KR20210043309A - Aged artificial skin, producing method of the same and screening method of anti-aging compound for skin using the same - Google Patents

Abstract

The present invention relates to an aged artificial skin, a producing method of the same, and a screening method of an anti-aging compound for skin using the same, wherein the aged artificial skin comprises a dermal mimic layer and an epidermal mimic layer located on the dermal mimic layer, the dermal mimic layer is formed by collagen and fibroblasts, and the fibroblasts are discolored when stained with a senescence marker. The present invention is to provide the aging artificial skin which has histological/physiological properties very similar to that of actual aging skin faster and easier than existing methods.

Description

Aging artificial skin, its manufacturing method, and skin anti-aging substance screening method using the same {AGED ARTIFICIAL SKIN, PRODUCING METHOD OF THE SAME AND SCREENING METHOD OF ANTI-AGING COMPOUND FOR SKIN USING THE SAME}

The present description relates to an aging artificial skin, a method for manufacturing the same, and a method for screening skin anti-aging substances 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 occupies most of the keratinocytes of the middle-layer squamous epithelium. The dermis, which is made of matrix proteins such as collagen fibers and elastic fibers, is located under the epidermis, and the dermis contains blood vessels, nerves, and sweat glands. The subcutaneous fat layer is composed of fat cells. Skin maintains its shape by interacting with various cells and constituents as described above, and exhibits various functions such as controlling body temperature and acting as a barrier to the external environment.

Artificial skin is a three-dimensional reconstruction of the above-described skin using skin cells and skin components such as collagen and elastin. It is composed of living fibroblasts and keratinocytes, and has a structural structure similar to that of real skin. It is also called skin equivalent or reconstructed skin because of its functional properties. 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 is also used in various areas such as skin physiology research, skin irritation evaluation, and skin efficacy evaluation. .

However, unlike actual skin, artificial skin has a flat structure in which the dermal-epidermal junction (DEJ) between the dermal and epidermal layers is flat, so there is a limitation in conducting anti-aging experiments with only artificial skin. This is because the interface between the dermal layer and the epidermal layer is not flat but curved in the actual skin.

For this reason, in recent years, attempts to screen for anti-aging substances and the like using an aging artificial skin model have been continued, and the aging artificial skin models reported to date mainly rely on aging skin cells. These artificial skin models are produced using aged skin cells, and aged skin cells are mainly obtained from the skin of the elderly, or young cells are obtained by inducing aging through thinning passages about 30 times. The problem is that the cells collected from the elderly skin have a significant difference in the degree of aging depending on the donor's condition, making it difficult to produce an aging artificial skin of uniform quality, and it is obtained through a donor's skin biopsy, so it has a limitation that it cannot continue to produce cells. will be. In addition, repeated passage culture requires a passage culture period of at least 3 to 6 months, and has a limitation in that it is difficult to artificially control the degree of aging.

Therefore, the present inventors induce aging of the fibroblasts in a very short time by treating the fibroblasts constituting the dermal mimetic layer, and then manufacturing artificial skin using this, compared with the existing aging artificial skin manufacturing method. However, it has come to develop a method for producing aging artificial skin of uniform quality in a short time.

It was very difficult to make aging artificial skin of uniform quality using only 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 specific drugs to induce their rapid aging, and by using the aged fibroblasts to prepare aging artificial skin, histology is easier and faster than conventional methods and very similar to actual aging skin. /To provide aging artificial skin with physiological properties.

Another embodiment is to provide a method of manufacturing the aging artificial skin.

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

According to one embodiment, it includes a dermal mimicking layer and an epidermal mimicking layer located on the dermal mimicking layer, wherein the dermal mimicking layer is formed by collagen and fibroblasts, and the fibroblasts are discolored when stained with an aging marker. Provide 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 fibroblasts with a DNA topoisomerase II inhibitor and insulin-like growth factor 1; Mixing the aging-induced fibroblasts with collagen to form a dermal mimetic layer; And it provides a method for manufacturing aging artificial skin comprising the step of forming an epidermal mimetic layer by applying keratinocytes on the dermal mimetic layer and then culturing it.

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 on the fibroblasts at a concentration ratio of 1:1 to 1:20.

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

After the dermal mimetic layer is formed and before the epidermal mimic layer is formed, the step of stabilizing the dermal mimetic layer by culturing in a fibroblast culture medium may be further included.

According to another embodiment, the step of forming a dermal mimicking layer by mixing fibroblasts with collagen; Inducing senescence of fibroblasts by treating the dermal mimetic 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 an epidermal mimetic layer by applying keratinocytes on the dermal mimetic layer and then culturing it.

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 on the fibroblasts at a concentration ratio of 1:1 to 1:20.

The step of inducing senescence of fibroblasts may be a step of treating 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 skin anti-aging substances, the step of applying the skin anti-aging substance to the artificial skin or artificial skin manufactured according to the manufacturing method of the artificial skin, and the skin anti-aging substance is applied It provides a method for screening a skin anti-aging substance comprising comparing the thickness of the dermal replica layer of artificial skin with the thickness of the dermal replica layer of artificial skin to which the skin anti-aging substance is not applied.

According to an embodiment, an aging artificial skin having a structure and properties most similar to that 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 and the anti-aging process of the skin.
Figure 2 is a photograph of artificial skin not treated with a DNA topoisomerase II inhibitor and insulin-like growth factor 1 (left) and a photograph of an aging artificial skin treated with a DNA topoisomerase II inhibitor and insulin-like growth factor 1 (right). to be.
3 is a cross-section of artificial skin not treated with a DNA topoisomerase II inhibitor and insulin-like growth factor 1, showing a micrograph after H&E tissue staining (left), a DNA topoisomerase II inhibitor, and an insulin-like growth factor 1. A cross-section of the treated aging artificial skin is a microscopic photograph after H&E tissue staining (middle) and a cross-section of the artificial skin treated with the aging artificial skin with a skin anti-aging substance after H&E tissue staining (right).
Figure 4 is a cross-section of artificial skin not treated with a DNA topoisomerase II inhibitor and insulin-like growth factor 1, showing a micrograph (left) after staining with collagen, a DNA topoisomerase II inhibitor, and an insulin-like growth factor 1. A cross-section of the treated aging artificial skin is a photomicrograph after staining of collagen (middle), and a cross-section of the artificial skin treated with the aging artificial skin with a skin anti-aging substance is a photomicrograph after staining of collagen (right).
Figure 5 is a graph showing the expression level of pro-collagen in 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 are treated A graph showing the expression level of pro-collagen in an aging artificial skin (middle) and a graph showing the expression level of pro-collagen in an artificial skin treated with the aging artificial skin with a skin anti-aging substance (right).
FIG. 6 is a photograph (left) and DNA in which fibroblasts in aging artificial skin treated with DNA topoisomerase II inhibitor and insulin-like growth factor 1 were stained blue by an aging marker (SA-β-gal). This is a picture of fibroblasts in artificial skin treated with only topoisomerase II inhibitors not stained blue by an aging marker (SA-β-gal) (right).
7 is a graph showing the number of fibroblasts positive for aging marker (SA-β-gal) in aging artificial skin treated with a DNA topoisomerase II inhibitor and insulin-like growth factor 1 (left) and DNA topo This is a graph showing the number of fibroblasts positive for the aging marker (SA-β-gal) in artificial skin treated with isomerase II inhibitor alone (right).

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art may easily implement the present invention. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

In the present specification, the term "artificial skin" refers to a three-dimensional reconstruction of skin using skin cells and collagen, which is a skin material, and a polymer composite that exhibits structural and functional properties similar to those of actual skin. This is the broadest meaning that includes all ramen noodles.

In the present specification, "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 portion such as a layer, film, region, plate, etc. is said to be “on” another portion, this includes not only the case where the other portion is “directly above”, but also the case where there is another portion in the middle. Conversely, when one part is "directly above" another part, it means that there is no other part in the middle.

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

The aging artificial skin according to an embodiment includes a dermal mimicking layer and an epidermal mimicking layer located on the dermal mimicking layer, wherein the dermal mimicking layer is formed by collagen and fibroblasts, and the fibroblasts are stained with aging markers. Discoloration.

Human skin consists of a dermal layer and an epidermal layer above the dermis layer, and 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 a basement membrane. The dermis provides a substrate that supports various appendages such as blood vessels and nerves that are inherent in its structure.

The dermal mimicking layer is formed using a mixture of fibroblasts and collagen from the viewpoint of human body correlation with actual skin. The dermal mimetic layer may consist of two or more layers, for example, and may be divided into a first dermal mimetic layer containing collagen and a second dermal mimetic layer containing fibroblasts, and the collagen and It may also consist of a single dermal mimetic layer containing fibroblasts. When the dermal replica layer is divided into a first dermal replica layer and a second dermal replica layer, it is possible to further alleviate the dermal contraction phenomenon of artificial skin, but the number of layers constituting the dermal replica layer is artificial aging according to one embodiment. It may not have a significant effect on the quality of the skin.

Since most of the artificial skin in the market today is artificial skin that mimics the skin of the human body before aging, it is practically impossible to screen for skin anti-aging substances with such artificial skin. Therefore, clinical trials, etc. are relied upon. There is no choice but to do, and there is too much loss in terms of time and cost.

In light of this problem, the elderly donate skin voluntarily (via biopsy, etc.) or young cells are repeatedly passaged dozens of times to obtain senescent cells and artificial skin from them. These are so different that uniform quality production is impossible, and it is too unproductive and uneconomical because it takes several months for dozens of repeated passages.

Accordingly, there have been attempts to manufacture aging artificial skin with a new method, that is, by saccharifying collagen, a component of the dermal layer, but it takes more than 1 month to saccharify collagen, and aging produced through saccharification of collagen. Since artificial skin itself is different from the actual aging skin of the human body, it is difficult to screen for accurate skin anti-aging substances. Specifically, the aging phenomenon of the skin is not caused by the aging of the collagen constituting the dermal layer, but rather changes in the shape of the fibroblasts due to the aging of the fibroblasts constituting the dermal layer (tight cells contract to form wrinkled cells. Change) occurs, and as the adhesion between fibroblasts and collagen decreases, collagen bound to fibroblasts is removed, and collagen also undergoes aging.If only the collagen linked thereto without aging of fibroblasts is glycosylated, collagen Although some may be eliminated from fibroblasts, since fibroblasts themselves do not undergo aging, it is very difficult to say that artificial skin manufactured using glycated collagen mimics actual aging skin (see Fig. 1).

Recently, a method of manufacturing artificial skin through mitocin treatment has been introduced. Specifically, after artificially stopping the growth of fibroblasts, artificial skin is manufactured using fibroblasts that have stopped growing, although it is a very advanced step in that aging artificial skin is manufactured using fibroblasts rather than collagen. , Skin aging is not simply stopped by the growth of cells, but is caused by degeneration that continues over time, and it is very difficult to say that this also mimics the actual aging skin. This is because, when cells that have simply stopped growth artificially are used, cells that have stopped growing under certain conditions may grow again, and it is difficult to regard this as aging only with 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 therefore, the development of artificial skin closest to actual aging skin has not been realized until now.

Therefore, the present inventors believe that when manufacturing artificial skin using aged fibroblasts, it is possible to manufacture artificial skin closest to actual aged skin. After a long study, the fibroblasts are simply treated with specific drugs. By using fibroblasts induced to age within a short period of time, rather than stopping growth, aging artificial skin very similar to actual aging skin has been produced.

The aging artificial skin according to one embodiment includes a dermal mimetic layer and an epidermal mimetic layer located on the dermal mimetic layer, and the dermal mimetic layer is formed by collagen and fibroblasts, the fibroblasts themselves within a short time, for example Since they are fibroblasts induced to become aging within about 3 to 10 days, such as about a week, the fibroblasts turn blue when stained with an aging marker. For example, since all conventional aging artificial skin does not use fibroblasts induced to become aging within the short-term as described above, fibroblasts are not stained blue even when stained with an aging marker, which is according to one embodiment. It can be one big difference between aging artificial skin and conventional aging artificial skin.

For example, the aging 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, it plays a role of inhibiting the growth of fibroblasts, but there is a limitation in using it alone in that it cannot induce aging of fibroblasts, and the insulin-like growth factor 1 is a fiber Although it induces aging of blast cells, when used alone, the aged fibroblasts do not form a dermal mimicking layer well together with collagen, or they make it impossible to recover the collagen used together. Accordingly, fibroblasts, which are components of the dermal mimetic layer of aging artificial skin according to an embodiment, may be treated using both a DNA topoisomerase II inhibitor and insulin-like growth factor 1. Simultaneous treatment of the above two substances (DNA topoisomerase II inhibitor and insulin-like growth factor 1) on fibroblasts can induce aging of fibroblasts within about a week, and has excellent compatibility with collagen to form a dermal mimetic layer. There is no problem with the skin, and when the produced artificial skin is treated with a skin anti-aging substance, the amount of collagen expression in the aging artificial skin increases, and thus it may be suitable for screening of skin anti-aging substances.

For example, the DNA topoisomerase II inhibitor may be treated at a concentration equal to or less than the insulin-like growth factor 1. When the DNA topoisomerase II inhibitor is treated at a higher concentration than 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 not be desirable because it induces death 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 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 are not limited thereto.

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

The dermal mimetic layer further contains collagen together with the fibroblasts, the collagen may be a collagen of any other origin among bovine origin, rat tail or fish, or natural collagen or collagen produced by genetic engineering, , It can contract in the presence of fibroblasts.

In addition to the fibroblasts and collagen, the dermal mimetic layer is an extracellular matrix constituting the dermis such as elastin, chitosan, glycosaminoglycans (GAGs), and hyaluronic acid (HA). extracellular matrix).

The thickness of the dermal mimicking layer is generally about 0.05 cm or more, such as about 0.05 to 2 cm, but may be increased or decreased as long as the advantageous properties of the artificial skin according to one embodiment are not impaired.

In another embodiment, fibroblasts are treated with a DNA topoisomerase II inhibitor and insulin-like growth factor 1 to induce senescence of fibroblasts; Mixing the aging-induced fibroblasts with collagen to form a dermal mimetic layer; And it provides a method for manufacturing aging artificial skin comprising the step of forming an epidermal mimetic layer by applying keratinocytes on the dermal mimetic layer and then culturing it.

In order to induce aging of the fibroblasts, DNA topoisomerase II inhibitor and insulin-like growth factor 1 must be treated as described above, and the DNA topoisomerase II inhibitor is at a concentration less than that of the insulin-like growth factor 1. Treatment, for example, the DNA topoisomerase II inhibitor and insulin-like growth factor 1 may be treated on the fibroblasts at a concentration ratio of 1:2 to 1:20, such as a concentration ratio of 1:10.

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

The method of manufacturing aging artificial skin according to an embodiment may further include stabilizing the dermal mimetic layer by culturing the dermal mimetic layer in a fibroblast culture medium after the dermal mimetic layer is formed and before the epidermal mimetic layer is formed.

A method for manufacturing aging artificial skin according to another embodiment includes the steps of forming a dermal mimicking layer by mixing fibroblasts with collagen; Inducing senescence of fibroblasts by treating the dermal mimetic layer with a DNA topoisomerase II inhibitor and insulin-like growth factor 1; And it may include the step of forming an epidermal mimetic layer by culturing after applying keratinocytes on the dermal mimetic layer. The above drugs (DNA topoisomerase II inhibitor and insulin-like growth factor 1) are treated even after forming a dermal mimetic layer by mixing fibroblasts that are not aging-inducing with collagen, and then treatment with DNA topoisomerase II inhibitor and insulin-like growth factor 1. Factor) selectively induces aging in fibroblasts.

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

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

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

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

The step of culturing after applying keratinocytes is a first step for forming an epidermal mimetic layer of artificial skin, and the keratinocytes used herein may be, for example, keratinocytes derived from humans. As the keratinocytes, any keratinocytes currently commercially used may be used, and keratinocytes derived from other cells as well as keratinocytes cultured after being isolated or isolated directly from humans may be used. Commercially available human keratinocytes include NHEK-Neo, Pooled (Neonatal Normal Human Epidermal Keratinocytes, Pooled: Product No. 00192906, Tissue Acquisition No. P867, Whites), NHEK-Neo (Product No. 00192907, Tissue) provided by Lonza. Acquisition number: 20647, white), NHEK-Adult (product number 00192627, tissue acquisition number: 21155, white), NHEK-Neo (product number 00192907, tissue acquisition number: 18080, black). And HEKn (Human Epidermal keratinocytes, neonatal: product number C0015C, tissue acquisition number 1781129, white) and HEKn (model number C0015C, tissue acquisition number 1803827, black) provided by Thermo Fisher can be mentioned. In order to maintain the property of keratinocytes attached 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 can be cultured in an incubator at 35-37° C., 5-10% CO ₂ , and can be subcultured at a density of about 70-80%.

The keratinocytes may be applied on the dermal mimicking 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 limited thereto. It is not.

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

Here, "keratinocyte medium" means a medium for culturing human keratinocytes, and any medium known in the art may be used. For example, the keratinocyte medium is 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 component. A commercially available example of the medium is CnT-3D-PR (CellnTEC).

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

By passing through the above-described steps, it is possible to manufacture an aging artificial skin in which an epidermal replica layer is formed on the dermal replica layer.

Another embodiment is a method for screening skin anti-aging substances, the step of applying the skin anti-aging substance to the aging artificial skin or the aging artificial skin manufactured according to the manufacturing method of the aging artificial skin, and It provides a method for screening skin anti-aging substances comprising comparing the dermal replica layer of the aging artificial skin to which the aging substance is applied and the dermal replica layer of the aging artificial skin to which the skin anti-aging substance is not applied.

The step of comparing the dermal replica layer of the aging artificial skin to which the skin anti-aging substance is applied and the dermal replica layer of the aging artificial skin to which the skin anti-aging substance is not applied may include the thickness of the dermal replica layer or the epidermal replica layer after tissue dyeing. It may be a step of comparing changes in proliferation/differentiation, or 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 limited thereto. It does not become.

Since the thickness of the dermal layer constituting the skin decreases as much as the aging skin is, the thickness of the dermal mimicking layer is also thinner in the aging artificial skin according to an embodiment. However, if the thickness of the dermal mimicking layer constituting the aging artificial skin is thickened by treatment with a specific substance, it can be seen that the specific substance functions as a skin anti-aging substance, and thus, screening of the skin anti-aging substance is possible.

In addition, aging skin has a characteristic that the expression of collagen in the dermal layer decreases.If the amount of collagen expression in the dermal mimetic layer constituting the aging artificial skin increases by treating a specific substance, the specific substance functions as a skin anti-aging substance. It can be seen that, thus, 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.

Manufacturing of artificial skin and confirmation of aging induction of fibroblasts

Example 1

Cellular senescence was induced by treating human neonatal fibroblasts (Human Dermal Fibroblasts, neonatal (HDFn), ThermoFisher Scientific) with Doxorubicin (100nM, Sigma) and IGF-1 (100nM, Sigma) for a week. Whether to induce fibroblast senescence was confirmed through β-gal staining, and the results are shown in FIGS. 6 and 7. Referring to Figure 6, it can be seen that the fibroblasts are stained blue (inducing aging of fibroblasts), and looking at Figure 7, the number of fibroblasts positively reacting to β-gal staining is large (inducing aging of fibroblasts). I can.

Thereafter, the fibroblasts and collagen were mixed to prepare a dermal mimetic layer, which was then cultivated in a fibroblast culture medium for a week to stabilize. Keratinocytes were seeded on the stabilized dermal mimetic layer and cultured for about 2 weeks to form an epidermal mimetic layer to prepare dermal-epidermal aging artificial skin. A photograph of the aging artificial skin according to Example 1 finally produced is shown in FIG. 2. Compared with the artificial skin photograph according to Comparative Example 1, it can be seen that the difference in shape is clearly revealed even with the naked eye.

Comparative Example 1

An 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 was finally shown in FIG. 2.

Comparative Example 2

An artificial skin was prepared in the same manner as in Example 1, except that IGF-1 was not treated. Whether fibroblast aging was confirmed through β-gal staining, and the results are shown in FIGS. 6 and 7. Referring to Figure 6, the fibroblasts are not stained blue, and looking at Figure 7, it can be seen that the number of fibroblasts positively reacting to β-gal staining is small. From this, if IGF-1 is not treated, the aging of fibroblasts It can be seen that it is not induced.

Evaluation: Realization of aging artificial skin and screening for skin anti-aging substances

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

3, compared to Comparative Example 1, it can be seen that the artificial skin of Example 1 has undergone aging and the thickness of the dermal mimetic layer is significantly thinner, and 3-Phosphoinositide-dependent protein kinase-1 inhibitor known as a skin anti-aging substance. After treatment of one of the compounds represented by the following formula 1 (selleckchem, USA), it can be confirmed that the thickness of the dermal mimicking layer is thickened again, and from this, the aging artificial skin according to one embodiment is made of a skin anti-aging substance. It can also be seen that it can be usefully used for screening.

[Formula 1]

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

Referring to Figure 5, which graphically shows the amount of collagen expression in the dermal mimicking layer of the artificial skin obtained in Example 1 and Comparative Example 1, the aging artificial skin according to Example 1 has a higher collagen expression level than that of the artificial skin according to Comparative Example 1. It can be seen that the amount is reduced, and it can be seen that the amount of collagen expression is significantly increased again when the compound represented by Formula 1 is treated. From this, it can be seen that the aging artificial skin according to the embodiment has properties very similar to the actual aging skin, and can be usefully used for screening skin anti-aging substances.

Although the 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 defined in the following claims are also present. It is within the scope of the invention's rights.

1 fibroblast
2 collagen
3 Bonding material that connects fibroblasts and collagen
4 Collagen aged by dropping from aged fibroblasts

Claims (13)

It includes a dermal replica layer and an epidermal replica layer located on the dermal replica layer,
The dermal mimetic layer is formed by collagen and fibroblasts,
The fibroblasts are aging artificial skin that is discolored when stained with an aging marker. In claim 1,
The aging marker is senescence associated beta galactosidase (SA-β-gal) of aging artificial skin. In claim 1,
The fibroblasts are aging artificial skin treated with DNA topoisomerase II inhibitor and insulin-like growth factor 1 (IGF-1). In paragraph 3,
The DNA topoisomerase II inhibitor is an aging artificial skin that is treated with a concentration equal to or less than the insulin-like growth factor 1. Inducing senescence of fibroblasts by treating fibroblasts with a DNA topoisomerase II inhibitor and insulin-like growth factor 1;
Mixing the aging-induced fibroblasts with collagen to form a dermal mimetic layer; And
Applying keratinocytes on the dermal mimetic layer and then culturing to form an epidermal mimetic layer
Aging artificial skin manufacturing method comprising a. In clause 5,
The DNA topoisomerase II inhibitor is a method for producing aging artificial skin that is treated at a concentration equal to or less than the insulin-like growth factor 1. In clause 5,
The DNA topoisomerase II inhibitor and insulin-like growth factor 1 is a method for producing aging artificial skin that is treated on the fibroblasts at a concentration ratio of 1:1 to 1:20. In clause 5,
The step of inducing senescence of fibroblasts is a step of treating fibroblasts with a DNA topoisomerase II inhibitor and insulin-like growth factor 1 for 3 to 10 days. Mixing fibroblasts with collagen to form a dermal mimetic layer;
Inducing senescence of the fibroblasts by treating the dermal mimetic layer with a DNA topoisomerase II inhibitor and insulin-like growth factor 1; And
Applying keratinocytes on the dermal mimetic layer and then culturing to form an epidermal mimetic layer
Aging artificial skin manufacturing method comprising a. In claim 9,
The DNA topoisomerase II inhibitor is a method for producing aging artificial skin that is treated at a concentration equal to or less than the insulin-like growth factor 1. In claim 9,
The DNA topoisomerase II inhibitor and insulin-like growth factor 1 is a method for producing aging artificial skin that is treated on the fibroblasts at a concentration ratio of 1:1 to 1:20. In claim 9,
The step of inducing aging of fibroblasts is a step of treating the dermal mimicking layer with a DNA topoisomerase II inhibitor and insulin-like growth factor 1 for 3 to 10 days. As a method for screening skin anti-aging substances,
Applying the skin anti-aging substance to the artificial skin of claim 1, the manufacturing method of claim 5, or the artificial skin manufactured according to the manufacturing method of claim 9, and
A method for screening skin anti-aging substances comprising the step of comparing the dermal replica layer of the artificial skin to which the skin anti-aging substance is applied and the dermal replica layer of the artificial skin to which the skin anti-aging substance is not applied.

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