Perkins S, Zimmermann U, Foung S K
Department of Pathology, Stanford University School of Medicine, CA.
Hum Antibodies Hybridomas. 1991 Jul;2(3):155-9.
Hypoosmolar conditions have permitted the development of electrofusion techniques capable of producing human hybridomas from as few as 10(5) B cells. A hybridoma formation efficiency of one hybrid for each 125 input B cells has been achieved with Epstein-Barr virus--activated B cells and mouse-human heteromyelomas. This is at least 100-fold higher in efficiency than with polyethylene glycol-induced cell fusion, as well as a 50- to 100-fold decrease in the required number of human B cells. The ability to fuse a small number of input B cells should lead to a greater success rate in immortalizing the rare antigen-specific B cells. The critical parameters include fusion voltages, the composition and number of wash steps used in cell preparation, the composition and duration of exposure to hypoosmolar fusion medium, fusion ratio, plating density, the use of growth medium without pH indicator, and the use of an irradiated human fibroblast feeder layer. By manipulating these parameters, a high hybrid yield can be achieved with different mouse-human heteromyelomas and Epstein-Barr virus-activated B cells.
低渗条件使得电融合技术得以发展，该技术能够从低至10⁵个B细胞中产生人杂交瘤。利用爱泼斯坦-巴尔病毒激活的B细胞和鼠-人异骨髓瘤，已实现每125个输入B细胞产生一个杂交瘤的杂交瘤形成效率。这一效率至少比聚乙二醇诱导的细胞融合高100倍，同时所需的人B细胞数量减少了50至100倍。融合少量输入B细胞的能力应会在使罕见的抗原特异性B细胞永生化方面带来更高的成功率。关键参数包括融合电压、细胞制备中使用的洗涤步骤的组成和数量、暴露于低渗融合培养基的组成和持续时间、融合比例、接种密度、使用不含pH指示剂的生长培养基以及使用经辐照的人成纤维细胞饲养层。通过操纵这些参数，使用不同的鼠-人异骨髓瘤和爱泼斯坦-巴尔病毒激活的B细胞可实现高杂交瘤产量。
