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Preparation method of human 3D structure epidermal cell sheet and clinical application thereof

A cell sheet, clinical application technology, applied in the field of culturing epidermal cell sheet with 3D stratified structure, can solve the problem of not seeing three kinds of cells co-cultured with 3D stratified structure epidermis, etc.

Inactive Publication Date: 2015-09-16
武汉北度生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007]In recent years, there have been many reports on the separate culture methods of epidermal cells, epidermal stem cells and melanocytes, but so far no three-dimensional co-cultured cells have been seen. Report on Stratified Epidermis

Method used

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  • Preparation method of human 3D structure epidermal cell sheet and clinical application thereof
  • Preparation method of human 3D structure epidermal cell sheet and clinical application thereof
  • Preparation method of human 3D structure epidermal cell sheet and clinical application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] 1. Co-isolation of human epidermal stem cells, melanocytes and keratinocytes

[0024] Aseptically place a sterile 2 x 2 cm 2 Normal full-thickness skin was placed in a petri dish containing 20ml of WBB serum-free medium, and the skin was prepared with a WBB cell separator to a diameter of 0.1mm×0.1mm 2 Tissue granules, put the tissue granules in WBB medium containing Wbbase mixed enzymes for enzymolysis, 4°C for 10-12 hours, filter, and centrifuge to obtain a variety of highly active seed cells, including basal keratinocytes and melanin stem cells , transient expansion cells, hair follicle stem cells, interfollicle epidermal stem cells (Interfollicle Epidermal Stem Cells, IFESCs) sebaceous gland stem cells, etc. Basal layer keratinocytes, melanin stem cells, transient expansion cells three kinds of cell viability reached above 95%, the total cell yield is greater than 1-5×10 7 / g skin.

[0025] 2. Serum-free mixed monolayer expansion culture of three kinds of cells ...

Embodiment 2

[0034] 1. Preparation of silk protein membrane

[0035] Place the aseptic silk fibroin film at the bottom of a 60mm culture dish and fix the silk protein film with a special stainless steel fixture for use.

[0036] 2. Co-isolation of human epidermal stem cells, melanocytes and keratinocytes

[0037] Aseptically place a sterile 2 x 2 cm 2 Normal full-thickness skin is placed in a petri dish containing 20ml of WBB serum-free medium, and the skin is prepared into a size of 0.1mm×0.1mm with a WBB cell separator 2 Tissue granules, put the tissue granules in WBB medium containing Wbbase mixed enzymes for enzymolysis, 4°C for 10-12 hours, filter, and centrifuge to obtain three kinds of high Active seed cells, including melanin stem cells, transient expansion cells, etc., the activity of the three types of cells reached over 95% respectively, and the total cell yield was greater than 1-6×10 7 / g skin.

[0038] 3. Serum-free mixed monolayer expansion culture of three kinds of cells ...

Embodiment 3

[0047] 1. Co-isolation of human epidermal stem cells, melanocytes and keratinocytes

[0048] Aseptically place a sterile 4 x 2 cm 2 Normal full-thickness skin was placed in a petri dish containing 20ml of WBB serum-free medium, and the skin was prepared with a WBB cell separator to a diameter of 0.1mm×0.1mm 2 Tissue granules, put the tissue granules in WBB medium containing Wbbase mixed enzymes for enzymolysis, 4°C for 10 hours, filter, and centrifuge to obtain a variety of highly active seed cells, including basal keratinocytes, melanin stem cells, ephemeral Expanding cells, hair follicle stem cells, interfollicle epidermal stem cells (Interfollicle Epidermal Stem Cells, IFESCs), sebaceous gland stem cells, etc. Basal layer keratinocytes, melanin stem cells, transient expansion cells three kinds of cell viability reached above 95% respectively, the total cell yield is greater than 1-4×10 7 / g skin.

[0049] 2. Serum-free mixed monolayer expansion culture of three kinds of ...

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Abstract

The invention discloses a preparation method of a human 3D structure epidermal cell sheet and a clinical application thereof. The preparation method is characterized by comprising the following steps that (1), human epidermal stem cells, base layer horn cells, melanocyte stem cells and the like are separated at a time through a Wbbase mixed enzyme; (2), two-step culture is carried out, wherein the two-step culture comprises a) non-serum mixing single-layer culture of three kinds of cells and b) induced differentiation of cells and culture of the 3D structure epidermal cell sheet; (3), the cell sheet is stripped; and (4), the cell sheet is clinically applied to treat leucoderma, burn and other skin pigment depigmentation diseases. The creativity of the preparation method of the human 3D structure epidermal cell sheet and the clinical application thereof is that three kinds of cells are separated simultaneously, the effective time is prolonged by 150-300 times compared with that of traditional trypsin separation methods, activity of the enzyme does not need to be neutralized, the activity of separated cells is greater than 95%, and the total yield is greater than 1-5*107 / g of skin; the two-step culture method amplifies the three kinds of cells at the same time, so that the amplification fold is improved by five times; the melanin forming unit is close to a normal person; and the compound color effect of transplanted skin is close to or consistent with the skin of a person.

Description

technical field [0001] The invention relates to a technology for cultivating epidermal cell membranes with 3D multilayer structure by using human autologous epidermal stem cells, melanocytes and basal keratinocytes as seed cells and its clinical application. Epidermal stem cells are a type of heterogeneous cells with different subtypes, and their molecular characteristics, differentiation ability and resident sites are different. Epidermal stem cells mainly include: hair follicle stem cells (HFSCs), hair follicle outer root sheath stem cells, hair follicle pluripotent stem cells (follicle pluripotent stem cells, fPSCs) expressing keratin-15, sebaceous gland stem cells (SGSCs), sweat stem cells (sweat stem cells) and melanin Stem cells (melancyte stem cells, EPI-NCSCs) and follicular epidermal stem cells (IFESCs), etc. [0002] The main technologies include the application of WBB cell culture medium, Wbbase mixed enzyme separation of epidermal stem cells, melanocytes and kerat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61L27/38A61L27/60C12N5/074C12N5/071
Inventor 杨国成邓勇杜佳静
Owner 武汉北度生物科技有限公司
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