[Primary cultures of various differentiated human cells and their transfer]

From our long experience with primary cultures of human differentiated cells, we have been able to come to the following conclusions. The culture media routinely employed have been developed using established cell lines but not primary cells as the growth marker. Therefore the culture media for primary cultures should be modified from those routinely employed. In addition the concentrated supplementation of serum to basal media dose not contribute to the growth of primary differentiated cells, and in fact on the contrary, is advantageous for the growth of non-target fibroblasts which would hamper the growth of the target cells. A hypoxic culture environment is more favorable for primary cultures especially when smaller cell numbers are used as the inoculum. The primary culture cells are more fragile in comparison with established cell lines. Therefore, the low-temperature cell diopersion procedure is recommended with the use of diluted crystalline trypsin saline. On the basis of the above findings, we have successfully carried out primary and transfer cultures of human esophageal and gall bladder epithelial cells, skin keratinocytes and endothelial cells. Their epithelial mature was proved by the presence of keratin in their cytoplasm, and the phenotype of endothelial cells was evident from the presence of Factor VIII-related antigen in their cytoplasm. Culture of endothelial cells requires the supplementation of a specific growth factor which we have utilized and isolated from conditioned medium of human diploid fibroblasts. Tiny amounts of the factor were shown to, induce the vascularization of rabbit eye cornea.