*2.3. Reagents and Instruments*

All experiments were carried out in sterile conditions and all instruments/drugs were kept sterile until use. The drugs, reagents, and instrumentation necessary to conduct this protocol are shown in Supplementary Materials (Tables S3 and S4).

## *2.4. Anesthesia, Surgery, and In-Situ Perfusion*

A schematic overview of all experimental procedures is provided in Supplementary Materials (Figure S1). Rats were anesthetized by intraperitoneal injection of 80 mg/kg of sodic thiopentale and maintained in spontaneous breathing with an O2 enriched air mixture by an open mask. The surgical procedure was carried on with a double-headed surgical microscope OPMI 1-F (Zeiss West Germany, Oberkochen, Germany). Procedure began with a xifo-pubic and bilateral subcostal incision. The hepatic

pedicle was then exposed, and the bile duct was dissected. It was distally ligated with a 8-0 nylon tie and a customized cannula (tip: 24 G cannula Braun, Bethlehem, PA, USA; tube: PE-50, BD-Clay Adams, Becton, UK) was inserted through a choledotomy; the cannula was secured with a proximal 8-0 nylon tie. Subsequently, the portal vein was cannulated. Then, the pyloric vein was ligated (8-0 nylon) and transacted, and the main trunk of the portal vein was dissected until distally to the splenic vein. The portal vein was encircled with three 5-0 silk tie and unfractioned heparin (2 IU/g diluted in 1 mL of normal saline) was administered by tail IV injection. After three minutes, we ligated the portal vein distally to the splenic vein insertion and a 16 G cannula was inserted by venipuncture until the tip of the cannula reached the portal bifurcation. A blood retrograde flush was performed, and the in-situ perfusion system was connected. Then, the cannula was secured with a two silk tie and gently retracted to avoid obstruction of the right lobe portal vein branch. Rapid sternotomy was performed, the heart was removed, and the inferior vena cava (IVC) was transected. In-situ cold perfusion (4 ◦C) was started with 35 mL of Celsior solution (IGL, Lissieu, France) at a pressure of 30 cmH2O. At the end of the perfusion, hepatectomy was completed and the liver graft was stored in 4 ◦C Celsior solution for 30 min. At the end of the cold storage and before NMP connection, the backtable was completed to free the superior vena cava (the liver was maintained on a refrigerated surface). All surgical times were recorded.
