CRCOs were cultured using previously described methods (10 (link), 11 (link), 14 (link)). The culture medium of CRCOs was refreshed every 3 days. CRCOs were subcultured every 3–14 days depending on the growth rate of organoids. CRCOs were passaged by mechanical dissociation into small fragments through shearing with 1% Bovine Serum Albumin (BSA)-coated glass pipette tip. For those dense organoids, they were resuspended in prewarmed TrypLE™ Express enzyme (1×) (GIBCO, 12605-010) before mechanical dissociation. After dissociation, CRCOs were washed with cold PBS several times to clear out the Matrigel. Finally, CRCO fragments were resuspended in fresh Matrigel, seeded into a prewarmed 24-multiwell plate, and cultured as described above.
For CRCO cryopreservation, organoids were harvested and mechanically dissociated into small fragments as described above. Then, organoid fragments were mixed with freezing medium (CELLBANKER™ 2, ZENOAQ, 170905) and frozen following standard procedures. As required, the frozen CRCOs were thawed according to standard procedures and cultured as mentioned before. The culture medium was supplemented with 10 μM RHOK inhibitor Y-27632 for the first 3 days of culture after thawing.
For CRCO cryopreservation, organoids were harvested and mechanically dissociated into small fragments as described above. Then, organoid fragments were mixed with freezing medium (CELLBANKER™ 2, ZENOAQ, 170905) and frozen following standard procedures. As required, the frozen CRCOs were thawed according to standard procedures and cultured as mentioned before. The culture medium was supplemented with 10 μM RHOK inhibitor Y-27632 for the first 3 days of culture after thawing.
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