1. Examine all flasks by inverted-phasemicroscopy. Cultures used for preservation should be grown free of antibiotics, show no signs of microbial contamination and should be subconfluent (confluent cultures may be less amenable to freezing).
2. Remove the growth medium and wash twice in PBS (B0.1 ml cm2).
3. Add prewarmed (37 1C) trypsin solution (0.5 ml per 25 cm2) to cover the cell monolayer, recap the flask and incubate atB37 1C for 5 min.
4. Examine the monolayer and tap the flask firmly on the bench to dislodge the cells. If the majority of the cells are not dislodged, continue the incubation for a further 5 min.
5. Harvest cells from each flask in 10 ml of medium containing serum or some other component that inactivates trypsin, bulk contents of all flasks and carry out a viable cell count by trypan blue dye exclusion or an alternative method, transfer to conical-based centrifuge tubes, mix by inversion and centrifuge at B100g for 5 min.
6. Aspirate the supernatant and resuspend the cell pellet in cryoprotectant medium to give a final cell concentration of at least 106 cells per ml(ideally between 2 and 5 106).
7. Mix the cell suspension by inversion in the cryoprotectant medium and divide into 1-ml aliquots in sterile labeled cryovials. Seal each vial immediately after it is filled.
8. Wrap the vials loosely in paper toweling (for insulation) inside a small polystyrene box and tape the box lid in place. Alternatively,proprietary devices such as the Mr Frosty (Invitrogen) can be used. Note that a variety of controlled rate freezing devices are marketed,which can help to minimize ice crystal damage and preserve greater cell viability. Nevertheless, the protocol described is adequate for most cell lines.
9. Transfer the box to a freezer overnight in a location where it will not be disturbed.
m CRITICAL STEP The freezer should be at 70 1C or lower.
10. The next day, place the box directly into the vapor phase of liquid nitrogen and then transfer all vials to a storage location in the vapor phase of liquid nitrogen. Alternatively, if vapor phase is not an option and there is no biohazard, transfer the vials to a small quantity of liquid nitrogen
and transfer to storage in liquid nitrogen. It is recommended to enclose the ampoules in case of explosion.! CAUTION Liquid nitrogen burns. Wear protective gloves and facial protection when using liquid nitrogen.
11. After overnight equilibration in nitrogen storage, recover one vial of cells and thaw rapidly at 37 1C in an incubator.
12. Gradually dilute the cell suspension by dropwise addition of prewarmed antibiotic-free growth medium.
13. Take a sample for a viable cell count and transfer the remaining cells to fresh growth medium in a T-25 culture flask and incubate and observe over several days to check for adequate adherence and growth.
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