Primary Murine Myotubes as a Model for Investigating Muscular Dystrophy

Figure 1

Flow chart for primary muscle fibers and satellite cells isolation and transduction. (a) Flow chart for primary muscle fibers transduction. Muscles after isolation underwent enzymatic digestion and then were triturated either in nonconcentrated LV or in DMEM supplemented with 20% FCS. After trituration in nonconcentrated LV (MOI 1), muscle fibers were transduced via nonconcentrated LV as well and were incubated with nonconcentrated LV for 5 min, 1.5 h, 3 h, or 4.5 h. Then muscle fibers were plated either directly to Geltrex-coated dish or cultivated on noncoated dish overnight and were then plated to Geltrex-coated dish. When cells were triturated in DMEM supplemented with 20% FCS, transduction was carried out either via nonconcentrated LV or via concentrated LV (MOI 100). Depending on LV type time of incubation varied, for nonconcentrated LV–5 min, 1.5 h, 3 h, and 4.5 h and for concentrated LV–5 min, 1.5 h, and 3 h. After transduction via concentrated LV cells were directly plated on Geltrex-coated dish. (b) Flow chart for satellite cells isolation, transduction, and cultivation. Satellite cells were isolated by means of enzymatic digestion and then centrifuged for 5 min at 400 ×g, and supernatant was discarded. Obtained cell pellet was twice resuspended in 2.5 mL of washing media (DMEM supplemented with 10% HS), suspension was settled for 5 min by gravity, and upper phase was transferred into fresh tube and spun down for 10 min at 1000 ×g. Cells pellet was dissolved in 0.5 mL of proliferation media (DMEM supplemented with 20% FCS, 10% HS, 1% CEE) and transduced via concentrated LV (MOI 20); polybrene at final concentration 8 μg/mL was added to cells. 72 hours after transduction positively transduced cells were observed. Seven days after isolation cells reached confluence and were induced to differentiation. 48 hours after differentiation first spontaneously contractile myotubes were detected. After seven days of differentiation myotubes were taken in analysis (immunocytochemistry, patch-clamp, and calcium measurement). Three weeks after isolation myotubes started to degrade. LV, lentivirus; MOI, multiplicity of infection; PM, proliferation media; WM, washing media.

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