WO2022131407A1 - Dermis-based artificial skin comprising basement membrane layer and manufacturing method therefor - Google Patents

Abstract

The present invention relates to a dermis-based artificial skin comprising a basement membrane layer and a manufacturing method therefor. The acellular dermis comprising the basement membrane layer according to the present invention comprises the basement membrane layer that facilitates adhesion of skin-derived cells within a structure of the acellular dermis, and has a preserved dermal layer structure. Thus, additional coating of animal-derived extracellular matrix is not required, skin-derived cells can be applied and cultured/differentiated under feeder-cell-free, xeno-free, and serum-free culturing factors, and also clinically, safety can be provided. In addition, an artificial skin manufactured by culturing skin-derived cells on an artificial skin support enables tissues to be regenerated safely and effectively when the artificial skin is grafted onto a skin defect.

Description

Dermis-based artificial skin including basement membrane layer and manufacturing method thereof

The present invention relates to a dermis-based artificial skin including a basement membrane layer and a method for manufacturing the same.

More specifically, the present invention minimizes the retention of cells and immune response-inducing factors in the epidermis and dermal layers, while preserving the basement membrane layer and includes a basement membrane layer that can be applied as artificial skin, and the cell-free dermis It relates to artificial skin prepared by culturing skin-derived cells in the dermis.

The skin is the largest organ in the human body and plays a primary role in defense against external stimuli and the risk of infection. The skin consists of three layers: the epidermis, dermis, and hypodermis. It is an important complex institution that carries out

As the need for replacement skin increases in the field of cosmetic surgery and reconstructive surgery for skin ulcers caused by extensive burns, diabetes, or skin damage and skin tissue dents due to accidents, trauma, aging, etc., a living body that can replace the skin The development of materials and artificial skin has been rapidly progressing, and recently, its application is expanding beyond this to alternative tests for animal testing in cosmetic development or toxicity tests for compounds used in new drug development.

Artificial skin is a form in which the dermis substitute and the stratum corneum existing thereon are differentiated. It is also involved in the wound healing process by controlling the formation of the epidermis. Therefore, artificial skin including a basement membrane layer is essential for skin regeneration and alternative tests using artificial skin.

The currently applied skin substitute is a form of directly applying biocompatible synthetic polymers such as PLGA and bio-derived degradable coating materials such as donated de-epidermal dermis, collagen, gelatin and small intestinal submucosa, or skin-derived collagen or polymer sponge. It is a form in which artificial skin cultured with cells is applied.

Collagen is a protein that occupies most of the dermal layer of the skin, and although it is marketed in various types of products, the price is high due to difficulties in the manufacturing process. There is a disadvantage that the dermal layer cannot be implemented as it is.

In addition, when manufacturing a substitute material or artificial skin in which skin-derived cells are cultured, animal-derived extracellular matrix is coated to form a basement membrane layer and cell adhesion, or culture elements containing animal-derived components (feeder cells, bovine serum, pig trypsin, etc.) There may be problems with safety such as contamination with adventitious viruses and mycoplasma by culturing cells.

In order to solve the above-mentioned limitations of the existing materials, in the present invention, the structure of the dermal layer including the basement membrane layer and the extracellular matrix, which facilitates the attachment of keratinocytes from the donor skin, is preserved, and an immune response can be generated. A cell-free dermis including the removed basement membrane layer is prepared. In addition, by applying skin-derived cells to the acellular dermis including the basement membrane layer and performing complex culture, a full-thickness artificial skin in which both the dermis and the epidermis exist is provided.

[Prior art literature]

[Patent Literature]

1. Korean Patent No. 10-0791502

An object of the present invention is to provide a dermis-based artificial skin including a basement membrane layer and a method for manufacturing the same.

More specifically, the present invention provides a cell-free dermis comprising a basement membrane layer that can be applied as artificial skin by minimizing the residual of cells and immune response-inducing factors in the epidermis and dermal layers, while preserving the basement membrane layer, and a method for manufacturing the same. intended to provide

In addition, the present invention provides feeder cell-free, xeno-free and serum-free formulations without coating of other animal-derived extracellular matrix on the cell-free dermis including the freeze-dried basement membrane layer. An object of the present invention is to provide an artificial skin prepared by applying skin-derived cells to a culture element and a method for manufacturing the same.

The present invention comprises the steps of culturing after seeding fibroblasts in the dermal direction of the acellular dermis including the basement membrane layer; and

Provided is a method for manufacturing a dermis-based artificial skin including a basement membrane layer, comprising the step of seeding keratinocytes on the basement membrane layer of the acellular dermis including the basement membrane layer, followed by culturing and differentiation.

In addition, the present invention is manufactured by the method for manufacturing artificial skin described above,

an acellular dermal scaffold comprising a basement membrane layer comprising fibroblasts; and

It provides a dermis-based artificial skin including a basement membrane layer formed on the basement membrane layer of the acellular dermis including the basement membrane layer, and composed of an epidermis in which keratinocytes are cultured and differentiated.

The cell-free dermis including the basement membrane layer according to the present invention includes a basement membrane layer that facilitates adhesion of skin-derived cells in its structure and preserves the structure of the dermal layer. Therefore, application and culture of skin-derived cells in culture elements of feeder cell-free, xeno-free and serum-free without requiring additional coating of animal-derived extracellular matrix. / Differentiation is possible and clinically safe can be given.

In addition, the artificial skin prepared by culturing skin-derived cells on the artificial skin support can safely and effectively regenerate tissue when transplanted into a skin defect.

1 shows a process of manufacturing the cell-free dermis including the basement membrane layer having a thin thickness.

2 shows the results of confirming cell proliferation by culturing human-derived fibroblasts (HDF) in acellular dermis including basement membrane layers having different thicknesses.

3(a) shows the results of confirming cell proliferation by culturing fibroblasts in the acellular dermis including the basement membrane layer and the acellular dermis not including the basement membrane layer.

3(b) and (c) show the results of confirming cell viability by period by culturing fibroblasts in the acellular dermis (B) including the basement membrane layer and the acellular dermis (C) not including the basement membrane layer.

4(a) shows the results of confirming cell proliferation by culturing keratinocytes (HEK) in the acellular dermis including the basement membrane layer and the acellular dermis not including the basement membrane layer.

Figure 4 (b) shows the results of confirming cell viability by period by culturing keratinocytes after coating CTS and collagen on the acellular dermis including the basement membrane layer.

Figure 4 (c) shows the results of seeding and culturing different numbers of keratinocytes in the acellular dermis including the basement membrane layer, and confirming the cell survival for each period.

5 shows the results of culturing human skin-derived keratinocytes and fibroblasts together in the acellular dermis including the basement membrane layer and proceeding with skin differentiation, and FIG. 6 shows the differentiation results through histological analysis.

The present invention comprises the steps of: A) seeding fibroblasts in the dermal direction of the acellular dermis including the basement membrane layer and then culturing; and

B) It relates to a method for manufacturing a dermis-based artificial skin including a basement membrane layer, comprising the step of seeding and culturing keratinocytes on the basement membrane layer of the acellular dermis including the basement membrane layer.

In an embodiment of the present invention, feeder cell free, xeno free and serum free without coating of animal-derived extracellular matrix on the cell-free dermis including the basement membrane layer prepared by the manufacturing method according to the present invention. By seeding and culturing skin-derived fibroblasts and/or keratinocytes in serum-free culture elements, the acellular dermis including the basement membrane layer has compatibility and affinity with skin-derived cells, making it suitable for artificial skin. was confirmed.

Hereinafter, a method for manufacturing a dermis-based artificial skin (hereinafter, referred to as artificial skin) including a basement membrane layer according to the present invention will be described in more detail.

In the present invention, the order of performing step A) and step B) is not limited, and step A) and step B) may be performed sequentially, or step B) and step A) may be performed sequentially, and also, step A ) and B) can be performed simultaneously.

In the present invention, artificial skin can be prepared by culturing and/or differentiating skin-derived cells in the acellular dermis including the basement membrane layer.

In the present invention, the acellular dermis including the basement membrane layer is prepared by delipidation and decellularization of the dermis including the basement membrane layer, and is an acellular dermal matrix (ADM).

The acellular dermis including the basement membrane layer may be applied to the same field as the general acellular dermis, and in the present invention may be applied to artificial skin, which is a skin substitute. Specifically, the acellular dermis including the basement membrane layer performs the role of the dermis in artificial skin, and may serve as a support for culturing and/or differentiation of skin-derived cells.

The cell-free dermis including the basement membrane layer is prepared by a) removing the epidermis from the skin tissue to prepare the dermis including the basement membrane layer having a thickness of 100 to 300 um;

b) removing the lipid component from the dermis comprising the basement membrane layer; and

c) it can be prepared through the step of removing cells from the dermis including the basement membrane layer from which the lipid component has been removed.

In the present invention, the skin tissue may be allogeneic or heterogeneous skin tissue. The homogeneous refers to a human, and the heterogeneous refers to animals other than humans, ie, mammals such as pigs, cattle, and horses.

That is, in the present invention, the acellular dermis including the basement membrane layer can be prepared according to the preparation method of the present invention using allogeneic or heterogeneous skin tissue.

In the present invention, step a) is a step of removing the epidermis from the skin tissue to prepare the dermis including the basement membrane layer with a thickness of 100 to 300 um.

In one embodiment, in the step, the skin including the epidermal layer may be excised to a desired thickness using a dermatome, and in particular, may be excised as thin as 300 um or less. In addition, by removing only the epidermal layer in a state in which the skin including the epidermal layer is thinly obtained, a thinner dermis can be easily secured.

In one embodiment, the thickness of the dermis including the basement membrane layer may be 100 to 300 um. In the above thickness range, the cell compatibility of the artificial skin support including the basement membrane layer is excellent. If the thickness exceeds 300 um, cell compatibility may decrease, and if it is too thin, it is unsuitable for use as artificial skin, so it is important to control the thickness within the above range.

In the present invention, since the dermis includes the basement membrane layer, the adhesion rate of skin-derived cells can be increased, and through this, artificial skin can be easily manufactured.

In the present invention, step b) is a step of removing the lipid component from the dermis including the basement membrane layer prepared in step a), and the dermis including the basement membrane layer may be de-fatty. The delipidation (delipidation) means removing the lipid component from the dermis including the basement membrane layer.

In one embodiment, defatification may be performed using a delipidation solution. The delipidation solution may include a polar solvent, a non-polar solvent, or a mixed solvent thereof. Water, alcohol, or a mixed solution thereof may be used as the polar solvent, and methanol, ethanol or isopropyl alcohol may be used as the alcohol. In addition, as the non-polar solvent, hexane, heptane, octane, or a mixed solution thereof may be used. Specifically, in the present invention, a mixed solution of isopropyl alcohol (IPA) and hexane may be used as the delipidation solution. At this time, the mixing ratio of isopropyl alcohol and hexane may be 20:80 to 80:20.

The treatment time of the delipidation solution may be 1 to 8 hours.

In the present invention, step c) is a step of removing cells from the dermis including the basement membrane layer from which the lipid component has been removed by step b), and the dermis including the basement membrane layer can be decellularized. Decellularization refers to removing other cellular components other than the extracellular matrix from the dermis including the basement membrane layer, for example, a nucleus, a cell membrane, and hexane. In the present invention, the dermis including the basement membrane layer that has undergone delipidation and delipidation may be expressed as acellular dermis including the basement membrane layer.

In one embodiment, decellularization can be performed using a decellularization solution. A basic solution may be used as the decellularization solution, and specifically, at least one selected from the group consisting of sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium carbonate, magnesium hydroxide, calcium hydroxide and ammonia may be used. In the present invention, sodium hydroxide (NaOH) may be used as a decellularization solution. Conventionally, decellularization was performed using a surfactant or enzyme. However, if the enzyme is used, it may damage the dermal layer itself, and if the enzyme remains and is transplanted into the body, it may damage the original tissue of the patient, and in severe cases, there is a problem of causing an immune response. In addition, when a surfactant is used, a washing operation is required to minimize the residue of the surfactant, and the residue of the surfactant may cause toxicity to cells and tissues in the body. Therefore, in the present invention, the above-mentioned problem can be solved by using a decellularization solution during decellularization, and also has the advantage that there is no cytotoxicity.

In one embodiment, the concentration of the decellularization solution may be 0.05 to 0.5 M. It is easy to remove the cells in the above concentration range.

Also, in one embodiment, the decellularization step may be performed for 30 minutes to 10 hours. Removal of cells in this time range is easy.

In addition, in the present invention, the step of freeze-drying the cell-free dermis including the basement membrane layer may be additionally performed. The freeze-drying removes moisture from the acellular dermis including the basement membrane layer, thereby improving the cell adhesion rate to the acellular dermis.

In one embodiment, freeze-drying may be performed at -60 to 90° C. below zero for 2 to 4 days.

In addition, in the present invention, the step of sterilizing the cell-free dermis including the basement membrane layer may be additionally performed.

In one embodiment, sterilization may be performed by irradiating radiation or electron beams, and the irradiation range may be 10 to 30 kGy.

The cell-free dermis including the basement membrane layer is prepared by the above-described manufacturing method, and the acellular dermis including the basement membrane layer may be composed of the basement membrane layer and the dermis. The cell-free dermis including the basement membrane layer minimizes the residual of immune response-inducing factors, while the basement membrane layer is preserved, so it can be suitably used as artificial skin.

The present invention prepares artificial skin using the acellular dermis including the basement membrane layer prepared by the above-described manufacturing method.

When manufacturing substitutes or artificial skin models containing cells, traditionally, cells are cultured with culture elements containing animal-derived ingredients or manufactured by coating animal-derived extracellular matrix. (Lamb R, Ambler CA (2013) Keratinocytes Propagated in Serum-Free, Feeder-Free Culture Conditions Fail to Form Stratified Epidermis in a Reconstituted Skin Model. PLoS ONE 8(1): e52494. doi:10.1371/journal.pone .0052494). However, when such animal-derived ingredients are used, they may cause contamination such as animal-derived viruses or mycoplasma, which may pose a safety problem.

The basement membrane layer of the skin is composed of components such as collagen, laminin, proteoglycan, and fibronectin, and serves to strongly attach epithelial cells to the tissue below.

Therefore, in the present invention, since acellular dermis including a basement membrane layer is used, a basement membrane layer that facilitates adhesion of skin-derived cells is included in the structure, and the structure of the dermis is preserved, so additional animal-derived cells No external coating is required. Through this, it is possible to culture and differentiate skin-derived cells on the acellular dermis including the basement membrane layer, and to provide clinical safety.

In addition, the acellular dermis including the basement membrane layer according to the present invention has a thin thickness of 100 to 300 um. Therefore, adhesion and culture efficiency of skin-derived cells can be further improved, and tissue can be safely and effectively regenerated when transplanted into a skin defect.

In the present invention, in step A) and step B), skin-derived cells can be seeded on the acellular dermis including the basement membrane layer without coating of the animal-derived extracellular matrix.

In addition, in the present invention, step A) and step B) may be performed in feeder cell free, xeno free and serum-free culture elements (environment). The feeder cell-free, xeno-free and serum-free culture elements are cultured and/or It means that differentiation is performed. Through this, it is possible to manufacture safe artificial skin by solving the problem of contamination of foreign animal-derived substances.

In the present invention, step A) is a step of seeding and culturing fibroblasts in the dermal direction of the acellular dermis including the basement membrane layer.

In the present invention, the dermal direction of the acellular dermis including the basement membrane layer refers to the dermis direction on which the basement membrane layer is not formed, and may be expressed as the dermal layer.

In the present invention, fibroblasts are the main constituent cells of the dermis, and can further strengthen the dermal tissue by secreting the extracellular matrix and growth factors.

In one embodiment, the number of fibroblasts to be seeded may be 1X 10 ⁴ to 1 X 10 ⁶ per cm ² .

In one embodiment, culturing of fibroblasts may be performed using a growth factor-based culture medium, and specifically, the culture medium may be a xeno-free and serum-free medium. Cultivation may be performed for 1 to 21 days.

In the present invention, step B) is a step of seeding keratinocytes on the basement membrane layer of the acellular dermis including the basement membrane layer, followed by culturing and differentiation.

The skin keratinocytes may be adhered to, cultured and differentiated to the acellular dermis including the basement membrane layer to perform the role of the epidermis. That is, in the present invention, it is possible to manufacture a full-thickness artificial skin in which both the dermis and the epidermis exist.

In one embodiment, the number of keratinocytes to be seeded may be 5 X 10 ⁴ to 1 X 10 ⁶ per cm ² .

In one embodiment, the culturing of keratinocytes may be performed using a keratinocyte culture medium generally used in the art, and specifically, the culture medium is a growth factor-based feeder cell free, It can be xeno free and serum-free media. Cultivation can be performed for 1 to 7 days.

In addition, in the present invention, after culturing keratinocytes, they can be differentiated for 7 to 21 days.

In this case, differentiation may be performed using a keratinocyte differentiation medium generally used in the art, and specifically, the differentiation medium may be a xeno-free and serum-free medium.

In addition, the present invention relates to a dermis-based artificial skin including a basement membrane layer manufactured by the method for manufacturing a dermis-based artificial skin including the basement membrane layer described above.

The artificial skin according to the present invention comprises: an acellular dermis comprising a basement membrane layer containing fibroblasts; and

It is formed on the basement membrane layer of the acellular dermis including the basement membrane layer, and may be composed of an epidermis in which keratinocytes are cultured and differentiated.

The artificial skin according to the present invention can be used as a skin substitute, and specifically, in the field of cosmetic and reconstructive surgery for skin ulcers due to extensive burns, diabetes, etc. or skin damage and accidents, trauma, and depression of skin tissue due to aging. It can be used as a skin substitute.

The present invention will be described in more detail through the following examples. However, the scope of the present invention is not limited to the following examples, and it will be understood by those skilled in the art that various changes, modifications or applications are possible within the scope without departing from the technical matters derived from the matters described in the appended claims. will be.

Example

Example 1. Fabrication of artificial skin including basement membrane layer

(1) Preparation of acellular dermis including basement membrane layer

Skin tissue (collected from a cadaver donated by a tissue bank for non-profit patient care) was prepared.

The dermis including the basement membrane layer was obtained by excising 100 to 300 μm including the epidermis from the skin tissue using a dermatom. After that, it was washed several times with sterile distilled water.

The dermis including the excised basement membrane layer was treated with a mixed solution of alcohol (IPA) and hexane (Hexane) to remove fat, and sodium hydroxide (NaOH) was treated to remove cells present in the epidermis and dermis (basement membrane layer). inclusion acellular dermis preparation).

Hydrogen peroxide (H ₂ O ₂ ) was used in the cell-free dermis including the basement membrane layer to remove the stain on the skin, and virus inactivation was performed. The washed skin tissue was lyophilized to remove moisture and sterilized.

(2) Manufacture of artificial skin

The dermis including the basement membrane layer was placed in a culture vessel and the dermal layer was positioned so that the fibroblasts suspended in the culture medium were seeded and cultured in the dermal layer direction to induce the fibroblasts to adhere and proliferate into the dermal layer.

After culturing for 1 to 21 days, the basement membrane layer of the acellular dermis, where fibroblasts are growing, was turned upside down and keratinocytes were seeded in the direction of the basement membrane layer and cultured for 1-7 days. Thereafter, artificial skin was prepared by inducing differentiation by maintaining it in a keratinocyte differentiation medium and an air-liquid interface (ALI) state for 14 days.

Comparative Example 1. Preparation of artificial skin without basement membrane layer

(1) Preparation of acellular dermis without basement membrane layer

In Example 1 (1), the epidermis and basement membrane layer were removed and 100 to 300 μm was excised to prepare the dermis not including the basement membrane layer. was prepared.

(2) artificial skin manufacturing

An artificial skin was prepared as in (2) of Example 1.

In the present invention, FIG. 1 shows a process for manufacturing acellular dermis including a thin basement membrane layer and acellular dermis not including a basement membrane layer.

As shown in Fig. 1, the dermis including the basement membrane layer (Example 1), 100 to 300 μm including the epidermis was excised with derma from the donated skin, and 100 to 300 μm from the donor skin, from which the epidermis and basement membrane layer were removed, derma Using the dermis without the basement membrane layer (Comparative Example 1) excised with a tom, the acellular dermis including the basement membrane layer and the acellular dermis not including the basement membrane layer were prepared through processes such as fat removal, decellularization and sterilization. .

Hereinafter, in the drawings, the dermis including the basement membrane shows a cell-free dermis including the basement membrane layer, and the dermis without the basement membrane uses the acellular dermis not including the basement membrane layer.

Experimental Example 1. Analysis of cell compatibility according to the thickness of acellular dermis

Cell adhesion and proliferation were confirmed according to the thickness of the acellular dermis including the basement membrane layer.

The cell-free dermis including the basement membrane layer, which had been washed, was prepared in a wet (hydration) state and a dry state by freeze-drying to a thickness of 1 mm or less and 2-3 mm. At this time, the cell-free dermis including the basement membrane layer used was prepared by the method (1) of Example 1 except for the thickness.

On the cell-free dermis containing the basement membrane layer, 2 X 10 ⁴ per cm ² of human dermal fibroblasts (human dermal fibroblasts, HDF) were sprayed and cultured. To check cell adhesion and survival, absorbance was measured with CCK-8 Kit on the 7th day of culture to confirm the degree of cell proliferation.

The results are shown in FIG. 2 .

FIG. 2 is a graph showing the results of confirming cell proliferation by culturing skin-derived fibroblasts in acellular dermis including basement membrane layers having different thicknesses. As shown in FIG. 2, it can be confirmed that the cell adhesion and cell proliferation are very excellent in the lyophilized formulation (dry state) having a thickness of 1 mm or less.

Experimental Example 2. Analysis of suitability of human skin-derived fibroblasts according to the inclusion of basement membrane layer

(1) Cell Compatibility Assay

Example 1 (1) and Comparative Example 1 (1) prepared in the acellular dermis containing the basement membrane layer (dermis including the basement membrane layer) and the acellular dermis without the basement membrane layer (dermis without the basement membrane layer) Cell compatibility was confirmed. The cell suitability was confirmed by culturing human skin-derived fibroblasts on the dermis. All cells used in the present invention were maintained and cultured under conditions from which animal-derived components were removed.

Specifically, using the dermis including the basement membrane layer prepared in Example 1 and the dermis without the basement membrane layer prepared in Comparative Example 1, coated and uncoated with extracellular matrix components (CTS, collagen), 1X10 per cm ² A cell suspension containing ⁵ human skin-derived fibroblasts was sprayed and cultured. In order to confirm cell adhesion and proliferation on the dermal surface, the cells were cultured in a cell incubator for 1, 4, 7, 14, and 21 days of culture.

Cell compatibility analysis was performed as follows. On days 1, 4, 7, 14, and 21, the absorbance was measured with a CCK-8 kit to confirm the degree of cell proliferation.

The results of cell compatibility analysis are shown in FIG. 3(a).

Figure 3 (a) shows the results of confirming the degree of proliferation of fibroblasts in the dermis with/without the basement membrane layer according to the presence or absence of the basement membrane layer. In the figure, Non coating is the dermis with/without the uncoated basement membrane layer, CTS is the dermis with/without the basement membrane layer coated with CTS, an extracellular matrix component, and Collagen is the dermis with/without the basement membrane layer coated with the extracellular matrix component, collagen. The results of absorbance measurements in the dermis are shown.

As shown in Fig. 3(a), proliferation of fibroblasts can be confirmed in all groups during the culture period, and it can be confirmed that the dermis with the basement membrane has superior cell adhesion than the dermis without the basement membrane.

(2) Cell adhesion and cell viability assay

In addition, human skin-derived fibroblasts were cultured in the dermis with the basement membrane layer prepared in (1) of Example 1 and the dermis without the basement membrane layer prepared in (1) of Comparative Example 1, and cultured for 1 day, 4 days, 7 days. Cell adhesion and survival were confirmed with a confocal microscope (LSM 700, Carl Zeiss, Germany) using Live/dead cell viability assay (Life Technology, USA) for 14 days and 21 days.

Cell adhesion and cell viability are shown in FIGS. 3(b) and (c).

3 (b) and (c) show the results of confirming cell survival (specifically, cell adhesion and cell proliferation) by culturing fibroblasts in the dermis with the basement membrane layer (B) and the dermis without the basement membrane layer (C) . After 7 days, both the seeded side and the opposite side were observed. Green indicates live cells and red indicates dead cells.

As shown in FIG. 3(b) , it can be confirmed that cell proliferation is active in both the dermis including the basement membrane layer and the dermis without the basement membrane layer. In particular, in the case of the dermis including the basement membrane layer, it can be seen that the cells proliferated in the opposite direction from which the cells were sprayed after 7 days.

Experimental Example 3. Analysis of compatibility of human skin-derived keratinocytes according to the presence or absence of basement membrane layer

(1) Cell Compatibility Assay

Cell compatibility for the dermis with/without the basement membrane layer prepared in Example 1 (1) and Comparative Example 1 (1) was confirmed. The cell suitability was confirmed by culturing human skin-derived keratinocytes (HEK) on the dermis with/without the basement membrane layer.

Specifically, using the dermis with the basement membrane layer and the dermis without the basement membrane layer, the cell suspension containing 1X10 ⁵ keratinocytes per cm ² is sprayed with and without the extracellular matrix component (CTS, collagen) coated. cultured. In order to confirm cell adhesion and proliferation on the dermal surface, the cells were cultured in a cell culture medium for a certain period of time.

Cell compatibility analysis was performed as follows. On the 1st, 3rd, and 7th days, the absorbance was measured with the CCK-8 kit to confirm the degree of cell proliferation.

The results of cell compatibility analysis are shown in FIG. 4(a).

4(a) shows the results of confirming the proliferation degree of keratinocytes in the dermis with/without the basement membrane layer according to the presence or absence of the basement membrane layer. In the figure, Non coating shows the absorbance measurement results in a state in which the dermis with/without basement membrane layer is not coated, CTS is in a state in which CTS, an extracellular matrix component, is coated, and Collagen is in a state in which collagen, an extracellular matrix component, is coated. .

As shown in Fig. 4(a), cells proliferated in the dermis including the basement membrane during the culture period regardless of whether or not the coating was applied, but in the dermis without the basement membrane layer, the absorbance decreased over time, thereby causing cell death.

(2) Cell adhesion and cell viability assay

In addition, keratinocytes were cultured in the dermis including the basement membrane layer prepared in Example 1, and cell adhesion and survival were observed using Live/dead cell viability assay (Life Technology, USA) on the 1st, 4th, and 7th days of culture. was confirmed with a confocal microscope (LSM 700, Carl Zeiss, Germany).

In this experimental example, the dermis including the basement membrane was not coated or coated with an extracellular matrix (CTS, collagen).

Cell adhesion and cell viability are shown in Fig. 4(b).

Figure 4(b) shows the results of confirming cell survival (specifically, cell adhesion and cell proliferation) by period by culturing keratinocytes in the dermis including the basement membrane layer. In FIG. 4(b), green indicates living cells and red indicates dead cells.

As shown in FIG. 4( b ), it can be confirmed that the proliferation rate of keratinocytes injected into the dermis including the uncoated basement membrane over time is superior to that of the coated artificial skin support.

(3) Analysis of differences in cell adhesion and proliferation according to the number of keratinocytes

In addition, in order to confirm the difference in cell adhesion and proliferation according to the number of keratinocytes, cells containing 1 to 3 X 10 ⁵ keratinocytes per cm ² on the dermis including the basement membrane prepared in (1) of Example 1 It was cultured by spraying the turbid solution. Cell adhesion and viability were confirmed with a confocal microscope (LSM 700, Carl Zeiss, Germany) on the 1st and 3rd days of culture using Live/dead cell viability assay (Life Technology, USA).

The results of cell adhesion and proliferation according to the number of keratinocytes are shown in FIG. 4(c).

4( c ) shows the results of seeding and culturing different numbers of keratinocytes in the dermis including the basement membrane layer and confirming the cell survival for each period. In this case, green indicates living cells and red indicates dead cells.

As shown in FIG. 4(c), it can be confirmed that keratinocytes proliferate according to the number of cells in the dermis including the basement membrane layer.

3 and 4, it can be confirmed that the dermis including the basement membrane has compatibility and affinity with skin-derived cells.

Experimental Example 4. Analysis of suitability of dermis including basement membrane as artificial skin

Using the dermis including the basement membrane layer prepared in Example 1 (1), fibroblasts labeled with DiO in the direction of the dermis were seeded and cultured, and keratinocytes labeled with DiI indicating red on the basement membrane layer were harvested. After seeding, culture was performed, and cell adhesion and morphology were confirmed after 1 day of culture.

The results of confirming cell adhesion and morphology are shown in FIG. 5 .

As shown in FIG. 5 , it can be confirmed that both keratinocytes and fibroblasts adhere to and proliferate in the dermal layer including the basement membrane layer.

In addition, differentiation of keratinocytes was induced through an air liquid interface (ALI).

Specifically, after culturing fibroblasts on the dermal layer of the dermis including the basement membrane layer, keratinocytes were seeded in the direction of the basement membrane layer and cultured, and then maintained in a keratinocyte differentiation medium and ALI (air-liquid interface) state for 14 days. differentiation was induced. Through this, artificial skin was manufactured.

6 shows the differentiation results through histological analysis. The skin-derived cells proliferated after seeding, and the differentiation-induced artificial skin was fixed with neutralized formalin for 14 days, frozen and sliced, and the entire specimen was observed with Hematoxylin & Eosin (H&E) staining.

6 , it can be confirmed that artificial skin in which keratinocytes are differentiated from the dermis including the basement membrane layer is identified.

Artificial skin prepared by culturing skin-derived cells on an artificial skin support can safely and effectively regenerate tissue when transplanted into a skin defect.

Claims (11)

culturing after seeding fibroblasts in the dermal direction of the acellular dermis including the basement membrane layer; and A method for manufacturing a dermis-based artificial skin comprising a basement membrane layer, comprising the step of seeding keratinocytes on the basement membrane layer of the acellular dermis including the basement membrane layer, followed by culturing and differentiation. The method of claim 1, The acellular dermis including the basement membrane layer is prepared by a) removing the epidermis from the skin tissue to prepare the dermis including the basement membrane layer having a thickness of 100 to 300 um; b) removing the lipid component from the dermis comprising the basement membrane layer; and c) The method for producing a dermis-based artificial skin comprising a basement membrane layer, which will be prepared through the step of removing cells from the dermis including the basement membrane layer from which the lipid component has been removed. 3. The method of claim 2, The skin tissue is an allogeneic or heterogeneous skin tissue, a method for producing a dermis-based artificial skin including a basement membrane layer. 3. The method of claim 2, Step b) is carried out using a delipidation solution, The delipidation solution comprises a polar solvent, a non-polar solvent, or a mixed solvent thereof, a method for producing a dermis-based artificial skin including a basement membrane layer. 3. The method of claim 2, Step c) is performed using a decellularization solution, The decellularization solution includes at least one selected from the group consisting of sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium carbonate, magnesium hydroxide, calcium hydroxide and ammonia, a method for producing a dermis-based artificial skin including a basement membrane layer. 3. The method of claim 2, The method for producing a dermis-based artificial skin comprising a basement membrane layer, further comprising the step of freeze-drying and sterilizing the cell-free dermis including the basement membrane layer. The method of claim 1, A method for producing a dermis-based artificial skin including a basement membrane layer, wherein the fibroblasts and keratinocytes are seeded without coating the animal-derived extracellular matrix in the acellular dermis including the basement membrane layer. The method of claim 1, Culturing of fibroblasts, and culturing and differentiation of keratinocytes are carried out in feeder cell free, xeno free and serum-free culture elements, respectively, comprising a basement membrane layer A method for manufacturing a dermis-based artificial skin. The method of claim 1, The number of seeded fibroblasts is 1 X 10 ⁴ to 1 X 10 ⁶ per cm ² The culturing of the fibroblasts will be performed for 1 to 21 days, a method for producing a dermis-based artificial skin comprising a basement membrane layer. The method of claim 1, The number of keratinocytes to be seeded is 5 X 10 ⁴ to 1 X 10 ⁶ per cm ² , Culturing of keratinocytes is performed for 1 to 7 days, and differentiation is performed for 7 to 21 days. It is manufactured by the method for manufacturing a dermis-based artificial skin comprising the basement membrane layer according to claim 1, acellular dermis comprising a basement membrane layer comprising fibroblasts; and A dermis-based artificial skin comprising a basement membrane layer that is formed on the basement membrane layer of the acellular dermis including the basement membrane layer, and is composed of an epidermis in which keratinocytes are cultured and differentiated.

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