From the Laboratory of Helen M. Blau, Stanford University
See references 1)Blau and Webster(PNAS) and 2)Webster et al.,(ECR)
1. Thaw frozen human myoblast cells rapidly in 37°C bath and dilute into 10 ml of GM in a 100 mm Falcon tissue culture dish (# 3003). For best long-term maintenance, cells should be grown on collagen coated dishes.
2. Incubate cells at 37°C in 5% CO2.
3. Change medium after all cells have adhered.
4. Routinely split the cells 1:4 when they get dense, usually every 2 to 3 days. Do not allow the cells to reach confluence or they will begin to differentiate, even in growth media. Cells are usually grown to about "80%" confluence. Refeed cells on second day if they are not split.
5. To differentiate, replate cells on collagen coated dishes and allow the cells to grow to about "80% - 90%" confluence. Switch dense cells into FM and feed every day. You will not get differentiation of 100% of the cells.
Ideally, cells are grown on collagen coated dishes.
These cells are primary cultures from human skeletal muscle tissue. They are not immortal. They have a limited life span in culture, and are unique and irreplaceable.