DMEM/F-12

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the growth factor reduced Matrigel in advance, on ice or in fridge	Replace growth factors every other day and Add fresh media every four days.

Upstream tips
Defrost the growth factor reduced Matrigel in advance, on ice or in fridge
Protocol tips
Replace growth factors every other day and Add fresh media every four days.

Publication protocol

Liver organoid growth medium: Cultures were performed as described with slight modifications (Huch et al., 2013). Liver organoid medium consists of DMEM/F12 medium (Invitrogen), 1× N2-supplement (Invitrogen), 1× B27 without vitamin A-supplement (Invitrogen), 10mM nicotinamide (Sigma-Aldrich), 0.001mM dexamethasone (Sigma-Aldrich), 10mM Hepes (Invitrogen) and 20μM Y27632 (Sigma-Aldrich). 50ng/ml rmEGF (R&D systems), 40ng/ml rmHGF (Peprotech), 100ng/ml rmWnt3a (Peprotech) and 500ng/ml rhRspo1 (R&D systems) were used as growth factor supplements. Growth factors were replaced every other day while fresh media was added every four days. Liver organoid generation: Isolated livers from newborn or adult mice were mechanically diced and digested. Filtered and pelleted cells were re-suspended in ice-cold growth factor reduced matrigel (BD Biosciences) with the growth factor cocktail. Polymerization of cell/matrigel mixture was performed at 37°C for 30min followed by addition of liver organoid growth medium. Liver organoid colonies were observed 7-10 days upon initial cell plating. To generate organoids from YAP Tg mice, YAP was induced for 3 weeks in the TetOYAP line and organoids were generated in +/- Dox conditions.

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Manufacturer protocol

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