*2.3. Cultivation of Hepatic Stellate Cells*

4 <sup>×</sup> 10<sup>5</sup> purified HSCs were seeded on an uncoated 6 well plate in Dulbecco's Modified Eagle Medium (DMEM) with 10% heat inactivated fetal calf serum (FCS) and 1% penicillin/streptomycin. After one, three, seven, or nine days, cells were then detached by accutase treatment for 10 min. Afterwards, the detached cells were washed once with cold phosphate-buffered saline (PBS) and pelleted by centrifugation at 570 rcf for 5 min in a cold centrifuge. Cells were then resuspended at 500 cells per μl in cold PBS with 0.1% bovine serum albumin (BSA) and directly subjected to the single cell RNA sequencing analysis, according to the manufacturers protocol.
