Protocol Citation: PMAT0001 2020. Cell cryopreservation. protocols.io https://protocols.io/view/cell-cryopreservation-bnmdmc26
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 20, 2020
Last Modified: October 21, 2020
Protocol Integer ID: 43397
Guidelines
- This protocol is for ADHERENT CELLS
Materials
MATERIALS
Cell counterThermo Fisher Scientific
DMSOJ.T. BakerCatalog #9224
Bright-Line HemacytometerFisher ScientificCatalog #02-671-5
Trypan Blue Stain 0.4% Invitrogen - Thermo Fisher
- Use a DMSO bottle only for cell culture work; Open only in laminar flow hood
Before start
- Asceptic techniques
- Wipe down hood and any item introduced into the hood with 70% ethanol
Gently detach cells from the tissue culture flask in the following manner:
Aspirate media
Wash with 2 mL PBS, then aspirate.
Wash with 2 mL trypsin.
Incubate for 00:05:00 at 37 °C .
5m
Resuspend the cells in 10 mL of DMEM with FBS. Aliquot into a centrifuge tube. Of course, if there is a T25 flask, resuspend in lesser volume.
Aliquot a small amount of the mixture (about 200 µL ) for cell counting. Ideally, cell viability should be in excess of 90% in order to achieve a good recovery after freezing. Keep at -4 °C
- We need 1 x 106 - 5 x 106 cells for freezing.
Centrifuge the cell suspensions at 1500rpm for 00:05:00
5m
Discard supernatant carefully
Add about 3 mL of freezing media into the tube and re-suspend.
Freezing media preparation:
- 10% DMSO and 90% appropriate cell culture medium
(e.g 500uL of DMSO + 4.5mL of cell culture medium with FBS and antibiotics)
Dispense the aliquots of cell suspension into the cryogenic storage vials.
Cool the vials gradually as follows:
-20 °C for 01:00:00
-80 °C overnight
Liquid Nitrogen
1h