Behavior of HepG2/C3A cell cultures in a microfluidic bioreactor

Abstract An important issue in toxicity studies is the development of pertinent new in vitro tests that will be able to provide an alternative to in vivo testing methods. Current developments in the fields of tissue engineering and microtechnology make it possible to propose the use of microfluidic bioreactors as a tool for enhanced in vitro investigations. However, both the cells’ behavior in complex environments and their response to chemicals need to be better understood, especially for future validation of any new assay. To characterize the sensitivity of this approach, we investigated the behavior of a liver cell model with respect to variations of two cell culture parameters in a microfluidic bioreactor: inoculated cell density (0.35 × 106, 0.45 × 106 and 0.65 × 106 cells/bioreactor) and microfluidic flow rates (0, 10 and 25 μL/min). We also investigated an environmental pollutant modeled with three ammonia concentrations (0, 5 and 10 mM). Proliferation in the bioreactor was found to be flow rate and inoculated cell density dependent. This led to a mean value of 1.2 ± 0.2 × 106 cells in the 3D microenvironment of the bioreactor without ammonia loadings after 96 h of cultures. Cell metabolism rates, such as glucose and glutamine consumption or CYP1A detoxification, were found to be higher in dynamic conditions than in static conditions. Furthermore, increased ammonium chloride concentration in turn increased glucose and glutamine consumptions and CYP1A activity. Inhibition of 50% of cell proliferation (IC50) during the ammonium chloride analysis was found at 5 mM when cell concentrations of 0.35 × 106 cells/bioreactor were inoculated. In contrast, no effect could be detected at 5 mM for larger cell densities of 0.65 × 106 cells/bioreactor, demonstrating concentration and cell density dependence in the bioreactors. This study highlighted the sensitivity of the HepG2/C3A cells to microfluidic culture conditions and illustrated the potential for larger in vitro toxicity studies using microfluidic bioreactors.

[1]  L. Griffith,et al.  Functional behavior of primary rat liver cells in a three-dimensional perfused microarray bioreactor. , 2002, Tissue engineering.

[2]  David J Beebe,et al.  Diffusion dependent cell behavior in microenvironments. , 2005, Lab on a chip.

[3]  F. Oesch,et al.  New Hepatocyte In Vitro Systems for Drug Metabolism: Metabolic Capacity and Recommendations for Application in Basic Research and Drug Development, Standard Operation Procedures , 2003, Drug metabolism reviews.

[4]  C L Alden,et al.  Survey of the QSAR and in vitro approaches for developing non-animal methods to supersede the in vivo LD50 test. , 1990, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[5]  Teruo Fujii,et al.  Perfusion culture of fetal human hepatocytes in microfluidic environments , 2004 .

[6]  Angelika Langsch,et al.  Development and characterization of a small‐scale bioreactor based on a bioartificial hepatic culture model for predictive pharmacological in vitro screenings , 2006, Biotechnology and bioengineering.

[7]  M. Yarmush,et al.  Evaluation of a microfluidic based cell culture platform with primary human hepatocytes for the prediction of hepatic clearance in human. , 2009, Biochemical pharmacology.

[8]  Nicolas Szita,et al.  Membrane‐aerated microbioreactor for high‐throughput bioprocessing , 2004, Biotechnology and bioengineering.

[9]  M. Ingelman-Sundberg,et al.  Transcriptional and post-translational regulation of CYP1A1 by primaquine. , 2001, The Journal of pharmacology and experimental therapeutics.

[10]  Laurent Griscom,et al.  Development of a Renal Microchip for In Vitro Distal Tubule Models , 2007, Biotechnology progress.

[11]  Teruo Fujii,et al.  Cell Culture in 3-Dimensional Microfluidic Structure of PDMS (polydimethylsiloxane) , 2003 .

[12]  Laurent Griscom,et al.  Trends in the development of microfluidic cell biochips for in vitro hepatotoxicity. , 2007, Toxicology in vitro : an international journal published in association with BIBRA.

[13]  A. Moorman,et al.  Hepatocyte heterogeneity in the metabolism of amino acids and ammonia. , 1992, Enzyme.

[14]  Teck Chuan Lim,et al.  A microfluidic 3D hepatocyte chip for drug toxicity testing. , 2009, Lab on a chip.

[15]  Melvin E. Andersen,et al.  The need for a new toxicity testing and risk analysis paradigm to implement REACH or any other large scale testing initiative , 2006, Archives of Toxicology.

[16]  Aaron Sin,et al.  Development of a Microscale Cell Culture Analog To Probe Naphthalene Toxicity , 2008, Biotechnology progress.

[17]  I. Marison,et al.  The importance of ammonia in mammalian cell culture. , 1996, Journal of biotechnology.

[18]  Ding‐Shinn Chen,et al.  Long-term culture of hepatocytes from human adults. , 1998 .

[19]  M. Toner,et al.  Effects of oxygenation and flow on the viability and function of rat hepatocytes cocultured in a microchannel flat-plate bioreactor. , 2001, Biotechnology and bioengineering.

[20]  S. A. Stern,et al.  Diffusion of Gases in Silicone Polymers: Molecular Dynamics Simulations , 1998 .

[21]  Helmut Greim,et al.  Toxicological comments to the discussion about REACH , 2006, Archives of Toxicology.

[22]  Clare Selden,et al.  Cells for bioartificial liver devices: The human hepatoma‐derived cell line C3A produces urea but does not detoxify ammonia , 2008, Biotechnology and bioengineering.

[23]  A. Meijer,et al.  Control of rat-liver glutaminase by ammonia and pH. , 1983, European journal of biochemistry.

[24]  Teruo Fujii,et al.  Microfluidic PDMS (Polydimethylsiloxane) Bioreactor for Large‐Scale Culture of Hepatocytes , 2004, Biotechnology progress.

[25]  Cecilia Clemedson,et al.  EDIT: A New International Multicentre Programme to Develop and Evaluate Batteries of In Vitro Tests for Acute and Chronic Systemic Toxicity , 1999, Alternatives to laboratory animals : ATLA.

[26]  D. Beebe,et al.  Microenvironment design considerations for cellular scale studies. , 2004, Lab on a chip.

[27]  A. Bader,et al.  Review of a flat membrane bioreactor as a bioartificial liver. , 2001, Annals of transplantation.

[28]  R. de Kanter,et al.  Precision-cut organ slices as a tool to study toxicity and metabolism of xenobiotics with special reference to non-hepatic tissues. , 2002, Current drug metabolism.

[29]  M. Toner,et al.  Microfabricated grooved substrates as platforms for bioartificial liver reactors. , 2005, Biotechnology and bioengineering.

[30]  Jong Hwan Sung,et al.  A microfluidic device for a pharmacokinetic-pharmacodynamic (PK-PD) model on a chip. , 2010, Lab on a chip.

[31]  F. Lestari,et al.  In vitro cytotoxicity of selected chemicals commonly produced during fire combustion using human cell lines. , 2005, Toxicology in vitro : an international journal published in association with BIBRA.

[32]  A. Guillouzo,et al.  Liver cell models in in vitro toxicology. , 1998, Environmental health perspectives.