Serum-free culture conditions for analysis of secretory proteinases during myogenic differentiation of mouse C2C12 myoblasts

We have been studying extracellular proteins such as proteinases and attachment factors under serum-free culture conditions. A number of studies on myogenesis using an in vitro culture system have reported that proteinases and ECM components play significant roles in muscle differentiation. However, most of the studies were performed in the presence of serum. Serum is abundant in the aforementioned proteins and its use in serum-free culture affects many cellular functions significantly. In this study, we tried to establish serum-free culture conditions for analyzing extracellular proteins involved in mouse myogenic differentiation. By evaluating media, supplements, and procedure of cell inoculation under serum-free conditions and by comparing the resultant conditions with conventional conditions on differentiated characteristics of the cells, it was revealed that serum-free Dulbecco's modified Eagle's medium/Ham's F-12 plus insulin more efficiently supported myogenesis morphologically and biochemically than conventional 2% horse serum-containing culture and that secretory proteinases obtained from our serum-free culture were different from those obtained utilizing conventional serum-free cultures in their activities and patterns. Since our serum-free medium consists of simple components, the medium is low cost and easy to prepare. Furthermore, the results suggest that our culture conditions are superior to conventional conditions biochemically and morphologically and will provide more precise and accurate information on extracellular proteins involved in myogenesis. (C) 1999 Academic Press.