Establishment of in vitro co-culture system for embryonic rat spinal motoneurons and C2C12 myotubes / 第二军医大学学报

ABSTRACT

Objective To identify the conditions for co-culturing embryonic rat spinal motoneurons and C2C12 myotubes, establish a stable co-culture system, and to form functional neuromuscular junction in vitro. Methods The C2C12 myoblasts were cultured to 60%-70% confluence and then were induced by differentiation medium. The embryonic spinal cord anterior horn motor neurons were obtained from 15-16 d pregnant SD rats, and were implanted in the myotubes after differentiating for 5 days; the products were co-cultured in the basic serum-free culture medium Neurobasal+2% B27. The neuronal morphology and projection length at each stage, myotubemorphological and contraction characteristics, and formation of the neuromuscular junction were observed under an inverted microscope. The crbungarotoxin (crBTX), which can specifically bind to acetylcholine receptor (AChR) of the postsynaptic membrane, was examined by immunofluorescence technique and the muscle contraction in the co-culture system was recorded by screen recording technology. Results Both the primal spinal motoneurons and the C2C12 myotubes survived in the co-culture system, with further differentiation and maturation. On day 3 the axons extended to the myotube membrane surface or surrounded the myotubes. On day 7 the myotubes were arranged in the same direction, with wide rhythmic contraction, and immunofluorescence showed that crBTX specifically bound to AChR of the postsynaptic membrane. On day 10 of co-culture, the motor neurons began to have apoptosis and the myotube cells gradually shrank. Conclusion Under in vitro culture condition, motor neurons and skeletal muscle cells can co-exist and grow, establishing synaptic connections, triggering a series of neuromuscular junction signal transduction, and causing rhythmic contraction of the myotubes.