Massimo Kubon

“Artificial micro organs”—a microfluidic device for dielectrophoretic assembly of liver sinusoids

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.

A microsensor system to probe physiological environments and tissue response

Foreign body responses and bio-fouling caused by the physiological environment impair sensor performance due to alteration of the sensor/tissue interface. For in vivo applications longterm stability is a critical prerequisite and often affected due to host response towards the implant. In order to assess tissue response towards implants, we propose continuous measurements at the implant/tissue interface employing a microsensor device placed in contact with the chorioallantoic membrane (CAM) of the avian embryo. We introduce a biostable microsensor implant (MSI) to measure oxygen, pH and electrical impedance in situ. These parameters were chosen for their sensitivity with respect to the composition and properties of biological tissue. Micro fabrication technology in combination with electrochemical electrode functionalization was used to combine all sensors in a small planar array. The chorioallantoic membrane assay (CAM-assay) of avian ex ovo cultures served as a quasi-in vivo environment. Here we established an immune-active and -deficient in vivo model, enabling comparison between weak and strong immune responses in the same organism. A miniaturized potentiostat unit (“MiniPot”) was developed for controlling the MSI in humid culture environments. Here we performed continuous measurements of all sensor parameters at the implant/tissue interface with the microsensor device placed in contact with the CAM of the avian embryo.

Numerical modelling and measurement of cell trajectories in 3‐D under the influence of dielectrophoretic and hydrodynamic forces

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.

Towards quantification of biocompatibility: Monitoring ingrowth behavior of biomaterials in tissue with a microsensor implant

We introduce a microsensor implant (MSI) comprising microsensors for dissolved oxygen, pH and electrical impedance to monitor ingrowth behavior of biomaterial coatings into tissue. The chorioallantoic membrane (CAM) of ex ovo transferred avian embryos served as a physiological tissue environment. Here, different signal progressions during ingrowth could be observed in a time period of 3–4 days. We tested biodegradable poly(DL-lactide-co-glycolide) and biostable plasma-polymerized coatings [1] compared to an uncoated MSI.

Multimodal Chemosensor‐Based, Real‐Time Biomaterial/Cell Interface Monitoring

Real‐time monitoring of the cell/biomaterial interface for biomedical applications can significantly improve the assessment of biomaterials. However, currently there are no multiparametric real‐time monitoring systems for the ex vivo assessment of biomaterials. This study presents a method for monitoring of cell and tissue response to biomaterials based on microsensor read‐outs of pH, O2, and impedance. In vitro, the sensors are compatible with cells implants. For ex vivo measurements, either a biodegradable polymer poly(lactic‐co‐glycolic acid) or a protein‐repellent plasma polymer nanofilm is coated onto sensor arrays and placed on the chorioallantoic membrane of avian embryo ex ovo cultures. Signals related to dissolved oxygen, pH, and electrical impedance are continuously acquired for up to 90 h. Optical observation, immunohistochemical examination of CD45, and Haematoxylin and Eosin (H&E) staining show different unspecific immune responses. Tissue inflammation, capsule formation, and neovascularization are observed. These results correlate well with the type of coating material and the signal patterns acquired by the chemosensors. In addition, tissue death can be inferred from sensor signal patterns. This system is intended to enable continuous monitoring of biomaterials and is envisioned as a prescreening tool to provide real‐time monitoring of cell/biomaterial interface and also to reduce animal testing for biomaterial assessment purposes.