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.
We developed a method to modify the surface in injection molded polymer microdevices prior to bonding and to pattern biomolecules in the completed microsystem in situ by a sequence of simple perfusion steps directly before utilization of the device. This method is compatible with production technology such as injection molding and bonding processes currently employed in the fabrication of polymer microsystems. It solves the problem of the inherent incompatibility of biomolecules with microfabrication technology as it allows for the biofunctionalization step to be performed after completion of the microsystem. Injection molded cyclic olefin copolymer (COC) microfluidic chips were modified by irradiating the surface with UV-light at lambda = 185 nm. This results in the formation of stable acidic groups which were further modified by binding of the extracellular matrix protein collagen type I. Non-irradiated surfaces were modified by binding of Pluronic® F-127 to become non-adhesive. Density of acid groups decreases to 50% within 45 days and to 25% within 19 weeks after irradiation. However, even then the remaining density of functional groups was shown to be sufficient to bind proteins and promote cell adhesion. Selective adhesion of primary hepatocytes on surfaces patterned by UV-irradiation and a biofunctional coating with collagen type I were demonstrated in injection molded microsystems.
This research is part of a program aiming at the development of a fluidic microsystem for in vitro drug testing. For this purpose, primary cells need to be assembled to form cellular aggregates in such a way as to resemble the basic functional units of organs. By providing for in vivo-like cellular contacts, proper extracellular matrix interaction and medium perfusion it is expected that cells will retain their phenotype over prolonged periods of time. In this way, in vitro test systems exhibiting in vivo type predictivity in drug testing are envisioned. Towards this goal a 3-D microstructure micro-milled in a cyclic olefin copolymer (COC) was designed in such a way as to assemble liver cells via insulator-based dielectrophoresis (iDEP) in a sinusoid-type fashion. First, numeric modelling and simulation of dielectrophoretic and hydrodynamic forces acting on cells in this microsystem was performed. In particular, the problem of the discontinuity of the electric field at the interface between the fluid media in the system and the polymer materials it consists of was addressed. It was shown that in certain cases, the material of the microsystem may be neglected altogether without introducing considerable error into the numerical solution. This simplification enabled the simulation of 3-D cell trajectories in complex chip geometries. Secondly, the assembly of HepG2 cells by insulator-based dielectrophoresis in this device is demonstrated. Finally, theoretical results were validated by recording 3-D cell trajectories and the Clausius-Mossotti factor of liver cells was determined by combining results obtained from both simulation and experiment.
HepaChip-MP: a 24-culture-chamber, automated microfluidic in vitro model of the liver sinusoid in multiwellplate format.
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