Hypothermic preservation of hepatocytes : I. Role of cell swelling

Hepatocytes from isolated rat livers were hypothermically incubated (5 degrees C) in an oxygenated environment with continuous shaking (to simulate organ perfusion preservation). The incubation solution was either a tissue culture medium (L-15), an organ preservation perfusate (UW gluconate), or a simple cold-storage solution used for organ preservation (UW lactobionate). Hepatocyte viability was assessed from the release of lactate dehydrogenase (LDH) into the incubation medium. Cell swelling (due to the uptake of water) was also measured. Within 24 hr, hepatocytes hypothermically stored in each of the three incubation solutions became swollen (30 to 40% water gain) and lost a significant amount of LDH (as much as 60%). The addition of polyethylene glycol (PEG; relative molecular mass 8000; 5 g%) to the solutions suppressed cell swelling and allowed the incubated hepatocytes to remain relatively well preserved (30% LDH release) for as long as 120 hr. Adding either dextran (relative molecular mass 10,000 to 78,000; 5 g%) or saccharides (100 mmol/liter) instead of PEG neither prevented cell swelling nor prevented the cells from dying. The results of this study suggest (i) there is a direct correlation (r = 0.873) between hypothermia-induced cell swelling and cell death (i.e., the suppression of cell swelling prevents cell death); (ii) the mechanism by which PEG prevents cell swelling (and thus maintains cell viability) is not related to the osmotic or oncotic properties of the molecule but instead is apparently related to some unknown interaction between PEG and the cell, an interaction that provides stability during hypothermic incubation; and (iii) hypothermia-induced cell swelling must be prevented if isolated hepatocytes are to be used as a model for studying the mechanism by which cell damage occurs during hypothermic organ preservation. By eliminating cell death due to cell swelling, the biochemical mechanisms of cell death can be studied.