The aim of this study was to select an effective and stable protocol for the differentiation of human satellite cells (Sc) and to identify the optimal time period for the experimental use of differentiated human Sc‐cultures. In order to identify the differentiation conditions
which give a good survival of myotubes and a high grade of differentiation, Sc‐cultures were induced to differentiate in media supplemented with either 2% fetal calf serum (FCS) 2% horse serum (HS) or 10% HS. Based on higher CK‐activities in cultures differentiating in FCS‐supplemented
media compared to horse sera, fetal calf serum was chosen to induce differentiation. The ATP, DNA and protein content increased during the first 4 days after induction of differentiation and was followed by a period with minor changes. The maximal differences of ATP, DNA and protein between
days 4–10 were evaluated and the differences in the three components were found to be less than 20% of the average value with a certainity of more than 0.9. Day 8‐myotubes were investigated morphologically and were found immunoreactive for fast myosin, and expressed areas with
clear cross striation. We recommend the use of differentiated Sc‐cultures in the period from day 4 to 8 after induction of differentiation as only minor differentation‐related changes will take place in the cells during this period of time.