Survival and function of isolated hepatocytes after cryopreservation

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Abstract

Cryopreservation in liquid nitrogen is presently the only way for long-term storage of isolated hepatocytes. Freeze–thaw conditions are not well defined yet; the most critical parameters appear to be the choice of the cryoprotectant, composition of the freezing medium, and cooling and thawing rates. Comparable results have been obtained with hepatocytes from various species, including man. Cryopreservation usually results in low cell recovery and early alterations of functional activities. However, both phase I and phase II xenobiotic metabolism is still active after thawing, at least during a short period. Moreover, survival and function of cryopreserved hepatocytes can be improved when these cells have a high energy status, are cryopreserved after immobilization in a gel, separated from dead cells on a Percoll gradient or placed in more favorable culture conditions (e.g. in coculture with liver non parenchymal cells). Additional studies are needed to improve freeze–thaw protocols and to better characterize liver parenchymal cells after storage, including evaluation of their responsiveness to specific inducers.

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).

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