Passaging and Conservation

In monolayer cultures, skeletal muscle cells will differentiate upon reaching confluence due to contact inhibition, even in high nutrition media. To prevent this, once cells have fused and exhibit irreversible withdrawal from the cell cycle, cells have to be detached from the growth surface by treatment with a trypsin solution, diluted, and plated in a fresh Petri dish. Although trypsinization is a standard procedure described in every culture manual (e.g. refs 53, 122), the optimal procedure depends on how firmly a specific cell type attaches to the surface and how sensitive the cells are to different concentrations of trypsin. Our human skeletal muscle cell cultures are passaged before the cultures reach confluence and before any myotubes are visible. The medium is aspirated from the cells and monolayers are treated with warm 0.5mM EDTA in DPBS for 1 minute. After aspiration of the EDTA solution, a thin layer of pre-warmed 0.025% trypsin (1:250) is added, and the cells are placed at 37°C for a few minutes until they detach from the surface and float. The action of trypsin is stopped by adding an equal volume of culture medium, and the cells are collected by centrifugation (6 min, 50g) in a 15 ml conical tube. After careful aspiration of the supernatant, the cell pellet is loosened by gently flicking the tube, the cells are resuspended in culture medium and plated in fresh culture dishes. A maximum subcultivation ratio of 1 :6 is recommended. If trypsinized cells are to be frozen, the cell pellet obtained after centrifugation has to be loosened, suspended in sterile freezing medium at a density between 2 x 105 and 5 x 106 cells per ml, and frozen as described above for skeletal muscle tissue fragments (see section 2.2). Freezing of skeletal muscle cells does not significantly alter their capacity to proliferate and differentiate if the frozen cells were not grown for a long period in culture [49, 50, 52]. It is, therefore, advisable to freeze large numbers of vials from the earliest passages of each individual culture.

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