Evaluation of three methods for curing hybridomas from mycoplasma contamination

Abstract

Hybridomas are as susceptible to mycoplasma contamination as animal cell cultures. Two in vitro methods and one in vivo passage into mice were compared for efficiency of curing hybridomas from mycoplasma contaminations. Four contaminated clones were treated with the combined action of 5-bromouracil (5-BrUra) and the Hoechst 33258 followed by photosensitization. The other in vitro method involved the use of BM-cycline. The success of overcoming the mycoplasma contamination was dependent on the level of the initial contamination of the individual hybridoma. BM-cycline was more efficient than the photosensitization method. For the most contaminated hybridomas, 10 successive treatments with 5-BrUra were necessary as compared to six treatments with BM-cycline. Moreover, the use of BM-cycline reduced the contamination by as much as 50% after the first treatment. After twenty passages following the curing of hybridomas with BM-cycline, cells were stable and retained their specificity and secretion of their respective immunoglobulins. Whereas, treatment with 5-bromouracil, recurrence of contamination was observed in one of the four hybridomas after 10 passages following treatment. Decontamination after one passage in peritoneal cavity in mice was not always sufficient since one of four hybridomas remained contaminated. BM-cycline appears to be the method of choice since it is more efficient, less time consuming, simpler and less expensive. Mycoplasma strains that could be identified were of bovine origin: Mycoplasma arginini and Acholeplasma laidlawii.