TITLE: Cryopreservation of organoid cultures
AUTHORS: Emily Souster, Hazel Rogers, Laura Letchford, Sara Vieira, Maria Garcia-Casado, Mya Fekry-Troll, Charlotte Beaver, Rachel Nelson, Hayley Francies, Mathew Garnett

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[STEPS]
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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).
If you plan to bank a specific number of cells, be sure to take a cell count at Step 7.The count can be taken from the cell suspension, or alterntively you could detach a representative well at this stage, from which to take the count.
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Transfer the organoid suspension to a 5ml Eppendorf or 15ml Falcon tube.
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Wash the wells with cold PBS (~) 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.
4 °C
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Centrifuge at  for 2 minutes (with the brake set on 4).
Centrifuge: 800 34
Centrifuge buckets must be seales using safety caps, which must only be opened inside a microbiological safety cabinet.
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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 the pellet is not clear, aspirate as close to the BME2 'haze' as possible and re-suspend in cold PBS, spin down again (800 x g for 2 minutes, brake set on 4) and then aspirate the supernatant.
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Resuspend the organoid pellet in as much Recovery Cell Freezing Media as required (1ml per cryovial) and transfer into the prelabelled cryovials.
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Place the cryovials into a CoolCell or appropriate freezing container and place into  freezer.
-80 °C
If there is an odd number of cryovials, use a 'balance' cryovial for better cryopreservation.
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After 24 hours (up to 72 hours) transfer cryovials to liquid nitrogen storage.
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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.
Organoids are typically counted as aggregates because they are difficult to break down to single cells.We take two aggregate cell counts which much be within 10% of each other, otherwise a third count is taken and an average is calculated between the three counts.We recommend freezing a cell density of 1x106 cells per cryovial.