Optimization of tissue-culture conditions for differentiation of muscle from dissociated single cells.

Chick embryo extract is widely used to induce differentiation in cultures of muscle and other tissues. As part of a systematic study of factors which promote maximal differentiation of muscle cells, we found that unmodified chick embryo extract could be quite toxic to cells. This report describes the effects of varying culture media composition and cell density on the rate and extent of differentiation as measured by biochemical and morphological criteria. Hind-leg muscles of 1-3-day-old NIH inbred strain mice were excised and dissociated with crude trypsin (Difco) in a balanced salt solution, pH 7.1, for 15 min at 37°C with gentle agitation. The procedure was repeated three times (Wilson et al., 1972). After the first and second dissociations, the mixtures were decanted and the supernatants discarded. The supernatant from the third trypsin treatment was used as a source of the cells. The maximal yields of nigrosin-negative cells were obtained at a concentration of 0.1 % (w/v) trypsin and were up to 4 0 ~ 1 0 ~ cells/g wet wt. of muscle. Unless noted otherwise, single cells were plated on rat tail collagen-coated Falconware dishes in Dulbecco’s medium with 10 % (v/v) foetal calf serum and 2 % (v/v) chick embryo extract and counted 24 h after plating.