Bio one black clear 384 well plates

Cells were plated in Greiner Bio-One black µClear 384-well plates (Alphen aan den Rijn, The Netherlands), at 18 000 cells per well in normal DMEM medium. Forty-eight hours after plating, cells switched from normal DMEM medium to AAGLY medium consisting of DMEM low glucose supplemented with 7.7% (v/v) FBS, 20 U/ml penicillin, 20 µg/ml streptomycin, 160 µl/ml MEM non-essential amino acids solution (100×), 80 µl/ml MEM essential amino acids (50×) solution and 2% glycine with a pH of 7 (20 (link)). To drive hepatic differentiation, cells were cultured for 30 days before treatment or RNA isolation. Medium was refreshed twice a week.

3D culturing was performed as described previously (17 (link),18 (link)). Briefly, cells were cultured in a layer of 5 mg/ml Matrigel matrix basement membrane, growth factor reduced (Corning, Cat#354230, Lot#6130005) in Greiner Bio-One black µclear 384-well plates (Alphen aan den Rijn, The Netherlands) at a density of 1000 cells per well to form liver spheroids in 21 days before treatment or RNA isolation. Spheroids were maintained in DMEM/F12 high glucose and phenol red free supplemented with 10% FBS and 25 U/ml penicillin and 25 µg/ml streptomycin (referred to as DMEM/F12 medium). Medium was refreshed twice a week.