Normothermic Ex Vivo Liver Platform Using Porcine Slaughterhouse Livers for Disease Modeling

Abstract

Metabolic and toxic liver disorders, such as fatty liver disease (steatosis) and drug-induced liver injury, are highly prevalent and potentially life-threatening. To allow for the study of these disorders from the early stages onward, without using experimental animals, we collected porcine livers in a slaughterhouse and perfused these livers normothermically. With our simplified protocol, the perfused slaughterhouse livers remained viable and functional over five hours of perfusion, as shown by hemodynamics, bile production, indocyanine green clearance, ammonia metabolism, gene expression and histology. As a proof-of-concept to study liver disorders, we show that an infusion of free fatty acids and acetaminophen results in early biochemical signs of liver damage, including reduced functionality. In conclusion, the present platform offers an accessible system to perform research in a functional, relevant large animal model while avoiding using experimental animals. With further improvements to the model, prolonged exposure could make this model a versatile tool for studying liver diseases and potential treatments.

Are slaughterhouse-obtained livers suitable for use in ex vivo perfusion research?

“…21 In LB, the ICG half-life increased two-fold between T0 and T300. When considered alongside the results of previous studies, in which tissue damage was assessed by measuring the ICG half-life up to 183 min, 16 , 31 this increase seemed not to be significant. However, the increase in ALT activity during the perfusion that occurred in LB might indicate initial tissue damage.…”

“…The liver was connected as described previously. 16 The gas provided to the oxygenator was composed of a mixture of O 2 (95% O 2 , 5% CO 2 ) and N 2 (95% N 2 , 5% CO 2 ) in a 1:3 ratio. The oxygen saturation of the blood was maintained at >95%.…”

“…To analyze liver functionality, indocyanine green (ICG) 5 mg/L (7695-2, Carl Roth, Karlsruhe, Germany) was added to the perfusate at the −15 and 285-minute time points and the distributions were analyzed as described previously. 16 Tissue samples were obtained using a biopsy punch (8 mm diameter, Megro GmbH & Co. KG, Wesel, Germany) for histologic evaluation following cold storage and 5 hours of perfusion. As a control, tissue samples were also obtained immediately after the harvesting of the livers.…”

“…The samples were further processed as described previously. 16 Histologic evaluation was performed by a board-certified veterinary pathologist.…”

“…The 3Rs principle guides the selection of methods for use in experiments that do not involve the use of animals, minimize their use, or involve less painful procedures. 8 Today, in addition to laboratory animal-derived organs, [9][10][11][12] slaughterhouse-derived organs are increasingly used in perfusion research, 5,[13][14][15][16] but standardized protocols for organ harvesting and the slaughter of the animals have not been established. The fixed processes used in slaughterhouses present a challenging environment for organ sampling, because the infusion of substances such as heparin prior to slaughter is not permitted, which increases the risk of intrahepatic thrombosis.…”