TITLE: Derivation of organoids from frozen tumour material
AUTHORS: 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 describes the cryopreservation and subsequent derivation of organoid models from tumour tissue. It has been developed by the organoid derivation team within the Cellular Generation and Phenotyping Group at the Wellcome Sanger Institute. We have used the process to derive organoids from colon and pancreas tumours. In our experience success rates were very low for derivation from frozen oesophageal tumour tissue. The team has extensive experience in organoid derivation and have successfully banked over 100 models.</div></div>

[STEPS]
?. [Process Diagram]
?. [Cryopreservation of primary tissue]
Pour or pipette tissue, and media sample has been transported in, into a glass petri dish.
If tissue has unknown infection status, only open the container the sample has been transported in within a microbiological safety cabinet.
We recommend using glass rather than plastic petri dishes as tissue can get stuck in grooves cut into the plastic dish whilst cutting up the sample.
?. [Cryopreservation of primary tissue]
Aspirate as much media as possible. Add PBS to wash the tissue sample. Aspirate PBS and repeat wash at least two more times (we perform 3 washes for pancreas and oesophagus and 5 washes for colon).
10 mL
If tissue is breaking up, making aspiration difficult without losing the sample, transfer tissue and media back to the tube and centrifuge ( 2 min). Asiprate supernatant and re-suspend in PBS to wash. Repeat these steps for appropriate number of washes.
?. [Cryopreservation of primary tissue]
Addof fresh PBS. Cut the sample up into as small pieces as possible (1-2 mm) using a scalpel.
10 mL
?. [Cryopreservation of primary tissue]
Transfer tissue and PBS to a tube. Centrifugefor 2 minutes.
15 mL
Centrifuge: 800 34
?. [Cryopreservation of primary tissue]
Aspirate supernatant and re-suspend pellet infreezing medium (chilled to) and transfer to a cryovial.
1 mL
4 °C
If there is alot of tissue, split across more than one cryovial. This is to ensure there is sufficient volume of freezing media to protect all tissue pieces during freezing.
?. [Cryopreservation of primary tissue]
Place cryovial(s) in a cell freezing container (chilled to) and transfer immediately to a freezer.
4 °C
-80 °C
?. [Cryopreservation of primary tissue]
After 24-72 hours transfer cryovials to liquid nitrogen storage.
?. [Derivation from cryopreserved tissue]
Before starting:Thaw BME2 aliquot overnight atand dilute 4:1 with appropriate organoid media (tissue specific) to make an 80% stock Ensure cell culture plates have been stored overnight inincubatorPre-warm organoid culture media to
4 °C
37 °C
0 Room temperature
?. [Derivation from cryopreserved tissue]
Aliquotappropriate culture media into atube.
8 mL
15 mL
?. [Derivation from cryopreserved tissue]
Thaw cyrovial(s) of frozen tissue atuntil only a small ice crystal remains.
37 °C
?. [Derivation from cryopreserved tissue]
Addorganoid media dropwise to the cryovial(s). Then transfer everything to thetube containing the remainder of the prepared media.
500 µl
15 mL
?. [Derivation from cryopreserved tissue]
Centrifuge atfor 2 minutes.
Centrifuge: 800 34
?. [Derivation from cryopreserved tissue]
Aspirate the supernatant. Re-suspend the pellet infreshly prepared digestion buffer.
10 mL
?. [Derivation from cryopreserved tissue]
Place sample in a tube rotator and incubate atfor 60-120 minutes.
37 °C
?. [Derivation from cryopreserved tissue]
Following incubation, assess tissue fragments under a microscope to confirm sufficient digestion. The sample should look cloudy to the eye and appear as single cells or small clumps under a microscope.
?. [Derivation from cryopreserved tissue]
Transfer digested sample to atube through a cell strainer. Use a pestle to pass any remaining tissue through the strainer.  Wash the tube withPBS and add to thetube through the cell strainer. Repeat the wash step.
50 mL
15 mL
10 mL
50 mL
?. [Derivation from cryopreserved tissue]
Centrifuge atfor 2 minutes.
Centrifuge: 800 34
?. [Derivation from cryopreserved tissue]
Aspirate supernatant and re-suspend pellet inPBS. Repeat spin atfor 2 minutes.
30 mL
Centrifuge: 800 34
When aspirating, you do not need to worry about getting too close to the pellet at this stage. The purpose of these washes is to dilute out any remainging Collagenase from the digestion buffer.
?. [Derivation from cryopreserved tissue]
Aspirate supernatant and re-suspend pellet inPBS. Transfer to atube (ortube). Washtube with anotherPBS and transfer totube. Repeat spin atfor 2 minutes.
2.5 mL
5 mL
15 mL
50 mL
2.5 mL
5 mL
Centrifuge: 800 34
Transferring to a smaller volume tube helps with re-suspension in a small volume of BME2 in the next step.
?. [Derivation from cryopreserved tissue]
Aspirate as much supernatant as possible. Re-suspend cell pellet in appropriate amount 80% BME2 (per well of a 6 well plate).
200 µl
BME2 must be dispensed as quickly as possible as it will begin to set at room temperature. A cool block could be used to help keep the temperature down while plating.
Volume of BME2 to re-suspend in must be determined from size of cell pellet. Aim to plate cells as close together as possible. If unsure re-suspend in a small volume. Pipette one or two - droplets and check under the microscope. If too dense increase BME2 volume.
?. [Derivation from cryopreserved tissue]
Using a P200 pipette, dispense organoid/BME2 suspension as small-droplets into a 6 well plate (seedper well).
15 µl
20 µl
200 µl
?. [Derivation from cryopreserved tissue]
Place in aincubator (5% CO2) for 15-30 minutes to allow BME2 to set.
37 °C
?. [Derivation from cryopreserved tissue]
Prepare media containing antibiotics and Y-27632 (rock inhibitor). Add volumes below per ml of appropriate culture media:primocin penicillin-streptomycin Y-27632 (10mM stock)
2 µl
10 µl
1 µl
?. [Derivation from cryopreserved tissue]
Addof appropriate prepared media per well of a 6 well plate.
2 mL
?. [Derivation from cryopreserved tissue]
Return to incubator. Media change twice a week until ready to passage. Keep in media containing antibiotics and Y-27632 until first passage.