KR20130056956A - Skin equivalent comprising melanocytes and the method preparing the same - Google Patents

Abstract

PURPOSE: A skin equivalent comprising melanocytes and a method preparing a same are provided to manufacture a epidermis layer containing melanocytes and similar to a real skin through a growth and a differentiation induction process without making a layer of melanocytes. CONSTITUTION: A skin equivalent comprises a fibroblast, a keratinocyte, and a melanocyte. The skin equivalent comprises a dermis layer and an epidermis layer. The dermis layer comprises the fibroblast. The epidermis layer comprises the keratinocyte and the melanocyte. The dermis layer comprises more than 2 layers. The dermis layer comprises a first layer containing a collagen and a second layer containing the fibroblast. [Reference numerals] (AA,FF) Epidermis layer; (BB) Dead skin forming cell; (CC,GG) Dermis layer; (DD) H&E dying; (EE) Fibroblasts; (HH) HMB45 dying; (II) Melanocytes

Description

Skin corresponding to melanocyte-forming cells and method for preparing the same {Skin equivalent comprising melanocytes and the method preparing the same}

The present invention relates to artificial skin containing fibroblasts, keratinocytes, and melanocytes, and artificial skin that contains melanocytes to simultaneously study various areas such as stimulation, whitening, and aging for skin research. A model can be provided. In addition, the artificial skin of the present invention provides an artificial skin, characterized in that it comprises two or more layers of the dermal layer is less histological deformation due to dermal shrinkage phenomenon, there is no physiological side effect, it can provide a high-correlation artificial skin model have.

Human skin is largely divided into dermal and epidermal layers. The dermal layer is composed of collagen and fibroblasts, and 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 a specific skin tone.

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 in three dimensions using skin cells and skin components (collagen, elastin, etc.), consisting of living fibroblasts and keratinocytes, similar to the actual morphology, It is also called a skin equivalent or reconstructed skin because it exhibits physiological properties. In Korean, 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 unlike artificial skin of the present invention, it does not exhibit life phenomena like skin. There is a difference.

Artificial skin was developed for the treatment of patients who could not be transplanted with their own skin due to severe burns or severe wounds in the 1980s. However, through continuous improvement and research, It is used in various fields such as physiology research, skin irritation evaluation and skin efficacy evaluation.

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, models of MatTek, a company specializing in the manufacture and sale of artificial skin, which dominates the global artificial skin market, do not have artificial skin models containing fibroblasts, melanocytes and keratinocytes. Because of the limited use of the model, there were limitations in purchasing and using different models in the areas of stimulation, whitening, and aging.

In order to solve these problems, the present inventors have improved the dermal contraction by using an artificial skin model including all of fibroblasts, melanocytes, and keratinocytes in the dermal-epidermal structure, so that physiological side effects due to tissue deformation are reduced. The present invention has been completed by focusing on high human correlation and providing artificial skin capable of simultaneously conducting 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 is to provide an artificial skin containing the epidermal layer comprising keratinocytes and melanocytes.

Another object of the present invention is 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) preparing an epidermal layer containing keratinocytes and melanocytes on top of the second dermal layer; It provides a method for producing artificial skin containing melanocytes comprising a.

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, and the keratinocytes and melanocytes may be included in the epidermal layer from the viewpoint of human correlation with the actual skin.

The dermal layer may include two or more layers to improve dermal shrinkage of artificial skin, and may preferably 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, thereby overcoming physiological side effects caused by tissue deformation, thereby providing artificial skin more suitable for skin research. can do.

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

The second dermal layer may be located above the first dermal layer and contain fibroblasts, elastin, chitosan, glycosaminoglycans (GAGs) and hyaluronic acid (HA). It may further contain an extracellular matrix constituting the dermis, such as).

The epidermal layer may be located above the second dermal layer, contain keratinocytes, and may 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 for research in each area such as stimulation, whitening, and aging. By overcoming the problem, it is possible to provide an artificial skin that can simultaneously perform whitening and photoaging research.

Also,

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) preparing an epidermal layer containing keratinocytes and melanocytes on top of the second dermal layer;

It relates to a method for producing artificial skin containing melanocytes comprising a.

In the step 1), the first dermal layer may be made of a layer containing only collagen, which may contribute to suppressing dermal contraction, and may be elastin (chilastan), chitosan, glycosaminoglucan ( GAGs; glycosaminoglycans), 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 using an aqueous collagen solution, elastin (Elastin), chitosan (Chitosan), glyco Samonoglucan (GAGs; glycosaminoglycans), 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 simultaneously seeding to include melanocytes in the epidermal layer. Subsequently, keratinocytes proliferate and differentiate to form an epidermal layer composed of multiple layers, but melanocytes remain at the base of the epidermis to form an epidermal layer containing melanocytes similar to the skin as shown in FIG. 3. . In other words, the artificial skin according to the present invention can produce an epidermal layer containing melanin-forming cells 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. The human skin has an advantage of increasing correlation.

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 the tissue culture process by containing two or more dermal layers. By suppressing dermal contraction appearing in the secured model stability 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 melanin-forming cells similar to the actual skin through an appropriate proliferation and differentiation induction process without the cumbersome process of separately creating a melanocyte-forming cell layer, thereby simplifying and resulting the manufacturing method. The human skin has an advantage of increasing correlation.

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 MMP-1 after irradiation with UVB in Example 1 and Comparative Example 2.

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 keratinocytes (HEKn), melanocytes (neonatal human epidermal melanocytes, HEMn), and fibroblasts (neonatal human dermal fibroblasts (HDFn)) were all purchased from Cascade Biologics (USA). The culture medium recommended by the vendor was used to incubate in 37 ° C., 5% CO ₂ incubator CO-. The medium used was EpiLife (keratinocytes), M254 (melanocytes), M106 (fibroblasts) medium to which supplements (supplement) were added, which were all purchased from GIBCO (Invitrogen, USA).

[ Example  1] Preparation of artificial skin containing melanocytes

Step 1. Preparation of the dermal layer

Sterilized with 3 Dulbeccos's Modified Eagle Medium (DMEM powder, Gibco, USA), 1 Ham's F-12 nutrient mixture (F-12 powder, Gibco, USA), 4 ml of antibiotic-antifungal (PSF, Gibco, USA) 40 μl of 10X P buffer prepared by dissolving in 400 ml of distilled water and 2.2 g of NaHCO ₃ and 4.77 g of HEPES were dissolved in 100 ml of 0.05 M NaOH, and 40 μl of the reconstitution buffer prepared by filtration was placed in an epi tube. Add and mix, add 320 μl of aqueous collagen solution (Sigma, USA) and mix using a pipette.Install a culture insert (12 mm Transwell®, Corning, USA) inside the 12-well cell culture dish. After the mixture is placed in the culture insert, the reaction is hardened for 1 hour in a 37 ° C. CO ₂ incubator to prepare a dermal layer free of fibroblasts.

Step 2. Preparation of the dermal layer with fibroblasts

Sterilized with 3 Dulbeccos's Modified Eagle Medium (DMEM powder, Gibco, USA), 1 Ham's F-12 nutrient mixture (F-12 powder, Gibco, USA), 4 ml of antibiotic-antifungal (PSF, Gibco, USA) 40 μl of 10X P buffer prepared by dissolving in 400 ml of distilled water and 2.2 g of NaHCO ₃ and 4.77 g of HEPES were dissolved in 100 ml of 0.05 M NaOH, and 40 μl of the reconstitution buffer prepared by filtration was placed in an epi tube. Add and mix, add 320 μl of aqueous collagen solution (Sigma, USA) and mix using a pipette Add 10 ⁴ fibroblasts to the mixture and mix well with a pipette to distribute the fibroblasts uniformly 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 ℃, 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 cultured overnight. Thereafter, the dermal layer containing fibroblasts was prepared by culturing for one week while changing the medium once every two days.

Step 3. Preparation of the Epidermal Layer

Seed by mixing 1.5X 10 ⁵ keratinocytes and 5x10 ⁴ melanocytes on the upper part of the dermal layer containing the fibroblasts prepared in step 2. 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. Then, incubate for one week while changing medium once every two days to induce proliferation of epidermal cells.

When a cell proliferative skin covered both the upper part of the dermis, and the medium was added to 1.2mM Ca ^{+ 2} and incubated in 5% CO _2, 37 ℃ incubator for two days to induce the differentiation of keratinocytes. At the end of two days of differentiation-inducing processes, thereby removing all of the medium and given into the culture medium outside the insertion only of the body (the keratinocyte culture medium containing 1.2mM Ca ^{+ 2)} exposing the keratinocytes in the air. The epidermal layer is produced by changing the medium every day for a week, followed by an air exposure culture.

 [ 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 one dermal layer containing fibroblasts as an existing 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), 4 ml antibiotic-antifungal (PSF, Gibco, USA) Was dissolved in 400 ml of sterilized distilled water and dissolved in 40 μl of 10X P buffer and 2.2 ml of NaHCO ₃ and 4.77 g of HEPES in 100 ml of 0.05 M NaOH, followed by filtration of 40 μl of Reconstitution buffer (Epi). tube), add 320μl of collagen aqueous solution (Sigma, USA) and mix using a pipette Put 10 ⁴ fibroblasts into the mixture and mix well with a pipette so that the fibroblasts are uniformly distributed in the solution. A culture insert (12 mm Transwell®, Corning, USA) was mounted in a 12-well cell culture dish, and the mixed solution was placed in the culture insert, and then in a 5% CO ₂ , 37 ° C. incubator. The dermal layer was prepared by hardening the collagen aqueous solution for 1 hour.

1.5x 10 ⁵ keratinocytes and 5x10 ⁴ melanocytes are mixed and seeded on top of the dermal layer. 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. Then, incubate for one week while changing medium once every two days to induce proliferation of epidermal cells.

When a cell proliferative skin covered both the upper part of the dermis, and the medium was added to 1.2mM Ca ^{+ 2} and incubated in 5% CO _2, 37 ℃ incubator for two days to induce the differentiation of keratinocytes. At the end of two days of differentiation-inducing processes, thereby removing all of the medium and given into the culture medium outside the insertion only of the body (the keratinocyte culture medium containing 1.2mM Ca ^{+ 2)} exposing the keratinocytes in the air. Existing artificial skin made of one dermal layer containing fibroblasts was produced by producing an epidermal layer by changing the medium daily for one week and then exposing the air to culture.

 [ Comparative example  2] Culture of artificial skin containing no melanocytes

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 It was.

[ Test Example  1] Histological observation of artificial skin

A cross section of the artificial skin including the melanocytes prepared in Example 1 was stained by Hematoxylin-Eosin (H & E) staining and observed by optical microscopy and stained with HMB45, a melanoma cell marker, 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 melanocytes are formed in the epidermal layer and the epidermal base formed of keratinocytes that proliferate and differentiate. It can be seen that the artificial skin of the present invention is very similar to the actual skin histologically.

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

In order to confirm the high human correlation of the 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, histological stability of the artificial skin model through the improvement of human correlation.

[ Test Example  3] UVB For Photoaging  Reactivity evaluation

After irradiating UVB of 60mJ on the artificial skin prepared in Example 1 and the artificial skin of Comparative Example 2 using a UVB irradiator (Vilber Lourmat, France), after 48 hours to collect the culture medium, MMP-1 The change of MMP-1 in the medium was measured by ELISA kit (Amersham, GE, USA). The detailed test method was based on the protocol provided by the manufacturer, and the significance of the results was analyzed using a statistical program (MINITAB, Minitab Inc., USA), and the result of MMP-1 expression was calculated in% of the control group. 5 is shown. 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 by increasing the expression of matrix metalloproteinase-1 (MMP-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 of MMP-1 by UVB was observed, whereas in Example 1 including melanocytes, significant increase of MMP-1 by UVB was observed. This shows that the artificial skin model including melanocytes of the present invention has secured physiological human correlation with UVB stimulation, and when the artificial skin model of the present invention is applied to photoaging evaluation, the human skin correlation can be derived. It can be seen that.

Claims (10)

Artificial skin containing fibroblasts, keratinocytes and melanocytes. According to claim 1, wherein the artificial skin dermal layer containing fibroblasts; And an epidermal layer comprising keratinocytes and melanocytes. 3. The artificial skin of claim 2, wherein the dermal layer comprises two or more layers. 3. The artificial skin of claim 2, wherein the dermal layer comprises a first layer containing collagen and a second layer containing fibroblasts. 3. The artificial skin of claim 2, wherein the melanocytes are present at the base of the epidermal layer. The artificial skin according to claim 1 or 2, wherein the artificial skin is for simultaneous study of skin whitening and skin aging. The artificial skin according to claim 1 or 2, wherein the artificial skin is for simultaneous study of skin whitening, skin aging and skin irritation. 1) preparing a first dermal layer containing collagen;
2) forming a second dermal layer containing fibroblasts on the first dermal layer; And
3) preparing an epidermal layer containing keratinocytes and melanocytes on top of the second dermal layer;
Method for producing artificial skin containing melanocytes comprising a. The method of claim 8, wherein the step 3) comprises artificial culture of the keratinocytes and melanocytes. The method of claim 8, further comprising the step of differentiating and proliferating keratinocytes after step 3).

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