Hypothernic storage of pig hepatocytes: influence of different storage solutions and cell density

For clinical use of bioartifical liver devices a constant supply of primary liver cells has to be provided. Hypothermic storage of isolated pig hepatocytes could support large-scale stocking of cells. Freshly isolated pig hepatocytes from slaughterhouse livers were stored at 4 degrees C for 24, 48, and 72 h three different solutions: Leibovitz L-15 + 5% polyethylene glycol (PEG), University of Wisconsin (UW) solution, and a simplified UW solution. After storage, cells were cultured for 2 weeks in the collagen sandwich configuration. Viability of hepatocytes was 65, 85, and 83% after 24 h storage, 21, 74, and 70% after 48 h, and 5, 65, and 59% after 72 h in Leibovitz L-15 medium, UW, and the simplified UW, respectively. After storage in L-15 medium, cells attached poorly to collagen matrices and exhibited ultrastructural lesions. Functional performance in this group, as judged by albumin secretion and cytochrome P450-dependent activity in subsequent culture, decreased rapidly as a function of storage time, with zero values after 48 h storage. In contrast, hypothermia of hepatocytes in both UW solutions resulted in well-preserved cells with respect to ultrastructural appearance, attachment rates, and functional performance during culture. No significant differences were observed between the original and the simplified UW solution. Higher cell concentrations up to 5 x 10(7) cells/ml improved viability of hepatocytes on warmup. In terms of cell supply for hybrid artificial liver support, hypothermic storage of hepatocytes at 4 degrees C could mean an alternative to the cryopreservation of cells, which usually results in a substantial loss of cells and vital function of cells. Thus, pig hepatocytes could be stored at 4 degrees C for several days and meet the logistical need of bioartificial liver devices while avoiding the hazards of cell freezing.