2. Add sterile PBS into a 60 mm Petri dish.
3. Under a dissection microscope, open the abdominal cavity, remove the liver tissue, which is dark pink colour under the septum transversum, and place it in sterile PBS.
4. Transfer all the liver tissues into a sterile 15 ml centrifuge tube containing 3 ml of PBS (see Note 3). Add 1 ml 0.25% trypsin solution.
5. Mix thoroughly by gently flicking the tube. Incubate at 37ýC for 30 min with a gentle mixture every 10 min.
6. After digestion, add 6 ml isolation medium to inactivate trypsin and pellet the cells by centrifugation at 100ýg for 5 min.
7. Aspirate the supernatant and resuspend the pelleted cells in 10 ml isolation medium. Mix the cells by gently pipetting up and down 5–6 times. Centrifuge at 100ýg for another 5 min.
8. Remove the supernatant and add 10 ml isolation medium to resuspend the cells.
9. Triturate the cell solution 6–8 times gently with a flame-polished Pasteur pipette to make single cell suspension.
10. After any remaining large clumps of tissue have settled down, perform cell counting using a haemocytometer.
11. Place the cells in collagen IV pre-coated culture vessels at the density of 3,000 per cm2 and culture the cells in growth medium.
12. Incubate the cells at 37℃ in 5% CO2.
