Aug 03, 2020

Public workspaceDerivation of organoids from frozen tumour material V.1

DOI
dx.doi.org/10.17504/protocols.io.biiakcae
  • 1Wellcome Sanger Institute
  • Cellular Generation and Phenotyping
  • Organoid and Assembloid
Hazel Rogers
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DOI: dx.doi.org/10.17504/protocols.io.biiakcae
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 frozen tumour material. protocols.io https://dx.doi.org/10.17504/protocols.io.biiakcae
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: July 13, 2020
Last Modified: August 03, 2020
Protocol Integer ID: 39202
Abstract
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.
Guidelines
General Information and tips

  • 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 frozen colon, oesphagus and pancreatic tumour tissue. Success rates were similar to derivation from fresh tissue for colon and pancreas. However, succes rates were very low for oesophagus. We therefore do not routinely freeze oesphagus tumour tissue.
  • You can make up your own feezing media (10% DMSO in media). Or you can use pre-prepared feezing media. We use RecoveryTM Cell Culture Freezing medium (Gibco). This should be aliquoted to avoid multiple freeze/thaws.
  • 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
ReagentDPBS (no Ca, no Mg)ThermofisherCatalog #14190144
ReagentPenicillin StreptomycinInvitrogen - Thermo FisherCatalog #15140 122
ReagentRecovery™ Cell Culture Freezing MediumThermo FisherCatalog #12648010
ReagentCollagenase, Type II, powderThermo FisherCatalog #17101015
ReagentART™ Barrier Specialty Pipette Tips, 1000, wide boreThermo FisherCatalog #2079GPK
ReagentNunc™ Biobanking and Cell Culture Cryogenic Tubes, 1.8mL, 48mm, external thread, printedThermo FisherCatalog #375418
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 LtdCatalog #732-2759
ReagentPestle for Cell StrainerSigma – AldrichCatalog #Z742105
ReagentPrimocinInvivoGenCatalog #ant-pm-1
ReagentY-27632 dihydrochlorideSigma – AldrichCatalog #Y0503
Equipment

  • Sterile cell culture hood
  • Centrifuge
  • 1000 µl and 200 µl pipettes and tips
  • Pipetteboy
  • Stripettes
  • Temperature37 °C waterbath
  • Temperature37 °C humidified incubator (5% CO2)
  • Light microscope
  • Tube rotator
  • Cell freezing container
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
  • Prepare Concentration100 mg/mL collagenase stock. Re-suspend Amount1 g collagenase II in Amount10 mL PBS. Aliquots can be stored at Temperature-20 °C for up to one year.
  • Place a cell freezing container (such as CoolCellTM or My FrostyTM) in the fridge so it is chilled to Temperature4 °C before commencing work.
  • Thaw appropriate amount of freezing media (Amount1 mL per cryovial) and keep at Temperature4 °C until use.
Process Diagram
Process Diagram
Cryopreservation
Derivation from cryopreserved tissue

Cryopreservation of primary tissue
Cryopreservation of primary tissue
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. Add Amount10 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
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 of fresh PBS. Cut the sample up into as small pieces as possible (1-2 mm) using a scalpel.
Tissue cut into small pieces
Examples of tissue samples with high (A) and low (B) cellularity (post cutting with a scalpel)

Transfer tissue and PBS to a Amount15 mL tube. CentrifugeCentrifigation800 x g for 2 minutes.

Aspirate supernatant and re-suspend pellet inAmount1 mL freezing medium (chilled toTemperature4 °C )) and transfer to a cryovial.

Note
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.

Place cryovial(s) in a cell freezing container (chilled toTemperature4 °C ) and transfer immediately to a Temperature-80 °C ffreezer.

After 24-72 hours transfer cryovials to liquid nitrogen storage.

Derivation from cryopreserved tissue
Derivation from cryopreserved tissue
Before starting:

  • 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 TemperatureRoom temperature
  • Prepare digestion buffer:



AliquotAmount8 mL appropriate culture media into aAmount15 mL tube.

Thaw cyrovial(s) of frozen tissue atTemperature37 °C until only a small ice crystal remains.

AddAmount500 µL organoid media dropwise to the cryovial(s). Then transfer everything to theAmount15 mL tube containing the remainder of the prepared media.

Centrifuge atCentrifigation800 x g for 2 minutes.

Aspirate the supernatant. Re-suspend the pellet inAmount10 mL freshly prepared digestion buffer.

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 Thikness100 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. The purpose of these washes is to dilute out any remainging Collagenase from the digestion buffer.


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 (10mM stock)
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.