Studies On Accelerating The Process Of Dissociating Human Keratinocytes From The Different Thickness Skin

Studies on Accelerating the Process of Dissociating Human Keratinocytes from the Different Thickness SkinPart one To choose the varieties of proteasesObjective: To choose the varieties of proteases that can dissociate human keratinocytes from the skin rapidly.Methods: Adult human full thickness skin from the legs of fresh corpses ( 20~30 years old ) were processed within 2h of collection . Epithelial sheets were separated with various proteases (1)0. 5%Dispase II (2)0. 5%Dispase II / 0.1%CollagenaseIV (3)0. 05%Thermolysin (4)0. 05%Thermolysin/0. 5%DispaseII, (5)0. 05%Thermolysin/ 0. l%CollagenaseIv\ ?. 05%Thermolysin/0. 5%Dispase II / 0. l%Collagenase) and basal epidermal cells were isolated by trypsinization (1)0.25% Trypsin, (2)0.25% Trypsin / 0. 1%EDTA). The yield of viable cells, cellular proliferation and viability, rates of adherence to dish and the time necessary to confluence of the isolated epidermal cells were investigated to determine their effects.Results: 0. 5%DispaseII/ 0.1%CollagenaseIV and 0.25% Trypsin / 0. 1%EDTA are the best choice in the dissociation of human keratinocytes.Conclusions: Dispasell/CollagenaselVand Trypsin /EDTA are fit for the studies on accelerating the process of dissociating human keratinocytes from the different thickness skin.Part two Studies on accelerating the process of separatingepithelial sheets from the ultrathin skinObjectives: To establish the optimal condition accelerating the process of separating epithelial sheets from the ultrathin skin.Methods: Adult human ultrathin skin from the legs of fresh corpses ( 20~ 30 years old ) were processed within 2h of collection . The thickness is 0. 2mm. Epithelial sheets were obtained by the separation with Dispasell/ Collagenase IV, and then keratinocytes were isolated by trypsinization at 37癈 30 minutes. The orthogonal trial of L16(4") was used to investigated five factors( the concentration of Dispase II and CollagenaselV -, the temperature the speed of the blender and the time at four different grades ). The yield of viable cells, cellular proliferation and viability and rates of adherence to dish of the isolated epidermal cells were investigated to determine their effects.Results: In the range investigated, the more the temperature of the proteases and the speed of the blender are, the less the time of dissociation is, but the harm to the keratinocytes is more rapidly increasing. The concentration of Dispase II impacts on the efficiency of Dispase II notably, but its impairment to the keratinocytes is trivial. The concentration of CollagenaselV has no marked impact on the efficiency of Dispasell, and its impairment to the keratinocytes is trivial. The size of the ultrathin skin doesn't counteract the speed of separating epithelial sheets from the ultrathin skin.Conclusions: Incubating the ultrathin skin (1.0cm2) with 0. l%Dispase II/O. !%CollagenaseIV for 16 minutes at 37癈> pH 7. 4 and ISOrpm of the blender is the optimal condition to separate epithelial sheets from the ultrathin skin.Part three Studies on accelerating the process of separatingepithelial sheets from the split thickness skinObjectives: To establish the optimal condition accelerating the process of separating epithelial sheets from the split thickness skin.Methods: Adult human split thickness skin from the legs of fresh corpses ( 20~30 years old ) were processed within 2h of collection .The thicknessis 0.4mm. Epithelial sheets were obtained by separation with Dispasell/ CollagenaselV, and then keratinocytes were isolated by trypsinizaton at 37 癈 30 minutes. The orthogonal trial of L16(4b) was used to investigated five factors( the concentration of Dispasell and CollagenaselV -. the temperature, the speed of the blender and the time at four different grades ). The yield of viable cells, cellular proliferation and viability and rates of adherence to dish of the isolated epidermal cells were investigated to determine their effects.Results: In the range investigated, the more the temperature of the proteases and the speed...