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Assessment of cold stored human hepatocytes for transplantation: Storage of liver tissue vs. isolated hepatocytes

Carl Jorns¹, Roberto Gramignoli², Mohammed Saliem¹, Helen Zemack¹, Lisa-Mari Nilsson¹, Greg Nowak¹, Bo-Göran Ericzon¹, Stephen Strom², Ewa Ellis^1,2

¹Transplantation Surgery, Karolinska Institute, Stockholm, Sweden; ²Pathology, University of Pittsburgh, Pittsburgh, PA, United States

Background: Hepatocyte transplantation is a useful treatment for patients with acute liver failure or metabolic disease. Repeated infusions over 1-2 days improve engraftment and clincial outcome and isolated hepatocytes are usually cold-stored during this time. This study aimed to determine the best way to cold preserve human hepatocytes for repeated transplantations.

Methods: Hepatocytes were isolated from tissue obtained from resections or organ donors by collagenase digestion. Hepatocytes were analysed directly after isolation (fresh) or stored for 48 hours at 4° C in University of Wisconsin Solution (UW cells). Liver tissue from the same donor was stored at 4° C in UW and hepatocytes were isolated after 48 hours (UW tissue cells). Hepatocyte functions were evaluated by trypan blue exclusion, plating efficiency, ammonia metabolism and drug-mediated metabolic activities including CYP1A1/2, 2C9, 3A7, 3A4 and conjugation, caspase and TUNEL assays.

Results: Mean viability of freshly isolated hepatocytes was 79%. Viability decreased after 48 hours of UW cold storage to 55% which was significantly less than UW tissue cells (71%). Plating efficiency decreased by 50% in UW cells whereas UW tissue cells retained 70% of the original plating efficiency. Fresh and UW tissue cells showed a similiar ammonia and drug metabolism activity which was significantly higher than in UW cells. Hepatocytes stored in 48 hrs in UW showed a strong increase of TUNEL positive cells whereas TUNEL staining in cold-stored liver tissue and hepatocytes isolated after 48 hrs was nearly unchanged, indicating an increased rate of apoptosis during cold storage of isolated hepatocytes.

Conclusions: Cold storage of liver tissue and hepatocyte isolation after 48 hrs is often superior to cold storage of isolated hepatocytes as measured by hepatocyte viability and function.