Separation and culture method of epidermal stem cells and cell suspension

Abstract

The invention relates to a separation and culture method of epidermal stem cells, which comprises the following steps: taking an initial skin graft greater than or equal to a preset size, and pretreating to obtain a first intermediate skin graft; cleaning to obtain a second middle skin graft; placing the second intermediate skin graft in mixed digestive juice and placing in a constant-temperature shaking table to be digested, obtaining a third intermediate skin graft, wherein the mixed digestive juice comprises trypsin and ethylenediamine tetraacetic acid, the mass volume ratio of the trypsin is 0.5%-1.25%, and the digestion time is 45-90 min; taking out the third intermediate skin graft, and scraping dissociated cells; collecting the dissociated cells to obtain a cell suspension, centrifuging, and discarding a supernatant; carrying out cell resuspension by using a completely serum-free keratinocyte culture medium to obtain a resuspended cell culture solution; and placing the resuspended cell culture solution in a culture dish with a human placenta IV type collagen planking, changing the solution according to preset time, and carrying out subculture when 70-80% of the culture dish is overgrown with cells. According to the method, both the separation speed and the separation ratio can be considered, and the cell proliferation capacity can be improved.

Description

technical field [0001] The invention relates to the technical field of tissue engineering, in particular to a method for separating and culturing epidermal stem cells. The invention also relates to the epidermal stem cell suspension prepared by the method. Background technique [0002] A wound is a surface formed after the integrity of normal skin (tissue) is disrupted and a certain amount of normal tissue is lost. At present, the materials used clinically to cover wounds mainly include patient's autologous skin, allogeneic skin or heterogeneous skin, and epidermis cultured from autologous epidermal cells. However, the above-mentioned skins from various sources have obvious shortcomings, such as the problem of insufficient skin source in autologous skin, the problem of rejection in allogeneic skin or heterogeneous skin, and the existence of cultured skin for a long time, susceptible to infection, expensive, poor elasticity, etc. question. Therefore, the construction of co...

AI Technical Summary

Problems solved by technology

[0004] The two enzymatic digestion methods have their own advantages and disadvantages. The epidermal dermal separation method has a relatively high proportion of stem cells in the epidermal cells, but it needs to be digested overnight. It takes a day from the sampling to the successful separation of the cells, and the digestion time is long; the direct digestion method separates The time is short, but a purification step is required after digestion, and the operation process is cumbersome

[0005] In addition, during the culture process after the isolated cells, it is necessary to add recombinant human epithelial growth factor (rhEGF) to the medium to stimulate cell proliferation, but the promotion effect is limited, and it is difficult to further improve the current technology. Improve cell proliferation

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