Overview: hepatocytes and cryopreservation—a personal historical perspective
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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Prolonged cultured human hepatocytes as an in vitro experimental system for the evaluation of potency and duration of activity of RNA therapeutics: Demonstration of prolonged duration of gene silencing effects of a GalNAc-conjugated human hypoxanthine phosphoribosyl transferase (HPRT1) siRNA
2021, Biochemical PharmacologyCitation Excerpt :The cryopreserved human hepatocytes used in the study (lot HH1142) were obtained from the liver of a 27-year old Caucasian female provided to our laboratory by the International Institute for the Advancement of Medicine (IIAM, Edison, NJ), with explicit donor/family consent and Institutional Review Board approval for research applications. Hepatocytes were isolated via collagenase digestion and cryopreserved immediately after isolation without culturing to retain in vivo liver functions as previously reported [11,12,16–18]. The cryopreserved human hepatocytes were thawed at 37 °C in Cryopreserved Hepatocyte Recovery Medium (CHRM™, AP Sciences Inc, Columbia, MD) and collected by centrifugation at 100 × g for 10 min.
Recent advances and novel strategies in pre-clinical formulation development: An overview
2011, Journal of Controlled ReleaseCitation Excerpt :These extrapolations are based on an assumption of a correlation between the exposure–response relationship in animals and man. Unfortunately, there is no single animal species that can serve as the ‘perfect surrogate’ for human subjects, and the appropriate surrogate species needs to be evaluated for each situation [123]. Selection of the animal species to be used for toxicity testing should factor the potential interspecies differences that can influence systemic drug exposure and target cell sensitivity.
In vitro co-metabolism of acetoacetate and ethanol in human hepatic mitochondrial and cytosolic fractions
2005, Chemico-Biological InteractionsCitation Excerpt :In vivo studies to evaluate methods to increase alcohol elimination are difficult to perform because of obvious ethic implications. Unfortunately animal models are often unsuitable as they do not exactly replicate the human metabolism [1]. However, the effects of toxic concentrations of xenobiotics may be evaluated in vitro using tissue samples collected shortly after death.
Inter-species differences in drug properties
2001, Chemico-Biological InteractionsCitation Excerpt :For example, rats have enormous amount of N-acetylating capacity, dogs have none, and humans are generally intermediate, but with wide intersubject variability to polymorphisms. As noted by Li [1], the field of interspecies comparisons was pre-occupied for a long time with the search for a “perfect” animal model. Eventually, there was a realization that “…not a single animal species would behave like (hu)man.
Ice Recrystallization Inhibition Activity of Protein Mimetic Peptoids
2021, Journal of Inorganic and Organometallic Polymers and Materials