KR102335878B1 - Aged artificial skin, producing method of the same and screening method of anti-aging compound for skin using the same - Google Patents

Abstract

Aging artificial skin comprising a dermal mimic layer and an epidermal mimicry layer located on the dermal mimicry layer, wherein the dermal mimicry layer is formed by collagen and fibroblasts, and the fibroblasts are discolored when stained with an aging marker, manufacturing method thereof And it provides a screening method for skin anti-aging substances using the same.

Description

Aging artificial skin, manufacturing method thereof, and skin anti-aging material screening method using the same

The present disclosure relates to aging artificial skin, a manufacturing method thereof, and a skin anti-aging material screening method using the same.

The skin is an organ that covers the outside of the body and consists of three layers: the epidermis, the dermis and the 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 below the epidermis, and the dermis contains blood vessels, nerves, and sweat glands. The subcutaneous fat layer is composed of fat cells. The skin maintains its shape by interacting with various cells and constituents as described above, and exhibits various functions such as body temperature regulation and a function as a barrier to the external environment.

Artificial skin is a three-dimensionally reconstructed skin as described above using skin cells and skin components such as collagen and elastin. Because it exhibits functional properties, it is also called skin equivalent or reconstructed skin. Artificial skin is not only used for replacement (permanent engraftment type) or regeneration (temporary covering type) of damaged skin such as burns or trauma, but also is used in various fields such as skin physiology research, skin irritation evaluation, and skin efficacy evaluation. .

However, unlike real skin, artificial skin has a flat structure at the dermal-epidermal junction (DEJ) layer, so there is a limit to conducting anti-aging experiments using only artificial skin. This is because the interface between the dermis and epidermis is curved rather than flat in real skin.

For this reason, in recent years, attempts have been made to screen anti-aging substances using an aged artificial skin model. This artificial skin model is manufactured using aged skin cells. Aged skin cells are mainly collected from elderly skin or young cells are obtained by inducing aging through subcultures that have been exfoliated about 30 times. The problem is that cells collected from the skin of the elderly differ in the degree of aging depending on the condition of the donor, making it difficult to manufacture artificial aging skin of uniform quality. will be. In addition, repeated subculture requires a subculture period of at least 3 to 6 months, and has a limitation in that it is difficult to artificially control the degree of aging.

Accordingly, the present inventors treat the fibroblasts constituting the dermal dermal layer with a specific substance to induce the aging of the fibroblasts in a very short time, and then use this to manufacture artificial skin, compared to the existing aging artificial skin manufacturing method, etc. , led to the development of a method for quickly manufacturing aging artificial skin of uniform quality.

It was very difficult to make artificial aging skin of uniform quality only by the methods known so far, and there was a problem that it took several months to manufacture aging artificial skin. However, in one embodiment, by treating fibroblasts with a specific drug to induce their rapid aging, and manufacturing aged artificial skin using the aged fibroblasts, it is easier and faster than existing methods, and histology very similar to actual aging skin. / To provide aging artificial skin with physiological characteristics.

Another embodiment is to provide a method for producing the aging artificial skin.

Another embodiment is to provide a method for screening a skin anti-aging material using the aging artificial skin.

According to one embodiment, it comprises a mimetic dermal layer and an epidermal mimic layer located on the dermal mimic layer, wherein the dermal mimic layer is formed by collagen and fibroblasts, and the fibroblasts are discolored when stained with a senescence marker. Provides artificial skin.

The aging marker may be senescence associated beta galactosidase (SA-β-gal).

The fibroblasts may be treated with a DNA topoisomerase II inhibitor and insulin-like growth factor 1 (IGF-1).

The DNA topoisomerase II inhibitor may be treated at a concentration equal to or less than the insulin-like growth factor 1 .

According to another embodiment, inducing senescence of fibroblasts by treating the fibroblasts with a DNA topoisomerase II inhibitor and insulin-like growth factor 1; forming a dermal mimic layer by mixing the aging-induced fibroblasts with collagen; And it provides a method for manufacturing aging artificial skin comprising the step of forming the epidermal mimetic layer by culturing after applying keratinocytes on the dermal mimic layer.

The DNA topoisomerase II inhibitor may be treated at a concentration equal to or less than the insulin-like growth factor 1 .

The DNA topoisomerase II inhibitor and insulin-like growth factor 1 may be treated to the fibroblasts in a concentration ratio of 1:1 to 1:20.

The step of inducing aging of the fibroblasts may be a step of treating the fibroblasts with a DNA topoisomerase II inhibitor and insulin-like growth factor 1 for 3 to 10 days.

After forming the dermal mimic layer and before the epidermal mimicry layer is formed, the method may further include stabilizing the dermal mimicry layer by culturing in a fibroblast culture medium.

According to another embodiment, mixing fibroblasts with collagen to form a dermal mimic layer; Inducing aging of fibroblasts by treating the dermal layer with a DNA topoisomerase II inhibitor and insulin-like growth factor 1; And it provides a method for manufacturing aging artificial skin comprising the step of forming the epidermal mimetic layer by culturing after applying keratinocytes on the dermal mimic layer.

The DNA topoisomerase II inhibitor may be treated at a concentration equal to or less than the insulin-like growth factor 1 .

The DNA topoisomerase II inhibitor and insulin-like growth factor 1 may be treated to the fibroblasts in a concentration ratio of 1:1 to 1:20.

The step of inducing aging of the fibroblasts may be a step of treating the fibroblasts with a DNA topoisomerase II inhibitor and insulin-like growth factor 1 for 3 to 10 days.

Another embodiment is a method for screening a skin anti-aging material, comprising the steps of applying the skin anti-aging material to the artificial skin or artificial skin prepared according to the method for manufacturing the artificial skin, and the skin anti-aging material is applied It provides a method for screening a skin anti-aging material comprising the step of comparing the thickness of the dermal imitation layer of artificial skin with the dermal imitation layer thickness of artificial skin to which the skin anti-aging material is not applied.

According to one embodiment, aging artificial skin having a structure and properties most similar to those of actual aging skin can be manufactured, and skin anti-aging substances can be easily screened using this.

1 is a schematic diagram showing changes in the shape of fibroblasts and collagen constituting the dermal layer of the skin during the aging process of the skin and the anti-aging process of the skin.
2 is a photograph of artificial skin not treated with DNA topoisomerase II inhibitor and insulin-like growth factor 1 (left) and a photograph of aged artificial skin treated with DNA topoisomerase II inhibitor and insulin-like growth factor 1 (right) am.
3 is a cross-section of artificial skin not treated with DNA topoisomerase II inhibitor and insulin-like growth factor 1, and micrographs after H&E tissue staining (left), DNA topoisomerase II inhibitor and insulin-like growth factor 1 A micrograph (middle) after H&E tissue dyeing as a cross section of the treated aged artificial skin, and a photomicrograph (right) after H&E tissue staining as a cross section of the artificial skin treated with the aging artificial skin with a skin anti-aging material.
4 is a cross-section of artificial skin not treated with DNA topoisomerase II inhibitor and insulin-like growth factor 1, and micrographs after collagen staining (left), DNA topoisomerase II inhibitor and insulin-like growth factor 1 A micrograph (middle) after dyeing collagen as a cross-section of the treated aged artificial skin, and a micrograph (right) after dyeing collagen as a cross-section of the artificial skin treated with the aging artificial skin with a skin anti-aging substance.
Figure 5 is a graph showing the pro-collagen expression level of artificial skin not treated with DNA topoisomerase II inhibitor and insulin-like growth factor 1 (left), DNA topoisomerase II inhibitor and insulin-like growth factor 1 treatment A graph showing the amount of pro-collagen expression of one aged artificial skin (middle) and a graph showing the amount of pro-collagen expression of artificial skin treated with the aging artificial skin with a skin anti-aging substance (right).
6 is a photograph (left) and DNA of fibroblasts in aged artificial skin treated with a DNA topoisomerase II inhibitor and insulin-like growth factor 1 stained blue by a senescence marker (SA-β-gal); It is a photograph (right) taken of fibroblasts in artificial skin treated only with topoisomerase II inhibitors that were not stained blue by the aging marker (SA-β-gal).
7 is a graph (left) and DNA topo A graph (right) showing the number of fibroblasts that respond positively to the aging marker (SA-β-gal) in artificial skin treated only with an isomerase II inhibitor.

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

As used herein, the term "artificial skin" refers to a three-dimensional reconstitution of skin using skin cells and collagen, which is a component of the skin, and a polymer complex exhibiting structural and functional properties similar to those of actual skin. It is the broadest meaning that includes all ramen noodles.

As used herein, "skin aging" refers to a state in which both fibroblasts and collagen constituting the dermal layer are damaged in the skin composed of the dermal layer and the epidermal layer.

In the present specification, when a part such as a layer, film, region, plate, etc. is "on" another part, it includes not only the case where the other part is "directly on" but also the case where there is another part in the middle. Conversely, when we say that a part is "just above" another part, we mean that there is no other part in the middle.

Hereinafter, aging artificial skin according to an embodiment will be described.

Aging artificial skin according to an embodiment includes a dermal imitation layer and an epidermal imitation layer located on the dermal imitation layer, wherein the dermal dermal layer is formed by collagen and fibroblasts, and the fibroblasts are stained with an aging marker discolored

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

The dermal mimic layer is formed using a material mixed with fibroblasts and collagen from the viewpoint of human correlation with the actual skin. The dermis mimic layer may consist of two or more layers, for example, may include, and may be divided into a first dermal mimic layer containing collagen and a second dermal mimicry layer containing fibroblasts, and the collagen and It may consist of one dermal mimic layer comprising fibroblasts. When the dermal imitation layer is divided into a first dermal imitation layer and a second dermal imitation layer, the dermal contraction phenomenon of the artificial skin can be further alleviated, but the number of layers constituting the dermal imitation layer is an aging artificial layer according to an embodiment It may not significantly affect the quality of the skin.

Most of the artificial skin currently distributed in the market is artificial skin that mimics the skin of the human body before aging. There is no choice but to do so, resulting in too much loss in terms of time and cost.

In view of these problems, skin is donated voluntarily from the elderly (via biopsy, etc.) or young cells are repeatedly passaged dozens of times to obtain senescent cells and manufacture artificial skin from them. This is so different that it is impossible to produce uniform quality, and it takes several months for several dozen repeated subcultures, so it is unproductive and uneconomical.

Accordingly, there was an attempt to manufacture aged artificial skin by saccharifying collagen, a component of the dermal layer, by a new method, that is, it takes more than a month to saccharify collagen, and aging produced through saccharification of collagen The artificial skin itself is different from the actual aging skin of the human body, so it is difficult to accurately screen anti-aging substances for the skin. Specifically, the aging phenomenon of the skin is not caused by the aging of collagen constituting the dermal layer, but changes in the shape of the fibroblasts due to the aging of the fibroblasts constituting the dermal layer (tight cells are contracted to form wrinkled cells) change) occurs, and as a result, the bonding strength between fibroblasts and collagen is lowered, and as the collagen bound to the fibroblasts falls off, it can be seen that the collagen also ages. Although some may be removed from the fibroblasts, since the fibroblasts themselves do not age, it is very difficult to see that the artificial skin manufactured using the glycated collagen mimics the aged real skin (see FIG. 1).

Recently, a method of manufacturing artificial skin through mitosin treatment has been introduced. Specifically, after artificially stopping the growth of fibroblasts, artificial skin is produced using fibroblasts that have stopped growing. , skin aging does not mean that the growth of cells simply stops, but is caused by a degeneration that continues over time, and it is also very difficult to see that it mimics aging skin. This is because, when cells whose growth has been artificially stopped is used, cells whose growth has been stopped under certain conditions may grow again, and it is difficult to view this as senescence based only on the event that cell growth has simply stopped.

As such, it is difficult to manufacture aging artificial skin that is very similar to actual aging skin, and thus, development of artificial skin closest to actual aging skin has not been realized until now.

Accordingly, the present inventors believe that when artificial skin is manufactured using aged fibroblasts, it is possible to manufacture artificial skin closest to actual aging skin. Instead of stopping growth, by using fibroblasts induced to age in a short time by themselves, it has come to manufacture artificial aging skin that is very similar to actual aged skin.

Aging artificial skin according to an embodiment includes a mimic dermis and an epidermal mimetic layer located on the mimetic dermal layer, wherein the mimetic dermis layer is formed by collagen and fibroblasts, which themselves within a short time, for example Since the fibroblasts are induced to age within about 3 to 10 days, for example, about a week, the fibroblasts are discolored blue when stained with a senescence marker. For example, since all conventional aging artificial skin does not use fibroblasts induced to age within a short span as described above, even if dyed with an aging marker, fibroblasts are not dyed blue, which is according to one embodiment. It can be one big difference that distinguishes aging artificial skin from conventional aging artificial skin.

For example, the senescence marker may include senescence associated beta galactosidase (SA-β-gal). For example, the aging marker may be senescence associated beta galactosidase (SA-β-gal).

The fibroblasts may be treated with a DNA topoisomerase II inhibitor and insulin-like growth factor 1 (IGF-1). In the case of the DNA topoisomerase II inhibitor, there is a limitation in using it alone in terms of inhibiting the growth of fibroblasts, but not inducing aging of fibroblasts, and the insulin-like growth factor 1 is Although it induces the aging of blast cells, when used alone, the aged fibroblasts do not form well the dermal mimic layer together with collagen, or have limitations such as making it impossible to recover the collagen used together. Accordingly, fibroblasts, which are one component constituting the dermal mimic layer of aging artificial skin according to an embodiment, may be processed using both a DNA topoisomerase II inhibitor and an insulin-like growth factor 1. When the above two substances (DNA topoisomerase II inhibitor and insulin-like growth factor 1) are simultaneously treated with fibroblasts, aging of fibroblasts can be induced within about a week, and the compatibility with collagen is excellent to form a dermal mimic layer. There is no problem in the aging artificial skin, and when a skin anti-aging material is treated on the manufactured aged artificial skin, the collagen expression level in the aged artificial skin increases, so it may be suitable for screening of skin anti-aging material.

For example, the DNA topoisomerase II inhibitor may be treated at a concentration equal to or less than that of the insulin-like growth factor 1. When the DNA topoisomerase II inhibitor is treated at a concentration higher than that of the insulin-like growth factor 1, the relative concentration of the DNA topoisomerase II inhibitor is too high and the relative concentration of the insulin-like growth factor 1 is too low. It may be undesirable because it induces apoptosis of fibroblasts rather than induction of senescence of blast cells. For example, the DNA topoisomerase II inhibitor may be treated at a concentration of 100 ng/ml or less, and at the same time, the insulin-like growth factor 1 may be treated at a concentration of 100 ng/ml or more. For example, within the concentration content range, the DNA topoisomerase II inhibitor and insulin-like growth factor 1 may be used in a concentration ratio of 1:1 to 1:20, but is not necessarily limited thereto.

For example, etoposide (VP-16), teniposide, daunorubicin, mitoxantrone, amsacrine, ellipticines, aurintricarboxylic acid, HU-331, quinolone, doxorubicin, EGCG, genistein, quercetin, resveratrol, etc. can be used as the DNA topoisomerase II inhibitor. However, it is not necessarily limited thereto.

The dermal mimic layer further contains collagen along with the fibroblasts, the collagen may be a collagen of bovine origin, from rat tail or from fish, or any other origin of natural collagen or collagen produced by genetic engineering; , which can contract in the presence of fibroblasts.

The dermal mimic layer is, in addition to the fibroblasts and collagen, elastin (Elastin), chitosan (Chitosan), glycosaminoglucans (GAGs; glycosaminoglycans), hyaluronic acid (HA; hyaluronic acid), such as the extracellular matrix constituting the dermis ( extracellular matrix) may be further contained.

The thickness of the dermal mimicry layer is generally about 0.05 cm or more, for example, about 0.05 to 2 cm, but may be increased or decreased as long as it does not damage the advantageous properties of the artificial skin according to an embodiment.

Another embodiment is to induce senescence of fibroblasts by treating the fibroblasts with a DNA topoisomerase II inhibitor and insulin-like growth factor 1; forming a dermal mimic layer by mixing the aging-induced fibroblasts with collagen; And it provides a method for manufacturing aging artificial skin comprising the step of forming the epidermal mimetic layer by culturing after applying keratinocytes on the dermal mimic layer.

In order to induce senescence of the fibroblasts, the DNA topoisomerase II inhibitor and insulin-like growth factor 1 should be treated as described above, wherein the DNA topoisomerase II inhibitor is administered at a lower concentration than the insulin-like growth factor 1. Treatment, such as the DNA topoisomerase II inhibitor and insulin-like growth factor 1, can be treated to the fibroblasts in a concentration ratio of 1:2 to 1:20, such as in a concentration ratio of 1:10.

For example, the step of inducing aging of the fibroblasts may be a step of treating the fibroblasts with a DNA topoisomerase II inhibitor and insulin-like growth factor 1 for about 3 to 10 days, such as for about a week.

Aging artificial skin manufacturing method according to an embodiment may further include stabilizing the dermal mimic layer by culturing the dermal mimicry layer in a fibroblast culture medium before the epidermal mimicry layer is formed after the dermal mimicry layer is formed.

Aging artificial skin manufacturing method according to another embodiment comprises the steps of: mixing fibroblasts with collagen to form a dermal mimic layer; Inducing aging of fibroblasts by treating the dermal layer with a DNA topoisomerase II inhibitor and insulin-like growth factor 1; And it may include the step of forming the epidermal mimetic layer by culturing after applying the keratinocytes on the dermal mimic layer. After forming the dermal mimic layer by mixing non-senescence-induced fibroblasts with collagen, the above drugs (DNA topoisomerase II inhibitor and insulin-like growth factor), only fibroblasts selectively induce aging.

Specifically, an aging artificial skin manufacturing method according to another embodiment comprises the steps of: forming a dermal mimic layer by mixing fibroblasts with collagen; inducing senescence of fibroblasts by treating the mimetic layer of the dermis with a DNA topoisomerase II inhibitor and insulin-like growth factor 1 for about 3 to 10 days, for example, about a week; And it may include the step of forming the epidermal mimetic layer by culturing after applying the keratinocytes on the dermal mimic layer.

Details of the DNA topoisomerase II inhibitor and insulin-like growth factor 1 are the same as described above.

When the dermal mimic layer is formed, keratinocytes are applied thereon and then cultured.

Keratinocytes constitute about 80% to 90% of the cells of the epidermal mimetic layer, and are formed through mitosis in the basal layer, which is the deepest layer of the epidermal mimetic layer, and then ascend to the upper layer toward the skin surface.

The step of culturing the keratinocytes after application is the first step for forming the epidermal mimic layer of the artificial skin, and the keratinocytes used herein may be, for example, keratinocytes derived from human. Any keratinocytes currently commercially available may be used as the keratinocytes, and keratinocytes derived from other cells as well as keratinocytes directly isolated or isolated from humans and cultured may be used. Commercially available human keratinocytes include NHEK-Neo, Pooled (Neonatal Normal Human Epidermal Keratinocytes, Pooled: Catalog No. 00192906, Tissue Acquisition No. P867, Caucasians) provided by Lonza, NHEK-Neo (Product No. 00192907, Tissue acquisition number: 20647, white), NHEK-Adult (product number 00192627, tissue acquisition number: 21155, white), and NHEK-Neo (product number 00192907, organization acquisition number: 18080, black). And HEKn (Human Epidermal keratinocytes, neonatal: product number C0015C, tissue acquisition number 1781129, white) and HEKn (product number C0015C, tissue acquisition number 1803827, black) provided by Thermo Fisher are mentioned. In order to maintain the property of keratinocytes to attach to the base membrane and proliferate, the dish is preferably coated with 0.1 to 0.2% gelatin or 0.1 to 0.2 mg/ml collagen type IV. The cells may be cultured in an incubator of 35-37° C., 5-10% CO ₂ and sub-cultured 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 not necessarily limited thereto. it is not

After the step of applying and culturing the keratinocytes, a step of culturing in a keratinocyte medium may be performed.

Herein, "keratinocyte medium" refers to a medium for culturing human keratinocytes, and any medium known in the art may be used. For example, the keratinocyte medium may include 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 so as to contact the lower surface of the dermal mimic layer, and the opposite surface of the surface on which the keratinocyte medium is installed may be exposed to air. Culturing in such a keratinocyte medium may be carried out 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.

By going through the above-described steps, it is possible to manufacture aged artificial skin in which the imitation epidermis layer is formed on the dermis imitation layer.

Another embodiment is a method for screening a skin anti-aging material, comprising the steps of applying the skin anti-aging material to the aging artificial skin or aging artificial skin prepared according to the manufacturing method of the aging artificial skin, and the skin anti-aging material It provides a method for screening a skin anti-aging material, comprising the step of comparing the dermal mimic layer of the aging artificial skin to which the aging material is applied and the dermal mimic layer of the aged artificial skin to which the skin anti-aging material is not applied.

Comparing the dermal imitation layer of the aging artificial skin to which the skin anti-aging material is applied and the dermal imitation layer of the aging artificial skin to which the skin anti-aging material is not applied, the thickness of the dermal imitation layer or the epidermal imitation layer after tissue dyeing It may be a step of comparing proliferation/differentiation changes, or it may be a step of comparing the expression levels of aging-related markers such as collagen or MMP-1 through molecular biological methods (ELISA, PCR), and the comparison method is not necessarily limited thereto. it is not going to be

Since the thickness of the dermal layer constituting the skin becomes thinner as the skin ages, the thickness of the dermal mimicry layer is also thin in the aged artificial skin according to an embodiment. However, if the thickness of the dermis imitation layer constituting the aging artificial skin is thickened by treating the specific material, it can be seen that the specific material functions as a skin anti-aging material, and thus, screening of the skin anti-aging material is possible.

In addition, aged skin has a characteristic that the expression of collagen in the dermal layer is decreased. If the amount of collagen expression in the dermal mimic layer constituting the aging artificial skin is increased by treating a specific substance, the specific substance also functions as a skin anti-aging substance. It can be seen that, therefore, it is possible to screen for skin anti-aging substances.

The embodiments of the present invention described above will be described in more detail through the following examples. However, the following examples are for illustrative purposes only and do not limit the scope of the present invention.

Manufacture of artificial skin and confirmation of aging induction of fibroblasts

Example 1

Cell senescence was induced by treating human neonatal fibroblasts (Human Dermal Fibroblasts, neonatal (HDFn), ThermoFisher Scientific) with Doxorubicin (100 nM, Sigma) and IGF-1 (100 nM, Sigma) for one week. Whether fibroblast senescence was induced was confirmed by β-gal staining, and the results are shown in FIGS. 6 and 7 . Referring to FIG. 6 , it can be seen that fibroblasts are stained blue (inducing senescence of fibroblasts), and referring to FIG. 7, it can be confirmed that the number of fibroblasts positively responding to β-gal staining is large (inducing senescence of fibroblasts). can

Thereafter, the dermal mimic layer was prepared by mixing the fibroblasts and collagen, and then cultured in a fibroblast culture medium for one week to stabilize it. By seeding keratinocytes on the stabilized dermal mimic layer and culturing for about 2 weeks, the epidermal mimetic layer was formed to prepare aged artificial skin with a dermal-epidermal structure. A photograph of the aged artificial skin according to Example 1 finally prepared is shown in FIG. 2 . It can be seen that the morphological difference is clearly revealed even with the naked eye compared with the artificial skin photograph according to Comparative Example 1.

Comparative Example 1

Artificial skin was prepared in the same manner as in Example 1, except that doxorubicin and IGF-1 were not treated. A photograph of the artificial skin according to Comparative Example 1 finally prepared is shown in FIG. 2 .

Comparative Example 2

Artificial skin was prepared in the same manner as in Example 1 except that IGF-1 was not treated. Whether fibroblast aging was confirmed by β-gal staining, the results are shown in FIGS. 6 and 7 . Referring to FIG. 6 , fibroblasts are not stained blue, and in FIG. 7 , it can be seen that the number of fibroblasts that respond positively to β-gal staining is small. It can be confirmed that it is not induced.

Evaluation: Whether to implement aging artificial skin and screen for skin anti-aging substances

The cross-sections of the artificial skin obtained in Example 1 and Comparative Example 1 were observed under a microscope (after H&E tissue staining), and the results are shown in FIGS. 3 and 4 .

Referring to FIG. 3 , compared to Comparative Example 1, it can be seen that the artificial skin of Example 1 is aging and the thickness of the dermal imitation layer is greatly reduced, and 3-Phosphoinositide-dependent protein kinase-1 inhibitor, known as a skin anti-aging material After treatment with one of the compounds represented by the following formula 1 (selleckchem, USA), it can be confirmed that the thickness of the dermal imitation layer is increased again, and from this, the aging artificial skin according to an embodiment is a skin anti-aging material. It can also be confirmed that it can be usefully used for screening.

[Formula 1]

Referring to FIG. 4 , compared to Comparative Example 1, it can be seen that the artificial skin of Example 1 is aging and the collagen expression level (blue color) in the dermal mitral layer is decreased, and 3-Phosphoinositide-dependent known as a skin anti-aging material. After treatment with the compound represented by Formula 1 (selleckchem, USA), which is one of the protein kinase-1 inhibitors, it can be confirmed that the collagen expression level in the dermal mitral layer is increased again, and from this, the aging artificial skin according to one embodiment is It can also be confirmed that it can be usefully used for screening skin anti-aging substances.

Referring to FIG. 5, which graphically shows the collagen expression level in the dermal mimic layer of the artificial skin obtained in Example 1 and Comparative Example 1, the aged artificial skin according to Example 1 had a higher collagen expression level than the artificial skin according to Comparative Example 1. It can be confirmed that the decrease, and also when the compound represented by the formula (1) is treated, it can be confirmed that the collagen expression level again significantly increased. From this, it can be confirmed that the aging artificial skin according to an embodiment has properties very similar to those of actual aged skin, and can be usefully used for screening skin anti-aging substances.

Although preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present invention as defined in the following claims are also presented. It belongs to the scope of the right of the invention.

1 Fibroblasts
2 Collagen
3 Bonding material that connects fibroblasts and collagen
4 Collagen that has fallen off from aged fibroblasts and aged

Claims (13)

Comprising a dermal mimic layer and an epidermal mimicry layer located on the dermal mimic layer,
The dermal mimic layer is formed by collagen and fibroblasts,
The fibroblasts are discolored when stained with a senescence marker,
The fibroblasts are treated with a DNA topoisomerase II inhibitor and insulin-like growth factor 1 (IGF-1),
Aging artificial skin treated with the DNA topoisomerase II inhibitor at a concentration of 100 ng/ml or less, and at the same time the insulin-like growth factor 1 is treated with a concentration of 100 ng/ml or more. In claim 1,
The aging marker is senescence associated beta galactosidase (SA-β-gal), artificial aging skin. delete delete Inducing senescence of fibroblasts by treating the fibroblasts with a DNA topoisomerase II inhibitor and insulin-like growth factor 1;
forming a dermal mimic layer by mixing the aging-induced fibroblasts with collagen; and
Forming an epidermal mimic layer by culturing after applying keratinocytes on the dermal mimic layer
including,
The DNA topoisomerase II inhibitor is treated at a concentration of 100 ng/ml or less, and at the same time, the insulin-like growth factor 1 is treated at a concentration of 100 ng/ml or more. delete In claim 5,
The DNA topoisomerase II inhibitor and insulin-like growth factor 1 are 1:1 to 1:20 in a concentration ratio of 1:20 to the fibroblasts in the artificial aging skin manufacturing method. In claim 5,
The step of inducing aging of the fibroblasts is a step of treating the fibroblasts with a DNA topoisomerase II inhibitor and insulin-like growth factor 1 for 3 to 10 days. Forming a dermal mimic layer by mixing fibroblasts with collagen;
Inducing aging of the fibroblasts by treating the dermal mimic layer with a DNA topoisomerase II inhibitor and insulin-like growth factor 1; and
Forming an epidermal mimic layer by culturing after applying keratinocytes on the dermal mimic layer
including,
The DNA topoisomerase II inhibitor is treated at a concentration of 100 ng/ml or less, and at the same time, the insulin-like growth factor 1 is treated at a concentration of 100 ng/ml or more. delete In claim 9,
The DNA topoisomerase II inhibitor and insulin-like growth factor 1 are 1:1 to 1:20 in a concentration ratio of 1:20 to the fibroblasts in the artificial aging skin manufacturing method. In claim 9,
The step of inducing aging of the fibroblasts is a step of treating the dermal layer with a DNA topoisomerase II inhibitor and insulin-like growth factor 1 for 3 to 10 days. A method for screening skin anti-aging substances, comprising:
The step of applying the skin anti-aging material to the artificial skin of claim 1 or the artificial skin manufactured according to the manufacturing method of claim 5 or the manufacturing method of claim 9, and
A method for screening a skin anti-aging material, comprising the step of comparing the dermal mimic layer of the artificial skin to which the skin anti-aging material is applied and the dermal mimic layer of the artificial skin to which the skin anti-aging material is not applied.

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