JPH08228768A - Hydrogel thin film containing extracellular matrix components - Google Patents

Abstract

PURPOSE: To obtain an extracellular matrix component-containing hydrogel thin film having mechanical strength enough to be useful as a cell culture substrate etc., easy to prepare and excellent in convenience, by hydrating a vitrified matrix gel thin film obtained from an extracellular matrix component. CONSTITUTION: A solution of an extracellular matrix component (e.g. collagen) gelates, and is dried and vitrified. The product is then hydrated into the objective hydrogel thin film. According as necessary, this thin film is integrated with a substrate. The vitrification is carried out by, for example, air-drying of an extracellular matrix component-contg. gel 0.05-5wt.% or so in the final concentration.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a hydrogel thin film containing extracellular matrix components. More specifically, the present invention is a novel extracellular matrix component-containing hydrogel thin film, which is useful in cell culture substrates, prevention of organ adhesion and the like, has appropriate strength, is easy to prepare, and can be conveniently used. It is about.

[0002]

2. Description of the Related Art Conventionally, cell culture has been carried out in various modes for the purpose of developing various medical techniques and pharmaceuticals. As one of the methods for cell culture, a method using an extracellular matrix component such as collagen is known. Also, in this method, as in the case of collagen, for example, relax the restrictions of ordinary cell culture as two-dimensional flat culture, and ensure three-dimensionality in a state as close as possible to the tissue morphology and function expression of the living body. Various attempts have been made.

However, the culturing method using collagen or the like, which has been attracting attention for its use, has a problem that, for example, collagen hydrogel is soft and difficult to handle, and it is used as a cell culture substrate. The fact is that the preparation is not always easy, and a simpler usage method has not been established. Therefore, the inventor of the present invention has studied the utilization of extracellular matrix components such as collagen from various viewpoints.
The problem at that time is to drastically improve the conventional culture method, to easily prepare it as a cell culture substrate, to use it easily, and to have good performance as a culture substrate, to prevent organ adhesion, etc. It was to realize a new matrix material utilization method that could be applied to

[0004]

MEANS FOR SOLVING THE PROBLEMS The present invention, which solves the above-mentioned problems, is a thin film comprising a hydrate of a vitrified matrix gel containing an extracellular matrix component. An outer matrix component-containing hydrogel thin film (claim 1) is provided. Further, according to the present invention, the extracellular matrix component-containing hydrogel thin film has a plurality of extracellular matrix components (Claim 2), the extracellular matrix component is collagen (Claim 3), and a cell culture medium. It is also provided as an embodiment that the component is included (Claim 4) and that the thin film is integrated with the holding body (Claim 5). Regarding the holding body, the holding body is an annular body or a mesh body (Claim 6), the holding body has a bioabsorber (Claim 7), the holding body has a circular opening, A mode in which the circular opening is made of a cylindrical body in which a thin film is held and integrated (claim 8) and the like is also adopted.

The present invention also provides an extracellular matrix component-containing gel dried glass body as a precursor of the extracellular matrix component-containing hydrogel thin film. Further, the present invention relates to the above-mentioned extracellular matrix component-containing hydrogel thin film, which comprises gelling a solution of the extracellular matrix component, further drying and vitrifying, and then hydrating. It also provides a method for producing a gel thin film (Claim 9) and the use of an extracellular matrix component-containing hydrogel thin film as a cell culture substrate (Claim 15).

Further, the present invention also provides a cell culture method using the above substrate.

[0007]

The extracellular matrix component-containing hydrogel thin film of the present invention as described above has a moderate strength that is easy to handle and a thin film shape as a thin film itself and as one integrated with a holder, Since it can have a composition as a cell culture substrate, it can be easily prepared for culturing and is extremely convenient.

Collagen is a typical extracellular matrix component, and its application is drawing attention. In addition to this collagen, there are fibronectin, vitronectin, laminin, proteoglycan, glycosaminoglycan, Matrigel (trade name) and the like, which are appropriately used. There is also no particular limitation on the components of the cell culture solution, and the optimum salt composition, concentration, pH
Etc. are selected.

The holding body may be any suitable body such as an annular body formed of wire, wire or the like, a mesh-like body made of gauze or other woven body, and may be a bioabsorbable body. The shape, size, material and the like may be selected according to the usage mode. It may be a cylinder having a circular opening, or a container as a precursor thereof. In the production as a cell culture substrate, for example, first, a composition having a medium, serum and the like is mixed with a matrix aqueous solution such as collagen, and various kinds of holders are put into this mixed solution, and the mixture is prepared. Incubate at appropriate temperature and gel.

When this gel body is further dried by air drying or the like, it vitrifies. This phenomenon of vitrification and further modification of the vitrified gel to be used, and further utilization as a cell culture substrate or the like by this modification are not known, and it was first realized by the present invention. That is. That is, in the production method of the present invention, the gel containing extracellular matrix components such as vitrified collagen is hydrated. As a result, a strong hydrogel thin film containing the extracellular matrix component is obtained. This becomes a cell culture substrate. It is also effective in preventing organ adhesions.

Regarding vitrification, in the present invention, a gel containing extracellular matrix components having a final concentration of about 0.05 to 5% is slowly and completely dried (for example, air dried). By doing this, it becomes vitrified. The hydration of this vitrified gel is easily carried out by treating it with PBS or the like. Hereinafter, examples using collagen as an example will be described in more detail, and the configuration and action and effect of the present invention will be described.

[0012]

【Example】

( Example 1 : Preparation of collagen hydrogel thin film) 2 ml of 5 times concentrated Dulbecco's modified Eagle medium (GIBCO # 31600-03) was placed in a sterilized conical tube (Falcon # 2070) with an internal volume of 50 ml cooled on ice.
4), 0.1 ml of 10,000 units / ml penicillin / 10,000 μg / ml streptomycin (GIBCO # 15140-031), 0.2 ml of 1
MHEPES (GIBCO # 15630-023),
0.493 ml of 7.5% sodium bicarbonate solution (GI
BCO # 25080-011), 1.407 ml of distilled water, and 1 ml of fetal bovine serum, and after mixing,
4.8 ml of 0.5% type I collagen aqueous solution (CELL
GEN I-AC or I-PC, manufactured by Koken Co., Ltd. was added and mixed uniformly. 2 ml of a final concentration 0.24% collagen mixed solution was placed in a hydrophobic polystyrene culture dish (φ35 mm: Falcon # 1008), and then 5% C was added.
The gelation was carried out by keeping in a humidified incubator at 37 ° C. in the presence of O ₂ /95% air for 3 hours. This final concentration 0.24%
The collagen gel was vitrified by slowly and completely air-drying with the lid covered. 2 ml to this
The vitrified collagen gel was hydrated by adding PBS. Further, the collagen gel hydrated with 2 ml of PBS was rinsed several times. The hydrated collagen gel was desorbed and collected from the petri dish as a strong thin-film collagen hydrogel as shown in the phase contrast micrograph of Fig. 1 by tracing the inner wall of the petri dish along the circumference with sharp tweezers. did it.

Example 2 Preparation of Thin-Film Collagen Hydrogel with Wire Circumferential Holder A circular holder having a knob portion (1) as illustrated in FIG. 2 was used as a stainless steel wire (size # 20, 0.9 mm). 2) prepared in Example 1
A final concentration of 0.24% collagen mixture solution was placed in a hydrophobic polystyrene culture dish (φ35 mm: Falcon # 1008) together with this wire holder. And
After gelling in the same manner as in Example 1 above, it was vitrified.
The vitrified collagen gel was hydrated by adding 2 ml of PBS thereto. In addition, 2 ml of PBS several times
The collagen gel hydrated with was rinsed. The hydrated collagen gel could be desorbed and collected from the petri dish as a strong collagen hydrogel thin film with a wire holder around the periphery as illustrated in FIG. 3 by gently lifting the knob of the stainless wire.

Example 3 Preparation of Gauze-Embedded Thin-Film Collagen Hydrogel A sterile gauze type III (Kepine, manufactured by Kawamoto Bandai Co., Ltd.) listed in the Japanese Pharmacopoeia is aseptically circular and has a knob. Cut into shape and culture medium (10% fetal bovine serum, 20 mM HEPES, 100 u
nits / ml penicillin, 100 μg / ml streptomycin-containing Dulbecco's modified Eagle medium). This gauze was mixed with 5 ml of a final concentration 0.24% collagen mixture solution prepared in Example 1 and a hydrophobic polystyrene culture petri dish (φ60 mm: Falcon # 1007).
In the same manner as in Example 1, the mixture was gelled and then vitrified.

Further, PBS was added in the same manner as in Example 1 to hydrate the vitrified collagen gel. The hydrated collagen gel was able to be detached and collected from the petri dish as a strong thin-film collagen hydrogel in which gauze was embedded throughout, by gently lifting the knob of the gauze, as illustrated in FIG. ( Example 4 : Preparation of tubular body-holding thin film collagen hydrogel) Sterilized conical tube (Falcon) with an internal volume of 50 ml
# 2070) was cut and processed to prepare a tubular body (10) device capable of fixing a thin film collagen hydrogel as a cell culture substrate as shown in FIG. 5 (a).

Subsequently, using the collagen hydrogel thin film (12) with a wire peripheral retainer (11) prepared in Example 2, similarly, as shown in FIG. 5 (b), a conical tube lid (13) was prepared. Pressing, wire holder (11)
Is removed. By doing so, the thin film collagen hydrogel can be easily transferred and fixed to the opening of the device (10). In this state, it can be used for cell culture. Furthermore, the gauze-embedded thin film collagen hydrogel shown in Example 3 could be fixed to this device by the same method.

( Example 5 : Cell culture on thin-film collagen hydrogel) 10 ml of cell culture solution was added to a petri dish for hydrophilic polystyrene culture (φ60 mm: Falcon # 300).
2), and the equipment to which the collagen hydrogel thin film prepared in Example 4 was fixed was put in this, and 2 m inside
1 cell suspension (3 × 10 ⁴ / ml) was seeded and 5%
It was cultured in a humidified incubator at 37 ° C. in the presence of CO ₂ /95% air. The cells are human dermis-derived fibroblasts (H
DF) and human cholangiocarcinoma epithelial cells (MEC) were used. After culturing for 5 days, the cells were fixed with formalin and directly stained with hematoxylin / eosin (HE). In the case of MEC, an image in which colonies were formed on the collagen hydrogel thin film was exhibited as shown in the photograph in FIG. A part of HDF scattered on the collagen hydrogel thin film appeared to infiltrate into the gel (Fig. 7 photograph). Therefore, when a frozen section perpendicular to the gel surface was prepared and stained with HE, M
EC showed no wet image in the gel, but HDF
A clear wet image in the gel was observed in.

Of course, the present invention is not limited to the above examples. In addition to the cells to be cultivated, the composition of the culture solution, and the culturing conditions, it is often possible to have various aspects regarding the type and matrix composition of the extracellular matrix component such as the collagen hydrogel thin film, and the thickness and strength of the thin film. I don't need a word.

[0019]

As described above in detail, according to the present invention, a culture matrix body of a hydrogel thin film containing an extracellular matrix component such as collagen and an organ adhesion preventing material which is easy to prepare and excellent in convenience can be obtained. .

[Brief description of drawings]

FIG. 1 is a phase-contrast microscope photograph as a substitute for a drawing showing the surface of a collagen hydrogel thin film as a reference example.

FIG. 2 is a perspective view illustrating a circular wire body as a holding body.

FIG. 3 is a photograph replacing a drawing, which illustrates a collagen hydrogel thin film matrix integrated with a circular wire holder.

FIG. 4 is a photograph replacing a drawing, which illustrates a collagen hydrogel thin film in which gauze is embedded.

FIG. 5 is a process drawing showing the production of the substrate body of the present invention in which the tubular body is a holding body.

FIG. 6 is a micrograph as a substitute for a drawing, which shows colony formation in the case of MEC.

FIG. 7 is a photomicrograph replacing the surface in the case of HDF.

[Explanation of symbols]

 1 Circular Wire Holder 10 Cylindrical Equipment 11 Circular Wire Holder 12 Collagen Hydrogel Thin Film 13 Lid

Claims (17)

[Claims] 1. A hydrogel thin film containing an extracellular matrix component, comprising a hydrate of a vitrified matrix gel thin film containing an extracellular matrix component. 2. An extracellular matrix component-containing hydrogel thin film according to claim 1, which contains a plurality of extracellular matrix components. 3. The extracellular matrix component-containing hydrogel thin film according to claim 1, wherein the extracellular matrix component is collagen. 4. An extracellular matrix component-containing hydrogel thin film according to claim 1, which has a cell culture medium component. 5. The extracellular gel component-containing hydrogel thin film according to claim 1, wherein the thin film is integrated with a support. 6. The extracellular matrix component-containing hydrogel thin film according to claim 5, wherein the support is a ring or a net. 7. The extracellular gel component-containing hydrogel thin film according to claim 5, wherein the support is made of a bioabsorbable material. 8. The holding body has a circular opening, and the circular opening is a cylindrical body holding and integrating the thin film.
Hydrogel thin film containing extracellular matrix components. 9. An extracellular matrix component-containing dried glass body comprising the dried body of the extracellular matrix component-containing gel according to any one of claims 1 to 7. 10. A method for producing an extracellular matrix component-containing hydrogel thin film, which comprises gelling a solution containing an extracellular matrix component, further drying and vitrifying, and then hydrating. 11. The method for producing an extracellular matrix component-containing hydrogel thin film according to claim 10, which has a plurality of extracellular matrix components. 12. The method for producing an extracellular matrix component-containing hydrogel thin film according to claim 10, wherein the extracellular matrix component is collagen. 13. The method for producing an extracellular matrix component-containing hydrogel thin film according to claim 10, which has a cell culture medium component. 14. An extracellular matrix component-containing hydrogel according to any one of claims 10 to 13, wherein a thin film formed by gelling in the presence of a support, drying and vitrifying and then hydrating is integrated with the support. Thin film manufacturing method. 15. A thin film, which is produced by the method of claim 14 and is held and integrated on an annular holding body, is transferred and fixed to a cylindrical body having a circular opening having a diameter smaller than that of the annular holding body. A method for producing a hydrogel thin film containing an extracellular matrix component, which is integrated with a tubular body comprising: 16. A cell culture substrate comprising the thin film according to claim 1. 17. A cell culture method using an extracellular matrix component-containing hydrogel thin film, which comprises culturing cells by seeding cells on the substrate according to claim 16.