Materials and equipment

• Eagle’s minimum essential medium, 10% foetal bovine serum, Eagle’s non-essential amino acids and other supplements as necessary
• Anchorage-dependent cell lin (e.g. MRC-5)
• Microcarrier (e.g. Cytodex3)
• PBS
• Spinner vessel adapted for microcarrier culture (Bellco or Techne)

Operations

1. 200ml complete medium to 1L spinner flask, 5%CO2,
2. Rinse Cytodex3 with PBS, and replace with medium (1g to 30-50 ml). Add cytodex 3 to spinner vessel (1-3g/L)
3. Put spinner on magnetic stirrer, 37C, equilibrate平衡for 1 hour
4. Add cell inoculum obtained by trypsinization of late log phase cells, 5-7cells/bead, 5-10 x104 /cm2. Cytodex3 at 2 g/l inoculated at six cells/bead. 8 x106 microcarriers=4.8 x 107 cells/l 9500 cm2=5 x 104 cells/cm2 200ml=2.5 x 105 cells/ml
5. Spinner flask 20-30rpm. Cells and carriers are limited to the 60-70% would be better. alternatively either: Inoculate in 50% of the final medium volume and add the rest of the medium after 4-8 hr. or stir intermittently (for 1min every 20min) for the first 4-8h.
6. After the cells have attached, the stirring speed increase to 40-60 rpm
7. Monitor progress by taking 1 ml samples daily
8. As the density increases there is often a tendency for microcarriers to clumping. So increasing the stirring speed to 75-90 rpm
9. After 3-4 days the culture will become acid. Re-gas the headspace or add sodium bicarbonnate(5.5% stock).
10. After 4-5 days, cells reach a maximum cell density. 1-2 x106 cell/ml (2-4 x 105 /cm2)
11. Harvest cells

• Harvest cells