Propagation and Characterization of a Rat Myeloma Cell Line Producing Immunoglobulin E in Vitro

Abstract

Mouse myeloma cell cultures have been found to be useful models for the study of immunoglobulin G, M and A synthesis, assembly, and secretion (1-4). Studies of myeloma-myeloma and myeloma-B-cell hybrids and their subclones have provided considerable information about the genetics and regulation of immunoglobulin production (5-6). Similar investigations concerning immunoglobulin E (IgE) have not been possible due to the unavailability of mouse IgE-myelomas and their cell lines. However, the spontaneous myeloma model in Louvain/Wsl rats, developed by H. Bazin and his colleagues (7, 8) does offer a ready source of IgE-producing myelomas. We report here on the establishment of one of these myelomas, IR 162, in cell culture, its characterization, and the development of a system for the assay of drug effects on cell growth and IgE production.Materials arid methods. Louvain/Wsl/M rats and the IR162 IgE-secreting tumor were obtained from H. Bazin, University of Louvain, Belgium. The tumor was maintained by subcutaneous passage (7), and ascites passages of the tumor were initiated by intraperitoneal injection of tumor cells. IgE secreting cell cultures were established by a procedure similar to that described by Burtonboy et al. (9). Ascites fluid was harvested aseptically; cells were sedimented by centrifugation and resuspended at a density of 3 x 105-3 x lo6 cells/ml in RPMI 1640 medium supplemented with penicillin (100 units/ml), streptomycin (0.1 mg/ml) and 20% fetal calf serum, followed by incubation at 37". Cultures were "fed" by addition of 1 or 2 ml nutrient medium every 24-48 hr depending on the number of cells and the pH of the culture fluid. Culture medium was changed at 7-to 10-day intervals by centrifuging the cells, then resuspending them in fresh medium in the original flask. Adherent cells from the ascites formed a feeder layer to which the tumor cells attached loosely. After 6 to 8 weeks the cell cultures were transferred to medium containing 10% fetal calf serum. Passages were made every 2 or 3 days by breaking up cell clumps with a pipet and diluting the cells 1 5 or 1:lO with fresh medium.Soft agar cloning of tumor cells was performed in agarose solidified RPMI 1640 medium having a final concentration of 17% fetal calf m u m , 5 x M 2-mercaptoethanol and 5 pg/ml gentamicin. 0.5% agarose was included in the base layer and 0.22% agarose in the cell layer. No cell feeder layer was required.Cells grown in vitro were washed twice in serum-free medium and injected subcutaneously or intraperitoneally into Lou/Wsl/M rats to determine whether they retained tumorigenicity. The animals were observed for a minimum of three months.