Influence of preculture on the prefreeze and postthaw characteristics of hepatocytes

Recent studies performed in our laboratory have shown that a brief period o f preculture prior to cryopreservation improves the postthaw viability of h epatocytes. The purpose of this investigation is to characterize specific m etabolic and biochemical characteristics of the hepatocytes (both frozen an d nonfrozen) to help elucidate the role of preculture on the postthaw viabi lity. Fresh and thawed hepatocytes were cultured in a bioartificial liver ( BAL) to determine albumin secretion as a function of time in culture. In ad dition, cell extracts were analyzed using nuclear magnetic resonance (NMR) spectroscopy to quantify changes in cell membrane composition and energetic s as a function of time in culture prefreeze and postthaw. The results of t hese studies showed an increase in albumin concentration in the culture med ium with time in culture for the period tested for both fresh and frozen an d thawed hepatocytes. NMR spectroscopy of lipid extracts indicates that in vitro culture of hepatocytes results in an increase in cholesterol relative to membrane phospholipid. Moreover, the NMR results also indicate phosphol ipid interconversion, via specific lipases in cultured hepatocytes, and the se changes are consistent with water permeability measurements performed pr eviously. Significant changes in phosphoenergetics were also observed, with the net energy charge for the cells increasing significantly with time in culture. In addition, NMR spectra show increased levels of g-phosphoglucona te, another indicator of the cellular response to the stresses of isolation and ex vivo culture. These results suggest that energetic considerations m ay be a significant factor in the ability of hepatocytes to survive the str esses of freezing and thawing. Significant shifts in membrane phospholipids may also influence membrane permeability and postthaw survival. (C) 2001 J ohn Wiley & Sons, Inc.