D hank s solution

MnCl₂•4H₂O, L-polylysine, D-Hank’s solution, and penicillin–streptomycin mixture were purchased from Sigma. DMEM-F12 medium was from HyClone. Neurobasal-A medium (without phenol red and serum) and fetal bovine serum (FBS) were purchased from Gibco. Human leukocyte antigen B27 (B27) was obtained from Invitrogen Life Technologies.

After anesthesia, 50 mL of D-hank's solution (Genview, Beijing, China) was perfused through the portal vein of mice at 37 °C, and 30 mL of D-hank's solution containing type IV collagens (Sigma-Aldrich, USA) was perfused. The infusion was discharged through the inferior vena cava. After stripping the liver, the hepatocytes were suspended in PBS solution at 4 °C and centrifuged in a gradient density Percoll solution (Pharmacia, GE). The liver cells deposited in the bottom layer were planted in petri dishes containing Collagen I (Corning, NY, USA).

The present work was approved by the Ethics Committee of the Second Hospital of Shandong University. All procedures were guided by the Care and Use of Laboratory Animals guidelines from the National Institutes of Health. We purchased C57BL/6 mice from Shanghai Experimental Animal Center of Chinese Academy of Sciences (China). Primary cardiomyocytes were isolated from C57BL/6 mice as previously reported (20 (link)). Briefly, ventricles were isolated from 2-day-old mice embryonic hearts, minced, and digested with 0.25% trypsin (Gibco, USA) and 0.2% collagenase type II (Sigma, USA) for two cycles. The extract was centrifuged at 45 g at room temperature for 5 min, and the supernatant was removed, followed by the addition of D-Hanks solution (Sigma). The sample was centrifuged at 100 g at room temperature for 5 min, the supernatant was removed, followed by adding the culture medium to obtain cell suspension. The obtained cells were seeded and incubated in DMEM with 1% penicillin/streptomycin, 10% fetal bovine serum (FBS) and 4 mM L-glutamine in an incubator with 5% CO₂ at 37°C.