Jul 07, 2020

Public workspacePassaging cancer organoid cultures

DOI
dx.doi.org/10.17504/protocols.io.bfe3jjgn
  • 1Wellcome Sanger Institute
  • Cellular Generation and Phenotyping
  • Organoid and Assembloid
Hazel Rogers
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DOI: dx.doi.org/10.17504/protocols.io.bfe3jjgn
Protocol Citation: Hazel Rogers, Laura Letchford, Sara Vieira, Maria Garcia-Casado, Mya Fekry-Troll, Charlotte Beaver, Rachel Nelson, Hayley Francies, Mathew Garnett 2020. Passaging cancer organoid cultures. protocols.io https://dx.doi.org/10.17504/protocols.io.bfe3jjgn
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: April 22, 2020
Last Modified: July 07, 2020
Protocol Integer ID: 36027
Keywords: Organoids, cancer, passage, propogation,
Abstract
This protocol describes the passaging 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 the passaging cancer organoids with successful propagation of organoids derived from colon, pancreas and oesophageal tumours.
Guidelines
General Tips

  • We generally use low split ratios of 1:3 or 1:2 with our organoid cultures. We will also passage to the same or a lower number of wells if organoids are not growing (see troubleshooting below).
  • 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.
  • Cold PBS is helpful for resolving some pelleting issues (see toubleshooting below). Therefore it is useful to keep a bottle of PBS in fridge.

Reasons for passaging organoids

Troubleshooting


Materials
MATERIALS
ReagentCostar® 6-Well Flat-Bottom Plate, Tissue Culture-Treated 50 Plates Stemcell TechnologiesCatalog #38015
ReagentTrypLE™ Express EnzymeThermo Fisher ScientificCatalog #12604013
ReagentFisherbrand™ Cell ScrapersFisher ScientificCatalog #11587692
ReagentFalcon™ 15mL Conical Centrifuge TubesFisher ScientificCatalog #14-959-53A
ReagentDPBS no calcium no magnesiumThermo Fisher ScientificCatalog #14190144
ReagentCultrex® Reduced Growth Factor Basement Membrane Matrix Type 2 (BME 2)TrevigenCatalog #3533-010-02
ReagentFalcon 50mL Conical Centrifuge TubesFisher ScientificCatalog #14-432-22
ReagentEppendorf Tubes 5.0 mlEppendorfCatalog #0030122321
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

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

Process Diagram
Process Diagram


Protocol
Protocol
Aspirate media from wells and addAmount1 mL orAmount2 mL TrypLE to each well.
Note
If any of the following are true collect organoids and media in a tube and centrifuge to pellet before adding TrypLE
  1. BME2 droplets detached from plate and floating in media
  2. Lots of cells or organoids floating in the media
  3. Lots of fibroblasts attached to the plate

Using a P1000 pipette, detach BME2 drops from the plate. (Optional - detach BME2 drops using a cell-scraper; recommended for passaging a large number of plates).
Pipette suspension up and down multiple times to dissociate organoids from BME2 and transfer to an approproate size tube using a P1000 pipette orAmount10 mL stripette.

Wash wells with TrypLE (approxAmount1 mL per well of a 6 well plate) to collect any remaining organoids and add to the collection tube.

Incubate atTemperature37 °C

Check organoid suspension under microscope after 5 minutes and then as required to assess the dissociation of organoids. Use a P1000 to pipette the cell suspension up and down to help dissociate organoids. Stop incubation when organoids broken down to small clumps of cells.
Note
Avoid breaking organoids down to single cells


Check if organoid suspension has broken down to small clumps of cells on a microscope.

Centrifuge atCentrifigation800 x g for 2 minutes.

Aspirate supernatant to leave organoid cell pellet.
Note
If the organoid suspension does not pellet during centrifugation or there is a grey haze above the pellet (residual BME2), aspirate as much supernatant as possible. Re-suspend in ice cold PBS and repeat centrifugation.


BME2 layer above prganoid pellet after centrifugation

Re-suspend pellet in appropriate amount of 80% BME2 (Amount200 µL per well of a 6 well plate).


Note
Start by re-suspending pellet in a small volume to break down, then add additional BME2 to required volume.

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
Avoid creating bubbles while re-suspending pellet.

Using a P200 pipette dispense a total ofAmount200 µL organoid/BME2 suspension per well of a 6 well plate asAmount10 µL -Amount15 µL droplets.





Place in aTemperature37 °C incubator (5% CO2) for 15-30 minutes to allow BME2 to set.
AddAmount2 mL of appropriate organoid media per well of a 6 well plate.

Return to incubator. Media change twice a week until ready to passage.