Abstract
Hepatic gene expression as a function of culture duration was evaluated in prolonged cultured human hepatocytes. Human hepatocytes from seven donors were maintained as near-confluent collagen-Matrigelsandwich 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, transferrin, and transthyretin; the hepatocyte-specific asialoglycoprotein receptor 1 cytochrome P450 isoforms CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A7; uptake transporter isoforms SLC10A1, SLC22A1, SLC22A7, SLCO1B1, SLCO1B3, and SLCO2B1; efflux transporter isoforms ATP binding cassette (ABC)B1, ABCB11, ABCC2, ABCC3, ABCC4, and ABCG2; and the nonspecific housekeeping gene hypoxanthine ribosyl transferase 1 (HPRT1). The well established dedifferentiation phenomenon was observed on day 2, with substantial (>80%) decreases in gene 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 This study reports that although 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 cytochrome 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 toward the evaluation of drug properties requiring an extended period of treatment and evaluation.
Introduction
Primary human hepatocytes are considered the gold-standard in vitro experimental system for the evaluation of human-specific drug properties, which, because of 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, 2016; Kenny et al., 2013; Li, 2015; Dvorak, 2016) during drug development for the selection 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 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 represent a useful experimental tool to evaluate the potency and duration of gene silencing effects of siRNA therapeutics (Yang et al., 2021).
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 et al., 2012; Bell et al., 2016; Chacko et al., 2019), cocultures of human hepatocytes with nonhepatic cells (Bhatia et al., 1997; Bonn et al., 2016; Ware et al., 2017; Cassidy and Yi, 2018), microfluidic cultures (Burkhardt et al., 2014; Kang et al., 2015; Ortega-Prieto et al., 2019; Shoemaker et al., 2020), and 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).
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., 2021). 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 hepatic functions versus culture duration in prolonged cultured human hepatocytes. Human hepatocytes from seven donors were cultured for 43 days with mRNA quantified by RT-PCR on days 0 (day of 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, SLC22A7, SLCO1B1, SLCO1B3, SLCO2B), and efflux transporters (ABCB1, ABCB11, ABCC2, ABCC3, ABCC4, ABCG2), as well as the nonspecific 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 seven 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 (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, 2007; Li et al., 1999; Hewitt and Li, 2015; Yang et al., 2021). Demographics of the donors are presented in Table 1.
Recovery and Culturing of Cryopreserved Human Hepatocytes.
Cryopreserved human hepatocytes from the seven donors were thawed at 37°C in cryopreserved hepatocyte recovery medium (AP Sciences Inc., Columbia, MD) and collected by centrifugation at 100g for 10 minutes. The cell pellet was resuspended in universal primary cell plating medium (In Vitro ADMET Laboratories Inc., Columbia, MD) followed by viability determination via trypan blue exclusion and cell concentration determination using a hemocytometer. Cell density was adjusted to 0.7 million viable cells per milliliter in Universal Cryopreservation Medium and plated in collagen-coated 24-well plates (CellAffix; AP Sciences Inc., Columbia, MD) at a volume of 0.5 ml (350,000 cells/well). Upon addition of the hepatocytes, the 24-well plates were placed in a cell culture incubator kept at 37°C in a humidified atmosphere of 5% carbon dioxide and 95% air. The hepatocytes were allowed to attach for approximately 4 hours, followed by replacement of the plating medium with 0.5 ml per well of hepatocyte induction medium (HIM; In Vitro ADMET Laboratories Inc., Columbia, MD) containing 0.25 mg/ml Matrigel (Corning Inc., PA) for the establishment of a collagen-Matrigel sandwich hepatocyte culture. Upon culturing for approximately 24 hours, medium was changed to HIM without Matrigel. HIM is a serum-free medium supplemented with 1% Insulin-Transferrin-Selenium (ITS) medium supplement (Sigma-Aldrich, St. Louis, MO) and dexamethasone (Sigma-Aldrich). The final concentrations of the supplements were as follows: recombinant human insulin (10 µg/ml), human transferrin (5.5 µg/ml), sodium selenite (0.005 μg/ml), and dexamethasone (0.1 µM).
Prolonged Culturing of the Hepatocytes.
Hepatocytes from each of the seven donors were cultured as independent single-donor cultures for 43 days with medium removed and replaced with fresh medium every Monday, Wednesday, and Friday. The hepatocytes from the seven donors were cultured in the same 24-well plate (three wells per donor), with each plate designated for a specific culture duration for mRNA isolation. mRNA quantification was performed at days 0 (4 hours after plating), 2, 7, 9, 16, 23, 26, 29, 36, and 43 of culture. For the duration of the study, cell morphology was monitored and recorded using phase contrast photomicrography (Axiovert 25; Carl Zeiss Microscopy Inc., White Plains, NY).
Real-Time Polymerase Chain Reaction.
mRNA was isolated from each of the seven independent cultures at the designated culture durations using an E-Z 96 Total RNA kit (Omega Bio-tek Inc., Norcross, GA) and quantitated using Quant-iT Ribogreen RNA Assay Kit (Life Technologies, Eugene, OR), and cDNA was synthesized from total RNA using the High-Capacity cDNA RT Kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s recommended protocols. cDNA was synthesized using a PTC-200 thermal cycler instrument (MJ Research, Watertown, MA). Real-time polymerase chain reaction (PCR) was done in triplicate in 96-well PCR plates using the Fast Universal PCR Master Mix (Quanta Biosciences Inc., Beverly, MA). The Master Mix was prepared by mixing per each well of the 96-well plate 5 µl of Fast Universal PCR Master Mix, 2 µl RNase-free water, and 1 µl primer/probe mix (commercial TaqMan Gene Expression Assays). In each well of the 96-well PCR plate, 2 µl cDNA solution and 8 µl Master Mix were pipetted. The 96-well plate was transferred into an ABI Prism 7500Fast RT-PCR instrument (version 2.0.6; Applied Biosystems, Foster City, CA). The primers used for mRNA expression analysis are shown in Table 2. The identities and key physiologic functions of the genes evaluated are shown in Table 3.
Data Analysis.
Results are reported as means and S.E.M. of mRNA expression values from the seven lots of human hepatocytes. Relative expression was calculated using the 2–ΔΔCt method (Rao et al., 2013), with the average cycle threshold (Ct) values of each of the target genes evaluated normalized to the average Ct value of the housekeeping gene, GAPDH, and expressed as ΔCt and with the ΔCt value at each culture duration normalized to the ΔCt value for that on the day of culture initiation (day 0) and expressed as ΔΔCt, with the results expressed as relative expression to that of day 0 using the following equations:
ΔCt = average Ct (target gene) – average Ct(GAPDH);
ΔΔCt = average ΔCt (culture duration) – average ΔCt (day 0); and
Relative Expression = 2-ΔΔCt.
Statistical Analysis.
Two-tailed ANOVA statistical analysis (GraphPad Prism 9.0) was employed to compare the mean relative expression values (mean of seven donors) at each culture duration versus that on the day of plating (day 0), with P values of 0.05 or less considered to be statistically significant. Pearson correlation analysis (GraphPad Prism 9.0) was used to evaluate the correlation of mRNA expression as a function of culture duration for the genes quantified.
Results
In this study, mRNA expression was quantified in human hepatocytes derived from livers of seven individual donors on days 0 (4 hours after plating), 2, 7, 9, 16, 23, 26, 29, 36, and 43 of culture. The average values of the seven lots of human hepatocytes (N = 7) are presented.
Cell Morphology.
On the day of plating (day 0), the hepatocytes attached after approximately 4 hours and assumed the cobblestone epithelial cell morphology but with minimal cell-cell contact. Near 100% confluency was observed on day 2 and throughout the duration of the study until day 30, when cell separation occurred, whereas the hepatocytes continued to exhibit extensive cell-cell contacts similar to that in the earlier confluent cultures (Fig. 1).
Hepatic Differentiation Markers.
Significant (>80%) decreases in relative expression were observed on day 2 cultures for the classic markers for hepatic differentiation, ALB, TR, TTR, and ASGR1. The relative expression values increased to within 50% of day 0 values on day 7, with the highest expression observed on day 9, followed by gradual decreases at the longer culturing durations. In contrast to the differentiation marker genes, expression of the housekeeping gene HPRT1 did not decrease on day 2 (Fig. 2).
Cytochrome P450 Isoforms.
As observed with ALB, TR, TTR, and ASGR1, significant (>80%) decreases in expression of all P450 isoforms evaluated were observed on day 2 of culturing, with increases observed on day 7. On days 7 and 9, expression was similar or higher than on day 0 for CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A7. Relative expression values for CYP2B6 and CYP2C8, although they increased to be higher than that for day 2, remained significantly lower than that on day 0. Decreased expression with culture duration was observed after day 9 for all isoforms except for CYP2C19 and CYP2D6, with relatively stable expression at levels similar to that for day 0 throughout the culture duration (Fig. 3).
Uptake Transporters.
Significant (>80%) decreases in expression of all uptake transporters SLC10A1, SLC22A1, SLC22A7, SLCO1B1, SLCO1B3, and SLCO2B1 were observed on day 2, with increases in expression on day 7. On days 7 and 9, the relative expression values were similar or higher than on day 0, and this was observed for all uptake transporter genes except SLCO1B3. Relative expression values for SLCO1B3, although increased to be higher on days 7 and 9 than on day 2, remained significantly lower than day 0 values (Fig. 4).
Efflux Transporters.
Significant (>80%) decreases in expression were observed on day 2 for ABCB11. Slight (≥50%) but statistically significant decreases in expression were observed for ABCC2 and ABCC3. Expression values higher than that on day 2 were observed on day 7 for these three isoforms. No decreases in relative expression values on day 2 were observed for ABCB1, ABCC4, and ABCG2. The relative expression values of ABCC4 were >4-fold of that for day 0 throughout the culture duration. Except for ABCC4, the relative expression values for the efflux transporters were generally within 50% of that for day 0 from day 7 to day 43 (Fig. 5).
Correlation Analysis of Gene Expression.
Pearson correlation analysis of gene expression versus culture duration was performed for the various genes. The results are as follows:
ALB, TR, TTR, ASGR1, and HPRT1. The expression of hepatic differentiation marker genes ALB, TR, TTR, and ASGR1 versus culture duration was found to be highly correlated among each other, with Pearson coefficient values of >0.8, with their expression negatively correlated with the housekeeping gene HPRT1 (Fig. 6A).
P450 isoforms. For P450 isoforms, positive correlation was observed among the various isoforms, with the exception of a negative correlation between CYP2C19 and CYP1A2 (Fig. 6B).
Uptake transporters. A positive correlation was observed among all transporter genes except for that between SLCO1B1 and SLCO2B1 (Fig. 6C).
Efflux transporters. A positive correlation was observed among ABCB1, ABCB11, ABCC2, and ABCC3 and among ABCC4, ABCB1, and ABCC3. Negative correlation was observed between ABCG2 and all efflux transporters (Fig. 6D).
All genes. A positive correlation was observed among all genes, with the following exceptions: HPRT1 has an overall negative correlation with all genes except CYP2B6, CYP2C8, and CYP2C9. CYP2C19 has a positive correlation with all genes except for CYP1A2, CYP2B6, and CYP2C9. ABCC4 has a positive correlation only with CYP2C19 and ABCG2. CYP2C19 had positive correlations with all genes except CYP1A2, CYP2B6, CYP2C8, and CYP2C9. ABCC4 had negative correlations with all genes except CYP2C19, SLCO2B1, ABCB1, and ABCC4. ABCG2 had negative correlations with all genes except HPRT1, CYP2B6, CYP2C8, CYP2C9, SLC22A1, and SLCO1B3 (Fig. 6E).
Discussion
We report here results of mRNA expression of genes responsible for key hepatic functions versus culture duration in prolonged cultured human hepatocytes. The hepatocyte-specific genes evaluated include hepatic proteins ALB, TR, and TTR, which are generally regarded as markers of mature hepatocytes (Lok and Loh, 1998; Ascoli et al., 2006; Buxbaum et al., 2008; Bal et al., 2013; Fujiwara and Amisaki, 2013; Lee and Wu, 2015; Alemi et al., 2016; Zorzi et al., 2019); the plasma membrane receptor ASGR1, used routinely for the delivery of therapeutic agents specifically to hepatocytes (Merwin et al., 1994; Kim et al., 2005; Thapa et al., 2015; Huang et al., 2017); P450 isoforms CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, which are considered the key isoforms responsible for drug metabolism (Rendic and Guengerich, 2010; Zanger and Schwab, 2013), as well as CYP3A7, a CYP3A isoform mainly expressed in fetal but also expressed in adult livers (Kamataki et al., 1995; Greuet et al., 1996; Okuyama et al., 2020); drug uptake transporters SLC10A1, SLC22A1, SLC22A7, SLCO1B1, SLCO1B3, and SLCO2B1 (Fenner et al., 2012; Barton et al., 2013; Bi et al., 2019) and efflux transporters ABCB1, ABCB11, ABCC2, ABCC3, ABCC4, and ABCG2 (Matsushima et al., 2005; Ishiguro et al., 2008; Pfeifer et al., 2014), which play key roles in the regulation of intracellular concentrations of drugs and their metabolites that are transporter substrates. Expression of the housekeeping gene HPRT1 (Nishimura et al., 2006) as a function of culture duration was also evaluated for comparison with the abovementioned hepatocyte-specific genes. The key attributes of the genes evaluated are shown in Table 3.
Our findings on dedifferentiation are consistent with the current opinion that hepatocyte dedifferentiation affects a multitude of hepatic functions. With the exception of five of the six efflux transporters (ABCB1, ABCC2, ABCC3, ABCC4, and ABCG2), all the hepatic functional genes evaluated demonstrated >80% decreases in gene expression after 2 days of culturing, an observation consistent with the well established phenomenon of dedifferentiation of cultured hepatocytes (Padgham and Paine, 1993). Interestingly, of the efflux transporters evaluated, only ABCB11 demonstrated the >80% decreased expression on day 2. The lack of decreased gene expression on day 2 for the housekeeping gene HPRT1 provides evidence that the decreased expression of the hepatocyte-specific genes is a function of dedifferentiation and not due to an overall decrease in RNA synthesis.
We report here a novel observation that all the genes with >80% decreased expression on day 2 (ALB, TR, TTR, ASGR1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, SLC10A1, SLC22A1, SLC22A7, SLCO1B1, SLCO1B3, SLCO2B1, and ABCB11) were found to have increased expression on day 7. As ALB, TR, and TTR are commonly used gene markers for hepatocyte differentiation (Page et al., 2007), the results suggest that redifferentiation occurs upon prolonged culturing of the hepatocytes. Positive correlations based on Pearson analysis of gene expression versus culture duration provide additional evidence that expression of P450 and transporter genes was similarly affected by the dedifferentiation and redifferentiation processes.
An interesting finding with uptake transporter and P450 isoform gene expression is that, although they all exhibit the dedifferentiation-redifferentiation phenomenon, the extent and duration of redifferentiation based on gene expression vary among the isoforms. This observation suggests that factors in addition to that present in the culture medium may be responsible for their respective levels of expression in vivo (presumably day 0 expression). A plausible explanation is the exposure of the liver donors to endogenous and exogenous inducers (Zanger et al., 2005; Zollner et al., 2006; Zheng et al., 2009; Inagaki et al., 2020; Zamek-Gliszczynski et al., 2020) that are absent in the culture medium.
Besides the housekeeping gene HPRT1, efflux transporter gene expression was less affected by the dedifferentiation/redifferentiation process in the prolonged cultured human hepatocytes, except for the bile salt efflux transporter ABCB11. ABCB11 expression had a >80% decrease in expression on day 2 of culturing and, on day 7, returned to a level near that of day 0. The other efflux transporter genes were expressed at levels within 50% of and, in the case for ABCB4 (multiple drug resistance protein 3), severalfold higher than that on day 0. The results suggest ABCB11 expression, but not the other efflux transporter genes evaluated, involves liver transcription factors that are downregulated and upregulated during dedifferentiation and redifferentiation, respectively, of the prolonged cultured human hepatocytes. Another interesting observation is that, in contrast to other efflux transporters, culture duration for ABCG2 (breast cancer resistance protein) expression was observed for hepatocytes from six of the seven donors (Fig. 10). Although the mechanism for this decrease is yet to be elucidated, it may be a result of the absence of biomolecules in the culture medium that are responsible for the maintenance of ABCG2 gene expression in vivo. It is interesting that ABCG2 has been reported to be regulated by aryl hydrocarbon receptor ligands (Tan et al., 2010; Sayyed et al., 2016) and that its downregulation with culture duration was similar to that observed for CYP1A2 in this study.
Examination of individual differences in gene expression provides additional insight on the expression of the various genes evaluated. Consistent responses among the seven donors were observed for the classic markers of hepatocyte differentiation—namely, ALB, TR, and TTR, as well as the hepatocyte-specific ASGR1—thereby providing strong evidence that confluent collagen-Matrigel human hepatocyte cultures undergo dedifferentiation on day 2 and redifferentiation on day 7 (Fig. 7). It is interesting that among the P450 isoforms (Fig. 8), the noninducible CYP2D6 expression was the most consistent among the seven donors, suggesting that its expression in cultured hepatocytes is mainly regulated by the differentiation process. Of the inducible isoforms, CYP2C19, CYP3A4, and CYP3A7 demonstrated more consistency among the seven donors than CYP1A2, CYP2B6, CYP2C8, and CYP2C19, which we will further explore to further improve our understanding of differences in P450 expression. Uptake transporter expression (Fig. 9) is consistent among all donors, with downregulation on day 2 and upregulation back to levels similar to that on day 0 (except for SLCO1B3, which returned to approximately 10% of that for day 0), suggesting that their expression is mainly regulated by the differentiation process. It is notable that CYP3A7, the fetal CYP3A isoform, also returned to a level similar but not significantly exceeding that for day 0, providing evidence supporting that the hepatocytes maintained their mature phenotypes throughout the prolonged cultured durations evaluated in this study. The comparatively lower expression of SLCO1B1 suggests that its expression may require factors present in vivo but absent in the culture medium. Expression of efflux transporters (Fig. 10) was consistent among the various donors, with an interesting observation made for ABCC4: expression on culture durations 2 days and longer was higher than that for day 0 for five of the seven donors, and one donor did not exhibit decreases in expression of ABCG2 with culture duration, as was observed for the other six donors. Elucidation of the mechanism of the observed individual differences may further our understanding of environmental and genetic factors regulating P450 and transporter expression in the human liver, thereby improving our ability to evaluate individual differences in drug properties in the human population.
The dedifferentiation and redifferentiation observed based on hepatic gene expression suggest that prolonged human hepatocyte cultures may represent an experimental tool to elucidate the mechanism of hepatocyte differentiation. Dedifferentiation of cultured hepatocytes has been attributed to the downregulation of transcription factors involved in liver-specific gene expression as a result of cell proliferation based on experimental findings with cultured rat hepatocytes (Padgham et al., 1993; Mizuguchi et al., 1998) and in vivo partial hepatectomy studies (Flodby et al., 1993; Eleswarapu and Jiang, 2005). As culture duration was the only variable in our study, reestablishment of gap junctions between hepatocytes is a likely mechanism. The current working hypothesis that we employ to guide further investigation is that hepatocyte dedifferentiation occurs in early hepatocyte cultures because of the lack of cell-cell communication via gap junctions that are disrupted during hepatocyte isolation, with redifferentiation occurring upon the reestablishment of cell-cell junctions upon prolonged culturing of the hepatocytes as confluent cultures. This hypothesis is consistent with the key roles established for cell-cell communication via gap junctions in the expression of hepatic functions (Hamilton et al., 2001; Stoehr and Isom, 2003; Vinken et al., 2006; Willebrords et al., 2015), as well as the findings that culturing of hepatocytes under experimental conditions allowing prolonged culturing with extensive cell-cell contact such as hepatocyte spheroids (Bell et al., 2017; Desai et al., 2017) and cocultures with nonhepatocytes (Ramsden et al., 2014; Ware et al., 2017) lead to enhanced hepatic functions. A potential implication of our findings is that in vivo conditions resulting in disruption of cell-cell junctions may lead to downregulation of hepatic functions including protein synthesis, uptake and efflux transport, and P450-dependent drug metabolism with recovery of the functions upon the reestablishment of cell-cell junctions.
The observed redifferentiation suggests that prolonged (>7 days) culturing of cryopreserved human hepatocyte may overcome the dedifferentiation phenomenon that has been a major challenge in the application of this gold-standard in vitro human experimental system. Prolonged (>7 days) cryopreserved human hepatocyte cultures may represent stabilized hepatocytes for the evaluation of certain aspects of drug properties requiring an extended period of treatment and evaluation, with the caveats that certain hepatic genes such as CYP2B6, CYP2C8, and SLCO1B3 remained substantially underexpressed compared with that at the initiation of the cultures and that the duration of the increased expression levels vary among the different genes. We have recently reported a proof-of-concept study successfully demonstrating the application of prolonged human hepatocyte cultures in the evaluation of potency and duration of siRNA therapeutics on target gene expression (Yang et al., 2021). Additional potential applications of the prolonged human hepatocyte cultures to aid drug development that we will evaluate in our laboratory include hepatitis viral replication, chronic drug toxicity, and investigation of the effects of prolonged drug treatment on the inhibition and induction of drug-metabolizing enzymes and transporters.
In our laboratory, we are investigating experimental approaches to overcome the phenomenon of dedifferentiation of primary human hepatocytes cultured as two-dimensional monolayers, a property that has been considered a limitation of this gold-standard in vitro experimental system for the evaluation of human drug properties. Our ultimate goal is to develop practical and reproducible culture conditions for stable and fully functional human hepatocytes to extend the utility of this valuable experimental system. Results from the current study provide convincing data based on mRNA expression that prolonged culturing may lead to hepatocytes with stable hepatic properties, at least for several days after day 7. As there may be differences between mRNA and protein expression (Wegler et al., 2019), it is necessary to confirm our results with functional evaluation. Research is ongoing in our laboratory to extensively evaluate drug-metabolizing enzyme and transporter activities as a function of culture duration. Our current investigation includes various cell culture plate formats (e.g., 24-well plates versus 96-well plates), donor-to-donor variations, and identification of medium supplements required to maintain hepatic functions.
Acknowledgments
The authors gratefully acknowledge Kirsten Amaral for her assistance in cell cultures and Linda Loretz, Ph.D., for her critical review of the manuscript.
Authorship Contributions
Participated in research design: Yang, Li.
Conducted experiments: Yang.
Contributed new reagents or analytic tools: Yang, Li.
Performed data analysis: Yang, Li.
Wrote or contributed to the writing of the manuscript: Yang, Li.
Footnotes
- Received February 16, 2021.
- Accepted June 10, 2021.
This work received no external funding.
The authors (Q.Y., A.P.L.) are employees of In Vitro ADMET Laboratories Inc., a commercial provider of cryopreserved human hepatocytes.
Abbreviations
- ABC
- AT P-binding cassette
- ALB
- albumin
- ASGR1
- asialoglycoprotein receptor 1
- Ct
- cycle threshold
- GAPDH
- glyceraldehyde 3-phosphate dehydrogenase
- HIM
- hepatocyte induction medium
- HPRT1
- hypoxanthine phosphoribosyl transferase 1
- ITS
- insulin transferrin selenium
- P450
- cytochrome P450
- PCR
- polymerase chain reaction
- RT-PCR
- real-time polymerase chain reaction
- siRNA
- small interfering RNA
- SLC
- solute carrier
- TR
- transferrin
- TTR
- transthyretin
- UCPM
- Universal Cryopreservation Plating Medium
- Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics