DMD Fast Forward. Published on June 16, 2021 as DOI: 10.1124/dmd.121.000424 This article has not been copyedited and formatted. The final version may differ from this version. Messenger RNA expression of albumin, transferrin, transthyretin, asialoglycoprotein receptor, P450 isoform, uptake transporter and efflux transporter genes as a function of culture duration in prolonged cultured cryopreserved human hepatocytes as collagen-matrigel sandwich cultures: Evidence for redifferentiation upon prolonged culturing Author names Qian Yang and Albert P. Li Downloaded from Author affiliations In Vitro ADMET Laboratories Inc., Columbia, MD dmd.aspetjournals.org at ASPET Journals on October 1, 2021 1 DMD Fast Forward. Published on June 16, 2021 as DOI: 10.1124/dmd.121.000424 This article has not been copyedited and formatted. The final version may differ from this version. Running title: Redifferentiation of prolonged human hepatocyte cultures Corresponding author: Albert P. Li, Ph. D., In Vitro ADMET Laboratories Inc., 9221 Rumsey Road Suite 8, Columbia, MD 21045, USA. Telephone: (410)869-9037. Fax: (410)869-9034. Email: [email protected]. Number of text pages: 26 Number of tables: 3 Downloaded from Number of figures: 10 Number of references: 78 dmd.aspetjournals.org Number of words (Abstract): 235 Number of words (Introduction): 520 at ASPET Journals on October 1, 2021 Number of words (Discussion): 1828 List of Abbreviations: ALB (albumin), ASGPR (asialoglycoprotein receptor), CYP (cytochrome P450), HPRT1 (hypoxanthine phosphoribosyl transferase 1), GAPDH (glyceraldehyde 3-phosphate dehydrogenase), RT-PCR (real time polymerase chain reaction), SLC (solute carrier), (TR) transferrin, TTR, (transthyretin). 2 DMD Fast Forward. Published on June 16, 2021 as DOI: 10.1124/dmd.121.000424 This article has not been copyedited and formatted. The final version may differ from this version. Abstract. Hepatic gene expression as a function of culture duration was evaluated in prolonged cultured human hepatocytes. Human hepatocytes from 7 donors were maintained as near-confluent collagen-matrigel sandwich cultures, with messenger RNA expression for genes responsible for key hepatic functions quantified by real time polymerase chain reaction at culture durations of 0 (day of plating), 2, 7, 9, 16, 23, 26, 29, 36 and 43 days. Key hepatocyte genes were evaluated including the differentiation markers albumin (ALB), transferrin (TR) and transthyretin (TTR); the hepatocyte-specific asialoglycoprotein Downloaded from receptor (ASGR1); cytochrome P450 isoforms CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A7; uptake transporter isoforms SLC10A1, SLC22A1, SLC22A7, SLCO1B1, SLCO1B3, dmd.aspetjournals.org SLCO2B1; efflux transporter isoforms ABCB1, ABCB11, ABCC2, ABCC3, ABCC4, ABCG2; as well as the nonspecific housekeeping gene hypoxanthine ribosyl transferase (HPRT1). The well-established dedifferentiation phenomenon was observed on day 2, with substantial (>80%) decreases in gene at ASPET Journals on October 1, 2021 expression in day 2 cultures observed for all genes evaluated except HPRT1 and efflux transporters ABCB1, ABCC2, ABCC3 ( <50% decrease in expression), ABCC4 ( >400% increase in expression), and ABCG2 (no decrease in expression). All genes with a >80% decrease in expression were found to have increased levels of expression on day 7, with peak expression observed on either day 7 or day 9, followed by a gradual decrease in expression up to the longest duration evaluated of 43 days. Our results provide evidence that cultured human hepatocytes undergo redifferentiation upon prolonged culturing. Significance Statement: We report that while human hepatocytes underwent dedifferentiation upon 2 days of culture, prolonged culturing resulted in redifferentiation based on gene expression of differentiation markers, uptake and efflux transporters, and P450 isoforms. The observed redifferentiation suggests that prolonged (>7 days) culturing of human hepatocyte cultures may represent an experimental approach to overcome the initial dedifferentiation process, resulting in “stabilized” hepatocytes that can be applied towards the evaluation of drug properties requiring an extended period of treatment and evaluation. 3 DMD Fast Forward. Published on June 16, 2021 as DOI: 10.1124/dmd.121.000424 This article has not been copyedited and formatted. The final version may differ from this version. Introduction Primary human hepatocytes are considered the “gold standard” in vitro experimental system for the evaluation of human-specific drug properties, which, due to species differences, may not be obtained from studies in laboratory animals. Human hepatocytes are used routinely in drug development for the definition of human hepatic drug metabolism, transporter-mediated drug uptake and efflux, drug-drug interactions, and hepatotoxic potential (LeCluyse et al., 2005; Hewitt et al., 2007; Zhang et al., 2009; Kenny et al., 2013; Li, 2015; Dvorak, 2016; Zhang et al., 2016) during drug development for the selection Downloaded from of drug candidates most likely to be successful in clinical trials. Primary cultured human hepatocytes have also been applied in drug discovery in the identification of potential therapeutic targets and the identification of new chemical entities for the treatment of various human liver diseases such as viral dmd.aspetjournals.org hepatitis and nonalcoholic steatohepatitis (Baktash and Randall, 2019; Ortega-Prieto et al., 2019; Suurmond et al., 2019; Xiang et al., 2019). Most recently, we reported that prolonged cultured human hepatocytes (PCHH) represent a useful experimental tool to evaluate the potency and duration of gene at ASPET Journals on October 1, 2021 silencing effects of siRNA therapeutics (Yang et al., 2020). Dedifferentiation of primary cultured hepatocytes, resulting in diminished hepatic functions, represents a major technical challenge limiting the utility of this experimental system (Elaut et al., 2006). Recently, several approaches have been applied successfully in the partial restoration and prolongation of hepatic functions. These approaches include three dimensional culturing of hepatocytes (Li et al., 1992; No da et al., 2012; Bell et al., 2016; Chacko et al., 2019), co-cultures of human hepatocytes with non-hepatic cells (Bhatia et al., 1997; Bonn et al., 2016; Cassidy and Yi, 2018; Ware et al., 2018), microfluidic cultures (Burkhardt et al., 2014; Kang et al., 2015; Ortega-Prieto et al., 2019; Shoemaker et al., 2020), as well as alterations of cell culture media composition and culturing conditions (Guo et al., 2017; Oorts et al., 2018; Xiang et al., 2019; Davidson and Khetani, 2020). 4 DMD Fast Forward. Published on June 16, 2021 as DOI: 10.1124/dmd.121.000424 This article has not been copyedited and formatted. The final version may differ from this version. Recently, we have optimized the cryopreservation conditions resulting in the preparation of cryopreserved human hepatocytes that can be maintained as near 100% confluent monolayer cultures for a prolonged culture duration of over 40 days (Yang et al., 2020). The longevity of the cultured hepatocytes allows investigation of the relationship between culture duration and hepatocyte functions, especially, if redifferentiation would occur upon prolonged culturing after initial dedifferentiation that has been previously reported by others. We report here the quantification of mRNA expression of as a function of genes responsible for key Downloaded from hepatic functions versus culture duration in prolonged cultured human hepatocytes. Human hepatocytes from 7 donors were cultured for 43 days with mRNA quantified by RT-PCR on days 0 (day of dmd.aspetjournals.org cell plating), 2, 7, 9, 16, 23, 26, 29, 36 and 43 for genes responsible for key hepatic functions including hepatic proteins (ALB, TR, TTR), plasma membrane receptor ASGR1, P450 isoforms (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A7), uptake transporters (SLC10A1, SLC22A1, at ASPET Journals on October 1, 2021 SLC22A7, SLCO1B1, SLCO1B3, SLCO2B), and efflux transporters (ABCB1, ABCB11, ABCC2, ABCC3, ABCC4, ABCG2), as well as the non-specific housekeeping gene HGPRT1. Our results provide evidence for redifferentiation upon prolonged culturing of human hepatocytes. Materials and methods Cryopreserved human hepatocytes. 999Elite™ Cryopreserved Human Hepatocytes (In Vitro ADMET Laboratories Inc., Columbia, MD) from 7 donors were used in the study. The cryopreserved human hepatocytes used were prepared from livers intended for but not used for transplantation, 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 (Loretz et al., 1989; Li, 1999; Li 5 DMD Fast Forward. Published on June 16, 2021 as DOI: 10.1124/dmd.121.000424 This article has not been copyedited and formatted. The final version may differ from this version. et al., 1999; Li, 2007; Hewitt and Li, 2015; Yang et al., 2020). Demographics of the donors are presented in Table 1. Recovery and culturing of cryopreserved human hepatocytes. Cryopreserved human hepatocytes from the 7 donors were thawed at 37°C in Cryopreserved Hepatocyte Recovery Medium (CHRM™, AP Sciences Inc, Columbia, MD) and collected