Jul 07, 2020

Public workspaceDerivation of organoids from primary tumour tissue

DOI
dx.doi.org/10.17504/protocols.io.bfvnjn5e
  • 1Wellcome Sanger Institute
  • Cellular Generation and Phenotyping
  • Organoid and Assembloid
Hazel Rogers
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DOI: dx.doi.org/10.17504/protocols.io.bfvnjn5e
Protocol Citation: Hazel Rogers, Laura Letchford, Sara Vieira, Maria Garcia-Casado, Mya Fekry-Troll, Charlotte Beaver, Rachel Nelson, Hayley Francies, Mathew Garnett 2020. Derivation of organoids from primary tumour tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.bfvnjn5e
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 01, 2020
Last Modified: July 07, 2020
Protocol Integer ID: 36494
Keywords: Organoid, Cancer, Derivation,
Abstract
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.

Guidelines
General Information and tips
  • The tissue samples we process are transported from clinical sites in Advanced DMEM-F12 containing primocin antibiotic (final concentration 100 ug/ml). Sampes are shipped chilled atTemperature4 °C .
  • 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.
  • We have experience of deriving organoid models from colon, oesophagus and pancreatic tumour tissue.
  • We recommend using glass rather than plastic petri dishes for tissue dissection as tissue can get stuck in grooves cut into the plastic dish.
  • Plate digested cells as close together as possible.
  • Be very cautious at initial passages after derivation. Organoids can grow well for a few passages and then significantly drop off. We generally keep organoids in the same number of wells or reduce the area plated in if growth is slow or some cellular material has died.
  • Not all derivations will be successful. Listed below are some common reasons we see for failure.


Trouble Shooting








Materials
MATERIALS
ReagentFalcon 15 mL Polystyrene Conical TubeFisher ScientificCatalog #352095
ReagentPenicillin StreptomycinInvitrogen - Thermo FisherCatalog #15140 122
ReagentDPBS no calcium no magnesiumThermo Fisher ScientificCatalog #14190144
ReagentCollagenase, Type II, powderThermo FisherCatalog #17101015
ReagentCultrex® Reduced Growth Factor Basement Membrane Matrix Type 2 (BME 2)TrevigenCatalog #3533-010-02
ReagentFalcon 50mL Conical Centrifuge TubesFisher ScientificCatalog #14-432-22
ReagentCostar 6-well Clear TC-treated Multiple Well Plates Bulk Packed SterileCorningCatalog #3506
ReagentEppendorf Tubes 5.0 mlEppendorfCatalog #0030122321
ReagentAnumbra Glass Petri Dish 100x15mmScientific Laboratory Supplies LtdCatalog #PET1008
ReagentSurgical Scalpel Blade No. 21Swann MortonCatalog #0507
ReagentCell Strainers 100 μm pore sizeVWR International (Avantor)Catalog #732-2759
ReagentPestle for Cell StrainerMerck MilliporeSigma (Sigma-Aldrich)Catalog #Z742105
ReagentPrimocinInvivoGenCatalog #ant-pm-1
ReagentY-27632 dihydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #Y0503
Equipment

  • Sterile cell culture hood
  • Centrifuge
  • 1000 µl and 200 µl pipettes and tips
  • Pipetteboy
  • Stripettes
  • 37oC waterbath
  • 37oC humidified incubator (5% CO2)
  • Light microscope
  • Tube rotator
Safety warnings

Note
For full safety information refer to individual COSHH and MSDS forms


  • Primocin can cause possible respiratory and skin sensitisation.
  • Penicillin Streptomycin can cause possible respiratory and skin sensitisation. May also damage fertility or the unborn child.
  • Rock inhibitor (Y-27632) is harmful if swallowed, inhaled or splashed on skin.
  • Organoids derived from primary samples may contain uncharacterised adventitious agents, including blood-borne viruses.

Before start
  • Thaw BME2 aliquot overnight atTemperature4 °C and dilute 4:1 with appropriate organoid media (tissue specific) to make an 80% stock
  • Ensure cell culture plates have been stored overnight inTemperature37 °C incubator
  • Pre-warm organoid culture media to room temperature
  • Prepare 100 mg/ml collagenase stock. Re-suspendAmount1 g collagenase II inAmount10 mL PBS. Aliquots can be stored at Temperature-20 °C for up to one year.
  • Prepare digestion buffer:





Process Diagram
Process Diagram


Protocol
Protocol
1h
1h
Pour or pipette tissue, and media sample has been transported in, into a glass petri dish.

Safety information
If tissue has unknown infection status, only open the container the sample has been transported in within a microbiological safety cabinet.

Note
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.

Aspirate as much media as possible. AddAmount10 mL 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).


Note
After last wash make sure to aspirate as much PBS as possible to avoid diluting the digestion buffer.

Note
If tissue is breaking up, making aspiration difficult without losing the sample, transfer tissue and media back to theAmount15 mL tube and centrifuge (Centrifigation800 x g 2 min). Asiprate supernatant and re-suspend in PBS to wash. Repeat these steps for appropriate number of washes.


AddAmount10 mL digestion buffer. Using a scalpel, cut sample into small pieces of approximately 1-2 mm in diameter.

Tissue cut into small pieces
Examples of tissue samples with high (A) and low (B) celullarity (post cutting with a scalpel)

Transfer tissue and digestion buffer to aAmount15 mL tube. Place sample in a tube rotator and incubate atTemperature37 °C for 60-120 minutes.
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.
Transfer digested sample to aAmount50 mL tube through a 100 μm cell strainer. Use a pestle to pass any remaining tissue through the strainer. Wash theAmount15 mL tube withAmount10 mL PBS and add to theAmount50 mL tube through the cell strainer. Repeat the wash step.


Centrifuge atCentrifigation800 x g for 2 minutes.
Aspirate supernatant and re-suspend pellet inAmount30 mL PBS. Repeat spin atCentrifigation800 x g for 2 minutes.
Note
When aspirating, you do not need to worry about getting too close to the pellet at this stage.


Aspirate supernatant and re-suspend pellet inAmount2.5 mL PBS. Transfer to aAmount5 mL tube (orAmount15 mL tube). WashAmount50 mL tube with anotherAmount2.5 mL PBS and transfer toAmount5 mL tube. Repeat spin atCentrifigation800 x g for 2 minutes.
Note
Transferring to a smaller volume tube helps with re-suspension in a small volume of BME2 in the next step.

Aspirate as much supernatant as possible. Re-suspend cell pellet in appropriate amount 80% BME2 (Amount200 µL per well of a 6 well plate).
Note
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.

Note
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 twoAmount15 µL -Amount20 µL droplets and check under the microscope. If too dense increase BME2 volume.


Using a P200 pipette, dispense organoid/BME2 suspension as smallAmount15 µL -Amount20 µL droplets into a 6 well plate (seedAmount200 µL per well).


Place in aTemperature37 °C incubator (5% CO2) for 15-30 minutes to allow BME2 to set.
Prepare media containing antibiotics and Y-27632 (rock inhibitor). Add volumes below per ml of appropriate culture media:

  • Amount2 µL primocin
  • Amount10 µL penicillin streptomycin
  • Amount1 µL Y-27632
AddAmount2 mL of appropriate prepared media per well of a 6 well plate.
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
Full Process Video
1h
1h