Canine muscle cell culture and consecutive patch-clamp measurements - a new approach to characterize muscular diseases in dogs

Muscular biopsies from 16 dogs of different age and breed were taken during standard surgical procedures and were stored for one to three days at 4°C in a transport medium in order to simulate shipping conditions. Afterwards biopsies were professionally processed, including harvesting of satellite cells, inducing their proliferation, differentiating them into myotubes and recultivating myotubes after long-term storage in liquid nitrogen. Myogenic origin of cultured cells was determined by immunofluorescence, immunohistology and by their typical morphology after inducing differentiation. Subsequent to the differentiation into myotubes feasibility of patch-clamp recordings of voltage gated ion channels was successfully.We have developed a canine muscle cell culture system, which allows sampling of biopsies from young and old dogs of different breeds under practical conditions. Patch clamp measurements can be carried out with the cultured myotubes demonstrating potential of these cells as source for functional research.In dogs, research into motor unit disorders is a growing field [1-3]. Up to now investigations regarding the pathogenesis are mainly based on electromyography, motor and sensory nerve conduction velocity measurements and histopathological examination of muscle and nerve biopsies or are based on the identification of genetic abnormalities [3-8]. Approaches to evaluate functional components of such disorders in cell culture systems have not been established in veterinary neurology so far.All previously mentioned procedures for muscle satellite cell culture systems of animals are described predominantly for use under laboratory conditions. Hereby, tissue processing such as harvesting of satellite cells takes place immediately. This limits the application of the technique for investigating spontaneously occurring disease. Only animals kept in the vicinity of or in the laboratory can be used for setting up the primary culture. Furthermore, applying these meth