RELATED APPLICATIONS

This application is a national stage application filed under 35 USC § 371 of PCT/JP2015/057917, filed Mar. 17, 2015, which claims the benefit of Japanese Patent Application No. 2014-088632, filed Apr. 22, 2014, Japanese Patent Application No. 2014-088634, filed Apr. 22, 2014, Japanese Patent Application No. 2014-088635, filed Apr. 22, 2014, and Japanese Patent Application No. 2014-181730, filed Sep. 5, 2014, all of which are incorporated herein, in entirety, by reference.

TECHNICAL FIELD

The present invention relates to a cell culture substrate, a cell culture vessel comprising such substrate, a method of cell culture using such substrate, and a method of formation of three-dimensional tissue from a cell using such substrate.

BACKGROUND ART

Cells constituting organs such as the liver, the pancreas, the skin, and the blood vessels form three-dimensional networks and function in vivo.

In the field of regenerative medicine aimed at restoration of functions of such organs, accordingly, it is necessary that cells constituting such organs be cultured in a manner such that cells can form a three-dimensional network (i.e., three-dimensional culture). The minimal tissue formed by the three-dimensional network of cells is a spheroid. When cells are to be cultured on the surface of a common resin-made cell culture substrate, however, cells spread and grow in planar direction. That is, a three-dimensional network is not formed.

A variety of substrates for three-dimensional culture have heretofore been developed.

For example, Patent Document 1 discloses a cell culture sheet comprising a substrate made of a thermoplastic organic polymer with columnar fine projections that extend from the substrate, which is used for culture by allowing cells to adhere to the columnar fine projections. Also, Patent Document 2 discloses a cell culture construct having a concave-convex structure and serving as a cell-adhering surface, which is composed of a plurality of unit structures each formed in a polygonal shape in a planar direction and having a minimum internal diameter of less than or equal to 3 μm.

Non-Patent Document 1 is focused on excellent biocompatibility of a fluorine-containing polyimide, and it discloses the following. That is, cell culture is conducted on the surface of the 6FDA-6FAP membrane, which is a type of fluorine-containing polyimide, cells two-dimensionally grow on the 6FDA-6FAP membrane with a flat surface, and a spheroid is not formed. When fine irregularities are provided on the surface via rubbing, however, a spheroid is formed. The term “6FDA-6FAP” used therein refers to a polyimide formed via polymerization of an acid dianhydride (i.e., 2,2′-bis(3,4-dicarboxyphenyl)hexafluoropropane dianhydride (6FDA)) and a diamine compound (i.e., 2,2′-bis(4-aminophenyl)hexafluoropropane (6FAP)). The authors of Non-Patent Document 1 disclose in Patent Document 3 the technique described below. That is, vascular endothelial cells are two-dimensionally cultured on a flat 6FDA-6FAP membrane that has not been subjected to rubbing, the cultured cells are transferred onto a gel to form vascular tissue, liver cancer cells are three-dimensionally cultured on a 6FDA-6FAP membrane with an irregular surface that has been subjected to rubbing to form a spheroid, and the resulting vascular tissue is then combined with the resulting spheroid.

Patent Documents 4 to 6 each disclose a fluorine-containing polyimide used for forming a structure with an irregular configuration and a film and a membrane obtained from a resin composition comprising a fluorine-containing polyimide.

Patent Document 7 discloses a technique of preparing a cell culture vessel comprising a gas permeable plastic material. Patent Document 7 is intended to provide a cell culture vessel that does not require an oxygen supply apparatus.

PRIOR ART DOCUMENTS

Patent Documents

Patent Document 1: JP Patent No. 4,897,192Patent Document 2: JP Patent No. 4,159,103Patent Document 3: JP 2009-213,716 APatent Document 4: JP 2014-83,783 APatent Document 5: JP 2014-210,404 APatent Document 6: JP 2015-17,232 APatent Document 7: JP Patent No. 3,761,676

Non-Patent Documents

Non-Patent Document 1: N. Matsumoto et al., Polymers for Advanced Technologies, 19, 1002, 2008

SUMMARY OF THE INVENTION

Problems to be Solved by the Invention

Cell culture substrates for three-dimensional culture disclosed in Patent Documents 1 and 2 have fine irregularities on their surfaces and thus are capable of three-dimensional culture. Such substrates are problematic in terms of process cost, because it was not easy to provide fine irregularities. When cell culture is to be performed on a substrate surface with fine irregularities, air bubbles are likely to remain on the substrate surface when a liquid medium is applied to the substrate surface. Thus, it is necessary to remove bubbles after a liquid medium has been applied to the substrate surface.

While the cell culture substrates comprising a 6FDA-6FAP membrane (i.e., a fluorine-containing polyimide) subjected to rubbing disclosed in Non-Patent Document 1 and Patent Document 3 can be prepared more easily than the cell culture substrates disclosed in Patent Documents 1 and 2, the cell culture substrates disclosed in Non-Patent Document 1 and Patent Document 3 still suffer from the same problems as those of Patent Documents 1 and 2. Accordingly, there is still scope for improvement.

Accordingly, the present invention is intended to provide a cell culture substrate comprising on its surface a polymer that enables three-dimensional tissue culture.

It should be noted that the vessel disclosed in Patent Document 7 is not aimed at the formation of three-dimensional tissue, and whether or not three-dimensional tissue formation can be achieved with the use of the vessel disclosed in Patent Document 7 is not examined.

Accordingly, the present invention is also intended to provide a cell culture substrate that enables three-dimensional tissue culture, a cell culture vessel comprising such substrate, and a method of cell culture using such substrate.

Means for Solving the Problems

The present inventors surprisingly found that cells could form three-dimensional tissue, such as a spheroid or three-dimensional cell aggregate, on a substrate comprising on its surface a resin composition containing a fluorine-containing polymer having one or more fluorine atoms in a repeating unit and exhibiting oxygen gas permeability of 219 cm3(STP)/(m2·24 h·atm) or higher.

Specifically, the cell culture substrate according to the present invention has a surface at least a part of which is composed of a resin composition comprising a fluorine-containing polymer having one or more fluorine atoms in a repeating unit, and the oxygen gas permeability of the cell culture substrate is 219 cm3(STP)/(m2·24 h·atm) or higher.

According to a preferable embodiment of the cell culture substrate, a fluorine-containing polymer comprises at least one type of fluorine-containing polyimide selected from the group consisting of:

(a) a fluorine-containing polyimide having one or more fluorine atoms in a repeating unit and having one or more ether bonds and thioether bonds in total in a polymerization repeating unit constituting the polyimide;

(b) a fluorine-containing polyimide resulting from thermal imidization of polyamide acid and having one or more fluorine atoms in a repeating unit; and

(c) a fluorine-containing polyimide comprising in its main chain a repeating unit represented by Formula (3):


wherein

X0represents a tetravalent organic group and Y0represents a divalent organic group;

X0and Y0contain one or more fluorine atoms in total; and

Y0represents a diamine compound comprising a biphenyl group with each of two benzene rings being substituted with an amino group and the amino group being substituted with a single bond to a nitrogen atom.

According to the embodiment described above, it is preferable that the fluorine content in the resin composition is 1% to 60% by mass, and the degree of imidization is 20% or higher.

According to another preferable embodiment of the cell culture substrate, the fluorine-containing polymer comprises a polymer having a fluorine-containing aromatic ring and having an ether bond in its main chain.

According to the embodiment described above, the fluorine content in the resin composition is preferably 1% to 60% by mass.

Regarding the cell culture substrate according to the present invention, the oxygen gas permeability coefficient of the resin composition is preferably 0.10×10−10cm3(STP)·cm/(cm2·s·cmHg) or higher.

The present invention also provides a cell culture vessel at least a part of which is composed of the aforementioned cell culture substrate.

Further, the present invention provides a cell culture vessel comprising, in at least in part, a substrate that is provided in a manner such that one surface of the substrate constitutes the bottom of a container portion for containing a cell and medium and the other surface is exposed to the outside of the vessel, wherein the substrate is the cell culture substrate described above and at least a part of the one surface is composed of the resin composition.

The present invention also relates to a method of cell culture comprising a step of culturing cells on the surface of a cell culture substrate composed of the resin composition.

The present invention further relates to a method of cell culture comprising a step of cell culture conducted with the use of a substrate with one surface at least a part of which is composed of a resin composition comprising a fluorine-containing polymer, while cells and a medium are in contact with the one surface of the substrate and oxygen-containing gas is in contact with the other surface of the substrate, wherein the fluorine-containing polymer has one or more fluorine atoms in a repeating unit and the oxygen gas permeability of the substrate is 219 cm3(STP)/(m2·24 h·atm) or higher.

According to the method of the present invention, the oxygen gas permeability coefficient of the resin composition is preferably 0.10×10−10cm3(STP)·cm/(cm2·s·cmHg) or higher.

The step of cell culture according to the method of the present invention may comprise a step of three-dimensional cell culture. In the step of three-dimensional cell culture, preferably, the cells are cultured to form a spheroid or three-dimensional cell aggregate.

Hereafter, other embodiments of the present invention are described. It should be noted that the technical idea constituted of any combination of a plurality of aspects or embodiments disclosed herein is within the scope of the present invention.

(1) First Aspect

The present inventors surprisingly found that cells could form three-dimensional tissue, such as a spheroid or three-dimensional cell aggregate, on a substrate comprising on its surface a fluorine-containing polyimide having one or more ether bonds and thioether bonds in total in a polymerization repeating unit (e.g., a polymerization repeating unit derived from acid dianhydride and diamine).

The cell culture substrate according to the first aspect of the present invention has a surface at least a part of which is composed of a polyimide-containing resin composition, wherein the polyimide is a fluorine-containing polyimide having one or more fluorine atoms in a repeating unit and the sum of the ether bonds and the thioether bonds in a polymerization repeating unit constituting the polyimide is at least 1.

In this aspect of the cell culture substrate according to the present invention, however, the polyimide is not at least one type of polyimide selected from the group consisting of:

a polyimide comprising in its main chain the repeating unit as shown in Formula (3), wherein X0represents a 4,4′-(hexafluoroisopropylidene)diphthalic anhydride residue and Y0represents a 2,2-bis(4-(4-aminophenoxyl)phenyl)hexafluoropropane residue;

a polyimide comprising in its main chain the repeating unit as shown in Formula (3), wherein X0represents a 4,4′-(hexafluoroisopropylidene)diphthalic anhydride residue and Y0represents a bis[4-(4-aminophenoxy)phenyl]sulfone residue; and

a polyimide comprising in its main chain the repeating unit as shown in Formula (3), wherein X0represents a 4,4′-(hexafluoroisopropylidene)diphthalic anhydride residue and Y0represents a bis[4-(3-aminophenoxy)phenyl]sulfone residue.



The cell culture substrate according to the present invention may have a surface at least a part of which is composed of a polyimide-containing resin composition, wherein the polyimide is obtained through the reaction of at least one acid dianhydride with at least one diamine, at least either the acid dianhydride or diamine comprises a fluorine atom in molecules thereof, at least either the acid dianhydride or diamine comprises an ether bond and/or a thioether bond in molecules thereof, and the sum of the ether bonds and the thioether bonds in a polymerization repeating unit derived from the acid dianhydride and the diamine constituting the polyimide is at least 1.

In this aspect of the cell culture substrate according to the present invention, however, the polyimide is not a polyimide obtained through the reaction between 4,4′-(hexafluoroisopropylidene)diphthalic anhydride and 2,2-bis(4-(4-aminophenoxyl)phenyl)hexafluoropropane, a polyimide obtained through the reaction between 4,4′-(hexafluoroisopropylidene)diphthalic anhydride and bis[4-(4-aminophenoxy)phenyl]sulfone, or a polyimide obtained through the reaction between 4,4′-(hexafluoroisopropylidene)diphthalic anhydride and bis[4-(3-aminophenoxy)phenyl]sulfone.

A cell culture substrate provided with the features described above has a surface with adequate degrees of flexibility and hydrophobic properties. Thus, cells can form three-dimensional tissue on such surface. In addition, the substrate according to the present invention does not need to have a steric structure on the surface that serves as a scaffold for cells, and it is thus easy to prepare such substrate.

According to a preferable embodiment of the cell culture substrate, the fluorine content in the resin composition is 1% to 60% by mass, and the degree of imidization is 20% or higher.

This embodiment is preferable since it allows three-dimensional tissue to be formed easily on the substrate surface.

The present inventors surprisingly found that cells could form three-dimensional tissue, such as a spheroid or three-dimensional cell aggregate, when culture was conducted with the use of a substrate comprising the polyimide on its surface while providing the substrate at the bottom of a single-well or multi-well plate or a petri dish and, preferably, when cell culture was conducted while bringing cells and a medium into contact with the surface of the substrate comprising the polyimide and bringing oxygen-containing gas such as air into contact with the other surface of the substrate, in addition to with the upper surface of the medium. This has led to the completion of the present invention.

Accordingly, the present invention relates to a cell culture vessel at least a part of which is composed of the aforementioned cell culture substrate.

The cell culture vessel according to the present invention has a surface provided with adequate degrees of flexibility and hydrophobic properties. With the use thereof, accordingly, cell growth and formation of three-dimensional tissue, such as a spheroid or three-dimensional cell aggregate, are likely to proceed. When cell culture is conducted while bringing cells and a medium into contact with the polyimide-containing surface of the substrate and bringing oxygen-containing gas such as air into contact with the other surface of the substrate, in addition to with the upper surface of the medium, cell growth and formation of three-dimensional tissue, such as a spheroid or three-dimensional cell aggregate, are likely to proceed.

The present invention also relates to a method of cell culture comprising a step of culturing cells on the surface of a cell culture substrate.

The present invention further relates to a method of cell culture comprising a step of three dimensionally culturing cells on the surface of the substrate at least a part of which is composed of a polyimide-containing resin composition, wherein the polyimide is a fluorine-containing polyimide having one or more fluorine atoms in a repeating unit, and the sum of the ether bonds and the thioether bonds in a polymerization repeating unit constituting the polyimide is at least 1.

The present invention also relates to a method of cell culture comprising a step of three dimensionally culturing cells on the surface of the substrate at least a part of which is composed of a polyimide-containing resin composition, wherein the polyimide is obtained through the reaction of at least one acid dianhydride with at least one diamine, at least either the acid dianhydride or diamine comprises a fluorine atom in molecules thereof, at least either the acid dianhydride or diamine comprises an ether bond and/or a thioether bond in molecules thereof, and the sum of the ether bonds and the thioether bonds in a polymerization repeating unit derived from the acid dianhydride and the diamine constituting the polyimide is at least 1.

According to the method of the present invention, cell culture and formation of three-dimensional tissue, such as a spheroid or three-dimensional cell aggregate, can be carried out via simple procedures.

(2) Second Aspect

Examples of methods for obtaining a polyimide via imidization of polyamide acid include a method in which polyamide acid is imidized in the presence of an imidization catalyst (i.e., chemical imidization) and a method in which polyamide acid is heated for imidization (i.e., thermal imidization). Polyimides used in the experiments disclosed in Non-Patent Document 1 and Patent Document 3 were obtained via chemical imidization involving the use of a tertiary amine compound as an imidization catalyst.

The present inventors found that, surprisingly, three-dimensional culture could be carried out on a flat surface of the 6FDA-6FAP membrane produced via thermal imidization while three-dimensional culture could not be carried out on a flat surface of the 6FDA-6FAP membrane produced via chemical imidization disclosed in Non-Patent Document 1 and Patent Document 3. In addition, they found that it would be difficult to carry out three-dimensional culture on a flat surface when a tertiary amine compound used as an imidization catalyst remained on the 6FDA-6FAP membrane.

The cell culture substrate according to the second aspect of the present invention comprises a surface at least a part of which is composed of a polyimide-containing resin composition, wherein the polyimide is a fluorine-containing polyimide resulting from thermal imidization of polyamide acid and having one or more fluorine atoms in a repeating unit.

The cell culture substrate of the present invention may comprise a surface at least a part of which is composed of a polyimide-containing resin composition, wherein the polyimide is obtained via thermal imidization of polyamide acid resulting from polymerization of at least one acid dianhydride and at least one diamine, and at least either the acid dianhydride or diamine comprises a fluorine atom in molecules thereof.

Regarding a cell culture substrate provided with the features described above, fluorinated polyimide is prepared via thermal imidization, which does not require the use of an imidization catalyst. Thus, a substrate surface can be free from an imidization catalyst, which may disturb three-dimensional culture, and cells can form three-dimensional tissue on such surface. In addition, the substrate according to the present invention does not need to have a steric structure on such surface that serves as a scaffold for cells, and it is thus easy to prepare such substrate.

According to a preferable embodiment of the cell culture substrate, the fluorine content in the resin composition is 1% to 60% by mass, and the degree of imidization is 20% or higher.

This embodiment is preferable since it allows three-dimensional tissue to be formed easily on the substrate surface.

According to a preferable embodiment of the cell culture substrate, the polyamide acid is subjected to thermal imidization in the absence of a tertiary amine compound.

According to this embodiment, a tertiary amine compound is not present on the substrate surface, and cells can thus form three-dimensional tissue on such surface.

In addition, the cell culture substrate of the present invention may have a surface at least a part of which is composed of a polyimide-containing resin composition, wherein the polyimide is a fluorine-containing polyimide resulting from thermal imidization of polyamide acid and having one or more fluorine atoms in a repeating unit, and the amount of the tertiary amine compound in the resin composition is 0.030% by mass or less relative to the total amount of the polyimide and the remaining polyamide acid in the resin composition.

The cell culture substrate according to another embodiment of the present invention may have a surface at least a part of which is composed of a polyimide-containing resin composition, wherein the polyimide is obtained through the reaction of at least one acid dianhydride with at least one diamine, the amount of the tertiary amine compound in the resin composition is 0.030% by mass or less relative to the total amount of the polyimide and the remaining polyamide acid in the resin composition, and at least either the acid dianhydride or diamine comprises a fluorine atom in molecules thereof.

According to the present invention, the amount of the tertiary amine compound remaining in the resin composition that would disturb three-dimensional culture is sufficiently low. Thus, three-dimensional culture can be carried out.

According to a preferable embodiment of the cell culture substrate, the fluorine content in the resin composition is 1% to 60% by mass, and the degree of imidization is 20% or higher.

This embodiment is preferable since it allows three-dimensional tissue to be formed easily on the substrate surface.

The present inventors surprisingly found that cells could form three-dimensional tissue, such as a spheroid or three-dimensional cell aggregate, when culture was conducted with the use of a substrate comprising the polyimide on its surface while providing the substrate at the bottom of a single-well or multi-well plate or a petri dish and, preferably, when cell culture was conducted while bringing cells and a medium into contact with the surface of the substrate comprising the polyimide and bringing oxygen-containing gas such as air into contact with the other surface of the substrate, in addition to with the upper surface of the medium. This has led to the completion of the present invention.

Accordingly, the present invention relates to a cell culture vessel at least a part of which is composed of the aforementioned cell culture substrate.

The cell culture vessel according to the present invention has a surface provided with adequate degrees of flexibility and hydrophobic properties. With the use thereof, accordingly, cell growth and formation of three-dimensional tissue, such as a spheroid or three-dimensional cell aggregate, are likely to proceed. When cell culture is conducted while bringing cells and a medium into contact with the polyimide-containing surface of the substrate and bringing oxygen-containing gas such as air into contact with the other surface of the substrate, in addition to with the upper surface of the medium, cell growth and formation of three-dimensional tissue, such as a spheroid or three-dimensional cell aggregate, are likely to proceed.

The present invention also relates to a method of cell culture comprising culturing cells on the surface of a cell culture substrate.

The present invention further relates to a method of cell culture comprising a step of three dimensionally culturing cells on the surface of a cell culture substrate.

According to the method of the present invention, cell culture and formation of three-dimensional tissue, such as a spheroid or three-dimensional cell aggregate, can be carried out via simple procedures.

The present invention also relates to a method for producing a cell culture substrate comprising a film made of a polyimide-containing resin composition on at least a part of its surface, which comprises:

a step of forming a membrane of a solution of polyamide acid containing one or more fluorine atoms in molecules thereof dissolved in a solvent; and

a step of heating the membrane to imidize polyamide acid in the membrane, thereby forming the film.

The present invention further relates to a method for producing a cell culture substrate comprising a film made of a polyimide-containing resin composition on at least a part of its surface, which comprises:

a step of forming a membrane of a solution of polyamide acid dissolved in a solvent, wherein the polyamide acid is obtained via polymerization of at least one acid dianhydride with at least one diamine, and wherein at least either the acid dianhydride or diamine comprises a fluorine atom in molecules thereof; and

a step of heating the membrane to imidize polyamide acid in the membrane, thereby forming the film.

According to this method, a cell culture substrate having a surface that is free from an imidization catalyst, which may disturb three-dimensional culture, can be produced.

According to a preferable embodiment of the method described above, the solution does not contain a tertiary amine compound.

According to this embodiment, a cell culture substrate having a surface that is free from a tertiary amine compound, which may disturb three-dimensional culture, can be produced.

(3) Third Aspect

The present inventors surprisingly found that cells could form three-dimensional tissue, such as a spheroid or three-dimensional cell aggregate, on a substrate comprising a polyimide, which is obtained through the reaction between acid dianhydride and diamine, which is aromatic diamine of a specific structure, on its surface.

According to the third aspect of the present invention, the cell culture substrate has a surface at least a part of which is composed of a polyimide-containing resin composition, wherein the polyimide is a fluorine-containing polyimide comprising in its main chain a repeating unit represented by Formula (3):


wherein

X0represents a tetravalent organic group and Y0represents a divalent organic group;

X0and Y0contain one or more fluorine atoms in total; and

Y0represents a diamine compound comprising a biphenyl group with each of two benzene rings being substituted with an amino group and the amino group being substituted with a single bond to a nitrogen atom.

Also, the cell culture substrate of the present invention has a surface at least a part of which is composed of a polyimide-containing resin composition, wherein the polyimide is obtained through the reaction of at least one acid dianhydride with at least one diamine. A diamine compound comprising a biphenyl group with each of two benzene rings being substituted with an amino group is within the scope of the diamine, and at least either the acid dianhydride or diamine comprises a fluorine atom in molecules thereof.

Cells can form three-dimensional tissue on the surface of a cell culture substrate provided with the features described above. In addition, the substrate according to the present invention does not need to have a steric structure on the surface that serves as a scaffold for cells, and it is thus easy to prepare such substrate.

According to a preferable embodiment of the cell culture substrate, the fluorine content in the resin composition is 1% to 60% by mass, and the degree of imidization is 20% or higher.

This embodiment is preferable since it allows three-dimensional tissue to be formed easily on the substrate surface.

The present inventors surprisingly found that cells could form three-dimensional tissue, such as a spheroid or three-dimensional cell aggregate, when culture was conducted with the use of a substrate comprising the polyimide on its surface while providing the substrate at the bottom of a single-well or multi-well plate or a petri dish and, preferably, when cell culture was conducted while bringing cells and a medium into contact with the surface of the substrate comprising the polyimide and bringing oxygen-containing gas such as air into contact with the other surface of the substrate, in addition to with the upper surface of the medium. This has led to the completion of the present invention.

Accordingly, the present invention relates to a cell culture vessel at least a part of which is composed of the aforementioned cell culture substrate.

The cell culture vessel according to the present invention has a surface provided with adequate degrees of flexibility and hydrophobic properties. With the use thereof, accordingly, cell growth and formation of three-dimensional tissue, such as a spheroid or three-dimensional cell aggregate, are likely to proceed. When cell culture is conducted while bringing cells and a medium into contact with the polyimide-containing surface of the substrate and bringing oxygen-containing gas such as air into contact with the other surface of the substrate, in addition to with the upper surface of the medium, cell growth and formation of three-dimensional tissue, such as a spheroid or three-dimensional cell aggregate, are likely to proceed.

The present invention also relates to a method of cell culture comprising a step of culturing cells on the surface of a cell culture substrate.

The present invention further relates to a method of cell culture comprising a step of three dimensionally culturing cells on the surface of a cell culture substrate.

According to the method of the present invention, cell culture and formation of three-dimensional tissue, such as a spheroid or three-dimensional cell aggregate, can be carried out via simple procedures.

(4) Fourth Aspect

The present inventors surprisingly found that cells could form three-dimensional tissue, such as a spheroid or three-dimensional cell aggregate, when culture was conducted with the use of a substrate comprising a fluorine-containing polyimide on its surface and comprising a resin composition with high oxygen gas permeability while bringing cells and a medium into contact with the surface of the substrate comprising the polyimide and bringing oxygen-containing gas such as air into contact with the other surface of the substrate, in addition to with the upper surface of the medium. This has led to the completion of the present invention.

The fourth aspect of the present invention relates to a cell culture vessel comprising, in at least in part, a substrate that is provided in a manner such that one surface of the substrate constitutes the bottom of a container portion for containing a cell and medium and the other surface is exposed to the outside of the vessel, wherein at least a part of the one surface of the substrate is composed of a polyimide-containing resin composition, the polyimide is a fluorine-containing polyimide having one or more fluorine atoms in a repeating unit, and the oxygen gas permeability of the substrate is 219 cm3(STP)/(m2·24 h·atm) or higher.

With the use of the cell culture vessel according to the present invention, oxygen is easily supplied to the cells through the bottom surface of the substrate from the outside of the vessel, in addition to the usual oxygen supply through the upper surface of the medium. Thus, cell growth and formation of three-dimensional tissue, such as a spheroid or three-dimensional cell aggregate, are likely to proceed.

In the cell culture vessel of the present invention, the oxygen gas permeability coefficient of the resin composition is more preferably 0.10×10−10cm3(STP)·cm/(cm2·s·cmHg) or higher.

In the cell culture vessel according to this embodiment of the present invention, a larger quantity of oxygen is easily supplied to cells, and cell growth and formation of three-dimensional tissue, such as a spheroid or three-dimensional cell aggregate, are likely to proceed.

In the cell culture vessel according to the present invention, more preferably, the fluorine content in the resin composition is 1% to 60% by mass, and the degree of imidization is 20% or higher.

According to this embodiment, cells can form three-dimensional tissue more easily on the substrate surface of the cell culture vessel according to the present invention.

The present invention also relates to a method of cell culture comprising a step of cell culture conducted with the use of a substrate having one surface at least a part of which is composed of a polyimide-containing resin composition, while keeping cells and a medium in contact with the one surface of the substrate and oxygen-containing gas in contact with the other surface of the substrate, wherein the polyimide is a fluorine-containing polyimide having one or more fluorine atoms in a repeating unit, and the oxygen gas permeability of the substrate is 219 cm3(STP)/(m2·24 h·atm) or higher.

According to the method of cell culture of the present invention, oxygen is easily supplied to the cells through the bottom surface of the substrate, in addition to the usual oxygen supply through the upper surface of the medium. Thus, cell growth is likely to proceed.

According to the method of cell culture of the present invention, more preferably, the oxygen gas permeability coefficient of the resin composition is 0.10×10−10cm3(STP)·cm/(cm2·s·cmHg) or higher.

In the method of cell culture according to this embodiment of the present invention, a larger quantity of oxygen is easily supplied to cells, and cell growth is likely to proceed.

According to the method of cell culture of the present invention, more preferably, the fluorine content in the resin composition is 1% to 60% by mass, and the degree of imidization is 20% or higher.

In the method of cell culture according to this embodiment of the present invention, cell growth is more likely to proceed.

In the method of cell culture according to the present invention, more preferably, the step described above is a step of three-dimensional cell culture. In the step of three-dimensional cell culture, more preferably, the cells are cultured to form a spheroid or three-dimensional cell aggregate.

According to the method of cell culture according to a more preferable embodiment of the present invention, cell culture and formation of three-dimensional tissue, such as a spheroid or three-dimensional cell aggregate, can be carried out via simple procedures.

The present invention also relates to a cell culture substrate having a surface at least a part of which is composed of a polyimide-containing resin composition, wherein the polyimide is a fluorine-containing polyimide having one or more fluorine atoms in a repeating unit, and the oxygen gas permeability of the substrate is 219 cm3(STP)/(m2·24 h·atm) or higher.

With the use of the cell culture substrate of the present invention, oxygen is easily supplied to the cells through the substrate, in addition to the usual oxygen supply through the upper surface of the medium. Thus, cell growth is likely to proceed.

Regarding the cell culture substrate of the present invention, more preferably, the oxygen gas permeability coefficient of the resin composition is 0.10×10−10cm3(STP)·cm/(cm2·s·cmHg) or higher.

Regarding the cell culture substrate according to this embodiment of the present invention, a larger quantity of oxygen is easily supplied to cells, and cell growth is likely to proceed.

Regarding the cell culture substrate of the present invention, more preferably, the fluorine content in the resin composition is 1% to 60% by mass, and the degree of imidization is 20% or higher.

According to this embodiment, cells can form three-dimensional tissue more easily on the surface of the cell culture substrate according to the present invention.

The fluorine-containing polyimide used in the present invention is typically a polyimide obtained through the reaction of at least one acid dianhydride with at least one diamine, and at least either the acid dianhydride or diamine comprises a fluorine atom in molecules thereof.

(5) Fifth Aspect

The present inventors surprisingly found that cells could form three-dimensional tissue, such as a spheroid or three-dimensional cell aggregate, on a substrate comprising on its surface a fluorine-containing polymer having a fluorine-containing aromatic ring and an ether bond in its main chain.

Accordingly, the fifth aspect of the present invention relates to a cell culture substrate with at least a part of its surface being composed of a resin composition comprising a fluorine-containing polymer, such fluorine-containing polymer being a polymer having a fluorine-containing aromatic ring and comprising an ether bond in its main chain.

Cells can form three-dimensional tissue on the surface of the cell culture substrate provided with the features described above. In addition, the substrate according to the present invention does not need to have a steric structure on the surface that serves as a scaffold for cells, and it is thus easy to prepare such substrate.

According to a preferable embodiment of the cell culture substrate, the oxygen gas permeability is 219 cm3(STP)/(m2·24 h·atm) or higher.

According to a preferable embodiment of the cell culture substrate, the oxygen gas permeability coefficient of the resin composition is 0.10×10−10cm3(STP) cm/(cm2·s·cmHg) or higher.

According to the embodiment described above, a larger quantity of oxygen is easily supplied to cells, and cell growth and formation of three-dimensional tissue, such as a spheroid or three-dimensional cell aggregate, are likely to proceed.

According to a preferable embodiment of the cell culture substrate, the fluorine content in the resin composition is 1% to 60% by mass.

This embodiment is preferable since it allows three-dimensional tissue to be formed easily on the substrate surface.

According to a preferable embodiment of the cell culture substrate, the fluorine-containing polymer is fluorine-containing aryl ether ketone polymer comprising a repeating unit represented by Formula (II-1):


wherein R42has any of the structures represented by formulae shown below.



According to this embodiment, the cell culture substrate of the present invention can provide a transparent scaffold to which cells can adequately adhere and on which three-dimensional tissue can be formed. In addition, a larger quantity of oxygen is easily supplied to cells, and cell growth and three-dimensional tissue formation are likely to proceed.

The present inventors surprisingly found that cells could form three-dimensional tissue, such as a spheroid or three-dimensional cell aggregate, when cell culture was conducted with the use of a substrate comprising a fluorine-containing polymer on one surface of the substrate and a resin composition with high oxygen gas permeability while keeping cells and a medium in contact with the one surface of the substrate containing the fluorine-containing polymer and keeping oxygen-containing gas such as air in contact with the other surface of the substrate, in addition to with the upper surface of the medium. This has led to the completion of the present invention.

Accordingly, the present invention relates to a cell culture vessel comprising, in at least in part, a substrate that is provided in a manner such that one surface of the substrate constitutes the bottom of a container portion for containing a cell and medium and the other surface is exposed to the outside of the vessel, wherein the substrate is the cell culture substrate described above.

With the use of the cell culture vessel according to the present invention, oxygen is easily supplied to the cells through the bottom surface of the substrate from the outside of the vessel, in addition to the usual oxygen supply through the upper surface of the medium. Thus, cell growth and formation of three-dimensional tissue, such as a spheroid or three-dimensional cell aggregate, are likely to proceed.

The present invention also relates to a method of cell culture comprising a step of culturing cells on the surface of a cell culture substrate composed of the resin composition.

The present invention further relates to a method of cell culture comprising a step of culturing cells with the use of a substrate, at least a part of one of the surfaces being composed of a resin composition comprising a fluorine-containing polymer, while cells and a medium are in contact with one of the surfaces of the substrate and oxygen-containing gas is in contact with the other surface of the substrate, wherein the fluorine-containing polymer has a fluorine-containing aromatic ring and comprises an ether bond in its main chain.

According to the method of cell culture of the present invention, oxygen is easily supplied to the cells through the bottom surface of the substrate, in addition to the usual oxygen supply through the upper surface of the medium. Thus, cell growth is likely to proceed.

According to a preferable embodiment of the method described above, the step of cell culture is a step of three-dimensional cell culture.

According to a preferable embodiment of the method described above, the step of three-dimensional cell culture is a step of culturing the cells to form a spheroid or three-dimensional cell aggregate.

According to the method of the present invention, cell culture and formation of three-dimensional tissue, such as a spheroid, can be carried out via simple procedures.

The term “resin composition” used in the present invention may be used interchangeably with the term “resin.” In general, a fluorine-containing polymer is composed of numerous fluorine-containing polymer molecules of different polymerization degrees. Thus, resin containing a fluorine-containing polymer is referred to as a “resin composition.” In the present invention, the “resin composition” or “resin” may or may not contain other components in an amount that would not adversely affect the effects of the present invention. Components or common additives used for polymerization can be used as other components in an amount that would not adversely affect the effects of the present invention.

Effects of the Invention

The surface of the cell culture substrate of the present invention that serves as a scaffold for cells is composed of a particular resin composition. Thus, cells can be three-dimensionally cultured.

According to the present invention, in addition, three-dimensional tissue, such as a spheroid or three-dimensional cell aggregate, can be prepared from cells.

