KR20210044763A - Skin equivalent manufactured using cd29 expressing cell and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a method for culturing skin cells capable of in vitro proliferation using only CD29-expressing cells. The skin cells expressing CD29 according to the present invention can be subcultured several times to produce a skin equivalent having a normal skin structure. Specifically, when only skin cells expressing CD29 are cultured, at least 15 passages can be cultured in vitro, have the properties of skin stem cells and proliferate in vitro. In addition, in the case of subculturing general skin-derived primary cells, the structure of various layers seen in normal skin is not found, whereas the skin cells expressing CD29 according to the present invention can be used to prepare a skin equivalent having a certain differentiation efficiency even after subculture through several passages.

Description

Artificial skin produced using CD29-expressing cells and its manufacturing method {SKIN EQUIVALENT MANUFACTURED USING CD29 EXPRESSING CELL AND MANUFACTURING METHOD THEREOF}

The present invention relates to a method of culturing skin cells capable of proliferating in vitro using only CD29-expressing cells.

Recently, the use of laboratory animals has been regulated to conduct safety evaluation on chemical substances such as cosmetics and pharmaceuticals that are easily exposed to human skin. In the case of the in vitro skin model used in the experimental animal replacement test method, products are released and sold by various companies. Most of them are primary cells isolated from human skin epidermis, and the isolated skin cells have the disadvantage that subculture is limited to 4 times or less. Accordingly, there is a need to mass-produce high-quality skin cells in order to manufacture artificial skin with a certain differentiation efficiency and high reproducibility.

On the other hand, there are attempts to artificially construct skin tissue. Such artificial skin is generally used for skin damage, skin ulcers, or toxicity and efficacy tests of pharmaceuticals or cosmetics. Artificial skin is a skin substitute that can replace damaged human skin, and is very important as an experimental material for skin treatment, development of pharmaceuticals for this, or testing the toxicity and efficacy of medical devices, daily necessities, and cosmetics that come into contact with the skin. .

Therefore, for such use, the development of artificial skin with a structure that mimics the natural skin layer is required, and a representative method among artificial skin developed so far is the method of culturing human keratin cells on the dermis from which the epidermis has been removed in three dimensions. , There is a method of culturing human keratinocytes three-dimensionally on a collagen matrix containing fibroblasts (Korean Laid-Open Patent Publication No. 10-2013-0056956). However, in the case of the artificial skin developed so far, there is a disadvantage that the dermal layer contraction occurs during the culturing process, and thus a technology for manufacturing an artificial skin similar to the natural skin layer is still required.

Korean Patent Application Publication No. 10-2013-0056956

Under the above background, the present inventors have made intensive research efforts to develop artificial skin similar to the human skin layer that can more stably be used for toxicity and efficacy experiments of pharmaceuticals or cosmetics, and as a result of separating proliferable cells expressing specific markers. The present invention was completed by confirming that the in vitro artificial skin produced by cultivation stably proliferates in vitro even in several passages, has a certain differentiation efficiency even in the later passages, and can overcome contraction and detachment of the dermal layer. .

Accordingly, an object of the present invention is to provide a method of culturing skin cells using only skin cells expressing CD29 to produce an artificial skin model, and to provide an artificial skin manufactured by the method.

In order to achieve the above object, the present invention comprises the steps of isolating only skin cells expressing CD29; And it provides a skin cell culture method comprising the step of culturing the separated skin cells.

In addition, the present invention comprises the steps of culturing fibroblast cells in a matrix; And it provides a method for producing an in vitro artificial skin comprising the step of culturing by adding the skin cells expressing CD29.

In addition, the present invention provides an extracorporeal artificial skin manufactured by the method of producing the extracorporeal artificial skin.

The skin cells expressing CD29 according to the present invention can stably proliferate in vitro even after several passages are performed, and artificial skin having a normal skin structure can be manufactured. Specifically, when only skin cells expressing CD29 are cultured, at least 15 passages can be cultured in vitro, have features of skin stem cells, and proliferate in vitro. In addition, when subculturing the skin-derived primary cells of a general later passage, the structure of various layers seen in normal skin is not visible, whereas the skin cells expressing CD29 according to the present invention have a constant differentiation efficiency even after several passages. Artificial skin can be manufactured. Accordingly, the present invention can reproduce a stable skin tissue model by isolating and purifying only skin cells expressing CD29 capable of proliferating in vitro and culturing them. In addition, in the manufacturing process of the in vitro artificial skin, the culture period is shortened compared to the conventional skin tissue culture, so that it is possible to manufacture an in vitro artificial skin having a normal structure without desorption of cells. The in vitro artificial skin according to the present invention can be applied to various fields as a substitute for animal testing, and can stably provide a large amount of in vitro experimental model.

1A shows the state of cell proliferation by passage (2nd, 4th, and 6th) after passage of human skin-derived primary cells in vitro.
2 shows the expression patterns of various skin stem cell markers in skin cells by passage.
Figure 3 shows the in vitro proliferation rate of the cells after separating and culturing only skin cells expressing CD29 through FACS.
Figure 4 shows the cell shape and proliferation state by passage after separation and cultivation of only skin cells expressing CD29 through FACS.
5 shows the protein expression patterns of skin-related markers and skin stem cell markers in skin cells expressing CD29 and control skin cells isolated through FACS.
6 shows the change in the proliferation rate of cells after 72 hours after treatment with 1 ug/ml and 5 ug/ml of antibodies that can specifically block only CD29 in the later passage of CD29 cells isolated through FACS. .
7 shows the proliferation rate of cells and expression patterns of proliferation-related markers 72 hours after treatment with siRNA capable of lowering the expression of mRNA encoding CD29 in the late passage of CD29 cells isolated through FACS.
8 shows a state in which only skin cells expressing CD29 are cultured using a multi insert dish.
FIG. 9 shows an artificial skin model produced using a control group of the initial passage (passage 2 and 3) and the mid-late passage (passage 6 and 10) and skin cells expressing CD29.
FIG. 10 shows the expression patterns of markers that can represent each skin layer of the artificial skin model produced in FIG. 8.
FIG. 11 shows an artificial skin model 48 hours after treatment with irritants to verify whether or not an artificial skin model produced using cells expressing CD29 is utilized in vitro.
12 is a table showing the cell viability of the artificial skin model tested in FIG. 11.

In one aspect, the present invention comprises the steps of isolating only skin cells expressing CD29; And it provides a skin cell culture method comprising the step of culturing the separated skin cells.

The skin cell culture method of the present invention may include the step of isolating only skin cells expressing CD29. In this case, the step of separating only skin cells expressing CD29 may be performed by a fluorescence-activated cell sorting (FACS) technique.

The term "CD29" as used herein, also known as Integrin beta-1 (ITGB1), is a cell surface receptor encoded by the ITGB1 gene in humans. The CD29 may be a polypeptide having the amino acid sequence of SEQ ID NO: 1.

The term "skin cell" used herein may be a cell present in the epidermis and the dermis.

The epidermis is divided into stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum and stratum germinativum (also referred to as "stratum basale"). , It has no blood vessels and diffuses from the dermis to receive nutrition. The epidermis is divided into several layers of cells formed through mitosis in the innermost layer. They are differentiated and filled with keratin and rise up into strata that change shape and composition. They eventually reach the top layer, called the stratum corneum (stratum corneum), which acts as the skin barrier layer, and peels or exfoliates. The outermost layer of the epidermis consists of 25 to 30 layers of dead cells.

The dermis is a layer of skin under the epidermis composed of epithelial tissue. The dermis is structurally divided into a superficial region adjacent to the epidermis, a papillary region, and a thicker region known as a reticular region. The dermis is closely connected to the epidermis by the basement membrane. It also contains many nerve endings that provide a sense of touch and heat, and contains hair follicles, sweat glands, sebaceous glands, apocrine glands, lymphatic vessels and blood vessels. The blood vessels in the dermis remove nutrients and waste from their own cells as well as the base of the epidermis.

The FACS is a differentiated type of flow cytometer, and provides a method of sorting heterogeneously mixed cells based on their respective specific light scattering and fluorescence characteristics. In one embodiment of the present invention, after confirming the expression pattern of various skin stem cell markers such as CD29, CK14, CD34, P63, YAP, CD44, CD49f and CD200 expressed in skin cells, CD29 among skin cells was identified through FACS. Only expressing skin cells were isolated.

Meanwhile, the present invention may include the step of primary culturing the skin cells before separating only the skin cells expressing CD29 from the skin cells. The term "primary culture" used herein refers to a culture starting with skin cells directly collected from an individual, and the skin cells may be derived from humans. The primary culture of skin cells is for 7 to 10 days. Can be done.

In addition, the culture of the skin cells expressing the isolated CD29 may be performed for 100 to 200 days, 120 to 180 days, 140 to 160 days, preferably 140 days. On the other hand, since one passage takes about 4 to 7 days, when only the skin cells expressing the isolated CD29 of the present invention are isolated and cultured, it can be cultured for at least 15 passages, specifically, for 15 to 30 passages. Can be cultured. This means that the cultivation period is significantly increased compared to that of the skin-derived primary cells (control group, CTL) without separating and culturing CD29 cells for 7 to 8 passages.

In another aspect, the present invention comprises the steps of culturing fibroblast cells in a matrix; And it provides a method for producing an in vitro artificial skin comprising the step of culturing by adding the skin cells expressing CD29.

The method of manufacturing an in vitro artificial skin of the present invention may include culturing fibroblast cells in a matrix. The matrix may comprise collagen and/or fibronectin. In this case, the step may be performed for 7 to 10 days. More specifically, a mesh-shaped insert was coated with 1 mg/ml to 3 mg/ml of type I collagen. Then, in a matrix containing 1 mg/ml to 3 mg/ml of type I collagen, 10 ul to 100 ul of Matrigel, and 5 ug/ml to 100 ug/ml of fibronectin. The step of culturing fibroblasts may be performed for 7 to 10 days.

The term "collagen" as used herein is a light protein present in both plants and animals, mainly present in the bones and skin of animals, and is also distributed in cartilage, organ membranes, hair, etc., and is also called collagen. In the present invention, collagen may be derived from humans, cattle, pigs, mice, rats, etc., but is not limited thereto. In the present invention, the collagen may be not only natural collagen but also biotechnologically modified or biotechnologically or chemically treated modified collagen, but is not limited to origin and modification.

As used herein, the term "fibronectin" is a large peripheral protein on the surface of most animal cells, and is present in the blood of animals, on the surface of cultured cells, or on the extracellular matrix of tissues.

As used herein, the term "fibroblast" refers to a cell constituting an important component of fibrous connective tissue, and particularly, in the skin related to the present invention, refers to a major cell constituting the dermal layer. In the present invention, fibroblasts may be derived from humans, mice, rats, etc., but are not limited thereto. For the purposes of the present invention, fibroblasts used in the manufacture of the dermal layer in the process of manufacturing the in vitro artificial skin of the present invention may in particular be human-derived fibroblasts. In addition, the fibroblasts of the present invention may be fibroblasts derived from normal tissues or pathogenic tissues such as chemical substances, burns, wounds, scars, ulcers, etc., but are not limited thereto.

In addition, the method of manufacturing an in vitro artificial skin of the present invention may perform the step of culturing by adding skin cells expressing CD29 to the culture cultured in the fibroblast culturing step. The skin cells expressing the CD29 may be cultured by the skin cell culture method according to the present invention. In this case, the step may be performed for 1 to 3 days, and specifically for 2 days. In addition, skin cells expressing CD29 are 1x10 ¹ cells/well to 1x10 ¹⁰ cells/well, 1x10 ² cells/well to 1x10 ⁸ cells/well, 1x10 ³ cells/well to 1x10 ⁷ cells It may be added at a concentration of number/well, 1×10 ⁴ cell number/well to 1×10 ⁶ cell number/well, and specifically, it may be added at a concentration of 1×10 ⁶ cell number/well.

In addition, after the step of culturing by adding the skin cells expressing CD29, the method of preparing the in vitro artificial skin of the present invention, the cultured cells are in a medium containing at least one selected from the group consisting of insulin, EGF, and hydrocortisone. It may further include a step of culturing. The medium may specifically contain insulin, EGF and hydrocortisone. In this case, the culturing step may be performed for 3 to 7 days, and specifically for 5 days.

In one embodiment of the present invention, an experiment was conducted to differentiate only skin cells expressing CD29 into an in vitro skin tissue model. First, fibroblasts were put in a matrix mixed with collagen, fibronectin, and matrigel, and then cultured in DMEM medium to which 10% FBS was added for 7 to 10 days. In the culture, a 24-well Multi Insert Dish was used. Thereafter, only the isolated CD29-expressing skin cells were ^{added at a concentration of 1×10 6} cells/well, and then cultured for 2 more days in a culture medium holding the skin cells. Thereafter, the Multi Insert Dish was transferred to a 24-well dish and cultured for 3 days in a medium to which 1% FBS, ITS, EGF, hydrocortisone, L-glutamine, and calcium chloride were added. At this time, the cultivation was performed using an air-liquid interface method in which culture medium was added to only the lower portion of the multi-insert dish and cultured. As a result, when a skin model was constructed using only skin cells expressing CD29 of the early (passage 3) and late (passage 10), in both cell groups, various cells in several layers similar to human skin tissues The organization model composed of was confirmed.

In another aspect, the present invention provides an in vitro artificial skin manufactured by the method for manufacturing an in vitro artificial skin according to the present invention.

The term "artificial skin in vitro" as used herein refers to a skin layer model similar to a natural skin layer manufactured through an artificial culture process in vitro. Skin cells constituting this may be derived from human skin.

In the in vitro artificial skin manufactured according to the method of manufacturing the in vitro artificial skin of the present invention, the expression of any one or more proteins selected from the group consisting of CD29, Cytokeratin 10, Cytokeratin 14, Yap, and P63 may be increased.

The in vitro artificial skin manufactured according to the method for manufacturing the in vitro artificial skin of the present invention can be usefully used in conducting physiological, molecular biology, or biochemical studies related to the skin of animals including humans. It can also be used for efficacy or toxicity tests on substances that are usually expected to come into contact with the skin. The substances expected to come into contact with the skin are generically referred to as all substances such as pharmaceuticals (especially external preparations), cosmetics, textiles, or detergents, and may be those that are typically used for efficacy or toxicity testing of pharmaceuticals or cosmetics. In addition, the in vitro artificial skin according to the present invention can be used not only for efficacy or toxicity tests in normal skin conditions, but also as a disease model for cases in which the skin is damaged by chemicals, burns, wounds, scars, ulcers, and the like. In addition, the in vitro artificial skin according to the present invention can be used as a substitute for animal testing. In addition, the in vitro artificial skin manufactured according to the present invention may be used not only for experiments but also as a drug for skin transplantation or wound treatment.

The in vitro artificial skin produced by the method of manufacturing the in vitro artificial skin according to the present invention was excellent in the formation and differentiation of the dermal layer and the epidermal layer, and the dermal layer and collagen contraction were suppressed, showing a structure similar to that of a real human skin layer.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited thereto.

Example 1. Subculture of skin-derived primary cells and production of artificial skin models

Human skin-derived primary cells (Applied Biological Materials and Biosolution Co., Ltd.) were cultured in a culture dish coated with 0.2% gelatin. At this time, as the culture medium, KGM-Gold keratinocyte growth medium (Lonza) was used. The number of cells was 40 cells / cm ² to 1,000 cells / cm ² put in a dish and cultured. The culture solution was changed once every 2 days, and passage was performed when the confluence reached 70% to 80%. In order to isolate CD29 cells, skin-derived primary cells aged 7 to 14 days of culture were removed from the culture dish. Thereafter, only cells expressing CD29 were stained using a CD29 antibody (Abcam) with a fluorescent dye attached, and then CD29 cells were isolated through FACS. Thereafter, the ^{culture was cultured in KGM TM} Gold ^TM keratinocyte growth medium (Lonza), and the culture medium was changed once every 2 days and subcultured when the density reached 70% to 80%.

After the human skin-derived primary cells were subcultured in vitro, a skin tissue model was produced using cells for each passage. The specific culture process is as follows. First, a mesh-type insert was coated with 1 mg/ml to 3 mg/ml of type I collagen. Then, fibroblasts (ATCC) irradiated with 6000 rad (gamma irradiation) of 1 mg/ml to 3 mg/ml of type I collagen, 5 ug/ml to 100 ug/ml of fibronectin and 10 ul to 100 ul of mat After putting in a matrix mixed with a rigel (matrigel), it was cultured in DMEM medium to which 10% FBS was added for 7 to 10 days. After 7 to 10 days, only skin-derived primary cells or skin cells expressing CD29 are ^{added at a concentration of 1x10 6} cells/well and then cultured to maintain skin-derived cells (KGM ^TM Gold ^TM , Lonza) Incubated for 2 more days. Thereafter, the insert dish was transferred to a 24-well dish, and in a medium containing 1% FBS, ITS, EGF, hydrocortisone, L-glutamine, and calcium chloride, 3 Incubated for days to 7 days. At this time, the cultivation was performed using an air-liquid interface method in which culture medium was added to only the lower portion of the multi-insert dish and cultured. A skin tissue model was produced using the cultured cells.

Among the produced models, each structure was confirmed by H&E staining a skin tissue model made of passage 2, 4, and 6 cells. The results are shown in FIG. 9. As shown in FIG. 9, in the case of the skin model made with the 6th cell of the passage, various cell layers were not observed, but in the case of the skin model made with the cells expressing CD29, the cells of the initial passage were Similar to the one used, various cell layers of the skin dermis were observed.

Example 2. Confirmation of expression of stem cell markers in skin cells by passage

In order to obtain cells that can proliferate in vitro, the expression patterns of various skin stem cell markers (CD29, CK14, CD34, P63, YAP, CD44, CD49f, CD200, Cytokeratin 10 and Cytokeratin 14) were confirmed in skin cells of each passage. I did. The results are shown in FIG. 2. In addition, only skin cells expressing CD29 were isolated and cultured through FACS, and the in vitro proliferation rate and cell shape by passage are shown in FIGS. 2 to 5.

As shown in FIG. 2, in the case of the skin stem cell marker CD29, it was observed that the expression amount decreased significantly after the proliferation rate of skin cells fell. In addition, as shown in FIGS. 3 and 4, the proliferation rate of only skin cells expressing CD29 was significantly increased compared to that of skin-derived primary cells (control, CTL) that were not separated and cultured, and the shape of the cells until the 15th passage And it was observed that the growth rate was maintained. In addition, as shown in FIG. 5, in skin cells expressing CD29, not only CD29 but also skin-related markers (Cytokeratin 10 and Cytokeratin 14) and other The expression of skin stem cell markers (Yap and P63) was remarkably increased.

Example 3. Confirmation of association between in vitro proliferation rate and CD29 expression

Antibodies capable of specifically binding to CD29 (Abcam #AB24693) to confirm whether CD29-expressing cells remarkably increased in vitro proliferation rate and skin stem cell marker expression have a direct correlation with CD29 expression (Abcam #AB24693) Alternatively, after reducing the expression of CD29 by treatment with siRNA, the expression patterns of cell proliferation and proliferation-related markers were confirmed. The results are shown in FIGS. 6 and 7.

As shown in Figure 6, as a result of confirming the proliferation rate of cells 48 hours after treatment with the CD29 antibody on the CD29-expressing cells, Ki67 in cells treated with 5 ug/ml of the antibody compared to the control (CTL) not treated with the antibody. It was observed that the amount of the cell group expressing the was significantly reduced. In addition, as a result of confirming the proliferation rate of cells by treatment with siRNA that can control the mRNA expression of CD29, the amount of KI67-expressing cell group not only decreased compared to the control group not treated with siRNA in the cell group treated with 1 nM siRNA. , It was confirmed that the expression of markers related to cell proliferation (cyclin D1 and cyclin B1) and skin stem cell markers (Yap and P63) were significantly reduced (FIG. 7). Through the above results, it was found that the expression of CD29 regulates the in vitro proliferation rate of skin-derived primary cells.

Example 4. Preparation of artificial skin model and identification of skin cell markers

In order to prepare skin tissue in vitro using only skin cells expressing CD29, SPL's 24-well Multi Insert Dish was used. At this time, the Multi Insert Dish and the state of culturing the cells using the same are shown in Fig. 8, and the culture results are shown in Figs. 9 and 10.

As shown in Figure 9, as a result of confirming each structure by H&E staining a skin tissue model made of skin-derived primary cells that were not separated and cultured for passage 2 and 6, passage 2 The structure of various layers seen in normal skin was not seen compared to the skin tissue model made with the first cell.

However, when a skin tissue model was constructed using only skin cells expressing CD29, various cells in several layers similar to human skin tissue were formed in both the early (passage 3rd) and late (passage 10th) cell groups. The structured organization model could be confirmed.

As shown in FIG. 10, as a result of confirming the expression of skin cell markers in several layers of the produced skin tissue model, various skin tissue-specific markers were found in both the early (passage 3rd) and late (passage 10th) cell groups. It was confirmed that it was expressed. Through the above results, it was confirmed that the produced skin tissue model was formed in a layer similar to that of the human body.

Example 5. In vitro skin irritation test of artificial skin model

To verify whether an in vitro artificial skin model produced using skin cells expressing CD29 can be used as an in vitro human model, an in vitro skin irritation test was performed. Diethyl phthalate was used as a control compound without skin irritation, the effect of tetrachlorethylene, which is known to irritate the skin, was examined, and 5% SDS was used as a control for cell death. The test results are shown in FIGS. 11 and 12.

As shown in FIG. 11, after performing an in vitro skin irritation test, the skin tissue was cut and H&E stained. As a result, in the case of a control compound not treated with the compound (CTL) and diethyl phthalate, a control compound without skin irritation, the skin tissue model was It showed the appearance of a normal skin model without being affected. On the other hand, in the case of tetrachloroethylene, which is known to irritate the skin, swelling of skin cells appeared to occur, resulting in cell death.

As shown in FIG. 12, as a result of confirming the death of cells after the in vitro skin irritation test through the MTT assay, about 70% of the cells died in the case of a skin tissue model treated with tetrachloroethylene, which can irritate the skin. Confirmed. Through the above results, it was proved that the produced skin tissue model can be used as an in vitro skin model.

From the above results, it is expected that the skin cell culture method including the step of isolating only skin cells expressing CD29 according to the embodiment can stably and effectively reproduce a skin tissue model. In addition, the in vitro artificial skin produced by the skin cell culture method is expected to be applicable to various fields as an alternative test method for experimental animals.

<110> Korea Research Institute of Bioscience and Biotechnology <120> SKIN EQUIVALENT MANUFACTURED USING CD29 EXPRESSING CELL AND MANUFACTURING METHOD THEREOF <130> FPD/202104-0004 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 798 <212> PRT <213> Artificial Sequence <220> <223> CD29 <400> 1 Met Asn Leu Gln Pro Ile Phe Trp Ile Gly Leu Ile Ser Ser Val Cys 1 5 10 15 Cys Val Phe Ala Gln Thr Asp Glu Asn Arg Cys Leu Lys Ala Asn Ala 20 25 30 Lys Ser Cys Gly Glu Cys Ile Gln Ala Gly Pro Asn Cys Gly Trp Cys 35 40 45 Thr Asn Ser Thr Phe Leu Gln Glu Gly Met Pro Thr Ser Ala Arg Cys 50 55 60 Asp Asp Leu Glu Ala Leu Lys Lys Lys Gly Cys Pro Pro Asp Asp Ile 65 70 75 80 Glu Asn Pro Arg Gly Ser Lys Asp Ile Lys Lys Asn Lys Asn Val Thr 85 90 95 Asn Arg Ser Lys Gly Thr Ala Glu Lys Leu Lys Pro Glu Asp Ile Thr 100 105 110 Gln Ile Gln Pro Gln Gln Leu Val Leu Arg Leu Arg Ser Gly Glu Pro 115 120 125 Gln Thr Phe Thr Leu Lys Phe Lys Arg Ala Glu Asp Tyr Pro Ile Asp 130 135 140 Leu Tyr Tyr Leu Met Asp Leu Ser Tyr Ser Met Lys Asp Asp Leu Glu 145 150 155 160 Asn Val Lys Ser Leu Gly Thr Asp Leu Met Asn Glu Met Arg Arg Ile 165 170 175 Thr Ser Asp Phe Arg Ile Gly Phe Gly Ser Phe Val Glu Lys Thr Val 180 185 190 Met Pro Tyr Ile Ser Thr Thr Pro Ala Lys Leu Arg Asn Pro Cys Thr 195 200 205 Ser Glu Gln Asn Cys Thr Ser Pro Phe Ser Tyr Lys Asn Val Leu Ser 210 215 220 Leu Thr Asn Lys Gly Glu Val Phe Asn Glu Leu Val Gly Lys Gln Arg 225 230 235 240 Ile Ser Gly Asn Leu Asp Ser Pro Glu Gly Gly Phe Asp Ala Ile Met 245 250 255 Gln Val Ala Val Cys Gly Ser Leu Ile Gly Trp Arg Asn Val Thr Arg 260 265 270 Leu Leu Val Phe Ser Thr Asp Ala Gly Phe His Phe Ala Gly Asp Gly 275 280 285 Lys Leu Gly Gly Ile Val Leu Pro Asn Asp Gly Gln Cys His Leu Glu 290 295 300 Asn Asn Met Tyr Thr Met Ser His Tyr Tyr Asp Tyr Pro Ser Ile Ala 305 310 315 320 His Leu Val Gln Lys Leu Ser Glu Asn Asn Ile Gln Thr Ile Phe Ala 325 330 335 Val Thr Glu Glu Phe Gln Pro Val Tyr Lys Glu Leu Lys Asn Leu Ile 340 345 350 Pro Lys Ser Ala Val Gly Thr Leu Ser Ala Asn Ser Ser Asn Val Ile 355 360 365 Gln Leu Ile Ile Asp Ala Tyr Asn Ser Leu Ser Ser Glu Val Ile Leu 370 375 380 Glu Asn Gly Lys Leu Ser Glu Gly Val Thr Ile Ser Tyr Lys Ser Tyr 385 390 395 400 Cys Lys Asn Gly Val Asn Gly Thr Gly Glu Asn Gly Arg Lys Cys Ser 405 410 415 Asn Ile Ser Ile Gly Asp Glu Val Gln Phe Glu Ile Ser Ile Thr Ser 420 425 430 Asn Lys Cys Pro Lys Lys Asp Ser Asp Ser Phe Lys Ile Arg Pro Leu 435 440 445 Gly Phe Thr Glu Glu Val Glu Val Ile Leu Gln Tyr Ile Cys Glu Cys 450 455 460 Glu Cys Gln Ser Glu Gly Ile Pro Glu Ser Pro Lys Cys His Glu Gly 465 470 475 480 Asn Gly Thr Phe Glu Cys Gly Ala Cys Arg Cys Asn Glu Gly Arg Val 485 490 495 Gly Arg His Cys Glu Cys Ser Thr Asp Glu Val Asn Ser Glu Asp Met 500 505 510 Asp Ala Tyr Cys Arg Lys Glu Asn Ser Ser Glu Ile Cys Ser Asn Asn 515 520 525 Gly Glu Cys Val Cys Gly Gln Cys Val Cys Arg Lys Arg Asp Asn Thr 530 535 540 Asn Glu Ile Tyr Ser Gly Lys Phe Cys Glu Cys Asp Asn Phe Asn Cys 545 550 555 560 Asp Arg Ser Asn Gly Leu Ile Cys Gly Gly Asn Gly Val Cys Lys Cys 565 570 575 Arg Val Cys Glu Cys Asn Pro Asn Tyr Thr Gly Ser Ala Cys Asp Cys 580 585 590 Ser Leu Asp Thr Ser Thr Cys Glu Ala Ser Asn Gly Gln Ile Cys Asn 595 600 605 Gly Arg Gly Ile Cys Glu Cys Gly Val Cys Lys Cys Thr Asp Pro Lys 610 615 620 Phe Gln Gly Gln Thr Cys Glu Met Cys Gln Thr Cys Leu Gly Val Cys 625 630 635 640 Ala Glu His Lys Glu Cys Val Gln Cys Arg Ala Phe Asn Lys Gly Glu 645 650 655 Lys Lys Asp Thr Cys Thr Gln Glu Cys Ser Tyr Phe Asn Ile Thr Lys 660 665 670 Val Glu Ser Arg Asp Lys Leu Pro Gln Pro Val Gln Pro Asp Pro Val 675 680 685 Ser His Cys Lys Glu Lys Asp Val Asp Asp Cys Trp Phe Tyr Phe Thr 690 695 700 Tyr Ser Val Asn Gly Asn Asn Glu Val Met Val His Val Val Glu Asn 705 710 715 720 Pro Glu Cys Pro Thr Gly Pro Asp Ile Ile Pro Ile Val Ala Gly Val 725 730 735 Val Ala Gly Ile Val Leu Ile Gly Leu Ala Leu Leu Leu Ile Trp Lys 740 745 750 Leu Leu Met Ile Ile His Asp Arg Arg Glu Phe Ala Lys Phe Glu Lys 755 760 765 Glu Lys Met Asn Ala Lys Trp Asp Thr Gly Glu Asn Pro Ile Tyr Lys 770 775 780 Ser Ala Val Thr Thr Val Val Asn Pro Lys Tyr Glu Gly Lys 785 790 795

Claims (13)

Separating only skin cells expressing CD29; And
Skin cell culture method comprising the step of culturing the separated skin cells. The method of claim 1,
The skin cell culture method further comprising the step of primary culturing the skin cells before separating only the skin cells expressing the CD29. The method of claim 1,
The isolated skin cells expressing the CD29 are capable of culturing at least 15 passages. The method of claim 1,
The CD29 is the amino acid sequence of SEQ ID NO: 1, skin cell culture method. The method of claim 1,
The step of isolating only the skin cells expressing CD29 is performed through FACS, skin cell culture method. Culturing fibroblast cells in a matrix; And
A method of producing an in vitro artificial skin comprising the step of culturing by adding skin cells expressing CD29. The method of claim 6,
The step of culturing by adding the skin cells expressing CD29 is performed for 1 to 3 days. The method of claim 6,
Skin cells expressing the CD29 will ^{be added at a concentration of 1x10 4} to 1x10 ⁶ cells/well, a method for producing artificial skin in vitro. The method of claim 6,
After the step of culturing by adding the skin cells expressing the CD29, the step of culturing in a medium containing at least one selected from the group consisting of insulin, EGF and hydrocortisone is further comprising the step of preparing an in vitro artificial skin Way. The method of claim 9,
The step of culturing in the medium is to be carried out for 3 to 7 days, the method of manufacturing an in vitro artificial skin. In vitro artificial skin manufactured by the method of claim 6. The method of claim 11,
In the in vitro artificial skin, the expression of any one or more proteins selected from the group consisting of CD29, Cytokeratin 10, Cytokeratin 14, Yap, and P63 is increased. The method of claim 11,
The in vitro artificial skin is for an animal test replacement, for skin transplantation, or for wound treatment, in vitro artificial skin.

Images