Primary Research On Strring Culture And Storing Of Human Dermal Fibroblasts

Objective:To observe the growth and proliferation status of human dermal fibroblasts cultured on microcarriers in strring model by magnetic stirring apparatus; To detect the viability and preservative time of human dermal fibroblasts under difference preservative conditions after propagating to grown platform stage in stirring culture; To observe the condition of migrate and proliferation from stirring culture and microcarriers in keeping time to usual culture flask;To investigate the proliferated characteristics of human dermal fibroblasts by stirring culture after imbedding artificial skin and to identify the cell.Methods: Human foreskin fibroblast cells and foreskin epidermal cells were proliferated by enzyme digestion. The six to ten passage human dermal fibroblasts were cultured on microcarrier cytodex-3 at cell density 4×10~4/ml in the modified medium, the stirring mode was set as 20 rpm, 50 min running, and 5 min pause. The viability of human dermal fibroblasts were detected under difference culture methods by CASY hemocytometer in stirring culture every 24 hours, and the fibroblasts shape was observed with inverted phase contrast microscpope and SEM at different point time. Cell viability and preservative period were detected in the different culture methods by MTT assay. Fibroblast microbeads were observed and removed from the bioreactor and cultivated in tissue culture flasks at the nineth day. TheⅠcollagen contents in the culture medium was detected by ELISA at different time point. The composite chitosan skin equivalent was prepared in our laboratory, the fibroblasts proliferation status was observed in it with HE.Result: The human dermal fibroblasts have normal shap when seeded in microcarrier at stirring culture condition. It needs less microcarriers (2.5g/L) or culture media (improved culture media 200ml per bottle) and can be quickly amplified in large scale compared with the culture in static state. The proliferation period is only 10~12 days from seeding to platform stage. The harvesting cell is five times the size of inoculums; The human dermal fibroblasts can be preserved about three weeks in room temperature after it was cultured and proliferated to platform stage. The activity of cell which was preserved in the third day reduced largely, then gradually recovered. Its activity is the best in the nineteenth day, which retained to 86.96%. At the twenty-third day, the activity decreased obviously, and the activity of cells which were preserved in 4℃is only 13.04% at the nineteenth day and the cell activity rapidly decreased; Fibroblast microbeads were removed from the bioreactor and cultivated the cell microbeads in tissue culture flasks at the nineth day. The stirring cultured fibroblasts were able to migrate from the microcarriers and showed normal shape with the capability of large scale expansion, theⅠcollagen content in the culture medium was detected and showed theⅠcollagen increasing with the proliferation of the fibroblasts in the limit period; The result shows that the human dermal fibroblasts were imbed in corium of tissue engineering after 2 weeks proliferated to be covered with corium and that the cells were HDF by being identified with HE.Conclusion: The human dermal fibroblasts have normal shape when seeded in microcarriers at stirring culture. It was amplified rapidly and well. Higher activity and more amount cells were acquired than those were cultured in static state. At room temperature the cells cultured in stirring may be preserved long time and have better viability than at 4°C. This preservative method is easy, which makes the cells have good viability and has long preservative time, as well as the superiority that the cells cultured in static state are inimitable. There are good practical value for skin seed cells to transportation and preserve. After imbedding in the corium of tissue engineering, the human dermal fibroblasts retain good proliferative characteristics. The stirring culture cells are the normal human dermal fibroblasts by being identified with HE.