Van de Griend R J, Van Krimpen B A, Bol S J, Thompson A, Bolhuis R L
J Immunol Methods. 1984 Feb 10;66(2):285-98. doi: 10.1016/0022-1759(84)90340-5.
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.
文中描述了可导致人源克隆细胞毒性T细胞快速扩增的培养条件。将同种异体淋巴细胞和经爱泼斯坦 - 巴尔病毒（EBV）转化的B细胞（B - LCL）作为辐照饲养细胞联合使用，其细胞产量比单独使用任何一种饲养细胞高出10倍。在相对较短时间内获得的大量细胞便于进行体外和体内实验。使用未预先热灭活的新鲜人血清可进一步提高细胞增殖。这表明人血清中存在一种热不稳定的生长刺激因子或多种因子。发现圆底微量滴定孔可获得最佳培养结果。一种专门设计的培养瓶便于在孔中培养的细胞进行铺板和收获。向培养基中添加白细胞凝集素（纯化的植物血凝素）可使细胞产量提高约3倍。当上述所有因素结合使用时可获得最佳培养结果。在大约30天内可将单个细胞毒性T细胞扩增至多达10⁹个细胞，同时完全保留细胞溶解活性和靶细胞特异性。目前T细胞克隆已培养超过70代。
