JPH1043213A - Manufacture of cultured skin, support and vessel used in the manufacture, and cultured skin base material and cultured skin obtained by the manufacture - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prevent shrinkage of collagen by forming a cultured skin on a support having a projection or in a vessel, and separating the same. SOLUTION: A plurality of wedge-like projections 1 are formed on the base of a support 2 and stored in a vessel 3. The upper part of the side of the support 2 is bent to form a grip part 61, and a grasping means hooking hole 62 is provided. On the support 2, a cultured skin base material is made, and epidermal cell and/or fibroblast derived from the skin is inoculated and cultivated to form the cultured skin. In this case, it is preferable to first inoculate and cultivate the fibroblast and subsequently culture epidermal cell from the viewpoint of preventing fallout of epidermal cell. The reverse is also all right. Dispersion solution is added to the cultured epidermal cell and let still standing at a designated temperature, for example, at 37 deg.C to strip the epidermal cell. This is added to a trypsin solution to disperse the epidermal cell and pick it. Thus, the support having the projection is used to restrain the shrinkage as compared with the case of using the support without a projection.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing cultured skin,
The present invention relates to a support and a container having projections used in the production method, a substrate for cultured skin and a cultured skin obtained by the production method.

More specifically, a method for producing a cultured skin for reconstructing or treating a defective tissue at an early stage by using it for a skin defective wound such as a burn, a wound, abrasion, a pressure sore or a skin ulcer, and a culture method. The present invention relates to a support having projections for producing skin, a container having projections on the bottom surface, a substrate for cultured skin and a cultured skin obtained by the production method.

[0003]

2. Description of the Related Art Conventionally, a method of applying ointment-applied gauze or the like has been generally used as a treatment for a skin defect wound up to a shallow part of the dermis such as superficial II degree burn, mild pressure sore or shallow skin ulcer. Was. However, deep II burns, II
Reconstruction of skin tissue by own epidermal cell proliferation when extensive skin deficits such as deep burns or deep dermal ulcers such as II or higher pressure ulcers or deep skin ulcers Can not expect.

Therefore, a technique for collecting epidermal cells or fibroblasts from a small amount of skin pieces and culturing them in a large amount in a culture flask has been developed, and various cultured skins using these cultured cells have been developed.

[0005] Howard Green,
James Reinwald
The cultured epidermis sheet developed by them et al. Is obtained by collecting stamp-sized skin and culturing epidermal cells in a culture flask in a large amount to obtain an epidermis sheet. The disadvantages are that the skin to be transplanted is the epidermis, so that when transplanted into a deep defect in the skin, unevenness remains even after closure of the wound, and excessively promotes wound contraction, resulting in scarring. It is easy.

[0006] The cultured epidermis sheet is prepared in a flask, and the basal cells are adhered to the bottom of the flask. However, these cells are the most important cells due to an enzyme treatment performed when the sheet is detached from the flask. It has been pointed out that autografts have low engraftment rates due to damaged basal cells (Rolling Ryu et al., The.
Journal of Trauma, Volume 34, NO. 5, 19
(See the May 1993 issue.)

[0007] Separately, a dermis-like tissue is constructed from collagen or the like as a cultured skin which compensates for such a drawback and can be used for skin diseases in which the dermis part has been lost as in the case of full thickness defect. Studies have been made to create a cell layer to provide a skin substitute.

[0008] Among them, a typical example is S. Boyce et al. (SU.
RGERY), 103, 421-431, 1988.
In a culture flask, a large amount of epidermal cells collected from the skin were cultured in a culture flask, and this was seeded on a collagen sponge-like substrate to which a small amount of chondroitin-6-sulfuric acid was added to form a stratified culture. Has developed skin. The sponge-like collagen base material shrunk during seed culture of epidermal cells and / or fibroblasts, and it was difficult to ensure a desired size with good reproducibility.

Further, Eugene Bell (E. Bell)
Et al. (Science), Volume 211, 105
Pp. 2-1054, 1981), separated from the skin,
The collected epidermal cells and fibroblasts were each cultured in large amounts in culture flasks, and a cultured skin in which epidermal cells were layered on a collagen gel substrate incorporating fibroblasts was developed. However, collagen, which has been used as a base material, has a disadvantage in that it contracts during the preparation of cultured skin, and particularly in a gel state, the contraction rate is large, and it is very difficult to secure a desired size. .

[0010] In addition, when the cultured skin thus prepared is transplanted or applied to a wound surface, it is lifted and affixed with tweezers or the like, but all of them are very fragile and often cause breakage. Was very difficult. For this reason, in practice, there have been many cases in which another biomaterial-derived coating material is superimposed or gauze is superimposed and applied to the wound surface while supporting the cultured skin.

[0011] Therefore, there has been a demand for the development of an inexpensive and simple alternative or method that can suppress shrinkage and safely support without causing breakage.

[0012]

As described above, among the current cultured skins, those using collagen as a base material shrink during the process of preparing the cultured skin, and it is difficult to obtain a cultured skin of a desired size. Was.

Further, there is a problem that the existing cultured skin is fragile and difficult to handle in use.

[0014] Therefore, there is a need for a container for preparing a cultured skin or a support for preparing a cultured skin, which can prevent contraction of collagen during preparation of the cultured skin. Further, there has been a demand for the development of a support or container for producing a cultured skin, which can support the produced cultured skin as it is and apply it to a wound surface.

[0015]

According to the present invention, a)
A step of preparing a substrate for cultured skin comprising a collagen sponge, a collagen sheet or a collagen gel on a support having protrusions or in a container in order to suppress shrinkage of the substrate; A step of seeding and culturing cells to obtain a cultured skin; and c) a step of peeling the cultured skin from the support or the container at the time of application, wherein steps a) and b) may be performed simultaneously. A support for culturing skin, wherein the support is provided with a protrusion, a side wall and a bottom surface, the protrusion is provided on the bottom surface, and at least a part of the bottom surface and the side wall are separable. Provided is a container for preparing a cultured skin, comprising: The support of the present invention preferably has flexibility so that the cultured skin is easily peeled off. The container of the present invention preferably has a flexible bottom surface so that the cultured skin is easily peeled off.

According to the present invention, there is provided a cultured skin substrate prepared on a support or a container having a projection having a thickness smaller than the thickness of the cultured skin substrate prepared so as not to provide a through-hole. Provided is a substrate for cultured skin comprising a collagen sponge, a collagen sheet or a collagen gel in which the content of the cultured skin is suppressed, and a cultured skin produced by the method using the substrate for cultured skin.

[0017]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In the present invention, a collagen solution such as an aqueous collagen solution is poured as a substrate into a container provided with a support for preparing a cultured skin having protrusions, and the resulting solution is formed into a sheet or gel. , Or later,
In the process of seeding and culturing fibroblasts and / or epidermal cells that have been lyophilized into sponge-like cultures, it enables the production of cultured skin while preventing contraction of the collagen base material. The cultured skin thus produced can be taken out of the container together with the support, and is a cultured skin producing system that can be directly transplanted or applied to a wound surface.

[0018] Alternatively, the present invention is a cultured skin preparation system which makes it possible to prepare a cultured skin while preventing shrinkage using a container for preparing a cultured skin having protrusions as described above. Further, the fibroblasts or (and) epidermal cells and the neutral collagen solution are mixed and poured into a container provided with the support for preparing cultured skin having the above-mentioned projections or a container for preparing cultured skin having projections, This is a cultured skin preparation system that allows a cultured skin to be prepared by gelation. In addition, the bottom surface of the container having protrusions also includes a cultured skin preparation system that can be easily separated from the cultured skin preparation container main body so that it can be directly transplanted or applied to a wound surface.

In the present invention, "on the support having the protrusion or in the container" means not only on the support having the protrusion and in the container having the protrusion, but also in the container on which the support having the protrusion is disposed. And the like including the support.

The substrate for cultured skin of the present invention includes collagen, gelatin, chitin, chitosan, polyglycolic acid,
Alternatively, mucopolysaccharides such as chondroitin sulfate and hyaluronic acid can be used, and the collagen is preferably excellent in biocompatibility such as atelocollagen.

The collagen sponge used in the present invention is obtained by homogenizing an acidicly prepared collagen solution with a homogenizer to sufficiently contain bubbles to form a means having projections, that is, a support having projections. Pour into a plastic container or container with protrusions, allow it to stand in an ammonia gas atmosphere, gel it, freeze-dry it, and then use UV irradiation or cross-linking agents to introduce intermolecular crosslinks. can do.

In the present invention, the collagen solution is prepared from collagen obtained from bovine dermis or the like, the pH is preferably adjusted to 2 to 4, and the concentration is 0.2 to 3 w / v%.
It is preferably obtained by setting the content to 0.5 to 2 w / v%. Gelation is carried out in a gas atmosphere such as ammonia for several minutes to 2 hours as necessary, and then freeze-drying is performed.

The main wavelength of ultraviolet light (UV) used for crosslinking is 2
It is preferably 50 to 270 nm, and the amount of ultraviolet rays is 500
１12000 mWsec / cm ² , preferably 1000
It is preferable to irradiate a dose of ５5000 mWsec / cm ² . Examples of the crosslinking agent used in the present invention include, for example,
Glutaraldehyde, ethylene glycol diglycidyl ether, polyglycerol polyglycidyl ether,
Glycerol polyglycidyl ether, hexamethylene diisocyanate and the like can be mentioned. Further, the size of the collagen sponge is appropriately selected according to the size of the wound surface. The thickness and shape of the collagen sponge vary depending on the condition of the wound surface to be applied.
The shape may be formed into a sheet shape, a plate shape, a rod shape, a spherical shape, a spindle shape, a lens shape having both sides convex, or the like.

The collagen sheet used in the present invention comprises:
The collagen solution can be produced by placing the collagen solution in the same container as used for the production of the sponge, air-drying in a drier to form a sheet, and then introducing ultraviolet rays or introducing an intermolecular crosslink with a crosslinking agent.

The collagen solution used for preparing the collagen sheet is prepared from collagen obtained from bovine dermis or the like, the pH is preferably adjusted to 2 to 4, and the concentration is 0.2 to 5 w / v%, preferably Is 0.5 to 2 w / v%
Is obtained. UV light used for crosslinking (U
The main wavelength of V) is preferably from 250 to 270 nm,
The amount of ultraviolet rays may be 500 to 12000 mWsec / cm ² , and preferably 1000 to 5000 mWsec / cm ² . Examples of the crosslinking agent used in the present invention include, for example, glutaraldehyde, ethylene glycol diglycidyl ether, polyglycerol polyglycidyl ether, glycerol polyglycidyl ether,
Hexamethylene diisocyanate and the like. The size and thickness of the collagen sheet are appropriately selected according to the condition of the wound surface.

The collagen gel used in the present invention can be produced by pouring a collagen solution into the same container as used for producing the sponge and applying heat or leaving it in an ammonia gas atmosphere to cause gelation. it can.

The collagen solution used for preparing the collagen gel is prepared from collagen obtained from bovine dermis or the like, and has a pH of preferably 2 to 4 and a concentration of 0.2 to 5 w / v%, preferably Is set to 0.5 to 2 w / v%, and gelation is performed in an atmosphere of an alkaline gas such as ammonia for several minutes to 2 hours as necessary. After gelation, pH
It is preferably washed with a buffer solution adjusted to pH 7.4 or the like or a neutral solution of collagen (pH
When 6.5 to 8) is gelled, the concentration is 0.1 to 1 w /
v%, preferably 0.2 to 0.5 w / v%, without using an alkaline gas or the like, leaving it at 36 to 40 ° C. to obtain a gel.

The main wavelength of ultraviolet light (UV) used for crosslinking is 2
It is preferably 50 to 270 nm, and the amount of ultraviolet rays is 500
１12000 mWsec / cm ² , preferably 1000
It is preferable to irradiate a dose of ５5000 mWsec / cm ² . Examples of the crosslinking agent used in the present invention include, for example,
Glutaraldehyde, ethylene glycol diglycidyl ether, polyglycerol polyglycidyl ether,
Glycerol polyglycidyl ether, hexamethylene diisocyanate and the like can be mentioned. The size of the collagen gel is appropriately selected according to the size of the wound surface. In addition, the thickness and shape of the collagen gel may be appropriately selected depending on the state of the wound surface to be applied, but the thickness is 0.2 to 30 mm, and the shape is a sheet, plate, rod, spindle, or both convex. It may be molded into a lens shape or the like.

When the cells are mixed to prepare a collagen gel culture skin, the cells are mixed with a collagen solution prepared at a low temperature from bovine dermis or the like, and the mixture is allowed to stand at 37 ° C.
It is made to gel by standing still.

The cultured skin of the present invention is prepared using a container having a projection or a container provided with a support having the projection so as to suppress shrinkage of the cultured skin. Close cultured skin can be obtained.

A support having projections according to the present invention;
The container including the support and the container having the protrusion are used when preparing a substrate, and the support or the container is removed from the substrate when the cultured skin is applied.

Further, a support having these projections,
The container or the like having the projections is designed so that the cultured skin can be supported so that it can be easily handled at the time of application.

Specifically, the projections can suppress the shrinkage of the cultured skin and can adhere the cultured skin to the support or the bottom of the container well, so that the fragile cultured skin can be easily handled. Therefore, the cultured skin is less likely to be damaged. In addition, since the container bottom portion having the support and the projections has an appropriate flexibility, it can be easily applied to a desired wound surface position. In order to further enhance the operability, preferably, the support is provided with a handle so that it can be easily taken out. More preferably, a hole is provided in the handle so that the support can be easily handled by gripping means such as tweezers.

It is necessary that the support or the container having the projections has a strength enough to prevent the shrinkage of the cultured skin, but the support or the container may be flexible so that it can be easily peeled off when the cultured skin is applied. is necessary.

The strength is required to prevent shrinkage of the cultured skin and to be hardly deformed by the shrinkage.
Further, the flexibility can be appropriately adjusted by the area, material, thickness, and shape.

The support having protrusions used in the present invention may be provided with protrusions on a sheet, a mesh-like sheet, a grid-like portion, or the like.

The shape, number, and size of the projections are not particularly limited in the present invention, but it is necessary that the shrinkage of the cultured skin be suppressed and that it can be easily peeled off at the time of application. The arrangement of the protrusions is preferably designed so as to have a structure in which the protrusions are arranged uniformly, whereby the shrinkage of the cultured skin can be favorably suppressed. For the same reason, it is necessary to dispose a projection at the periphery.

The height of the projection may be less than the thickness of the obtained substrate, but it is necessary to select the shape and form of the projection so that the entire surface of the substrate is not cut. In the case where the base material is in the form of a beam, a wall, or the like, it is necessary to provide the base material with a thickness smaller than the thickness of the base material so that the base material is not completely partitioned by these. The preferred height of the protrusion is not more than 2/3 of the thickness of the substrate. Further, the height of the projections is preferably at least ５ of the thickness of the substrate in order to effectively suppress shrinkage of the cultured skin.

As the shape, specifically, a beam shape, a wall shape, a pin shape, a wedge (v) shape, a cylindrical shape, a conical shape, a polygonal prism shape, a polygonal pyramid shape and the like are suitably used.

The protrusions may be arranged vertically upward with respect to the support, or may be inclined toward the periphery.

In addition, it is preferable that both ends of the support are bent so that the cultured skin is easily detached from the bottom surface of the support when the cultured skin is applied, and that the side surface is a handle. In the container for preparing cultured skin in which the projections are provided on the bottom surface, only the lower portion of the outer side wall, which is an extension of the bottom surface of the container, is located outside the inner side wall of the container so that the cultured skin can be easily taken out from the bottom surface of the container. Adhered to the lower part, the upper part of the outer side wall of the container is not adhered to the inner side wall, it is preferable that the container bottom part and the inner side wall of the container can be separated by holding the upper part of the outer side wall and tilting it outward. .

In the present invention, the "side wall" preferably comprises an inner side wall and an outer side wall which is an extension of the bottom surface, but at least a part thereof is separable from the bottom surface and is adhered to the bottom surface, or at least a part thereof. May be any of the side walls that are extensions of the bottom surface and can be separated from the bottom surface. Further, these side walls may be further modified so that the cultured skin obtained can be easily taken out.

Further, when a support is installed on the container and used, it is preferable that a hole is formed in the support as a handle so that the tip of a gripping means such as tweezers can be hooked. .

After placing such a support in a container suitable for preparing cultured skin and preparing a cultured skin substrate,
A cultured skin is prepared, and the support together with the cultured skin is removed from the container and applied to the wound surface.

On the other hand, a container having projections used for preparing cultured skin has the same projections on the bottom surface as those arranged on the support. The bottom surface is adapted to be separable from at least a portion of the side wall upon application of the cultured skin. Or
A portion of the side wall (for example, in the case of a rectangular container, a connection portion between the four side walls) can be separated so that the side wall can be tilted outward with respect to the bottom surface.

The support having the protrusions and the container in which the support is provided are made of, for example, plastic, stainless steel, aluminum, glass, ceramic, or the like. The support is preferably made of a material that is easily bent, such as a plastic sheet, stainless steel, or an aluminum sheet, so that the cultured skin can be easily separated from the support when the cultured skin is applied to the wound surface. Further, the container having the projections is preferably made of a plastic or aluminum sheet.

FIG. 1 shows an embodiment of the support of the present invention. A plurality of wedge-shaped projections 1 are provided on the bottom surface of the support 2. The support 2 is used while housed in the container 3. The upper side of the support 2 is bent to form a handle 61, and the handle 61 is provided with a hole 62 for hooking a gripping means.

FIG. 2 shows another embodiment, in which a plurality of square pillar-shaped projections 11 are provided on the bottom surface of a support 12 having a handle 71. The handle is provided with a hole 72 for catching the gripping means. The support is used while housed in the container 13.

FIGS. 4 to 6 show a case where a projection is provided on the bottom surface.
FIG. 4 shows various embodiments of the container (FIG. 4) or support (FIGS. 5-6) of the present invention. The projections in FIGS. 4 to 6 have a cylindrical shape, a lattice shape and a wall shape, respectively. FIG.
6, reference numerals 31, 41 and 51 denote protrusions, in FIG. 4, 35 denotes an outer side wall of the container 33, 36 denotes a bottom surface of the container 33, and 37 denotes an inner side wall of the container 33, respectively. When applied, the outer side wall 35 of the container, which is part of the bottom extension glued at the bottom with the inner side wall 37 of the container, is turned outward, separating the inner side wall 37 of the container from the bottom of the container,
Apply cultured skin. Also, in FIGS.
2 is a support, and 91 and 101 are handle parts.

The cultured skin substrate of the present invention is seeded with epidermal cells and / or fibroblasts derived from skin, preferably from mammalian skin, and cultured and propagated. The term "epidermal cells" refers to keratinocytes including basal cells and other cells usually present in the epidermal cell layer. Among them, keratinocytes are cultured and proliferated. The fibroblast is a major cell in the dermis, and refers to a cell that produces connective tissue components such as collagen and binds to these components to form connective tissue. The following three types of the cultured skin of the present invention can be prepared according to the cells to be seeded: Cultured skin in which only fibroblasts are seeded and cultured on the collagen substrate of the present invention (cultured dermal substitute) A cultured skin in which epidermal cells are seeded and cultured on the collagen substrate of the present invention (a cultured epidermal substitute), and a cultured skin in which fibroblasts are seeded and cultured on the collagen substrate of the present invention, and then epidermal cells are seeded and cultured (cultured skin) Alternative).

In this case, it is more preferable to first carry out the step of seeding and culturing fibroblasts, and then to carry out the step of seeding and culturing epidermal cells, since it is possible to prevent the falling off of the epidermal cells. It is also possible to carry out the step of seeding and culturing fibroblasts after seeding culture and propagation.

The above three types of cultured skins have a wound surface area,
The effect can be more effectively exhibited by properly using them according to the depth and the symptoms of the patient. In addition, these cultured skins can be properly used at home and at other homes.

For example, in the case of a widespread severe burn, it is necessary to reconstruct the dermis-deficient tissue after removing the necrotic burn site. This is to form a tissue rich in capillaries called benign granulation tissue in order to increase the engraftment rate of the autograft skin (reconstruction of the transplant bed). To this end, the graft bed is first reconstructed using the dermal substitute described above. At this time, it is preferable to reconstruct the transplanted bed with an in-house dermis substitute, but it is rational to use another dermis substitute in view of urgency and economy. Thereafter, the skin tissue including the epidermis is reconstructed using the autocultured skin substitute or the autogenous epidermis substitute. At this time, treatment can be performed by selecting a cultured skin substitute if the defect is deep, and selecting an epidermal substitute if not too deep.

The epidermal cells are prepared by the following procedure.
The skin (the epidermis and part of the dermis or the entire dermis) collected in a clean environment is disinfected and immersed in a buffer solution such as a saline solution containing an antibiotic or Hank's solution. This skin is treated with a dispase concentration of 1000 U
/ ML of Dulbecco's modified Eagle's minimum essential medium (DMEM) (hereinafter referred to as “dispase solution”), and then separated into epidermis and dermis. The obtained epidermis is immersed in a Hanks solution (hereinafter, referred to as “trypsin solution”) prepared at a trypsin concentration of 0.25 w / v% and a concentration of sodium ethylenediaminetetraacetate (EDTA) of 0.005 mM / mL at 37 ° C. for about 15 minutes. After that 10v / v%
DMEM containing fetal calf serum (FCS) (hereinafter referred to as "DM
EM + 10% FCS) and shake to disperse the cells.
It can be obtained by centrifuging for minutes for collection. The obtained epidermal cells are, for example, green (Gr).
een) A medium, NCTC168 medium, MCDB153 medium, particularly preferably a green medium is added to obtain an epidermal cell suspension. The green medium is DMEM and Ham '
s F-12 mixed 3: 1 with hydrocortisone (0.4 μg / mL), insulin (5 μg / mL),
Transferrin (5 μg / mL), triiodothyronine (0.0013 μg / mL), cholera toxin (0.01
μg / mL), adenine (24.3 μg / mL), epidermal growth factor (0.01 μg / mL) and antibiotics,
Epidermal cell growth medium containing 10% FCS (Cell, Vol. 40, pp. 677-683, 1985)
March).

To grow the epidermal cells with high efficiency,
For example, 3T which is a mouse-derived fibroblast whose proliferation ability has been stopped by mitomycin treatment or irradiation.
Culture is preferably performed in a culture flask in which three cells or the like are established as supporting cells.

Specifically, after culturing the 3T3 cells in a culture flask, the medium is removed, and the medium is rinsed with Hanks' solution to remove it. Then 4 μg of mitomycin C
The DMEM solution containing / mL is added so that the whole cells are sufficiently immersed (in a 75 cm ² flask, about ² to 4 mL is preferable). The mixture is allowed to stand at 37 ° C. for about 2 hours, and then washed with a buffer to remove mitomycin C. Thus, 3T3
The cells remain alive and only proliferative capacity is stopped. The 3T3 cells having no proliferation ability are collected, suspended in the green medium, and 1 × 10 ^{3 to} 5 × 10 ⁴ cells / c.
m ² , preferably 5 × 10 ^{3 to} 3 × 10 ⁴ cells / c
After adjusting to a density of m ^2, the cells are seeded in a culture flask. After inoculating 3T3 cells in this culture flask, the epidermal cells were added to 5 × 10 ^{3 to} 5 × 10 ⁵ cells / c.
m ² , preferably 1 × 10 ⁴ to 2 × 10 ⁵ cells / c
Seed at a cell density of m ² .

For example, the above-mentioned epidermal cells obtained from about 2 × 2 cm of separated skin were placed in a 5% CO ₂ incubator at 37 ° C.
Culture at Since the 3T3 cells float in the medium from the bottom of the culture flask in the process of forming the colonies while the culture continues and are removed when the medium is replaced, the 3T3 cells are hardly contained in the finally obtained epidermal cells. .

The cultured and grown epidermal cells are detached by adding a dispase solution and allowing to stand at 37 ° C. for about 2 hours.
This is added to the trypsin solution to disperse the epidermal cells, and then collected, and a green medium is added to obtain a cell suspension. Epidermal cells are subcultured as needed. Specifically, the required number of the supporting cells are prepared, and the epidermal cells cultured and grown as described above are collected and seeded and cultured on the supporting cells. At this time, subculture is performed so that epidermal cells are not keratinized. The obtained epidermal cells were generated by suppressing the shrinkage in the container equippedwith a support having a projection, for example of about 6 × 9.5cm, the collagen sponge substrate with a thickness of 3mm, 5 × 10 ⁴ ~ Seed at a cell density of 2 × 10 ⁵ cells / cm ² .

After the epidermal cells have adhered to the substrate, a green medium is added and the cells are cultured in a 5% CO ₂ incubator at 37 ° C. for 7 to 21 days while changing the medium every three days. In this way, a cultured epidermal substitute is obtained.

The fibroblasts are obtained by separating the collected skin into epidermis and dermis in the same manner as described above, crushing the obtained dermis using scissors, a homogenizer, etc., to obtain 0.5 w / v.
% Collagenase DMEM solution (hereinafter referred to as “collagenase solution”), shaken at about 37 ° C. for about 6 hours to dissolve the connective tissue, and then about 400 × g to about 1,000 × g, preferably about 600 × g to about 800 ×
Collect by centrifugation at g. The fibroblasts obtained are D
The cells are cultured in a culture flask at 37 ° C. in a CO ₂ incubator using MEM + 10% FCS or the like as a medium at 37 ° C., and subcultured to obtain as many fibroblasts as necessary.

The cultured fibroblasts were detached from the culture flask using a trypsin solution, collected, and collected in DMEM + 1.
Prepare in 0% FCS. 5 × this fibroblast suspension
10 ^{3 to} 5 × 10 ⁵ cells / cm ² , preferably 1 ×
The cells are seeded on a collagen substrate at a cell density of 10 ^{4 to} 2 × 10 ⁵ cells / cm ² . Alternatively, fibroblasts are suspended in an aqueous solution of neutral collagen, poured into the container, gelled at 37 ° C., and incorporated into a collagen gel. After the cells adhere to the substrate by such an operation, DMEM + 1
Add 0% FCS, 37 in 5% CO ₂ incubator
Cultivation is performed for 1 to 21 days at 3 ° C. while changing the medium every 3 days. In this way, a cultured dermal substitute can be obtained. The cultured dermal substitute is obtained by culturing and growing fibroblasts on a substrate.

A cultured skin substitute having both epidermal cells and dermal cells is prepared as follows. After fibroblasts are inoculated in the above-described step of preparing a cultured dermis substitute and the cells are well adhered to a substrate, the step of preparing a cultured epidermal substitute is performed to obtain a cultured skin substitute.
The cultured skin substitute thus obtained is preferably washed with DMEM or the like before being applied to the wound surface, and replaced with a similar medium without FCS.

FIG. 3 shows the most useful embodiment of the method for producing cultured skin of the present invention. A plastic support 22 provided with a projection 21 is provided on a transparent plastic container 23 (with a lid 9), and the height of the projection is about 2/3 of the thickness of the cultured skin, for example, the cultured skin. If the thickness is 5 mm, the thickness is 1 to 4 mm, preferably 2 to 3.5 mm. The arrangement is always provided at the periphery of the support, that is, at the periphery of the cultured skin. Four square pillars each having a cross section of 1 mm on each side are provided on a square having a side of 15 mm. The long side end of the support is bent at the top of the case,
The handle portion 81 is prepared so that the support can be easily taken out by hand, and a hole of about 3 mm (φ) is provided in the upper portion of the handle so that the support portion can be easily taken out by a gripping means such as tweezers. The support is, for example, 0.7 mmP
Select a material having an appropriate flexibility and a thickness such as a VC sheet.

Atelocollagen aqueous solution 4 containing 1 w / v% (pH 4), homogenized and containing air bubbles was placed in a container provided with a support for preparing cultured skin provided with such projections.
(See FIG. 3 (a)), gelled in an ammonia gas atmosphere, freeze-dried to produce an atelocollagen sponge 7, and then sterilized with ethylene oxide gas (see FIG. 3 (b)).

The fibroblast suspension 8 was seeded on the atelocollagen sponge prepared in the container (FIG. 3 (c)).
), The medium 10 is added, the lid 9 is closed, and the cells are cultured in a CO ₂ incubator at 37 ° C for 2 weeks (see (d) in FIG. 3). At the time of application, after the medium is removed and replaced with DMEM, the holding means 63 such as tweezers is inserted into the hole 8 of the handle 81.
2, the obtained cultured dermis substitute is taken out of the container together with the support (see FIG. 3 (e)), the support is bent, and the cultured dermis substitute is moved to the wound surface in the direction of the arrow using the gripping means 63. Apply (see FIG. 3 (f)).

The hyaluronic acid used in the cultured skin of the present invention is a physiologically active substance produced from cells at an early stage when a wound or the like is inflicted. In the process of healing the wound surface, epidermal cells and fibroblasts are produced. All are said to be necessary for good movement on the collagen substrate. Hyaluronic acid is thought to play a function of facilitating cell migration by loosening the cell scaffold.

Hyaluronic acid is used at a concentration of 0.1 w / w% to 5 w / w% of collagen when preparing a collagen base material.

For better adhesion of cells to the substrate, fibronectin, laminin, or the like as a cell adhesion factor is coated on the substrate of the present invention to obtain a more favorable cultured skin.

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

[0070]

【Example】

Examples 1 to 3 and Comparative Example 1 Preparation of Cultured Dermal Replacement (Base Material: Gel) Using a Support or Container Having a Protrusion According to the Present Invention A piece of human skin collected in a clean environment ( About 2 × 2c
m) is immersed in an isodine solution (manufactured by Meiji Seika Co., Ltd.), washed with Hanks solution, and then streptomycin (1000 μg /
mL), penicillin (1000 U / mL) and amphotericin B (2.5 μg / mL) mixed in a Hanks solution at room temperature for 30 minutes.

Next, dispase (Kodo Shusei Co., Ltd.)
Immersed in 10 ml of the solution at 4 ° C. for 12 hours, separated into epidermis and dermis using tweezers, and the obtained dermis portion was crushed into a paste with scissors to give a collagenase (manufactured by Wako Pure Chemical Industries, Ltd.) solution. After treatment with 10 ml for about 6 hours at 37 ° C. to remove connective tissue, fibroblasts were obtained by centrifugation at about 700 × g for 5 minutes. The fibroblasts obtained are D
5% C using MEM (manufactured by Gibco) + 10% FCS
The cells were subcultured and grown in plastic culture flasks at 37 ° C. in an O ₂ incubator while changing the medium every three days.

[0072] After this, peeled fibroblasts from culture flasks using trypsin solution, was collected to allow seeded at 1 × 10 ⁵ cells / cm ² cell density in the cultured skin fabrication vessel.

The collected fibroblasts were added to a 0.2 w / v% atelocollagen DMEM solution (pH 7.4, manufactured by Koken Co., Ltd.), and mixed to prepare an atelocollagen suspension of fibroblasts. 20 mL of each was poured into the cultured skin preparation containers of 2), 3) and 4).

1) Square-shaped projections each having a height of 2 mm and a side of 1 mm are arranged at intervals of 15 mm parallel to the long side and 1 mm parallel to the short side.
4mm interval, the center of the outermost projection is 1.5 from the periphery
mm, a plastic container (6 × 10 mm) provided with a support (59 × 93 mm) as a plastic sheet
0 × 95 mm, height 21 mm) (see FIG. 2).

2) A plastic container (60 × 95 mm, height 21 mm) provided with a support (59 mm × 93 mm) provided with a V-shaped projection on a plastic sheet. Make a V-shaped cut of 2 mm on each side of the plastic sheet,
At 45 ° to 60 ° with respect to the sheet, the projection was raised toward the periphery to form a projection. As shown in FIG. 1, the V-shaped projections were alternately arranged at intervals of about 14 mm parallel to the long side and at intervals of about 17 mm parallel to the short side, and the center of the outermost projection was located at a position about 4 mm from the periphery (see FIG. 1). ).

3) The lower portion of the outer side wall, which is an extension of the bottom surface of the container provided with columnar projections, is adhered to the lower portion of the inner side wall of the container. Is separated from the plastic container (60 x 95m
m, height 21 mm) (see FIG. 4).

4) The plastic container (60 × 95) used in the above 1) or 2) which does not include a support having protrusions
mm, height 21 mm).

After that, the mixture was allowed to stand at 37 ° C. for 1 hour to form a gel, and an atelocollagen gel into which fibroblasts having a thickness of about 3 mm were incorporated was obtained. The DMEM + 10% FCS15
ml in a 5% CO ₂ incubator at 37 ° C.
Culture for 14 days, changing the medium every 3 days,
A cultured dermis substitute was obtained.

The cultured dermis substitutes (Examples 1, 2 and 3 respectively) of the present invention prepared in the containers 1) to 3) are the containers in which the projections of 4) are not provided. As shown in Table 1 below, shrinkage was markedly suppressed as compared with the cultured dermis substitute obtained in Comparative Example 1 (Comparative Example 1). The area change was determined by comparing the area of the upper surface of the substitute parallel to the bottom surface of the container with respect to the area (a) at the time of preparing the collagen gel mixed with fibroblasts after 7 days of culture and 14 days after culture (after 7 days). Area (b), area after 14 days (c))
Percentage of

[0080]

(Equation 1)

Are shown. Table 1 shows the area change for the cultured dermal substitute.

[0082]

[Table 1]

The cultured dermis substitute prepared in the above containers 1) and 2) can be easily taken out together with the support by hooking tweezers on a hole formed in a portion where the upper side of the support is bent and lifting it. Was. In addition, the cultured dermis substitute prepared in the container of the above 3) can be easily prepared by turning a part of the upper portion of the outer side wall of the container, which is an extension of the container bottom surface, outward, and separating the inner side wall and the bottom surface of the container. I took it out.

The cultured dermis substitute prepared in the containers 1), 2) and 3) above is tightly supported by a support or a projection provided on the bottom surface of the container up to the position where the wound surface is to be applied. Therefore, it was easy to carry, and at the time of application, one end of the cultured dermis substitute was easily pinched with forceps, and at the same time, the support or the bottom of the container could be easily peeled off by bending, so that it could be placed at a desired wound surface position.

On the other hand, the cultured dermis substitute prepared in the container described in 4) was partially damaged when picked up with tweezers from the container, and was difficult to handle because it could not be lifted.

Examples 4 to 6 and Comparative Example 2 Preparation of atelocollagen sponge using support having protrusions according to the present invention Aqueous solution of atelocollagen (manufactured by Koken Co., Ltd.) prepared at 1 w / v% (hydrochloric acid) Adjust to pH 4 with) 10
0 mL was stirred for 1 minute using a homogenizer (manufactured by Nippon Seiki Seisaku-sho, Ltd.) to introduce air bubbles. After allowing to stand in a gas atmosphere for 2 hours, freeze-drying was performed to obtain an atelocollagen sponge having a thickness of about 3 mm (Example 4, Example 5, Example 6, and Comparative Example 2 respectively). Next, one side of the sponge was irradiated with UV at 1 mW / cm ² for 30 minutes, and then sterilized with an ethylene oxide gas in a plastic container.

Examples 7 to 9 and Comparative Example 3 Preparation of cultured dermal substitute (substrate: sponge-like) using container having protrusions according to the present invention 1 was added to the collagen sponge of each container prepared in Examples 4 to 6 and Comparative Example 2.
The cells were collected so that they could be seeded at × 10 ⁵ cells / cm ² , and suspended in the DMEM + 10% FCS.

The collagen sponge prepared in each container was
5 mL of DMEM + 10% prior to seeding fibroblasts
Dipped in FCS.

Thereafter, DMEM + 1 of the fibroblast was used.
5 mL of the 0% FCS suspension was dropped on the collagen sponge prepared in each container (seeding density: 1 × 10 ⁵ cell)
s / cm ² ). After leaving this to stand in a clean bench for 5 hours, DMEM + 10% FCS was added and 5% CO2 was added.
_The cells were cultured in a ₂ incubator at 37 ° C for 14 days while changing the medium every three days to obtain a cultured dermal substitute.

When the area change was calculated and compared in the same manner as in Examples 1 to 3 and Comparative Example 1, the cultured dermal substitutes prepared in the containers 1), 2) and 3) were used (Examples 7 and 8 respectively). In (9) and (9), the shrinkage was suppressed as compared with the cultured dermis substitute (Comparative Example 3) prepared in the container of (4).
Table 2 shows the calculated change in area.

The cultured dermis substitute prepared in the containers 1) and 2) can be easily taken out together with the support by hooking tweezers on a hole formed in a portion where the upper side of the support is bent and lifting it. Was. In addition, the cultured dermis substitute prepared in the container of the above 3) can be easily prepared by turning a part of the upper portion of the outer side wall of the container, which is an extension of the container bottom surface, outward, and separating the inner side wall and the bottom surface of the container. I took it out. The cultured dermis substitute prepared in the above containers 1), 2) and 3) is easy to carry because it is tightly supported by a support or a projection provided on the bottom surface of the container to the position where the wound surface is to be applied when applied. At the time of application, one end of the cultured dermis substitute was easily pinched with forceps, and at the same time, the support or the bottom surface of the container was bent to be easily peeled off and placed at a desired wound surface position.

On the other hand, the cultured dermis substitute prepared in the container of 4) was difficult to pick up from the container with tweezers and was difficult to handle.

Example 10 and Comparative Example 4 Preparation of cultured skin substitute (substrate: sponge-like) using container having projections according to the present invention Epidermal cells were collected from humans collected in a clean environment. The epidermis separated from the skin (about 2 × 2 cm) in the same manner as described in Example 1 was transferred to 10 ml of a trypsin solution, treated therein at 37 ° C. for 15 minutes, and then used in Example 1. The cells were dispersed in the same DMEM + 10% FCS, shaken to disperse the cells, collected by sedimentation by centrifugation at about 400 × g for 5 minutes, and suspended in a green medium having the above composition. In order to grow epidermal cells with high efficiency, the following feeder cells were used. 3T3 cells, which are mouse-derived fibroblasts, were DMEM + 10
5% CO _{2 in} % FCS until sub-confluent
The cells were cultured at 37 ° C. in an incubator. Then, the medium was removed and rinsed with Hanks' solution, and the same DM as in Example 1 was used.
A physiological saline solution (0.1 mg / mL) containing mitomycin C (manufactured by Wako Pure Chemical Industries, Ltd.) was added so that the final concentration was 0.0004% by adding EM. After the culture flask was allowed to stand at 37 ° C. for 2 hours, it was washed with Hanks' solution to remove mitomycin C, and 3T3 cells whose growth ability was stopped were collected. The obtained cells were suspended in a green medium, counted, adjusted to a density of 2 × 10 ⁴ cells / cm ² , and seeded in a culture flask.

On the day after the 3T3 cells thus prepared were seeded, the above-mentioned epidermal cells were seeded on the 3T3 cells.
Cultures were grown in a ₂ % CO ₂ incubator.

On the other hand, fibroblasts were added to the atelocollagen sponge obtained in each of the containers 1) and 4) in the same manner as in Example 4 and Comparative Example 2 by 2 × in the same manner as in Example 7 and Comparative Example 3. The cells were seeded at a density of 10 ⁴ cells / cm ² , added with the above-mentioned DMEM + 10% FCS, and cultured in a CO ₂ incubator for 2 days. Next, the medium was removed, and the epidermal cells grown and cultured were placed thereon at 1 × 10 ⁵ ce.
Seeded at a density of 11 s / cm ² . After leaving this on a clean bench for 5 hours, the green medium was added thereto for 5 hours.
The cells were cultured for 14 days in a% CO ₂ incubator at 37 ° C. while changing the medium every three days to obtain a cultured skin substitute.

When the area change was calculated and compared in the same manner as in Examples 1 to 3 and Comparative Example 1, the cultured skin substitute (Example 10) produced in the container of 1) was replaced with the container of 4). Shrinkage was suppressed as compared to the prepared cultured skin substitute (Comparative Example 4). Table 2 shows the calculated change in area.

The cultured skin substitute prepared in the container described in 1) above was easily taken out together with the support by hooking tweezers on a hole formed in a handle having a bent upper side of the support and lifting the same. In addition, the cultured skin substitute prepared in the container of the above 1) is easy to carry because it is tightly supported by the projections set on the support up to the position where the wound surface is applied when applied, and the cultured skin substitute is applied when applied. The one end of the object was gently pinched with tweezers, and at the same time, the support was bent by bending the support so that the object could be easily peeled off and placed at a desired wound surface position.

On the other hand, the cultured skin substitute prepared in the container of 4) was difficult to pick up from the container with tweezers and was very difficult to handle, as in Comparative Example 3.

Example 11 and Comparative Example 5 Substrates for cultured skin containing hyaluronic acid (sponge-like)
Preparation of Cultured Dermal Substitute Using Ethanol 1 g of atelocollagen (manufactured by Koken Co., Ltd.) was dissolved in distilled water adjusted to pH 3 with hydrochloric acid, and an aqueous solution adjusted to pH 4.5 with an aqueous sodium hydroxide solution was obtained. Then sodium hyaluronate (molecular weight about 2,000,000) 0.01
g to this atelocollagen aqueous solution,
Distilled water adjusted to 4.5 was added to make 100 mL (atelocollagen 1 w / v%, hyaluronic acid 0.01 w / v).
v% solution). After homogenizing this for 1 minute, an atelocollagen-hyaluronic acid sponge containing hyaluronic acid was prepared in the containers 1) and 4) in the same manner as in Example 4 and Comparative Example 2.

A cultured dermis substitute (Example 11 and Comparative Example 5, respectively) was prepared in the same manner as in Example 7 and Comparative Example 3. Shrinkage and ease of handling were almost the same as in Example 10 and Comparative Example 4. Table 2 shows the area change calculated in the same manner as in Example 7 and Comparative Example 3.

[0101]

[Table 2]

[0102]

According to the present invention, cultured skin can be prepared inexpensively and easily by suppressing the shrinkage of the substrate for cultured skin, and can be safely supported without causing damage to the obtained cultured skin and applied to the wound surface. Facilitates handling when applying

[Brief description of the drawings]

FIG. 1 is an explanatory view of one embodiment of a support of the present invention,
(A) is a plan view, (b) is a cross-sectional view taken along line AA of (a), (c) is a side view of a container in which a support for preparing cultured skin is stored, and (d) is It is a side explanatory view showing the state where the above-mentioned support was installed in the container.

FIG. 2 is an explanatory view of another embodiment of the support of the present invention, wherein (a) is an explanatory plan view, (b) is an explanatory sectional view taken along the line BB of (a), and (c) is a culture. It is a side view of a container which stores a support for skin preparation, and (d) is a side view showing a state where the support is installed in the container.

FIG. 3 is an explanatory diagram showing a procedure for producing a cultured skin (cultured dermal substitute) using a container provided with a support of the present invention.

FIG. 4 is an explanatory view of one embodiment of the container of the present invention,
(A) is an explanatory plan view, (b) and (c) are C in (a).
FIG. 4 is a sectional view taken along line C. (C) shows a step of removing the cultured skin from the container of the present invention.

FIG. 5 is an explanatory view of one embodiment of the support of the present invention,
(A) is a plane explanatory view, (b) is a DD sectional view explanatory drawing of (a).

FIG. 6 is an explanatory view of one embodiment of the support of the present invention,
(A) is a plane explanatory view, (b) is an EE line sectional explanatory view of (a).

[Explanation of symbols]

 1, 11, 21, 31, 41, 51 Protrusion 2, 12, 22, 42, 52 Support 3, 13, 23, 33 Container 4 Atelocollagen aqueous solution 7 Atelocollagen sponge 8 Fibroblast suspension 9 Lid 10 Medium 35 Outer side wall of container 36 Bottom surface of container 37 Inner side wall of container 61, 71, 81, 91, 101 Handle 62, 72, 82 Hole 63 Gripping means

Claims (7)

[Claims] 1. a) A cultured skin substrate comprising a collagen sponge, a collagen sheet or a collagen gel,
Preparing on a support having a protrusion or in a container to suppress shrinkage of the substrate, b) seeding and culturing skin-derived cells on the substrate to obtain a cultured skin, and c) culturing at the time of application A method for producing a cultured skin, comprising a step of peeling the skin from the support or the container, and the steps a) and b) may be performed simultaneously. 2. A support for preparing a cultured skin, wherein a support is provided on the surface. 3. The support according to claim 2, which has flexibility so that the cultured skin is easily peeled off. 4. A container for producing cultured skin, comprising a side wall and a bottom surface, a projection provided on the bottom surface, and at least a part of the bottom surface and the side wall being separable. 5. The container according to claim 4, wherein the bottom surface has flexibility so that the cultured skin is easily peeled off. 6. A cultured skin substrate comprising a collagen sponge, a collagen sheet or a collagen gel produced on a support or in a container having projections smaller than the thickness of the cultured skin substrate to be produced and having reduced shrinkage. . 7. A cultured skin produced by the method according to claim 1 using the substrate for cultured skin according to claim 6.