Katrin Zeilinger

HepaRG human hepatic cell line utility as a surrogate for primary human hepatocytes in drug metabolism assessment in vitro.

INTRODUCTION Primary human hepatocytes are considered as a highly predictive in vitro model for preclinical drug metabolism studies. Due to the limited availability of human liver tissue for cell isolation, there is a need of alternative cell sources for pharmaceutical research. METHODS In this study, the metabolic activity and long-term stability of the human hepatoma cell line HepaRG were investigated in comparison to primary human hepatocytes (pHH). Hepatocyte-specific parameters (albumin and urea synthesis, galactose and sorbitol elimination) and the activity of human-relevant cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) were assayed in both groups over a period of 14 days subsequently to a two week culture period in differentiated state in case of the HepaRG cells, and compared with those of cryopreserved hepatocytes in suspension. In addition, the inducibility of CYP enzymes and the intrinsic clearances of eleven reference drugs were determined. RESULTS The results show overall stable metabolic activity of HepaRG cells over the monitored time period. Higher albumin production and galactose/sorbitol elimination rates were observed compared with pHH, while urea production was not detected. CYP enzyme-dependent drug metabolic capacities were shown to be stable over the cultivation time in HepaRG cells and were comparable or even higher (CYP2C9, CYP2D6, CYP3A4) than in pHH, whereas commercially available hepatocytes showed a different pattern The intrinsic clearance rates of reference drugs and enzyme induction of most CYP enzymes were similar in HepaRG cells and pHH. CYP1A2 activity was highly inducible in HepaRG by β-naphthoflavone. DISCUSSION In conclusion, the results from this study indicate that HepaRG cells could provide a suitable alternative to pHH in pharmaceutical research and development for metabolism studies such as CYP induction or sub-chronic to chronic hepatotoxicity studies.

Use of primary human liver cells originating from discarded grafts in a bioreactor for liver support therapy and the prospects of culturing adult liver stem cells in bioreactors: a morphologic study.

Introduction. The development of a bioreactor providing a three-dimensional network of interwoven capillary membranes with integrated oxygenation and decentralized mass exchange enables the culture of primary human liver cells from discarded donor organs for extracorporeal liver support. Methods. Primary liver cells were isolated from 54 discarded organs (donor age 56.7±13.2 years). Between 2.8×1010 and 6.4×1010 parenchymal cells (PC) were cocultured with nonparenchymal cells (NPC) of the same organ in bioreactors (n=36). The metabolic activity of the cells was regularly determined during culture. The cell morphology and ultrastructure were investigated after culture periods of 1 to 5 weeks. Results. Cell metabolism was maintained over at least 3 weeks after a phase of adaptation lasting 2 to 3 days. Through the use of transmission electron microscopy and immunohistochemistry, it was demonstrated that PC and NPC spontaneously formed tissue-like structures. Vascular cavities (CD 31 immunoreactivity [IR]) and bile duct-like channels (CK 19 IR), both exhibiting proliferation activity (Ki-67 IR), were regularly distributed. Some of the bile duct-like channels showed similarities to the Canals of Hering found in the natural liver. Cells expressing morphologic and antigenic characteristics of adult liver stem cells (CD 34 IR and c-kit IR) and areas with cells that showed both hepatocyte and biliary characteristics were detected. Conclusion. The results show that primary human liver cells obtained from discarded donor organs recover and can be maintained in bioreactors for clinical liver support therapy. In addition, initial observations on adult liver stem-cell culture in bioreactors are presented.

Bioartificial liver systems: why, what, whither?

Acute liver disease is a life-threatening condition for which liver transplantation is the only recognized effective therapy. While etiology varies considerably, the clinical course of acute liver failure is common among the etiologies: encephalopathy progressing toward coma and multiple organ failure. Detoxification processes, such as molecular adsorbent recirculating system (MARS) and Prometheus, have had limited success in altering blood chemistries positively in clinical evaluations, but have not been shown to be clinically effective with regard to patient survival or other clinical outcomes in any Phase III prospective, randomized trial. Bioartificial liver systems, which use liver cells (hepatocytes) to provide metabolic support as well as detoxification, have shown promising results in early clinical evaluations, but again have not demonstrated clinical significance in any Phase III prospective, randomized trial. Cell transplantation therapy has had limited success but is not practicable for wide use owing to a lack of cells (whole-organ transplantation has priority). New approaches in regenerative medicine for treatment of liver disease need to be directed toward providing a functional cell source, expandable in large quantities, for use in various applications. To this end, a novel bioreactor design is described that closely mimics the native liver cell environment and is easily scaled from microscopic (<1 ml cells) to clinical ( approximately 600 ml cells) size, while maintaining the same local cell environment throughout the bioreactor. The bioreactor is used for study of primary liver cell isolates, liver-derived cell lines and stem/progenitor cells.

Cytochrome P450-Dependent Metabolism in HepaRG Cells Cultured in a Dynamic Three-Dimensional Bioreactor

Reliable and stable in vitro cellular systems maintaining specific liver functions important for drug metabolism and disposition are urgently needed in preclinical drug discovery and development research. The cell line HepaRG exhibits promising properties such as expression and function of drug-metabolizing enzymes and transporter proteins, which resemble those found in freshly isolated human hepatocytes. In this study, HepaRG cells were cultured up to 68 days in a three-dimensional multicompartment capillary membrane bioreactor, which enables high-density cell culture under dynamic conditions. The activity of drug-metabolizing cytochrome P450 (P450) enzymes was investigated by a cocktail of substrates for CYP1A1/2 (phenacetin), CYP2C9 (diclofenac), CYP2B6 (bupropion), and CYP3A4 (midazolam). The model P450 substrates, which were introduced to the bioreactor system mimicking in vivo bolus doses, showed stable metabolism over the entire experimental period of several weeks with the exception of bupropion hydroxylase, which increased over time. Ketoconazole treatment decreased the CYP3A4 activity by 69%, and rifampicin induced the CYP3A4- and CYP2B6-dependent activity 6-fold, which predicts well the magnitude of changes observed in vivo. Moreover, polarity of transporter expression and formation of tissue-like structures including bile canaliculi were demonstrated by immune histochemistry. The long-lasting bioreactor system using HepaRG cells thus provides a promising and stable liver-like in vitro model for continuous investigations of the hepatic kinetics of drugs and of drug-drug interactions, which well predict the situation in vivo in humans.

Effect of human patient plasma ex vivo treatment on gene expression and progenitor cell activation of primary human liver cells in multi‐compartment 3D perfusion bioreactors for extra‐corporeal liver support

Cultivation of primary human liver cells in innovative 3D perfusion multi‐compartment capillary membrane bioreactors using decentralized mass exchange and integral oxygenation provides in vitro conditions close to the physiologic environment in vivo. While a few scale‐up bioreactors were used clinically, inoculated liver progenitors in these bioreactors were not investigated. Therefore, we characterized regenerative processes and expression patterns of auto‐ and paracrine mediators involved in liver regeneration in bioreactors after patient treatment. Primary human liver cells containing parenchymal and non‐parenchymal cells co‐cultivated in bioreactors were used for clinical extra‐corporeal liver support to bridge to liver transplantation. 3D tissue re‐structuring in bioreactors was studied; expression of proteins and genes related to regenerative processes and hepatic progenitors was analyzed. Formation of multiple bile ductular networks and colonies of putative progenitors were observed within parenchymal cell aggregates. HGF was detected in scattered cells located close to vascular‐like structures, expression of HGFA and c‐Met was assigned to biliary cells and hepatocytes. Increased expression of genes associated to hepatic progenitors was detected following clinical application. The results confirm auto‐ and paracrine interactions between co‐cultured cells in the bioreactor. The 3D bioreactor provides a valuable tool to study mechanisms of progenitor activation and hepatic regeneration ex vivo under patient plasma treatment. Biotechnol. Bioeng. 2009;103: 817–827.

Analysis of drug metabolism activities in a miniaturized liver cell bioreactor for use in pharmacological studies.

Based on a hollow fiber perfusion technology with internal oxygenation, a miniaturized bioreactor with a volume of 0.5 mL for in vitro studies was recently developed. Here, the suitability of this novel culture system for pharmacological studies was investigated, focusing on the model drug diclofenac. Primary human liver cells were cultivated in bioreactors and in conventional monolayer cultures in parallel over 10 days. From day 3 on, diclofenac was continuously applied at a therapeutic concentration (6.4 µM) for analysis of its metabolism. In addition, the activity and gene expression of the cytochrome P450 (CYP) isoforms CYP1A2, CYP2B6, CYP2C9, CYP2D6, and CYP3A4 were assessed. Diclofenac was metabolized in bioreactor cultures with an initial conversion rate of 230 ± 57 pmol/h/106 cells followed by a period of stable conversion of about 100 pmol/h/106 cells. All CYP activities tested were maintained until day 10 of bioreactor culture. The expression of corresponding mRNAs correlated well with the degree of preservation. Immunohistochemical characterization showed the formation of neo‐tissue with expression of CYP2C9 and CYP3A4 and the drug transporters breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) in the bioreactor. In contrast, monolayer cultures showed a rapid decline of diclofenac conversion and cells had largely lost activity and mRNA expression of the assessed CYP isoforms at the end of the culture period. In conclusion, diclofenac metabolism, CYP activities and gene expression levels were considerably more stable in bioreactor cultures, making the novel bioreactor a useful tool for pharmacological or toxicological investigations requiring a highly physiological in vitro representation of the liver. Biotechnol. Bioeng. 2012; 109: 3172–3181.

Isolation and Characterization of Adult Human Liver Progenitors from Ischemic Liver Tissue Derived from Therapeutic Hepatectomies

Recent evidence suggests that progenitor cells in adult tissues and embryonic stem cells share a high resistance to hypoxia and ischemic stress. To study the ischemic resistance of adult liver progenitors, we characterized remaining viable cells in human liver tissue after cold ischemic treatment for 24-168 h, applied to the tissue before cell isolation. In vitro cultures of isolated cells showed a rapid decline of the number of different cell types with increasing ischemia length. After all ischemic periods, liver progenitor-like cells could be observed. The comparably small cells exhibited a low cytoplasm-to-nucleus ratio, formed densely packed colonies, and showed a hepatobiliary marker profile. The cells expressed epithelial cell adhesion molecule, epithelial-specific (CK8/18) and biliary-specific (CK7/19) cytokeratins, albumin, alpha-1-antitrypsin, cytochrome-P450 enzymes, as well as weak levels of hepatocyte nuclear factor-4 and gamma-glutamyl transferase, but not alpha-fetoprotein or Thy-1. In vitro survival and expansion was facilitated by coculture with mouse embryonic fibroblasts. Hepatic progenitor-like cells exhibit a high resistance to ischemic stress and can be isolated from human liver tissue after up to 7 days of ischemia. Ischemic liver tissue from various sources, thought to be unsuitable for cell isolation, may be considered as a prospective source of hepatic progenitor cells.

Featured Article: Isolation, characterization, and cultivation of human hepatocytes and non-parenchymal liver cells.

Primary human hepatocytes (PHH) are considered to be the gold standard for in vitro testing of xenobiotic metabolism and hepatotoxicity. However, PHH cultivation in 2D mono-cultures leads to dedifferentiation and a loss of function. It is well known that hepatic non-parenchymal cells (NPC), such as Kupffer cells (KC), liver endothelial cells (LEC), and hepatic stellate cells (HSC), play a central role in the maintenance of PHH functions. The aims of the present study were to establish a protocol for the simultaneous isolation of human PHH and NPC from the same tissue specimen and to test their suitability for in vitro co-culture. Human PHH and NPC were isolated from tissue obtained by partial liver resection by a two-step EDTA/collagenase perfusion technique. The obtained cell fractions were purified by Percoll density gradient centrifugation. KC, LEC, and HSC contained in the NPC fraction were separated using specific adherence properties and magnetic activated cell sorting (MACS®). Identified NPC revealed a yield of 1.9 × 106 KC, 2.7 × 105 LEC and 4.7 × 105 HSC per gram liver tissue, showing viabilities >90%. Characterization of these NPC showed that all populations went through an activation process, which influenced the cell fate. The activation of KC strongly depended on the tissue quality and donor anamnesis. KC became activated in culture in association with a loss of viability within 4–5 days. LEC lost specific features during culture, while HSC went through a transformation process into myofibroblasts. The testing of different culture conditions for HSC demonstrated that they can attenuate, but not prevent dedifferentiation in vitro. In conclusion, the method described allows the isolation and separation of PHH and NPC in high quality and quantity from the same donor.

In‐depth physiological characterization of primary human hepatocytes in a 3D hollow‐fiber bioreactor

As the major research focus is shifting to three‐dimensional (3D) cultivation techniques, hollow‐fiber bioreactors, allowing the formation of tissue‐like structures, show immense potential as they permit controlled in vitro cultivation while supporting the in vivo environment. In this study we carried out a systematic and detailed physiological characterization of human liver cells in a 3D hollow‐fiber bioreactor system continuously run for > 2 weeks. Primary human hepatocytes were maintained viable and functional over the whole period of cultivation. Both general cellular functions, e.g. oxygen uptake, amino acid metabolism and substrate consumption, and liver‐specific functions, such as drug‐metabolizing capacities and the production of liver‐specific metabolites were found to be stable for > 2 weeks. As expected, donor‐to‐donor variability was observed in liver‐specific functions, namely urea and albumin production. Moreover, we show the maintenance of primary human hepatocytes in serum‐free conditions in this set‐up. The stable basal cytochrome P450 activity 3 weeks after isolation of the cells demonstrates the potential of such a system for pharmacological applications. Liver cells in the presented 3D bioreactor system could eventually be used not only for long‐term metabolic and toxicity studies but also for chronic repeated dose toxicity assessment. Copyright

Recovery of preservation-injured primary human hepatocytes and nonparenchymal cells to tissuelike structures in large-scale bioreactors for liver support: an initial transmission electron microscopy study.

This study investigated large-scale regeneration and tissue reorganization of adult human liver cells from preservation injured transplant organs. The use of basement membrane protein gels and growth factor enriched culture medium in standard culture flasks promotes liver tissue formation in isolated rat and pig hepatocytes, resulting in prolongation of phenotypic stability and metabolic competence of primary cells in vitro. A special bioreactor construction for high-density three-dimensional cell recovery was developed and isolation of cells from discarded human donor livers was enabled. In vitro regeneration of adult human liver cells isolated from preservation-injured organs took place over a period of 2 weeks in a purpose-built bioreactor. Basement membrane protein and growth factors were avoided. Reorganization of tissue structures was studied using transmission electron microscopy (TEM). This showed regeneration and tissue reorganization of adult human cells from preservation-injured organs by coculture with nonparenchymal cells in the bioreactor. The majority of the aggregated hepatocytes in the bioreactors showed morphological similarities to those in vivo (although not re-formed to hepatocyte plates), exhibiting cell-cell junctions and reconstituted bile canaliculi-like spaces between neighboring hepatocytes. Perfusion channels appeared regularly between cell aggregates. The arrangement of nonparenchymal cells between the hepatocyte aggregates exhibited similarities to liver sinusoids. Endothelial cells often covered the aggregates and formed a borderline to the perfusion channels between the capillaries. Similar to the space of Disse, further nonparenchymal cells were located between the endothelial cells and the parenchymal aggregates. Deposits of biomatrix fibers occurred spontaneously. The regenerated cell mass was close to that of a single liver lobe. In conclusion, the further optimization of bioreactors that enable cell recovery from preservation injury may lead to the utilization of cells from discarded whole or split transplants for extracorporeal temporary liver support therapy or hepatocyte transplantation.