TITLE: Derivation of organoids from primary tumour tissue
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 derivation of organoid models from primary 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, pancreas and oesophageal tumours. The team has extensive experience in organoid derivation and have successfully banked over 100 models.</div></div>

[STEPS]
?. [Process Diagram]
?. [Protocol]
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.
?. [Protocol]
Aspirate as much media as possible. AddPBS 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
After last wash make sure to aspirate as much PBS as possible to avoid diluting the digestion buffer.
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.
?. [Protocol]
Adddigestion buffer. Using a scalpel, cut sample into small pieces of approximately 1-2 mm in diameter.
10 mL
?. [Protocol]
Transfer tissue and digestion buffer to atube. Place sample in a tube rotator and incubate atfor 60-120 minutes.
15 mL
37 °C
?. [Protocol]
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.
?. [Protocol]
Transfer digested sample to atube through a 100 μm cell strainer. Use a pestle to pass any remaining tissue through the strainer.  Wash thetube withPBS and add to thetube through the cell strainer. Repeat the wash step.
50 mL
15 mL
10 mL
50 mL
?. [Protocol]
Centrifuge atfor 2 minutes.
Centrifuge: 800 34
?. [Protocol]
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.
?. [Protocol]
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.
?. [Protocol]
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.
?. [Protocol]
Using a P200 pipette, dispense organoid/BME2 suspension as small-droplets into a 6 well plate (seedper well).
15 µl
20 µl
200 µl
?. [Protocol]
Place in aincubator (5% CO2) for 15-30 minutes to allow BME2 to set.
37 °C
?. [Protocol]
Prepare media containing antibiotics and Y-27632 (rock inhibitor). Add volumes below per ml of appropriate culture media:primocinpenicillin streptomycinY-27632
2 µl
10 µl
1 µl
?. [Protocol]
Addof appropriate prepared media per well of a 6 well plate.
2 mL
?. [Protocol]
Return to incubator. Media change twice a week until ready to passage. Keep in media containing antibiotics and Y-27632 until first passage.
?. [Full Process Video]