Overview: hepatocytes and cryopreservation—a personal historical perspective

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Abstract

Hepatocytes represent an important tool for the investigation of species differences in drug metabolism and toxicity. Data obtained with hepatocytes from multiple animal species, including man, allow better prediction of the effects of xenobiotics in man. Cryopreservation of hepatocytes extends the use of this important experimental system by enhancing the convenience of its use. Also, it allows the researchers to perform experiments not plausible with freshly isolated hepatocytes, such as the direct comparison of xenobiotic toxicity and metabolism in hepatocytes from multiple human donors in a single experiment.

Introduction

Approximately 15 years ago, after years of working on chemical toxicity [1] and genotoxicity [2] with rodent cells, I finally saw the light. The light came on during one of the presentations in a symposium. A slide was shown—I can still see it very clearly—on the organ-specific toxicity of myriad pharmaceuticals in man and various laboratory animals. It struck me clearly that there was not a single animal species that would behave like man. For one drug, the rat would behave like man. For another drug, it would be the dog, or the monkey. If different animal species exhibited different responses to a toxic chemical, how would one logically evaluate which animal species would predict human effects? I then realized that a bridge was needed to allow one to extrapolate logically data from animals to human. This bridge needed to have human-specific properties that would be critical to the manifestation of the toxicological effects.

Section snippets

Bridging animals and human

I researched on the different approaches to bridge laboratory animals and man. I read publications on scaling based on body weight [3] or surface area [4], assuming that size, and thereby dose to the organism, was the critical determinant of species differences. Although there were plentiful examples of successful applications of these approaches, I could not accept this concept entirely. I simply thought: if this were true, then any animals with a size similar to human (e.g. pig) would behave

Experimental models of the liver

I searched for an appropriate experimental model. At that time, liver S-9, a 9000×g postmitochondrial supernatant of homogenized liver, was commonly used in mutagenicity studies as an exogenous activating system. I worked on S-9 and found something disturbing, that the relative potency of mutagens depended on how much S-9 I used [6]. For benzo(a)pyrene, for instance, lower S-9 concentrations yielded higher mutagenicity in Chinese hamster ovary cells at the hypoxanthine–guanine phosphoribosyl

Research on hepatocytes

The rest is history. My scientific endeavor for the past two decades has revolved around animal and human hepatocytes, with emphasis on human hepatocytes. I learned how to make rat hepatocytes from Carol Green [7] and, after trial and error with human liver biopsies, I modified the procedures for human hepatocyte isolation [8]. My research centered on species comparison and in vitro–in vivo correlation. My colleagues and I found that the sex dimorphism in acetaminophen metabolism observed in

Cryopreservation

To overcome the problem of the limited availability of human livers, we initiated the development of procedures for the cryopreservation of hepatocytes [19]. Cryopreservation of hepatocytes has always been controversial. When I first started working with hepatocytes in the early 1980’s, there was a common belief that hepatocytes could not be cryopreserved. What was troubling to me was that researchers tended not to believe published reports on successful hepatocyte cryopreservation. We

Final comments on cryopreservation

The purpose of this Special Issue on Hepatocyte Cryopreservation, which represents mainly the papers presented in the 1998 Annual Symposium of the Hepatocyte Users Group of North America, is to gather thoughts and observations made by researchers from independent laboratories on the cryopreservation of hepatocytes. The general conclusion of the symposium is that limited success has been achieved with cryopreserved hepatocytes and that cryopreserved hepatocytes are appropriate for some

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