GoKawiilEthical approval and sample collection
All necessary ethical approvals to conduct this work and to ensure that the derived organoids could be used for academic and commercial purposes were obtained from each participating clinical site for the collection of patient samples as well as for the Wellcome Sanger Institute for organoid derivation (IRAS ID:203519; REC 16/LO/1110). Further details are provided in Supplementary Information.
Availability of Organoid Biobank
Organoids have ethical approval for use in academic and commercial purposes. Models are being distributed by American Type Tissue Culture (ATCC) as part of the HCMI collection (https://www.atcc.org/hcmi) and EMD Millipore. Repository details and model availability are provided in Supplementary Table 2.
Organoid derivation
Detailed protocols for organoid derivation, cryopreservation and routine culturing are published on protocols.io and include embedded demonstration videos and workflow diagrams62,63,64. Tumour samples were washed 3 times with PBS, minced, and either cryopreserved after centrifugation (800g, 2 min)63 or enzymatically digested for 1–2 h at 37 °C. The suspension was filtered (100 μm), centrifuged and washed to remove debris and digestion buffer62. Isolated cells were embedded in approximately 15 μl droplets of extracellular matrix (80:20 basement membrane extract (BME):medium; 6.4–9.6 mg ml−1 protein; Cultrex BME Type 2 Select 3532-001-02) and plated in pre-warmed 6-well plates following established protocols64. After polymerization (15–20 min, 37 °C), 2 ml of organoid medium prepared using established recipes39,65,66,67 was added, supplemented with antibiotics and 1 μl ml−1 ROCK inhibitor (Y-27632, Staratech Scientific S1049-SEL-5mg).
After expansion to ≥25 million cells, 25 cryovials were banked and pellets collected for sequencing. Post-thaw viability was confirmed by re-culturing for four passages with freeze–thaw quality control assessment.
Organoid culture
Organoids were maintained either in 80% BME-2 droplets or in 5% BME-2 suspension culture38. In the 80% BME-2 culture, organoids were cultured as described above. For the 5% suspension method, cancer organoids were suspended in a medium/extracellular matrix (ECM) dilution. For example, combining 10 ml of medium with 500 μl of BME-2 achieved the desired concentration, and organoids were then cultured in ultra-low-adherent flasks or plates.
For passaging, the medium, cells and ECM were collected and centrifuged at 800g for 2 min. After discarding the supernatant, organoids were dissociated using TrypLE (Gibco 12604013), an enzymatic reagent. The suspension was incubated for 10–60 min, allowing dissociation into small clumps. The cells were then pelleted and replated as described64.
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