Gibco™Advanced DMEM/F-12

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost in advance the BME gel before use	Split primary organoids after 14 days and for maintenance every 7-10 days

Upstream tips
Defrost in advance the BME gel before use
Protocol tips
Split primary organoids after 14 days and for maintenance every 7-10 days

Publication protocol

Tissue processing, cell isolation and organoid culture. Liver cells were dissociated from the human biopsy by collagenase digestion. Cells were washed with cold Advanced DMEM/F12, filtered with a 70 μm filter and the cell pellet resuspendend in BMER (Amsbio). Isolation medium was based on AdDMEM/F12 (Invitrogen) supplemented with: 1X B27 (both from GIBCO), 1.25 mM N-Acetylcysteine (Sigma), 10 nM gastrin (Sigma), 50 ng/ml EGF (Peprotech), 10% RSPO1 conditioned media (homemade), 100 ng/ml FGF10 (Peprotech), 25 ng/ml HGF (Peprotech), 10 mM Nicotinamide (Sigma), 5 μM A83.01 (Tocris), 10 μM FSK (Tocris), 25 ng/ml Noggin (Peprotech), 30% Wnt conditioned media (homemade), and 10 uM Y27632 (Sigma Aldrich). After 3-4 days, the isolation medium was changed into a medium without Noggin, Wnt conditioned media, Y27632. Organoids could be splitted after ca. 14 days by mechanical shearing and re-plated into fresh BMER. Passage was performed in a 1:5 split ratio once every 7–10 days for at least 6 months.

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Manufacturer protocol

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