Protocol #1: Medium Replacement –
For Adherent Independent Cells (Suspensions)
Approximate time required: 2 weeks – 6 weeks
Culture conditions:
- Mammalian cells: 95% air, 5% CO2, 35°C–37°C
- Invertebrate cells: air, 25°C–27°C
- Begin with cultures at maximum cell density.
- NOTE: Attachment dependent cells that are exposed to trypsin during subculturing should be converted to serum-free growth using Protocol #2.
- Split cells 1:2 using serum-free medium as the diluent.
- Incubate cells until the maximum cell density is achieved.
- Split cells 1:5 or to 3.0 × 105 cells/mL for attachment independent cells or 30% confluency for attachment dependent cells using serum-free medium as the diluent.
- Incubate cells until the maximum cell density is achieved.
- If the cell viability is >85% at this point, and the generation time is similar to that observed with serumcontaining medium, the culture may be maintained in serum-free medium using a similar split schedule as originally optimized for serum-containing medium.
- If the cells exhibit slow growth or low viability, maintain the split ratio at 1:2 or 1:5 for 3 successive splits. The minimum cell density should be above 3.0 × 105 cells/ mL or 30% confluency during this period.
- Gradually increase the split ratio to obtain a maximum value for the cell type being used.
NOTE: Some cells may require a small amount of serum for growth. If the cells have not adapted to serum-free cultivation using the above protocol, add 0.1%–0.5% serum to the culture or contact Scientifc Support.