Ethylene glycol tetraacetic acid (egta)

Primary hepatocyte isolation was performed using two-step collagenase perfusion technique, as previously described [27] . Briefly, a C57BL6/J mouse was anesthetized with isoflurane (Wako) and the inferior vena cava was exposed and cannulated with a 25-gauge needle. After clamping the superior vena cava and cutting the hepatic portal vein, the liver was perfused with 50 mL of Ca 2+ -free HBSS containing 0.5 mM EGTA (Wako) and 2 U/mL heparin (Novo-Heparin; Mochida, Tokyo, Japan) for 7 min, followed by 100 mL of collagenase solution containing 0.2% dispase II (Sanko Junyaku, Tokyo, Japan), 0.2% collagenase type II (Gibco, Palo Alto, CA, USA), 0.1 mg/mL DNase I (from bovine pancreas, Sigma), heparin 2 U/mL, 150 mmol/L NaCl, 5.4 mmol/L KCl, 0.34 mmol/L NaHPO 4 , 0.1 mmol/L MgSO 4 , 5.0 mmol/L CaCl 2 , 4.2 mmol/L, NaHCO 3 , 5.6 mmol/L glucose, and 10 mmol/L HEPES (all of the chemical reagents other than those indicated were purchased from Wako) for 7 min at 37°C. The liver was then resected out and gently shaken to retrieve the cells.
After filtering the isolated cells through a 100 µm strainer (BD Falcon, Franklin Lakes, NJ, USA), the cell suspension was washed three times by centrifugation at 50 × g for 3 min at 4°C. The isolated primary hepatocytes were immediately used for RNA extraction or the recellularization procedure.

D-glucose, diazoxide (DZ), diltiazem, and EGTA were purchased from Wako Pure Chemicals (Osaka, Japan). Fluo3 acetoxymethylester (Fluo3-AM), NNC55-0396, tolbutamide (TB), and u-conotoxin (u-CgTX) GVIA were purchased from Sigma-Aldrich.