Transplantation/Immunology
Subnormothermic Machine Perfusion at Both 20°C and 30°C Recovers Ischemic Rat Livers for Successful Transplantation

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Background

Utilizing livers from donors after cardiac death could significantly expand the donor pool. We have previously shown that normothermic (37°C) extracorporeal liver perfusion significantly improves transplantation outcomes of ischemic rat livers. Here we investigate whether recovery of ischemic livers is possible using sub-normothermic machine perfusion at 20°C and 30°C.

Methods

Livers from male Lewis rats were divided into five groups after 1 h of warm ischemia (WI): (1) WI only, (2) 5 h of static cold storage (SCS), or 5 h of MP at (3) 20°C, (4) 30°C, and (5) 37°C. Long-term graft performance was evaluated for 28 d post-transplantation. Acute graft performance was evaluated during a 2 h normothermic sanguineous reperfusion ex vivo. Fresh livers with 5 h of SCS were positive transplant controls while fresh livers were positive reperfusion controls.

Results

Following machine perfusion (MP) (Groups 3, 4, and 5), ischemically damaged livers could be orthotopically transplanted into syngeneic recipients with 100% survival (N ≥ 4) after 4 wk. On the other hand, animals from WI only, or WI + SCS groups all died within 24 h of transplantation. Fresh livers preserved using SCS had the highest alanine aminotransferase (ALT), aspartate aminotransferase (AST), and the lowest bile production during reperfusion, while at 28 d post-transplantation, livers preserved at 20°C and 30°C had the highest total bilirubin values.

Conclusions

MP at both 20°C and 30°C eliminated temperature control in perfusion systems and recovered ischemically damaged rat livers. Postoperatively, low transaminases suggest a beneficial effect of sub-normothermic perfusion, while rising total bilirubin levels suggest inadequate prevention of ischemia- or hypothermia-induced biliary damage.

Introduction

It is estimated that approximately 15% of deaths due to chronic liver disease and cirrhosis in the United States could be prevented by whole organ transplantation [1], however, the lack of donor organs limits the prevalence of this treatment option. A significant number of donors experience cardiac death (DCD). Organs from these donors are rarely considered for transplantation as static cold storage (SCS) [2], the current gold standard of organ preservation, is incapable of reversing the ischemic damage these organs have sustained. Encouraging results in animal models have shown that both normothermic (37°C) extracorporeal machine perfusion (37MP) 3, 4, 5, and hypothermic (4°C) machine perfusion (4MP) that aims to augment SCS, can improve that state of ischemically damaged organs 6, 7. However, 4MP has been shown to compromise graft integrity with cold-induced damage [8], and 37MP requires a labor-intensive and complex heated perfusion system, hindering its translation into the clinical setting. Further, it is unknown whether normothermic conditions are necessary or, in fact, optimal.

Reducing perfusion temperature into the subnormothermic range may enable a less complex system to be used by reducing the need for strict temperature control as well as lowering oxygen demand during perfusion. Therefore, we investigated whether MP-based recovery of warm ischemic (WI) livers was also possible at both 20°C and 30°C and evaluated survival in a rat model of orthotopic liver transplantation, as well short-term graft function in an ex vivo perfusion setup. After orthotopic liver transplantation into syngeneic recipients, long-term graft performance was evaluated over the span of 28 d. Short-term graft function was determined using a 2 h ex vivo reperfusion model of 50% saline and 50% whole blood post-MP recovery. Early graft evaluation during reperfusion revealed comparable performance by WI+MP livers to fresh livers, while long-term post-transplantation evaluation showed 100% survival in WI+MP livers. It is conceivable, therefore, that MP may be reduced in complexity without compromising function, thereby bringing it a step closer to translation into standard clinical practice.

Section snippets

Groups

Experiments were performed using male Lewis rats weighing 250–300 g (Charles River Labs, Wilmington, MA, USA). The animals were maintained in accordance with National Research Council guidelines and the experimental protocols were approved by the Subcommittee on Research Animal Care, Committee on Research, Massachusetts General Hospital. Animals were divided into five groups based on how they were treated after 1 h of WI: (1) WI only; (2) 5 h of SCS; 5 h of machine perfusion at (3) 20°C, (4)

Survival after Transplantation

Transplantation of all livers was successful and the animals recovered from anesthesia rapidly. One hundred percent of the animals that received MP livers survived beyond 1 mo in good health and without any external signs of jaundice (Table 1). Within 12 h of transplantation, all recipients of SCS-preserved livers died. Autopsy revealed non-homogeneously perfused livers and serous fluid in the abdomen. All recipients of directly transplanted ischemic livers died later, within 24 h of

Discussion

Machine perfusion of donor livers is gaining recognition as an organ preservation technique capable of both expanding the current donor pool and producing grafts superior to conventional static cold storage methods 16, 17, 18, 19, 20. Recovery of damaged donor organs such as those which are ischemic 5, 21, 22, 23, 24 or steatotic has been demonstrated in animal models 25, 26, enabling clinical application of machine perfusion at 4°C 27, 28. To our knowledge, this study is the first to

Acknowledgments

This work was supported by grants from the National Institutes of Health (R01DK59766, R00DK080942) and the Shriners Hospitals for Children.

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    These authors contributed equally to this work.

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