Behavior of HepG2/C3A cell cultures in a microfluidic bioreactor

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 x 10(6), 0.45 x 10(6) and 0.65 x 10(6) cells/bioreactor) and microfluidic flow rates (0, 10 and 25 mu L/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 x 10(6) 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 x 106 cells/bioreactor were inoculated. In contrast, no effect could be detected at 5 mM for larger cell densities of 0.65 x 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. (C) 2010 Elsevier B.V. All rights reserved.