Serum-free medium allows chicken myogenic cells to be cultivated in suspension and separated from attached fibroblasts

Abstract

When cells obtained by trypsinization of embryonic chick muscle tissue are cultured in an insulin-supplemented, serum-free medium, most cells attach to the substratum. Later, many cells detach. The suspended cells are myogenic: they remain viable and, over a period of days, aggregate and then fuse to form syncytial “myoballs”. If, however, the medium is supplemented with serum, the suspended cells attach to, and spread upon, a gelatinized substratum, yielding cultures that are nearly free of fibroblasts and in which approx. 85 % of all nuclei are in myotubes. The remaining uninucleated cells are bipolar, elongated, and—like the myotubes but unlike fibroblasts—show positive immunofluorescent staining for the MM isoenzyme of creatine kinase; this indicates that, though unfused, they have initiated terminal differentiation. During the first day in serum-free medium the suspended cells include many proliferative myogenic precursors, as indicated by the fact that more than 50% of these cells incorporate [ 3H]thymidine into their DNA. By the end of the second day, almost all of the suspended cells are post-mitotic myoblasts: only about 5 % of the cells incorporate [ 3H]thymidine. The cells which remain attached after 3 days or so are non-myogenic (presumably fibroblasts): few myotubes form when such cells are given a serum-containing medium known to allow extensive myotube formation in primary cultures of cells from embryonic chicken muscle. The reattachment of suspended cells is being used to assay for substances in serum which promote myoblast attachment and spreading.