Protocol Citation: Emily Souster, Hazel Rogers, Laura Letchford, Sara Vieira, Maria Garcia-Casado, Mya Fekry-Troll, Charlotte Beaver, Rachel Nelson, Hayley Francies, Mathew Garnett 2020. Cryopreservation of organoid cultures. protocols.io https://dx.doi.org/10.17504/protocols.io.bh4ij8ue
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: June 30, 2020
Last Modified: August 03, 2020
Protocol Integer ID: 38762
Abstract
This protocol defines the procedure for cryopreservation of organoid cultures. It has been developed by the organoid derivation team within the Cellular Generation and Phenotyping Group at the Wellcome Sanger Institute. The team has extensive experience passaging and expanding organoid models. The method described has mainly been used for cancer organoids derived from colon, pancreas and oesophageal tumours.
Guidelines
We use 5 ml Eppendorf tubes to help with sterility. However, if you do not have access to these tubes any alternative sterile tubes of appropriate volume can be used.
It is useful to keep a bottle of cold PBS in the fridge as this can be used to resolve pelleting issues. Resuspending a 'hazy' pellet in cold PBS can help to re-melt the BME2, resulting in a more distinct cell pellet after centrifugation.
Appropriate freezing containers will ensure that the liquid freezes at a controlled rate of around -1 °C per minute at -80 °C .
Materials
MATERIALS
DPBS, no calcium, no magnesiumThermo FisherCatalog #14190136
Before cryopreservation of the line, ensure the organoids are of good quality and avoid banking organoids that have become very large or dark.
Place a labelled CoolCell or appropriate freezing container in the fridge and make sure it is at 4 °C before commencing work.
Thaw an appropriate amount of freezing media (1ml per cryovial) and keep at 4 °C before use.
Process diagram
Process diagram
Protocol
Protocol
Using a P1000 pipette, or a cell-scraper, detach BME2 drops from as many wells as required (generally 1 well into 1 cryovial- we recommend aiming for a density of 1x106 cells per cryovial).
Transfer the organoid suspension to a 5ml Eppendorf or 15ml Falcon tube.
Wash the wells with cold PBS (~4 °C) to collect any remaining organoids and add to the collection tube.
Make sure organoids are dissociated from BME2 drops by aspirating up and down multiple times with a stripette.
Centrifuge at 800 x g for 2 minutes (with the brake set on 4).
Aspirate off the supernatant, leaving the organoid cell pellet at the bottom of the tube. Organoid cell pellets are loose pellets so take care when aspirating.
If you do not want to bank a specific number of cells, proceed to Step 8.
If banking a specific number of cells, either take a cell count from your whole organoid suspension, or from a 'representative well' by resuspending the pellet in an appropriate amount of organoid media. Take a cell count from this suspension.
From this cell count you can calculate how much organoid suspension to use to freeze the desired cell density.
Resuspend the organoid pellet in as much Recovery Cell Freezing Media as required (1ml per cryovial) and transfer into the prelabelled cryovials.
Place the cryovials into a CoolCell or appropriate freezing container and place into -80 °C freezer.
After 24 hours (up to 72 hours) transfer cryovials to liquid nitrogen storage.