Abstract
Skeletal (voluntary) muscle is the most abundant tissue in the body, thus making it an important biomedical research subject. Studies of neuromuscular transmission, including disorders of ion channels or receptors in autoimmune or genetic neuromuscular disorders, require high-spatial-resolution measurement techniques and an ability to acquire repeated recordings over time in order to track pharmacological interventions. Preclinical techniques for studying diseases of neuromuscular transmission can be enhanced by physiologic ex vivo models of tissue-tissue and cell-cell interactions. Here, we present a method, which allows tracking the development of primary skeletal muscle cells from myoblasts into mature contracting myotubes over more than 2 months. In contrast to most previous studies, the myotubes did not detach from the surface but instead formed functional networks between the myotubes, whose electrical signals were observed over the entire culturing period. Primary cultures of mouse myoblasts differentiated into contracting myotubes on a chip that contained an array of 26,400 platinum electrodes at a density of 3,265 electrodes per mm2. Our ability to track extracellular action potentials at subcellular resolution enabled study of skeletal muscle development and kinetics, modes of spiking and spatio-temporal relationships between muscles. The developed system in turn enables creation of a novel electrophysiological platform for establishing ex vivo disease models.
Highlights
Skeletal muscle cells are excitable, multinucleated cells that generate electrical signals as a result of depolarization and, once mature, contract
After 1–2 days in differentiation medium, myoblasts were seen fusing into myotubes
During the first few weeks of cell culture, we observed electrical activity in the form of isolated spikes or isolated “islands” of spikes, which slowly organized into much larger spiking areas that eventually were seen over most of the chip
Summary
Skeletal muscle cells are excitable, multinucleated cells that generate electrical signals as a result of depolarization and, once mature, contract. The beginning of prominent contractions, marks the true beginning of a mature cell culture that resembles physiological function in the body. It is not until an age of about 14 days that myotubes become highly developed with well-organized A- and I-bands and Zlines along with well-organized mitochondria, and the majority of myotubes contracts in unison (Nag and Foster, 1981). It may not be until almost 1 month in vitro that the formation of the excitation-contraction apparatus is fully developed (Flucher et al, 1994; Das et al, 2009)
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