Establishment of a serum free medium for human muscle cells and its application of studyefor muscle disease

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1. Establishment of a chemically defined medium for humuan muscles. We prepared a chemically defined medium for human muscles. Briefly, the medium consisted of F12 medium intensified with insulin, transferin and etc.The medium allowed to grow human muscle cells and to generate myotubes with poly nucliei. These myotubes were able to contract spontaneously.This medium was also useful for in vitro studies of muscles from patients with cytochrome c oxidase difficiency and for selective induction of muscle cells from undifferentiated malignant fibrous histiocytoma.2. Enzyme immune assay for Thy-1 antigens expressed on cultured muscle cells and quantative immunohistchemical study on frozen sections of biopsied muscle tissues.From the qualitative studies of histochemistry, Thy-1 antigens have been suggested to be a surface marker of muscle differentiation.In the present study, we developed an enzyme immuno assay for quantitation of extracellular and intracellular Thy-1 antigens expressed by muscle cells cultured in 96 well-microplates. CK and LDH activities were also measured as muscle differentiation markers. Thy-1 antigens could be detected as early as 24 hours after trypsinization of muscle cells by our assay method but not by the immunofluorescence staining. The decrease of extracellular Thy-1 antigens but the increase of intracellular Thy-1 antigens were observed in parallel with the development of myotubes. When this procedure was applied for frozen sections of biopsied muscle tissues, several muscle enzymes were successfully quantitated.