Cryopreserved Primary Hepatocytes as a Constantly Available in Vitro Model for the Evaluation of Human and Animal Drug Metabolism and Enzyme Induction*

Abstract

The use of primary hepatocytes is now well established for both studies of drug metabolism and enzyme induction. Cryopreservation of primary hepatocytes decreases the need for fresh liver tissue. This is especially important for research with human hepatocytes because availability of human liver tissue is limited. In this review, we summarize our research on optimization and validation of cryopreservation techniques. The critical elements for successful cryopreservation of hepatocytes are (1) the freezing protocol, (2) the concentration of the cryoprotectant [10% dimethyl-sulfoxide (DMSO)], (3) slow addition and removal of DMSO, (4) carbogen equilibration during isolation of hepatocytes and before cryopreservation, and (5) removal of unvital hepatocytes by Percoll centrifugation after thawing. Hepatocytes of human, monkey, dog, rat, and mouse isolated and cryopreserved by our standard procedure have a viability > or = 80%. Metabolic capacity of cryopreserved hepatocytes determined by testosterone hydroxylation, 7-ethoxyresorufin-O-de-ethylase (EROD), 7-ethoxycoumarin-O-deethylase (ECOD), glutathione S-transferase, UDP-glucuronosyl transferase, sulfotransferase, and epoxide hydrolase activities is > or = 60% of freshly isolated cells. Cryopreserved hepatocytes in suspension were successfully applied in short-term metabolism studies and as a metabolizing system in mutagenicity investigations. For instance, the complex pattern of benzo[a]pyrene metabolites including phase II metabolites formed by freshly isolated and cryopreserved hepatocytes was almost identical. For the study of enzyme induction, a longer time period and therefore cryopreserved hepatocyte cultures are required. We present a technique with cryopreserved hepatocytes that allows the induction of testosterone metabolism with similar induction factors as for fresh cultures. However, enzyme activities of induced hepatocytes and solvent controls were smaller in the cryopreserved cells. In conclusion, cryopreserved hepatocytes held in suspension can be recommended for short-term metabolism or toxicity studies. Systems with cryopreserved hepatocyte cultures that could be applied for studies of enzyme induction are already in a state allowing practical application, but may be further optimized.

Highlight report: perspectives in stem cell research—unbiased quantification of the similarity between in vitro generated and primary hepatocytes

“…Primary human cells still represent a gold standard in toxicological research (Heise et al 2012;Ghallab 2013Ghallab , 2014a. However, their use is hampered by difficult availability (Hewitt et al 2007;Hengstler et al 2000). Theoretically, stem cells offer a possibility to overcome this limitation.…”

Highlight report: towards the replacement of in vivo repeated dose systemic toxicity testing

“…It is also well established that induction or inhibition of detoxifying enzymes (Gebhardt et al 2003;Hengstler et al 2000;Hewitt et al 2007) or protective factors (Ilowski et al 2010(Ilowski et al , 2011 plays an important role in repeated dose toxicity. Moreover, much progress has been achieved in physiologically based toxicokinetic modelling to predict relevant concentrations for in vitro testing (Mielke et al 2011).…”

Can drinking tea prevent cancer?

“…Today, prevention of oxidative stress (Beyersmann and Hartwig 2008;Glahn et al 2008;Arivarasu et al 2008;Matés et al 2008;Hengstler and Bolt 2008a, b;Sivalingam et al 2008;Do Amaral et al 2008;Abraham and Sugumar 2008), modulation of carcinogen metabolism (Strassburg et al 2008;Pelkonen et al 2008;Naraharisetti et al 2008;Höhme et al 2007;Hewitt et al 2007;Hengstler et al 2000) and prevention of DNA damage (Florl and Schulz 2008;Zhang et al 2008;Hengstler and Bolt 2008a, b), mechanisms most frequently addressed in our journal, have also been suggested to contribute to the anti-carcinogenic eVects of tea polyphenols. The editors are pleased that Chung S. Yang (University of New Jersey), Joshua D. Lamberg (The Pennsylvania State University) and Shengmin Sang (North Carolina Central University), some of the most recognized experts in the Weld of tea polyphenols, have accepted our invitation and contributed a comprehensive review about the anti-carcinogenic activities of tea ).…”