Gene Expression in Human Hepatocytes in Suspension After Isolation Is Similar to the Liver of Origin, Is Not Affected by Hepatocyte Cold Storage and Cryopreservation, but Is Strongly Changed After Hepatocyte Plating

Abstract

ABSTRACT:Isolated primary human hepatocytes are a well accepted system for evaluating pharmacological and toxicological effects in humans. However, questions remain regarding how culturing affects the liver-specific functions of the hepatocytes. In addition, cryopreservation could also potentially affect the differentiation state of the hepatocytes. The first aim of the present study was to compare gene expression in freshly isolated primary hepatocytes to that of the liver of origin and to evaluate the expression changes occurring after cryopreservation/thawing, both when maintained in suspension and after plating. The second aim of the present study was to evaluate gene expression in hepatocytes after cold storage of suspensions up to 24 h compared with freshly isolated hepatocytes in suspension. Our results show that the gene expression in freshly isolated human hepatocytes in suspension after isolation is similar to that of the liver of origin. Furthermore, gene expression in primary human hepatocytes in suspension is not affected by hepatocyte cold storage and cryopreservation. However, the gene expression is profoundly affected in monolayer cultures after plating. Specifically, gene expression changes were observed in cultured relative to suspensions of human hepatocytes that are involved in cellular processes such as phase I/II metabolism, basolateral and canalicular transport systems, fatty acid and lipid metabolism, apoptosis, and proteasomal protein recycling. An oxidative stress response may be partially involved in these changes in gene expression. Taken together, these results may aid in the interpretation of data collected from human hepatocyte experiments and suggest additional utility for cold storage and cryopreservation of hepatocytes.The liver serves as the primary site of detoxification of exogenous and endogenous compounds in the systemic circulation. Other biological and physiological functions include the production and secretion of critical blood and bile components, such as albumin, bile salts, and cholesterol. The liver is also involved in protein, steroid, and fat metabolism, as well as vitamin, iron, and sugar storage.One of the most complex functions specific to liver is its ability to metabolize an enormous range of xenobiotics. Many drugs present in the blood are absorbed by hepatocytes, where they can be metabolized via phase I and II biotransformation reactions. Information concerning hepatic drug uptake and metabolism, phase I and phase II induction, drug interactions affecting hepatic metabolism, and hepatotoxicity are essential for the pharmacology and toxicology of a given drug. Because of major species differences both in the catalytic activities and regulation of enzymes involved in drug metabolism, many of these evaluations can only be accurately investigated with human tissue. Because intact cells more closely reflect the environment to which drugs are exposed in the liver, isolated primary human hepatocytes are a well accepted system for evaluating pharmacological and to...

Upcyte Human Hepatocytes: a Potent In Vitro Tool for the Prediction of Hepatic Clearance of Metabolically Stable Compounds

“…Hence, various methodologies based on in vitro data have been reported for the prediction of metabolic CL (Ring et al, 2011), routinely assessed early during drug discovery. Primary hepatocyte suspensions are commonly preferred for the in vitro determination of intrinsic CL (CL int ) , representing a fully competent metabolic model (Blanchard et al, 2006;Hewitt et al, 2007) with an expression pattern of drug-metabolizing enzymes (DMEs) comparable to the liver of origin (Richert et al, 2006). However, suspended hepatocytes lose viability and metabolic activity within a few hours (Skett, 1994;Gebhardt et al, 2003), which frequently averts reliable and accurate CL int determination, in particular for slowly cleared compounds (Di and Obach, 2015;Hutzler, et al, 2015).…”

Thyroid Hormone Is Necessary for Expression of Constitutive Androstane Receptor in Rat Hepatocytes

“…In addition, the duration of cryopreservation (1 week to 1 year) did not affect this phenomenon, and MHs that were cryopreserved by the same procedure as SHs possessed CYP2B1 protein (data not shown). Moreover, cryopreserved human hepatocytes were reported to maintain the activity and the expression of many metabolic enzymes (Shitara et al, 2003;Richert et al, 2006). These results indicate that the loss of the CAR expression may result from the procedure of cryopreservation of SHs.…”

“…These results indicate that the loss of the CAR expression may result from the procedure of cryopreservation of SHs. Although it is well known that cryopreservation subjects cells to intensive stress, most damaged functions may be recovered with time if suitable culture conditions are used (Richert et al, 2006). The reason why only CAR was repressed in cryo-SH is not clear.…”

A replication study of genome-wide CNV association for hepatic biomarkers identifies nine genes associated with liver function

“…Therefore, chronic ethanol consumption suppressed proteasome activity in the liver (15,16). Richert et al reported that PSMC6 (ATPase activity subunit) and PSMD12 (a non-ATPase subunit) were significantly overexpressed in human hepatocytes (31). The enriched Gene Ontology terms included programmed cell death (DFFA, CIDEB and SIAH1; P = 0.04) and protein binding (all 9 genes; P = 0.0071).…”