Clonal growth of lymphoid cells in serum-free media requires elimination of H2O2 toxicity

Abstract

We have recently described the development of a serum-free medium that contains casein, insulin, testosterone, transferrin, and linoleic acid and that supports the long-term growth of a wide variety of lymphoid cells. A problem of culturing cells in this medium is the difficulty of cloning cells or growing cells at low density. We now describe the formulation of a chemically defined medium that supports the clonal growth of the murine S49 T lymphoma cell line. This medium contains catalase, insulin, transferrin, testosterone, Na₂SeO₃, and dilinoleoyl phosphatidylcholine and contains less than 50 μg/ml total protein. The two novel additions in this medium are catalase, which replaces casein and dilinoleoyl phosphatidylcholine, which substitutes for linoleic acid in this defined medium. In addition to S49 cells, the medium described above supports the long-term growth of other lymphoid cells, including human and murine hybridomas. We propose that catalase functions to degrade H₂O₂ that is present in the cultures and that casein, bovine serum albumin, and other proteins commonly included in media for cultured cells may also scavenge H₂O₂. Na₂SeO₃ also partially protects against the death of cells at clonal density and this protection may, like catalase, be due to removal of H₂O₂. Our results suggest that H₂O₂ is an important cytotoxic agent that prevents growth of lymphoid cells during culture in serum-free media and perhaps in serum-containing media as well.