In order to study possible toxic side effects of potential drug compounds in vitro a reliable test system is needed. Predicting liver toxicity presents a major challenge of particular importance as liver cells grown in a cell culture suffer from a rapid loss of their liver specific functions. Therefore we are developing a new microfluidic test system for liver toxicity. This test system is based on an organ-like liver 3D co-culture of hepatocytes and endothelial cells. We devised a microfluidic chip featuring cell culture chambers with integrated electrodes for the assembly of liver sinusoids by dielectrophoresis. Fluid channels enable an organ-like perfusion with culture media and test compounds. Different chamber designs were studied and optimized with regard to dielectrophoretic force distribution, hydrodynamic flow profile, and cell trapping rate using numeric simulations. Based on simulation results a microchip was injection-moulded from COP. This chip allowed the assembly of viable hepatocytes and endothelial cells in a sinusoid-like fashion.
Robust bubble-free priming of complex microfluidic chips represents a critical, yet often unmet prerequisite to enable their practical and widespread application. Towards this end, the usage of a network of capillary stop valves as a generic design feature is proposed. Design principles, numerical simulations, and their application in the development of a microfluidic cell culture device are presented. This chip comprises eight parallel chambers for the assembly and cultivation of human hepatocytes and endothelial cells. The inlet channel divides into cell chambers, after which the flows are reunited to a single chip outlet. Dimensions and geometry of channels and cell chambers are designed to yield capillary burst pressures sequentially increasing towards the chip outlet. Thus, progress of liquid flow through the device is predefined by design and enclosure of air bubbles inside the microfluidic structures is efficiently avoided. Capillary stop valves were designed using numerical simulations. Devices were fabricated in cyclic olefin polymer. Pressure during filling was determined experimentally and is in good agreement with data obtained from simulation.
We have developed a microfluidic system--microPrep--for subcellular fractionation of cell homogenates based on dielectrophoretic sorting. Separation of mitochondria isolated from a human lymphoblastoid cell line was monitored by fluorescence microscopy and further characterized by western blot analysis. Robust high throughput and continuous long-term operation for up to 60 h of the microPrep chip system with complex biological samples became feasible as a result of a comprehensive set of technical measures: (i) coating of the inner surfaces of the chip with BSA, (ii) application of mechanical actuators to induce periodic flow patterns, (iii) efficient cooling of the device to ensure integrity of organelle, (iv) a wide channel to provide for high fluidic throughput, and (v) integration of a serial arrangement of 10 dielectrophoretic deflector units to enable separation of samples with a high particle load without clogging. Hence, microPrep yields tens of micrograms of enriched and purified mitochondria within hours. Western blots of mitochondria fractions showed that contaminating endoplasmatic reticulum was reduced by a factor 6 when compared with samples prepared by state of the art centrifugation.
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