The Experimental Study Of Culturing Human Oral Keratinocytes With Human Autologous Serum

Hypospadias is a congenital genitourinary deformity, The main manifestation is the defects of distal urethra. Reconstruction of the missing urethra is the only one choice at present,which is a traditional treatment model to repair the tissue defects by adding new traum, Its clinical application is restrained due to the dilemma of larger tissue demand and little donor tissue. How to reconstruct the missing urethra with minimal tissue traum? Tissue engineering makes it into practice. Epithelium is a essential constitution of urethra, so the epithelium culturing is very important to engineering urethra. The seed cell of engineering urethra is usually derived from urinary mucosa so far, which could exert more traum on donor site. The less epithelium cell containing and slow proliferation contrasted with the oral keratinocyte was investigated by the previous study. Therefore we select human oral keratinocyte(HOK) as a seed cell for engineering urethra.It is paramount premise for clinical application of engineering mucosa to establish a steady and high successful rate system of culturing oral mucosa by tissue engineering techique. The oral mucosal tissue were treated with 2.47ml Dispase II and 0.25% Trypsin to separate mucosa cell in different time of 5, 10, 15, 30, 45 minuts. The cell viability was demonstrated by Trypan blue exclusion, the total number of exclusion cell were 35×10~4/ml, 59×10~4/ml, 63×10~4/ml, 65.5×10~4/ml, 54×10~4/ml, and the 24 hours clone rate were 9.76×10~3/cm~2,, 7.6×10~3/cm~2, 7.13×10~3/cm~2, 4.2×10~3/cm~2, 2.5×10~3/cm~2, for each time point respectively. 15 minuts was the optimal cell disassociated time demonstrated by the statistics analysis of cell viability and the 24 hours clone rate.The difference of total cell number obtained per mm~2 and the 24 hours clone rate, 2.52×10~4/mm2, 2.38×10~4mm~2 and 7.4×10~3/cm~2,5.2×10~3/cm~2,between groups below 10 years old and 11 to 30 years old respectively, were not significant.In order to avoid the immunoreaction and infection disease caused by heterogeneous serum and apply the engineering tissue to clinical patients directly, we have cultured the HOK using human autologous serum(HAS) and plasm(HAP) as a adjuvant instead of fetal calf serum(FCS). The results have shown that the HOK could proliferate well in medium containing HAS or HAP. The difference of the 24 hours clone rate, population doubling time(PDT) and confluence time at different seeded cell densities, were not significant, PDT...