KR20200031018A - Epidermis and dermis matrix similar to the real skin and artificial skin comprising the same - Google Patents

Abstract

The present invention relates to an artificial epidermal layer, an artificial dermal layer, and artificial skin comprising the same which are similar to biological skin. The present invention comprises an artificial epidermal layer having physical properties similar to those of the biological skin, an artificial dermal layer having biological properties similar to those of the biological skin, and artificial skin in which the artificial epidermal layer and the artificial dermal layer are stacked, thereby providing an artificial skin model having high human correlation.

Description

Artificial epidermis layer, artificial dermis layer and artificial skin comprising the same physically and biologically {Epidermis and dermis matrix similar to the real skin and artificial skin comprising the same}

The present invention relates to an artificial epidermal layer having physical properties similar to that of a biological skin, an artificial dermal layer having biological properties similar to a biological skin, and an artificial skin in which the artificial epidermal layer and the artificial dermal layer are stacked.

Skin acts as a barrier that protects internal organs from chemical agents such as cosmetics and surfactants, as well as biological agents such as ultraviolet rays and bacteria, so reactions such as inflammation, allergies, and hypersensitivity to these substances may occur. You can. For the above reasons, after the development of drugs used for cosmetics or skin, it is necessary to evaluate their effectiveness and safety, and the currently used evaluation method is an animal test method using animals such as rats and rabbits.

However, in recent years, many studies have been conducted on the in vitro evaluation platform that simulates human skin tissue due to a number of failures caused by the difference between the real human and animal skin and the trend of emphasizing animal ethics. It has been developed and commercialized [F. Groeber et al, Advanced drug delivery reviews 63 (4), 352-366 (2011), C.M. Reijnders et al, Tissue engineering. Part A 21 (17-18), 2448-59 (2015)].

Human skin is largely composed of epidermal and dermal layers. The epidermal layer is a thin layer constituting the outer side of the skin (about 90 μm), composed of a stratum corneum, a transparent layer, a granular layer, a polar layer, and a base layer, and composed of keratinocytes, which are cells that differentiate. Cells are constantly replaced with new cells to protect the skin from damage.

The dermal layer located beneath the epidermal layer is formed of a layer (about 1500 μm) in which the extracellular matrix such as collagen is about 15-40 times thicker than the epidermis, and includes various structures such as blood vessels and nerves. Despite having these structural features, most models are actually two-dimensional cultures using fibroblasts or keratinocytes constituting the epidermal layer, or because they mimic only the epidermis or dermis. And shows a lot of difference.

Accordingly, in the present invention, in order to improve these limitations, as a result of earnest research efforts based on the characteristic structure of the actual biological skin, the membrane has a physical and biological property similar to the membrane and dermal layer of a porous structure using a polymer having a structure and properties similar to the epidermal layer. The epidermal layer and the dermal layer simulation platform, which is a composite of a 3D tissue made of hydrogel, were completed.

Republic of Korea Registered Patent No. 1874790 Republic of Korea Registered Patent No. 1846138

An object of the present invention is to provide an artificial epidermal layer having properties similar to biological skin.

In addition, another object of the present invention is to provide an artificial dermal layer having biological properties similar to biological skin.

In addition, another object of the present invention is to provide an artificial skin having high human correlation, including the epidermal layer and the dermal layer.

In addition, another object of the present invention to provide a method for manufacturing the artificial skin.

An artificial epidermal layer similar to the bioskin of the present invention for achieving the above object is an electrospun porous biodegradable polymer; A collagen coating layer coated on the biodegradable polymer; And keratinocytes cultured with keratinocytes on the collagen coating layer.

The concentration of the keratinocytes may be 1X10 ⁵ to 5X10 ⁶ cells / cm ² , and the keratinocytes may be cultured at 32 to 39 ° C. for 7 to 28 days.

The thickness of the electrospun biodegradable polymer may be 50 to 120 μm.

The biodegradable polymer is polycaprolactone (PCL), lactide, polyglycolic acid (PGA), poly (D, L-lactic acid-co-glycolic acid) (poly (D, Llactide-co-glycolide); PLGA), Diol / acid-based aliphatic polyester, polyester-amide / polyester-urethane, poly (valerolactone), poly (hydroxy butyrate), poly (hydroxy valerate), polyanhydride, polyorthoester ( polyorthoesters), polyvinyl alcohol, polyethylene glycol, polyurethane, polyacrylic acid, and poly-N-isopropylacrylamide.

The biodegradable polymer may be dissolved in an organic solvent and electrospun.

The organic solvent may be at least one selected from the group consisting of hexafluoroisopropanol, THF (tetrahydrofuran), DMF (dimethylformamide), DMSO (dimethylsulfoxide), chloroform, dichloromethane, hexane, diethyl ether and acetonitrile. .

The electrospinning may be performed under conditions of a radiation distance of 15 to 20 cm, an injection speed of 1 to 10 ml / h and a voltage of 5 to 15 kV.

The biodegradable polymer may be post-treated with oxygen plasma after electrospinning.

In addition, the artificial dermal layer similar to the bioskin of the present invention for achieving the other object described above may be formed by artificial blood vessels by encapsulating fibroblasts and endothelial cells in a hydrogel containing collagen.

The hydrogel may be a collagen, fibrin, heparin, catechol, and one or more additional components selected from the group consisting of chitosan in a weight ratio of 1: 0.5-3.

The concentration of the mixture of the collagen and the additional component may be 1 to 10 mg / ml.

The fibroblasts and endothelial cells may be mixed in a weight ratio of 1: 0.2-5.

The concentration of the mixed cells in which the encapsulated fibroblasts and endothelial cells are mixed may be 1X10 ⁶ to 9X10 ⁷ cells / ml.

In addition, the artificial skin similar to the bioskin of the present invention for achieving another object described above is encapsulated fibroblasts and endothelial cells in a hydrogel containing collagen, a dermal layer formed with artificial blood vessels; And an epidermal layer formed on an upper surface of the dermal layer and a keratinocyte cultured keratin layer formed on collagen coated on an electrospinning porous biodegradable polymer.

In addition, the method for producing an artificial skin of the present invention for achieving the above another object is (A) electrospinning (electrospinning) a biodegradable polymer to form a membrane having a porous structure; (B) coating the surface of the membrane with collagen; (C) obtaining a layer of epidermis by culturing keratinocytes on the collagen coated membrane to form a stratum corneum; (D) forming a dermal layer having artificial blood vessels by encapsulating and stacking fibroblasts and endothelial cells on a hydrogel containing collagen; (E) laminating the surface cultured in the epidermal layer and the hydrogel of the dermal layer to contact each other; And (F) gelling the artificial skin in which the dermal layer and the epidermal layer are stacked.

After the step (F), (G) forming a storage layer on the gelled artificial skin, adding media to the storage layer, and then completely gelling the artificial skin; (H) forming a channel inside the gelled dermal layer and then coating the channel by adding endothelial cells; (I) filling the storage layer with a culture medium and culturing.

The endothelial cells may be added at a concentration of 1X10 ⁶ to 9X10 ⁷ cells / ml.

In addition, the artificial skin similar to the biological skin of the present invention for achieving the above another object is at least one additional component selected from the group consisting of collagen, fibrin, heparin, catechol and chitosan in a weight ratio of 1: 0.5-3 The mixed cells in which the fibroblasts and endothelial cells were mixed in a weight ratio of 1: 0.2-5 in a hydrogel containing a mixture of 1 to 10 mg / ml were mixed at a concentration of 1X10 ⁶ to 9X10 ⁷ cells / ml. A dermal layer formed by encapsulation to form artificial blood vessels; It includes; And epidermal layer formed on the upper surface of the dermal layer, the electrospinned porous biodegradable polymer having a thickness of 50 to 120 ㎛ treated with oxygen plasma and coated with collagen;

The epidermal layer was cultured for 15 to 25 days at 32 to 39 ° C. by adding keratinocytes to a collagen coated biodegradable polymer at a concentration of 1 X 10 ⁵ to 5 X 10 ⁶ cells / cm ² , and the electrospinning Radiation distance 15 to 20 cm, injection speed of 1 to 10 ml / h and voltage 5 to 15 kV may be performed under the conditions.

Since the artificial epidermal layer of the present invention includes a stratum corneum, it can be used as an in vitro evaluation platform for in vitro evaluation of animal experiments and an evaluation platform for wound coating.

In addition, since the artificial dermal layer of the present invention includes artificial vascular tissue, it can be used for screening new drugs and evaluating image treatment platforms through induction of skin disease models.

In addition, the artificial skin of the present invention has physical and biological properties similar to that of the biological skin, and also has artificial vascular tissue, so it can be used as wound dressing and implanted skin with improved engraftment rate and regeneration rate. In addition, when developing a new drug, it can be used not only for complex screening, but also for skin whitening verification, skin aging verification, or skin irritation verification.

In addition, since the artificial skin of the present invention has a tissue similar to that of an actual biological skin, it is possible to minimize physiological deformation or side effects due to tissue deformation.

1 is a fluorescence image showing the stratum corneum provided in the artificial epidermal layer prepared according to Examples 1 to 9 of the present invention.
2 is a cross-sectional image of the stratum corneum provided in the artificial epidermal layer prepared according to Examples 1 to 9 of the present invention by TUNEL assay.
Figure 3a is a photograph taken by SEM of the surface and cross-section of the membrane used in the artificial epidermal layer prepared according to an embodiment of the present invention, Figure 3b is a fluorescent image of the membrane coated with the surface of the membrane of Figure 3a, collagen, Figure 3c is a fluorescent image of the surface and cross-section of keratinocytes cultured for 5 days on a membrane coated with collagen.
Figure 4A is a photograph showing the form of the hydrogel used in the artificial dermal layer of the present invention, Figure 4B is a graph showing the elastic modulus of the hydrogel used in the artificial dermal layer of the present invention, Figure 4C is a figure showing the figure 4B It is a ticket.
Figure 5A is a fluorescent image showing the cell viability of fibroblasts (HDFs) in artificial dermal layer tissue prepared in Preparation Example (green = live cells, red = dead cells), Figure 5B quantitatively shows the cell viability of Figure 5A It is the graph shown.
6A is a fluorescence image showing the degree of artificial blood vessel formation in an artificial dermal layer prepared according to an embodiment of the present invention (1 day of culture), and FIG. 6B shows the degree of artificial blood vessel formation in an artificial dermal layer prepared according to an embodiment of the present invention. It is a fluorescent image shown (4 days of culture), Figure 6C is a fluorescent image showing the degree of artificial blood vessel formation of the artificial dermal layer prepared according to an embodiment of the present invention (culture 7 days), Figure 6D is the artificial observed in Figure 6C It is a graph measuring the length of the blood vessel, and FIG. 6E is a graph measuring the area of the artificial blood vessel observed in FIG. 6C.
7 is a schematic view showing a process of manufacturing an artificial skin according to an embodiment of the present invention.
8A is a fluorescence image showing artificial vascularized tissue (RFP-HUVECs), dermal layer (HDF) and epidermal layer (HaCaT) of artificial skin prepared according to an embodiment of the present invention, and FIG. 8B is according to an embodiment of the present invention. It is a fluorescence image showing the cross-sectional area of the manufactured artificial skin.
9A is an image showing a cross-section of FITC-Toner on an artificial skin manufactured according to an embodiment of the present invention, and FIG. 9B is an FITC after processing FITC-Toner on an artificial skin manufactured according to an embodiment of the present invention. -This is an image showing the penetration depth of the toner.

The present invention relates to an artificial epidermal layer having physical properties similar to that of a biological skin, an artificial dermal layer having biological characteristics similar to a biological skin, and an artificial skin in which the artificial epidermal layer and the artificial dermal layer are stacked.

Hereinafter, the present invention will be described in detail.

Artificial epidermal layer

The artificial epidermal layer of the present invention includes an electrospun porous biodegradable polymer; A collagen coating layer coated on the biodegradable polymer; And a stratum corneum cultured with keratinocytes on the collagen coating layer. In particular, the artificial epidermal layer of the present invention simulates physical properties similar to biological skin using an electrospun porous biodegradable polymer, and uses a keratinous layer to simulate biological properties similar to biological skin.

The artificial epidermal layer is formed by treating a biodegradable polymer with electrospinning under a specific condition to produce a porous membrane having a thickness of 50 to 120 µm, preferably 80 to 100 µm, and having a small pore size, thereby providing a compact structure of the actual epidermal layer. To simulate. At this time, the average particle diameter of the pores is 0.01 to 0.04 mm.

The biodegradable polymer is dissolved in an organic solvent and electrospun, wherein the organic solvent is hexafluoroisopropanol (THF), tetrahydrofuran (THF), dimethylformamide (DMF), dimethylsulfoxide (DMSO), chloroform, dichloromethane, hexane, di It is preferable to use one or more selected from the group consisting of ethyl ether and acetonitrile to prepare a membrane having a desired thickness and pore size.

The electrospinning distance is 15 to 20 cm, preferably 16 to 18 cm; Injection rate 1 to 10 ml / h, preferably 2 to 5 ml / h; And a voltage of 5 to 15 kV, preferably 8 to 12 kV. When the radiation distance, the injection speed and the voltage during electrospinning are less than the lower limit of the preferred range, the membrane may not be formed in a porous structure, and the thickness may not be thin enough to simulate real skin, and the upper limit is exceeded. In this case, not only a membrane having a low hardness is provided, but the pore size is also large, and thus the structure of the actual epidermal layer cannot be simulated.

The biodegradable polymer according to the present invention is treated with electrospinning and then treated with oxygen plasma to increase the adhesion accuracy of keratinocytes, increase the adhesion amount of keratinocytes, and increase the adhesion retention time due to an increase in surface activity. When the biodegradable polymer is directly treated with oxygen plasma instead of electrospinning, the adhesion rate of keratinocytes is lower than that treated with oxygen plasma after electrospinning, and unlike actual skin, keratinocytes are not cultured in the epidermal layer. It may not.

In addition, collagen is coated on the surface of the biodegradable polymer treated with oxygen plasma after electrospinning or electrospinning to improve the adhesion rate of keratinocytes. The collagen is coated by an EDC / NHS chemical method, specifically, N- (3-dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride (EDC) in the presence of N-hydroxysuccinimide (NHS) is used. Thereby chemically coated.

The collagen-coated biodegradable polymer is cultured by adding keratinocytes at a concentration of 1X10 ⁵ to 5X10 ⁶ cells / cm ² , preferably 7X10 ⁵ to 2X10 ⁶ cells / ml. When the concentration of keratinocytes is outside a desired range, an stratum corneum layer is not formed, and thus an artificial epidermal layer biologically similar to an actual epidermal layer cannot be formed.

In addition, the keratinocytes are cultured at 32 to 39 ° C, preferably 35 to 37 ° C for 7 to 28 days, preferably 21 to 28 days. When the culture temperature and time of keratinocytes are less than the lower limit of the preferred range, the stratum corneum may not be formed, and when it exceeds the upper limit, the stratum corneum may be easily removed.

The biodegradable polymer includes polycaprolactone (PCL), lactide, polyglycolic acid (PGA), and poly (D, L-lactic acid-co-glycolic acid) (poly (D, Llactide-co-glycolide); PLGA) , Diol / Esidic aliphatic polyester, polyester-amide / polyester-urethane, poly (valerolactone), poly (hydroxy butyrate), poly (hydroxy valerate), polyanhydride, polyorthoester (polyorthoesters), polyvinyl alcohol, polyethylene glycol, polyurethane, polyacrylic acid, and one or more selected from the group consisting of poly-N-isopropylacrylamide, preferably, polycaprolactone (PCL) and polylactide It may be a mixture of, and more preferably, a mixture of polycaprolactone (PCL) and lactide 1: 1-5 in a weight ratio, more preferably polycaprolactone (PCL) and lactide 1: 1 Can be a mixture mixed in a weight ratio of -2 The. When the biodegradable polymer according to the present invention satisfies the above-mentioned preferred range, a membrane having a desired thickness and pore size can be prepared, thereby providing an epidermal mimetic having similar tissue, physical and biological properties to the actual epidermal layer. can do.

Artificial dermal layer

The artificial dermal layer of the present invention is an artificial blood vessel formed by encapsulating fibroblasts and blood vessels constituting human dermis into a hydrogel containing collagen.

The hydrogel is a mixture of collagen, fibrin, heparin, catechol, and one or more selected from the group consisting of chitosan in a weight ratio of 1: 0.5-3, preferably 1: 1-2. When the content of at least one selected from the group consisting of fibrin, heparin, catechol, and chitosan based on collagen is less than the lower limit of the preferred range, physical properties and biological properties similar to the actual dermal layer cannot be exhibited. Physical properties may be lower than that of the dermal layer.

The concentration of the mixture of one or more additional ingredients selected from the group consisting of collagen, fibrin, heparin, catechol, and chitosan is 1 to 10 mg / ml, preferably 3 to 5 mg / ml. When the concentration of the mixture is less than the lower limit of the preferred range, a dermal layer having properties similar to the actual dermal layer cannot be obtained, and when it exceeds the upper limit, the survival of dermal cells contained in the dermal layer may be difficult.

The fibroblasts can be used with the hydrogel to exert physical and biological properties similar to the actual dermal layer, and the endothelial cells form artificial blood vessels to secure cell viability in the long term and are used with fibroblasts to achieve biological properties. I can show it more.

The fibroblasts and endothelial cells are encapsulated together and stacked on the hydrogel. Fibroblasts and endothelial cells are encapsulated by mixing in a weight ratio of 1: 0.2-5, preferably in a weight ratio of 1: 0.3-3. In the case of deviating from the above-mentioned preferred range of endothelial cells based on fibroblasts, blood vessels are not formed, and the contraction of the artificial dermal layer is not suppressed, thereby ensuring the stability of the model, thereby minimizing physiological or side effects due to tissue deformation. Not only is there no side effects, such as allergic reactions.

 The artificial dermal layer of the present invention has a cross-sectional area reduction rate of 25% or less, preferably 15% or less, more preferably 11% or less, based on 100% of the initial dermal layer cross-sectional area, which enables more stable in vitro culture for a long time. In addition, it can suppress scars when applied in vivo for wound healing.

In addition, the concentration of the mixed cells in which the encapsulated fibroblasts and endothelial cells are mixed is 1X10 ⁶ to 9X10 ⁷ cells / ml, preferably 2X10 ⁶ to 9X10 ⁶ cells / ml. When the concentration of the mixed cells is outside the above-mentioned preferred range, angiogenesis is not promoted, and the penetration level of the agent is low, and when used as an implanted skin, blood supply to the wound is not smooth and adheres to the actual skin. This can be difficult.

In addition, the present invention can be vascularized only when fibroblasts and endothelial cells are mixed in a weight ratio of 1: 1-3 and the mixed cells have a concentration of 2X10 ⁶ to 9X10 ⁶ cells / ml.

The artificial skin of the present invention is formed including the epidermal and dermal layers.

In addition, the present invention is to provide a method for producing artificial skin having similar physical properties to biological skin.

The method of manufacturing the artificial skin of the present invention comprises the steps of (A) electrospinning a biodegradable polymer to form a membrane having a porous structure; (B) coating the surface of the membrane with collagen; (C) obtaining a layer of epidermis by culturing keratinocytes on the collagen-coated membrane to form a stratum corneum; (D) forming a dermal layer having artificial blood vessels by encapsulating and stacking fibroblasts and endothelial cells on a hydrogel containing collagen; (E) laminating the surface cultured in the epidermal layer and the hydrogel of the dermal layer to contact each other; And (F) gelling the artificial skin in which the dermal layer and the epidermal layer are stacked.

In addition, after the step (F), (G) forming a storage layer on the gelled artificial skin, adding a medium to the storage layer, and then completely gelling the artificial skin; (H) forming a channel inside the gelled dermal layer and then coating the channel by adding endothelial cells; (I) filling the storage layer with a culture medium and culturing.

First, in steps (A) and (B), the biodegradable polymer is dissolved in an organic solvent, and then electrospinning to form a membrane having a thin porous structure, and then treating the membrane with oxygen plasma, followed by The surface is coated with collagen.

Next, in step (C), the epidermal layer is obtained by spraying keratinocytes on the collagen-coated membrane and incubating at 32 to 39 ° C for 7 to 28 days to form a stratum corneum.

Next, in step (D), the mixed cells in which fibroblasts and endothelial cells encapsulated on a hydrogel containing one or more selected from the group consisting of collagen, fibrin, heparin, catechol, and chitosan are mixed. Laminates form a dermal layer.

Next, in the steps (E) and (F), the surfaces cultured in the epidermal layer and the hydrogels of the dermal layer were laminated so as to contact each other, and then gelled at 35 to 40 ° C. for 10 to 50 minutes.

Next, in steps (G) and (H), a storage layer is formed on the gelled artificial skin, media is added to the storage layer, and then gelled at 35 to 40 ° C. for 20 to 26 hours to completely gel the artificial skin. After forming a channel inside the gelled dermal layer and adding the endothelial cells having a concentration of 1X10 ⁶ to 9X10 ⁷ cells / ml, preferably 6X10 ⁶ to 9X10 ⁶ cells / ml, to the channel to rotate for 0.5 to 2 hours. After coating the channel with endothelial cells, the storage layer is filled with a culture solution and incubated at 35 to 40 ° C for 20 to 26 hours.

In particular, the artificial skin of the present invention is a mixture of collagen, fibrin, heparin, catechol, and one or more additional components selected from the group consisting of chitosan in a weight ratio of 1: 0.5-3 in a concentration of 1 to 10 mg / ml A dermal layer encapsulated in a concentration of 1X10 ⁶ to 9X10 ⁷ cells / ml of mixed cells in which fibroblasts and endothelial cells are mixed in a weight ratio of 1: 0.2-5 to a hydrogel containing; It includes; And epidermal layer formed on the upper surface of the dermal layer, the electrospinned porous biodegradable polymer having a thickness of 50 to 120 ㎛ treated with oxygen plasma and coated with collagen;

The epidermal layer is cultured by adding epidermal cells to a collagen-coated biodegradable polymer at a concentration of 1X10 ⁷ to 9X10 ⁸ cells / ml, and the electrospinning distance is 15 to 20 cm, and the injection rate is 1 to 10 ml / h. And a voltage of 5 to 15 kV. When all of the above conditions are satisfied at the same time, biological characteristics that are more similar to actual skin are exhibited compared to when all of the above conditions are not satisfied at the same time or when only a part of all the conditions are satisfied. Since it is more similar to the actual skin structure, the human correlation can be further improved, resulting in more accurate results when used in experiments.

Hereinafter, a preferred embodiment is provided to help the understanding of the present invention, but the following examples are merely illustrative of the present invention, and it is apparent to those skilled in the art that various changes and modifications are possible within the scope and technical scope of the present invention. It is no wonder that such variations and modifications fall within the scope of the appended claims.

[art Epidermal layer]

Examples 1 to 9: Preparation of artificial epidermal layer

After dissolving PLCL having a weight ratio of 50:50 of L-Lactide and ε-Caprolactone in 9% by weight in 1,1,1,3,3,3-Hexafluoroisopropanol (HFIP), the radiation distance is 18 cm, injection A membrane having a thickness of 100 μm was fabricated by electrospinning at a rate of 3 ml / h and a voltage of 9 kV, and this was cut into a size of 1 X 1 cm. After the oxygen plasma treatment (CUTE-B system, FEMTO Science, Korea) was performed on the prepared membrane, collagen type I was coated with a covalent bond using an EDC / NHS chemical method (ccPLCL-NFM). The HaCaT cells in the keratin cells on the membrane is the collagen-coated with each of 4X10 ⁵ cells / cm ^2, a concentration of 7X10 ⁵ cells / cm ² and 10X10 ⁵ cells / cm ² seeded (seeding) by 1, 2, and 3 weeks after haejun While incubating at 37 ° C in an incubator to form a stratum corneum, an artificial epidermal layer with a stratum corneum was obtained.

Test Example 1: Fluorescence image and TUNEL analysis image of the stratum corneum provided on the artificial epidermal layer

1 is a fluorescent image showing the stratum corneum provided in the artificial epidermal layer prepared according to Examples 1 to 9 of the present invention, Figure 2 TUNEL the stratum corneum provided in the artificial epidermal layer prepared according to Examples 1 to 9 of the present invention This is a cross-sectional image performed by assay.

1 and 2, it was confirmed that as the concentration of HaCaT cells increased, the number of HaCaT cells attached to ccPLCL-NFM increased and proliferation increased. It was confirmed that the stratum corneum was formed. When cultured for 1 or 2 weeks, the stratum corneum was not formed.

In the present invention, it was confirmed that HaCaT cells were seeded at a concentration of 10 × ^{10 5} cells / cm ² to obtain a desired stratum corneum when cultured for 3 weeks.

Test Example 2: SEM imaging

Figure 3a is a photograph taken by SEM of the surface and cross-section of the membrane used in the artificial epidermal layer prepared according to an embodiment of the present invention, Figure 3b is a fluorescent image of the membrane coated with the surface of the membrane of Figure 3a, collagen, Figure 3c is a fluorescent image of the surface and cross-section of keratinocytes cultured for 5 days on a membrane coated with collagen.

3A to 3C, it was confirmed that the membrane used for the artificial epidermal layer of the present invention is porous and has a thickness of 100 μm, which is similar to that of the human epidermal layer, and the entire front and interior of the collagen coated membrane. It was confirmed that the keratinocytes were cultured over.

[art Dermis layer]

Preparation Examples 1 to 6: Preparation of artificial dermal layer (omit endothelial cells)

Collagen and fibrin at a concentration of 3 mg / ml and 5 mg / ml, respectively, in a PDMS chip with two 500 µm-diameter tubes in a weight ratio of 25:75, a weight ratio of 50:50, and a weight ratio of 75:25 (collagen: fibrin) After mixing with), the skin fibroblast HDF was encapsulated at a concentration of 1X10 ⁶ cells / ml to measure the elastic modulus, and cultured at 37 ° C for 1 and 7 days to confirm cell viability.

Test Example 3: Measurement of the elastic modulus of the hydrogel

Figure 4A is a photograph showing the form of a hydrogel used in the dermal layer, Figure 4B is a graph showing the elastic modulus (using universal testing machine (UTM)) of the hydrogel used in the dermal layer, Figure 4C is a numerical value of Figure 4B It is shown in the table. * Indicates p <0.05 for the collagen50 / fibrin50 group, ** indicates p <0.001 for the collagen50 / fibrin50 group.

4A to 4C, the shape and elastic modulus of the hydrogel were confirmed to be excellent in mixing collagen and fibrin at a concentration of 3 mg / ml and 5 mg / ml in a weight ratio of 50:50 and a weight ratio of 75:25. Did.

Test Example 4: Measurement of the cell viability of the hydrogel

Figure 5A is a fluorescent image showing the cell viability of fibroblasts (HDFs) in the dermal layer tissue prepared in Preparation Example (green = live cells, red = dead cells), Figure 5B quantitatively shows the cell viability of Figure 5A It is a graph. Scale bar = 50 μm. * Is collagen 50 / fibrin 50, p <0.05 for day 1 group, + is collagen 50 / fibrin 50, p <0.05 for day 7 group, ++ is collagen 50 / fibrin 50, p <0.001 for day 7 group Indicates that

As shown in Figures 5A to 5B, it was confirmed that the viability of fibroblasts at a concentration ratio of 3 mg / ml and fibrin at a weight ratio of 50:50 is maintained at 90% or more even after 1 and 7 days of culture.

That is, it was confirmed that the hydrogel in which the collagen and fibrin at a concentration of 3 mg / ml was mixed in a weight ratio of 50:50 was excellent.

Vascularization was not observed in the dermal layer prepared in the preparation example.

Example  10 to 18: Artificial dermal layer production (including endothelial cells)

HDF, a skin fibroblast tagged with Vybrant CFDA SE Cell Tracer Kit on 3 mg / ml collagen / fibrin hydrogel (collagen: fibrin = 50:50 weight ratio) on a PDMS chip with two 500 μm diameter tubes And red fluorescent protein expressing human umbilical vein endothelial cells (RFP-HUVECs), a type of endothelial cell, with a weight ratio of 25:75, a weight ratio of 50:50 and a weight ratio of 75:25 (HDF: RFP-HUVECs) of 2X10 ⁶ Encapsulation to a concentration of cells / ml and incubation for 1, 4 and 7 days.

Test example  5: In dermal layer tissue Angiogenic ability  Measure

Figure 6A is a fluorescent image showing the degree of artificial blood vessel formation of the dermal layer prepared according to an embodiment of the present invention (1 day of culture), Figure 6B is a fluorescence showing the degree of artificial blood vessel formation of the dermal layer prepared according to an embodiment of the present invention It is an image (4 days of culture), Figure 6C is a fluorescent image showing the degree of artificial blood vessel formation of the dermal layer prepared according to an embodiment of the present invention (7 days of culture), Figure 6D is the length of the artificial blood vessel observed in Figure 6C Is a graph measuring, and FIG. 6E is a graph measuring the area of the artificial blood vessel observed in FIG. 6C. red = endothelial cells (HUVECs), green = fibroblasts (HDFs), Scale bar = 200 μm. * Indicates p <0.05, ** indicates p <0.001. The isolated branch represents the immature vascular structure, the segment represents the mature vascular structure, and the capillary-like tubular structure area represents the stable vascular network structure.

6A to 6C, HDF and RFP-HUVECs are 2X10 ^{6 in} a weight ratio of 50:50. When the cells / ml concentration was encapsulated to a concentration, after 7 days of culture, it was confirmed that the new artificial vascular structures were well formed and both vascular endothelial cells (RFP-HUVECs) and fibroblasts (HDFs) survived well.

In addition, as shown in FIGS. 6D to 6E, it was confirmed that when the weight ratio of HDF: RFP-HUVECs = 25: 75 is a mature artificial vascular structure, there are many segments and a large artificial vascular area. However, it was confirmed that the degree of formation of dermal structure was insufficient compared to the case of HDF: RFP-HUVECs = 75: 25 and 50:50.

[Artificial skin]

Example  19: Artificial skin manufacturing

Artificial epidermal layer

After dissolving PLCL having a weight ratio of 50:50 of L-Lactide and ε-Caprolactone in 9% by weight in 1,1,1,3,3,3-Hexafluoroisopropanol (HFIP), the radiation distance is 18 cm, injection A membrane having a thickness of 100 μm was fabricated by electrospinning at a rate of 3 ml / h and a voltage of 9 kV, and this was cut into a size of 1 X 1 cm. After the oxygen plasma treatment (CUTE-B system, FEMTO Science, Korea) was performed on the prepared membrane, collagen type I was coated with a covalent bond using an EDC / NHS chemical method (ccPLCL-NFM). HaCaT cells, keratinocytes, were seeded on the collagen-coated membrane at a concentration of 10X10 ⁵ cells / cm ² , and then cultured in an incubator at 37 ° C. for 3 weeks to form an artificial epidermal layer.

Artificial dermal layer

The collagen / fibrin hydrogel (collagen: fibrin = 50:50 weight ratio) at a concentration of 3 mg / ml was added to the PDMS chip with two 500 μm-diameter tubes, and the skin fibroblast HDF and endothelial cell RFP-HUVECs were 50. 2X10 ^{6 at} a weight ratio of 50 An artificial dermal layer was formed by encapsulation to a concentration of cells / ml and incubation for 7 days.

Artificial skin

The prepared epidermal layer is placed on the hydrogel of the dermal layer, so that the cultured surface of the epidermal layer makes contact with the hydrogel. Thereafter, gelation was carried out in an incubator at 37 ° C for 30 minutes, and when the hydrogel gelled, media was added to a reservoir and incubated for 24 hours in a 37 ° C incubator for complete gelation of the hydrogel. When the hydrogel is completely gelled, remove the 500 µm diameter tube that has been inserted to make a channel inside the hydrogel, seed HUVECs at a concentration of 7 x 10 ⁶ cells / ml in the channel, and rotate for 1 hour. After coating on the channel, the storage layer was filled with the culture solution and cultured in a 37 ° C incubator to obtain artificial skin (FIG. 7).

In the present invention, blood vessels naturally form capillaries such as capillaries by endothelial cells contained inside the dermis layer, and the blood vessels extend into the dermis layer as if the branches extend from the micro-sized vessels simulated through the channels to form blood vessels. .

Test example  6: Fluorescence image of artificial skin

8A is a fluorescence image showing vascularized tissue (RFP-HUVECs), dermal layer (HDF) and epidermal layer (HaCaT) of artificial skin prepared according to an embodiment of the present invention, and FIG. 8B is prepared according to an embodiment of the present invention This is a fluorescence image showing the cross-sectional area of artificial skin (red = vascularized tissue, green = dermal tissue, blue = epidermal tissue).

8A and 8B, it was confirmed that the skin-like complex tissue of the human body in which the blood vessels, the dermis and the capillaries in the dermis and the epidermal tissue are complexed is well formed and maintained during the culture period.

Test example  7: Toner test

Figure 9A is an image showing a cross-section treated with FITC-Toner on artificial skin prepared according to an embodiment of the present invention (red: HUVEC tagged with CM-Dil, blue: HaCaT tagged with CMAC, green: FITC-Toner ), Figure 9B is an image showing the penetration depth of the FITC-Toner after processing FITC-Toner on the artificial skin prepared according to an embodiment of the present invention. Scale bars = 200 μm (FIG. 9A), 50 μm (FIG. 9B).

9A and 9B, as a result of processing FITC-Toner to check the absorption rate of the actual cosmetic in the artificial epidermal layer prepared according to the embodiment, most of the FITC-Toner is distributed in the epidermal layer and in the vascular layer It was confirmed that it was hardly found. This is a result of showing that the artificial skin layer of the developed artificial skin acts as a barrier similar to that of the actual skin, so that the toner is not absorbed into the subcutaneous tissue in which blood vessels exist. It can be said that it has potential as a safety evaluation platform.

Claims (19)

An electrospun porous biodegradable polymer;
A collagen coating layer coated on the biodegradable polymer; And
An artificial epidermal layer comprising; a stratum corneum cultured with keratinocytes on the collagen coating layer. The artificial epidermal layer according to claim 1, wherein the concentration of the keratinocytes is 1X10 ⁵ to 5X10 ⁶ cells / cm ² . The artificial epidermal layer according to claim 1, wherein the keratinocytes are cultured at 32 to 39 ° C for 7 to 28 days. The artificial epidermal layer according to claim 1, wherein the thickness of the electrospun biodegradable polymer is 50 to 120 μm. The method of claim 1, wherein the biodegradable polymer is polycaprolactone (PCL), lactide, polyglycolic acid (PGA), poly (D, L-lactic acid-co-glycolic acid) (poly (D, Llactide-co- glycolide); PLGA), diol / acid-based aliphatic polyester, polyester-amide / polyester-urethane, poly (valerolactone), poly (hydroxy butyrate), poly (hydroxy valerate), polyanhydride Artificial skin layer characterized by at least one selected from the group consisting of lead, polyorthoesters, polyvinyl alcohol, polyethylene glycol, polyurethane, polyacrylic acid and poly-N-isopropylacrylamide. The artificial epidermal layer according to claim 1, wherein the biodegradable polymer is dissolved in an organic solvent and electrospun. The method of claim 6, wherein the organic solvent is hexafluoroisopropanol (Hexafluoroisopropanol), THF (tetrahydrofuran), DMF (dimethylformamide), DMSO (dimethylsulfoxide), chloroform, dichloromethane, hexane, diethyl ether and acetonitrile in the group consisting of Artificial epidermal layer, characterized in that at least one selected. The artificial epidermal layer according to claim 1, wherein the electrospinning is performed under conditions of a radiation distance of 15 to 20 cm, an injection speed of 1 to 10 ml / h, and a voltage of 5 to 15 kV. The artificial epidermal layer according to claim 1, wherein the biodegradable polymer is post-treated with oxygen plasma after electrospinning. An artificial dermal layer characterized by forming an artificial blood vessel by encapsulating fibroblasts and endothelial cells in a hydrogel containing collagen. The artificial dermal layer of claim 1, wherein the hydrogel is a mixture of collagen, fibrin, heparin, catechol, and chitosan and at least one additional component selected in a weight ratio of 1: 0.5-3. The artificial dermal layer according to claim 10, wherein the concentration of the mixture of the collagen and the additional component is 1 to 10 mg / ml. 11. The bio-artificial dermal layer according to claim 10, wherein the fibroblasts and endothelial cells are mixed in a weight ratio of 1: 0.2-5. The artificial dermal layer according to claim 10, wherein the concentration of the mixed cells in which the encapsulated fibroblasts and endothelial cells are mixed is 1X10 ⁶ to 9X10 ⁷ cells / ml. An artificial skin comprising an artificial dermal layer and an artificial epidermal layer,
The dermal layer is an artificial blood vessel formed by encapsulating fibroblasts and endothelial cells in a hydrogel containing collagen;
The epidermal layer is formed on an upper surface of the dermal layer, and includes an electrospun porous biodegradable polymer, a collagen coating layer coated on the biodegradable polymer, and a keratinocyte cultured keratinocyte on the collagen coating layer. Artificial skin, characterized in that. (A) electrospinning the biodegradable polymer to form a membrane having a porous structure;
(B) coating the surface of the membrane with collagen;
(C) obtaining a layer of epidermis by culturing keratinocytes on the collagen coated membrane to form a stratum corneum;
(D) forming a dermal layer having artificial blood vessels by encapsulating and stacking fibroblasts and endothelial cells on a hydrogel containing collagen;
(E) laminating the surface cultured in the epidermal layer and the hydrogel of the dermal layer to contact each other; And
(F) gelling the artificial skin in which the dermal layer and the epidermal layer are laminated; a method for manufacturing an artificial skin comprising a. 17. The method of claim 16, wherein after step (F), (G) forming a storage layer on the gelled artificial skin, adding media to the storage layer, and then completely gelling the artificial skin;
(H) forming a channel inside the gelled dermal layer and then coating the channel by adding endothelial cells;
(I) filling the storage layer with a culture solution and culturing; artificial skin manufacturing method comprising a. 18. The method of claim 17, wherein the endothelial cells are added at a concentration of 1X10 ⁶ to 9X10 ⁷ cells / ml. Fibroblasts in a hydrogel containing a mixture of collagen, fibrin, heparin, catechol, and one or more additional components selected from the group consisting of chitosan in a weight ratio of 1: 0.5-3 at a concentration of 1 to 10 mg / ml. And a dermal layer in which artificial blood vessels are formed by encapsulating mixed cells in which endothelial cells are mixed at a weight ratio of 1: 0.2-5 at a concentration of 1X10 ⁶ to 9X10 ⁷ cells / ml; And
It is formed on the upper surface of the dermal layer, the thickness of 50 to 120 ㎛ electrospinned porous biodegradable polymer layer treated with oxygen plasma and coated with collagen; includes,
The epidermal layer was cultured for 15 to 25 days at 32 to 39 ° C. by adding keratinocytes to a collagen coated biodegradable polymer at a concentration of 1 X 10 ⁵ to 5 X 10 ⁶ cells / cm ² , and the electrospinning Artificial skin characterized in that it is performed under the conditions of a radiation distance of 15 to 20 cm, an injection rate of 1 to 10 ml / h and a voltage of 5 to 15 kV.

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