JP2020105083A - Crosslinked fibrotic collagen gel for oral mucosal epithelial cell culture - Google Patents

Abstract

To provide cell culture substrates, whose component are collagen, having suppressed contraction during culturing oral mucosal epithelial cells including gas-liquid phase interface culture.SOLUTION: The cell culture substrate is a crosslinked fibrotic collagen gel that satisfies all the conditions (i) to (iii): (i) being composed of not-damaged (intact) crosslinked fibrotic collagen; (ii) having a region including a concave shape and/or a convex shape on at least a part of its surface; and (iii) being what uncrosslinked fibrotic collagen gel, after being chemically crosslinked by a carbodiimide crosslinking agent, is crosslinked by γ-ray irradiation in the presence of an aqueous solvent.SELECTED DRAWING: Figure 3

Description

The present invention relates to a crosslinked fibrotic collagen gel used as a cell culture substrate in the culture of oral mucosal epithelial cells including air-liquid interface culture.

Collagen accounts for 30% of proteins in the body and is an important protein having functions such as skeletal support and cell adhesion. For example, bone/cartilage, ligament/tendon, corneal stroma, skin, liver, muscle, dentin. Tissue such as periodontal tissue including is made of collagen fibers. Collagen fibers in a living body are aggregates in which collagen molecules having a triple helix structure are oriented substantially regularly.

Conventionally, using collagen obtained from biological tissue, cell culture substrate, scaffold material for regenerative medicine (for example, regeneration of cartilage, bone, spine, nucleus pulposus, ligament, corneal stroma, skin, blood vessel, nerve, liver tissue) Materials), transplant materials, wound dressing materials, bone filling materials, hemostatic materials, anti-adhesion materials, drug delivery carriers and the like, various technical developments have been carried out. In the present specification, "collagen" means a collagen molecule having a triple helix structure, and an aggregate or an aggregate of the collagen molecule. The concept of "collagen" in the present specification does not include heat-denatured collagen (gelatin) in which the triple helix structure is dissolved and collagen peptide.

As a method of solubilizing collagen contained in a biological tissue to obtain a solubilized collagen aqueous solution, a method of solubilizing with an enzyme, a method of extracting with a dilute acid, a method of solubilizing with an alkali, and the like are known. In the present specification, unless otherwise specified, the “solubilized collagen aqueous solution” refers to a collagen aqueous solution solubilized by any treatment method.

When a solubilized collagen aqueous solution is added with a fibrating agent such as a buffer solution so that the solubilized collagen aqueous solution has an appropriate ionic strength and pH, the collagen molecules associate and orient to form a structure similar to the collagen fibers in the living body. By taking it, a collagen gel having a certain shape can be obtained. This collagen gel is called "fibrotic collagen gel".

In Patent Document 1, porcine type I atelocollagen acidic aqueous solution is injected into a template in which a plurality of ice particles are arranged, and the ice particles are lyophilized to remove the ice particles, and then crosslinked with glutaraldehyde to obtain a surface. There is disclosed a technique for producing a porous collagen molded body in which a plurality of recesses are provided. Patent Document 2 discloses a technology relating to a crosslinked collagen fiber porous body which is composed of collagen fibers, has a porous structure capable of three-dimensional cell culture, and is crosslinked.

The porous collagen molded bodies described in Patent Documents 1 and 2 are considered to be porous bodies having pores (recesses) having a considerably large pore diameter for cells. When this porous collagen molded body is used as a cell culture substrate, cells tend to fall inside the pores of the molded body, making it difficult to stay on the surface of the molded body. Therefore, it is not preferable as a culture substrate for cell types such as oral epithelial cells that form tissue on the surface of the cell culture substrate.

In Patent Document 3, uncrosslinked fibrillated collagen gel, fibrillated collagen film or non-fibrillated collagen film is crosslinked by γ-ray irradiation, electron beam irradiation, UV irradiation or plasma irradiation in the presence of an aqueous solvent. A body, at least a part of the surface of which has a concave shape and/or a convex shape, and whose main constituents are intact fibrotic collagen or collagen molecules. A technique relating to a surface-treated collagen molded body is disclosed.

In Patent Document 4, uncrosslinked fibrillated collagen gel, fibrillated collagen film or non-fibrillated collagen film is crosslinked by γ-ray irradiation, electron beam irradiation, UV irradiation or plasma irradiation in the presence of an aqueous solvent. A body, at least a part of the surface of which has a concave shape and/or a convex shape, and whose main constituents are intact fibrotic collagen or collagen molecules. A technique relating to a cell culture method using a surface-treated collagen molded body is disclosed.

Patent No. 5822266 JP, 2005-213676, A JP, 2017-149814, A JP, 2017-147951, A

According to the knowledge of the present inventors, when the γ-ray cross-linked fibrotic collagen gel obtained in Production Example 2 of Patent Document 4 is used as a cell culture substrate, liquid phase culture of fibroblast cell line L929 is performed, Even at the end of the culture, almost no contraction of the cell culture substrate is observed. On the other hand, in Example 2 of Patent Document 4, when the γ-ray cross-linked fibrotic collagen gel obtained in Production Example 2 was used as a cell culture substrate for human oral mucosal epithelium-derived primary cultured cells. It is described that the cell culture substrate contracted with the lapse of culture days.

The contraction during culture makes it difficult to secure a stable number of cultured cells and a size of a cultured tissue at the time of transplantation. Further, it becomes difficult to obtain a cultured tissue having an expected concave structure and/or a convex structure at the end of the culture. Furthermore, when the γ-ray crosslinked fibrous collagen gel itself is transplanted as a scaffolding material for regenerative medicine to a site of oral mucosal defect, defects such as tightness (scarring contracture) due to contraction after transplantation occur, and QOL may be impaired. Under such circumstances, there has been a demand for a culture substrate for oral mucosal epithelial cells in which the contraction during cell culture is suppressed in the material containing collagen as a constituent element.

Here, in Example 2 of Patent Document 4, gas phase-liquid phase interface culture is performed after liquid phase culture of oral mucosal epithelial cells to induce differentiation formation of the stratum corneum. However, it is considered that the cause of the contraction of the γ-ray cross-linked fibrotic collagen gel that occurs during the culture of the oral mucosal epithelial cells is the gas-liquid interface culture, or the oral mucosal epithelial cells have it. It is not clear whether it is due to cell traction or other factors. Therefore, a cell culture substrate containing collagen as a constituent, in which contraction during oral mucosal epithelial cell culture is suppressed, has not yet been proposed.

An object of the present invention is to develop a cell culture substrate whose constituent element is collagen and whose contraction is suppressed during the culture of oral mucosal epithelial cells including gas phase-liquid phase interface culture.

As a result of intensive studies on the above problems, the present inventors have conducted a chemical cross-linking treatment using a carbodiimide-based cross-linking agent before the non-cross-linked fibrous collagen gel is cross-linked by γ-ray irradiation in the presence of an aqueous solvent. , A cell culture substrate that suppresses the contraction of the oral mucosal epithelial cells at the end of the vapor-liquid phase interface culture is obtained while taking advantage of the properties of fibrotic collagen gel with excellent cell adhesion and habitability. Based on such findings, the present invention has been completed.

The present invention is as follows.
[1] A crosslinked fibrotic collagen gel used as a cell culture substrate in the culture of oral mucosal epithelial cells including gas-liquid interface culture, which crosslinks all of the following conditions (i) to (iii): Fibrotic collagen gel.
(i) The crosslinked fibrotic collagen gel is composed of intact crosslinked fibrotic collagen.
(ii) The crosslinked fibrous collagen gel has a region including a concave shape and/or a convex shape on at least a part of its surface.
(iii) The cross-linked fibrillated collagen gel is obtained by chemically cross-linking the non-cross-linked fibrillated collagen gel with a carbodiimide-based cross-linking agent and then γ-irradiating it in the presence of an aqueous solvent.
[2] The method for producing a crosslinked fibrous collagen gel according to [1], which comprises the following steps.
(1) A transfer member having a concave shape and/or a convex shape on at least a part of its surface is prepared, and the collagen in the solubilized collagen aqueous solution is fibrillated in a state of being in contact with the transfer member, and the surface thereof is formed. A first step of obtaining a fibrillated collagen gel having a region including a concave shape and/or a convex shape in at least a part of the above.
(2) A second step in which the fibrillated collagen gel obtained in the first step is chemically crosslinked with a carbodiimide-based crosslinking agent.
(3) A third step in which the chemically crosslinked fibrotic collagen gel obtained in the second step is crosslinked by γ-ray irradiation in the presence of an aqueous solvent.
[3] A cell culture method in which oral mucosal epithelial cells are subjected to liquid phase culture using the crosslinked fibrous collagen gel according to [1] as a cell culture substrate, and then vapor-liquid interface culture.
[4] A medical material for oral mucosa regeneration, which comprises the crosslinked fibrous collagen gel according to [1].
[5] The medical material according to [4], which comprises oral mucosal epithelial cells and/or oral mucosal epithelial-like tissue adhered to the crosslinked fibrous collagen gel.
[6] A drug evaluation material comprising the crosslinked fibrotic collagen gel according to [1] and the oral mucosal epithelial cells and/or oral mucosal epithelial-like tissue adhered to the crosslinked fibrotic collagen gel.

In the culture of oral mucosal epithelial cells including gas phase-liquid phase interface culture, when the crosslinked fibrotic collagen gel of the present invention is used as a cell culture substrate, contraction of the cell culture substrate is suppressed, and oral mucosal epithelial tissue of a living body is suppressed. It is possible to form a tissue similar to that (hereinafter, also referred to as "oral mucosa epithelial tissue"). INDUSTRIAL APPLICABILITY The crosslinked fibrous collagen gel and the cultured tissue containing the same as a cell culture substrate according to the present invention can be useful as a medical material useful for treating patients with defective oral mucosal epithelium.

FIG. 1 is a schematic view ((a): a plan view, (b): a cross-sectional view taken along the line X-X of (a)) showing a transfer member provided with a region having a convex shape on the surface. 3 is a photograph of the appearance of the cell culture substrate after completion of culture in Examples and Comparative Examples. 3 is a histological image of cultured tissue subjected to HE staining in Examples and Comparative Examples.

Hereinafter, the present invention will be described in detail based on preferred embodiments, but the present invention is not limited to the following embodiments, and various modifications can be made within the scope of the claims. In the present invention, the notation ``numerical value 1 to numerical value 2'' relating to the numerical value range means a numerical value range including numerical values 1 and 2 at both ends, with numerical value 1 being the lower limit value and numerical value 2 being the upper limit value, It is synonymous with "1 or more and 2 or less".

[Crosslinked fibrous collagen gel]
INDUSTRIAL APPLICABILITY The crosslinked fibrillated collagen gel of the present invention is suitable for use as a cell culture substrate in the culture of oral mucosal epithelial cells including gas phase-liquid phase interface culture. This crosslinked fibrillated collagen gel satisfies all the following conditions (i) to (iii).
(i) The crosslinked fibrotic collagen gel is composed of intact crosslinked fibrotic collagen.
(ii) The crosslinked fibrous collagen gel has a region including a concave shape and/or a convex shape on at least a part of its surface.
(iii) The cross-linked fibrillated collagen gel is obtained by chemically cross-linking the non-cross-linked fibrillated collagen gel with a carbodiimide-based cross-linking agent and then γ-irradiating it in the presence of an aqueous solvent.

(Oral mucosal epithelial cells and culture method)
Oral mucosal epithelial cells are also called oral mucosa-derived epithelial cells, and the category thereof includes cell lines, iPS cells, primary cultured cells derived from human oral mucosal epithelium, and the like. Oral mucosal epithelial cells can be cultivated only by liquid phase culturing, but the culturing method using the crosslinked fibrous collagen gel according to the present invention as a cell culture substrate is an oral mucosa containing at least gas-liquid phase interface culturing. This is a method for culturing epithelial cells. As long as the effect of the present invention is not impaired, culture methods other than the gas-liquid phase interface culture, for example, liquid phase culture are also acceptable. An example of a preferred embodiment is a culture method in which oral mucosal epithelial cells are liquid-phase-cultured, and then gas-liquid phase interface culture is performed. That is, this culturing method is a method in which oral mucosal epithelial cells are fixed on a cell culture substrate by liquid phase culturing, and then the formation of the stratum corneum is induced by gas phase-liquid phase interface culturing.

(Condition (i))
The crosslinked fibrotic collagen gel according to the present invention is composed of intact crosslinked fibrotic collagen. As used herein, "intact crosslinked fibrotic collagen" means crosslinked fibrous collagen that has not been damaged by processing such as cutting. In other words, this condition (i) defines that the crosslinked fibrotic collagen gel has not been subjected to processing such as cutting that damages the crosslinked fibrous collagen.

(Condition (ii))
On the surface of the crosslinked fibrous collagen gel according to the present invention, one or more concave portions and/or convex portions are formed. Hereinafter, in the present specification, one or more concave portions on the surface of the crosslinked fibrous collagen gel are referred to as a concave shape, and one or more convex portions are referred to as a convex shape. In addition, the concave shape and/or the convex shape may be collectively referred to as a “pattern shape”. The surface of the crosslinked fibrous collagen gel means the outer surface. In addition, a region having a pattern shape on the surface of the crosslinked fibrous collagen gel may be referred to as a “pattern region”, and a region having no pattern shape may be referred to as a “non-pattern region”. The concave shape and the convex shape in the present invention are strictly the surface shapes of the non-porous crosslinked fibrous collagen gel of the present invention. Therefore, the depth of the concave portion and the height of the convex portion, which will be described later, are measured with the non-patterned region of the non-porous crosslinked fibrotic collagen gel of the present invention as a reference plane, and, for example, Patent Document 1 and The average pore size of the porous body described in Patent Document 2 is a technical matter that is essentially different.

The crosslinked fibrous collagen gel of the present invention may have a pattern shape on the entire surface thereof, or may have a part of the surface thereof having a pattern shape. For example, when the crosslinked fibrous collagen gel of the present invention has a flat film-like outer shape, it is preferable that at least one of the upper surface and the lower surface thereof has a pattern shape. Further, a plurality of pattern areas may be formed in different areas on one surface.

The number of pattern shapes in the crosslinked fibrous collagen gel of the present invention may be one or plural. From the viewpoint of use as a cell culture substrate, a plurality of cells is preferable. In the case of a plurality of patterns, the array of the plurality of pattern shapes may be a regular pattern or an irregular pattern. The arrangement is preferably a regular pattern.

When the pattern shape is a concave shape, the shape is not particularly limited as long as the shape has a depression. For example, as a plan view shape of the concave portion having a concave shape, a polygon such as a square, a rectangle, a triangle, a hexagon, and an octagon, a rounded polygon with rounded corners, a circle, an ellipse, and the like can be illustrated. The vertical cross-sectional shape of the concave recessed portion is a semicircle, a square, a rectangle, a triangle, a trapezoid, a square with a rounded bottom, a rectangle with a rounded bottom, a triangle with a rounded bottom, a bottom. Examples thereof include a trapezoid with rounded corners and a papillary shape. When the pattern shape is a convex shape, the shape is not particularly limited as long as it has a protrusion. As the plan view shape of the convex portion having the convex shape, the same shape as the concave shape can be exemplified. Moreover, as for the sectional view shape of the convex portion forming the convex shape, a shape obtained by inverting the concave shape and the vertical direction can be exemplified. In the crosslinked fibrillated collagen gel of the present invention, a concave shape and a convex shape may coexist on one surface, or two or more kinds of the respective shapes may coexist.

The size of the pattern, for example, is preferably in the range of 1000μm ² ~300000μm ² as a plan view area of each recess or each protrusion. Also, the depth of the recess (when the surface of the non-pattern area is the reference surface, the difference between this reference surface and the deepest part of the recess) or the height of the projection (when the surface of the non-pattern area is the reference surface) The difference between the reference surface and the highest part of the convex portion is preferably in the range of 50 to 500 μm.

From the viewpoint of obtaining oral mucosa epithelial-like tissue by culturing oral mucosal epithelial cells, one particularly preferable form of the pattern shape is a concave shape constituted by a plurality of concave portions.
Further, it is preferably a concave pattern shape satisfying at least one of the following requirements 1 to 6, more preferably 2 or more, and particularly preferably all of the requirements 1 to 6.
Requirement 1: A plurality of concave portions are regularly arranged.
Requirement 2: The shape of each recess in plan view is a circle, a square, or a rounded square.
Requirement 3: The cross-sectional shape of each recess is a square, a rectangle, a trapezoid, a square with a rounded bottom, a rectangle with a rounded bottom, and a trapezoid with a rounded bottom or a papillary.
Requirement 4: a plan view area of each recess is in the range of 1500μm ² ~250000μm ^2.
Requirement 5: The depth of each recess is in the range of 50 to 300 μm.
Requirement 6: The shortest distance between adjacent recesses in plan view (the shortest distance between the peripheral edges of the recesses) is in the range of 50 to 500 μm.

At a defined range of requirements 4, the lower limit of the plan view area of each recess is more preferably 2000 .mu.m ^2, more preferably from 5000 .mu.m ^2, still more preferably 7000μm ^2. The upper limit is more preferably 100000 ^2, more preferably from 70000Myuemu ^2, still more preferably 50000 ^2.

In the range defined in Requirement 5, the lower limit of the depth of each recess is more preferably 75 μm, and the upper limit thereof is more preferably 250 μm.

In the range defined in Requirement 6, the lower limit of the shortest distance between adjacent recesses in plan view (the shortest distance between the peripheral edges of the recesses) is more preferably 100 μm, and the upper limit thereof is more preferably 300 μm.

(Condition (iii))
The crosslinked fibrillated collagen gel according to the present invention is obtained by subjecting an uncrosslinked fibrillated collagen gel to a crosslinking treatment by a predetermined method. First, the reason for the provision of the cross-linking treatment in specifying the cross-linked fibrillated collagen gel of the present invention will be described. As a method for crosslinking collagen, a physical crosslinking method and a chemical crosslinking method are known. Typical examples of the physical crosslinking method are irradiation crosslinking (crosslinking by γ-ray irradiation, electron beam irradiation, UV irradiation, plasma irradiation, etc.) and thermal dehydration crosslinking, and a typical example of the chemical crosslinking method is a water-soluble chemical crosslinking agent. Alternatively, there is crosslinking by a chemical crosslinking agent having vaporizing ability. Hereinafter, regardless of the cross-linking method, the cross-linked fibrous collagen gel is also referred to as “cross-linked body”.

First, regarding the physical cross-linking method, it is extremely difficult to find a difference in appearance between the cross-linked product obtained by irradiation cross-linking and the cross-linked product obtained by thermal dehydration cross-linking, Further, it is extremely difficult to distinguish which cross-linking method has been cross-linked by analysis. Furthermore, it is extremely difficult to distinguish between a crosslinked product obtained by γ-ray irradiation and a crosslinked product obtained by other irradiation crosslinking.

Next, it is extremely difficult to find a difference in appearance between the crosslinked products obtained by irradiation crosslinking and the crosslinked products obtained by the chemical crosslinking method, even if the crosslinked products are compared. Of the chemical cross-linking methods, when a chemical cross-linking agent such as glutaraldehyde or a polyepoxy compound (ethylene glycol diglycidyl ether, glycerol polyglycidyl ether, etc.) is used, the chemical cross-linking agent binds to collagen. Since a cross-linking reaction occurs, if a chemical cross-linking agent can be detected, the two can be distinguished. However, when a chemical cross-linking agent such as carbodiimide cross-linking agent that can be removed from the collagen by washing after cross-linking is used, traces of the chemical cross-linking agent are hardly found even if the cross-linked product is analyzed. It is impossible.

Further, it is extremely difficult to distinguish an uncrosslinked fibrillated collagen gel (hereinafter also referred to as “uncrosslinked body”) from a crosslinked body. For example, it is extremely difficult to find a difference between an uncrosslinked body and a crosslinked body by analysis, especially in an irradiation crosslinked body, because there is only a difference in the number of crosslinking points. The uncrosslinked product generally has weaker strength than the crosslinked product and tends to have lower storage stability in water, but it is also extremely difficult to prove that the difference in the physical tendencies is due to the presence or absence of crosslinking treatment. Is.

Because of the above difficulty of distinction, the type of cross-linking treatment is defined as an item for specifying the invention of the cross-linked fibrous collagen gel of the present invention.

The uncrosslinked fibrotic collagen gel is a gel-shaped molded product made of fibrotic collagen that has not been crosslinked. The overall appearance shape of the uncrosslinked fibrotic collagen gel is not particularly limited, and examples thereof include various shapes such as a film shape, a cube shape, and a column shape. Preferably, this uncrosslinked fibrotic collagen gel has a region including a concave shape and/or a convex shape on at least a part of its surface.

The type of the carbodiimide-based cross-linking agent is not particularly limited as long as it has a function of cross-linking the uncross-linked fibrillated collagen gel. From the viewpoint of easy removal of unreacted substances after the crosslinking treatment, a water-soluble carbodiimide crosslinking agent is preferable. Specific examples of the water-soluble carbodiimide-based cross-linking agent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 1-ethyl-3-(3-trimethylaminopropyl)carbodiimide (ETC), 1-cyclohexyl -3-(2-morpholinoethyl)carbodiimide (CMC) and the salts thereof, and mixtures thereof can be mentioned.

The aqueous solvent is preferably, for example, water, a buffer solution, an aqueous solution of an alkali metal hydrogen carbonate, or the like, and may be a mixed solvent obtained by adding an organic solvent to these. As the buffer, phosphate buffer, Tris buffer, HEPES buffer, acetate buffer, carbonate buffer, citrate buffer, phosphate buffered saline (PBS), Dulbecco's phosphate buffered saline (D- PBS), Tris buffered saline, HEPES buffered saline and the like. Examples of the aqueous solution of alkali metal hydrogen carbonate include sodium hydrogen carbonate aqueous solution, potassium hydrogen carbonate aqueous solution and the like.

The collagen concentration contained in the crosslinked fibrous collagen gel according to the present invention is preferably in the range of 0.3 to 3 mass% from the viewpoint of obtaining a certain strength as a cell culture substrate.

The overall appearance shape of the crosslinked fibrous collagen gel of the present invention is preferably a circular membrane shape suitable for the size of the well, from the viewpoint of use as a cell culture substrate. When the crosslinked fibrous collagen gel is in the form of a film, its thickness is preferably in the range of 0.5 to 5 mm from the viewpoint of handleability, for example. When the cross-linked fibrillated collagen gel is in the form of a circular film, its size is preferably in the range of 1 to 20 mm in diameter in plan view from the viewpoint of use as a cell culture substrate.

(Evaluation of contractility)
When the cross-linked fibrillated collagen gel according to the present invention is used as a cell culture substrate for culturing oral mucosal epithelial cells including at least gas phase-liquid phase interface culture, the degree of shrinkage of the cell culture substrate after the completion of the culture test is ,small. Although the detailed mechanism is unknown, according to the fibrillated collagen gel of the present invention which is γ-ray crosslinked after the chemical treatment with the carbodiimide-based crosslinking agent, it is crosslinked only by γ-ray irradiation obtained in Production Example 2 of Patent Document 4. Compared with the fibrillated collagen gel, the contraction during the culture test is significantly suppressed.

In evaluating the contractility of the crosslinked fibrotic collagen gel of the present invention, for example, by using human oral mucosal epithelium-derived primary cultured cells, in the following cell culture test, the crosslinked fibrotic collagen gel of the present invention is used as a cell culture medium. When used as a material, when the area shrinkage ratio (%) of the cross-sectional fibrous collagen gel after the test in plan view to the plan view area before the test is 10% or less, it is evaluated that the shrinkage is suppressed. can do. The area shrinkage ratio is preferably 7% or less, more preferably 5% or less, more preferably 3% or less, and ideally 0%. Here, the area shrinkage ratio is calculated by the following formula.
Area shrinkage (%)=(AB)/A×100
(Here, A is a plan view area of the crosslinked fibrous collagen gel before the cell culture test, and B is a plan view area of the crosslinked fibrous collagen gel after the cell culture test.)
[Cell culture test]
Step 1: Mold the cross-linked fibrillated collagen gel into a circular shape in plan view (diameter 19 mm), and use it as a cell culture substrate in a well of a 12-well plate so that the region including the concave and/or convex regions faces upward. After coating with a mixed solution of 25 μL of 1 μg/μL type IV collagen aqueous solution and 500 μL of phosphate buffer, the mixture is left to stand overnight at 4° C.
Step 2: A medium (hereinafter referred to as “medium A”) prepared by adding EDGS to EpiLife (registered trademark, Thermo Fisher Scientific) and having 1.2 mM Ca++(high calcium) was prepared. A is mixed with A to prepare a cell suspension. 1 mL of the cell suspension was used to inoculate the surface of the crosslinked fibrotic collagen gel of step 1 at 1×10 ⁶ cells/well, then 3.8 mL of medium A was injected into the well, and the total medium was added. Adjust the volume to 4.8 mL (1st day of culture).
Procedure 3: Inoculate the seeded cells of Procedure 2 by liquid phase culture, changing the medium every day until the 4th day of culture.
Procedure 4: On the 4th day of culture, transfer to gas phase-liquid phase interface culture, and perform culture by changing the medium every other day until the 11th day of culture.
Procedure 5: On day 11 of culture, the cross-linked fibrotic collagen gel containing the cultured cells is taken out of the medium, and the area B in plan view is measured in a wet state.

(Other components)
As long as the object of the present invention is not impaired, various additives may be added to the crosslinked fibrous collagen gel of the present invention as other constituent elements depending on the purpose of use. Examples of other components include fibrin, thrombin, gelatin, hyaluronic acid, chondroitin sulfate, alginic acid and the like.

[Production method]
The method for producing a crosslinked fibrillated collagen gel of the present invention includes the following steps.
(1) The collagen in the aqueous solution of solubilized collagen is fibrillated in a state in which at least a part of the surface is in contact with a transfer member having a concave shape and/or a convex shape, and at least a part of the surface has a concave shape and And/or a first step of obtaining a fibrotic collagen gel having a region including a convex shape.
(2) A second step in which the fibrillated collagen gel obtained in the first step is chemically crosslinked with a carbodiimide-based crosslinking agent.
(3) A third step in which the chemically crosslinked fibrotic collagen gel obtained in the second step is crosslinked by γ-ray irradiation in the presence of an aqueous solvent.

(First step)
The first step is a step of obtaining, from the solubilized collagen aqueous solution, an uncrosslinked fibrotic collagen gel having a region including a pattern shape on at least a part of its surface.

The type of collagen contained in the solubilized collagen aqueous solution is not particularly limited, but type I collagen, which is abundant in the living body, is preferable, and atelocollagen from which telopeptide that is an antigenic determinant is removed is more preferable. Usually, collagen derived from biological materials such as mammals, seafood, birds and reptiles is preferably used. Collagen derived from fish and shellfish that does not have a virus in common with humans is more preferable, and collagen derived from fish is particularly preferable. Examples of the site for collecting fish-derived collagen include scales and skins. Scales are advantageous in that they contain few impurities such as lipids that cause fishy odor, and collagen with high purity can be obtained. One preferable embodiment is type I atelocollagen derived from fish, and more preferably type I atelocollagen derived from fish scale. Further, from the viewpoint of convenience of operation during production, a good example of the fish species is the genus Oleochromis, which can provide collagen with a high denaturation temperature. Among the genus Oreochromis, tilapia, which is mainly cultivated for food from China to Southeast Asia and is easily available, is particularly preferable. Classifying by extraction method from collagen-containing tissue, it is obtained by acid-solubilized collagen obtained by dilute acid extraction method, enzyme-solubilized collagen obtained by enzyme solubilization treatment, and alkali solubilization treatment method Examples include alkali-solubilized collagen. Any extraction method may be used as long as the effect of the present invention can be obtained.

The collagen concentration in the solubilized collagen aqueous solution is preferably in the range of 0.3 to 3% by mass from the viewpoint of obtaining a certain strength as a cell culture substrate and the handleability of the solubilized collagen aqueous solution.

By adding a fibrating agent such as a buffer to the solubilized collagen aqueous solution and adjusting the ionic strength and pH of the aqueous solution to conditions suitable for fibrosis, the collagen molecules associate and orient, and the fibrosis (refibrillation) occurs. ) Occurs. At this time, gelation also progresses as the fibrosis of collagen progresses. Thereby, a fibrillated collagen gel can be obtained. In the process of fibrosis, it is also a preferred embodiment to promote fibrosis by maintaining a predetermined temperature (below the denaturation temperature of collagen) for a certain period of time. For example, the predetermined temperature may be 15 to 30° C., and the holding time may be 6 to 24 hours.

The fibrating agent is not particularly limited as long as it is suitable for collagen fibrosis depending on the type of collagen. Examples thereof include physiological saline, buffer solutions, buffered saline, acidic salt aqueous solutions, neutral salt aqueous solutions, alkaline salt aqueous solutions, and the like. Specific examples of buffer solution and buffered saline solution include phosphate buffer solution, Tris buffer solution, HEPES buffer solution, acetate buffer solution, carbonate buffer solution, citrate buffer solution, phosphate buffered saline solution (PBS), and Dulbeccoline. Examples include acid buffered saline (D-PBS), Tris buffered saline, HEPES buffered saline and the like.

In the fibrillated collagen gel, for example, when observing with a scanning electron microscope at a magnification of 10,000, if there are innumerable fibrous structures, it can be confirmed that fibrillated collagen is present. Although it is generally not easy to confirm that fibrillated collagen has a D cycle by a scanning electron microscope, if a D cycle is confirmed even in a part of fibrillated collagen, the whole fibrillated collagen has a D cycle. It is safe to judge that.

In the first step of the production method according to the present invention, a pattern shape is imparted to the surface of the fibrillated collagen gel by utilizing the fibrillation process of collagen in the solubilized collagen aqueous solution. One preferable method is to prepare a transfer member having a concave shape and/or a convex shape on at least a part of its surface, and to transfer the transfer member installed so that the concave shape and/or the convex shape faces upward. A method of pouring collagen by pouring an aqueous solution of solubilized collagen mixed with a fibrating agent. When the transfer member cannot store the poured aqueous solution, it is preferable to place an appropriate frame on the transfer member. Another preferable method is to pour the solubilized collagen aqueous solution mixed with a fibrating agent into a suitable container, and then press the concave and/or convex surface of the transfer member against the liquid surface. Then, it is a method of fibrillating collagen. By the way, since the pattern shape imparted to the fibrillated collagen gel may be any suitable for culturing oral mucosal epithelial cells, it is necessary to completely reflect the concave shape and/or convex shape of the transfer member. There is no.

The shape of the transfer member is not particularly limited. It may be a flat member having irregularities such as a net, a woven cloth or a non-woven cloth, or a perforated plate such as punching metal. Further, it may be of a container type and have a concave shape and/or a convex shape on its inner side surface or bottom surface. Further, it may have a concave shape and/or a convex shape in a specific portion like a stamp.

As a cell culture substrate suitable for forming an oral mucosal epithelium-like tissue, a transfer member having a convex shape on at least a part of its surface is preferable from the viewpoint of obtaining crosslinked fibrotic collagen having a region containing a concave shape on its surface. .. A part of this transfer member is shown as a schematic view in FIG. 1A is a plan view of the transfer member 15, and FIG. 1B is a sectional view taken along the line X-X. The transfer member 15 includes a plurality of convex portions 33. The shape of each convex portion 33 in a plan view is a square, the length of one side thereof is L, and the distance between adjacent convex portions is D. The height of each convex portion 33 from the reference surface 21 is H. Note that the dotted line on the outer contour of the transfer member 15 in FIG. 1 is for showing that it is a partial view.

The manufacturing method of the transfer member is not particularly limited, and examples thereof include known molding methods such as injection molding, extrusion molding, and pressure molding, and manufacturing methods such as surface processing of existing molded articles.

Examples of the material of the transfer member include thermoplastic resin and thermosetting resin. Specific examples are acrylic resin, polyethylene, polypropylene, ABS resin, polycarbonate, polyethylene terephthalate, polyamide, styrene resin, silicone resin, urethane resin, phenol resin, epoxy resin and the like. It is also possible to select an inorganic material such as metal (stainless steel, aluminum, etc.), glass or the like as the material. It is preferable that the material is such that collagen does not easily adhere thereto. In the present manufacturing method, the transfer member may be removed from the fibrous collagen gel after the first step, or may be subjected to the second step and the third step while being in contact with the transfer member. When the transfer member is brought into contact with the second step and the third step as well, it is possible to select a material that does not crosslink with collagen by the crosslinking treatment of each step and has high durability against each crosslinking treatment. desirable. An example of such a material is silicone resin. Polydimethylsiloxane is a good example of the silicone resin. Further, the material of the transfer member may have a property such as air permeability.

(Second step)
The second step is a step of chemically cross-linking the uncrosslinked fibrotic collagen gel obtained in the first step using the carbodiimide-based cross-linking agent described above. The method for chemically crosslinking the uncrosslinked fibrillated collagen gel with the carbodiimide-based crosslinking agent is not particularly limited, and a known method can be appropriately adopted. A method of immersing the fibrillated collagen gel in a solution of a carbodiimide-based crosslinking agent is preferably used. Examples of the solvent of the carbodiimide-based crosslinking agent solution include water, an organic solvent miscible with water, and a mixture thereof. Examples of the organic solvent miscible with water include methanol, ethanol, isopropanol, dimethylsulfoxide (DMSO), dimethylformamide (DMF) and the like. Ethanol is preferred.

When a non-crosslinked fibrillated collagen gel is immersed in a solution of a carbodiimide-based crosslinking agent for chemical treatment, the immersion conditions are appropriately selected depending on the size of the fibrillated collagen gel and the type of the carbodiimide-based crosslinking agent. .. The preferred immersion time is 1 to 72 hours. From the viewpoint of accelerating the crosslinking reaction, the solution may be heated or stirred to such an extent that the pattern shape on the surface of the fibrillated collagen gel is not impaired.

The amount of the carbodiimide-based cross-linking agent used is, for example, 0.01 to 30 parts by mass with respect to 100 parts by mass of the fibrillated collagen gel. The upper limit of the above range is preferably 27.5 parts by mass, more preferably 25 parts by mass. The lower limit of the above range is preferably 0.05 part by mass, more preferably 0.1 part by mass, and further preferably 1 part by mass. Further, from the viewpoint of reducing the amount of the carbodiimide-based crosslinking agent remaining in the chemically crosslinked fibrotic collagen gel in advance, the upper limit of the above range is, for example, preferably 20 parts by mass, and 15 parts by mass. Is more preferable. The amount of the carbodiimide-based cross-linking agent used was specified based on the fibrillated collagen gel having a collagen concentration of 1% by mass.

In a preferred embodiment in the second step, after the fibrous collagen gel is chemically cross-linked with a carbodiimide-based cross-linking agent, a washing operation is performed in order to remove the carbodiimide-based cross-linking agent remaining in the chemically cross-linked fibrous collagen gel. Is to do. Preferably, the washing operation is performed until the concentration of the carbodiimide type crosslinking agent remaining in the chemically crosslinked fibrotic collagen gel becomes lower than the concentration at which no cytotoxicity is exhibited. Regarding the cytotoxicity, the IC50 value of the carbodiimide cross-linking agent may be referred to. Specific examples of the washing work include washing with water and immersion in water, but are not limited thereto. In addition, the operation in the presence of the aqueous solvent in the third step may be positioned as one of the cleaning operations. One suitable embodiment is to use the carbodiimide-based cross-linking agent within a concentration range that does not show cytotoxicity, and in this case, the washing operation can be omitted.

(Third step)
The third step is a step of γ-ray crosslinking the fibrotic collagen gel after the chemical crosslinking treatment in the presence of an aqueous solvent. The type of aqueous solvent is as described above. The amount of the aqueous solvent used is not particularly limited and may be adjusted according to the outer shape and size of the chemically crosslinked fibrotic collagen gel obtained in the second step. For example, the amount is such that at least the entire surface of the fibrillated collagen gel is covered with the aqueous solvent, and preferably the amount is such that the fibrillated collagen gel is completely immersed in the aqueous solvent. Further, the fibrillated collagen gel is not completely immersed in the aqueous solvent, for example, even if a part of the fibrillated collagen gel is not immersed in the aqueous solvent, if the infiltrate in the portion can be ensured, It can be said that it is immersed in an aqueous solvent. In the present specification, the term "in the presence of an aqueous solvent" is used inclusive of the states exemplified above.

Gamma-ray irradiation is a cross-linking method that has high penetrating power and can uniformly cross-link fibrotic collagen gel. In γ-ray irradiation, a predetermined irradiation dose can be easily obtained by appropriately setting conditions such as irradiation time using a radiation source with a constant dose rate. For example, when a cobalt 60 radiation source is used, the crosslinking treatment can be performed with an irradiation dose of 5 to 75 kGy. The irradiation dose is preferably 5 to 50 kGy, more preferably 10 to 50 kGy, and further preferably 15 to 30 kGy. The irradiation time is preferably set appropriately so that the crosslinking reaction proceeds sufficiently. Furthermore, if the irradiation conditions are appropriately set, the sterilization treatment can be performed simultaneously with the crosslinking treatment. Therefore, by maintaining a sealed state during the cross-linking treatment and after the cross-linking treatment, it is possible to circulate the sterilized product as it is on the market.

(Combining other components)
When the above-mentioned other components are blended with the crosslinked fibrous collagen gel of the present invention, it is preferable to appropriately set the blending timing of the other components according to the type of the other components, the intended use, and the like. Examples of the compounding timing include before the fibrosis in the first step, before the chemical crosslinking treatment in the second step, and before the γ-ray crosslinking treatment in the third step.

(Use)
One suitable use of the crosslinked fibrous collagen gel of the present invention is as a medical material or a drug evaluation material. A preferred configuration of the material is one in which the crosslinked fibrotic collagen gel of the present invention is an essential component, and oral mucosal epithelial cells and/or oral mucosal epithelial-like tissue adhered to the crosslinked fibrotic collagen gel are included as optional components. ..

For example, when the crosslinked fibrous collagen gel itself according to the present invention is transplanted to a site of oral mucosal defect, the crosslinked fibrous collagen gel becomes a scaffold material for oral mucosal cells that have migrated from the periphery. Therefore, this crosslinked fibrous collagen gel can contribute to repair of the affected area as a medical material for oral mucosa regeneration. At this time, it can be said that the transplanted crosslinked fibrotic collagen gel of the present invention functioned as a cell culture substrate for oral mucosal epithelial cells in the gas-liquid interface culture in the affected area.

Further, the material containing the crosslinked fibrotic collagen gel of the present invention and the oral mucosal epithelial cells and/or the oral mucosal epithelial-like tissue adhered to the crosslinked fibrotic collagen gel is, for example, a medical material for transplantation into an oral mucosal defect site. Can be used as Further, the material can be suitably used as a drug evaluation material for investigating the drug efficacy and side effects of a drug on oral mucosal epithelial cells and/or oral mucosal epithelial tissue.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

(Preparation of solubilized collagen aqueous solution)
"Cell campus FD-08G" (freeze-dried product) manufactured by Taki Kagaku Co., Ltd., which was manufactured from tilapia scales, was dissolved in a pH 3 HCl solution, and then the collagen concentration was adjusted to 1.1% and pH 3 to obtain a colorless and transparent solution. A solubilized collagen aqueous solution was obtained.

(Transfer members A and B)
The materials forming the transfer members A and B are both polydimethylsiloxane. The transfer members A and B were produced by pouring polydimethylsiloxane (trade name SILPOT 184, manufactured by Dow Corning Toray Co., Ltd.) into a Si mold (square with one side of 30 mm in plan view) and curing it under known conditions. The pattern shapes shown in FIG. 1 were formed in the square regions of 10 mm on each side of the central portions of the obtained transfer members A and B in plan view. This pattern shape is a convex shape in which a plurality of convex portions having a square shape in plan view are regularly arranged.
In the transfer member A, L=100 μm, D=100 μm, and H=100 μm.
In the transfer member B, L=200 μm, D=200 μm, and H=200 μm.

(Transfer member C)
The transfer member C was produced by pouring polydimethylsiloxane into a Si mold (square having a side of 30 mm in plan view) and curing it under known conditions. The transfer member C has a flat plate shape having no pattern shape on its surface.

[Example 1]
(First step)
A silicon plate (thickness: 2.5 mm) having a hole in the center was placed on the transfer member A installed so that the region having the convex shape faces upward. This hole was circular with a diameter of 19 mm, and a silicon plate was placed so that the region having the convex shape of the transfer member A was arranged at the center of the hole.
Next, 0.78 mL of a mixed solution obtained by mixing 9 parts by volume of the solubilized collagen aqueous solution and 1 part by volume of 10 times concentrated Dulbecco's phosphate buffered saline (D-PBS) was poured into the hole of the silicon plate. It is. The mixed liquid easily entered the depressions between the protrusions of the transfer member A. Then, the mixture was kept at 25° C. for 12 hours to obtain a fibrillated collagen gel (0.78 g, collagen concentration 1% by mass). It was easy to remove the fibrillated collagen gel from the transfer member A, and the transfer member A had no deposits.
When the surface of the fibrous collagen gel that was in contact with the region having the convex shape of the transfer member A was confirmed, it was found that the convex region of the transfer member A had a region including the concave shape that was almost reflected. confirmed.

(Second step)
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)·hydrochloride was dissolved in ethanol to a concentration of 1 w/v% to prepare an EDC solution. The fibrillated collagen gel having the concave shape obtained in the first step was completely immersed in 10 mL of the EDC solution, and was rotatively stirred at room temperature for 24 hours to cause a crosslinking reaction. Next, the chemically crosslinked fibrotic collagen gel was completely immersed in D-PBS, and was stirred by rotation at room temperature for 3 hours to wash.

(Third step)
After the chemically crosslinked fibrotic collagen gel was taken out from the washing D-PBS, it was irradiated with 25 kGy of γ-ray in a state of being completely immersed in new D-PBS. A crosslinked fibrillated collagen gel was obtained. It was confirmed that the crosslinked fibrillated collagen gel retained the concave shape confirmed in the first step.

[Example 2]
A crosslinked fibrous collagen gel of Example 2 was obtained in the same manner as in Example 1 except that the transfer member B was used instead of the transfer member A. It was confirmed that the crosslinked fibrous collagen gel had a region on its surface including a concave shape in which the convex shape of the transfer member B was reflected almost as it was.

[Comparative Example 1]
A crosslinked fibrous collagen gel of Comparative Example 1 was obtained in the same manner as in Example 1 except that the transfer member C was used instead of the transfer member A. It was confirmed that the crosslinked fibrillated collagen gel had a flat plate shape without any pattern shape on its surface.

[Comparative Example 2]
By performing the first step of Example 1 in the same manner, a fibrillated collagen gel having a concave shape was obtained. Next, this fibrotic collagen gel was completely immersed in D-PBS and irradiated with 25 kGy of γ-ray to obtain a crosslinked fibrous collagen gel of Comparative Example 2. It was confirmed that the crosslinked fibrous collagen gel had a region on its surface including a concave shape in which the convex shape of the transfer member A was almost directly reflected.

[Comparative Example 3]
A crosslinked fibrous collagen gel of Comparative Example 3 was obtained in the same manner as Comparative Example 2 except that Transfer Member B was used instead of Transfer Member A. It was confirmed that the crosslinked fibrous collagen gel had a region on its surface including a concave shape in which the convex shape of the transfer member B was reflected almost as it was.

[Cell culture test]
A cell culture test was carried out by the following procedure using each of the crosslinked fibrous collagen gels of Examples 1 and 2 and Comparative Examples 1 to 3 formed in a circular shape in plan view (diameter 19 mm) as the cell culture substrate. The thickness of each cell culture substrate was about 2.5 mm. The cells used were primary cultured cells derived from the oral mucosal epithelium of patients who underwent oral surgery at the Niigata University Medical and Dental General Hospital, which was used in the experiment after being approved by the Ethics Committee of the Faculty of Dentistry, Niigata University.

(Day 0)
Each cell culture substrate was placed in a 12-well plate. In each of the crosslinked fibrous collagen gels of Examples 1 and 2 and Comparative Examples 2 and 3, the surface having the concave shape was set as the upper surface. This was coated with a mixed solution of 25 μL of a 1 μg/μL type IV collagen aqueous solution per well and 500 μL of a phosphate buffer solution, and then allowed to stand at 4° C. overnight.
(Day 1)
The oral mucosal epithelium-derived primary culture cells were mixed with medium A to prepare a cell suspension. 1 mL of the cell suspension was used to inoculate the surface of the crosslinked fibrotic collagen gel of step 1 at 1×10 ⁶ cells/well, then 3.8 mL of medium A was injected into the well, and the total medium was added. The volume was 4.8 mL. Then, the medium was exchanged every day until the fourth day of culture (Day 4) by submerged culture.
(Day 4)
The medium was changed to air-liquid interface culture, and the medium was replaced every other day. The culture was continued until the 11th day of culture (Day 11).
(Day 11)
Each cell culture substrate was taken out and immersed in 4% paraformaldehyde overnight (4° C.) to fix the obtained culture tissue.
Then, the paraffin-embedded specimen was stained with hematoxylin-eosin (HE) by a conventional method, and subjected to morphological observation with an optical microscope.

(result)
FIG. 2 is a photograph of the appearance of each wet culture substrate taken out on Day 11 before fixation with 4% paraformaldehyde. From the appearance photograph, in Examples 1 and 2 and Comparative Example 1 obtained by γ-ray crosslinking after chemical crosslinking, the shrinkage is remarkable as compared with Comparative Examples 2 and 3 obtained by only γ-ray crosslinking. You can see that it was suppressed.
Based on the appearance photograph of FIG. 2, the planar view area B of each cell culture substrate after the test was determined. Next, before the cell culture test test, the area shrinkage (%)=(AB)/A×100 from the area A of the cell culture substrate having a circular shape (19 mm in plan view) in plan view The rate was calculated. The respective area shrinkage rates thus calculated were Example 1:-0.6%, Example 2:1%, Comparative Example 1:-5.3%, Comparative Example 2:35.5%, Comparative Example 3:60.1%. It is considered that the crosslinked fibrous collagen gels of Example 1 and Comparative Example 1 in which the area shrinkage ratio showed a negative value were swollen.
In addition to the appearance photograph of FIG. 2, the shrinkage was remarkably suppressed in Examples 1 and 2 and Comparative Example 1 obtained by γ-ray crosslinking after chemical crosslinking, not only by the area shrinkage value, It was shown that significant shrinkage occurred in Comparative Examples 2 and 3 obtained only by linear crosslinking.

The above results indicate that, in the culture of oral mucosal epithelial cells including at least the gas phase-liquid phase interface culture, a sufficient contraction inhibitory effect cannot be obtained only by γ-ray irradiation cross-linking in the presence of an aqueous solvent, and γ-ray irradiation cross-linking Previously, it was shown that chemical cross-linking with a carbodiimide-based cross-linking agent is effective in suppressing shrinkage.

FIG. 3 is a histological image showing a cross section of a HE-stained cultured tissue obtained from each of the cell culture substrates of Example 1, Example 2 and Comparative Example 1. In all the histological images of Example 1, Example 2 and Comparative Example 1, formation of a continuous epithelial layer was observed on the entire surface of the cell culture substrate.
Regarding the histological images of Example 1 and Example 2, it can be said that cells having epithelial leg-like cells that proliferated in the recesses of the cell culture substrate were observed, and that the structure was similar to that of the oral mucosal epithelial tissue of a living body having a papillary structure. there were. In addition, Example 1 was more similar to the oral mucosa epithelial tissue of the living body.
On the other hand, the histological image of Comparative Example 1 had a flat structure and was not similar to the oral mucosa epithelial tissue of the living body. Moreover, the formed cultured tissue was easily separated from the substrate.

From the above results, using the crosslinked fibrous collagen gel obtained in Example 1 and Example 2 as a cell culture substrate, oral mucosal epithelial cells were subjected to liquid phase culture, and then gas phase-liquid phase interface culture was performed. It was shown that a cultured tissue similar to the oral mucosa epithelial tissue of the living body having a papillary structure was obtained when the above-mentioned were obtained.

15... Transfer member 21... Reference surface 33... Convex portion

Claims (6)

A cross-linked fibrotic collagen gel used as a cell culture substrate in the culture of oral mucosal epithelial cells including gas phase-liquid phase interface culture,
A crosslinked fibrotic collagen gel that satisfies all the following conditions (i) to (iii).
(i) The crosslinked fibrotic collagen gel is composed of intact crosslinked fibrotic collagen.
(ii) The crosslinked fibrous collagen gel has a region including a concave shape and/or a convex shape on at least a part of its surface.
(iii) The cross-linked fibrillated collagen gel is obtained by chemically cross-linking the non-cross-linked fibrillated collagen gel with a carbodiimide-based cross-linking agent and then γ-irradiating it in the presence of an aqueous solvent. The method for producing a crosslinked fibrotic collagen gel according to claim 1, comprising the following steps.
(1) A transfer member having a concave shape and/or a convex shape on at least a part of its surface is prepared, and the collagen in the solubilized collagen aqueous solution is fibrillated in a state of being in contact with the transfer member, and the surface thereof is formed. A first step of obtaining a fibrillated collagen gel having a region including a concave shape and/or a convex shape in at least a part of the above.
(2) A second step in which the fibrillated collagen gel obtained in the first step is chemically crosslinked with a carbodiimide-based crosslinking agent.
(3) A third step in which the chemically crosslinked fibrotic collagen gel obtained in the second step is crosslinked by γ-ray irradiation in the presence of an aqueous solvent. A cell culture method comprising using the crosslinked fibrous collagen gel according to claim 1 as a cell culture substrate to culture oral mucosal epithelial cells in a liquid phase, and then performing a gas-liquid phase interface culture. A medical material for oral mucosal regeneration comprising the crosslinked fibrous collagen gel according to claim 1. The medical material according to claim 4, comprising oral mucosal epithelial cells and/or oral mucosal epithelial-like tissue adhered to the crosslinked fibrous collagen gel. A material for drug evaluation comprising the crosslinked fibrotic collagen gel according to claim 1 and the oral mucosal epithelial cells and/or oral mucosal epithelial-like tissue adhered to the crosslinked fibrotic collagen gel.