KR20230115654A - Photo-aging Artificial Skin Model - Google Patents

Abstract

The present invention relates to a method for manufacturing an artificial skin model having the same photoaging marker expression profile as a photoaging skin cell tissue, and an artificial skin model manufactured by the method. It has similar characteristics, so it can be usefully used in the development of dermatological treatments or cosmetics.

Description

Photo-aging Artificial Skin Model}

The present invention relates to a photoaging artificial skin model having the same photoaging marker expression profile as a photoaging skin cell tissue and a manufacturing method thereof.

With the improvement of tissue culture technology and cell culture technology, tissue engineering techniques that are used for various diseases by manufacturing cells and tissues similar to actual living tissues are used. Skin is an organ that covers the outside of the body and is composed of three layers: epidermis, dermis, and subcutaneous fat layer from the outside. The epidermis is composed mostly of keratinocytes of the stratified squamous epithelium. The dermis composed of matrix proteins such as collagen fibers and elastic fibers is located under the epidermis, and the dermis contains blood vessels, nerves, sweat glands, and the like. The subcutaneous fat layer is composed of fat cells. The skin maintains its shape by interacting with various cells and components as described above, and exhibits various functions such as regulating body temperature and functioning as a barrier against the external environment.

Artificial skin is a three-dimensional reconstruction of the skin using skin cells and skin constituents such as collagen and elastin. Because it shows functional characteristics, it is also called skin equivalent or reconstructed skin. Artificial skin is a polymer composite that exhibits similar physical properties to skin, such as elasticity, strength, and material permeation, and is different in that it does not exhibit life phenomena such as skin. Artificial skin is not only used for replacement (permanent engraftment type) or regeneration (temporary covering type) of damaged skin such as burns and trauma, but also in various fields such as skin physiology research, skin irritation evaluation, and skin efficacy evaluation. .

In the currently used manufacturing method of artificial skin, fibroblasts constituting the dermal mimic layer are treated with a DNA topoisomerase II inhibitor and insulin-like growth factor 1 to induce aging of the fibroblasts, and then artificial skin is produced using the same. A manufacturing method (Korean Patent Publication 2021-0043309) is known, and in the case of artificial skin produced by this method, it shows positive for the aging marker SA-β-gal.

However, according to recent research results, it has been revealed that in the case of artificial skin manufactured by processing compounds and substances, there is a large difference in characteristics from photoaging skin tissue caused by ultraviolet rays. In photoaging skin, it was confirmed that along with the SA-β-gal aging marker, the expression of p16 ^INK4A increased, the expression of SDF1, a protein that inhibits skin pigmentation, decreased, and the expression of GDF-15, a protein that promotes skin pigmentation, increased. (Yoon, JE et al ., Theranostics , 8: 4620, 2018, Kim, Y et al., J. Invest. Dermatol ., 140:2478, 2020).

Therefore, the need for a photoaging artificial skin model in which the above markers are identified for simulating the photoaging skin is emerging.

Therefore, the present inventors cultured keratinocytes on the dermal layer in which fibroblasts photoaged by UV treatment and normal fibroblasts were co-cultured and induced differentiation through three-dimensional air-liquid interface culture, photoaging skin of the human body. It was confirmed that an artificial skin model having the same photoaging marker expression profile as the cell tissue could be prepared, and the present invention was completed.

An object of the present invention is to provide a method for producing artificial skin having the same photoaging marker expression profile as that of human photoaged skin cells.

Another object of the present invention is to provide artificial skin having the same photoaging marker expression profile as photoaging skin cell tissue.

In order to achieve the above object, the present invention provides a method for manufacturing photoaging artificial skin comprising the following steps:

(a) preparing photoaged fibroblasts by treating fibroblasts with UV;

(b) constructing an artificial dermis layer by co-cultivating the photoaged fibroblasts and normal fibroblasts;

(c) constructing an artificial dermal-epidermal layer by culturing keratinocytes on the constructed artificial dermal layer; and

(d) manufacturing artificial skin by additionally culturing the artificial dermal layer and the artificial dermal-epidermal layer by stacking them.

The present invention also provides a photoaging artificial skin model comprising;

(i) an artificial dermis layer formed by photoaging fibroblasts in which photoaging is induced by UV treatment; and

(ii) an artificial epidermal layer formed by keratinocytes.

The photoaging artificial skin model according to the present invention has characteristics similar to skin aging tissue by ultraviolet rays, and can be usefully used in the development of dermatological treatments or cosmetics.

1 shows the result of confirming the expression of the aging marker SA-β-gal in photoaged fibroblasts induced by UV treatment.
2 is a schematic diagram showing a method of manufacturing a photoaging artificial skin model according to the present invention.
4 shows the result of confirming p16 ^INK4A expression in the photoaging artificial skin model by immunostaining.
5 shows the results of confirming the expression of SDF1 and GDF15 in the photoaging artificial skin model by immunostaining.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement the present invention. However, the present invention may be embodied in many different forms and is not limited to the embodiments set forth herein.

Human skin consists of a dermis layer and an epidermis layer above the dermis layer. The dermis is a skin layer below the epidermis made of connective tissue, which acts as a buffer to protect the body from stress and strain. The dermis is tightly connected to the epidermis through a basement membrane. The dermis provides a matrix that supports the various appendages, such as blood vessels and nerves, that are inherent in its structure.

In the present specification, "artificial skin" means a three-dimensional reconstruction of skin using skin cells and skin components, etc. it means.

In the present specification, "skin aging" means that fibroblasts and collagen constituting the dermis layer are damaged in the skin composed of the dermis layer and the epidermis layer, and "photoaging" means that the dermis layer of the skin is damaged by ultraviolet rays. do.

During photoaging of skin tissue by ultraviolet rays, along with the SA-β-gal aging marker, the expression of p16 ^INK4A increases, the expression of SDF1, a protein that inhibits skin pigmentation, decreases, and the expression of GDF-15, a protein that promotes skin pigmentation. increases (Yoon, JE et al ., Theranostics , 8: 4620, 2018, Kim, Y et al., J. Invest. Dermatol ., 140:2478, 2020). In the present invention, an attempt was made to develop an artificial skin model similar to skin tissue in photoaging. After photoaging fibroblasts by irradiating ultraviolet rays, culturing them together with normal fibroblasts to form an artificial dermal layer, and culturing keratinocytes on the artificial dermal layer After forming the artificial epidermis layer, the expression profile of photoaging markers similar to photoaging tissue was confirmed in the artificial skin model obtained through 3D culture.

Accordingly, in one aspect, the present invention relates to a method for manufacturing a photoaging artificial skin comprising the following steps:

(a) preparing photoaged fibroblasts by treating fibroblasts with UV;

(b) constructing an artificial dermis layer by co-cultivating the photoaged fibroblasts and normal fibroblasts;

(c) constructing an artificial dermal-epidermal layer by culturing keratinocytes on the constructed artificial dermal layer; and

(d) manufacturing artificial skin by additionally culturing the artificial dermal layer and the artificial dermal-epidermal layer by stacking them.

In the present invention, the UV treatment may preferably be performed with UV of 200 to 400 nm, and more preferably with UV of 320 to 400 nm.

In one aspect of the present invention, UV of 320 ~ 400 nm wavelength was treated for 29 minutes and 46 seconds (5 J/cm ² ), but is not limited thereto.

In the present invention, the photoaged fibroblasts and the normal fibroblasts may be mixed and co-cultured at a ratio of 1:2 to 1:20, preferably at a ratio of 1:2 to 1:10.

In the present invention, the culture of step (b) is preferably performed for 28 days.

In the present invention, the culturing in step (c) is preferably performed for 7 days.

In the present invention, the culturing in step (d) is preferably performed for 14 to 21 days, more preferably for 14 days.

In the present invention, 1 to 5 artificial dermal layers in step (d) may be overlapped, and the additional culture in step (d) is a differentiation induction process through a three-dimensional air-liquid interface (ALI). there is.

In the present invention, the "three-dimensional air-liquid interface (ALI)" is an air-exposure culture method, through which keratinocytes are exposed to the air to create an epidermal layer to produce artificial skin. Cultivation proceeds on a stainless steel lattice net and a polycarbonate membrane.

In one aspect of the present invention, it was confirmed that the expression of p16 ^INK4A , an aging marker, was increased in the photoaging artificial skin model prepared by the method of the present invention, compared to the normal artificial skin model (FIG. 3). In addition, while SDF1, a protein that inhibits skin pigmentation, was confirmed in the normal artificial skin model, it was not confirmed in the photoaging artificial skin model (FIG. 4). In addition, while expression of GDF15, a protein that promotes skin pigmentation, was not confirmed in the normal artificial skin model, it was confirmed that GDF15 was expressed in the photoaging artificial skin model (FIG. 4).

In the present invention, keratinocytes constitute about 80% to 90% of the cells of the epidermal mimic layer and are formed through mitosis in the basal layer, which is the deepest layer of the epidermal layer, and then rise to the upper layer toward the skin surface. The step of culturing after applying the keratinocytes is the first step for forming an artificial epidermal layer of artificial skin, and the keratinocytes used herein may be, for example, human-derived keratinocytes. As the keratinocytes, any keratinocytes that are currently commercially available may be used, and keratinocytes derived from other cells as well as keratinocytes directly isolated or separated from humans and then cultured may be used. Commercially available human keratinocytes include NHEK-Neo, Pooled (Neonatal Normal Human Epidermal Keratinocytes, Pooled: product number 00192906, tissue access number P867, Caucasians), NHEK-Neo (product number 00192907, tissue number) provided by Lonza. Acquisition number: 20647, Caucasian), NHEK-Adult (product number 00192627, tissue accession number: 21155, Caucasian), NHEK-Neo (product number 00192907, tissue accession number: 18080, black). and HEKn (Human Epidermal keratinocytes, neonatal: product number C0015C, tissue accession number 1781129, Caucasian) and HEKn (product number C0015C, tissue accession number 1803827, black) provided by Thermo Fisher. In order to maintain the property of keratinocytes adhering to the base membrane and proliferating, the dish is preferably coated with 0.1-0.2% gelatin or 0.1-0.2 mg/ml collagen type IV. The cells can be cultured in a 35-37 ° C., 5-10% CO2 incubator and can be subcultured when the density is about 70-80%.

The keratinocytes may be applied on the dermal mimic layer by, for example, seeding, and the culture period may be, for example, 1 to 4 days, 1 to 3 days, or 1 to 2 days, but is necessarily limited thereto It is not.

After the steps of applying and culturing the keratinocytes, a step of culturing in a keratinocyte medium may be performed. Here, "keratinocyte medium" refers to a medium for culturing human keratinocytes, which is not known in the art. Any known medium may be used. For example, the keratinocyte medium includes Bovine Pitutary Extract (BPE), human epidermal growth factor (hEGF), bovine insulin, hydrocortisone, and gentamicin and It may be a medium containing amphotericin-B (GA-1000 (Gentamicin, Amphotericin-B)). In addition, it may be a medium further containing epinephrine and transferrin in the above components. A commercially available example of the medium is CnT-3D-PR (CellnTEC).

The keratinocyte medium may be installed to touch the lower surface of the dermal mimic layer, and the surface opposite to the surface on which the keratinocyte medium is installed may be exposed to air. Culturing in such a keratinocyte medium may be performed for a period of, for example, 3 to 10 days, 4 to 10 days, 5 to 9 days, or 4 to 8 days, but is not necessarily limited thereto.

In another aspect, the present invention relates to a photoaging artificial skin model comprising:

(i) an artificial dermis layer formed by photoaging fibroblasts in which photoaging is induced by UV treatment; and

(ii) an artificial epidermal layer formed by keratinocytes.

In the present invention, the UV treatment may preferably be performed with UV of 200 to 400 nm, and more preferably with UV of 320 to 400 nm.

In one aspect of the present invention, UV of 320 ~ 400 nm wavelength was treated for 29 minutes and 46 seconds (5 J/cm ² ), but is not limited thereto.

The photoaging artificial skin model of the present invention shows increased expression of the aging marker p16 ^INK4A compared to the normal artificial skin model, has a reduced expression of SDF1, a protein that inhibits skin pigmentation, and a protein that promotes skin pigmentation. It can be characterized by increased expression of GDF15.

Hereinafter, the present invention will be described in more detail by examples. These examples are only for explaining the present invention in more detail, whereby it will be apparent to those skilled in the art that the technical scope of the present invention is not limited to these examples.

Example 1: Construction of photoaged fibroblasts

To prepare photoaged fibroblasts, fibroblasts (Human Dermal Fibroblasts, (IRB no: AJIRB-BMR-SMP-17-438)) were treated with 25 ng/ml 8-methoxy psoralen (Sigma), and after 16 hours, 320 UV of ~400 nm wavelength was irradiated for 29 minutes and 46 seconds (5 J/cm2).

After fixing the photoaged fibroblasts obtained by the ultraviolet irradiation with 4% formalin (Formalin, Sigma) for 15 minutes, β-galactosidase solution (1 mg/ml X-gal, 40 mM pH 5.8 citric acid/sodium phosphate, 5 mM Potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl ₂ ) at 37 ° C. for 12 hours, and the expression of the aging marker SA-β-gal marker was confirmed.

As a result, as shown in FIG. 1, it was confirmed that the expression of the SA-β-gal marker was markedly increased in the prepared photoaged fibroblasts compared to the UV-untreated control group.

Example 2: Preparation of photoaging artificial skin

Photoaging artificial skin was prepared using photoaging fibroblasts.

Photoaged fibroblasts prepared in Example 1 and fibroblasts not subjected to UV treatment (Human Dermal Fibroblasts, (IRB no: AJIRB-BMR-SMP-17-438)) were seeded by mixing 1.25X10 ⁴ and 2.5X10 ⁴ respectively, and 10% fetal bovine serum (FBS, Gibco) and 50 μg/ml ascorbic acid were added to the fibroblast medium (Dublbeccos's Modified Eagle Medium, DMEM, Gibco). (Ascorbic acid, Sigma) was co-cultured for 28 days in a medium containing artificial dermal layer.

Keratinocytes (Primary epidermal keratinocytes; Normal, Human, Neonatal, HEKn, ATCC ) were seeded on top of the fabricated artificial dermal layer at 2.2 x 10 ⁵ and mixed with DMEM and Ham's F-12 medium at a ratio of 3:1. 5% FBS (Gibco), 24.3 μg/ml Adenine (Adenin, Sigma), 5 μg/ml Insulin (Insulin, Sigma) 0.4 μg/ml Hydrocortisone (Sigma), 0.212 μg/ml Isoproterenol hydrochloride , Sigma), 10 ng/ml human epidermal growth factor (hEGF, Sigma), and 50 μg/ml Ascorbic acid (Sigma), followed by culturing for 7 days to prepare an artificial dermal-epidermal layer.

After overlapping the obtained artificial dermis layer 2 layer and the artificial dermal-epidermal layer 1 layer, differentiation was induced through a three-dimensional air-liquid interface (ALI) to prepare a photoaging artificial skin model (FIG. 2).

The three-dimensional air-liquid interface is an air exposure culture method, through which keratinocytes are exposed to the air to create an epidermal layer to produce artificial skin. Culture was carried out on a stainless steel grid type net and a polycarbonate membrane. Cultivate so that the medium does not come into contact with the epidermal layer. The medium is a 3:1 mixture of DMEM and Ham's F-12 medium, 5% FBS (Gibco), 24.3 μg/ml adenine (Adenin, Sigma), 5 μg/ml insulin (Insulin, Sigma) 0.4 μg/ml They were cultured for 14 days in a medium containing hydrocortisone (Sigma), 0.212 μg/ml isoprenaline (Isoproterenol hydrochloride, Sigma), and 50 μg/ml Ascorbic acid (Sigma).

Example 3: Immunochemical staining of photoaged artificial skin

It was confirmed whether the photoaging artificial skin model prepared in Example 2 had an expression profile substantially similar to that of photoaging-related markers in photoaging skin tissue.

Using only normal fibroblasts, the expression of p16 ^INK4A , SDF1, and GDF15 in the artificial dermis layer was immunochemically stained for the normal artificial skin model prepared by the method of Example 2 and the photoaged artificial skin model prepared in Example 2. confirmed through.

Immunohistochemical staining is a method of detecting the presence of an antigen in tissue using an antigen-antibody reaction. By using this, photoaging artificial skin tissue is treated with p16 ^INK4A (Ventana), an aging marker, and SDF1 (SDF1, a protein that inhibits skin pigmentation). R&D), after reacting with the primary antibody of GDF15 (Novus Biologicals), a protein that promotes skin pigmentation, the expression was confirmed by reacting with each secondary antibody.

As a result, as shown in FIG. 3, it was confirmed that the expression of p16 ^INK4A , an aging marker, was increased in the photoaging artificial skin model compared to the normal artificial skin model.

As shown in FIG. 4, SDF1, a protein that inhibits skin pigmentation, was confirmed in the normal artificial skin model, but not in the photoaging artificial skin model. In addition, while the expression of GDF15, a protein that promotes skin pigmentation, was not confirmed in the normal artificial skin model, it was confirmed that GDF15 was expressed in the photoaging artificial skin model.

As above, specific parts of the content of the present invention have been described in detail, and for those skilled in the art, these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. It will be clear. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents. Simple modifications or changes of the present invention can be easily used by those skilled in the art, and all such modifications or changes can be considered to be included in the scope of the present invention.

Claims (7)

A manufacturing method of photoaging artificial skin comprising the following steps:
(a) preparing photoaged fibroblasts by treating fibroblasts with UV;
(b) constructing an artificial dermis layer by co-cultivating the photoaged fibroblasts and normal fibroblasts;
(c) constructing an artificial dermal-epidermal layer by culturing keratinocytes on the constructed artificial dermal layer; and
(d) manufacturing artificial skin by additionally culturing after stacking the artificial dermal-epidermal layer on the artificial dermal layer.
The method of claim 1, wherein the UV treatment is performed with UV of 320 to 400 nm.
The method of claim 1, wherein the photoaged fibroblasts and the normal fibroblasts are mixed and co-cultured at a ratio of 1:2 to 1:20.
The method of claim 1, wherein 1 to 5 artificial dermal layers are used in step (d).
The method of claim 1, wherein the additional culture in step (d) is a differentiation induction process through a three-dimensional air-liquid interface (ALI).
photoaging artificial skin models including;
(i) an artificial dermis layer formed by photoaging fibroblasts in which photoaging is induced by UV treatment; and
(ii) an artificial epidermal layer formed by keratinocytes.
The photoaging artificial skin model according to claim 6, wherein the UV treatment is performed with UV of 320 to 400 nm.