Corning® Hepatocyte Culture Media Kit, 500 mL

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the Matrigel in advance on ice or in fridge	Use pre-coated 6-well plate (Corning) with 60% Matrigel	Possibility to generate stocks of organoids to culture in a future

Upstream tips
Defrost the Matrigel in advance on ice or in fridge
Protocol tips
Use pre-coated 6-well plate (Corning) with 60% Matrigel
Downstream tips
Possibility to generate stocks of organoids to culture in a future

Publication protocol

Tissue dissociation and organoid culture: Organoid culture was performed as previously described (Chua et al., 2014), with minor modifications to the tissue dissociation protocol to improve cell viability. For tissue dissociation, tumor tissues from patients or xenografts were washed in organoid culture media (hepatocyte media with 10 ng/ml EGF, 5% CS-FBS, 10 μM Y-27632 (STEMCELL Technologies), and 1X Glutamax (Gibco)), supplemented with 100 μg/ml Primocin, and minced with scissors. Tumor tissues were then incubated in 10 ml of the organoid culture media supplemented with 100 μg/ml Primocin and 1:10 dilution of collagenase/hyaluronidase (STEMCELL Technologies) at 37 °C for 15 min. Dissociated tissues were spun down at 350 g for 5 min, resuspended in 10 ml of PBS, and spun down again. The tissues were resuspended in 5 ml of TrypLE Express (Invitrogen), followed by incubation at room temperature for 3 min. Trypsinization was stopped by addition of 10 ml modified Hank’s balanced salt solution (HBSS; STEMCELL Technologies) supplemented with 5% CS-FBS, 10 μM of the ROCK inhibitor Y-27632 and 100 μg/ml Primocin, followed by centrifugation at 350 g. Dissociated tissues were resuspended with 10 ml of HBSS supplemented with 5% CS-FBS, 10 μM Y-27632 and 100 μg/ml Primocin, and passed through a 100 μm cell strainer (Corning). Dissociated cell clusters (approximately 2–10 cells per cluster, and 1 x 106 cells in total) were spun down and resuspended in 60% Matrigel (Corning)/organoid culture media, and plated in a 250 μl drop in the middle of one well of a pre-coated 6-well plate (Corning) with 60% Matrigel. The drop was solidified by a 30-minute incubation at 37°C and 5% CO2. After solid drops formed, 1.5 ml of the organoid culture media was added to the well, and the medium was changed every 3–4 days. Typically, approximately 50–80% of the cell clusters would form organoids, although there was considerable variation between lines and not all organoids could propagate after passaging. For passaging, 1 mg/ml dispase (STEMCELL Technologies) was added to the medium followed by incubation for 60 min at 37°C to digest the Matrigel. Subsequently, organoids were centrifuged at 350 g for 5 min, washed in PBS and spun down. 5 ml TrypLE Express (Invitrogen) was added, and organoids were incubated at room temperature for 3 min, followed by mechanical dissociation to small cell clusters by pipetting. Organoids were passaged at a 1:2-3 dilution every 2–3 weeks. To generate stocks, organoids were snap-frozen in 90% CS-FBS and 10% DMSO and stored in liquid nitrogen. Cryopreserved stocks have been successfully recovered for up to at least 18 months after freezing.

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Manufacturer protocol

Download the product protocol from Corning for Corning® Hepatocyte Culture Media Kit, 500 mL below.

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