Supercooling preservation and transplantation of the rat liver

Abstract

The current standard for liver preservation involves cooling of the organ on ice (0-4 °C). Although it is successful for shorter durations, this method of preservation does not allow long-term storage of the liver. The gradual loss of hepatic viability during preservation puts pressure on organ sharing and allocation, may limit the use of suboptimal grafts and necessitates rushed transplantation to achieve desirable post-transplantation outcomes. In an attempt to improve and prolong liver viability during storage, alternative preservation methods are under investigation. For instance, ex vivo machine perfusion systems aim to sustain and even improve viability by supporting hepatic function at warm temperatures, rather than simply slowing down deterioration by cooling. Here we describe a novel subzero preservation technique that combines ex vivo machine perfusion with cryoprotectants to facilitate long-term supercooled preservation. The technique improves the preservation of rat livers to prolong storage times as much as threefold, which is validated by successful long-term recipient survival after orthotopic transplantation. This protocol describes how to load rat livers with cryoprotectants to prevent both intracellular and extracellular ice formation and to protect against hypothermic injury. Cryoprotectants are loaded ex vivo using subnormothermic machine perfusion (SNMP), after which livers can be cooled to -6 °C without freezing and kept viable for up to 96 h. Cooling to a supercooled state is controlled, followed by 3 h of SNMP recovery and orthotopic liver transplantation.

Figure 1

Rat liver subnormothermic machine perfusion system. Illustration of various components (a); black lines represent the perfusate circuit; blue lines the antifreeze circuit running through the jackets of the organ chamber, bubble trap and oxygenator; green line the bile duct drain cannula. Red * indicates where the circuit can be interrupted to open the system and stop recirculation. Image of the SNMP system (b) and schematic representation (c). Section of tubing that runs between the bubble trap and the liver portal vein, interrupted to include an section of tubing to act as a hydrostatic manometer (d). Liver in a supercooling bag (e).

Figure 2

Fashioning of vascular anastomosis cuffs. A 4 mm section is cut from a 16 and 14G catheter (a, b). A quarter section is removed from the piece, leaving a tail and body of the cuff (c). A groove is made fully around the body for proper fixation of the suture (d).

Figure 3

Flow and pressure regimen for the first 30 minutes of SNMP recovery. Temperature is slowly increased in the first 17 minutes to 21 °C (grey), while the flow is increased in parallel to a maximum of 12 ml/min (green). The pressure should not exceed the temperature-dependent maximum (black)

Figure 4

Real time observable perfusion parameters (a–c) and transplantation outcome (d, e). Vascular resistance of fresh livers and liver stored for increasing durations of static cold storage (SCS) (a). Bile production in fresh livers and liver preserved by SCS and supercooling for 72 or 96 hours (b), vascular resistance of livers preserved by supercooling for 72 or 96 hours (c). Long–term (30 days) survival for various groups (d). Supercooling controls include all groups in which a component of the supercooling protocol was omitted. Body weight of recipients of fresh livers and livers supercooled for 96 hours (e).