Estimation of Heterokaryon Formation and Hybridoma Growth in Murine and Human Cell Fusions

Abstract

Four mouse myelomas commonly used for cell fusions (X63.Ag8.653, SP2/0, NS1, P3U1), 3 human myeloma-like cell lines (ARH77, U-266, GM1500) and 3 human x mouse hybridomas (SPAZ4, SA2, SA3) were compared for their heterokaryon formation and successful hybridoma growth after cell fusion with polyethylene glycol. The cells were stained with different fluorescent dyes which do not alter hybridoma growth or antibody secretion. After fusion myeloma cells containing at least 1 nucleus from a lymphocyte (heterokaryons) were counted from fluorescence photomicrographs and the heterokaryon frequency was calculated. Mouse myelomas fused at a frequency of 1-7%, whereas human myeloma lines showed a higher heterokaryon frequency ranging from 3-25%. In mouse fusions almost every well contained growing hybridomas showing a minimum hybridoma frequency of 2/10(6) lymphocytes. In human fusions the SPAZ4 and SA2 lines showed a heterokaryon frequency nearly as good as mouse myelomas, whereas U-266 yielded no growing hybridomas despite 20% heterokaryon frequency. Furthermore, human cell lines showed a high tendency of multikaryon formation whereas this phenomenon was rarely observed with murine and murine x human heterohybrids. In individual fusion experiments no correlation was found between heterokaryon formation and the number of growing hybridomas. Thus, our study shows that defects in hybridoma growth may not always result from lack of a successful fusion and human hybridomas might be more sensitive to post-fusion conditions.

Human anti-anthrax protective antigen neutralizing monoclonal antibodies derived from donors vaccinated with anthrax vaccine adsorbed

“…The antibody producing cells in the chimeric mice were recovered from the spleen and peritoneal washes in sufficient numbers to permit fusion with a standard mouse myeloma P3X63Ag8.653 [21] to form hybridomas. Others [25,26] and we have noted that the formation of mouse/human hybridomas using a murine fusion partner with human derived plasma cells results in unstable hybrids, usually these are difficult to clone, expand and isolate. We circumnavigate this problem by rescuing the transcripts encoded by mRNA from a small cluster of cells and generating stable recombinant CHO cell lines and testing these for the activity.…”

Autoantibodies to Tumor-Associated Antigens as Cancer Biomarkers

“…Methods for generation of human hybridomas include the fusion of human B cells with human or mouse myelomas, transformation of human B cells by Epstein-Barr virus (EBV) or combination of EBV transformation with the fusion [36]. The main limitations for the development of hybridomas, producing fully human immunoglobulins (Ig) are: 1) low levels of Ig production when human myeloma cell lines are used as fusion partners [37], 2) cessation of Ig production due to segregation and loss of human chromosomes from the genome when mouse myelomas are used as fusion partners [38] and 3) low stability and clonability in the case of EBV transformation [36]. These limitations may be overcome by the development of improved partner cell lines.…”