Polystyrene conical centrifuge tube

Fresh CRC tissue samples were cut into small pieces using a scalpel, washed with ice-cold PBS containing antibiotic 3∼5 times, and subsequently digested with 0.05% trypsin, 0.02% EDTA (Thermo Fisher Scientific, Waltham, MA, USA) for 12 min at 37 °C with shaking every 15 min. The remaining fragments were additionally treated with Collagenase NB 4G (SERVA Electrophoresis GmbH, Heidelberg, Germany) at 37 °C for 20 min. The pellet was re-suspended in 24 ml 40% Percoll PLUS/Percoll, placed in 50-ml polystyrene conical centrifuge tube (BD Biosciences) and overlaid with 9 ml 70% Percoll solution. Centrifuge immediately at 2500 rpm (Eppendorf 5810R centrifuge) for 20 min (brake off), at room temperature. The cell fraction was carefully and gently collected above the interphase band (above 1.065 g ml⁻¹) by using a sterile Pasteur pipet, then pelleted at 1500 rpm (Eppendorf, Hamburg, Germany) for 7 min at 4 °C. The cell pellet was suspended with Matrigel (growth factor reduced; BD Biosciences) and dispensed into 48-well culture plates (25 ml Matrigel per well), which have also cover with single layer of MSC. The basal culture medium for human intestinal organoids was prepared as recently described (Fujii et al, 2016 ).

Liver cells were isolated by a modification of the method described by Troutman et al.^[30] Mouse livers were digested by retrograde perfusion with liberase through the inferior vena cava. The dissociated cell mixture was placed into a 50‐ml conical tube and centrifuged twice at 50g for 2 min to pellet hepatocytes. The non‐parenchymal cells (NPC)‐containing cell supernatant was further used to isolate Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), and HSCs. The cell suspension was pelleted by centrifugation (700g, 10 min, 4°C) and resuspended in phosphate‐buffered saline (PBS) and OptiPrep (Sigma) to a final concentration of 17%. Afterward, 5 ml of the indicated suspension was placed in a 15‐ml polystyrene conical centrifuge tube (BD Biosciences) and overlaid with 5 ml of a 9% OptiPrep solution followed by 2 ml of PBS. After centrifugation at 1400g for 20 min at 4°C with decreased acceleration and without breaks, the various cell types were arranged according to their density. HSCs were enriched in the upper cell layer, whereas KCs and LSECs were separated as a second layer of higher density. Cell fractions were collected separately by pipetting. HSC purity over 99% was confirmed by retinoid‐based fluorescence‐activated cell sorting.