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Cryopreservation of Mammalian Cells
Karmella Haynes & Ben Nyer, 2014
- 70% ethanol spray bottle
- 1x Phosphate-buffered saline (PBS)
- Trypsin-EDTA buffer/ media
- Complete cell culture medium (e.g., 10% FBS, 1% pen-strep)
- Fetal Bovine Serum (FBS) with 10% DMSO (in freezer)
- For MCF 10A cell lines: prepare complete cell medium w/ 7.5% DMSO added
- 15mL Falcon tubes
- 2mL cryo vials
- 100% confluent (dense) cell culture in T-75 flask(s) (check confluency under the light microscope)
- Warm up the PBS, Trypsin-EDTA and complete cell culture medium to 37°C
- Thaw the FBS/DMSO solution and then place on ice.
- Label one 15mL conical per T-75 flask you are turning into frozen stock.
- Turn on the vacuum pump.
- Label five 2mL cryo vials per T-75 flask you are turning into frozen stock. Label them with the cell line, date, and your initials.
- After reagents are warmed, spray bottles down with 70% ethanol and prepare the biosafety hood as for routine work. Do all work in the biosafety hood.
- Harvest cells from the flask in the same manner you would for passaging cells. You will end up with cells suspended in 10ml growth medium (includes the trypsin-EDTA).
- Transfer the 10 mL of cells into the 15 mL Falcon tube. Pellet the cells at room temp for 5 min at ~200 g (typical spin). Use a balance if you have an odd number of tubes.
- Aspirate off the growth medium from the pellet, but leave a bit of liquid covering the pellet. Flick the tube with your finger to break up the pellet.
- Sterilize the chilled FBS/DMSO bottle with the ethanol spray bottle and place it in the biological safety cabinet.
- Resuspend the cells in 5 mL chilled FBS/DMSO. Pipet up and down a couple of times to make sure there are no cells left in a pellet at the bottom.
- Aliquot 1 mL of cells into each cryo vial. Place closed vials on ice. Chill for 5 min.
- Get a styrofoam box with a lid and place a multi-tube insert from a cardboard freezer box into it. This will serve as a slow-cooling unit for the cells.
- Place cell vials into the slow cooling unit. Place this in the -80 C freezer for two days (48 hrs).
- Transfer tubes to -150 C for long term storage.