maintain a function closer to the state in the living body, but spheroids are prone to necrosis from the inside when oxygen is not supplied, and in the long run There is a drawback that can not be cultured. On the other hand, when cells are cultured using the support of the present invention, the cells can be cultured while supplying oxygen, so that even when spheroids (cell aggregates) are formed, culture can be performed over a long period of time.

  Moreover, it is preferable that the silicone film which comprises the support body of this invention has high transparency, and transparency is maintained also in the support body of this invention. In this case, there is also an advantage that it is easy to observe cells temporally with an inverted microscope or the like. The support of the present invention more preferably has a transmittance of 85% or more, more preferably 90% or more, at visible light of 400 to 800 nm.

  The support of the present invention may include a layer other than the silicone membrane surface-modified with a polymer layer containing an amphiphilic polymer and a cytophilic substance, as long as the gas permeability, cell adhesion and the like are not impaired. A preferred embodiment is a support comprising a silicone membrane surface-modified with a polymer layer comprising the amphiphilic polymer and the cytophilic substance. A support composed of a silicone film surface-modified (coated) with a polymer layer containing the amphiphilic polymer and the cytophilic substance is unlikely to be peeled off between layers, and has excellent gas permeability and cell adhesion, Furthermore, it can be set as a support which has high transparency.

  Hereinafter, the silicone membrane and the polymer layer which comprise the support body of this invention are demonstrated.

[Silicone film]
The support of the present invention has a silicone membrane.
The silicone membrane constituting the support of the present invention is a sheet-like or film-like membrane which is gas permeable and liquid impermeable. That is, the silicone membrane is a sheet-like or film-like membrane that can permeate gas such as oxygen and carbon dioxide but can not permeate liquid.

As such a silicone membrane, a silicone membrane having an oxygen permeability coefficient of 1.000 to 10000.000 cm ³ · cm / (m ² · day · atm) can be used.
By using such a silicone membrane, cells can be cultured while supplying oxygen and discharging carbon dioxide, so that cells can be cultured in an environment closer to the inside of a living body. Moreover, it is suitably used also for long-term culture.
In addition, the silicone membrane is also suitable in terms of ease of use such as attachment as a part of the cell culture plate (in particular, the bottom surface) or shape adjustment, and also of low toxicity to the living body and excellent biocompatibility.

As the silicone film, for example, silicone rubber manufactured by the method described below can be used.
First, various additives are blended with a raw rubber-like high polymerization degree dimethylpolysiloxane and fine powder silica as a reinforcing material as basic components to obtain a silicone rubber compound. Then, a vulcanizing agent such as an organic peroxide and a catalyst is added to this compound at the time of use, and the silicone rubber obtained by heat curing is formed into a film.
In the molding process of the silicone rubber, low molecular weight siloxane may be contained even after the vulcanization process and the like derived from the raw materials and the like. Since this low molecular weight siloxane may exhibit toxicity to living cells, it is preferable to use a cell culture plate of the present invention in which such low molecular weight siloxane is reduced as much as possible. Volatilization and the like by heating are generally performed to remove low molecular weight siloxanes. Furthermore, in the present invention, in order to further reduce low molecular weight siloxane contained in silicone rubber, it is preferable to carry out treatment under reduced pressure, and the environment under this reduced pressure is equivalent to a vacuum of about 1000 to 0.01 Pa, Preferably, the vacuum equivalent is about 100 to 0.1 Pa or so. This treatment is preferably used in combination and heat treatment (eg, about 80 to 200 ° C.) under reduced pressure (vacuum condition, about 1000 to 0.01 Pa), and the treatment time is 1 hour or more, preferably 10 hours It is particularly preferable to set the above to more preferably 1 day or more. The upper limit of the treatment time may be set because the low molecular weight siloxane can be reduced as the treatment time becomes longer, but the effect may be saturated for a certain time or more. Therefore, the upper limit may be 10 days or less, or about 7 days.

  Specifically, as the silicone film, C6 series manufactured by S.I.R. Inc. or the like can be used.

  The thickness and the like of the silicone membrane constituting the support of the present invention are appropriately designed depending on the shape of the whole cell culture plate, the specific culture object, the detailed structure of the silicone membrane and the like. The upper limit of the thickness of the silicone film is preferably 1 mm or less. More preferably, it is 0.5 mm (500 micrometers) or less, More preferably, it is 0.3 mm (300 micrometers) or less. If the thickness of the silicone membrane is more than 1 mm, the gas permeability may be reduced and it may not be suitable for cell culture. In addition, as the thickness is larger, the relative rigidity is too high, and the formability as a cell culture plate may be reduced. On the other hand, the lower limit of the thickness of the silicone film is preferably 50 μm or more, more preferably 100 μm or more. If the silicone membrane layer is too thin, mechanical properties such as toughness as part of the cell culture plate may be insufficient.

[Polymer layer]
In the present invention, the polymer layer is a layer containing an amphiphilic polymer and a cytophilic substance. The polymer layer is configured to include a cytophilic substance on the surface thereof, thereby improving cell adhesion.

The thickness and the like of the polymer layer are appropriately designed depending on the specific culture object, the composition of the polymer layer, and the like. The upper limit of the thickness of the polymer layer is preferably 5 μm or less. More preferably, it is 1 μm or less. On the other hand, the lower limit of the thickness of the polymer layer is preferably 0.05 μm or more, more preferably 0.1 μm or more. If the thickness of the polymer layer is too thick, supply of oxygen to cells may not be efficiently performed. When the polymer layer is too thin, the cell adhesion of the support of the present invention may be insufficient.
The thickness of the polymer layer may be determined from the difference between the thickness of the polymer layer and that of the silicone film before and after laminating the polymer layer, or may be determined from the thickness obtained by observing the cross section of the support with a microscope or the like. It can also be calculated from the concentration of the amphiphilic polymer and the cytophilic substance contained in the coating solution when forming the polymer layer and the amount applied.

One embodiment of the polymer layer is a polymer layer containing a mixture of amphiphilic polymer and cytophilic material. At this time, as long as the cytophilic substance is contained in the surface of the polymer layer, the distribution of the cytophilic substance in the polymer layer may be uniform or non-uniform.
Preferably, it is a polymer layer formed by applying and drying a coating solution containing an amphiphilic polymer and a cytophilic substance on a silicone film, in which case the cytophilic substance is distributed throughout the polymer layer As a result, the cytophilic substance is difficult to peel off, and the surface with excellent cell adhesion can be maintained for a long time. In the case of a configuration in which the cytophilic substance is distributed throughout the polymer layer, the support has a more excellent peel strength (for example, a peel strength such that the polymer layer does not peel from the silicone membrane in normal medium such as DMEM medium for 1 month or more). , Long-term culture is more suitable

  In the polymer layer containing a mixture of an amphiphilic polymer and a cytophilic substance, the ratio of the amphiphilic polymer to the cytophilic substance in the polymer layer is appropriately adjusted according to the specific culture object, the composition of the polymer layer, etc. It is The amount of the cytophilic substance is preferably 5 parts by mass or more, and more preferably 10 parts by mass or more with respect to 100 parts by mass of the amphiphilic polymer. If the amount of the cytophilic substance is too small relative to the amphiphilic polymer, the cell adhesion of the support tends to be insufficient. On the other hand, the polymer layer is preferably 100 parts by mass or less, more preferably 50 parts by mass or less, and preferably 30 parts by mass or less based on 100 parts by mass of the amphiphilic polymer. Is more preferred. When the amount of the cytophilic substance is too large relative to the amphiphilic polymer, the adhesion between the cytophilic substance and the silicone membrane is reduced, and the cytophilic substance is easily peeled off from the silicone membrane.

The polymer layer may have a single-layer structure or a laminated structure having a plurality of layers having different compositions, as long as a cytophilic substance is contained on the surface of the polymer layer.
For example, the polymer layer may have a laminated structure in which a layer made of a cytophilic substance is laminated on a layer made of an amphiphilic polymer.

(Amphiphilic polymer)
An amphiphilic polymer (Amphiphilic Polymer) is a polymer which has characteristics of both hydrophobicity and hydrophilicity, as a polymer has a hydrophobic part and a hydrophilic part.
In the present invention, among the amphiphilic polymers, at least one selected from the group consisting of a structural unit derived from polyurethane, a structural unit derived from polyethylene, a structural unit derived from nylon, and a structural unit derived from silicone among the amphiphilic polymers Structural units derived from polyamino acids, structural units derived from water-soluble collagen, structural units derived from gelatin, structural units derived from cellulose, structural units derived from starch, structural units derived from polysulfonic acid A copolymer having at least one structural unit selected from the group consisting of units and structural units derived from polyethylene glycol is used.
The amphiphilic polymers constituting the polymer layer may be used alone or in combination of two or more amphiphilic polymers.

  Also, the polymer form of the amphiphilic polymer is not particularly limited, and may be in the form of block copolymer, random copolymer, graft copolymer, etc., but block copolymer is preferable. .

  Among them, the hydrophobic portion of the amphiphilic polymer preferably comprises a structural unit derived from polyurethane.

The polyurethane can be generally obtained by the condensation polymerization reaction of a polyol and a polyvalent isocyanate compound, and the structural unit derived from polyurethane is composed of a structure in which a polyol and a polyvalent isocyanate are linked by a urethane bond.
Examples of the polyol include polyethylene glycol, polytetramethylene glycol, trimethylolpropane and trimethylolethane.
As an isocyanate compound, tolylene diisocyanate, diphenyl diisocyanate, isophorone diisocyanate etc. can be mentioned.

  The hydrophilic part of the amphiphilic polymer preferably comprises one or more structural units selected from the group consisting of structural units derived from polyamino acids, structural units derived from polysulfonic acids and structural units derived from polyethylene glycol.

  Polyamino acids can generally be obtained by condensation polymerization of amino acids, and structural units derived from polyamino acids consist of a structure in which amino acids are linked by an amide bond. Polyamino acids can be obtained by condensation polymerization of monomers such as α-amino acid-N-carboxylic acid anhydride. Examples of amino acids that constitute the polyamino acid include glutamic acid, aspartic acid, lysine and the like.

  The polysulfonic acid is a polymer having a sulfonic acid group, and examples thereof include polystyrene sulfonic acid and polyvinyl sulfonic acid.

In particular, the hydrophobic part is composed of a structural unit derived from polyurethane, and the hydrophilic part is at least one structure selected from the group consisting of a structural unit derived from polyamino acid, a structural unit derived from polysulfonic acid and a structural unit derived from polyethylene glycol It is preferable that it is a copolymer which consists of units.
Examples of suitable amphiphilic polymers include block copolymers of polyurethane and polyamino acid and block copolymers of polyurethane, polysulfonic acid and polyethylene glycol.
Such a copolymer is excellent in the adhesiveness of the silicone membrane and is also excellent in the compatibility with the cell-friendly substance, so it is difficult to peel off from the silicone membrane, and a polymer layer to which cells to be cultured easily adhere is easily formed. .

  The molecular weight (weight average molecular weight Mw) of the amphiphilic polymer may be appropriately determined, and is preferably 10,000 to 50,000.

(Cell-friendly substance)
The cytophilic substance may be any substance having cell adhesiveness and may be a natural substance or a synthetic substance. The cytophilic substance may be used alone or in combination of two or more kinds of cytophilic substances. Specific examples of the cytophilic substance include collagens, growth factors, mucopolysaccharides, fibronectin, laminin, proteins such as fibrin, oligosaccharides, gelatin and the like. Among these, one or more selected from the group consisting of collagens, mucopolysaccharides and growth factors is preferable.

  Although the origin of collagens is not particularly limited, for example, collagen derived from animals and fish can be used. Examples of collagens include collagen hydrolysates and collagen derivatives in which the side chain of collagen is modified.

Mucopolysaccharides are not particularly limited, and include, for example, dextran sulfate, hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, heparin derivatives or metal salts thereof.
Examples of suitable mucopolysaccharides include heparin derivatives, and examples of heparin derivatives include heparin, sodium heparin, potassium heparin, lithium lithium heparin, calcium heparin, zinc zinc salt, heparin ammonium salt and the like.

  The growth factor is not particularly limited, and for example, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), bone The formation protein (BMP) etc. are mentioned.

  The growth factor may also be a binding growth factor. That is, a bound growth factor in which a growth factor and a collagen-binding fusion protein are bound may be used. Bound growth factors can be synthesized by methods known in the art.

Examples of synthetic substances that can be used as the cytophilic substance include polymers or copolymers of monomers having a functional group having cell adhesiveness. As a functional group having cell adhesion, for example, carboxylic acid group and its salt, anhydride, sulfonic acid group and its salt, sulfonic acid ester, sulfonic acid amide, phosphoric acid group and its salt, amino group, hydroxyl group, length Examples thereof include chain alkyl group, mercapto group, ether group, thioether group, polyether group, ketone group, aldehyde group, acyl group, acyl group, cyano group, nitro group, acylamino group, halogen group, glycidyl group, allyl group and the like.
In addition, as long as it is a substance having cell adhesion, an amphiphilic polymer may be used as a cytophilic substance.

Although the manufacturing method of the support of the present invention is not particularly limited, it can be obtained, for example, by the following method.
(I) A manufacturing method including a step (a) of applying a solution containing an amphiphilic polymer and a cytophilic substance to a silicone membrane.
(Ii) A manufacturing method comprising: applying a solution of an amphiphilic polymer to a silicone membrane (b1); and applying a solution of a cytophilic substance (b2) after the step (b1).

  According to the method (i), it is possible to obtain a support in which the surface of the silicone membrane is coated with a mixture of an amphiphilic polymer and a cytophilic substance.

  According to the method (ii) above, it is possible to obtain a support having a configuration in which the surface of the silicone membrane is coated with the amphiphilic polymer, and the surface of the amphiphilic polymer layer is further coated with the cytophilic material. . That is, the polymer layer which covers a silicone membrane can obtain the support which is a laminated structure of the layer which consists of amphiphilic polymer, and the layer which consists of cytophilic substances.

  By employing such a production method as in (i) or (ii), it is possible to easily produce a support having stable adhesion as compared with the conventional method using plasma or the like.

The method for applying the coating solution (a solution containing an amphiphilic polymer and a cytophilic substance, a solution of an amphiphilic polymer, a solution of a cytophilic substance) to the silicone film is not particularly limited, and a known method may be used. it can. For example, a spin coat method, a bar coat method, a dip coat method, a spray coat method, a roll coat method, a die coat method and the like can be mentioned.
Moreover, as in the method (ii), when the application is performed a plurality of times, each application method may be the same or different.

  Examples of the solvent for preparing the coating solution include dimethylformamide (DMF), dimethyl sulfoxide (DMSO), methyl ethyl ketone (MEK), tetrahydrofuran (THF) and the like.

  The concentration of the coating solution may be appropriately adjusted depending on the composition of the coating solution, the solvent, the coating method, and the like, but the solid content concentration is preferably adjusted to 1.0 to 15% by mass.

  When preparing a solution containing an amphiphilic polymer and a cytophilic substance, the concentration of the amphiphilic polymer is 1.0 to 10% by mass, and the concentration of the cytophilic substance is 0.1 to 2.0 mass. %, Preferably adjusted to 5 to 100 parts by mass (more preferably 10 to 50 parts by mass) of the cytophilic substance with respect to 100 parts by mass of the amphiphilic polymer.

The method (i) may include steps other than the step (a), and may include a drying step and a washing step.
Likewise, the method (ii) may include steps other than the step (b1) and the step (b2), and may include a drying step and a washing step.

[Cell culture plate]
The support of the present invention is a cell culture plate (hereinafter sometimes referred to as "the cell culture plate of the present invention") disposed at the bottom of the plate so that the polymer layer is the culture surface (upward). can do.
The specific shape of the cell culture plate of the present invention can be a dish or well shape.
In the case of a well shape, even if a silicone membrane provided with a polymer layer only on the portion to be a cell culture surface is attached to the opening at the bottom of the well, a silicone membrane provided with a polymer layer on the entire surface is used. It may be attached to the opening at the bottom of the well.
In addition, this cell culture plate can be subjected to EOG (ethylene oxide) sterilization or the like, and is appropriately used after being subjected to a treatment such as sterilization.

  When culturing cells using the cell culture plate of the present invention, culture conditions and the like can be selected from conventionally known methods depending on the specific culture object.

In addition, since the support of the present invention is capable of supplying oxygen to the cultured cells and discharging carbon dioxide from both sides of the support, cells requiring oxygen (eg, liver cells, pancreatic cells, neurons, skin) Cells, lung cells etc.). The support of the present invention is capable of culturing a culture period of about 2 times or more, as compared to a generally commercially available plastic plate.
Therefore, the cell culture plate of the present invention can be suitably used for long-term culture of cells. Since supply of oxygen and discharge of carbon dioxide are performed, cells are stably cultured, for example, even in a culture period of 10 days or more.

  Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to the following examples as long as the gist thereof is not changed.

Example 1
<Material>
(A) Silicone membrane (1)
・ "C6-530 (PDMS-based silicone film, thickness 300 μm)" manufactured by S.I.R.
(B) Amphiphilic polymer · APP (1): polyurethane · polysulfonic acid · polyethylene glycol copolymer (hydrophobic block: polyurethane, hydrophilic block: polysulfonic acid · polyethylene glycol, hydrophobic block Mw about 20,000, hydrophilic Block Mw about 20,000, total molecular weight Mw: about 40,000 (C) cell-friendly substance, natural factor: EGF (Epedermal Growth Factor-epithelial cell growth factor), WAKO human recombinant, 059-07873
FNBCD-EGF: Collagen-binding growth factor (EGF-collagen-binding peptide fusion) The FNBCD-EGF used is described in Polymer Journal Vol. 69 (2012), No. 1, P1-10, binding property The fusion protein of the collagen binding site of fibronectin and EGF was synthesized by the recombinant expression of insect cells according to the method described in "Generation and application of cell growth factor, Takashi Kitajima, Seiichi Tada, Yoshihiro Ito".
Collagen: porcine-derived atelocollagen, manufactured by KOKEN CO., LTD., APN-221
The collagen was freeze-dried and used after removing the stabilizing preservative phenoxyethanol.

Example 1-1
(1) Preparation of Support A solution of solute concentration 5% by mass was prepared by dissolving APP (1) and FNBCD-EGF in dimethyl sulfoxide in a ratio of 10: 2 (w / w). I got 1)).
The silicone membrane was immersed in solution (1) at 25 ° C. for 1 hour, removed from the solution, and immersed in 2,000 mL of pure water at room temperature for 2 hours. Thereafter, it was further dried with hot air at 70 ° C. for 2 hours to obtain a support (1) in which APP (1) and FNBCD-EGF were coated on the surface of the silicone film.

(2) Production of cell culture plate On the bottom of a 96-well plate having holes in the bottom of 96 wells, the surface coated with APP (1) and collagen on the support (1) is facing up As attached, cell culture plate (1) was obtained.

[Example 1-2]
A solution (solution (2)) with a concentration of 5% by mass was prepared by dissolving APP (1): FNBCD-EGF: collagen in dimethyl sulfoxide at a ratio of 10: 1: 2 (w / w) The
A cell culture plate (2) was obtained in the same manner as in Example 1-1 except that the solution (2) was used instead of the solution (1).

[Example 1-3]
A solution (solution (3)) with a concentration of 2% by mass was obtained by dissolving APP (1): collagen at a ratio of 10: 2 (w / w) in dimethyl sulfoxide.
A cell culture plate (3) was obtained in the same manner as in Example 1-1 except that the solution (3) was used instead of the solution (1).

Example 1-4
APP (1) was dissolved in dimethyl sulfoxide to obtain a 2% by mass solution (solution (4)).
A cell culture plate (4) was obtained in the same manner as in Example 1-1 except that the solution (4) was used instead of the solution (1).

Comparative Example 1-1
(1) Preparation of Support EGF was dissolved in dimethyl sulfoxide to obtain a solution with a concentration of 5% by mass (EGF solution).
A cell culture plate (1 ′) was obtained in the same manner as in Example 1-1 except that an EGF solution was used instead of the solution (1).

Comparative Example 1-2
A cell culture plate (2 ′) was obtained in the same manner as in Comparative Example 1-1 except that collagen was used instead of EGF.

  The composition of the polymer layer of the cell culture plate manufactured below is shown.

<Evaluation>
HepG2 cells are seeded on the culture surface at 4 × 10 4 cells / well (96 wells) using the cell culture plates prepared in Examples 1-1 to 1-4 and Comparative examples 1-1 and 1-2. The cells were cultured in a culture solution (WE medium). Moreover, culture medium exchange was performed on the first and fifth culture days. Cell growth activity was assessed relative to the number of days of culture. The cell proliferation activity was determined by the cell proliferation verification method based on intracellular ATP. Cell proliferation was measured with the GloMax instrument using CellTiTerGlo Assay from Promega.

The result at the time of using the cell culture plate of Example 1-1, 1-2, comparative example 1-1 in FIG. 1 is shown. As shown in FIG. 1, in Comparative Example 1-1 (a cell culture plate coated only with EGF), a large improvement in cell growth with respect to the number of culture days is not observed. It is considered that this is because adhesion to the silicone membrane is weak and EGF is exfoliated over time by simply adding EGF.
On the other hand, in Example 1-1 (a cell culture plate coated with APP (1) and FNBCD-EGF), cell proliferation by EGF was promoted as compared with Comparative Example 1-1. It is considered that this is probably because APP (1) firmly adheres to the silicone membrane, and furthermore, APP (1) and FNBCD-EGF are bound to some extent.
In addition to the adhesion of FNBCD and collagen to the silicone membrane by APP (1), Example 1-2 (a cell culture plate coated with APP (1), FNBCD-EGF, and Collagen) further exhibited FNBCD- It is thought that EGF was fixed by collagen, so that cell proliferation by EGF was promoted.

Moreover, the result at the time of using the cell culture plate of Example 1-3, 1-4, and Comparative Example 1-2 in FIG. 2 is shown. As shown in FIG. 2, in the cell culture plate of Comparative Example 1-2 to which only collagen was added, improvement of cell proliferation was not seen with the passage of culture days. This is because collagen is not stably immobilized on the surface of the silicone membrane only by addition of collagen, and the adhesiveness of the support used for the cell culture plate decreases with the passage of culture days. it is conceivable that.
In Example 1-3, cell growth with respect to progress of culture | cultivation days is improving, and it turns out that collagen adheres firmly to the silicone membrane through APP (1). Moreover, Example 1-4 also shows cell growth equivalent to Example 1-3, and shows that APP (1) has cell adhesiveness and also has cell proliferation.

Example 2
Polyurethane-polyamino acid copolymer (PAU) was used as an amphiphilic polymer.
In the same manner as in the method described in Examples of JP-A-2001-136960, PAU is urethane prepolymer (terminal: isocyanate group, copolymer of polytetramethylene ether glycol and triresin isocyanate) and γ -Methyl-L-glutamate-N carboxy anhydride was reacted at 1: 1 (w / w) for synthesis.
A cell culture plate (5) was obtained in the same manner as in Example 1-3 except that PAU was used instead of APP (1).
The cell proliferation activity was evaluated by the same method as in Example 1 using the cell culture plate (5), and the cell activity was improved as compared to Comparative Example 1-2 (in which only collagen was added to the silicone membrane). Was confirmed.

  Since the support of the present invention is excellent in gas permeability and cell adhesion, culturing the cells using the support of the present invention makes it possible to stably culture the cells in an environment closer to in vivo, It is useful.

Claims (7)

It has a silicone membrane surface-modified by a polymer layer containing an amphiphilic polymer and a cytophilic substance,
The amphiphilic polymer has a hydrophobic portion and a hydrophilic portion,
The hydrophobic portion has one or more structural units selected from the group consisting of structural units derived from polyurethane, structural units derived from polyethylene, structural units derived from nylon, and structural units derived from silicone,
The said hydrophilic part is a structural unit derived from polyamino acid, a structural unit derived from water-soluble collagen, a structural unit derived from gelatin, a structural unit derived from cellulose, a structural unit derived from starch, a structural unit derived from polysulfonic acid and a structural unit derived from polyethylene glycol A support for cell culture which is a copolymer having one or more structural units selected from the group consisting of units. In the amphiphilic polymer,
The hydrophobic portion is composed of a structural unit derived from polyurethane,
The copolymer according to claim 1, wherein the hydrophilic portion is a copolymer comprising one or more structural units selected from the group consisting of structural units derived from polyamino acids, structural units derived from polysulfonic acids and structural units derived from polyethylene glycol. Support for cell culture.   The support for cell culture according to claim 1 or 2, which comprises a silicone membrane surface-modified with the polymer layer.   The support for cell culture according to any one of claims 1 to 3, wherein the cytophilic substance is distributed throughout the polymer layer.   The support for cell culture according to any one of claims 1 to 4, wherein the cytophilic substance is one or more selected from the group consisting of collagens, growth factors and mucopolysaccharides.   The support for cell culture according to any one of claims 1 to 5, wherein the support is a support for use in long-term culture.   A cell culture plate in which the support for cell culture according to any one of claims 1 to 6 is disposed at the bottom of the plate such that the polymer layer is a culture surface.