Dynamic 3D Bioreactor Culture Model For Long-Term Maintenance Of Human Hepatocyte Metabolic Function

1. Human Liver Bioreactor CD68 CK19 MDR1 Dynamic 3D Bioreactor Culture Model For Long-Term Maintenance Of Human Hepatocyte Metabolic Function Thomas Schreiter1, Malin Darnell3, Therese Söderdahl2, Nils Bohmer1, Daniel Knobeloch4, Birgitta Dillner2, Anna-Lena Berg2, Andreas K.N. Nüssler5, Jörg C. Gerlach1,6, Katrin Zeilinger1, Tommy B. Andersson2,3 1Div. of Experimental Surgery, BCRT and 4Department of Surgery, Charité Universitätsmedizin Berlin, Germany; 2AstraZeneca R&D, Sweden; 3Karolinska Institute, Stockholm, Sweden; 5Department of Traumatology, TU Munich, MRI, Germany; 6McGowan Institute for Regenerative Medicine, University of Pittsburgh, PA, USA Introduction A major requirement in the development of new potential drug candidates is adequate safety testing. The liver represents the central target for human drug metabolism, and hepatic drug toxicity is one of the main reasons for withdrawal of drugs from clinical use. In vitro models based on human liver cells would be useful to provide human-predictive information on the metabolism and possible side-effects of drugs. We focus on bioreactor technologies that address the cellular needs of 3D tissue organisation in a highly physiological environment. Primary human hepatocytes as well as hepatic cell lines survive and can be maintained in the bioreactor over several weeks. In this study the pattern and long-term stability of various cytochrome P 450 (CYP) activities was investigated in primary human hepatocytes (pHHC) and two different liver cell lines (C3A, Huh7) and cultured in a miniaturized bioreactor prototype. Results Comparison of CYP activities in primary human hepatocytes, C3A and Huh7 cells Primary human hepatocytes cultured in bioreactors show more than 10x higher CYP activities than the liver cell lines for all three CYP isoforms tested (Fig. 4). Among the cell lines investigated, the Huh7 cell line shows distinctly higher CYP activities, in particular regarding CYP1A1/2 and CYP2C9. Fig. 4: Course of CYP metabolite formation from phenacetin (CYP1A1/1A2), diclofenac (CYP2C9) and midazolam (CYP3A4) in human hepatocytes (blue), Huh7 cells (red) and C3A cells (orange) cultured in bioreactors. Methods Bioreactor system The bioreactor (Fig. 1) consists of two independent bundles of microfiltration capillaries for the transport of culture medium (red and yellow), interwoven with one bundle of oxygenation capillaries (blue). The capillaries provide a 3D scaffold for the cells residing in the extra-capillary space (cell compartment). The bioreactor is integrated into a perfusion system equipped with pump units for medium supply, pressure and flow control, a gas mixing unit and an electronically controlled heating unit (Fig. 2). Immunohistochemical stainings demonstrate cell reorganisation and formation of tissue like aggregates in the system, including biliary and vascular structures as well as expression of hepatic transporters (Fig. 3). Evaluation of the long-term stability of CYP activities of primary human hepatocytes cultured in bioreactors All three CYP isoforms investigated exhibit distinct and reproducible activities in primary human hepatocyte bioreactors after 3-5 days (Fig. 5, blue curves). In one bioreactor maintained over 23 days human hepatocytes still show marked CYP activities at the end of the culture period (Fig. 5, red curves). Fig. 5: Course of CYP1A1/1A2, CYP2C9 and CYP3A4 activities in primary human hepatocyte bioreactors. The blue curves each represent the mean of five bioreactors cultured with human hepatocytes from five donors for 3-5 days. The red curves show CYP activities of human hepatocytes cultured in a bioreactor for 23 days. CYP activity is correlated with urea production We observed that as long as human hepatocytes produce urea above detection limit they exhibit CYP activity. The heat map (Fig. 6) was created to compare urea production and CYP activities for several bioreactors at different time points of bioreactor culture. The fact that urea and CYP activities show the same color at most time points representing either low or high levels indicates a correlation. The calculated correlation coefficient was highest for CYP1A1/1A2 (0.9) and less evident for CYP2C9 and CYP3A4 (0.63 and 0.51, respectively). Fig. 1: Bioreactor (schematic) Fig. 2: Bioreactor perfusion system Fig. 3: Immunohistochemical stainings of human liver tissue and liver cell material from a bioreactor Cell preparation / Bioreactor operation A total of 6.0 to 8.0 x 107 cells of the human hepatoma cell line C3A or Huh7 were inoculated in bioreactors with a cell compartment volume of 2 ml, respectively. Cells were allowed to adapt to the 3D environment and to reach a stable functional level before starting experiments. Primary human hepatocytes (pHHC) were prepared by collagenase digestion of healthy tissue obtained from partial liver resections and between 5.0 and 12.0 x 107 cells were inoculated into one bioreactor, respectively. During the entire run biochemical parameters for cell viability, including glucose consumption and production of urea, lactate and ammonia were measured daily. Cell damage was surveyed by release of LDH, AST, ALT and GGT into the culture medium. CYP activity analysis CYP1A1/1A2, CYP2C9 and CYP3A4 activities were determined by incubation of the cultures with suitable model substrates (phenacetin, diclofenac and midazolam). Concentrations of parent substances and their metabolites were analyzed at ten time points over 24 h by liquid chromatography/ mass spectrometry (LC/MS). Fig. 6: Heat map illustrating levels of urea production and degrees of CYP activity (low – yellow, high – orange) for six human hepatocyte bioreactors at different time points of culture. Conclusion The results show that the analysed CYP activities are well expressed and can be maintained in human liver cell bioreactors over several weeks. CYP dependent metabolism was markedly higher in primary hepatocytes than in liver cell lines. CYP activity correlated with urea production in primary human hepatocyte bioreactors. The 3D four-compartment bioreactor technology allows long-term cell maintenance for repeated studies in the same experimental setup enabling drug metabolism studies and long running drug exposure on human liver cells in a highly controlled, dynamic environment. 11th European Regional Meeting of the International Society for the Study of Xenobiotics (ISSX), 17 to 20 May 2009, Lisbon, Portugal