24 gauge polyethylene tube

Whole liver grafts were retrieved as detailed elsewhere.27, 28 Briefly, rats were anesthetized with isoflurane (Escain, Mylan, Osaka, Japan) via a small animal anesthetizer (MK‐A110; Muromachi Kikai Co., Ltd., Tokyo, Japan). After heparinization (200 IU/rat; Mochida Pharmaceutical Co., Ltd., Tokyo, Japan), the common bile duct was cannulated with a 24‐gauge polyethylene tube (TERUMO, Tokyo, Japan). A 14‐gauge catheter (Argyle, COvidIEN, Tokyo, Japan) was inserted into the PV trunk, followed by a blood washout with 40 mL of ice‐cold University of Wisconsin (UW) solution (Viaspan, Astellas, Tokyo, Japan). A 24‐gauge polyethylene tube (TERUMO, Tokyo, Japan) was inserted into the HA through the celiac trunk. The suprahepatic caval vein was cannulated with a 14‐gauge short stent. The liver was immediately flushed with 20 mL of ice‐cold UW solution and then preserved for 24 hours at 4°C in 40 mL of UW solution. Sham‐operated rats underwent the same procedure, but they were not cold‐stored and were directly subjected to isolated ex vivo perfusion for reference.

Under anesthesia with isoflurane, the donor rat’s abdomen was opened by a bilateral subcostal incision. After the liver was mobilized, the infrahepatic inferior vena cava (IHIVC) was separated from the right adrenal vein. The rat was subsequently heparinized via the penile vein with 300 IU of heparin (Mochida Pharmaceutical Co., Ltd., Tokyo, Japan) in 1 mL of Ringer solution. The bile duct was then divided and cannulated with a 24-gauge polyethylene tube (TERUMO, Tokyo, Japan) for stenting. Next, the gastroduodenal artery was ligated and divided. The portal vein (PV) was isolated by transecting the pyloric and splenic vein. In situ liver perfusion from aortic bifurcation with 100 mL of cold histidine-tryptophan-ketoglutarate (HTK) solution was performed, just as clinical procurement. Next, the IHIVC, PV, common hepatic artery (CHA), and suprahepatic inferior vena cava (SHIVC) were divided and then the graft was immediately immersed in a basin filled with HTK solution at 4°C.