—Protocol—

Human Myoblast Culture

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.

Growth Media:

• F10
• 15% FBS —OR— 10% FBS + 5% defined, supplemented" calf serum (Hyclone, iron supplemented), to save money on FBS.
• Pen-Strep
• 0.5% Chick Embryo Extract

  Ideally, cells are grown on collagen coated dishes.

Fusion Media:

• DME
• 2% horse serum
• 10-6 M Insulin
• 2.5 X 10-6 M Dexamethasone

Insulin: Sigma I5500; 100 X stock (10-4 M) = 60 mg/ 100 ml dH2O + 85 µl conc. HCl

Dex: Sigma D1756; stock = 10-2 M in 95% ethanol; use 25 µl/100 ml.

Notes:

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.