Rapid expansion of human cytotoxic T cell clones: growth promotion by a heat-labile serum component and by various types of feeder cells.

Culture conditions are described that result in rapid expansion of cloned cytotoxic T cells of human origin. A combination of allogeneic lymphocytes and Epstein-Barr virus (EBV) transformed B cells (B-LCL) as irradiated feeder cells resulted in a 10-fold higher cell yield than obtained by use of either feeder cell alone. The large cell numbers obtained in a relatively short period of time facilitate in vitro and in vivo experimentation. Further enhancement of cell proliferation was obtained by the use of fresh human serum not heat-inactivated before use. This suggests the presence of a heat-labile growth stimulating factor or factors in human serum. Round-bottomed microtitre wells were found to give best culture results. Plating and harvesting of cells cultured in wells was facilitated by a specially designed culture flask. Addition of leucoagglutinin (purified phytohaemagglutinin) to the culture medium resulted in an approximately 3-fold higher cell yield. Optimal culture results were obtained when all the above factors were combined. It was possible to expand single cytotoxic T cells to up to 10(9) cells in about 30 days with full retention of cytolytic activity and target cell specificity. T cell clones have now been cultured for more than 70 generations.