Ischemia-reperfusion injury
Experimental: Liver
Hepatocyte Viability and Adenosine Triphosphate Content Decrease Linearly Over Time During Conventional Cold Storage of Rat Liver Grafts

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Abstract

Introduction

The gold standard in organ preservation is static cold storage (SCS) using University of Wisconsin solution (UW). Although it is well-known that there is a finite limit to SCS preservation, and that there is a correlation between the adenosine triphosphate (ATP) levels and organ function post-preservation, a quantitative relationship has not been established, which is important in understanding the fundamental limitations to preservation, minimizing cold ischemic injury, and hence maximizing use of the donor organ pool.

Aim

This study determines the time limits of cellular viability and metabolic function during SCS, and characterizes the relationship between cellular viability and energetic state using clinically relevant techniques in organ preservation.

Methods

Rat livers were procured and stored using conventional storage in UW solution at 4°C. Viability was assessed by determining the amount of viable hepatocytes and intracellular ATP content after 0, 24, 48, 72, and 120 hours of storage.

Results

Numbers of viable hepatocytes that were isolated from these livers decreased steadily during SCS. After 5 days, viable hepatocytes decreased from 25.95 × 106 to 0.87 × 106 cells/gram tissue. Intracellular ATP content decreased from 9.63 to 0.93 moles/g tissue. Statistical analysis of variance established a linear relation for both parameters as a function of time (P < .05).

Conclusion

The linear correlation between hepatocyte viability, ATP content, and storage time suggests a shared physiological foundation. These findings confirm ATP as direct predictor for organ quality in the context of liver preservation, which will aid quantitative assessment of donor organs for various applications.

Section snippets

Procurement of Rat Livers

Experiments were performed using female Lewis rats (n = 30) weighing 150 to 200 g (Charles River Labs, Wilmington, Mass). The animals were maintained in accordance with National Research Council guidelines and the experimental protocols were approved by the Subcommittee on Research Animal Care, Massachusetts General Hospital. Subjects were anesthetized with isoflurane (Forane, Baxter, Deerfield, IL) using a Tech 4 vaporizer (Surgivet, Waukesha, Wis). The liver was harvested using the technique

Cell Yield During SCS

To assess the viability of each graft, the live hepatocyte fraction was determined at various SCS storage times. Figure 1A shows the results of the hepatocyte isolations for each interval. Cell yields decreased consistently with the increase in storage time; the 120-hour control interval showed a yield of 0.87 ± 0.42 × 106 hepatocytes per gram of liver tissue, or 3.3% of the amount of freshly isolated hepatocytes (25.95 × 106 cells/g liver). Linear regression analysis revealed a correlation

Discussion

In this study we have shown that when a healthy liver is stored ex vivo there is a strong association between the amount of viable hepatocytes and the ATP level, both of which decrease linearly with time. The dynamic correlation of this process supports further exploration into the use of cellular energy levels as a quantitative criterion of graft viability. For example, previous studies have shown that rat livers can be stored and transplanted after an average SCS time of 18 to 24 hours15, 16,

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    Tim Berendsen was funded by the Professor Michael-van Vloten Foundation, National Institutes of Health (R01 DK59766, R01 EB 008678, R00 DK080942), Shriners Hospitals for Children.

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