KR19990040309A - Three-dimensionally regenerated artificial skin tissue and its manufacturing method - Google Patents

Abstract

In order to accomplish the above object, the present invention provides a method for producing a fibrous cell, which comprises culturing fibroblasts at a concentration of 80-90%, treating the cells with mitomycin C, dispensing the cells in a culture dish to prepare a feeder layer, separating the epithelium and connective tissue with tweezers, Followed by centrifugation in 0.1% trypsin / EDTA for 15 min. Subsequently, the epithelial cells were collected and dispensed into the feeder layer, and subcultured to a density of 70-80% by the medium. ; 1 type collagen was neutralized with 2.2% NaHCO ₃ , 0.05N NaOH and 200 mmol / l HEPES as a neutralizing solution, mixed with fibroblasts and allowed to stand to induce polymerization, and primary cultured epithelial cells And cultured in medium for 7-10 days to form a monolayer. The culture medium above the epithelial cells was removed, and the outer culture medium of the ring was maintained at the height of the dermis to allow the epithelial cells to be exposed to the air. A step of performing histotyping raft culture comprising the steps of: And laminin and type 4 collagen are applied to the upper part of the gel and polymerization is carried out in an incubator at 30 ° C. until polymerization to provide a basement membrane component, and a three-dimensionally reproduced artificial epithelial tissue is provided do.

Description

Three-dimensionally regenerated artificial skin tissue and its manufacturing method

The present invention relates to an artificial skin tissue and a method for producing the same, and more particularly, to an artificial skin tissue regenerated three-dimensionally by binding of a skin cell and a basement membrane and a method for producing the same.

The epithelium is composed of highly proliferating stem cells and committed precursor cells and an extremely low proliferating cell population (Lajtha LG: Stem cell concepts. Differentiation 14 : 23-34, 1979). Histologically, the basal cell layer composed of undifferentiated cells, the suprabasal cell layer composed of highly proliferative progenitor cells, the spinous cell layer in which differentiation begins, (keratinzed layer), and each cell group has a polarizing structure based on the basement membrane at the upper part of the connective tissue (Wright NA: The kinetics of human epidermal cell populations in health and disease Recent advances in dermatology. Edited by Rooke, Savin J. London, Churchill Livingston, 1980, pp 317-343).

Morphologically, basal membrane, which is the basis of epithelial polarity, is formed from epithelial cells and lower connective tissues and can be observed under optical microscope through periodic acid schiff (PAS) and silver stain (si1ver stain). The electron microscopic microstructure of the basement membrane includes lamina lucida and lamina densa directly under the epithelium. The basement membrane is a hemidesmosome with the epithelium and anchoring filament with the lower collagen fibers. ) (Timpl and Dzladek, M. Structure, development, and molecular pathology of basement membranes. Int Rev Exp Pathol 1986, 29: 1-112). Recently, extracellular matrix of the basement membrane has been known, and type IV collagen is the constituent of the basal membrane and constitutes the major extracellular matrix (Tsilibary EC and Charonis AS. The role of the main noncollagenous domain (NCI) in type IV collagen self-assembly. J Cell Biol 1986, 103: 2467-2473). Type IV collagen plays a role in binding epithelium and connective tissue with other extracellular matrix, such as laminin, fibronectin, etc. (Bell E, Ivarsson B, Merrill C. Production of a tissue-like structure by contraction of collagen lattices by human fibroblast of different proliferative potential in vitro. Proc Natl Acad Sci USA 1979, 76; 1274-1279). In this way, the basement membrane has a variety of complex structures, and it supports the epithelial function and morphological characteristics (Lajtha LG: Stem cell concepts. Differentiation 14: 23-34, 1979, Timpl and Dzladek, M. Structure, development , and molecular pathology of basement membranes. Int Rev Exp Pathol 1986, 29: 1-112, Fine JD, The skin basement membrane zone, Adv Dermatol 1987, 2: 283-304).

In addition, each epithelial cell is densely packed at a distance of about 20-30 nm and is connected to the desmosome to form a rigid structure, which is resistant to various stimuli from the outside. One of the other features of epithelial cells is the fact that they have an intermediate filament consisting of a protein family called "cytokeratin" that acts as a skeleton in the cytoplasm (Steinert PM, Steven AC , Roop DR. The molecular biology of intermediate filaments. Cell 1985 42: 411-419). To date, it has been known that cell keratin consists of more than 20 protein families, and relatively small proteins (CK 8, 18, 7, 17, 19) are present in simple epithelia or basal cells, CK 5, 14). (Steinert PM, Steven AC, Roop DR. The molecular biology of intermediate filaments. Cell 1985, Wang et al. In addition, CK 3, 12, etc. exist in the keratinized part, and various manifestations are observed according to the process of epithelial proliferation and differentiation, that is, the polar differentiation process of epithelium (Wang E, Fishchman D, Liem RKH, Sun TT. filament. Ann NY Acad Sci 455, 1985).

The epithelium performs various complex functions of the epithelium through various morphological and structural features such as the above. Therefore, in addition to the evaluation of the biological properties of the oral mucosal epithelium, it is essential for all related studies to reproduce complex and diverse characteristics based on epithelial polarity as described above.

The cell culture method is one of the most widely used experimental methods recently because it is easier to control physiological-chemical conditions than an animal experiment, can study human cells, and can obtain objective research results (Prunieras M. Recent advances in epidermal cell cultures. Arch Dermatol Res 264: 243-247, 1979). The epithelial cell culture requires very strict conditions compared to other connective tissue cultures. Current methods for culturing epithelial cells include low-calcium medium, which provides a physiological environment for inhibiting differentiation and inducing proliferation, And a method using a mouse fibroblast feeder (Rheinwald JG and Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6: 331 -343, 1975). Among them, the culturing method using irradiated 3T3 fibroblast feeder (irradiated mouse 3T3 fibroblast feeder) can selectively cultivate only epithelial cells (1), and (2) because of various growth factors provided from feeder cells, Which is most commonly used because it has no problem in forming the layer. However, using such a conventional method, a large amount of epithelial cells can be obtained, but (1) overgrowth of undifferentiated epithelial cells due to differentiation inhibition, (2) lack of control between proper epithelial-epithelium and epithelial-connective tissue And there is a problem that the differentiation function is lost. Therefore, the more the subculture of epithelial cells is cultured, the more phenotypes of cultured epithelial cells are changed, resulting in different morphological, biochemical and functional characteristics from the original biotissue. In addition, although it can induce proliferation or differentiation through administration of a specific biochemical substance or change in physiological conditions, it has a problem that it can not achieve a three-dimensional structure in which proliferation and differentiation are balanced as in vivo. The limitations of such conventional epithelial cell culture methods are largely due to two reasons. First, there is no interaction between the epithelium and connective tissues due to the lack of connective tissue below the epithelium, so that polarity of the basement membrane and epithelium is not formed. In this study, we investigated the effect of air-liquid interface on the epithelium, which is exposed to the air, in the basement membrane and in the epithelium. . Thus, under conditions similar to that of living organisms, ie type I collagen and fibroblast, providing basement membrane with dermis or submucosa to provide epithelial polarity and provide culture conditions that can be maintained on the air-liquid surface Is very important.

To overcome this problem, a method of applying extracellular matrix such as fibronectin, laminin, and type IV collagen to the bottom of the culture dish has been introduced, but it is helpful for the attachment and proliferation of epithelial cells However, induction of differentiation or stratification was not achieved. The importance of epithelial-mesenchymal interactions between epithelial and connective tissues has been well established in adult tissues as well as in mammalian developmental processes, but this concept has not been actively introduced into cell culture (Peault 1995, Limat et al., 1995). However, in 1979, Bell and others first introduced a three-dimensional culture method using a collagen lattice, and then a full-scale study has been conducted. First, a method of using dead dermis (dead de-epidermized dermis) and a method of using a dermal equivalent using collagen-fibroblast lattice is described as a method of providing connective tissue below the epithelium Recently, a method using a dermal equivalent has been preferred for the simplicity and reproducibility of the experiment. Until recently, it has been possible to regenerate the epithelium with similar morphological, physiological and chemical characteristics similar to normal epithelium through many improvements of the experimental method. Using this, epithelial epidemiology, induction of epithelial cell differentiation by biochemical substance, This study has been applied to many fields such as tumorigenesis research, viral growth and epidemiology in epithelial tissues, surgical restoration of cultured epithelium, pharmacological effects and mechanism studies. However, despite the variability of the disease and the usefulness of the three - dimensionally regenerated epithelium, there were few results.

As described above, the conventional epithelial culture method is far from the process of reproducing the characteristics of the in vivo epithelial cells. Thus, it is possible to reproduce biological, tissue, physiological and biochemical characteristics by maintaining the equilibrium relationship between (1) proliferation and differentiation, (2) interaction between epithelium and epithelial-connective tissue, and (3) polarity of epithelial tissue It is essential to be able to.

In order to solve the above-mentioned problems, the present invention provides a method of culturing a mammal by using a histotypic raft culture under culture conditions capable of maintaining an epithelial collagenous tissue and an air-liquid surface using a type 1 collagen fiber and fibroblasts And an object of the present invention is to provide an artificial skin tissue regenerated three-dimensionally and a culturing method thereof.

In order to accomplish the above object, the present invention provides a method for producing a fibrous cell, which comprises culturing fibroblasts at a concentration of 80-90%, treating the cells with mitomycin C, dispensing the cells in a culture dish to prepare a feeder layer, separating the epithelium and connective tissue with tweezers, Followed by centrifugation in 0.1% trypsin / EDTA for 15 min. Subsequently, the epithelial cells were collected and dispensed into the feeder layer, and subcultured to a density of 70-80% by the medium. ; 1 neutralizing collagen is neutralized with 2.2% NaHCO ₃ , 0.05N NaOH and 200 mmol / 1 HEPES as a neutralizing solution, mixed with the fibroblasts and allowed to stand to induce polymerization, and the primary cultured epithelial cells And cultured in medium for 7-10 days to form a monolayer. The culture medium above the epithelial cells was removed, and the outer culture medium of the ring was maintained at the height of the dermis to allow the epithelial cells to be exposed to the air. A step of performing histotyping raft culture comprising the steps of: And applying the laminin and the type-4 collagen on the upper part of the gel, respectively, and polymerizing the mixture in an incubator at 30 캜 until polymerization to provide a basement membrane component.

Also, type 1 collagen; A basement membrane flatly coated on the collagen type I; A basal cell layer on which a nucleus is arranged to have a polarity structure on the basal membrane and which is flatly formed as one or two layers, a polar cell layer formed by three to five layers on the basal cell layer, And a keratin layer formed on the polar cell layer. The present invention also provides a three-dimensionally regenerated artificial epithelial tissue.

Hereinafter, the present invention will be described in detail with reference to the drawings and the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a process chart showing a method for regenerating artificial epithelial tissues three-dimensionally according to the present invention;

2 is an optical microscope photograph showing the histological characteristics of the artificial epithelial tissue according to the calcium concentration in the culture medium according to the present invention,

FIG. 3 is an optical microscope photograph showing the histological characteristics of the artificial epithelial tissue according to the concentration of fetal bovine serum in the culture medium according to the present invention,

FIG. 4 is an optical microscope photograph showing the histological characteristics of the basement membrane of the artificial epithelial tissue according to the present invention,

FIG. 5 is an electron micrograph showing a stereoscopic view of the artificial epithelium according to the present invention. FIG.

6 shows optical microscope photographs showing immunohistochemical results of differentiation and proliferation markers of artificial epithelial tissues provided with a basement membrane according to the present invention, respectively.

A method of manufacturing an artificial epithelial tissue will be described with reference to FIG.

First, primary culturing of epithelial cells is carried out by the following procedure.

When swiss 3T3 derived from mouse fibroblast was grown at 80-90%, it was treated with mitomycin C (8 μg / ml) and dispensed in a 100 mm culture dish to form a feeder layer. . Then, in the epithelial tissues, the area without inflammation is removed when the supernatant is removed, and the primary cultivation of the epithelial cells is performed after being divided into three equal parts. The tissue for culturing is washed with PBS 3 times or more, and finely cut into a size of 1-2 mm and treated with 0.15% collagenase-dispase for 30 minutes. After the epithelium and connective tissue are separated by tweezers under stereoscopic microscope, the epithelium is centrifuged for 15 minutes in 0.1% trypsin / EDTA. The epithelial cells were collected and dispensed into the feeder layer. The medium contained 0.4 μg / ml hydrocortison, 5 μg / ml insulin, 0.1 μg / ml cholera toxin, 5 μg / ml human transferrin, (3'-5-triiodo-L-thyronine) and 5ng / ml epidermal growth factor. The medium is changed every 3-4 days. When the density reaches 70-80%, the medium is frozen or subcultured.

Next, the histotyping raft culture is carried out through the following procedure.

One type of collagen (type 1 collagen, pH 3) was used as a three-dimensional reconstruction of the epithelial tissue, and this was neutralized with 2.2% NaHCO ₃ , 0.05N NaOH and 200 mmol / l HEPES as a neutralizing solution, To induce polymerization. As dermal cells, swiss 3T3 fibroblasts or submucosal fibroblasts are used. Before the fibroblasts and collagen are polymerized, they are mixed and homogeneously distributed to a concentration of 1.2 x 10 ⁵ / ml. The primary cultured epithelial cells above the polymerized gel are dispensed at 5 × 10 ⁴ / cm ² and cultured in E medium for 7-10 days to form a monolayer. Thereafter, the culture medium above the epithelial cells is removed, and the outer culture medium of the ring is kept at the height of the dermis, so that the epithelial cells are exposed to the air and maintained for 7-10 days. After removing the culture medium, a portion of the epithelium was fixed to 10% neutral formalin, and histological and immunohistochemical staining was performed. Some of the epithelium was fixed to buffered glutaraldehyde-paraformaldehyde and subjected to electron microscopic examination do.

Basal membrane components for binding primary cultured epithelial cells to the basement membrane are provided as follows.

When the primary cultured epithelium is dispensed on top of the polymerized gel, the polarity of the epithelium is maintained by providing the components of the basement membrane. Namely, laminin and type 4 collagen were applied to the upper part of the gel to a concentration of 0.33 mg / ml (7 μg / cm ² ) and 2.6 mg / ml (40.0 μg / cm ² ), respectively. . Through the above process, three-dimensionally regenerated epithelial tissue is formed.

The three-dimensional structure of the skin tissue obtained through culture was confirmed by the following method. The skin tissue obtained from the operation and the skin tissue obtained from the culture are fixed to 10% neutral formalin, embedded in the usual method, and then serial sections are obtained and histological and immunohistochemical detection are performed. That is, serial sections were stained with hematoxylin / eosin and pyridic acid scaffold to search for tissue-pathological structures and immunohistochemical staining was performed using the following avidin-biotin method .

Continuous sections of 4 μm are obtained to remove paraffins from xylene and function stepwise in 95%, 90%, and 70% ethanol. After removing the endogenous peroxidase from 0.5% hydrogen peroxide, the cells were treated with normal goat serum for 30 minutes. The primary antibody was suspended in phosphate buffered saline (PBS) containing 3% bovine serum albumin (BSA) ) According to the manufacturer's instructions and allowed to react for 1 hour. CK 14 as a marker of basal cell layer, involucrin as marker of extracellular layer and AE1 as marker of keratinization layer are used as the primary antibodies and Proliferating Cell Nuclear Antigen , PCNA). After the primary antibody reaction, the cells were washed 3 times in Tris-buffered saline (TBS) and then incubated with 5 μg / ml biotinylated anti-mouse / anti-rabbit immunoglobulin diluted in 3% -mouse / anti-rabbit IgG) for 30 min and washed three times in Tris-buffer saline. After incubation for 30 minutes with 3 μg / ml horseradish peroxidase streptoavidin, color was developed using diaminobenzidine (DAB) and hydrogen peroxide, and then compared with Meyer's hematoxylin in Meyer's hematoxylin Dye. The tissues obtained by histopathological examinations were compared with those obtained by histopathological examinations using optical microscope to determine the characteristics of the in vivo epithelium and the morphological characteristics of the regenerated epithelium And proliferation-differentiation markers.

The method of examining the artificial epithelium structure by electron microscopy is as follows.

The tissues obtained from the surgery and the tissue obtained from the cultures were fixed in a glutaraldehyde-paraformaldehyde solution, washed in a 1% cacodylate buffer for 12 hours, washed with 1% osmium tetroxide Fixed. Stepwise dehydration in ethanol then embedded in Epon 812 resin. 1 mu m samples were stained with toluidine blue and examined with an optical microscope. Ultrathin sections were stained with uranyl acetate / lead citrate. (1) the continuity and microstructure of the basement membrane, (2) epithelial contact areas such as desmosomes, and (3) intracellular sites such as tonofilament. Observe the microstructure. The results of confirmation of the regenerated epithelial tissues by the above methods are as follows.

(1) Three-dimensional culture according to the kind of medium (see FIGS. 2 and 3)

Laminin and type 4 collagen were added to the connective tissues and then cultured. When cultured in "E" medium, it formed 5 ~ 6 layers of epithelium, and polarity of basal cells, formation of extracellular layer, formation of hypocalcified layer was observed, and cell junction was well developed. When cultured in 1% fetal bovine serum, formation of basal cell and acinar cell layer and formation of each class were observed in 5 ~ 6 layer epithelial layers. Some cells contained granules. In 0.5% fetal bovine serum, formation of basal cells and extracellular layer and formation of keratinization layer were observed in 5 ~ 6 layers of epithelium. Partial granular layer was formed and intrinsic keratinization was observed. At the concentration of 0.15 mM calcium, the epithelium did not grow because the epithelium did not adhere to the lower connective tissues. At 0.5 mM calcium concentration, the thickness of the epithelial layer was decreased compared to the "E" medium. When cultured in serum-free KGM, two to three layers of epithelium were formed, but no distinction was made between basal and acinar cell layers.

Characteristics of Artificial Epithelial Tissue According to Media Condition

[Table 1]

As a result, the three-dimensional culture of oral mucosa was performed in the "E" medium containing 1.5 mM Ca ⁺⁺ . When laminin and type 4 collagen were used as the basement membrane in the collagen matrix, . Therefore, the 'E' medium preferably contains Ca ⁺⁺ of 0.7 to 1.5 mM.

(2) three-dimensional culture according to basement membrane configuration (see FIG. 4)

When both type 4 collagen and laminin were provided, the epithelium was composed of 5 ~ 6 layers, and polarity of basal cells, formation of extracellular layer and formation of subcortical layer were similar to normal epithelium. The artificial epithelium is composed of type 1 collagen, a basal membrane flattened on the basal membrane, a basal cell layer in which the nucleus is arranged to have a polar structure on the basal membrane and the cell nucleus is arranged flatly in one to two layers, And a keratinization layer formed on the polar cell layer. At this time, the basal cell layer consists of one to two layers, the polar cell layer consists of three to five layers, and the keratin layer consists of one or two layers. However, unlike living tissues, basal cell layers are arranged flat without dermal papilla formation. In addition, although the thickness of the extracellular layer varies according to the curved shape of the dermal papilla in the living tissue, the thickness of the extracellular layer according to the present invention is almost constant. When only one type of collagen or laminin was provided, the epithelial layer consisted of two or three layers, but the polarity of the basal cells, the spinous cell layer was not observed, and the keratinized layer was not present. Particularly, when laminin was reconstructed, epithelial cells invaded into the lower connective tissue. When the lower connective tissue was formed without reconstruction of the basement membrane, 5 ~ 6 layers of epithelium were formed and basal cell layer, extracellular layer and partial keratinization layer were formed, but the cells of the basal cell layer showed reverse polarity.

Histological evaluation of artificial epithelium according to basement membrane components

[Table 2]

5 shows the artificial epithelium tissue regenerated according to the present invention observed with an electron microscope. As shown in FIG. 5, when cultured after formation of both type 4 collagen and laminin, the cells were flattened with a flat shape and formed with a slight discontinuity in the basal layer. Semi - collagen, collagen, and mesenchyme fibers were well developed, and other organelles such as mitochondria and ribosomes were well developed. When the basement membrane was not formed, the shape of the cell was flattened and formed well, and the formation of the basement membrane was only slight in the surface layer. The collaterals, filaments, and other organelles are also well developed and keratinized granules are observed on the surface. The structure similar to the myofibers in the cytoplasm of the basal cell nucleus is observed, which is thought to be related to the reverse polarity of the basal cell observed in the optical microscope. When only 4-type collagen or laminin is formed, the cells on the surface layer are not flattened, but have a round or polygonal shape while the nuclei are dissolved or reduced, and in some, denatured cells are also observed.

Electron Microscopic Findings of Three Dimensional Regenerated Artificial Epithelial Tissue

[Table 3]

Figure 6 shows the results for differentiation markers and proliferation markers of artificial epithelial tissue regenerated according to the present invention. This is explained as follows.

(1) Markers of differentiation

1) Involucrin dyeing

Boleucrin, a water-soluble protein in the cytoplasm, is a protein thought to be a precursor of keratin and is not present in the basal cell layer, which divides and proliferates, and is expressed in the cell layer and granule layer. In all cases of formation of both type 4 collagen and laminin, formation of lower connective tissue without basement membrane reconstruction, and formation of laminin only, involucrin was not stained in the basal cell layer. Strongly positive staining was observed from the basal layer to the surface, . When only type 4 collagen was formed, most of the basal layer cells were not stained, but some positive cells were observed and staining was seen from the basal layer to the surface layer. In the case of not forming the basement membrane or forming both type 4 collagen and laminin, the differentiation layer and the undifferentiated layer are clearly distinguished, whereas in the case of forming only one of the two components, the two cells tend to be mixed slightly gave.

Results of in vitro expression

[Table 4]

2) CK14

CK14 is known to be a cytokeratin expressed in the basal layer of all epithelial cells. CK14 was expressed only in the basal lamina in the epidermis of intact keratinized epithelium. All tissues obtained from three - dimensional cell culture were expressed in the cytoplasm from the basal layer to the keratinization layer.

3) CK10 / 13

CK10 / 13 is an antibody that reacts with cellular keratinocytes of 53 kD and 56.5 kD. It is known that CK10 / 13 is expressed in the basal lamina except the undifferentiated basal cell layer in the keratinized middle layer epithelium and non - keratinized middle layer epithelium. In the present study, normal epithelium was expressed positively in the upper cell layer except for the basal cell layer. When both type 4 collagen and laminin were formed, a negative reaction was observed in the basal cell layer and some upper basal layer of the basal cell layer, and all the upper epithelial layer was expressed . When only one component of laminin or type 4 collagen was added, it was positive only in some cells in the uppermost layer and negative in most of the remaining cells, suggesting that the presence of only one of the two components did not result in differentiation.

In the case of the different culture media, all the tissues except for the basal cell layer except the basal cell layer were positive like the normal oral mucosal epithelium in the "E" medium. When cultured in serum-free KGM, the cells were positive only in some cells in the upper layer. When cultured in 0.5% and 1% fetal bovine serum, the cells were negative in the basal cell layer and one or two layers of polar cells, I showed you. When cultured in 0.5 mM Ca, negative reaction was observed in all but the one layer of the keratinization layer. As a result, it was found that all of the cells were not differentiated when cultured in a medium having a low concentration of fetal calf serum or calcium.

Expression of CK10 / 13

[Table 5]

(2) Markers of proliferation

In the staining for proliferating nuclear antigen (PCNA) used as a proliferative marker, proliferative ferritin was found in the basal cell layer and was similar to the biotissue. There was no significant difference in the changes depending on each media condition and basement membrane formation.

The three-dimensionally regenerated epithelial tissue according to the present invention can be positively utilized in a number of research and clinical fields as follows.

end. Research Field

(1) Studies on the proliferation and differentiation of epithelium by administration of specific components in medium, growth factors, chemical administration, etc.

(2) a study on the genetic background of epithelial diseases, precancerous lesions of oral mucosa

(3) Epithelial changes and toxicity by drug administration using systemic administration via topical application or in the medium

(4) Changes in epithelial cell proliferation and differentiation during carcinogenesis by carcinogen application and administration

(5) Evaluation of invasiveness of connective tissue through culture of carcinoma cells

(6) Study on epidermal epidemiology

(7) Studies on the biochemical metabolism of epithelial cells against various toxic substances

(8) Epithelial-mesenchymal relationship of functional cells

I. Clinical field

(1) Artificial mucosa and skin for protection of wound, infection prevention and epithelial regeneration after surgery or trauma

(2) Plastic surgery for removal of burns and scars, regeneration of mucous membranes

All. Industry

(1) Toxicity evaluation of various synthetic and natural substances such as cosmetics and skin drugs to epithelial cells

(2) Analysis of effects on the treatment of skin diseases

(3) commercialization of artificial skin

Claims (5)

When the fibroblasts were grown at 80-90%, they were treated with mitomycin C to prepare a feeder layer. The epithelium and connective tissues were separated by tweezers, and the epithelium was treated with 0.1% trypsin / EDTA for 15 minutes Centrifuging, collecting the epithelial cells, dispensing them into the feeder layer, and subculturing the medium until reaching a density of 70-80% by the medium; 1 type collagen was neutralized with 2.2% NaHCO ₃ , 0.05N NaOH and 200 mmol / l HEPES as a neutralizing solution, mixed with fibroblasts and allowed to stand to induce polymerization, and primary cultured epithelial cells And culturing the medium for 5-7 days to form a monolayer; removing the culture medium above the epithelium; keeping the outer culture medium of the ring at the height of the dermis to allow the epithelial cells to be exposed to the air for 14 to 25 days; A step of performing histotyping raft culture comprising the steps of: And applying the laminin and the type-4 collagen on the upper part of the gel, respectively, and polymerizing the mixture in an incubator at 30 캜 until polymerization to provide a basement membrane component. The method according to claim 1, wherein the basement membrane is composed of laminin having a concentration of 0.33 mg / ml (7 μg / cm ² ) and a type 4 collagen having a concentration of 2.6 mg / ml (40.0 μg / cm ² ) Lt; RTI ID = 0.0 > artificial < / RTI > epithelial tissue. 2. The method according to claim 1, wherein the medium comprises 0.7 to 1.5 mM Ca < ⁺⁺ > as the 'E' medium. Type 1 collagen; A basement membrane flatly coated on the collagen type I; A basal cell layer on which the nucleus is arranged and flatly formed as one or two layers so as to have a polar structure on the base film; A polar cell layer uniformly formed on the basal cell layer in three to five layers; And a keratin layer formed on the extracellular matrix layer. The three-dimensionally regenerated artificial epithelium tissue. 5. The method according to claim 4, wherein the basement membrane is composed of laminin having a concentration of 0.33 mg / ml (7 μg / cm ² ) and quadriformal collagen having a concentration of 2.6 mg / ml (40.0 μg / cm ² ) A three-dimensionally regenerated artificial epithelium.