Peter Ertl

Lab-on-a-chip technologies for stem cell analysis.

The combination of microfabrication-based technologies with cell biology has laid the foundation for the development of advanced in vitro diagnostic systems capable of analyzing cell cultures under physiologically relevant conditions. In the present review, we address recent lab-on-a-chip developments for stem cell analysis. We highlight in particular the tangible advantages of microfluidic devices to overcome most of the challenges associated with stem cell identification, expansion and differentiation, with the greatest advantage being that lab-on-a-chip technology allows for the precise regulation of culturing conditions, while simultaneously monitoring relevant parameters using embedded sensory systems. State-of-the-art lab-on-a-chip platforms for in vitro assessment of stem cell cultures are presented and their potential future applications discussed.

Detection of viruses with molecularly imprinted polymers integrated on a microfluidic biochip using contact-less dielectric microsensors

Rapid detection of viral contamination remains a pressing issue in various fields related to human health including clinical diagnostics, the monitoring of food-borne pathogens, the detection of biological warfare agents as well as in viral clearance studies for biopharmaceutical products. The majority of currently available assays for virus detection are expensive, time-consuming, and labor-intensive. In the present work we report the creation of a novel micro total analysis system (microTAS) capable of continuously monitoring viral contamination with high sensitivity and selectivity. The specific interaction between shape and surface chemistry between molecular imprinted polymer (MIP) and virus resulted in the elimination of non-specific interaction in the present sensor configuration. The additional integration of the blank (non-imprinted) polymer further allowed for the identification of non-specific adsorption events. The novel combination of microfluidics containing integrated native polymer and MIP with contact-less dielectric microsensors is evaluated using the Tobacco Mosaic Virus (TMV) and the Human Rhinovirus serotype 2 (HRV2). Results show that viral binding and dissociation events can be readily detected using contact-less bioimpedance spectroscopy optimized for specific frequencies. In the present study optimum sensor performance was achieved at 203 kHz within the applied frequency range of 5-500 kHz. Complete removal of the virus from the MIP and device reusability is successfully demonstrated following a 50-fold increase in fluid velocity. Evaluation of the microfluidic biochip revealed that microchip technology is ideally suited to detect a broader range of viral contaminations with high sensitivity by selectively adjusting microfluidic conditions, sensor geometries and choice of MIP polymeric material.

Development of a disposable microfluidic biochip for multiparameter cell population measurements.

An under recognized cause of preventable mortality is healthcare-associated (nosocomial) infections such as biofilms found on implants and catheters. About 5% of U.S. and E.U. patients acquire nosocomial infections leading to prolonged hospitalization, increased patient suffering, and mortality rates. To date, no satisfactory solutions are available to monitor biofilm formation under near-native conditions. As a consequence, in the present work, we report the development of a disposable microfluidic biochip capable of continuously monitoring cell population dynamics under physiological shear force conditions. We demonstrate the simultaneous application of contactless bioimpedance spectroscopy and amperometric measurements to monitor fungal biofilm growth rates and metabolic activities. Quantitative cell analysis is accomplished by the use of high-density interdigitated capacitors (microIDC) isolated by a 700 nm epoxy (SU-8 resist) based passivation layer to noninvasively assess biofilm formation in predefined proliferation chambers. Additionally, biofilm respiration activity is measured using redox-mediators oxidized at band electrodes located downstream within microchannels. The disposable biofilm analysis platform is used to continuously monitor the dynamic responses of C. albicans to different glucose and galactose concentrations.

Recent advances and future applications of microfluidic live-cell microarrays

Microfluidic live-cell microarrays show much promise as screening tools for biomedical research because they could shed light on key biological processes such as cell signaling and cell-to-cell and cell-to-substrate dynamic responses. While miniaturization reduces the need for expensive clinical grade reagents, the integration of functional components including micropumps, biosensors, actuators, mixers and gradient generators results in improved assay reliability, reproducibility and well-defined cell culture conditions. The present review addresses recent technological advances in microfluidic live-cell microarray technology with a special focus on the applications of microfluidic single-cell, multi-cell and 3D cell microarrays.

Microfluidic platforms for advanced risk assessments of nanomaterials

Abstract In the past few years, promising efforts to utilize microfabrication-based technologies have laid the foundation for developing advanced, and importantly more physiologically-realistic, microfluidic methods for risk assessment of engineered nanomaterials (ENMs). In the present review, we discuss the wave of recent developments using microfluidic-based in vitro models and platforms for nanotoxicological assays, such as determination of cell viability, cellular dose, oxidative stress and nuclear damage. Here, we specifically highlight the tangible advantages of microfluidic devices in providing promising tools to tackle many of the current and ongoing challenges faced with traditional toxicology assays. Most importantly, microfluidic technology not only allows to recreate physiologically-relevant in vitro models for nanotoxicity examinations, but also provides platforms that deliver an attractive strategy towards improved control over applied ENM doses. In a final step, we present examples of state-of-the-art microfluidic platforms for in vitro assessment of potential adverse ENM effects.

Interdigitated impedance sensors for analysis of biological cells in microfluidic biochips

ZusammenfassungEin neuartiges miniaturisiertes Analysesystem zur quantitativen Zellanalyse, auf Basis dielektrischer Mikrosensoren und Mikrofluidik, wird in dieser Arbeit vorgestellt. Das realisierte Lab-on-a-Chip beinhaltet in Kammern eingebettete, passivierte interdigitale Elektrodensysteme. Die Einführung einer Multilagen-Passivierung ermöglicht, im Gegensatz zu herkömmlichen Bioimpedanz-Systemen, die Isolation und somit die räumliche Trennung der dielektrischen Mikrosensoren von der Flüssigkeitsumgebung in den Analysekammern. Anhand von unterschiedlichen, in vitro cultivated cells. The overall performance of the system is demonstrated on various bacterial and yeast strains. Due to the high sensitivity of the contact-less dielectric microsensors it is possible to directly identify microbial strains, based on morphological differences and biological composition in the absence of any indicators or labels. Additionally, dielectric changes occurring in sub-cellular structures such as membranes can be directly monitored over a wide frequency range. As a result, microfluidic biochips are developed to continuously monitor cell morphology changes in a non-invasive manner over long periods of time.SummaryIn the presented work we describe the novel combination of contact-less dielectric microsensors and microfluidics for quantitative cell analysis. The lab-on-a-chip system consists of microfluidic channels and chambers together with integrated and passivated interdigitated electrode structures. In contrast to existing bioimpedance methods implemented for cell analysis, the dielectric microsensors are completely insulated and physically removed from the liquid sensing environment using defined multi-passivation layer of distinct size and composition. Consequently, these structures act as contact-less microsensors for the characterization of in vitro kultivierten, Bakterien und Hefezellen wird das Lab-on-a-Chip charakterisiert. Es zeigt sich, dass mikrobiologische Substanzen aufgrund morphologischer Unterschiede bzw. ihrer biologischen Zusammensetzung ohne Verwendung von Markern oder Indikatoren identifiziert werden können. Dielektrische Variationen in subzellularen Strukturen, wie beispielsweise der Membranen, sind über einen weiten Messfrequenzbereich beobachtbar. Der präsentierte mikrofluidische Biochip wurde speziell für die kontinuierliche und nicht-invasive Beobachtung der Zellmorphologie über lange Zeiträume entwickelt.

Monitoring Dynamic Interactions of Tumor Cells with Tissue and Immune Cells in a Lab-on-a-Chip

A complementary cell analysis method has been developed to assess the dynamic interactions of tumor cells with resident tissue and immune cells using optical light scattering and impedance sensing to shed light on tumor cell behavior. The combination of electroanalytical and optical biosensing technologies integrated in a lab-on-a-chip allows for continuous, label-free, and noninvasive probing of dynamic cell-to-cell interactions between adherent and nonadherent cocultures, thus providing real-time insights into tumor cell responses under physiologically relevant conditions. While the study of adherent cocultures is important for the understanding and suppression of metastatic invasion, the analysis of tumor cell interactions with nonadherent immune cells plays a vital role in cancer immunotherapy research. For the first time, the direct cell-to-cell interactions of tumor cells with bead-activated primary T cells were continuously assessed using an effector cell to target a cell ratio of 10:1.

Rapid liposome quality assessment using a lab-on-a-chip

Although liposomes have many outstanding features such as biocompatibility, biodegradability, low toxicity and structural diversity, and are successfully applied in many areas of chemistry and biotechnology, a lack of characterization standards and quality control tools are still inhibiting the translation of liposome technology into clinical routine. The greatest obstacle to clinical scale commercialization is the inability to ensure liposome formulation stability because small size variations or altered surface chemistries can significantly influence in vivo distribution and excretion kinetics that could in turn lead to unpredictable therapy outcomes. To enhance the product development process we have developed a microfluidic biochip containing embedded dielectric microsensors capable of providing quantitative results on formulation composition and stability based on the monitoring of the unique electric properties of liposomes. Computational fluid dynamic (CFD) simulations confirmed that microfluidics offer reproducible and well-defined measurement conditions where a moving liposome suspension within a microchannel behaves like a bulk material. Results of this study demonstrate the ability of microfluidics, in combination with dielectric spectroscopy and multivariate data analysis methods, to identify nine different liposomes. We also show that various liposome modifications such as membrane-bound surface proteins, lipid bilayer soluble drugs, as well as protein and dye encapsulations, can be detected in the absence of any labels or indicators. Since shelf-life stability of a liposome formulation is regarded of prime importance for regulatory approval and clinical application, we further provide a possible practical application of the developed liposome analysis platform as a high-throughput tool for industrial quality insurance purposes.

Automated, Miniaturized, and Integrated Quality Control-on-Chip (QC-on-a-Chip) for Cell-Based Cancer Therapy Applications

The combination of microfabrication-based technologies with cell biology has laid the foundation for the development of advanced in vitro diagnostic systems capable of evaluating cell cultures under defined, reproducible and standardizable measurement conditions. In the present review we describe recent lab-on-a-chip developments for cell analysis and how these methodologies could improve standard quality control in the field of manufacturing cell-based vaccines for clinical purposes. We highlight in particular the regulatory requirements for advanced cell therapy applications using as an example dendritic cell-based cancer vaccines to describe the tangible advantages of microfluidic devices that overcome most of the challenges associated with automation, miniaturization and integration of cell-based assays. As its main advantage lab-on-a-chip technology allows for precise regulation of culturing conditions, while simultaneously monitoring cell relevant parameters using embedded sensory systems. State-of-the-art lab-on-a-chip platforms for in vitro assessment of cell cultures and their potential future applications for cell therapies and cancer immunotherapy are discussed in the present review.

Microfluidic Migration and Wound Healing Assay Based on Mechanically Induced Injuries of Defined and Highly Reproducible Areas

All cell migration and wound healing assays are based on the inherent ability of adherent cells to move into adjacent cell-free areas, thus providing information on cell culture viability, cellular mechanisms and multicellular movements. Despite their widespread use for toxicological screening, biomedical research and pharmaceutical studies, to date no satisfactory technological solutions are available for the automated, miniaturized and integrated induction of defined wound areas. To bridge this technological gap, we have developed a lab-on-a-chip capable of mechanically inducing circular cell-free areas within confluent cell layers. The microdevices were fabricated using off-stoichiometric thiol-ene-epoxy (OSTEMER) polymer resulting in hard-polymer devices that are robust, cost-effective and disposable. We show that the pneumatically controlled membrane deflection/compression method not only generates highly reproducible (RSD 4%) injuries but also allows for repeated wounding in microfluidic environments. Performance analysis demonstrated that applied surface coating remains intact even after multiple wounding, while cell debris is simultaneously removed using laminar flow conditions. Furthermore, only a few injured cells were found along the edge of the circular cell-free areas, thus allowing reliable and reproducible cell migration of a wide range of surface sensitive anchorage dependent cell types. Practical application is demonstrated by investigating healing progression and endothelial cell migration in the absence and presence of an inflammatory cytokine (TNF-α) and a well-known cell proliferation inhibitor (mitomycin-C).