When adapting cells from serum-containing to serum-free media, is the direct or sequential adaptation method better?
The sequential adaptation method is recommended. When cells are transferred to serum-free medium from a serum-rich environment, the cells are deprived of components in the serum-rich environment. Sequential adaptation allows the cells to gradually adapt to their new serum-free environment. Below is an example protocol. Cell types vary. Contact us for a tailored solution.
Suggested Sequential Adaptation Protocol:
- Start with cultures (suspension or adherent) that are at maximum cell density and high viability (>80%). If cells are attachment-dependent, trypsinization procedures should be used.
- Split cells 1:2 using the desired final serum-free medium as the diluent.
- Incubate cells until the maximum cell density is achieved.
- Split cells again. The ratio may vary depending on whether the cells are growing attached or in suspension culture. For attached cells, try 1:2 or 1:3 split based on confluency. For suspensions, try 1:3 to 1:5 or approximately 3-4x105 cells per mL.
- Continue incubation until maximum cell density is achieved.
- Monitor the growth and viability of the cells relative to cells cultured in the original or control medium. Cells that have been successfully adapted to serum-free media should have a viability greater than 85%, with a doubling time equivalent to the control medium (serum-containing or other serum-free medium) from which the culture was adapted.
- Low viability or poor doubling times indicate that the culture may not yet be adapted. 3 to 5 successive splits using 1:2 to 1:3 may be necessary. It is not uncommon for some cells to continue to require 0.1 to 0.5% serum.
- Time for adaptation to serum-free conditions will vary with the growth characteristics of the particular cell type. If at any time during the weaning process the culture viability drops below 80%, or the time to double increases significantly, weaning may have occurred too quickly. The cells may require several passages at the previous dilution before renewal of the sequential weaning process resumes.