My primary cells survive thawing but do not proliferate. What should I check?

Good post-thaw viability does not necessarily mean that primary cells will immediately proliferate. If the cells survive thawing and attach but show little or no growth, first check the culture conditions and handling steps.

Quick checks:
Growth medium and supplements
Make sure you are using the recommended growth medium for your specific cell type and that all required supplements have been added correctly. The recommended media are important for achieving the expected proliferation potential.
Thawing procedure
Review the thawing procedure. For anchorage-dependent primary cells, the established protocol specifies thawing followed by plating at the recommended seeding density. Depending on the protocol, cells can be directly plated or centrifuged before seeding.
Seeding density
Check that the cells were seeded at the density recommended in the cell-specific Instruction Manual. The established thawing protocol specifically requires plating cells at their recommended seeding density.
Cell attachment and morphology
Examine the culture microscopically and monitor whether the cells attach and develop the morphology expected for the respective primary cell type.
Post-thaw lag phase and population doubling time (PDT)
Keep in mind that some primary cells or cell types may have a prolonged lag phase after thawing before active proliferation becomes apparent. Cells with a longer population doubling time (PDT) may also initially appear to grow very slowly, even when the culture is developing normally. Check the lot-specific PDT stated in the Certificate of Analysis (CoA) when assessing whether the observed growth rate is within the expected range.
First medium change
Avoid changing the medium too early unless the cell-specific protocol specifies otherwise. For cells centrifuged before plating, the general published thawing protocol specifies changing the medium no earlier than 24 hours after seeding. Some cell types have different requirements.
Cell-type-specific requirements
Always check the Instruction Manual for the respective cell type, as thawing, seeding, coating, medium change, and culture requirements can differ.

If proliferation remains impaired after the recommended culture conditions and the expected PDT have been considered, please contact our Scientific Support team for assistance with troubleshooting.