CELL THAWING AND RECOVERY
1) Immediately after removal from liquid nitrogen, place cryogenic vial in a warm water bath at 37 ° C,
agitating gently until completely thawed. (about 1-2 minutes)
- Rapid thawing reduces or prevents ice crystals from forming within cells during rehydration.
2) Transfer cells to 15 mL conical tube containing 5 mL warm (37 ° C) media (DMEM, 20% FBS, 1%
Pen/Strep). Gently triturate cells and centrifuge at 500 x g x 1 minute (adjust time if needed to pellet
cells). Aspirate off the media, add back 5 mL of fresh media, and gently triturate cell suspension.
- Cryoprotective agents used in the freezing buffer may damage cells upon prolonged exposure and should be removed as quickly and as gently as possible.
- Freezing causes cell membranes to become fragile, treat the cells gently.
- Cells brought out of freezer are initially seeded into media containing 2 x FBS to encourage recovery and growth.
3) Transfer the cell suspension to a suitable culture dish, usually a T-75 flask. Add additional media
proportionate with dish size, if needed, and incubate normally (5% CO2, 37 ° C).
- We use 15 mL media for a T-75 flask.
4) Check cells in 3-4 days. If cells are growing well and look healthy, they can be
switched to normal cell culture media containing 10% FBS (DMEM, 10% FBS, 1% Pen/Strep).
5) After initial media switch from 20% FBS to 10% FBS, media is usually changed every 3-4 days, by
aspirating off old media and replacing with 15 mL of fresh media.
6) Allow cells sufficient time to recover from freezing, at least 7 days, before splitting and reseeding. We
prefer that the dish is completely confluent before the cells are reseeded.
Remember to use only sterile supplies and reagents and to work under a cell culture hood to maintain sterile conditions.
Supplies Reagent Vendor catalog #
DMEM Invitrogen 11965
FBS Atlanta Biologicals S11550H
Pen/Strep Invitrogen 15140-122
