Viability and primary culture of rat hepatocytes after hypothermic preservation: The superiority of the leibovitz medium over the university of wisconsin solution for cold storage

Abstract

Hepatocytes isolated from adult rat livers were hypothermically preserved for 24 or 48 hr before being plated under conventional culture conditions. They were stored either in the Leibovitz medium, a cell culture medium with and without polyethylene glycol (PEG), a compound known to suppress ischemia-induced cell swelling, or in the University of Wisconsin solution, the most effective solution for cold organ preservation. After 24 or 48 hr of storage at 4.5 degrees C in Leibovitz medium, cell viability and adherence efficiency to plastic dish, were only slightly reduced, whereas University of Wisconsin hepatocytes had a decreased viability and (especially after 48-hr storage) lost their adhesion ability; they did not survive in vitro. The metabolic competence of hepatocytes maintained in Leibovitz medium was retained over the 3 days of culture, as shown by low extracellular levels of the membrane-bound and cytosolic hepatic enzymes, as well as by intracellular glutathione content, albumin secretion rate and several phase I and phase II drug metabolic reactions very close to those found with fresh hepatocytes maintained under similar culture conditions. Addition of polyethylene glycol to the Leibovitz medium resulted in slightly higher viability and function of hepatocytes after cold storage. These results clearly demonstrate that viability of a transplanted liver does not correlate with long-term in vitro viability of isolated hepatocytes after hypothermic preservation in University of Wisconsin solution. They also suggest that nutritional and energy substrates as found in the Leibovitz medium are probably required to define a suitable solution for cold preservation of isolated parenchymal cells. The findings with Leibovitz medium favor the conclusion that hypothermically preserved hepatocytes could be used for various metabolic studies and for the treatment of liver insufficiency.

Liver cell models in in vitro toxicology.

“…When they recover levels of functional activities stored at 40C eirher in the Leibovitz medium close to those found in fresh hepatocyte (Gibco Laboratories, Cergy-Pontoise, cocultures after a few days (45). A recent France) or in the University of Wisconsin study has shown that when entrapped in solution (Dupont) (the most effective solu-alginate beads, hepatocytes exhibit only tion for cold organ preservation), rat hepato-limited reduced cell viability and retain cytes survived for 1 or 2 days (34)(35)(36). their functions at high levels (46).…”

“…Moreover, conspicuous individual variations in cellular functions are cells preserved in this medium are able to frequently observed in human hepatocytes correctly attach to plastic and survive in (28) ( Table 2). In addition to age, sex, liver culture (36). diseases, and genetic polymorphisms, preVarious procedures for cryopreservation medication and the duration and conditions of hepatocytes have been described; the of liver preservation before cell disruption importance of the nature and concentramay greatly influence the metabolic activities tion of cryoprotectant, cooling rate, and of freshly isolated human hepatocytes conditions of thawing have been stressed (29,30).…”

The Optimization of Short-Term Hepatocyte Preservation Before Transplantation

“…However, in this study, we found that both UW solution and ETK solution, which has been shown to be useful for pancreatic islet preservation, 37 appeared to be undesirable for maintaining the isolated hepatocytes during a short-term preservation period. Although it was previously reported that UW solution seemed to be beneficial to some extent in terms of maintaining the viability of hepatocytes, 13,14 the effects on the survival rate remain uncertain. In accordance with previous studies, the viability of the hepatocytes that were preserved in UW solution remained at the same level as the other groups in the present study.…”

Interleukin‐1β antagonizes phenobarbital induction of several major cytochromes P450 in adult rat hepatocytes in primary culture

“…activity assays EROD and PROD activities, mainly supported by CYPIA and CYP2B subfamilies, respectively, were measured essentially according to Burke and Mayer [29,30], with slight modifications for living hepato¢yte monolayers [31]. Briefly, the medium was removed after 12, 24, 72 and 96 h of treatment, and cells were washed with 100 mM PBS and then incubated with ethoxyresorufin or pentoxyresorufln (50/~M) and udi~lamide (I.5 raM) in PBS at 37"C, Kinetic reading with a spectrofluorimeter was performed over 15 min.…”