JPH05317406A - Artificial skin - Google Patents

Abstract

PURPOSE:To produce an artificial skin base material allowed to implant onto a part of a lost skin by making a non-proliferative fibroblast derived from an animal on a porous membrane base material while a signal layer of a skin base cell yet to be differentiated is arranged. CONSTITUTION:A 3T3 cell which is made non-prolefirative by irradiation with gammarays is seeded onto a porous membrane base material to be cultivated and then, a skin cell is seeded to be incubated at a specified temperature. As a result, the skin cell yet to be differentiated is arranged to be a signal layer to obtain an artificial skin that allows the implantation of a cell portion onto a wound face of a part of a lost skin without peeling cells off the porous membrane. An example of the porous membrane is that made of polyolefin or the like produced by polymerizing one polymer or more selected from alkoxy alkyl acrylates. A sample of the fibroblast existing on the porous membrane is mouse lung fibroblast, (3T3)., human skin fibroblast or rat skin fibroblast.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel artificial skin used to heal skin wounds such as burns and a method for producing the same.

[0002]

2. Description of the Related Art Animal cell culture technology is used as a means of producing interferon, lymphokines, physiologically active substances such as various growth hormones and cell growth factors, biologically derived materials and therapeutic vaccines, or as a basis for cell-incorporated artificial organs. It is an indispensable technique, and in recent years, a high-density culture method for producing these substances with high efficiency and in a large amount has attracted attention.

In animal cells, particularly in high-density culture of "adhesion-dependent cells", which adhere to an artificial or natural substrate for survival, proliferation, or production of a target substance, the surface of the substrate on which the cells are cultured is the cell. It is necessary that the surface has excellent adhesiveness and proliferation. Also, in order to maintain the survival of cells and the ability to produce target substances, it is important to maintain a favorable environment for cells. Therefore, it is necessary to continuously supply nutrients and enzymes and remove waste products. Will be.

Regarding the former cell adhesion to the surface of the base material, it is known that the polystyrene-based material usually used as a plastic dish for tissue culture and the surface having an appropriate charge density are excellent. JP-A-2-169
No. 72 discloses a porous film having a cross-linked styrene-divinylbenzene polymer retained on the surface thereof, and a surface having an appropriate charge density is described in Journal of
Theological Biology (Journal of Theological
Biology), Vol. 49, pages 417-424 (19).
75): Adhesion and Spreading of Cells on Charged Surfaces
and Spreading of Cells on Charged Sarfaces). In addition, various polymer materials whose surfaces are subjected to surface treatments such as low temperature plasma treatment, sputtering treatment, ultraviolet treatment, electron beam treatment, and coating treatment of a monomolecular accumulated film (Japanese Patent Publication No. 58-32589, JP-A No. 63-19896).
7, JP-A-63-198977, JP-A-1-15133.
Etc.), or materials treated with cell adhesion factors or cell growth factors such as collagen, fibronectin, hydronectin, and laminin (for example, Trans American Society of Artificial Organs (Trans A
American Society of Artificial Organs (1987),
Volume 33, Page 6, Scholarly Reviews (Schola
rly Reviews) “Cells Adhesion to Bimaterials”) is also being considered.

However, when polystyrene is used as the polymer material and subjected to the above-mentioned surface treatment, polystyrene has good cell adhesiveness, but its physical properties are weak, so that a thin film-like material or The drawback is that it is difficult to form a porous membrane suitable for high-density culture. In addition, when the cell culture substrate has a special shape of “porous membrane” with a thickness of about 100 μm, the surface treatment that is generally performed causes “deterioration of physical properties of membrane” and “blocking of pore diameter”. There were many problems.

Further, treatment of the surface of the base material with collagen, an intercellular matrix (ECM), a cell adhesion factor or the like improves the adhesion and spreadability of cells such as hepatocytes.
These methods have a drawback that the coating process must be performed aseptically before use, which makes the operation complicated. Further, the above method is not preferable also from the viewpoint of increasing the possibility of contamination by various bacteria. Furthermore, considering the production of useful substances by high-density culture, a large amount of membranes is used, so that the required amounts of collagen and cell adhesion factors increase, which poses a problem from an economical point of view.

On the other hand, a method using a porous membrane is considered to be excellent for maintaining the growth environment of high density cultured cells. That is, by stacking porous flat membranes or bundling hollow fibers, it is possible to secure a large surface area within a limited volume. It is possible to maintain a good growth environment by continuously diffusing through the pores or by forcing by the pressure difference between the membranes. In particular, in the case of cells having polarity such as epithelial cells and endothelial cells, it is preferable for the cells that absorption and metabolism of nutrients be performed from the side of the adhesion substrate as in physiological conditions.

[0008]

The skin is the only organ that is in direct contact with the outside world, and has a basal cell layer having a dividing ability in the lowermost layer and a barbed layer formed by gradually differentiating the basal cells in the upper layer.・ It is composed of a granular layer and the outermost stratum corneum. As the basal cells differentiate, the cells flatten, accumulate keratin in the cytoplasm, and finally become keratin completely, and are excreted to the outside world. Thus, in basal cells of epithelial system such as skin, there is an intracellular polarization in which nutrients are supplied from the dermis side and excreted toward the stratum corneum. Due to such characteristics, the epidermal basal cells cannot be supplemented with nutrients from the adhesive surface with the substrate when cultured on a plastic petri dish, and gradually peel off from the substrate and die when the culture is continued. Therefore, in order to take advantage of the characteristic of the epidermal cell, which is the intracellular polarization, it is necessary to be able to take in nutrients from the part where the cells are attached. Therefore, a substance-permeable substrate for cell culture is desired. It has been reported that a membrane made of collagen was capable of retaining epidermal basal cells for a long period of time, compared to a collagen substrate (a plastic petri dish coated with collagen) that does not have substance permeability (Journal Cell Science, Journal Cell Science). , No. 91, pages 491-499 (1988)). However, the collagen membrane is not permeable to nutrients to supplement nutrients, is physically unstable, and may be degraded by collagenase produced by epidermal basal cells.

[0009] In recent years, there has been performed a culture transplantation method or a transplantation method of a material similar to skin which can be called cultured skin.
This is for severe patients, and the skin of the remaining normal site of the patient is collected, and the isolated cells are cultured in a test tube to grow to several tens to several hundred times of the original cells. Later,
It is intended to be cured by being transplanted to a wound site of a patient together with a coating film to form epidermis. As an example of clinical application of this method, a study by [GGGallico et al.,] (New England Journal of Medicine, 311, No. 7, 448-451) [New En
gland Journal Medicine, vol 311, No7, p448〜451 (19
84)].

On the other hand, a cell culture substrate using an artificial material is commercially available as a material that compensates for the above-mentioned drawbacks of the collagen membrane. However, the adhesiveness of epidermal basal cells is low, and the advantage of the substance-permeable membrane in the artificial material. There is not yet a supplement.

[0011]

In view of the above circumstances, the present invention presents an artificial skin substrate capable of culturing the epidermal cells of a transplanter on a substrate in vitro and directly transplanting it to a skin defect site. The purpose is to do.

The above object is achieved by the present invention having the following constitution.

(1) A non-proliferative animal-derived fibroblast is present on a porous membrane substrate, and undifferentiated epidermal basal cells are present in a monolayered state, and the cells are porous. An artificial skin that can be transplanted from the cell side to the wound surface of the skin defect without peeling from the membrane.

(2) The artificial skin according to (1), wherein the fibroblasts are selected from at least one of mouse lung fibroblasts (3T3), human skin fibroblasts and rat skin fibroblasts.

(3) At least one selected from the group consisting of a polyolefin in which the porous film is graft-polymerized with one or more polymers selected from alkoxyalkyl acrylates and a polyolefin in which some or all of the hydrogen is halogenated. The artificial skin according to (1) or (2), which is mainly composed of species.

(4) The average pore diameter of the porous membrane is 0.01 to
The artificial skin according to any one of (1) to (3), which has a thickness of 1.0 μm.

(5) 0.0 gamma rays were applied to the porous membrane substrate.
3T3 cells that had been irradiated with 3 Mrad to make them non-proliferative were seeded and cultured at 1 × 10 ⁴ to 1 × 10 ⁵ cells / cm ² ,
Further, the epidermal cells were added to 1 × 10 ⁴ to 1 × 10 ⁵ cells / cm ² (1
% Suspension in calcium-free DME medium containing fetal bovine serum) and incubating at 37 ° C to undifferentiate epidermal cells into a monolayer without removing the cells from the porous membrane. A method for producing artificial skin capable of transplanting the cell side to the wound surface of a skin defect.

The present invention can also be achieved by the following constitution.

(6) Undifferentiated epidermal basal cells are present as a monolayer on the porous membrane substrate, and the cell side is transplanted to the wound surface of the skin defect without peeling the cells from the porous membrane. Artificial skin that can be.

(7) The porous film is selected from the group consisting of polyolefins graft-polymerized with one or more polymerizable monomers selected from alkoxyalkyl acrylates and polyolefins in which some or all of the hydrogen is halogenated. The artificial skin as described in (6), which contains at least one kind as a main component.

(8) The average pore diameter of the porous membrane is 0.01 to
The artificial skin according to (6) or (7), which has a thickness of 1.0 μm.

(9) The artificial skin as described in any one of (6) to (8), wherein the porous film is subjected to plasma discharge treatment.

(10) 1 × 10 ⁴ to 1 × 10 ⁶ cells / cm ² (suspended in calcium-free DME medium containing 1% fetal bovine serum) of epidermal basal cells on a porous membrane substrate. By seeding at a concentration and further incubating at 37 ° C, undifferentiated epidermal cells can be monolayered, and the cell side can be transplanted to the wound surface of the skin defect without peeling the cells from the porous membrane. Method for producing simple artificial skin.

In the present invention, the substance-permeable membrane is preferably a porous membrane having through holes having an average pore size of 0.01 to 1.0 μm. Such a film is not particularly limited, but is preferably a polyolefin such as polypropylene or a halogenated polyolefin such as polyvinylidene fluoride, and the above-mentioned porous film is hydrophobic, so that it is hydrophilized. Is desirable. Other membranes include nylon, polysulfone, polyvinyl terephthalate, nitrocellulose, regenerated cellulose, cellulose acetate and the like.

In the artificial skin according to the present invention, non-proliferative animal-derived fibroblasts treated with γ-rays or mitomycin C are present on a porous membrane substrate, and undifferentiated epidermal basal cells are present. It is a monolayer, and enables the cell side to be transplanted to the wound surface of the skin defect portion without peeling the cell from the porous membrane. It is desirable that the porous membrane used in this case has good cell adhesion, fragility, and hydrophilicity by being surface-grafted with a polyalkoxyalkyl acrylate having excellent cell growth property, hydrophilicity, and flexibility. In addition, since polyalkoxyalkyl acrylate has moderate hydrophilicity without having a cationic or anionic polar group in the molecule, the protein adsorbed on the surface of the material also has electrostatic interaction or hydrophobic interaction. The action becomes mild, so that a good growth environment for cells is easily maintained.

Further, when a polymer having an alkoxyalkyl acrylate as a constituent component is introduced as a graft chain onto the surface of the substrate by a covalent bond, the polymer layer is different from coating or insolubilization using a crosslinking agent. It does not elute or peel off. In addition, if the base material is a hydrophilized porous membrane, it can be used without prior hydrophilization, and the culture solution can penetrate and pass through the pores of the membrane. It enables high-density culture on the membrane.

In the presence of non-proliferative animal-derived fibroblasts treated with γ-rays or mitomycin C, a small amount of epidermal basal cells may be seeded. In order to produce this artificial skin, first, a part of the patient's skin to be transplanted is exfoliated with a dermatome to divide it into epidermis and dermis, and it is preliminarily made to have a nonproliferative state on a porous membrane substrate by γ-ray treatment. 3T3 cells were seeded at 10 ⁴ to 1 × 10 ⁵ cells / cm ² ,
After culturing for 1 day, the epidermal basal cells were further added to 10 ⁴ to 1 × 10 ^5.
Cells were seeded in the range of cells / cm ² , and particularly when calcium-free DME medium containing 1% fetal bovine serum was used and cultured at 37 ° C for about 3 to 7 days, 3T3 cells spontaneously formed a porous membrane substrate. Undifferentiated epidermal cells, which have detached from the top and directly formed into a monolayer, adhere to the porous membrane substrate so that the cell side can be transplanted to the wound surface of the skin defect without peeling the cells from the porous membrane. It is possible to obtain artificial skin.

In the artificial skin according to the present invention, undifferentiated epidermal basal cells are monolayered on the porous membrane substrate,
It is possible to transplant the cell side to the wound surface of the skin defect portion without peeling the cell from the porous membrane. The porous membrane used in this case is a polyolefin which is graft-polymerized with one or more polymerizable monomers selected from alkoxyalkyl acrylates and a part or all of them in order to impart cell growth property, hydrophilicity and flexibility. It is preferable that the main component is at least one selected from the group consisting of polyolefins in which hydrogen is halogenated. To prepare the artificial skin, a portion of the patient first to be transplanted were detached with dermatome, divided into epidermis and dermis, further epidermal basal cells in the range of ^{1 × 10 4 ~1 × 10 6} cells / cm 2 Undifferentiated epidermal cells can be monolayered by seeding on a porous membrane substrate and incubating at 37 ° C, especially when using calcium-free DME medium containing 1% fetal bovine serum, It is possible to obtain an artificial skin in which the cell side can be transplanted to the wound surface of the skin defect portion without peeling the cell from the porous membrane.

The fibroblasts used in the present invention are not particularly limited, but preferably mouse lung fibroblasts (3T
3) Human dermal fibroblasts and rat dermal fibroblasts are good.

The epidermal basal cells used in the present invention are not particularly limited, but preferably human skin, particularly preferably the skin of the patient himself / herself who is transplanted with the artificial skin of the present invention.

Hereinafter, the present invention will be specifically described with reference to examples.

[0032]

【Example】

(Example 1) Melt flow index was 30 and 0.
Polypropylene mixture of 3 (mixing weight ratio 100: 40)
400 parts by weight of liquid paraffin (number average molecular weight 324) and 0.3 parts by weight of 1,3,2,4-bis (p-ethylbenzylidene) sorbitol as a crystal nucleating agent per 100 parts by weight of biaxial type. It was melted by an extruder and pelletized. The pellets are heated to 150-
Melted at 200 ° C, extruded into the air from a T-die having a slit width of 0.6 mm, guided into a cooling and solidifying liquid by rotation of a guide roller for a cooling liquid phase placed immediately below the T-die, cooled and solidified and wound up. ..

The wound film-like material is cut into a certain length, fixed in both longitudinal and lateral directions, and 1,1,2-trichloro-
Liquid paraffin was extracted by immersing it in 1,2,2-trifluoroethane for a total of 4 times for 10 minutes, and then for a total of 4 times for 10 minutes in air at 135 ° C. to extract a liquid puffin, and then 135 After treating in air at ℃ for 2 minutes,
A polypropylene porous film having a pore size of 0.45 μm and a film thickness of 120 μm was obtained.

The porous film thus obtained was irradiated with low-temperature plasma (argon, 0.1 ton) for 15 seconds, and then a methoxyethyl acrylate monomer was supplied in a gaseous state,
Surface graft polymerization was carried out at a temperature of 288K for 5 minutes. After the graft polymerization, the unreacted monomer was degassed under reduced pressure to 0.0
1ton or less, and plasma discharge treatment 0.1ton, 2
The operation was performed for 0 seconds (100 W). The membrane thus obtained was washed with a dichloromethane / methanol azeotropic solvent for 2 days and then dried.

On the other hand, 10% C of 3T3 cells (type NIH, obtained from Dainippon Pharmaceutical Co., Ltd., ATCC No. CRL1658) were previously prepared.
S (Calf Serum) -DME (Dulbecoo's Modified Eagle
Medium) suspension to 3 × 10 ⁵ cells / ml,
After irradiating with γ-ray for 0 second, it was centrifuged at 1000 rpm for 5 minutes, and finally seeded at 8 × 10 ⁴ cells / cm ² on the porous membrane substrate prepared above. Furthermore, after culturing for 1 day, the culture solution was removed, and human keratinocytes collected by enzymatic treatment from human neonatal skin were suspended in calcium-free DME medium containing 1% fetal bovine serum, and 1 × 10 ⁴ cells / The seeds were sown in cm ² , and cultured artificial skin was produced for 7 days in an incubator under a carbon dioxide atmosphere.

When the artificial skin was transplanted to a nude mouse having a skin defect wound with undifferentiated epidermal cells on the wound surface side, it was confirmed histologically that it was engrafted on the 21st day.

Example 2 A solution prepared by dissolving 18 parts by weight of polyvinylidene fluoride powder (Kynar K301, manufactured by Mitsubishi Petrochemical Co., Ltd.) in 73.8 parts by weight of acetone and 8.2 parts by weight of dimethylformamide was prepared. After casting on a polyethylene terephthalate film, it was immersed in a 1,1,2-trichlorotrifluoroethane bath for 5 minutes and dried to obtain a polyvinylidene fluoride porous film having a thickness of 125 μm and an average pore diameter of 0.45 μm. ..

The porous membrane thus obtained was subjected to surface graft polymerization and plasma discharge treatment in the same manner as in Example 1, and the obtained sample was seeded with human epidermal cells in the same manner as in Example 1. Then, it was cultured for 7 days to prepare artificial skin.

As a result of transplanting the artificial skin into a nude mouse, epidermal cells were engrafted.

Example 3 A polypropylene mixture having a melt flow index of 30 and 0.3 (mixing weight ratio: 1)
00:40) 400 parts by weight of liquid paraffin (number average molecular weight 324) and 0.3 parts by weight of 1,3,2,4-bis (p-ethylbenzylidene) sorbitol as a crystal nucleating agent per 100 parts by weight. Was melted by a twin-screw extruder and pelletized. The pellets were melted at 150 to 200 ° C. using the above extruder, and the T having a slit width of 0.6 mm was used.
It was extruded into the air from the die, guided to the cooling and solidifying liquid by the rotation of the guide roller for the cooling liquid phase placed immediately below the T die, cooled and solidified, and then wound.

The wound film-like material is cut into a certain length, fixed in both longitudinal and lateral directions, and 1,1,2-trichloro-
The liquid paraffin was extracted by immersing it in 1,2,2-trifluoroethane for a total of 4 times for 10 minutes, and then in the air at 135 ° C for a total of 4 times for 10 minutes to extract the liquid paraffin, and then 135 A heat treatment was performed for 2 minutes in air at 0 ° C. to obtain a polypropylene porous film having a pore size of 0.45 μm and a film thickness of 120 μm. The porous film thus obtained was irradiated with low temperature plasma (argon, 0.1 Torr) for 15 seconds, and then a monomer of methoxyethyl acrylate was supplied in a gaseous state, and surface graft polymerization was performed at a temperature of 288K for 5 minutes. I went. After the graft polymerization, the unreacted monomer was degassed under reduced pressure to 0.01 ton or less, and further plasma discharge treatment was conducted to 0.02.
The operation was performed at 1 ton for 20 seconds (100 W). The membrane thus obtained was washed with dichloromethane / methanol azeotropic solvent
It was washed for a day and dried.

On this porous membrane substrate, human keratinocytes collected from human skin by enzyme treatment were suspended in calcium-free Dulbecco's modified Eagle medium (DME) containing 1% fetal bovine serum, and 1 × 10 6. Seed at ⁵ cells / cm ² ,
It was cultured in an incubator for 7 days under a carbon dioxide atmosphere to prepare artificial skin.

When the artificial skin was transplanted to a nude mouse having a skin defect wound with undifferentiated epidermal cells on the wound side, it was recognized histologically that it had engrafted on the 21st day.

Example 4 18 parts by weight of polyvinylidene fluoride powder (Kynar K301, manufactured by Mitsubishi Petrochemical Co., Ltd.) was added to 73.8 parts by weight of acetone and 8.2 parts of dimethylformamide.
After casting a solution prepared by dissolving 1 part by weight on a polyethylene terephthalate film, it is immersed in a 1,1,2-trichlorotrifluoroethane bath for 5 minutes and dried to give a film thickness of 125 μm and an average pore diameter of 0.45 μm. A porous film made of polyvinylidene fluoride was obtained.

The porous membrane thus obtained was subjected to surface graft polymerization and plasma discharge treatment in the same manner as in Example 1, and the obtained sample was seeded with human epidermal cells in the same manner as in Example 1. Then, it was cultured for 7 days to prepare artificial skin. As a result of transplanting the artificial skin 4 into a nude mouse, epidermal cells were engrafted.

Comparative Example 1 Polypropylene film (film thickness: 60 μm, manufactured by Nimura Chemical Co., Ltd., FOP #
60) is placed in the chamber and the pressure is reduced to 0.01 tons or less, then low temperature plasma (0.1 ton of argon) is irradiated for 15 seconds, the pressure is reduced again to 0.01 tons or less, and further alkoxyalkyl acrylate such as methoxyethyl acrylate is added. The monomer was supplied in a gaseous state, and surface graft polymerization was performed at a temperature of 288K for 5 minutes. After the graft polymerization, the unreacted monomer was degassed under reduced pressure to 0.01 ton or less, and then plasma discharge treatment was performed for 0.1 ton for 20 seconds (100 tons).
W) I went. The membrane thus obtained was washed with a dichloromethane / methanol azeotropic solvent for 2 days and then dried. Human epidermal cells were seeded on the obtained samples in the same manner as in Examples 1 and 3, and the epidermal cells adhered to the substrate, but they were hard to engraft even when transplanted to nude mice.

[0047]

The artificial skin of the present invention comprises a porous membrane substrate,
There are non-proliferative animal-derived fibroblasts treated with γ-rays or mitomycin C, and undifferentiated epidermal basal cells are monolayered. Can be transplanted to the wound surface of the skin defect. In addition, the artificial skin of the present invention has a monolayer of undifferentiated epidermal basal cells on a hydrophilized porous membrane substrate, and the cell side does not have to be peeled off from the porous membrane to cause a skin defect. It was made possible to be transplanted to the wound surface of the part.

When these artificial skins of the present invention are transplanted into a skin defect, the healing process can be shortened remarkably.

Claims (2)

[Claims] 1. An artificial skin characterized in that non-proliferative animal-derived fibroblasts are present on a porous membrane substrate, and undifferentiated epidermal basal cells are present as a monolayer. .. 2. An artificial skin in which undifferentiated epidermal basal cells are present as a monolayer on a porous membrane base material whose surface has been hydrophilized.