After 1 week of seeding the organoids, isolation media were changed to human liver expansion media (EM; Ad+++ supplemented with 1X B27 supplement without retinoic acid (Gibco), 1X N2 supplement (Gibco), 1.25 mM N-acetyl-L-cysteine (Sigma), 20% (vol/vol) Rspo-1 conditioned medium, 1.25% (vol/vol) Wnt3a conditioned medium [Barker et al., 2010 (link)], 10 mM nicotinamide (Sigma), 10 nM recombinant human (Leu15)-gastrin I (Sigma), 50 ng/ml recombinant human EGF (Peprotech), 100 ng/ml recombinant human FGF10 (Peprotech), 25 ng/ml recombinant human HGF (Peprotech), 10 μM forskolin (Sigma), and 5 μM A8301 (Tocris)) (Huch et al., 2015 (link)).
EM was changed twice a week, and cultures were split every 7–10 days according to organoid density. For passaging (1:4-1:8, depending on growth rate of the culture), organoids were resuspended in 10 ml Ad+++, incubated in ice for 10 min, and collected by centrifugation (5 min at 200 xg). Subsequently, organoids were incubated for 1–2 min in TrypLE Express at RT and mechanically disrupted by pipetting. After a further wash in Ad+++, cells were resuspended in BME solution and seeded in 24- or 48-well suspension plates. After BME solution had solidified, wells were filled with 500 µl (24 wells) or 250 µl (48 wells) of human liver organoid expansion medium.
EM was changed twice a week, and cultures were split every 7–10 days according to organoid density. For passaging (1:4-1:8, depending on growth rate of the culture), organoids were resuspended in 10 ml Ad+++, incubated in ice for 10 min, and collected by centrifugation (5 min at 200 xg). Subsequently, organoids were incubated for 1–2 min in TrypLE Express at RT and mechanically disrupted by pipetting. After a further wash in Ad+++, cells were resuspended in BME solution and seeded in 24- or 48-well suspension plates. After BME solution had solidified, wells were filled with 500 µl (24 wells) or 250 µl (48 wells) of human liver organoid expansion medium.