| The demands always were large for skin tissue in clinical treatment to the patients with large area skin injuries or dermatogic disease, but the amount of skin obtained is limited . In traditional, the treatment approach is grafting autologous skin, which often results in heavy pigmentation and scarring in donor sites. Since the culture method of epidermal cells in vitro was founded successfully and the epidermal cellswere used to treat skin injury by Green eta in 1975, it became a milestoneof the burn therapeutics domain .After developing for several decades, there are some corporations which can produce artifical skin, composed of the skin cells and some matrix material. A lot of seed cells, including epidermal cell and fibroblast, so it is very important to culture the seed cells reasonable and fast. Up to now, The culture technique of fibroblast already is very successful and available, nevertheless it is still a difficult problem how to culture Keratinocyte, which is determined by the characteristics of Keratinocyte, because the Keratinocyte is a kind of committed stem cell, it is easy to differentiation, furthermore its life span is very short.Isolation and culture of Keratinocyte, it is the first step to isolate the epidermal basal layer cells. So far, we isolate the epidermal basal layer cells with 0. 25%Dispase II solution and/orO. 25%Trypsin solution, pH value of 7. 2-7. 4. There are such culture methods: cultivation with 3T3 fibroblast or not, and air-exposed cultivation et al, and two different mediums: serum medium and serum free medium. The methods of epidermalcells for clinical application are sheets of epidermal cells and composite artifical skin grafting. Therefore it is still difficult to obtain more keratinocyte with strong proliferative competence, as well as to culture epidermal cells quickly for clinical application. In this experiment, we will study on isolation of human epidermal cells, optimization of the serum culture method and the condition of subculture and application of the allogenic keratinocytes sheets grafted to the donor sites.PART I Effect of pHvalue on isolation and culture of human epidermal basal cellsObjective To observe the effect of pH value on isolation and cultivation of human epidermal basal cells'.Methods We isolated the epidermal with 0. 25%Dispase II solution of three level pH value in 37癈, the status of epidermal isolation was observed and scored until one group of them was isolated completely, Then we isolated epidermal basal cells with 0. 25%Trypsin solution. The cells were detected the activity, we cultured them and inspected the proliferation and growth competence separately.Results The epidermis was isolated completely after the skin was digested for 90 minutes with 0. 25%Dispase II solution of pH value 6-7 in 37. There were differences between three groups in statistics. But there were no difference in the proliferation and growth competence of cells, those were proved through cultivation.Conclusion It was faster to isolate human epidermis with 0. 25%Dispase II solution of pH value 6-7 in 37than that pH value 7-8. The proliferation and growth competence of cells were normal.PART II Optimization of the method for the serum culture of epidermal cellsObjective We used the DF12 culture medium and adjust calcium ionicconcentration; The growth states of epidermal cells were observed in medium with different calcium ionic concentration; We did subculture two kinds digest juices, 0. 25% Trypsin and 0. 25%Dispase associated 0. 2%EDTA, the effects of them were compared.Methods We determined the concentration of calcium ions of 4 kinds medium and calf serum; Then the calcium ions concentration of 4 kinds blood serum culture medium were calculated. We adjusted the calcium ions concentration of culture medium, the growth states of passage epidermal cells were observed in medium with different calcium ions concentration. The two groups epidermal cells were isolated with 0. 25%dispase associated 0.2%EDTA or 0.25%Trypsin separately, and cultured... |
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