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:

  1. 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.
  2. Split cells 1:2 using the desired final serum-free medium as the diluent.
  3. Incubate cells until the maximum cell density is achieved.
  4. 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.
  5. Continue incubation until maximum cell density is achieved.
  6. 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.
  7. 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.
  8. 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.