Detection and control of occult mycoplasma contamination in human tumor cell lines

Summary

Mycoplasma contamination of established cell lines is a well-known but often poorly controlled artefactual problem in immunological studies of human tumor cell lines. We have evaluated four methods for detecting mycoplasmas in cell lines, namely direct culture, DNA staining, uridine phosphorylase assay, and a fourth technique based on our finding that the supernatant medium of mycoplasma-infected cell cultures inhibits thymidine uptake of mitogen-stimulated peripheral blood lymphocytes. In our hands the simplest, most reliable, and least expensive means of monitoring cell cultures for mycoplasma proved to be DNA staining. The uridine phosphorylase assay was unsuitable for use with melanoma cell lines, as six of eight lines that were negative with the other three techniques were positive with this assay.

Of 14 contaminated cell lines injected to nude mice, eitht produced tumors, five of which were shown to be mycoplasma-free after one to five passages, confirming the usefulness of this approach for salvaging contaminated cell lines.