The scarcity of viable hepatocytes is a significant bottleneck in cell transplantation, drug discovery, toxicology, tissue engineering, and bioartificial assist devices, where trillions of high-functioning hepatocytes are needed annually. We took the novel approach of using machine perfusion to maximize cell recovery, specifically from uncontrolled cardiac death donors, the largest source of disqualified donor organs. In a rat model, we developed a simple 3 hour room temperature (20±2°C) machine perfusion protocol to treat non-premedicated livers exposed to 1 hour of warm (34°C) ischemia. Treated ischemic livers were compared to fresh, fresh-treated and untreated ischemic livers using viable hepatocyte yields and in vitro performance as quantitative endpoints. Perfusion treatment resulted in both a 25-fold increase in viable hepatocytes from ischemic livers, and a 40% increase from fresh livers. While cell morphology and function in suspension and plate cultures of untreated warm ischemic cells was significantly impaired, treated warm ischemic cells were indistinguishable from fresh hepatocytes. Further, a strong linear correlation between tissue ATP and cell yield enabled accurate evaluation of the extent of perfusion recovery. Maximal recovery of warm ischemic liver ATP content appears to be correlated with optimal flow through the microvasculature. These data demonstrate that the inclusion of a simple perfusion-preconditioning step can significantly increase the efficiency of functional hepatocyte yields and the number of donor livers that can be gainfully utilized.

Background High-quality human hepatocytes form the basis of drug safety and efficacy tests, cell-based therapies, and bridge-to-transplantation devices. Presently the only supply of cells derives from an inadequate pool of suboptimal disqualified donor livers. Here we evaluated whether machine perfusion could ameliorate ischemic injury that many of these livers experience prior to hepatocyte isolation. Methods Non-heparinized female Lewis rat livers were exposed to an hour of warm ischemia (34°C) and then perfused for 3 hours. Five different perfusion conditions that utilized the cell isolation apparatus were investigated, namely: (1) modified Williams Medium E and (2) Lifor, both with active oxygenation (95%O2/5%CO2), as well as (3) Lifor passively oxygenated with ambient air (21%O2/0.04%CO2), all at ambient temperatures (20±2°C). At hypothermic temperatures (5±1°C) and under passive oxygenation were (4) University of Wisconsin solution (UW) and (5) Vasosol. Negative and positive control groups comprised livers that had ischemia (WI) and livers that did not (Fresh) prior to cell isolation, respectively. Results Fresh livers yielded 32±9 million cells/g liver while an hour of ischemia reduced the cell yield to 1.6±0.6 million cells/g liver. Oxygenated Williams medium E and Lifor recovered yields of 39±11 and 31±2.3 million cells/g liver, respectively. The passively oxygenated groups produced 15±7 (Lifor), 13±7 (Vasosol), and 10±6 (UW) million cells/g liver. Oxygenated Williams Medium E was most effective at sustaining pH values, avoiding the accumulation of lactate, minimizing edematous weight gain and producing bile during perfusion. Conclusions Machine perfusion results in a dramatic increase in cell yields from livers that have had up to an hour of warm ischemia, but perfusate choice significantly impacts the extent of recovery. Oxygenated Williams Medium E at room temperature is superior to Lifor, UW and Vasosol, largely facilitated by its high oxygen content and low viscosity.