Biotin, B 12, and other vitamin requirements of a strain of mammalian cells grown in chemically defined medium

Abstract

A strain of mouse cells designated NCTC2071 has been cultured for years in chemically defined medium NCTC 109 free of antibiotics or any undefined supplements. This strain, derived from clone NCTC 929-L, and free of pleuropneumonia-like organisms, was examined with respect to vitamin requirements. The effects of the 18 vitamins in medium 109 on cell survival, rates of proliferation, and morphology were determined in long-term studies. Inter-relationships between the vitamins and other components of the medium such as the nucleic acid derivatives were explored. In a coenzyme-free modification of medium 109, the cells required 5 vitamins for survival, namely, niacinamide (or niacin), thiamine, riboflavin, pantothenate, and choline chloride. In addition, pyridoxal (or pyridoxine) prevented destruction of cells when subjected to mechanical agitation, and folic acid and biotin slightly increased rates of cell proliferation but were not required for continuous cell propagation. Eight vitamins appeared to have no significant effects on the cells, namely, vitamins A, D, K, E, C, p-aminobenzoic acid, B 12, and i-inositol. When cells were cultured in a medium lacking all the nucleic acid derivatives except deoxycytidine, a requirement for folic acid was demonstrated. Neither thymidine nor vitamin B 12 completely substituted for this requirement. In a medium containing the seven vitamins and only two of the five nucleic acid derivatives, the cells required biotin for survival, a requirement that was not detected when cells were grown in the complete medium. The lots and/or levels of cysteine in medium 109 were found to be toxic for single well-isolated cells. A toxicity of glutathione was counteracted by vitamin C. However, all three reducing agents could be eliminated without detectable reduction in cell growth. A stimulatory effect of vitamin B 12 on cell proliferation was observed under one set of conditions. Cells that had been cultured for a long period in a medium lacking all nucleic acid derivatives exhibited a marked stimulatory response to the vitamin. However, cells cultured for months in an identical medium except that it contained thymidine and deoxycytidine failed to show any response to the vitamin. Interpretations of these results are discussed. The influence of the type of chemically defined medium on the transplantability and/or malignancy of the cells was tested. Several new formulations of chemically defined media with evaluations of their action on these cells are presented.