Jul 23, 2020
Freezing cancer cell lines
- Emily Souster1,
- Verity Goodwin1,
- Adam Jackson1,
- Charlotte Beaver1,
- Rizwan Ansari1,
- Fiona Behan1,
- Mathew Garnett1
- 1Wellcome Sanger Institute
Protocol Citation: Emily Souster, Verity Goodwin, Adam Jackson, Charlotte Beaver, Rizwan Ansari, Fiona Behan, Mathew Garnett 2020. Freezing cancer cell lines. protocols.io https://dx.doi.org/10.17504/protocols.io.bgtyjwpw
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: May 25, 2020
Last Modified: July 23, 2020
Protocol Integer ID: 37464
Abstract
This protocol outlines routine banking of cancer cell lines and Ca9 transduced cancer lines.
Process diagram:
Guidelines
- As a guideline, we usually bank 5 cryovials from a 70% confluent T150 flask, each containing 1ml cell suspension.
Materials
MATERIALS
Falcon™ 15mL Conical Centrifuge TubesFisher ScientificCatalog #14-959-53A
TrypLE™ Express Enzyme (1X), no phenol redThermo FisherCatalog #12604021
Nunc™ Biobanking and Cell Culture Cryogenic Tubes, 1.8mL, 48mm, external thread, printedThermo FisherCatalog #375418
DMSOSigma AldrichCatalog #D2650
DPBSInvitrogen - Thermo FisherCatalog #14190
Select an appropriate culture media for your cell line. Common culture medias used for cancer cell lines are serum supplemented Advanced DMEM F-12 or RPMI, in the presence of pen-strep.
Equipment
Light Microscope
Microbiological Safety Cabinet (MSC)
Pipette Boy
Stripettes
Pipettes and tips
37 °C , 5% CO2 incubator
Centrifuge
CoolCell or appropriate freezing container
-80C freezer
Liquid Nitrogren storage
Before start
- Pre-warm complete culture media to room-temperature.
- Check the cells under the microscope and record percentage confluency. Cancer cells should be banked when ~70% confluent.
Prepare 1 mL freezing media per vial as follows: complete culture media + 10% DMSO.
Detach and collect cells from a flask, by following Steps 1-6 of the protocol: Passaging adherent cancer cell lines.
Aspirate the supernatant, taking care to avoid disturbing the cell pellet. Resuspend the pellet in an appropriate volume of freezing media- depending on the number of vials being frozen. Mix well to ensure a single cell suspension.
For example, if 5 vials are being frozen from a T150, resuspend the cell pellet in 5 mL of freezing media.
Transfer 1 mL aliquots of the cell suspension to pre-labelled 1.8ml cryovials.
Place vials in a 'CoolCell' or appropriate freezing container and store at -80 °C overnight.
Note
Appropriate freezing containers will ensure that the liquid freezes at a controlled rate of around
-1 °C per minute at -80 °C .
Transfer the vials to liquid nitrogen for long-term storage.