KR20020008676A - Skin equivalent and manufacturing method of same - Google Patents

Abstract

PURPOSE: A method is provided to produce artificial skin by culturing keratinocyte on the demal substrate obtained by combining the de-epidermized dermis on collagen matrix containing fibroblast therein, wherein the artificial skin is similar to actual human being so that it is used to a skin virulence inspection and a skin transplant such as a burn. CONSTITUTION: A demal substrate is obtained by combining the de-epidermized dermis on collagen matrix containing fibroblast therein. The collagen matrix contains fibroblast ranging from 1x10¬5 to 5x10¬5 per collagen mixture mL. The artificial skin is provided by inoculating and culturing a keratinocyte on the demal substrate obtained by combining the de-epidermized dermis on the collagen matrix containing fibroblast therein. The culture of the artificial skin is performed by culturing the inoculated keratinocyte with sufficiently immersed it within a culture fluid, and exposing the same. The artificial skin is used in detecting a skin virulence. The artificial skin is grafted to a portion which skin is damaged through the use of a skin graft operation.

Description

Artificial skin and its manufacturing method {Skin equivalent and manufacturing method of same}

The present invention relates to an artificial skin and a method of manufacturing the same, and more particularly, to an artificial skin prepared by culturing keratinocytes on top of the collagen matrix containing fibroblasts and then removing the epidermis.

The skin is composed of the uppermost epidermis and the dermis existing in the lower epidermis. The epidermis is the keratinized stratified squamous epithelium, which is mainly composed of keratinocytes. The epidermis is composed of four layers of basal layer, pole layer, granule layer, and stratum corneum. After division of keratinocytes in the basal layer, keratinocytes move from the basal layer to the surface of the skin and undergo different stages of differentiation. Keratinocytes are developed and established as cells suitable for performing their own functions during the differentiation process. This differentiation can be seen by the formation of histologically distinctive layers and by biochemically observing unique proteins expressed in the cells at each stage of differentiation.

The characteristics that can be observed in the layers forming each epidermis are as follows.

In the polar layer, keratinocytes elongated in the basal layer were expanded, and keratin K1 and K10 having high molecular weight were expressed instead of the small-molecular keratin K5 and K14 expressed in the basal layer. (Nelson W, Sun TT J Cell Biol. 1983; 97: 244-251; Fuchs E, Green H, Cell. 1980; 19: 1033-1042; Eicher R, Bonitz P, Sun TT, J Cell Biol. 1984; 78: 423-427).

In the granulation layer, keratinhailin granules are observed in the cytoplasm of keratinocytes, which contain profilaggrin. Propyllagrin acts to agglomerate keratin filaments in the stratum corneum when the keratinocytes migrate into the stratum corneum and break down the cytoplasm. (Ford MJ, Int J Dermatol. 1986 25: 547-551) In the final stage of differentiation, transglutaminase is activated to induce cross-linking of several proteins, forming a cornified envelop inside the keratinocyte membrane. The cornipid envelope is an important factor for the skin to act as a protective barrier against external harmful substances (Thacher SM, Rice RH, Cell. 1985; 40: 685-695). Among them, involucrin, a major protein, is already expressed in the polar layer and loricrin is expressed in the granule layer (Watt FM, J Invest Dermatol. 1983) . 81: 100-103; Mehrel T, Hohl D, Rothnagel JA, Cell. 1990; 61: 1103-1112)

In the stratum corneum, the outermost layer of the epidermis, keratinocytes lose their nucleus and cytoplasm, leaving only keratin filaments and filagrins, and the surrounding cornipid envelope replaces the function of the cell membrane with the loss of the cell membrane.

Since keratinocytes of the skin can be cultured in vitro early, they have been widely used to study skin properties and to utilize them in histology. The initial method of culturing keratinocytes is a culture of keratinocytes submerged in a culture medium. The keratinizing squamous epithelium, which is a characteristic of the skin, cannot be formed, and most cells have the properties of cells corresponding to the basal layer of the epidermis. . Therefore, it was not possible to reflect the characteristics of human skin as it is, and to observe the biochemical changes occurring in the differentiation process in particular.

However, in recent years, a three-dimensional culture method of keratinocytes, that is, an artificial skin model, has been developed that can develop epidermal tissue similar to that of human skin by developing existing cell culture methods.

Representative examples of currently developed artificial skin models include three-dimensional culturing of human keratinocytes on the epidermis from which the epidermis has been removed (reconstructed epidermis on de-epidermized dermis (RE-DED)) and on the collagen matrix containing fibroblasts. There is a method of three-dimensional culturing keratinocytes (living skin equivalent, LSE) (Regnier M, Prunieras M, Woodley D. Front Matrix Biol . 1981; 9: 4-35; Bell E, Sher S, Hull B). , Merrill C, Rosen S, Chamson A, Asselineau D, Dubertret L, Coulomb B, Lapiere C, Nusgens B, Neveux Y. J Invest Dermatol. 1983; 81: 2s-10s.)

As the culture conditions are gradually improved after the development of artificial skin, the epidermis, the most important component of artificial skin, is not only very similar to human skin morphologically, including the stratum corneum, but also biochemically and functionally. (Asselineau D, Bernard BA, Bailly C, Darmon M, Prunieras M. J Invest Dermatol. 1986; 86: 181-186; Ponec M. Toxicol in Vitro. 1991; 5: 597-606; Ponec M, Weerheim A, Kempenaar J, Mulder A, Gooris GS, Bouwstra J, Mommaas AM.T. J Invest Dermatol. 1997; 109: 348-355; Ponec M, Gibbs S, Weerheim A, Kempenaar J, Mulder A, Mommaas AM. Arch Dermatol Res. 1997; 289: 317-326; Gibbs S, Vicanova J, Bouwstra J, Valstar C, Kempenaar J, Ponec M. Arch Dermatol Res. 1997; 289: 585-595; Gibbs S, Boelsma E, Kempenaar J , Ponec M. Cell Tissue Res. 1998; 292: 107-114; Stark HJ, Baur M, Breitkreutz D, Mirancea N, Fusenig NE.J Invest Dermatol. 1999; 112: 681-691)

However, the artificial skin developed to date is not the same as the human skin, and the skin of the artificial skin is different from the skin of the human skin in terms of expression of differentiation markers, lipid composition, and protective function. In addition, the method of culturing human keratinocytes three-dimensionally on the dermis from which the epidermis was removed does not have cellular components in the dermis, and the method of three-dimensionally culturing human keratinocytes on the collagen matrix containing fibroblasts is known as fibroblasts. It is included, but unlike the actual human dermis, it contains only type 1 collagen.

Also, a biotissue equivalent containing collagen of Korean Patent Publication No. 90-14547, a method of manufacturing a chitin-based interpenetrating structure film for artificial skin of Korean Patent Publication No. 96-6788, and a biocompatible perforated membrane in Korean Patent Publication No. 91-8999 And a method of manufacturing the same, artificial skin tissue such as three-dimensional regenerated artificial skin tissue and a method of manufacturing the same in Korean Patent Publication No. 97-60646, but using collagen or a biodegradable polymer, but only artificial skin It is not provided with artificial skin that can reproduce the appearance of human skin, confined to only three-dimensional culture.

Therefore, there is still a need to develop artificial skin culture methods that are suitable for academic research or medical treatment by maintaining artificial skin cultured in vitro.

Therefore, an object of the present invention is to provide an artificial skin that can reproduce the appearance of human skin as it is.

In addition, an object of the present invention is to provide a method for producing artificial skin that can reproduce the appearance of human skin as it is.

Figure 1 illustrates an artificial skin manufacturing method of the present invention,

Figure 2 is a comparison of the dermal counterpart of the artificial skin of the present invention and the existing artificial skin (RE-DED),

3 is a photograph showing that the artificial skin of the present invention dyed with hematoxylene and eosin,

Figure 4 is a photograph staining the distribution of keratin of artificial skin of the present invention by antigen antibody reaction,

Figure 5 is a photograph of the distribution of the involukrin of artificial skin of the present invention by antigen antibody reaction,

Figure 6 is a photograph of the distribution of the lorryrin of the artificial skin of the present invention by antigen antibody reaction,

7 is a photograph of the distribution of the filaggrin of artificial skin of the present invention by antigen antibody reaction.

In order to achieve the above object, the present invention provides a dermal counterpart combining the dermis from which the epidermis has been removed on a collagen substrate containing fibroblasts.

The present invention provides artificial skin cultured by inoculating keratinocytes into the dermal counterpart.

Hereinafter, the present invention will be described in detail.

The present inventors cultured the epidermal cells, preferably keratinocytes, in the dermal counterpart to produce artificial skin that can reproduce the appearance of human skin as it is.

The dermal counterpart is the most important factor in the cultivation of artificial skin as a substitute for nutrients, growth factors, signaling substances, etc. present in the epidermis of the human skin.

Preferred dermal counterparts of the artificial skin of the present invention are preferably collagen containing fibroblast and dermis from which the epidermis has been removed.

The artificial skin obtained by culturing keratinocytes on the dermis of collagen containing fibroblasts, the dermal counterpart for culturing the artificial skin of the present invention, is morphologically very similar to the epidermis of human skin, basal layer, pore layer, granule layer. , Epidermis with stratum corneum, and epidermis in the epidermis-dermis boundary, as in the epidermis of human skin. In addition, fibroblasts were observed in the dermis where the epidermis was removed from the artificial skin as in the human dermis, which is the result of migration of fibroblasts in the collagen matrix into the dermis.

In addition, the artificial skin of the present invention confirmed the differentiation pattern of keratinocytes by keratin 1 (Keratin 1), Involucrin, Loricrin and Filaggr in differentiation markers. It was found to have a similar differentiation pattern.

On the other hand, using the artificial skin of the present invention having similar properties to human skin, not only can perform physiological and molecular studies of the skin, but can also perform skin toxicity test on skin contact substances in a conventional manner. . The skin contact material is a material that comes into contact with the skin such as materials, cosmetics, fibers, and detergents.

In addition, the artificial skin of the present invention can be used for the purpose of treatment by performing a conventional method and implanting in the site where the skin is defective due to burns and skin ulcers.

Artificial skin manufacturing method of the present invention is as follows.

(1) Cell culture and final cell preparation step: separating and culturing fibroblasts, keratinocytes from skin tissue.

(2) Dermal counterpart preparation step: mixing the dermis with the epidermis removed on the collagen containing fibroblasts.

(3) keratinocyte inoculation step: inoculating the keratinocytes prepared in the cell preparation step on the dermal counterpart.

(4) artificial skin culture step: culturing keratinocytes in the state exposed to the atmosphere from 2 days after the culture solution so that the keratinocytes inoculated in step (3) is sufficiently submerged in the culture medium.

Skin tissue that can separate the fibroblasts, keratinocytes in step (1) is preferably any skin, the young skin is most preferred. The keratinocytes and fibroblasts are separated from the skin tissue and then cultured in the keratinocyte culture medium and the fibroblast culture medium. Collagen is preferably collagen type I, III and IV, more preferably type I collagen. Collagen solution: Reconstitution buffer: 10-fold concentrated P culture solution (DMEM: Ham's nutrient mixture F12 = 3: 1) in a ratio of 8: 1: 1 to prepare a collagen mixture.

In the preparation of the dermal counterpart of step (2), the fibroblasts cultured above are preferably included as 1 X 10 ⁵ to 5 X 10 ⁵ per mL of the collagen mixture prepared above. The fibroblast and collagen mixtures were mixed and put into the collagen mixture containing fibroblasts in millicells.

In the artificial skin culture of step (4), the culture solution is added so that the keratinocytes are sufficiently immersed in the culture solution, and cultured at 37 ° C. After 2 days, the culture solution in the millicell is removed to remove the keratinocytes from the air at 37 ° C. Incubate for 7 to 14 days.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

Example 1

Artificial skin preparation of the present invention was obtained by proceeding as shown in FIG. A detailed description with reference to FIG. 1 is as follows.

Cell Culture and Final Cell Preparation Steps

1) Collagen Preparation

As shown in a of FIG. 1, type 1 collagen solution (Nita gelatin, Tokyo, Japan) was dissolved in 0.05 N NaOH in a reconstitution buffer (2.2 g NaHCO _3, 4.77 g HEPES (200 mM) and finally The volume was adjusted to 100 ml) and 10 times P culture medium (DMEM and Ham's nutrient mixture F12 were mixed in a 3: 1 ratio) in a ratio of 8: 1: 1 to form a collagen mixture.

2) Primary culture of fibroblasts

As shown in b of FIG. 1, the skin tissue was washed three or more times with Solution A buffer (30 mM Hepes, 10 mM glucose, 3 mM KCl, 130 mM NaCl, 1.0 mM Na ₂ HPO₄.7H ₂ O, 0.0033 mM phenol red, pH 7.4). The washed tissue was placed in a solution A buffer containing 0.25 vol% of collagenase and incubated for 90 minutes in a CO 2 incubator. Remove skin tissue and separate epidermis and dermis using forceps. The isolated dermis was incubated in trypsin-EDTA (0.025%-0.01%) solution at 37 ° C for 5 minutes and then reacted for 10 minutes by adding 10% FBS (fetal bovine serum) to inactivate trypsin. After the reaction, the aspirate and discharge were repeated with a 5 ml pipette to release the cells. The cell suspension was centrifuged at 200 g for 5 minutes to remove the supernatant and washed again with Solution A for 5 minutes. The cells were mixed in fibroblast culture medium and the number of cells was measured on a hemocytometer. The cells were inoculated to about 1 × 10 ⁴ cfu / cm 2 and cultured at 37 ° C. Cell culture medium was cultured fibroblasts using DMEM medium containing 10% FBS. After 2 days of inoculation, the cells were changed and washed once with solution A after 5 days, and then changed again. After that, the cells were washed with solution A once every 2 days and ground. When fibroblasts were 60-80% full in the culture vessel, the fibroblasts were removed and passaged or cryopreserved.

3) Primary culture of keratinocytes

As shown in c of FIG. 1, the epidermis was isolated and cultured in the same manner as the fibroblasts of 2), and KGM (Clonetics, USA) was used as a keratinocyte culture medium, and the modified MCDB (modifed MCDB) 153 medium was used. Epidermal growth factor 10 ng / ml, bovine pituitary extract 70 ㎍ / ml, hydrocortisone 0.5 ㎍ / ml, insulin 5 ㎍ / ml, penicillin A culture medium containing 100 µg / ml, streptomycin 100 µg / ml, and fungizone 0.25 µg / ml was used. Once the cells have grown 60-80% in the culture vessel, detach and freeze the cells.

4) Preparation of dermis with epidermis removed

As shown in FIG. 1 d, normal skin of the obtained interlayer skin graft was washed, and then cultured for 7 days at 37 ° C. in a PBS solution without addition of calcium and magnesium. The epidermis was then separated from the dermis using forceps. Freezing and thawing were repeated several times using liquid nitrogen to remove various cellular components in the epidermis from which the epidermis was separated.

Dermal counterpart manufacturing step

The fibroblasts cultured above were mixed at 3 × 10 ⁵ per mL of the collagen mixture prepared above. 0.2 mL of the collagen mixture containing fibroblasts was divided into millicells in a 6-well plate and incubated for 30 minutes in a 37 ° C. incubator to form a collagen matrix containing fibroblasts. After 2 days, the epidermis from which the epidermis prepared above was removed was placed on the collagen matrix in millicells, with the basement membrane facing upward.

Keratinocyte inoculation step

The keratinocytes subcultured at 5 × 10 ⁵ cells / millicells on the epidermis with fibroblasts containing fibroblasts in millicells were subcultured at a ratio of 3: 1 in DMEM and Ham's nutrient mixture F12. 10% FBS, 5 ug / mL insulin, 1 X 10 ^-10 M Choleratoxin, 0.4 ug / mL hydrocor tisone, 5 ug / mL transferrin, 1 X 10 ^-11 M triiodothyronine (triiodothyronine added) cultures were mixed and inoculated.

Artificial skin culture stage

In the artificial skin culture of the present invention, as shown in Figure 1e, 10% FBS, 5 ug / in a mixture in which the inoculated keratinocytes were sufficiently mixed in a culture solution (DMEM and Ham's nutrient mixture F12 3: 1 ratio) mL insulin, 1 X 10 ^-10 M choleratoxin, 0.4 ug / mL hydrocortisone, 5 ug / mL transferrin, 1 X 10 ^-11 M triiodothyronine The culture solution was added to submerged in the culture solution) and incubated at 37 ° C. After 2 days of culture, keratinocytes were cultured in the state of being exposed to the air as f, and cultured for 1 week or more while changing the culture solution every 2 days.

Comparative Example 1

Reconstructed epidermis on de-epidermized method for culturing human keratinocytes on the dermis from which the most representative epidermal skin is currently developed to compare the tissue characteristics of the artificial skin with the existing artificial skin. dermis, RE-DED) to prepare artificial skin. 2 a) shows that the artificial skin culture method of the present invention is inoculated and cultured in the order of the dermis (2), keratinocytes (1) in which the epidermis was removed from the collagen matrix (3) containing the lower fibroblasts, 2 b) shows that the keratinocytes 1 were cultured on the dermis 2 from which the epidermis was removed by the RE-DED method.

Experimental Example

Comparison of Morphological Properties of Skin

Artificial skin tissue and normal skin tissues of the examples and comparative examples were fixed in 5% formalin and then embedded in paraffin to make blocks. Hematoxylin and Eosin staining was performed after the block was set to 4-6 μm to observe the morphological characteristics of artificial skin under a microscope.

The results are shown in Figure 3, hematoxylin and eosin stained pictures are shown. a, b and c are skin tissues of the artificial skin of the present invention, a is the entire skin tissue, b is the upper part of a, c is an enlarged photograph of the lower part of a, and arrows b and c indicate fibroblasts. . In addition, d, e, f is a picture of the artificial skin, RE-DED skin, human skin tissue picture of the present invention.

As a result of observing with hematoxylin-eosin staining, in the artificial skin of the present invention, the epidermis of the basal layer, the polar layer, the granule layer, and the stratum corneum was formed very much like the epidermis of the human skin, and also the epidermis of the human skin. Epidermal capacity was observed at the epidermal-dermal boundary as in. In the artificial skin of the present invention, fibroblasts (arrows in b and c of FIG. 3) were observed in the dermis from which the epidermis was removed, which can be said to be a result of the migration of fibroblasts in the collagen matrix into the dermis. Compared to the skin, it can be seen that the artificial skin of the present invention is almost similar to human skin.

Expression Pattern of Differentiation Markers of Skin Tissues

In the epidermis of the artificial skin of the above embodiment, immunohistochemical staining was performed to determine the expression patterns of the differentiation markers. As the primary antibody of immunohistochemical staining, antibodies to keratin 1, Involucrin, Loricrin, and Filagg rin were used. Immunohistochemical staining using the antibodies was performed as follows using the LSAB kit.

Deparaffinization was carried out using alcohol and xylene, followed by reaction for 30 minutes with a solution containing 3% H ₂ O ₂ in methanol to inhibit the activity of endogenous peroxidase . Thereafter, the resultant was reacted with a blocking reagent (commercially available) for 5 minutes, and the primary antibody was diluted and reacted at room temperature for 1 hour or overnight at 4 ° C. After completion of the reaction, a biotin-fused secondary antibody (Biotinylated secondary antibody) was added and reacted with skin tissue for 20 minutes, followed by another 20 minutes in Streptoavi din peroxidase. Subsequently, a time-dependent color reaction was induced with DAB, and after staining with Meyer's hematoxylin, the expression patterns of the differentiation markers were observed under an optical microscope.

(1) keratin 1 staining

4, a, b, and c show skin tissues stained with keratin, a is normal human skin tissue, b is artificial skin of an example, and c is a photograph of stained artificial skin of a comparative example of RE-DED.

In the RE-DED artificial skin, it was expressed in all layers including the stratum corneum except for the lower two or three layers of the epidermis, but in the artificial skin of the present invention, keratin 1 started to be expressed more from the lower epidermis and was more similar to the epidermis of human skin. have.

(2) Involukrin Dyeing

5, a, b, and c show skin tissues stained with involuclin, a is normal human skin tissue, b is artificial skin of an example, and c is a photograph of stained RE-DED artificial skin of a comparative example. to be.

In the artificial skin of RE-DED artificial skin, Involukrin was expressed in all layers except the stratum corneum, but in the artificial skin of Example, Involukrin began to be expressed later except the lower one or two layers of the epidermis, which is more similar to the epidermis of human skin. have.

(3) dyeing of lyochlorine

6, a, b, and c show skin tissues stained with lyochlorine, a is normal human skin tissue, b is artificial skin of an example, and c is a photograph of stained RE-DED artificial skin of a comparative example. .

In the artificial skin of RE-DED, lyochlorine was discontinuously weakly expressed in the granule layer, but in the artificial skin of the example, the lyochlorine was linearly expressed in the granular layer almost the same as the epidermis of the human skin.

(4) dyeing of filaggrin

7, a, b, and c show skin tissues stained with filaggrin, a is normal human skin tissue, b is artificial skin of an example, and c is a photograph of stained RE-DED artificial skin of a comparative example. .

In the RE-DED artificial skin, the filaggrin is partially weakly expressed in the upper part of the polar layer, the granule layer, and the stratum corneum, but in the artificial skin of the example, the filaggrin was expressed in the granule layer and the stratum corneum very similarly to the epidermis of the human skin.

As mentioned above, the artificial skin of the present invention maintains the shape of the human skin with the epidermis of the middle layer having a basal layer, a polar layer, a granular layer, and a stratum corneum, in contrast to the existing artificial skin. In addition, the expression of differentiation markers specifically expressed in the epidermal layer of normal human skin was similarly generated in the artificial skin of the present invention. Therefore, the artificial skin of the present invention can be used as a material for skin biology research or skin toxicology research, as well as in the clinical field can be used for skin transplantation.

Claims (6)

Dermal counterparts that combine the epidermis with the epidermis removed on the collagen substrate containing fibroblasts. The dermal counterpart according to claim 1, wherein the collagen matrix containing fibroblasts comprises 1 × 10 ⁵ to 5 × 10 ⁵ fibroblasts per mL of the collagen mixture. An artificial skin cultured by inoculating keratinocytes into a dermal counterpart to which the epidermis from which the epidermis has been removed on a collagen substrate containing fibroblasts. The artificial skin according to claim 3, wherein the artificial skin culture is performed after culturing the inoculated keratinocytes in a state sufficiently immersed in the culture medium and then exposing it to the atmosphere. 4. The artificial skin of claim 3, wherein the artificial skin is used for skin toxicity test. The artificial skin according to claim 3, wherein the artificial skin is used as a skin graft method on a site where the skin is defective.