Improvement of EBV transformation and cloning efficiency of human B cells using culture supernatants from lymphoblastoid cell lines.
Exposure of human B cells to Epstein-Barr virus (EBV) usually results in low frequencies of transformed cells. The transformed cells can be cloned poorly by limiting dilution, even when feeder cells are used. In recent years it has become clear that growth and antibody production of EBV-transformed cells are influenced by auto- and paracrine growth factors. Therefore, supernatants from the lymphoblastoid B cell lines JY and Raji were used as a source of growth factors to investigate their effect during EBV transformation of human B cells and consequently on cloning by limiting dilution of these transformed cells. Initial experiments to clone three established EBV-transformed B cell lines showed a strong increase in outgrowth of the number of cells in the presence of the supernatant (range: 1 per 2-8 of the originally plated cells) as compared to cells cultured without the supernatant (range: 1 per 17-100 of the originally plated cells). Transformation efficiencies of freshly isolated tonsil B cells were not influenced by the supernatant and were generally less than 1%. In contrast, transformation efficiency was increased up to 9.4% if B cells were both transformed and cultured in the presence of the supernatant. Cloning efficiencies increased if the cells used were transformed in the presence of the supernatant. Best results were seen when the supernatant was present during transformation and cloning of the cells. The presence of the supernatant during transformation and/or cloning of the B cells dramatically enhanced the number of B cells secreting IgG. Cloning of two established tonsil B cell lines resulted in a large number of B cell clones.(ABSTRACT TRUNCATED AT 250 WORDS)
Laboratory of Cell Biology and Histology, University of Amsterdam, Netherlands.
M L
ML
Mevissen
J
J
Kwekkeboom
E
E
Goormachtig
E
E
Lindhout
C
C
de Groot
D000917:Antibody Formation / Q000187:drug effects; D001402:B-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000382:microbiology; D002455:Cell Division / Q000187:drug effects; D002461:Cell Line, Transformed / Q000378:metabolism*; D002472:Cell Transformation, Viral / Q000187:drug effects*; D002478:Cells, Cultured; D002648:Child; D002999:Clone Cells / Q000166:cytology / Q000187:drug effects; D017077:Culture Media, Conditioned / Q000494:pharmacology*; D016207:Cytokines / Q000096:biosynthesis; D006133:Growth Substances / Q000378:metabolism / Q000494:pharmacology; D004854:Herpesvirus 4, Human / Q000502:physiology*; D006801:Humans; D014066:Palatine Tonsil / Q000166:cytology; D013268:Stimulation, Chemical
D017077:Culture Media, Conditioned; D016207:Cytokines; D006133:Growth Substances