Demand for human primary hepatocytes is increasing, particularly for clinical trials of hepatocyte transplantation. However, due to the severe shortage of organ transplant donors, the source of cells for these endeavors is restricted to untransplantable livers, such as those from non-heart-beating donors and surgically resected liver tissues. To improve cell recovery from such sources after warm ischemia, we evaluated the efficacy of applying perfusion solutions, focusing on improvement of hepatocyte recovery. Warm ischemia was induced by clamping both portal vein and hepatic artery for 10 or 15 min in rats. The liver was perfused with either Euro-Collins (EC) or extracellular-type trehalose-containing Kyoto (ETK) solutions supplemented with an anticoagulant, either heparin or citrate phosphate dextrose solution (CPD), compared to Ca(2+), Mg(2+)-free Hanks solution. While the viability of recovered cells was 81.5 ± 4.2% and cell yield was 2.27 ± 0.53 × 10(8) in nonwarm ischemia controls (n = 11), these values were only 74.7 ± 2.9% and 0.38 ± 0.17 × 10(8), respectively, in the 10-min warm ischemia group, using the Hanks as the perfusion solution. Although the addition of heparin increased the live cell number only twofold (0.71 ± 0.40 × 10(8), n = 4), the best improvement was achieved by adding CPD to EC. This resulted in a recovery of 1.41 ± 0.50 × 10(8) in the 10-min ischemia group (n = 7) and 1.37 ± 0.28 × 10(8) in the 15-min group (n = 3). Macroscopic observation showed that blood had been completely flushed out by the solution, suggesting good restoration of the microcirculation in ischemic liver. Using ETK instead of EC resulted in a slight decrease in efficacy. These results demonstrate that CPD, as opposed to heparin, is effective in ensuring liver microcirculation and flushing out the blood and that EC is the best perfusion solution for obtaining hepatocytes from ischemic liver.
Keywords: Citrate; Hepatocyte isolation; Warm ischemia.