Method for preparing a three-dimensionally cultured skin comprising dermis and epidermis, and the cultured skin made therefrom
The present invention includes a method for preparing a three-dimensionally cultured skin model comprising dermis and epidermis, which comprises: a step of preparing the dermis using a composition comprising murine fibroblasts; and a combination of native collagen and atelocollagen; and a step of forming the epidermis using keratinocytes. Also, the present invention relates to a three-dimensionally cultured skin model which comprises: a dermis prepared by a composition comprising murine fibroblasts, native collagen, or a combination of native collagen and atelocollagen; and epidermis formed from keratinocytes.
1. A method for preparing a three-dimensionally cultured skin model comprising dermis and epidermis, comprising:
(a) inoculating a composition, which comprises murine fibroblasts or human fibroblasts; and a combination of native collagen and atelocollagen, into a culture dish and solidifying the same to prepare the dermis, wherein atelocollagen and native collagen are combined in a ratio of 1:1 to 1:10;
(b) inoculating keratinocytes on the dermis prepared in step (a) and culturing until a cell sheet is formed thereon; and
(c) raising the cell sheet in step (b) to the air/liquid interface to form the upper layers of epidermis, thereby preparing said three-dimensionally cultured skin model comprising dermis and epidermis, wherein said dermis formed in step (a) by a combination of native collagen and atelocollagen exhibits at least one of the following characteristics compared to when atelocollagen is used alone:
i) inhibition of dermal contraction,
ii) increase in epidermal differentiation;
iii) decrease in the level of MMP expression; and
iv) inhibition of collagen degradation.
2. The method of claim 1 , wherein, in step (a),
the native collagen comprises alpha-1 chain consisting of the amino acid sequence of SEQ ID NO: 1 and alpha-2 chain consisting of the amino acid sequence of SEQ ID NO: 2; and
the atelocollagen comprises alpha-1 chain consisting of the amino acid sequence of SEQ ID NO: 3 and alpha-2 chain consisting of the amino acid sequence of SEQ ID NO: 4.
3. The method of claim 1 , wherein, in step (a), the composition, which comprises murine fibroblasts or human fibroblasts; and a combination of native collagen and atelocollagen, comprises 1×10 4 to 1×10 7 murine fibroblasts or human fibroblasts per cm 2 .
4. The method of claim 1 , wherein, in step (a), the composition further comprises at least one cell selected from the group consisting of fibroblasts, dermal papilla cells, endothelial cells, adipocytes, and mesenchymal stem cells.
5. The method of claim 1 , wherein step (a) is performed for 1 hour to 48 hours.
6. The method of claim 1 , wherein 1×10 4 to 1×10 7 keratinocytes per cm 2 of the dermis prepared in step (a) are inoculated in step (b).
7. The method of claim 1 , wherein the keratinocytes in step (b) are derived from the skin, mucosa, cornea, or hair follicles.
8. The method of claim 1 , wherein step (b) further comprises inoculating at least one cell selected from the group consisting of melanocytes, Merkel cells, and Langerhans cells.
9. The method of claim 1 , wherein step (c) is performed for 7 days to 21 days.
10. A three-dimensionally cultured skin model prepared by the method of claim 1 .