Dynamic culture of human liver equivalents inside a micro-bioreactor for long-term substance testing
© Materne et al.; licensee BioMed Central Ltd. 2013
Published: 4 December 2013
Current in vitro and animal tests for drug development are failing to emulate the organ complexity of the human body and, therefore, to accurately predict drug toxicity. In this study, we present a self-contained, bioreactor based human in vitro tissue culture test system aiming to support predictive substance testing at relevant throughput. We designed a microcirculation system interconnecting several tissue culture spaces within a PDMS-embedded microfluidic channel circuit. The bioreactor is reproducibly perfused by a peristaltic on-chip micro-pump, providing a near physiologic fluid flow and volume to liquid ratio.
Materials and methods
Liver microtissue aggregates containing 4.8 × 104 HepaRG cells and 0.2 × 104 human hepatic stellate cells (HHSteC) were formed in Perfecta3D® 384-Well Hanging Drop Plates (3D Biomatrix, USA). After two days of hanging drop culture, 20 aggregates were loaded into a single tissue culture compartment of the micro-bioreactor. Each circuit of the micro-bioreactor device contained 700 μl medium in total. During the first 7 days, a 40% media exchange rate was applied at 12 h intervals. From day 8 onwards, a 40% exchange rate was applied at 24 h intervals. Daily samples were collected for respective analyses. Experiments were stopped at day 14 and 28 and tissues were subjected to immunohistochemical stainings and qRT-PCR analyses. Experiments were conducted with four replicates. To expose the chip-cultures to troglitazone, liver microtissues were cultured for one day in normal medium and were, subsequently, treated with 0 μM, 5 μM and 50 μM substance, respectively. Application of troglitazone was repeated at 12 h or 24 h intervals simultaneously with the medium change.
A promising tool for long term culture of human liver equivalents has been developed. The simple MOC design presented, assisted the culture of human liver equivalents over a period of up to 28 days. The cultures, operated at a total on-chip volume of 700 μl medium at recirculation rates of 40 μl/min assisted by an on-chip micropump, stabilize approximately within a week at a metabolic steady state. The prediction of toxicology profiles of compounds metabolised by the liver was demonstrated possible by exposing the cells to different concentrations of troglitazone. This platform is designed to generate high-quality in vitro data predictive of substance safety in humans. Tissue cultures can be exposed to pharmaceutical substances at regimens relevant to respective guidelines, currently used for subsystemic substance testing in animals.
The work has been funded by the German Federal Ministry for Education and Research, GO-Bio Grand No. 0315569.
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