Primary skeletal muscle culture and differentiation

Skeletal muscle culture for the aneural system has been previously described (Falcone et al., 2014). Skeletal muscle cultures for the co-culture system were performed as previously described (Falcone et al., 2014) and with the following alterations. Tibialis anterior, extensor digitorum longus, gastrocnemius and quadriceps from thigh muscles were sampled from P7 pups. Muscles were minced manually then digested in 0.5 mg/ml collagenase and 3.5 mg/ml dispase at 37°C for 1.5 h (Fig. 1, day −6). Digestion was stopped with dissection medium consisting of IMDM with Glutamax (Gibco, 31980-022), 1% penicillin/streptomycin (Gibco, 15140-122) and 10% fetal calf serum (Eurobio, CVFFCSF00-01). Cells were centrifuged at 600 rpm (72 g) for 6 min and the supernatant was recovered and centrifuged at 1300 rpm (340 g) for 7 min. Cell pellet was re-suspended in dissection medium and filtered on a 40 µm cell strainer (Falcon Corning, 352340) and re-suspended in dissection medium in 100 mm Petri dishes (Falcon Corning, 353025) for pre-plating. Pre-plating was carried out over 3 h. Fluorodishes suitable for immunofluorescence (3.5 mm; World Precision Instruments, FD35-100) were coated with Matrigel. After pre-plating, cells were recovered, centrifuged at 1500 rpm (453 g) for 10 min and re-suspended in proliferation medium consisting of IMDM with Glutamax, 1% penicillin/streptomycin, 20% fetal calf serum and 1% chicken embryo extract. Once primary myoblasts had reached 60-70% confluence, cells were switched to differentiation medium consisting of IMDM with Glutamax, 1% penicillin/streptomycin and 2% horse serum (Gibco, 26050088) (Fig. 1, day −3). The day after (Fig. 1, day −2), myotubes were coated with Matrigel and kept in fresh differentiation medium for 48 h at 37°C in a 5% CO₂ incubator before plating of spinal cord explants.