Establishment of primary myoblast cell cultures from cryopreserved skeletal muscle biopsies to serve as a tool in related research & development studies.

BACKGROUND: Primary myoblast cell cultures display the phenotypic characteristics and genetic defects of the donor tissue and represent an in vitro model system reflecting the disease pathology. They have been generated only from freshly harvested tissue biopsies. Here, we describe a novel technique to establish myoblast cell cultures from cryopreserved skeletal muscle biopsy tissues that are useful for diagnostic and research purposes.

METHODS AND RESULTS: This protocol was performed on seven gradually frozen muscle biopsy specimens from various neuromuscular disorders that were stored in dimethylsulfoxide (DMSO)-supplemented freezing media at -80 °C for up to one year. After storage for varying periods of time, primary myoblast cultures were successfully established from all cryopreserved biopsy tissues without any chromosomal abnormality. Desmin immunoreactivity confirmed that the cell cultures contained >90% pure myoblasts. The myoblasts differentiated into multinucleated myotubes successfully. Furthermore, there were no statistically significant differences in cell viability, metabolic activity, population doubling time, and myocyte enhancer factor 2 (MEF2C) expression between cell cultures established from freshly harvested and one year-stored frozen tissue specimens.

CONCLUSIONS: This protocol opens up new horizons for basic research and the pre-clinical studies of novel therapies by using cryopreserved skeletal muscle biopsies stored under suitable conditions in tissue banks.