IntestiCult™ Organoid Growth Medium (Human)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the Matrigel in advance on ice or in fridge	Use of 24-well tissue 37 °C pre-warmed culture plate

Upstream tips
Defrost the Matrigel in advance on ice or in fridge
Protocol tips
Use of 24-well tissue 37 °C pre-warmed culture plate

Publication protocol

Organoid culture from primary tumor tissues and co-culture: Surgical specimens were minced into 1 mm3 fragments in a sterile tissue culture dish with sterile forceps and #10 scalpel. Minced specimens were transferred to 8 mL of 37 °C Digestion Media in a 15 mL conical tube and placed on a rocker at 37 °C for 2 h. Tubes were allowed to settle upright at room temperature for 2 min. Supernatants were transferred to a new 15 mL conical tube containing 6 mL cold Wash Media, leaving any solids behind. Tubes were centrifuged at 150 x g, 4 °C, 5 min. Supernatants were aspirated and pellets washed twice with 6 mL Wash Media and centrifugation as above. Pellets were resuspended in 1.5 mL 37 °C TryplE (ThermoFisher). If visible clumping was observed, 10 μL of DNAse I (Sigma) 5 mg/mL in sterile DPBS was added. Tubes were placed on a rocker at 37 °C for 15 min. Tubes were centrifuged as above and washed once in Wash Media as above. Supernatants were thoroughly aspirated without disturbing the pellet, leaving up to 20 μL of Wash Media. Pellets were resuspended in 100 μL liquefied Matrigel (Corning) at 4 °C. Aliquots of 35 μL of resuspended cells in Matrigel were plated in triplicate as droplets in the centers of wells of a 24-well tissue culture plate, warmed to 37 °C and kept on a 37 °C warming block. Plates were incubated at 37 °C, 5% CO2 for 15 min to solidify Matrigel droplets. Droplets were overlaid with 500 μL of 37 °C OGM with Rho kinase inhibitor and incubated at 37 °C, 5% CO2 in a humidified incubator. For established and primary cell lines (Panc1, MCW670, A549) organoid suspensions were overlaid with either OGM or RPMI1640 with 10% FBS. Cultures were media changed every 2-3 days by careful aspiration of the media overlay and gentle administration of fresh media to the side of the well. For ascites, up to 150 mL was centrifuged at 150 x g, 4 °C, 5 min. Pellets were resuspended in 5 mL ACK lysing buffer (ThermoFisher) and incubated at 4 °C, 5 min to lyse red blood cells. No digestion was performed. Pellets were washed in 6 mL Wash Media twice, removing mucinous or solid components with a 1000 μL pipette and tip. Cell pellets were suspended in Matrigel and plated in 35 μL droplets as described above. The volume of Matrigel that pellets were resuspended in depended on the size of the pellets, with the pellet volume not exceeding 25% of the total resuspended volume. For fibroblast co-culture, 5 × 105 patient-matched CAFs per well were suspended in Matrigel and plated with organoids. For lymphocyte co-culture, 500,000 CD3+ T lymphocytes per well were suspended in 500 μL OGM and added to organoids in Matrigel domes or empty Matrigel domes. After 72 h in culture, lymphocytes in media were removed for flow analysis and immunofluorescence was conducted on remaining organoids and lymphocytes in Matrigel domes as described below.
Organoid splitting and freezing: Cultures were split when organoids became large (~ 300 uM) or over-confluent (once every few days to weeks depending on growth rate). Media was aspirated away from Matrigel domes. Matrigel was broken up and dissolved in 500 μL cold (4 °C) PBS with a 1000 μL pipette and tip and added to a 15 mL conical tube with 8 mL cold PBS. Tubes were centrifuged at 300 x g, 4 °C, 5 min. All but 2 mL supernatant was aspirated. Visible organoids were broken up by gentle passage through a 23 g 1″ needle on a 3 mL syringe. Tubes were centrifuged at 300 x g, 4 °C, 5 min. Supernatants were thoroughly aspirated, leaving the pellet and up to 20 μL of supernatant. If cells were to be frozen, pellets were resuspended in 1 mL Restore freezing media (ThermoFisher) and frozen in 0.5 mL cryovial aliquots at − 80 °C for at least 4 h before transferring to liquid nitrogen. If cells were to be re-plated, pellets were resuspended in Matrigel and plated as above. Wells were overlaid with 500 μL of 37 °C OGM with Rho kinase inhibitor and incubated at 37 °C, 5% CO2 in a humidified incubator.

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