Preclinical validation of a customized circuit for ex situ uninterrupted cold-to-warm prolonged perfusion of the liver.

CONTEXT: Clinical adoption of ex situ liver perfusion is growing. While hypothermic perfusion protects against ischemia-reperfusion injury in marginal grafts, normothermic perfusion enables organ viability assessment and therefore selection of borderline grafts. The combination of hypothermic and normothermic perfusion, known as "cold-to-warm," may be the optimal sequence for organ preservation, but is difficult to achieve with most commercial perfusion systems. We developed an adaptable customized circuit allowing uninterrupted "cold-to-warm" perfusion and conducted preclinical studies on healthy porcine livers and discarded human livers to demonstrate the circuit's efficacy. METHODS: In collaboration with bioengineers, we developed a customized circuit that adapts to extracorporeal circulation consoles used in cardiovascular surgery and includes a proprietary reservoir enabling easy perfusate change without interrupting perfusion. This preclinical study was conducted on porcine and human livers. Perfusion parameters (pressures, flows, oxygenation) and organ viability were monitored. RESULTS: The customized circuit was adapted to a LivaNova S5® console, and the perfusions were flow-driven with real-time pressure monitoring. Ten porcine liver and 12 discarded human liver perfusions were performed during 14 to 18 h and 7 to 25 h, respectively. No hyperpressure was observed (porcine and human portal pressure 2-6 and 2-8 mm Hg; arterial pressure 10-65 and 20-65 mm Hg, respectively). No severe histological tissue injury was observed (Suzuki score ≤ 3 at the end of perfusion). Seven (70%) porcine livers and five (42%) human livers met the UK viability criteria. CONCLUSION: The customized circuit and system design enables smooth uninterrupted "cold-to-warm" perfusion not present in current commercial perfusion systems.

Immunofluorescent Staining of Human Hepatic Multicellular Spheroids: A Model for Studying Liver Diseases.

In vitro studies on liver diseases, such as non-alcoholic fatty liver disease, fibrosis, and hepatocellular carcinoma, are traditionally performed in two-dimensional (2D) cultures of isolated primary cells or immortalized cell lines. However, this approach has limitations, as 2D cultures inadequately replicate the cell-cell and cell-extracellular matrix interactions found in three-dimensional (3D) environments. To overcome this limitation, various 3D models, such as spheroids, have been developed. These spheroids serve as simplified biomimetic in vitro models for studying liver diseases. They can be generated using a variety of cells from healthy and pathological tissues, including liver cancer. Here, we present a comprehensive protocol for performing immunofluorescent staining and confocal imaging on whole human hepatic multicellular spheroids, utilizing primary cells or cell lines. The immunofluorescence technique is a potent tool to understand the spatial distribution of different cell types within the spheroids and define the interactions that occur among these cells.