Method for making three-dimensional cultured skin model including dermis and epidermis, and three-dimensional cultured skin model made thereby

一种三维培养、真皮的技术，应用在表皮细胞/皮肤细胞、生物化学设备和方法、3D培养等方向，能够解决不完全、形成基底膜、真皮收缩等问题，达到抑制收缩和降解、优异形成和分化的效果

Inactive Publication Date: 2017-04-19
全世华
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the three-dimensional cultured skin developed so far has the disadvantage of incomplete formation of basement membrane between the dermis and epidermis prepared with collagen and dermal shrinkage during the culture process.

Method used

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  • Method for making three-dimensional cultured skin model including dermis and epidermis, and three-dimensional cultured skin model made thereby
  • Method for making three-dimensional cultured skin model including dermis and epidermis, and three-dimensional cultured skin model made thereby
  • Method for making three-dimensional cultured skin model including dermis and epidermis, and three-dimensional cultured skin model made thereby

Examples

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preparation example Construction

[0051] In the method for preparing a three-dimensional cultured skin model of the present invention, the composition comprising mouse fibroblasts and natural collagen or a combination of natural collagen and atelocollagen in step a) may comprise 1×10 4 -1×10 7 mouse fibroblasts / cm 2 , especially 1×10 5 -1×10 6 a fibroblast. In addition, in the above step a), the composition may also contain at least one cell selected from human fibroblasts, dermal papilla cells, endothelial cells, adipocytes and mesenchymal stem cells.

[0052] In a specific embodiment of the present invention, in the step of preparing the dermis according to the preparation method of the three-dimensional cultured skin model, by using 0.5×10 5 fibroblasts / cm 2 An amount containing murine fibroblasts was used to prepare a composition comprising murine fibroblasts; native collagen; or a combination of native collagen and atelocollagen.

[0053] In addition, the step a) of the preparation method of the thr...

Embodiment 1

[0067] Example 1. Materials for preparing a three-dimensional cultured skin model comprising dermis and epidermis

[0068] In the present invention, experiments were conducted for preparing a three-dimensional cultured skin model comprising dermis and epidermis and having properties most similar to human skin. Materials used to prepare basic three-dimensional cultured skin models are as follows.

[0069] ※Experimental Materials

[0070] Mouse fibroblasts (3T3 cell line) or human fibroblasts

[0071] human keratinocytes

[0072] Collagen (atelocollagen and / or native collagen)

[0073] Transwell chamber (6 wells)

[0074] maintenance plate

[0075] DMEM / F12 medium containing 10% FBS

[0076] Epidermal Growth Factor (EGF)

[0077] A three-dimensional cultured skin model comprising dermis and epidermis was prepared using the materials described above.

Embodiment 2

[0078] Example 2. Preparation of a three-dimensional cultured skin model comprising dermis and epidermis

[0079] 2-1. Preparation of dermis (collagen dermis)

[0080] Mix mouse fibroblast 3T3 cells or human fibroblasts with 2.0 mg / mL collagen solution and 10X reconstitution buffer (0.2M sodium bicarbonate, 0.2M HEPES, 0.05N NaOH) to prepare cell mixture solution at a concentration of 1X 10 5 cells / mL. Add the cell mixture solution into the 6-well Transwell chamber at an amount of 2 mL / well. The mixture solution was placed in an incubator (37°C, 10% CO 2 ) to cure overnight (1 hour to 24 hours).

[0081] 2-2. Preparation of skin (skin arrangement)

[0082] Keratinocytes were divided into 2.5×10 5 Concentrations of cells were seeded on the dermis prepared in Example 2-1. Keratinocytes were incubated in an incubator (37°C, 10% CO 2 ) for about 5 to 6 days until the keratinocytes form a sheet, while replacing the medium with fresh medium containing 10 ng / mL EGF at int...

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Abstract

The present invention relates to a method for making a three-dimensional cultured skin model comprising a dermis and an epidermis, which comprises: a step of preparing the dermis using a composition comprising a mouse-derived fibroblast, a native collagen or a combination of a native collagen and an atelocollagen; and a step of forming the epidermis using a keratin cell. Also, the present invention relates to a three-dimensional cultured skin model which comprises: a dermis made by a composition comprising a mouse-derived fibroblast, a native collagen, or a combination of a native collagen and an atelocollagen; and an epidermis formed from a keratin cell. The three-dimensionally cultured skin model of the present invention may be used widely in toxicity and efficacy experiments of medicines or cosmetics, and in the field of alternative experiments for animal experiments since the three-dimensionally cultured skin model is excellent in formation and differentiation of a dermis and an epidermis through use of a mouse-derived 3T3 cell for making the skin model and use of a mixture of an atelocollagen and a native collagen, and has a structure similar to a natural skin layer by inhibiting collagen shrinkage and degradation phenomenon of the dermis.

Description

technical field [0001] The present invention relates to a method for preparing a three-dimensional cultured skin model comprising dermis and epidermis, comprising the steps of preparing the dermis using mouse fibroblasts and native collagen alone or a combination of native collagen and atelocollagen, and using keratinocytes The step of forming the epidermis. The present invention also relates to a three-dimensional cultured skin model consisting of a dermis prepared using a composition comprising murine fibroblasts and native collagen alone or a combination of native collagen and atelocollagen, and an epidermis formed by keratinocytes. Background technique [0002] Human skin is the largest organ covering the entire surface of the body. The skin has various functions including temperature control, protection from the external environment, etc., and has about 1.2m in adults 2 to about 2.3m 2 surface area. Skin is composed of epidermis and dermis. The dermis is composed o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61L27/60A61L27/38A61L27/24
CPCA61L27/24A61L27/38A61L27/60C12N5/0062C12N5/0629C12N5/0698C12N2502/1323C12N2533/54
Inventor 全世华金伦希文玹阿
Owner 全世华
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