Isolation and Culture of Grass Carp Hepatocytes

In this study, primary hepatocytes of grass carp were isolated and cultured according to a previous study [64 (link)]. Briefly, prior to the isolation of hepatocytes, the blood of the fish was drawn with a syringe. Then, the liver was rapidly isolated and washed several times in ice-cold phosphate-buffered saline (PBS) (Servicebio, Wuhan, China) containing 500 U/mL penicillin and streptomycin. After removal of PBS using sterile pipettes, the samples were cut into small pieces (about 1 mm³). The small pieces of liver were digested with trypsin at 28 °C for 10 min, then the cells were collected, and the process was repeated 3 times. Thereafter, the cell suspension was centrifuged at 400 g for 10 min and washed twice. The harvested cell pellets were resuspended in M199 medium (Gibco, NY, USA) with 10% fetal bovine serum (Gibco, NY, USA) and 100 U/mL penicillin and streptomycin (Gibco, NY, USA) at a density of 1 × 10⁶ cells/mL. Finally, primary hepatocytes were kept at 28 °C in a 5% CO₂ environment.