Freezing and thawing of cell lines
Hybridoma and myeloma cell lines are stored by freezing the cells at a controlled rate (approximately 1ºC per minute) in an appropriate cryoprotectant. This procedure allows the cell line to be preserved indefinitely.
- Only cells that are healthy and rapidly dividing should be frozen.
- One or two days before freezing, split the cells 1:10 into fresh medium and maintain in culture.
- On the day of freezing, count the cells.
- You need to have between 2 to 5x106 cells in each freezing vial.
- Transfer the appropriate volume of cells to a sterile centrifuge tube.
- Spin at 300g for 5 minutes.
- Carefully remove as much supernatant as possible without disturbing the pellet.
- Gently resuspend the cell pellet in 1.5ml of freezing medium containing 10% Dimethylsulphoxide (DMSO, from Sigma D2650) in 90 % FCS previously heat-inactivated).
- Transfer 1.5ml of the resulting cell suspension into a freezing vial (Thermo 375418).
- Seal the vial (finger tight) to prevent liquid nitrogen from entering the vial.
- Place the sealed vials in a special freezing container (Thermo Scientific 5100-0001) which allows the cells to cool down slowly.
- Close the freezing container and place at -80ºC for at least 4 hours.
- Transfer the vials of frozen cells to liquid nitrogen.
- Extended storage of cells at -80ºC is not recommended.
- Prolonged exposure to DMSO is toxic to cells. For example, handling more than 10-20 vials at any one time will lead to extended exposure of the cells to DMSO prior to freezing.
- Remove the frozen vial from the liquid N2 storage.
- Loosen the cap of the vial slightly to release the pressure inside.
- Thaw the cells in a 37ºC water bath.
- Keep the lid of the freezing vial above the surface of the water to lessen the chances of contamination.
- When the cells are almost thawed (only a small piece of ice) move the vial to the tissue culture hood.
- Wipe the outside of the vial with 70% ethanol and remove the top.
- Carefully remove the cell suspension using a sterile Pasteur pipette.
- Transfer the contents to a centrifuge tube containing 10 ml of appropriate culture médium (See Appendix I – remember myeloma medium should not contain HAT).
- Spin the cell suspension gently at 300g for 5 min.
- Carefully remove the medium without disturbing the pellet.
- Gently resuspend the cells in 10ml of fresh appropriate culture medium and place in a small T25 flask (Corning 3056).
- Take 1ml from this flask and add to 9mls of complete culture medium in another small flask. This step ensures that at least one concentration of cells is suitable for continued culture.
- Place the flasks in a 5% CO2 incubator with their tops loosened enabling gaseous exchange to occur.