The methods in the following sections describe 1) the growth of cloned hybridoma cell lines, 2) high-density culture systems required for bulk production of MAbs, and 3) the generation of validated cell banks where hybridomas can be stored for future retrieval.
All procedures that involve manipulation of cell lines are conducted within the confines of a class II microbiological safety cabinet using sterile equipment. Cell cultures are maintained at 37 °C in an atmosphere of 5% CO2 in air and 100% humidity. It is preferable to use tissue culture flasks that have filter-vented caps to minimize the chances of contamination. Several hybridoma cell lines were used in the following procedures: 36F (3); VPM 20, VPM 21, VPM 22 (4); 73B (5); 3C2, and 8D8 (6). These cell lines have individual characteristics, but the procedures described are generic in nature and can be applied to all hybridoma cell lines, taking into account the comments in the Notes section.
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