WO2013077597A1

WO2013077597A1 - Artificial skin containing melanin producing cells, and method for manufacturing same

Info

Publication number: WO2013077597A1
Application number: PCT/KR2012/009710
Authority: WO
Inventors: 민대진; 이성훈; 나용주; 배지현; 조준철
Original assignee: 주식회사 아모레퍼시픽
Priority date: 2011-11-23
Filing date: 2012-11-16
Publication date: 2013-05-30
Also published as: KR20130056956A; KR101874790B1

Abstract

The present invention relates to artificial skin containing fibroblasts, keratinocytes, and melanin producing cells, and can provide an artificial skin model that contains melanin producing cells, in order to simultaneously perform research in various areas such as the irritation, whitening, and ageing of skin. Also, the artificial skin of the present invention provides an artificial skin characterized by comprising a dermis including at least two layers and which has little histological change due to dermal contraction, so that no physiological side effects can occur, thereby providing an artificial skin model having high correlation with the human body.

Description

【Specification】

[Name of invention]

Artificial skin containing melanocytes and preparation method thereof

Technical Field

The present invention relates to an artificial skin containing fibroblasts, keratinocytes and melanocytes, which contains melanocytes, which can simultaneously study various areas such as irritation, whitening and aging for skin research. Skin model can be provided. In addition, the artificial skin of the present invention provides an artificial skin, characterized in that the dermal layer includes two or more layers, so that there is little physiological side effect due to dermal shrinkage phenomenon, and there is no physiological side effect. have. Background Art

Human skin is largely divided into dermal and epidermal layers. The dermal layer is composed of collagen and fibroblasts. The epidermal layer is composed of keratinocytes and melanocytes.

Fibroblasts play an important role in the maintenance and repair of the dermal layer by producing collagen, and keratinocytes play a key role in maintaining the barrier function of the skin by creating an epidermal layer surrounded by the stratum corneum. In addition, melanocytes protect the skin from UV damage by absorbing ultraviolet rays, and play a role in expressing specific skin tones. The skin is a complex of these various cells and components that maintain their form and function by their constant interaction. Due to the structural and functional complexity of the skin, skin research using one type of skin cell has its limitations, and the artificial skin is a three-dimensional skin model developed to overcome this problem. Artificial skin in the present invention by reconstructing the skin three-dimensionally using skin cells and skin components (collagen, elastin, etc.), consisting of living fibroblasts and keratinocytes morphologically similar to the actual skin, It is also called a skin equivalent or reconstructed skin because of its physiological properties. In our words, artificial skin, which is used as an artificial skin, refers to polymer complexes that exhibit physical properties similar to skin (elasticity, strength, material permeation, etc.), and do not exhibit life phenomena like skin unlike the artificial skin of the present invention. There is a difference. Artificial skin was developed for the treatment of patients who suffered severe burns in the 1980s, but the degree of burn was too severe or the lesions were too wide to be transplanted with their own skin. Menstruation It is used in various areas such as evaluation of skin irritation and evaluation of skin efficacy. The artificial skin model currently used in most laboratories is a dermal-epidermal artificial skin model, and is composed of dermal fibroblasts and epidermal keratinocytes without melanocytes due to its complexity. In addition, the model of MatT Dasa, a company specializing in the manufacture and sale of artificial skin, which dominates the world's artificial skin market, does not currently have artificial skin models containing fibroblasts, melanocytes and keratinocytes. As a result, the use of the model was limited, and there was a limit to purchasing and using different models in the areas of stimulation, whitening, and aging.

[Detailed Description of the Invention]

[Technical problem]

In order to solve these problems, the inventors of the present invention used an artificial skin model including fibroblasts, melanocytes, and keratinocytes in the dermal-epidermal structure to improve dermal contraction, thereby reducing physiological side effects due to tissue deformation. The present invention has been completed in view of the fact that it can provide artificial skin that has high human correlation and can simultaneously perform integrated research on skin irritation, whitening and aging.

Accordingly, an object of the present invention is to provide an artificial skin containing fibroblasts, keratinocytes and melanocytes. More specifically, an object of the present invention is a dermal layer containing fibroblasts; And it provides an artificial skin containing the epidermal layer comprising keratinocytes and melanocytes.

Another object of the present invention is to prepare a first dermal layer containing collagen;

2) preparing a second dermal layer containing fibroblasts in contact with the first dermal layer; And 3) preparing a skin layer containing keratinocytes ⁱ and melanin forming cells on top of the second dermal layer; It provides a method for producing artificial skin containing melanocytes, including.

Technical Solution

The present invention provides an artificial skin containing fibroblasts, keratinocytes and melanocytes.

In addition, the present invention

1) preparing a first dermal layer containing collagen;

2) preparing a second dermal layer containing fibroblasts in contact with the first dermal layer step; And

3) It provides a method of manufacturing artificial skin containing melanocytes comprising the step of preparing the epidermal layer containing keratinocytes and melanocytes on the upper part of the second dermal layer.

Advantageous Effects

Artificial skin of the present invention can increase the human correlation of the artificial skin model by including all three constituent cells of the human skin, fibroblasts, melanin forming cells and keratinocytes, and tissue culture process by containing two or more dermal layers By suppressing the dermal contraction appearing in the ensure the stability of the model can provide artificial skin with minimal physiological deformation or side effects due to tissue deformation. In addition, by containing melanocytes, not only normal skin physiology studies but also whitening and aging studies, which are not possible in the existing models, can be simultaneously performed, and artificial skin suitable for research on photoaging can be provided. Therefore, by using such an artificial skin model has the advantage that the comprehensive efficacy evaluation of the skin active material.

In addition, the artificial skin according to the present invention can produce an epidermal layer containing melanocytes similar to the real skin through an appropriate proliferation and differentiation induction process without the cumbersome process of separately creating a melanocyte-forming cell layer, thereby simplifying the manufacturing method and The human body of the resulting artificial skin also has the advantage of being improved.

【Brief Description of Drawings】

Figure 1 shows the structure of a typical artificial skin (skin equivalent).

Figure 2 shows the type and use of existing artificial skin model.

Figure 3 shows the histological observation results staining the cross section of the artificial skin of the present invention.

Figure 4 shows the results of observing the dermal shrinkage of Example 1 and Comparative Example 1.

5 is a graph showing whether the expression change of 丽 P-1 after UVB irradiation in Example 1 and Comparative Example 2.

[Best form for implementation of the invention]

Hereinafter, the present invention will be described in more detail. The present invention relates to artificial skin containing fibroblasts, keratinocytes and melanocytes.

The artificial skin may be composed of a dermal layer and an epidermal layer. Preferably, the fibroblasts are included in the dermal layer from the viewpoint of human correlation with the actual skin, and the keratinocytes and melanocytes may be included in the epidermal layer.

The dermal layer may include two or more layers in order to improve the dermal contraction phenomenon of the artificial skin, and preferably may include a first dermal layer containing collagen and a second dermal layer containing fibroblasts. By forming the dermal layer in two or more layers, the artificial skin of the present invention improves the dermal contraction phenomenon of the existing artificial skin model, and overcomes the physiological side effects caused by tissue deformation, thereby making the skin more suitable for skin study. Can be provided.

The first dermal layer may contain collagen, and extracellular matrix constituting the dermis such as elastin, chitosan, glycosaminoglycans, and hyaluronic acid. extracel lular matrix).

The second dermal layer can be located on the first dermal layer, containing fibroblast cells, and elastin (Elastin), chitosan (Chitosan), glycolate four diamino-glucan (GAGs; glycosaminoglycans), ^'hyaluronic acid (HA; hyaluronic It may further contain an extracellular matrix that makes up the dermis.

The epidermal layer may be located above the second dermal layer and may contain keratinocytes and may also contain melanocytes. This enhances human correlation with the actual skin, and because it does not contain melanocytes in the existing artificial skin model consisting of epidermis and dermis, it is necessary to purchase and use different models in each area research such as stimulation, whitening, and aging. By overcoming the problem, it is possible to provide an artificial skin that can simultaneously conduct a whitening and photoaging research.

In addition, the present invention

1) preparing a first dermal layer containing collagen;

2) preparing a second dermal layer containing fibroblasts in contact with the first dermal layer; And

3) It relates to a method for producing artificial skin containing melanocytes comprising the step of preparing the epidermal layer containing keratinocytes and melanocytes on the upper part of the second dermal layer. In the step l), the first dermal layer may be made of a layer containing only collagen, which may contribute to suppressing dermal contraction, elastin, chitosan, glycosaminoglucan ( GAGs; g lycosami nog 1 yeans), hyaluronic acid (HA; hyaluronic acid) may further contain one or more selected from the group consisting of.

In the step 2), the second dermal layer is formed on top of the first dermal layer and contains fibroblasts, and can be prepared by hardening with aqueous collagen solution, elastin (Elastin), chitosan (Chitosan), Glycosaminoglucan (GAGs; g 1 ycosami nog 1 yeans), may further contain one or more selected from the group consisting of hyaluronic acid.

In step 3), the epidermal layer may be formed on the second dermal layer, and may contain keratinocytes and melanocytes.

Artificial skin manufacturing method containing melanocytes of the present invention is characterized in that in the step 3) by mixing the keratinocytes and melanocytes in the upper part of the dermal layer and seeding at the same time to include melanocytes in the epidermal layer. . After this, keratinocytes proliferate and differentiate, forming a multi-layered epidermal layer. Melanogenic cells remain at the base of the epidermis and form an epidermal layer containing melanocytes in a form similar to the actual skin as shown in FIG. 3. Namely according to the present invention _. Artificial skin can produce epidermal layer containing melanin-forming cells similar to the actual skin through the proper proliferation and differentiation induction process without the cumbersome process of separately creating melanin-forming cell layers, which simplifies the manufacturing method and results in human correlation of the resulting artificial skin It also has the advantage of being higher.

[Form for implementation of invention]

Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples, but the scope of the present invention is not limited thereto.

Reference Example 1 Culture of keratinocytes, melanocytes and fibroblasts

Neonatal human epidermal kerat inocytes (HEKn), melanocytes (neonatal human epidermal melanocytes, HEMn), and fibroblasts (neonatal human dermal fibroblasts (HDFn)) were all purchased from Cascade Biologies (USA). ， 37 ^° C, 5% C 0 ₂ Cultured in an incubator. The medium used was EpiLife (keratinocytes), M254 (melanocytes), M106 (fibroblasts) medium to which supplements (supplement) were added, and all were purchased from GIBCO (Invitrogen, USA).

Example 1 Preparation of Artificial Skin Containing Melanogenic Cells

Step 1. Preparation of the dermal layer

Three DMEM (Dulbeccos's Modified Eagle Medium (DMEM powder, Gibco, USA)) 1 Ham 's F-12 nutrient mixture (F-12 powder, Gibco, USA), antibiotic—antifungal (PSF, Gibco, USA) ) Reconstitution buffer 40μ1 prepared by dissolving 2.2 g of NaHC0 ₃ and 4.77 g of ^' HEPES in 40 μ 1 of 10X P buffer and 0.05 μ NaOH 100 mL prepared by dissolving 4 ml in 400 ml of sterile distilled water. After mixing the mixture into an Epi tube, mix it with 320 µ1 of collagen aqueous solution (Sigma, USA), and mix using a pipette, and insert the culture insert into a 12-well cell culture dish (12 mm Transwell®,). Corning, USA), and the mixed solution in the culture insert, and then reacted for 1 hour in a 37 ^° C0 ₂ incubator to prepare a dermal layer free of fibroblasts.

Step 2. Preparation of the dermal layer with fibroblasts

3 DMEKDulbeccos's Modified Eagle Medium (DMEM powder, Gibco, USA), 1 Ham's F-12 nutrient complex (F-12 powder, Gibco, USA), 4 ml antibiotic-antifungal (PSF, Gibco, USA) Was dissolved in 400ml of sterilized distilled water, dissolved 2.2 g of NaHC0 ₃ and 4.77 g of HEPES in 10 μl P buffer 40 μ 1 and 0.05 μ NaOH lOral, and filtered and reconstituted 40yl of reconstitution buffer 40yl. After mixing and mixing in (Epi tube), add 320μ1 of collagen aqueous solution (Sigma, USA) and mix using a pipette. Add 10 ⁴ fibroblasts to the mixture and mix well with a pipette so that the fibroblasts are evenly distributed in solution. .

Put 400ul of the fibroblast mixture mixed above into the culture insert including the dermal layer prepared in step 1, and solidify in 37 ^° C, 5% CO ₂ incubator for 2 hours.

When the collagen is completely hardened, the fibroblast culture medium (M106; GIBCO, Invitrogen, USA) is placed inside and outside the culture insert and incubated overnight. After that, the culture medium was exchanged once a week and cultured for one week to prepare a dermal layer containing fibroblasts. Multisystem 3. Manufacture of Cuticle Gun

1.5X 10 ⁵ keratinocytes and 5xl0 ⁴ melanogenesis cells are mixed and seeded on top of the dermal layer containing the fibroblasts prepared in step 2 above. Put keratinocyte culture medium (EpiLife; GIBCO, Invitrogen, USA) into both the inside and outside of the culture insert and check the attachment state of the cells after overnight culture. After this, the culture medium is exchanged once a week, thereby inducing proliferation of epidermal cells.

When the proliferated epidermal cells covered the upper part of the dermal layer, 1.2niM Ca ²⁺ was added to the medium, followed by culturing in a 5% CO ₂ , 37 ^° C incubator to induce differentiation of keratinocytes. After two days of induction of differentiation, the medium is removed and the keratinocytes are exposed to the air by adding the medium (1.2mM Ca ²⁺ -added keratinocyte culture medium) only to the outside of the culture insert. The epidermal layer is created by air exposure culture, changing the medium every day for a week.

Comparative Example 1 Culture of Existing Artificial Skin

Corresponding to the artificial skin of the present invention, the artificial skin of Comparative Example 1 prepared as a dermal layer containing fibroblasts as a conventional artificial skin model was prepared as follows. 3 culture inserts DMEM (Dulbeccos's Modified Eagle Medium (DMEM powder, Gibco, USA)), 1 Ham's F-12 nutrient mixture (F-12 powder, Gibco, USA), antibiotic-antifungal (PSF , Gibco, USA) Reconstitution buffer prepared by dissolving 2.2 g of NaHC0 ₃ and 4.77 g of HEPES in 40 μl of 10X P buffer and 100 μl of 0.05 Μ NaOH dissolved in 400 ml of sterile distilled water. After mixing 40jj l into an Epi tube, mix, add 320μ1 of collagen aqueous solution (Sigma, USA), and mix using a pipette.Add 10 ⁴ fibroblasts to the mixture and shake well with a pipette. The fibroblasts are then distributed evenly in the solution A culture insert (12 'Transwell®, Corning, USA) is placed inside a 12-well cell culture dish, and the mixed solution is placed inside the culture insert. After soaking in the collagen aqueous solution for 1 hour in a 5% C0 _2> 37 ^° C incubator. Tongue dermal layers were prepared.

1.5X 10 ⁵ keratinocytes and 5xl0 ⁴ melanocytes are mixed and seeded on top of the dermal layer. Culture of keratinocytes inside and outside the culture insert Insert the medium (EpiLife; GIBCO, Invitrogen, USA) and check the adherence state of the cells after overnight culture. After this, the culture medium is exchanged once a week, thereby inducing proliferation of epidermal cells. 、

When the proliferated epidermal cells covered the upper part of the dermal layer, 1.2mM Ca ²⁺ was added to the medium, followed by culturing in a 37 ^° C. incubator with 5> C0 ₂₎ to induce differentiation of keratinocytes. After the differentiation induction process, all of the medium is removed, and the keratinocytes are exposed to the air by adding a medium (a keratinocyte culture medium with 1.2mM Ca ²⁺ added) only to the outside of the culture insert. Existing artificial skin made of one dermal layer containing fibroblasts was produced by advancing the air exposure culture while changing medium every day for one week.

Comparative Example 2 Culture of Artificial Skin without Melaninocytes

Artificial skin containing no melanin forming cells of Comparative Example 2 was cultured artificial skin consisting of two layers of dermis cultured in the same manner as in Example 1, except that the step of seeding melanocytes Produced.

Test Example 1 Histological Observation of Artificial Skin

A cross section of the artificial skin containing the melanocytes prepared in Example 1 was stained by Hematoxylin-Eosin (H & E) staining and observed by optical microscopy, and HMB45, a melanoma cell marker, was stained by tissue immunostaining. One result is shown in FIG. 3.

As shown in FIG. 3, the artificial skin of the present invention is composed of a dermal layer and an epidermal layer, as in actual skin, and fibroblasts are observed in the dermal layer, and melanin formation is formed in the epidermal layer and the epidermal base consisting of keratinocytes which proliferate and differentiate It can be seen that the cells are observed, the artificial skin of the present invention can be seen that histologically very similar to the actual skin.

[Test Example 2] Improvement of dermal contraction of artificial skin

In order to confirm the high human correlation of artificial skin according to the present invention, the dermal contraction of Example 1 and Comparative Example 1 was observed.

The artificial skin prepared in Example 1 and the artificial skin of Comparative Example 1 were visually observed at the end of incubation, and the result photograph is shown in FIG. 4. As shown in FIG. 4, Comparative Example 1 corresponding to the existing artificial skin showed severe contraction of the dermis as compared with Example 1, and it can be seen that morphological deformation of the artificial skin model occurs. Artificial skin of the present invention by suppressing the dermal layer shrinkage by making the dermal layer in two layers, it can be seen that through the morphological and histological stability of the artificial skin model through the improvement of human correlation. Test Example 3 Evaluation of Photoaging Reactivity to UVB

After irradiating the UVB of 60mJ on the top of the artificial skin prepared in Example 1 and the artificial skin of Comparative Example 2 using a UVB irradiator (Vilber Lourmat, France), the culture medium was collected after 48 hours, 醒 pl ELISA kit (Aniersham, GE, USA) was used to measure the change in the 醒 P-1 in the medium. The detailed test method followed the protocol provided by the manufacturer, and analyzed the significance of the result using a statistical program (MINITAB, Minitab Inc., USA). Calculated as shown in FIG. As a control, except that the UVB was not irradiated, the artificial skin of Example 1 and Comparative Example 2 was cultured for 48 hours under the same conditions.

UVB is widely known to cause photoaging reactions by increasing the expression of matrix metalloproteinase-1 (P-1), which degrades dermal collagen in both cells and the human body.

As shown in FIG. 5, in the model of Comparative Example 2, no increase in 醒 P-1 by UVB was observed, whereas in Example 1 including melanocytes, significant increase in 醒 P-1 by UVB was observed. It became. This shows that the artificial skin model including melanocytes of the present invention has secured physiological human correlation with UVB stimulation, and results in higher human correlation when applying the artificial skin model of the present invention to photoaging evaluation. It can be seen that it can be derived.

Industrial Applicability

The artificial skin of the present invention can increase the human correlation of the artificial skin model by including all three constituent cells of the human skin, fibroblasts, melanocytes and keratinocytes, and culture the tissue by containing two or more dermal layers. By suppressing dermal contraction during the process, the stability of the model can be secured, thereby providing artificial skin with minimal physiological and side effects caused by tissue deformation. In addition, by containing melanocytes, whitening and aging that are not possible with conventional skin physiology studies and existing models Simultaneous research can be performed and artificial masses suitable for the study of photoaging can be provided. Therefore, by using such an artificial skin model it is possible to evaluate the overall efficacy of the skin active material.

In addition, the artificial skin according to the present invention can produce an epidermal layer containing melanocytes similar to the real skin through an appropriate proliferation and differentiation induction process without the cumbersome process of separately creating a melanocyte-forming cell layer, thereby simplifying the manufacturing method and The human body of the resulting artificial skin is also highly correlated.

Claims

[Range of request]

[Claim 1]

Artificial skin containing fibroblasts, keratinocytes and melanocytes.

[Claim 2]

According to claim 1, wherein the artificial skin dermal layer containing fibroblasts; And an epidermal layer comprising the epidermal layer comprising keratinocytes and melanocytes.

[Claim 3]

3. The artificial skin of claim 2, wherein the dermal layer comprises two or more layers.

[Claim 4]

3. The artificial skin of claim 2, wherein the dermal layer comprises a first layer containing collagen and a second layer containing fibroblasts.

[Claim 5]

The artificial skin of claim 2, wherein the melanocytes are present at the base of the epidermal layer. ·

[Claim 6]

The artificial skin according to claim 1 or 2, wherein the artificial skin is used for the simultaneous study of skin whitening and skin aging.

[Claim 7]

The artificial skin according to claim 1 or 2, wherein the artificial skin is intended for the simultaneous study of skin whitening, skin aging and skin irritation.

[Claim 8]

1) preparing a first dermal layer containing collagen;

2) forming a second dermal layer containing fibroblasts on the first dermal layer;

3) Method of manufacturing artificial skin containing melanogenesis tax i comprising the step of preparing an epidermal layer containing keratinocytes and melanogenesis cells on top of the second dermal layer.

[Claim 9]

The method of claim 8, wherein the step 3) comprises mixing and culturing keratinocytes and melanocytes at the same time. [Claim 10]

The method of claim 8, further comprising the step of differentiating and proliferating keratinocytes after step 3).