Survival and function of isolated hepatocytes after cryopreservation
Introduction
Isolated hepatocytes are now widely used as a model system in various research areas. Human hepatocytes are highly desirable to answer questions relative to the human situation since various hepatic functions exhibit qualitative and quantitative interspecies variations, particularly when comparisons involve experimental animals and human beings. However, major problems are encountered with hepatocytes from human beings and some animal species such as non human primates. Although large cell numbers can be obtained from a single organ, their sources are limited. Moreover availability of human liver samples is erratic and unpredictable and still reduced by legal and ethical considerations in certain countries [1]. Because of the progress in liver transplantation human hepatocytes are now mostly prepared from surgical tissue pieces resected from primary and secondary tumors. Therefore there is a need for long-term preservation of isolated hepatocytes and for the establishment of liver parenchymal cell banks. Various cryopreservation protocols have been proposed, mostly for rat hepatocytes, over the last 25 years. However in a number of studies cell recovery was low and limited information was brought on maintenance of liver-specific functions after thawing. Only the most recent and convincing studies on cryopreservation of adult hepatocytes from various species will be discussed here.
Section snippets
Freeze–thaw conditions
Freeze–thaw protocols have been designed for liver cell suspensions. The experimental conditions used in our laboratory for rat hepatocytes [2], [3] are the following: to 6×106 rat hepatocytes in 1 ml of Leibovitz L-15 medium supplemented with 0.2% bovine serum albumin. an equal volume of the same medium containing 32% dimethylsulfoxide (DMSO) and 20% fetal calf serum (FCS) is slowly added. Then the cell suspension is distributed in freezing vials (1.6 ml of the suspension per vial) and kept
Survival of hepatocytes after cryopreservation
After cryopreservation, hepatocyte viability, usually based on the trypan blue exclusion test, is decreased by 10–25% depending on the species and the study (Table 2). Much lower viability rates have been reported in some studies on rat hepatocytes: e.g. 90±3% and 53±9% for fresh and cryopreserved cells, respectively [20]. The duration of storage in liquid nitrogen has limited, if any, influence on cell viability, even if it reaches several years [3]. Most dead cells can be eliminated by
Functional activities of hepatocytes after cryopreservation
A number of studies have dealt with functional activities of cryopreserved hepatocytes (Table 3); however the majority concerns only a limited number of functions measured within a few hours following thawing and cells of rat origin. In cryopreserved isolated rat hepatocytes having a high ATP/ADP ratio metabolism of benzo(a)pyrene was quite similar to that observed in their unfrozen counterparts whether phase 1 or phase 2 metabolizes were considered [21]. However, functional alterations are
Conclusions
Prolonged storage of cryopreserved isolated hepatocytes is now possible and such cells have been used with success for drug metabolism and toxicity studies and for liver support [20], [30], [31]. After thawing, hepatocytes retain in vitro, for at least a short period, functional activities including phase I and phase II drug metabolizing enzyme activities, making it possible for studies on identification of interspecies metabolism pathways of a new drug. However, cell recovery is generally low
Acknowledgements
The authors thank A. Vannier for typing the manuscript. Personal studies were supported in part by EEC (contract no. 11277-95-10 Field ISP NL and BIOMED (contract BMH4 CT96-02-254).
References (32)
- et al.
Report on the international workshop on the use of human in vitro liver preparations to study drug metabolism in drug development
Biochem. Pharmacol.
(1995) - et al.
Cryopreservation of isolated rat hepatocytes: a critical evaluation of freezing and thawing conditions
Cryobiology
(1988) - et al.
Cryopreservation of adult human hepatocytes: the influence of deep freezing storage on the viability, cell seeding, survival, fine structure and albumin synthesis in primary cultures
J. Hepatol.
(1986) - et al.
Thawed human hepatocytes in primary culture
Cryobiology
(1992) - et al.
Insights into the cryoprotective mechanism of demethyl sulfoxide for phospholipid bilayers
Cryobiology
(1991) - et al.
Characterization of cryopreserved rat liver parenchymal cells by metabolism of diagnostic substrates and activities of related enzymes
Biochem. Pharmacol.
(1992) - et al.
Development of bioartificial liver: bilirubin conjugation in Gunn rats
J. Surg. Res.
(1990) - et al.
A new approach to the cryopreservation of hepatocytes in a sandwich culture configuration
Cryobiology
(1990) - et al.
Influence of alginate gel entrapment and cryopreservation on survival and xenobiotic metabolism capacity of rat hepatocytes
Toxicol. Appl. Pharmacol.
(1996) - et al.
Long-term maintenance of drugmetabolizing enzyme activities in rat hepatocytes after cryopreservation
Toxicol. Appl. Pharmacol.
(1997)