WO2021256377A1

WO2021256377A1 - Cell culture substrate

Info

Publication number: WO2021256377A1
Application number: PCT/JP2021/022134
Authority: WO
Inventors: Zhe XU; Ichiro Hirahara; Takao Anzai
Original assignee: Terumo Kabushiki Kaisha
Priority date: 2020-06-18
Filing date: 2021-06-10
Publication date: 2021-12-23
Also published as: JP2023072702A

Abstract

The present invention is to provide a means for improving cell adhesion to a hydrophobic polymer substrate. Provided is a cell culture substrate comprising a coating layer containing a polymer having a structural unit (1) derived from furfuryl (meth)acrylate represented by the following Formula (1) and having a weight average molecular weight of 200,000 or more, on at least one surface of a polymer substrate having surface free energy of less than 33 mJ/m².

Description

CELL CULTURE SUBSTRATE

The present invention relates to a cell culture substrate having excellent cell adhesion activity, a bioreactor using the cell culture substrate, and a method for culturing a stem cell.

Background

In recent years, a cell culture technology has been used in the development of regenerative medicine or drug design. In particular, attention has been paid to use of stem cells, and technology for repairing and replacing a damaged or defective tissue has been actively studied by using stem cells expanded from a donor cell. Most of cells of animals including human are adherent (scaffold-dependent) cells which cannot survive in a floating state and survive in a state of being adhered to something. For this reason, various developments of functional culture substrates for culturing adherent (scaffold-dependent) cells at high density to obtain a cultured tissue similar to a living tissue have been conducted.

As a cell culture substrate, a plastic (for example, polystyrene) or glass vessel has been used, but it has been reported that surfaces of these cell vessels are subjected to a plasma treatment or the like. A substrate subjected to the treatment has excellent adhesion to cells, and can be used to grow cells and maintain their function.

Meanwhile, regarding a structure of the cell culture substrate (cell culture vessel), in addition to a conventional flat dish (plate) structure, various structures, such as a structure in which a porous body is inserted as a culture scaffold in a bag, a hollow fiber structure, a sponge structure, a flocculent (glass wool) structure, and a structure in which a plurality of dishes are laminated, have been developed. It is difficult or impossible to perform the plasma treatment to culture vessels having such various or complicated structures.

Therefore, as a method other than the plasma treatment, a technique using a polymer compound that promotes adhesion to cells (cell adhesion) has been proposed. For example, Non Patent Literature 1 discloses that cell adhesion to a cell culture substrate is improved by applying a solution containing a homopolymer of tetrahydrofurfuryl acrylate (PTHFA; polytetrahydrofurfuryl acrylate) to a polyethylene terephthalate (PET) film.

Colloids and Surfaces B; Biointerfaces 145 (2016) 586-596.

Summary of the Invention

In a general cell dish, as described in Non Patent Literature 1, a hydrophilic substrate such as of PET or the like is used for medium exchange. On the other hand, a hollow fiber bioreactor is required to have high gas exchange capacity. From this viewpoint, it is preferable to use a substrate formed of a hydrophobic polymer for a hollow fiber membrane. Therefore, excellent cell adhesion to a hydrophobic polymer substrate is demanded.

Accordingly, the present invention has been made in view of the above circumstances, and an object thereof is to provide a means for improving cell adhesion to a hydrophobic polymer substrate.

The present inventors have conducted intensive studies to solve the above-described problems. As a result, the present inventors have found that the above problems can be solved by coating a surface of a hydrophobic polymer substrate with a polymer having a structural unit (1) derived from furfuryl (meth)acrylate having a specific structure and having a specific molecular weight or more, thereby completing the present invention.

That is, the object can be achieved by a cell culture substrate including a coating layer containing a polymer comprising a structural unit (1) derived from furfuryl (meth)acrylate represented by the following Formula (1) and having a weight average molecular weight of 200,000 or more, the coating layer being formed on at least one surface of a polymer substrate having surface free energy of less than 33 mJ/m².

Chemical Formula 1

in the Formula (1), R¹ is a hydrogen atom or a methyl group, and R² is a group represented by the following Formula (1-1) or the following Formula (1-2):

Chemical Formula 2

in the Formula (1-1) and the Formula (1-2),R³ represents an alkylene group having 1 to 3 carbon atoms.

Fig. 1 is a partial side view illustrating an embodiment of a bioreactor (hollow fiber type bioreactor) of the present invention. Fig. 2 is a partially cut-away side view of the bioreactor of Fig. 1.

Detailed Description

The cell culture substrate of the present invention includes on at least one surface of a polymer substrate having surface free energy of less than 33 mJ/m², a coating layer containing a polymer which has a structural unit (1) derived from furfuryl (meth)acrylate represented by the following Formula (1) and has a weight average molecular weight (Mw) of 200,000 or more. By the cell culture substrate of the present invention, excellent cell adhesion can be imparted to a hydrophobic polymer substrate.

Chemical Formula 3

In the Formula (1), R¹ represents a hydrogen atom or a methyl group, and R² represents a group represented by the following Formula (1-1) or (1-2):

Chemical Formula 4

In the Formula (1-1) or (1-2), R³ represents an alkylene group having 1 to 3 carbon atoms.

In the present specification, a polymer substrate having surface free energy of less than 33 mJ/m² is also simply referred to as a “polymer substrate”, a “substrate”, a “hydrophobic polymer substrate”, or a “hydrophobic substrate”. In addition, in the present specification, a weight average molecular weight is also simply referred to as a “molecular weight” or “Mw”.

In the present specification, furfuryl (meth)acrylate represented by the Formula (1) is also simply referred to as "furfuryl (meth)acrylate", and a structural unit (1) derived from furfuryl (meth)acrylate represented by the Formula (1) is also simply referred to as a "structural unit (1)". Further, a polymer comprising a structural unit (1) is also simply referred to as a "polymer" or a "polymer according to the present invention".

In the present specification, the term “(meth)acrylate” includes both acrylate and methacrylate. Similarly, the term "(meth)acrylic acid" includes both acrylic acid and methacrylic acid, and "(meth)acryloyl" includes both acryloyl and methacryloyl.

In the present specification, the case when a structural unit is defined as "derived from" a monomer means that the structural unit is a divalent structural unit generated by cleavage of one of polymerizable unsaturated double bonds of the corresponding monomer.

The cell culture substrate of the present invention has a feature in that a coating layer containing a polymer having a specific structure (Formula (1)) and a specific molecular weight (Mw = 200,000 or more) is formed on at least one surface of a polymer substrate having specific hydrophobicity (surface free energy = less than 33 mJ/m²). The coating layer formed using the polymer can exhibit excellent cell adhesion. Here, a mechanism for exhibiting the effect(s) by the present invention is presumed to be as follows. Incidentally, the present invention is not limited to the following presumption.

Conventionally, cell culture has been performed on a cell culture plate such as a cell culture dish (Petri dish), 96 wells, or the like, and these cell culture vessels have been formed of a hydrophilic material such as polystyrene, PET, or the like in order to perform medium exchange. On the other hand, in recent years, cell culture can be automatically performed in a closed environment using a hollow fiber bioreactor in which separate facilities (for example, an incubator, a safety cabinet, and a clean room) are integrated. In such a hollow fiber bioreactor, it is preferable to use a substrate formed of a hydrophobic polymer for a hollow fiber membrane from the viewpoint of securing high gas exchange capacity. As a result of conducting intensive studies on various material, the present inventors have found that polytetrahydrofurfuryl acrylate (PTHFA) having a specific molecular weight or more is particularly effective. PTHFA having such a molecular weight can coat a hydrophobic substrate stably with a uniform thickness. Further, it is presumed that a cell adhesion factor (cell adhesive protein) contained in a medium is preferably adsorbed on a PTHFA coating layer (PTHFA film) having a uniform thickness as described above, and cells can readily adhere via the cell adhesion factor (that is, cell adhesion activity can be improved). In addition to the above, it is presumed that the use of a polymer having a large molecular weight increases the number of protein adsorption functional groups exposed on the surface, which also can promote adsorption of a cell adhesion factor (cell adhesive protein) to improve cell adhesion activity. Further, it is considered that an integrin-binding site of a cell adhesive protein adsorbed on the surface is directed to a cell to exhibit excellent cell proliferation (that is, cell proliferation activity can be improved).

Therefore, the cell culture substrate according to the present invention can achieve excellent cell adhesion. In addition, the cell culture substrate according to the present invention can achieve excellent cell proliferation.

Hereinafter, a preferred embodiment(s) of the present invention will be described. It should be noted that the present invention is not limited only to the following embodiment(s).

In the present specification, the term "X to Y" which indicates a range means "X or more and Y or less" including X and Y. Unless otherwise specified, operation(s) and measurement(s) of physical property(ies) and the like are conducted under conditions of room temperature (20 to 25°C)/relative humidity of 40 to 50% RH.

<Cell culture substrate>
The cell culture substrate of the present invention comprises a coating layer containing the polymer formed on at least one surface of a polymer substrate having surface free energy of less than 33 mJ/m².

When the coating layer containing the polymer according to the present invention is formed on the surface of the hydrophobic polymer substrate, excellent cell adhesion can be exhibited. The cell culture substrate according to the present invention also has excellent cell proliferation (cell elongation).

(Coating Layer)
The coating layer contains a polymer described below.

(Polymer)
The polymer according to the present invention has a structural unit (1) derived from furfuryl (meth)acrylate represented by the following Formula (1). By using the polymer, a coating layer having a uniform thickness can be stably formed on a hydrophobic substrate. Therefore, it is possible to impart excellent cell adhesion (further, cell proliferation) to the hydrophobic polymer substrate. In addition, by applying a solution of the polymer to a surface of the hydrophobic polymer substrate, a coating layer can be easily formed even for substrates having various shapes. Therefore, when the polymer according to the present invention is used, a coating layer excellent in cell adhesion (further, cell proliferation) can be formed on cell culture substrates (cell culture vessels) having various shapes and designs.

The polymer according to the present invention essentially includes the structural unit (1). It may further have a structural unit derived from another monomer as well as the structural unit (1). Here, the another monomer is not particularly limited as long as it does not inhibit cell adhesion. Specific examples of the another monomer may include acrylamide, N,N-dimethylacrylamide, N,N-diethylacrylamide, methacrylamide, N,N-dimethylmethacrylamide, N,N-diethylmethacrylamide, ethylene, propylene, N-vinylacetamide, N-isopropenyl acetamide, N-(meth)acryloyl morpholine, and the like. These monomers may be used singly or in combination of two or more kinds thereof. A composition of the structural unit derived from another monomer in a case where the polymer further has the structural unit derived from another monomer is not particularly limited as long as it does not inhibit cell adhesion. The composition of the structural unit derived from another monomer is preferably more than 0% by mole and less than 10% by mole and more preferably about 3 to 8% by mole with respect to the structural unit (1).

From the viewpoint of further improving cell adhesion (further, cell proliferation), the polymer preferably does not have a structural unit derived from another monomer, that is, the polymer is preferably composed of only the structural unit(s) (1). That is, according to a preferred embodiment of the present invention, the polymer is composed of the structural unit(s) (1).

(Structural Unit (1))
The structural unit (1) is derived from furfuryl (meth)acrylate of the following Formula (1). Incidentally, the structural unit (1) constituting the polymer may be used singly or in combination of two or more kinds thereof. That is, the structural unit (1) may be composed of only one kind of the structural unit derived from furfuryl (meth)acrylate of the following Formula (1) or may be composed of two or more kinds of the structural units derived from furfuryl (meth)acrylate of the following Formula (1). In the latter case, each structural unit may be present in the form of block or random.

Chemical Formula 5

In the Formula (1), R¹ is a hydrogen atom or a methyl group.

R² is a group represented by the following Formula (1-1) or the following Formula (1-2). Among them, R² is preferably a group represented by the following Formula (1-1) from the viewpoint of further improving cell adhesion (further, cell proliferation) or the like.

Chemical Formula 6

In the Formulae (1-1) and (1-2), R³ is an alkylene group having 1 to 3 carbon atoms. Herein, the alkylene group having 1 to 3 carbon atoms may include a methylene group (-CH₂-), an ethylene group (-CH₂CH₂-), a trimethylene group (-CH₂CH₂CH₂-), and a propylene group (-CH(CH₃)CH₂- or -CH₂CH(CH₃)-). Among them, R³ is preferably a methylene group (-CH₂-) or an ethylene group (-CH₂CH₂-), and more preferably a methylene group (-CH₂-), from the viewpoint of further improving cell adhesion (further, cell proliferation).

That is, the furfuryl (meth)acrylate may include tetrahydrofurfuryl acrylate, tetrahydrofurfuryl methacrylate, furfuryl acrylate, furfuryl methacrylate, 5-[2-(acryloyloxy)ethyl]tetrahydrofuran, 5-[2-(methacryloyloxy)ethyl]tetrahydrofuran, 5-[2-(acryloyloxy)ethyl]furane, 5-[2-(methacryloyloxy)ethyl]furane, and the like. These may be used singly or in combination of two or more kinds thereof. Among them, tetrahydrofurfuryl (meth)acrylate is preferable, and tetrahydrofurfuryl acrylate (THFA) is more preferable, from the viewpoint of further improving cell adhesion (further, cell proliferation).

A weight average molecular weight (Mw) of the polymer is 200,000 or more. Here, when the weight average molecular weight of the polymer is less than 200,000, uniformity of a thickness of a coating layer is insufficient. In addition, the number of protein adsorption functional groups exposed on the surface is small, and a sufficient amount of cell adhesion factors (cell adhesive proteins) cannot be adsorbed. Therefore, sufficient cell adhesion cannot be achieved. The weight average molecular weight (Mw) of the polymer is preferably more than 300,000, more preferably 350,000 or more, and particularly preferably 400,000 or more, from the viewpoint of further improving cell adhesion (further, cell proliferation). In addition, an upper limit of the weight average molecular weight (Mw) of the polymer is preferably 900,000 or less, more preferably 800,000 or less, still more preferably 700,000 or less, and particularly preferably 600,000 or less, from the viewpoint of improving solubility of the polymer in a solvent, improving coating film formability, and further uniformizing a thickness of a coating film (that is, coating layer). That is, in a preferred embodiment of the present invention, the weight average molecular weight (Mw) of the polymer is 200,000 or more and 900,000 or less. In a more preferred embodiment of the present invention, the weight average molecular weight (Mw) of the polymer is more than 300,000 and 800,000 or less. In a still more preferred embodiment of the present invention, the weight average molecular weight (Mw) of the polymer is 350,000 or more and 700,000 or less. In a particularly preferred embodiment of the present invention, the weight average molecular weight (Mw) of the polymer is 400,000 or more and 600,000 or less.

In the present specification, as the "weight average molecular weight (Mw)," a value measured by gel permeation chromatography (GPC) using polystyrene as a standard substance and tetrahydrofuran (THF) as a mobile phase respectively is adopted. Specifically, the polymer is dissolved in tetrahydrofuran (THF) so as to have a concentration of 10 mg/ml, thereby preparing a sample. Regarding the sample prepared as above, GPC column LF-804 (manufactured by Showa Denko K.K.) is attached to a GPC system LC-20 (manufactured by SHIMADZU CORPORATION), THF is supplied as a mobile phase, and polystyrene is used as a standard substance, to measure GPC of the polymer. After preparing a calibration curve with a standard polystyrene, the weight average molecular weight (Mw) of the polymer is calculated on the basis of this curve.

The polymer according to the present invention is not particularly limited, and for example, can be produced by employing a conventionally known polymerization method such as bulk polymerization, suspension polymerization, emulsion polymerization, solution polymerization, a living radical polymerization method, a polymerization method using a macroinitiator, a polycondensation method, or the like.

In the method, a polymerization solvent which can be used in the preparation of a monomer solution is not particularly limited as long as it can dissolve the monomer used above. Examples thereof may include aqueous solvents such as water, alcohols such as methanol, ethanol, propanol, and isopropanol, and polyethylene glycols; aromatic solvents such as toluene, xylene, and tetralin; halogen-based solvents such as chloroform, dichloroethane, chlorobenzene, dichlorobenzene, and trichlorobenzene; and the like. Among these, taking in consideration of easy dissolution of the monomer, or the like, methanol or ethanol is preferable. In addition, a monomer concentration in the monomer solution is not particularly limited, but the monomer concentration in the monomer solution is generally 5 to 60% by mass, preferably 10 to 50% by mass, more preferably 15% by mass or more and less than 50% by mass, further preferably 20 to 45% by mass, and particularly preferably 30 to 40% by mass. The concentration of the monomer is a total concentration of the furfuryl (meth)acrylate of the Formula (1) and if being used, a monomer which is copolymerizable therewith (another monomer, copolymerizable monomer).

A polymerization initiator is not particularly limited, and a known polymerization initiator may be used. From the viewpoint of high polymerization stability, the polymerization initiator is preferably a radical polymerization initiator. Specific examples thereof may include persulfates such as potassium persulfate (KPS), sodium persulfate, and ammonium persulfate; peroxides such as hydrogen peroxide, t-butyl peroxide, and methyl ethyl ketone peroxide; and azo compounds such as azobisisobutyronitrile (AIBN), 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile), 2,2'-azobis(2,4-dimethylvaleronitrile), 2,2'-azobis[2-(2-imidazoline-2-yl)propane]dihydrochloride, 2,2'-azobis[2-(2-imidazoline-2-yl)propane]disulfate dihydrate, 2,2'-azobis(2-methylpropionamidine)dihydrochloride, 2,2'-azobis[N-(2-carboxyethyl)-2-methylpropionamidine)]hydrate, 3-hydroxy-1,1-dimethylbutyl peroxyneodecanoate, α-cumylperoxy neodecanoate, 1,1,3,3-tetrabutyl peroxyneodecanoate, t-butyl peroxyneodecanoate, t-butyl peroxyneoheptanoate, t-butyl peroxypivalate, t-amyl peroxyneodecanoate, t-amyl peroxypivalate, di(2-ethylhexyl)peroxydicarbonate, di(secondary butyl)peroxydicarbonate, and azobiscyanovaleric acid. Further, for example, a reducing agent such as sodium sulfite, sodium hydrogen sulfite, or ascorbic acid may be used in combination with the radical polymerization initiator as a redox type initiator. A blending amount of the polymerization initiator is preferably 0.5 to 5 mmol with respect to 1 mol of a total amount of the monomer(s). With such a blending amount of the polymerization initiator, polymerization of the monomers is efficiently proceeded.

The polymerization initiator as it is may be mixed with the furfuryl (meth)acrylate of the Formula (1), if being used, a monomer which is copolymerizable with this component (another monomer, copolymerizable monomer), and a polymerization solvent, or alternatively a solution of the polymerization initiator obtained by being dissolved in another solvent in advance may be mixed with the monomer(s) and the polymerization solvent. In the latter case, another solvent used to dissolve the polymerization initiator is not particularly limited as long as it can dissolve the polymerization initiator. The same solvents as the polymerization solvents described above can be exemplified. Further, another solvent may be the same as or different from the polymerization solvent, but in consideration of easy control of polymerization, and the like, another solvent is preferably the same as the polymerization solvent. Further, in this case, a concentration of the polymerization initiator in another solvent is not particularly limited, but in consideration of easy mixing, and the like, the addition amount of the polymerization initiator is preferably 0.1 to 10 parts by mass and more preferably 0.5 to 5 parts by mass with respect to 100 parts by mass of another solvent.

Further, in the case of using the polymerization initiator in the solution state, a deaeration treatment may be performed in advance before adding a solution in which the monomer(s) (furfuryl (meth)acrylate, and a copolymerizable monomer which is used as optionally) is dissolved in the polymerization solvent, to the polymerization initiator solution. For the deaeration treatment, for example, an inert gas such as nitrogen gas or argon gas may be blown for about 10 seconds to 5 hours into the above-described solution. In the deaeration treatment, the solution may be adjusted to about 30°C to 80°C, preferably to a polymerization temperature in a polymerization step as described below.

Next, the monomer solution is heated to polymerize the monomer(s). Here, as the polymerization method, for example, a known polymerization method such as radical polymerization, anionic polymerization, or cationic polymerization can be adopted, and radical polymerization which facilitates production is preferably used.

Polymerization conditions are not particularly limited as long as the furfuryl (meth)acrylate of the Formula (1), and if being used, a monomer which is copolymerizable therewith (another monomer, copolymerizable monomer) can be polymerized. Specifically, a polymerization temperature is preferably 30°C to 80°C and more preferably 40°C to 55°C. Further, a polymerization time is preferably is 1 to 24 hours and more preferably 5 to 12 hours. Under such conditions, polymerization of the monomer(s) can efficiently proceed. Further, it is possible to effectively suppress or prevent gelation in the polymerization step and to achieve high production efficiency.

As necessary, a chain transfer agent, a polymerization rate-adjusting agent, a surfactant, and other additive(s) may be appropriately used during the polymerization.

An atmosphere under which the polymerization reaction is carried out is not particularly limited, and the reaction can be carried out under an air atmosphere, an inert gas atmosphere such as nitrogen gas or argon gas, and the like. Further, during the polymerization reaction, the reaction solution may be stirred.

The polymer after polymerization can be purified by a general purification method such as a reprecipitation method (precipitation method), a dialysis method, an ultrafiltration method, or an extraction method.

The purified polymer can be dried by an arbitrary method such as freeze drying, vacuum drying, spray drying, or heat drying. Freeze drying or vacuum drying is preferred from the viewpoint that physical properties of the polymer are less affected.

(Hydrophobic Polymer Substrate)
In the present invention, the coating layer containing the polymer is formed on at least one surface of the hydrophobic polymer substrate. Here, the hydrophobic polymer substrate according to the present invention has surface free energy of less than 33 mJ/m². Here, surface free energy of a material is known depending on a kind of the material forming the substrate. In this case, the surface free energy of the substrate is surface free energy of the material. The surface free energy of the substrate can also be measured by a known method such as contact angle measurement, the Wilhelmy method, or the like. When the surface free energy of the substrate is unknown, the surface free energy (mJ/m²) in the present specification is measured by the following contact angle measurement method.

(Method for Measuring Surface Free Energy)
The surface free energy is measured by determining a contact angle between a substrate and a standard liquid (pure water, nitromethane, and methylene iodide) using a contact angle meter (manufactured by Kyowa Interface Science Co., Ltd., contact angle meter DMs-401) at 23°C and a relative humidity of 55%RH (measurement method, in accordance with JIS R 3257:1999 (sessile drop method)) (3 times in total). An average contact angle is determined from the average of the measured values. Next, three components of the surface free energy of the substrate are calculated based on the following Young-Dupre equation and extended Fowkes equation.

Incidentally, the surface free energy (mJ/m²) of the standard liquid is shown in Table 1. Based on the contact angle, each of the component values (γ_s ^d, γ_s ^p, and γ_s ^h) of the surface free energy of the surface of the substrate is calculated.

As described above, the surface free energy of the substrate correlates with the contact angle of the substrate surface with respect to water. The contact angle of the substrate surface with respect to water is usually more than 78° and preferably 100° or more.

Examples of a material constituting the substrate may include polypropylene (surface free energy = 29 mJ/m², contact angle = 100 to 110°), polymethylpentene (surface free energy = 19 mJ/m², contact angle = 108°), polytetrafluoroethylene (surface free energy = 18 mJ/m², contact angle = 105 to 115°), polycarbonate (surface free energy = 46.7mJ/m², contact angle = 90°), and the like. The materials may be used singly or in combination of two or more kinds thereof. Among them, polypropylene, polytetrafluoroethylene, or polycarbonate is preferable, and polypropylene is particularly preferable, from the viewpoint of higher gas exchangeability, easy uniformization of a coating film, and the like. That is, in a preferred embodiment of the present invention, the polymer substrate contains at least one selected from the group consisting of polypropylene, polytetrafluoroethylene, and polycarbonate. In a more preferred embodiment of the present invention, the polymer substrate contains polypropylene. In a particularly preferred embodiment of the present invention, the polymer substrate is formed of polypropylene. Incidentally, surface free energy of polyethylene terephthalate (PET) described in Non Patent Literature 1 and surface free energy of polystyrene generally used as a cell culture vessel are 43 mJ/m² (contact angle = 65 to 75°) and 33 mJ/m²(contact angle = 78°), respectively.

A structure of the hydrophobic polymer substrate is not limited, but the hydrophobic polymer can be designed into various structures (shapes) such as a structure in which a porous body is inserted, a hollow fiber structure, a porous membrane structure, a sponge structure, a cotton-like (glass wool) structure, and the like, in addition to a planar structure. As described later, the cell culture substrate of the present invention can be suitably used in a bioreactor, particularly, a hollow fiber type bioreactor. Therefore, the hydrophobic polymer substrate preferably has a hollow fiber, and is more preferably a porous membrane (hollow fiber membrane) constituted by a plurality of hollow fibers. Here, the porous membrane (hollow fiber membrane) as the polymer substrate is preferably a porous membrane at least partially formed of polypropylene. Here, a part of the polymer substrate may be formed of polypropylene and the other part of the polymer substrate may be formed of a material other than polypropylene (another material), or the polymer substrate may be partially or entirely formed of polypropylene and another material. Preferably, the hollow fiber membrane as the polymer substrate is formed of only polypropylene. Examples of the another material can include polymer materials such as polyethylene, polysulfone, polyacrylonitrile, polytetrafluoroethylene, and cellulose acetate, and the like. When the hollow fiber membrane is formed of polypropylene and another material as described above, a content of the polypropylene is not particularly limited as long as the surface free energy of the polymer substrate is less than 33 mJ/m². The content of the polypropylene is usually more than 80% by mass (in terms of solid content), preferably 90% by mass (in terms of solid content) or more, and more preferably 95% by mass (in terms of solid content) or more (upper limit: less than 100% by mass) with respect to a total content of materials forming the polymer substrate (hollow fiber membrane).

An inner diameter (diameter) of the hollow fibers is not particularly limited, but is preferably 50 to 1,000 μm, more preferably 100 to 500 μm, and particularly preferably about 150 to 350 μm. An outer diameter (diameter) of the hollow fibers is not particularly limited, but is preferably 100 to 1,200 μm, more preferably 150 to 700 μm, and particularly preferably about 200 to 500 μm. A length of the hollow fibers is not particularly limited, but is preferably 50 to 900 mm, more preferably 100 to 700 mm, and particularly preferably about 150 to 500 mm. The number of the hollow fibers constituting the hollow fiber membrane is not particularly limited, but is, for example, about 1,000 to 100,000, more preferably 3,000 to 50,000, and particularly preferably about 5,000 to 25,000. In one embodiment, the hydrophobic polymer substrate is constituted by about 9,000 hollow fibers having an average length of about 295 mm, an average inner diameter of 215 μm, and an average outer diameter of 315 μm. Herein, the coating layer may be formed on the inner side or the outer side of the hollow fiber membrane, but is preferably formed on the inner (lumen) surface.

An outer layer of the hollow fiber may have an open pore structure with a certain surface roughness. An opening (diameter) of the pore is not particularly limited, but is in the range of about 0.5 to about 3 μm, and the number of pores on the outer surface of the hollow fiber may be in the range of about 10,000 to about 150,000 per 1 square millimeter (1 mm²). A thickness of the outer layer of the hollow fiber is not particularly limited, and for example, is in the range of about 1 to about 10 μm. The hollow fiber may have an additional layer (second layer) on the outer side, and at this time, the additional layer (second layer) preferably has a sponge structure having a thickness of about 1 to about 15 μm. The second layer having such a structure can serve as a support for the outer layer. Further, in this embodiment, the hollow fiber may have a further additional layer (third layer) at the outer side of the second layer. In this embodiment, the further additional layer (third layer) preferably has a finger-like structure. With the third layer having such a structure, mechanical stability is obtainable. Further, a high void volume with low resistance to membrane transfer of molecules can be provided. In this embodiment, during use, the finger-like voids may be filled with a fluid and the fluid lowers resistance for diffusion and convection as compared with a matrix with a sponge-filled structure having a lower void volume. This third layer has a thickness of, preferably, about 20 to about 60 μm.

A method for producing a hollow fiber and a porous membrane is not particularly limited, and a known production method can be applied similarly or appropriately modified. For example, it is preferable that micro fine holes are formed on a wall of hollow fiber by a stretching method or a solid-liquid phase separation method.

A method for producing the hydrophobic polymer substrate is not particularly limited, but examples thereof may include a method for producing a hydrophobic polymer substrate using a conventionally known method using the material or a mixture of the material and the another material and the like.

(Method of Forming Coating Layer)
A method for forming the coating layer containing the polymer according to the present invention on the surface of the hydrophobic polymer substrate is not particularly limited. For example, in a case where the surface of the hydrophobic polymer substrate has a flat dish (plate) structure, it is possible to use a method in which a polymer-containing solution in which the polymer according to the present invention is dissolved is applied (for example, added to wells) to a predetermined surface and then drying is performed. In addition, for example, in a case where the hydrophobic polymer substrate is a hollow fiber or a porous membrane, it is possible to use a method in which a polymer-containing solution in which the polymer according to the present invention is dissolved is brought into contact with a cell contact portion of a hollow fiber (for example, distributed to an inner surface (lumen) or an outer surface of the hollow fiber) and then drying is performed. Incidentally, in a case where the hydrophobic polymer substrate is a porous membrane constituted by a plurality of hollow fibers, the coating with the polymer-containing solution may be performed on one hollow fiber and then the hollow fibers may be bundled, or may be performed after a plurality of hollow fibers are bundled to produce a porous membrane.

Here, a solvent for dissolving the polymer according to the present invention is preferably a solvent capable of dissolving the polymer. According to this, the polymer-containing solution can be more stably applied to the polymer substrate to form a coating layer having a more uniform thickness. In addition, as the solvent for dissolving the polymer according to the present invention, a solvent having a small influence on a cell culture substrate (deformation, cracking, destruction, or the like) is suitable. Examples of such a solvent may include aqueous solvents such as water, alcohols such as methanol, ethanol, propanol, isopropanol, and the like, polyethylene glycols, and the like; ketone-based solvents such as acetone and the like; and ether solvents such as 1,4-dioxane, 1,3-dioxolane, methyl cellosolve, ethyl cellosolve, butyl ether, diethyl ether, diisopropyl ether, 1,2-dimethoxyethane, cyclopentyl methyl ether, and tetrahydrofuran (THF), and the like. The solvent(s) may be used singly or in the form of a mixture of two or more kinds thereof. In particular, the solvent is preferably a mixed solvent of alcohol and an ether solvent in consideration of improvement in solubility of the polymer according to the present invention and the like. The alcohol used in the mixed solvent is preferably a lower alcohol having 1 to 3 carbon atoms (methanol, ethanol, propanol, or isopropanol) from the viewpoint of improving solubility of the polymer, and among them, methanol or ethanol is more preferable, and methanol is particularly preferable. In addition, the ether solvent used in the mixed solvent is preferably tetrahydrofuran (THF) or acetone, and particularly preferably tetrahydrofuran (THF), from the viewpoint of improving solubility of the polymer. That is, it is preferable that the solvent consists of methanol and tetrahydrofuran. Here, a mixing ratio of the alcohol (particularly methanol) to the ether solvent (particularly tetrahydrofuran) is not particularly limited, but for example, the mixing ratio (volume ratio) of alcohol : ether solvent is preferably 1 to 10 : 1, and more preferably 3 to 7 : 1. With such a mixing ratio, the polymer can be more easily dissolved in the mixed solvent. Therefore, a coating layer that has a more uniform thickness and is flat (with less or no coating unevenness) can be formed on the polymer substrate. In addition, the polymer-containing solution can be easily applied to the polymer substrate (excellent in coatability).

A concentration of the polymer in the polymer-containing solution is preferably low. Specifically, the concentration of the polymer in the polymer-containing solution is preferably 0.0001 to 5% by mass, more preferably 0.001% by mass or more and less than 5% by mass, still more preferably 0.05 to 4% by mass, further still more preferably 0.1 to 2.5% by mass, and particularly preferably 0.2% by mass or more and less than 2.5% by mass. When the polymer-containing solution has such a concentration, a coating layer that has a more uniform thickness and is flat (with less or no coating unevenness) can be formed on the polymer substrate. In addition, the polymer can be easily applied to the polymer substrate (excellent in coatability).

A method of forming the coating film of the polymer is not particularly limited, and a conventionally known method such as filling, dip coating (immersion method), spraying, spin coating, dropping, doctor blade, brush coating, roll coating, air knife coating, curtain coating, wire bar coating, gravure coating, or mixed solution-impregnated sponge coating can be applied.

Conditions for forming the coating film of the polymer are not particularly limited. For example, a contact time between the polymer-containing solution and the hydrophobic polymer substrate (for example, a time for distribution of the polymer-containing solution to the lumen or the outer surface of the hollow fiber) is preferably 1 to 5 minutes, and more preferably 1 to 3 minutes, in consideration of ease of forming the coating film (that is, the coating layer), effect of reducing coating unevenness, and the like. A contact temperature between the polymer-containing solution and the hydrophobic polymer substrate (for example, a temperature at which the polymer-containing solution is distributed to the lumen or the outer surface of the hollow fiber) is preferably 5 to 40°C, and more preferably 15 to 30°C, in consideration of ease of forming the coating film (that is, the coating layer), effect of reducing coating unevenness, and the like.

An application amount of the polymer-containing solution to the surface of the hydrophobic polymer substrate is not particularly limited, but is preferably an amount in which a ratio of a mass of the polymer per unit area in the coating layer after drying is within the above range. In a case where such an amount cannot be obtainable by single contact (application), a contact (application) step (or a application step and a drying step described later) may be repeated until a desired application amount is attained.

Next, after the contact between the hydrophobic polymer substrate and the polymer-containing solution, the coating film is dried to form a coating layer (coating) of the polymer according to the present invention on the surface of the hydrophobic polymer substrate. Herein, drying conditions are not particularly limited as long as the coating layer (coating) of the polymer according to the present invention can be formed. Specifically, a drying temperature is preferably 5 to 50°C and more preferably 15 to 40°C. A drying step may be performed under a single condition or may be performed stepwise under different conditions. A drying time is not particularly limited, but is, for example, about 1 to 60 hours, and preferably 240 minutes to 18 hours. In addition, in a case where the hydrophobic polymer substrate is a porous membrane (hollow fiber membrane), the coating film may be dried by continuously or stepwisely distributing a gas at 5 to 40°C and more preferably 15 to 30°C on a surface of the hollow fiber on which the polymer-containing solution is applied. Herein, the gas is not particularly limited as long as it has no influence on the coating film (coating layer) and can dry the coating film. Specific examples thereof include air, an inert gas such as nitrogen gas or argon gas, and the like. Further, a circulation amount of the gas is not particularly limited as long as the coating film can be sufficiently dried. The circulation amount of the gas is preferably 5 to 150 L/min and more preferably 30 to 100 L/min.

According to such a method, the coating layer containing the polymer according to the present invention can be efficiently formed on the hydrophobic polymer substrate. Incidentally, depending on the type of cells to be adhered, the hydrophobic polymer substrate may be further treated with a cell adhesion factor such as fibronectin, laminin, collagen, or the like. With such a treatment, adhesion of cells to the substrate surface and growth of cells thereon can be further promoted. Incidentally, in a case where the hydrophobic polymer substrate is a porous membrane constituted by a plurality of hollow fibers, the treatment with the cell adhesion factor may be performed on one hollow fiber and then the hollow fibers may be bundled, or may be performed after a plurality of hollow fibers are bundled to produce a porous membrane. Further, the treatment with a cell adhesion factor may be performed after the coating layer containing the polymer according to the present invention is formed, before the coating layer containing the polymer according to the present invention is formed, or at the same time the coating layer containing the polymer according to the present invention is formed.

In the present invention, a thickness of the coating layer (dry film thickness) formed on the hydrophobic polymer substrate is 0.005 to 20 μm, preferably 0.1 to 2 μm, and more preferably 0.2 to 1.5 μm.

<Bioreactor>
The cell culture substrate of the present invention is excellent in cell adhesion (further, cell proliferation). Therefore, the cell culture substrate of the present invention can be suitably used for a bioreactor. That is, the present invention provides a bioreactor including the cell culture substrate of the present invention. Here, the bioreactor may be a plane type bioreactor or a hollow fiber type bioreactor, but is particularly preferably a hollow fiber type bioreactor. Therefore, in the following description, although a hollow fiber type bioreactor will be described as a preferred embodiment, the bioreactor of the present invention may be a plane type bioreactor, and in this case, the following embodiment can be appropriately changed and applied. Further, dimensional ratios in the drawings are exaggerated for the sake of explanatory convenience and may differ from actual ratios.

The bioreactor in which the cell culture substrate of the present invention can be suitably used is not particularly limited, but the cell culture substrate and the bioreactor of the present invention can be applied to, for example, a cell culture/proliferation system described in JP-T-2010-523118 (JP 5524824 B) (WO 2008/124229 A2), JP-T-2013-524854 (JP 6039547 B) (WO 2011/140231 A1), JP-T-2013-507143 (JP 5819835 B) (WO 2011/045644 A1), JP 2013-176377 A (WO 2008/109674 A), JP-T-2015-526093 (WO 2014/031666 A1), JP-T-2016-537001 (WO 2015/073918 A1), JP-T-2017-509344 (WO 2015/148704 A1), or the like; and a Quantum cell proliferation system manufactured by TERUMO BCT, INC. Conventionally, in the cell culture, facilities such as an incubator, a safety cabinet, and a clean room are separately needed, but the culture system as described above has all of those functions so that the facility can be very simplified. Further, by controlling temperature or gas during the cell culture using the system as described above, a functionally closed system can be ensured and the cell culture can be performed automatically and in a closed environment.

Hereinafter, an embodiment of the bioreactor of the present invention will be described with reference to the drawings, but the present invention is not limited to the following embodiment.

Fig. 1 is a partial side view illustrating an embodiment of a bioreactor (hollow fiber type bioreactor) of the present invention. Further, Fig. 2 is a partially cut-away side view of the bioreactor of Fig. 1. In Figs. 1 and 2, a bioreactor 1 has a cell culture substrate 2 of the present invention provided in a cell culture chamber 3. The cell culture chamber 3 has four openings, that is, four ports (an inlet port 4, an outlet port 6, an inlet port 8, and an outlet port 10). Herein, a culture medium including cells flows to a hollow fiber intracapillary (IC) space of the cell culture substrate 2 in the cell culture chamber 3 through the inlet port 4, and discharged from the outlet port 6. According to this, cells are efficiently adhered (attached) to and cultured on the surface of the hollow fiber lumen. Meanwhile, a culture medium or gas (such as oxygen or carbon dioxide) flows through the inlet port 8 to be in contact with a hollow fiber extracapillary (EC) space of the cell culture substrate 2 in the cell culture chamber 3, and discharged from the outlet port 10. According to this, in the cell culture chamber 3, small molecules such as culture medium components flow into the hollow fibers or unnecessary components are discharged from the inside of the hollow fibers, and cells adhered onto the surface of the hollow fibers are cultured. Further, after culturing for a predetermined time, a liquid (for example, PBS) containing trypsin is introduced into the hollow fiber intracapillary (IC) space of the cell culture substrate 2 in the cell culture chamber 3 through the inlet port 4, and then is held for a predetermined time (for example, about 5 to 10 minutes). Next, a culture medium or an isotonic solution such as PBS flows in the hollow fiber intracapillary (IC) space of the cell culture substrate 2 in the cell culture chamber 3 through the inlet port 4 to apply a shear force to cells, the cells are released from the inner wall of the hollow fiber, and the cells are recovered from the bioreactor through the outlet port 6. Incidentally, although the cells are adhered to the hollow fiber intracapillary (IC) space in the above embodiment, the present invention is not limited to the above embodiment, and cells may be cultured in such a manner that a culture medium containing cells flows from the inlet port 8 to the outlet port 10, the cells are efficiently adhered (attached) to an outer surface of the hollow fiber, and a culture medium flows from the inlet port 4 to the outlet port 6 in an hollow fiber lumen. Further, the fluid from the inlet port 4 to the outlet port 6 may flow in either a co-current or counter-current direction with respect to flow of fluid from the inlet port 8 to the outlet port 10.

(Use of Bioreactor)
As described above, the bioreactor of the present invention includes a cell culture substrate excellent in cell adhesion (further, cell proliferation). Herein, cells which can be cultured in the bioreactor of the present invention may be adherent (scaffold-dependent) cells, non-adherent cells, or any combination thereof. Since the bioreactor is provided with the cell culture substrate excellent in cell adhesion, the bioreactor of the present invention can be particularly suitably used in culturing of adherent (scaffold-dependent) cells. Herein, as the adherent (scaffold-dependent) cells, there are animal cells such as stem cells including mesenchymal stem cell (MSC) or the like, fibroblast cells, and the like. As mentioned above, attention has been paid to stem cells in development of regenerative medicine or drug discovery. Therefore, the bioreactor of the present invention can be suitably used in culturing of stem cells. That is, the present invention is to provide a method for culturing a stem cell using the bioreactor of the present invention. Herein, the method for culturing a stem cell is not particularly limited, and a general culturing method can be applied similarly or appropriately modified.
Examples

The effects of the present invention will be described using the following examples and comparative examples. However, the technical scope of the present invention is not limited to only the following examples. Incidentally, in the following examples, operations were carried out at room temperature (25°C) unless otherwise specified. In addition, unless otherwise specified, "%" and "part(s)" mean "% by mass" and "part(s) by mass," respectively.

<Production of Polymer>
(Production Example 1: Synthesis of Polymer 1 (Mw = 200,000))
To a 300 mL four-neck round-bottom flask, 190 g of methanol and 60 g of tetrahydrofurfuryl acrylate (THFA) (manufactured by FUJIFILM Wako Pure Chemical Corporation) were sequentially added, and then nitrogen gas was bubbled. Subsequently, a solution obtained by dissolving 0.06 g of 2,2’-azobis(4-methoxy-2,4-dimethylvaleronitrile) (manufactured by FUJIFILM Wako Pure Chemical Corporation, V-70) as a polymerization initiator in 2 mL of acetone was added to the flask to prepare a monomer solution (monomer concentration: 24% by mass). The monomer solution was polymerized at 45°C (liquid temperature) for 6 hours to obtain a polymerization liquid. The polymerization liquid was transferred to a beaker and left in a draft for 2 hours, and then the supernatant was removed to recover a polymer component. Acetone was added to the polymer component to dissolve the polymer component, the solution was added to cyclohexane, and the precipitated polymer component was recovered and dried under reduced pressure to obtain polytetrahydrofurfuryl acrylate (PTHFA) (polymer 1). A weight average molecular weight of the polymer 1 was 200,000.

(Production Example 2: Synthesis of Polymer 2 (Mw = 400,000))
To a 300 mL four-neck round-bottom flask, 120 g of methanol and 60 g of tetrahydrofurfuryl acrylate (THFA) (manufactured by FUJIFILM Wako Pure Chemical Corporation) were sequentially added, and then nitrogen gas was bubbled. Subsequently, a solution obtained by dissolving 0.06 g of 2,2’-azobis(4-methoxy-2,4-dimethylvaleronitrile) (manufactured by FUJIFILM Wako Pure Chemical Corporation, V-70) as a polymerization initiator in 2 mL of acetone was added to the flask to prepare a monomer solution (monomer concentration: 33.3% by mass). The monomer solution was polymerized at 45°C (liquid temperature) for 6 hours to obtain a polymerization liquid. The polymerization liquid was transferred to a beaker and left in a draft for 2 hours, and then the supernatant was removed to recover a polymer component. Acetone was added to the polymer component to dissolve the polymer component, the solution was added to cyclohexane, and the precipitated polymer component was recovered and dried under reduced pressure to obtain polytetrahydrofurfuryl acrylate (PTHFA) (polymer 2). A weight average molecular weight of the polymer 2 was 400,000.

(Production Example 3: Synthesis of Polymer 3 (Mw = 600,000))
To a 300 mL four-neck round-bottom flask, 90 g of methanol and 60 g of tetrahydrofurfuryl acrylate (THFA) (manufactured by FUJIFILM Wako Pure Chemical Corporation) were sequentially added, and then nitrogen gas was bubbled. Subsequently, a solution obtained by dissolving 0.06 g of 2,2’-azobis(4-methoxy-2,4-dimethylvaleronitrile) (manufactured by FUJIFILM Wako Pure Chemical Corporation, V-70) as a polymerization initiator in 2 mL of acetone was added to the flask to prepare a monomer solution (monomer concentration: 40% by mass). The monomer solution was polymerized at 45°C (liquid temperature) for 6 hours to obtain a polymerization liquid. The polymerization liquid was transferred to a beaker and left in a draft for 2 hours, and then the supernatant was removed to recover a polymer component. Acetone was added to the polymer component to dissolve the polymer component, the solution was added to cyclohexane, and the precipitated polymer component was recovered and dried under reduced pressure to obtain polytetrahydrofurfuryl acrylate (PTHFA) (polymer 3). A weight average molecular weight of the polymer 3 was 600,000.

(Production Example 4: Synthesis of Polymer 4 (Mw = 100,000))
To a 300 mL four-neck round-bottom flask, 220 g of methanol and 30 g of tetrahydrofurfuryl acrylate (THFA) (manufactured by FUJIFILM Wako Pure Chemical Corporation) were sequentially added, and then nitrogen gas was bubbled. Subsequently, a solution obtained by dissolving 0.06 g of 2,2’-azobis(4-methoxy-2,4-dimethylvaleronitrile) (manufactured by FUJIFILM Wako Pure Chemical Corporation, V-70) as a polymerization initiator in 2 mL of acetone was added to the flask to prepare a monomer solution (monomer concentration: 12% by mass). The monomer solution was polymerized at 45°C (liquid temperature) for 6 hours to obtain a polymerization liquid. The polymerization liquid was transferred to a beaker and left in a draft for 2 hours, and then the supernatant was removed to recover a polymer component. Acetone was added to the polymer component to dissolve the polymer component, the solution was added to cyclohexane, and the precipitated polymer component was recovered and dried under reduced pressure to obtain polytetrahydrofurfuryl acrylate (PTHFA) (polymer 4). A weight average molecular weight of the polymer 4 was 100,000.

<Coating to Hydrophobic Polymer Substrate>
(Example 1)
The polymer 1 (Mw = 200,000) obtained in Production Example 1 was dissolved in a mixed solvent of tetrahydrofuran (THF) and methanol (THF : methanol = 1 : 3 (volume ratio)) so as to have a concentration of 0.2% by mass, thereby preparing a polymer solution (concentration = 0.2% by mass).

50 μL of this polymer solution was added to each of 96 wells of a commercially available polypropylene dish for tissue culture (no plasma treatment, manufactured by Caplugs Evergreen, surface free energy = 29 mJ/m², contact angle of surface with respect to water = 100 to 110°), and drying was performed in a draft at 25°C for 240 minutes to form a coating film of the polymer 1 (thickness when dried: 0.3 μm) on a well surface, thereby obtaining a cell culture dish (1).

(Example 2)
A cell culture dish (2) was obtained by forming a coating film of the polymer 2 (thickness when dried: 0.3 μm) on the well surface in the same manner as that of Example 1, except that the polymer 2 (Mw = 400,000) obtained in Production Example 2 was used instead of the polymer 1 in Example 1.

(Example 3)
A cell culture dish (3) was obtained by forming a coating film of the polymer 3 (thickness when dried: 0.3 μm) on the well surface in the same manner as that of Example 1, except that the polymer 3 (Mw = 600,000) obtained in Production Example 3 was used instead of the polymer 1 in Example 1.

(Comparative Example 1)
A comparative cell culture dish (1) was obtained by forming a coating film of the polymer 4 (thickness when dried: 0.3 μm) on the well surface in the same manner as that of Example 1, except that the polymer 4 (Mw = 100,000) obtained in Production Example 4 was used instead of the polymer 1 in Example 1.

(Example 4)
The polymer 1 (Mw = 200,000) obtained in Production Example 1 was dissolved in a mixed solvent of tetrahydrofuran (THF) and methanol (THF : methanol = 1 : 3 (volume ratio)) so as to have a concentration of 1.0% by mass, thereby preparing a polymer solution (concentration = 1.0% by mass).

50 μL of this polymer solution was added to each of 96 wells of a commercially available polypropylene dish for tissue culture (no plasma treatment, manufactured by Caplugs Evergreen, surface free energy = 29 mJ/m², contact angle of surface with respect to water = 100 to 110°), and drying was performed in a draft at 25°C for 240 minutes to form a coating film of the polymer 1 (thickness when dried: 1 μm) on a well surface, thereby obtaining a cell culture dish (4).

(Example 5)
A cell culture dish (5) was obtained by forming a coating film of the polymer 2 (thickness when dried: 1 μm) on the well surface in the same manner as that of Example 4, except that the polymer 2 (Mw = 400,000) obtained in Production Example 2 was used instead of the polymer 1 in Example 4.

(Example 6)
A cell culture dish (6) was obtained by forming a coating film of the polymer 3 (thickness when dried: 1 μm) on the well surface in the same manner as that of Example 4, except that the polymer 3 (Mw = 600,000) obtained in Production Example 3 was used instead of the polymer 1 in Example 4.

(Comparative Example 2)
A comparative cell culture dish (2) was obtained by forming a coating film of the polymer 4 (thickness when dried: 1 μm) on the well surface in the same manner as that of Example 4, except that the polymer 4 (Mw = 100,000) obtained in Production Example 4 was used instead of the polymer 1 in Example 4.

(Example 7)
The polymer 1 (Mw = 200,000) obtained in Production Example 1 was dissolved in a mixed solvent of tetrahydrofuran (THF) and methanol (THF : methanol = 1 : 3 (volume ratio)) so as to have a concentration of 2.5% by mass, thereby preparing a polymer solution (concentration = 2.5% by mass).

50 μL of this polymer solution was added to each of 96 wells of a commercially available polypropylene dish for tissue culture (no plasma treatment, manufactured by Caplugs Evergreen, surface free energy = 29 mJ/m², contact angle of surface with respect to water = 100 to 110°), and drying was performed in a draft at 25°C for 240 minutes to form a coating film of the polymer 1 (thickness when dried: 2 μm) on a well surface, thereby obtaining a cell culture dish (7).

(Example 8)
A cell culture dish (8) was obtained by forming a coating film of the polymer 2 (thickness when dried: 2 μm) on the well surface in the same manner as that of Example 7, except that the polymer 2 (Mw = 400,000) obtained in Production Example 2 was used instead of the polymer 1 in Example 7.

(Example 9)
A cell culture dish (9) was obtained by forming a coating film of the polymer 3 (thickness when dried: 2 μm) on the well surface in the same manner as that of Example 7, except that the polymer 3 (Mw = 600,000) obtained in Production Example 3 was used instead of the polymer 1 in Example 7.

(Comparative Example 3)
A comparative cell culture dish (3) was obtained by forming a coating film of the polymer 4 (thickness when dried: 2 μm) on the well surface in the same manner as that of Example 7, except that the polymer 4 (Mw = 100,000) obtained in Production Example 4 was used instead of the polymer 1 in Example 7.

(Example 10)
The polymer 1 (Mw = 200,000) obtained in Production Example 1 was dissolved in a mixed solvent of tetrahydrofuran (THF) and methanol (THF : methanol = 1 : 3 (volume ratio)) so as to have a concentration of 5.0% by mass, thereby preparing a polymer solution (concentration = 5.0% by mass).

50 μL of this polymer solution was added to each of 96 wells of a commercially available polypropylene dish for tissue culture (no plasma treatment, manufactured by Caplugs Evergreen, surface free energy = 29 mJ/m², contact angle of surface with respect to water = 100 to 110°), and drying was performed in a draft at 25°C for 240 minutes to form a coating film of the polymer 1 (thickness when dried: 4 μm) on a well surface, thereby obtaining a cell culture dish (10).

(Example 11)
A cell culture dish (11) was obtained by forming a coating film of the polymer 2 (thickness when dried: 4 μm) on the well surface in the same manner as that of Example 10, except that the polymer 2 (Mw = 400,000) obtained in Production Example 2 was used instead of the polymer 1 in Example 10.

(Example 12)
A cell culture dish (12) was obtained by forming a coating film of the polymer 3 (thickness when dried: 4 μm) on the well surface in the same manner as that of Example 10, except that the polymer 3 (Mw = 600,000) obtained in Production Example 3 was used instead of the polymer 1 in Example 10.

(Comparative Example 4)
A comparative cell culture dish (4) was obtained by forming a coating film of the polymer 4 (thickness when dried: 4 μm) on the well surface in the same manner as that of Example 10, except that the polymer 4 (Mw = 100,000) obtained in Production Example 4 was used instead of the polymer 1 in Example 10.

<Cell Adhesion Activity Assay>
Using the cell culture dishes (1) to (12) obtained in Examples 1 to 12 and the comparative cell culture dishes (1) to (4) obtained in Comparative Examples 1 to 4, cells were cultured according to the following process, and cell adhesion activity (cell adhesion) was evaluated.

As cells, human adipose tissue-derived mesenchymal stem cells (Lonza, Walkersville, Maryland, U.S.A.) were used. The donor was a 22-year-old man and the prepared cells were expressed CD13, CD29, CD44, CD73, CD90, CD105, SD166 ≧ 90%, CD14, CD31, CD45 ≦ 5%

The human adipose tissue-derived mesenchymal stem cells were seeded in each well of each cell culture dish at 8 × 10³ cells/well, and then, the cells were cultured for 1 day in Mesenchymal Stem Cell Growth Medium 2 (PromoCell, Bedford, Massachusetts, U.S.A.) under humidified condition at 37°C in the presence of 5% CO₂. After completion of the culture, the culture medium was exchanged with Mesenchymal Stem Cell Growth Medium 2 containing 10% WST-1 (Premix WST-1 Cell Proliferation Assay System, Takara Bio Inc., Shiga, Japan), the cells were incubated for 4 hours under humidified condition at normal pressure (37°C, 5% CO₂), and then, the absorbance (450 nm, comparison 600 nm) was measured by a microplate reader (Corona Multi-grating Microplate Reader SH-9000, manufactured by Hitachi High-Tech Science Corporation) and regarded as cell adhesion activity. The results are shown in Table 2.

<Cell Proliferation Activity Assay >
Using the cell culture dishes (1) to (12) obtained in Examples 1 to 12 and the comparative cell culture dishes (1) to (4) obtained in Comparative Examples 1 to 4, cells were cultured and proliferated according to the following process, and cell proliferation activity (cell proliferation) was evaluated.

The same human adipose tissue-derived mesenchymal stem cells as those used in the <Cell Adhesion Activity Assay> were seeded in each well of each cell culture dish at 4 × 10³ cells/well, and then, the cells were cultured for 2 days in Mesenchymal Stem Cell Growth Medium 2 (PromoCell, Bedford, Massachusetts, U.S.A.) under humidified condition at 37°C in the presence of 5% CO₂. After the completion of culture, the culture medium was exchanged with Mesenchymal Stem Cell Growth Medium 2 containing 10% WST-1 (Premix WST-1 Cell Proliferation Assay System, Takara Bio Inc., Shiga, Japan) and then cultured for 4 hours under humidified condition at normal pressure (37°C, 5% CO₂), and then absorbance (450 nm, comparison 600 nm) of culture supernatant was measured by a microplate reader and regarded as cell proliferation activity. The results are shown in Table 2.

As shown in Table 2, it is noted that in the cell culture dishes (1) to (12) of Examples, the cell adhesion activity and the cell proliferation activity are higher than those in the comparative cell culture dishes (1) to (4). In addition, by comparison between Example 1 and Examples 2 and 3, between Example 4 and Examples 5 and 6, between Example 7 and Examples 8 and 9, or between Example 10 and Examples 11 and 12, it is noted that, particularly in the cell culture dish having the coating film containing a polymer having a weight average molecular weight of 400,000 or more, the cell adhesion activity is excellent.
The entire disclosure of Japanese Patent Application No. 2020-105423, filed on June 18, 2020, is incorporated herein by reference in its entirety.

1 Bioreactor
2 Cell Culture Substrate
3 Cell Culture Chamber
4, 8 Inlet Port
6, 10 Outlet Port

Claims

A cell culture substrate comprising a coating layer containing a polymer having a structural unit (1) derived from furfuryl (meth)acrylate represented by the following Formula (1) and having a weight average molecular weight of 200,000 or more, on at least one surface of a polymer substrate having surface free energy of less than 33 mJ/m²:

wherein R¹ represents a hydrogen atom or a methyl group; and R² represents a group represented by the following Formula (1-1) or (1-2):

wherein R³represents an alkylene group having 1 to 3 carbon atoms.
[Claim 2] The cell culture substrate according to claim 1, wherein theweight average molecular weight of the polymer is more than 300,000 and 800,000 or less.
[Claim 3] The cell culture substrate according to claim 1 or 2, wherein the polymer substrate contains at least one selected from the group consisting of polypropylene, polytetrafluoroethylene, and polycarbonate.
[Claim 4] A bioreactor comprising the cell culture substrate set forth in any one of claims 1 to 3.
[claim 5] A method for culturing a stem cell, comprising culturing a stem cell using the bioreactor set forth in claim 4.