Liver Nonparenchymal Cell Isolation

Fresh liver tissues were stored in GEXSCOPE^® Tissue Preservation Solution (Singleron Biotechnologies, Nanjing, China) and transported to the Singleron lab on ice as soon as possible. Specimens were washed thrice with Hank’s Balanced Salt Solution (HBSS) and minced into pieces of size 1–2 mm. Then, the tissue pieces were digested with 2 mL GEXSCOPE^® Tissue Dissociation Solution (Singleron Biotechnologies, Nanjing, China) for 15 min at 37 °C in a 15 mL centrifuge tube with sustained agitation. After digestion, 70 um sterile strainers were employed to filter samples. Next, we centrifuged samples at 50× g for 5 min at 4 °C to remove hepatocytes [24 (link),25 (link),26 (link),27 (link)]. Then, the precipitation was discarded, and the supernatant was resuspended in 1 mL of phosphate-buffered saline (PBS; HyClone, Logan, UT, USA). This action was followed by centrifuging the samples at 300× g for 5 min at 4 °C. Then, the supernatant was discarded, and the precipitation was resuspended in 1 mL of PBS. To remove red blood cells, 2 mL GEXSCOPE^® red blood cell lysis buffer (Singleron Biotechnologies, Nanjing, China) was added for 10 min at 25 °C. The solution was then centrifuged at 500× g for 5 min at 4 °C and resuspended in PBS. The samples were stained with trypan blue (Sigma, Darmstadt, Germany) and evaluated under a microscope. These cells were hepatic NPCs with few hepatocytes.