Biosensors for label-free continuous monitoring of microfluidic cell cultures

Mikrofluidik ist eine aufstrebende Technologie, die sich besonders für die Entwicklung neuartiger Systeme für zellbasierte Testung eignet.[1] Während heutzutage bereits zahlreiche ausgeklügelte und spezialisierte mikrofluidische Plattformen existieren, ist die Zellanalyse zumeist noch auf mikroskopische Beobachtung beschränkt.[2] Mikroskopische Aufnahmen können zwar wichtige qualitative Informationen liefern, eine Quantifizierung der Messung ist jedoch meist nicht möglich. Um mikrofluidische Zellkulturexperimente optimal auswerten zu können braucht es quantitative Analysenmethoden, die idealerweise kontinuierliche Messungen ermöglichen und ohne zusätzliche Arbeitsschritte automatisierbar sind. All diese Anforderungen werden von markerfreien Biosensoren erfüllt, da diese für invasive Langzeitmessungen von lebenden Zellpopulationen geeignet sind. Das Ziel dieser Arbeit war daher die Entwicklung markerfreier Zellsensoren für die Integration in mikrofluidische Systeme. Drei verschiedene Plattformen wurden designet, hergestellt, optimiert, charakterisiert und zur Überwachung von Zellkulturen verwendet. Als erstes wird ein optischer Sensor zur 2D Sauerstoffbestimmung präsentiert. Die Sauerstoffmessung wird dabei mittels einer fluoreszierenden Schicht bestehend aus einem sauerstoffsensitiven und einem nicht sauerstoffsensitiven (Referenz-) Fluoreszenzfarbstoff bewerkstelligt. Der Sensor erlaubt es Sauerstoffgradienten mit hoher zeitlicher und örtlicher Auflösung zu verfolgen, was insbesondere für 3D Kulturen mit potentieller Sauerstofflimitierung relevant ist. Als nächstes wird ein System mit kombinierter optischer und elektrischer Zellmessung präsentiert. Während Lichtstreuungsmessung Auskunft über Zellzahle und -morphologie gibt, erlauben Impedanzsensoren die Messung der Zell-Substratinteraktion. Zusammengenommen, kann durch die komplementären Sensoren die Verlässlichkeit von Zellexperimenten verbessert und Klarheit bei uneindeutigen Resultaten geschaffen werden. Schließlich wurde das duale Messsystem noch erfolgreich verwendet um dynamische Zell-Zellinteraktionen in einem mikrofluidischen Ko-Kulturexperiment zu beobachten. Das dritte in dieser Arbeit entwickelte System ist eine integrierte Messstation für hochfrequente (bis 20 MHz) Impedanzmessungen. Das System wird verwendet um verschiedene Zellantworten wie Zelltod, Serumentzug, [beta] -Adrenorezeptoraktivierung und Entzündungsreaktionen zu identifizieren. Abschließend werden die generellen Vorteile aber auch Nachteile der neu entwickelten markerfreien Biosensoren für mikrofluidische Systeme präsentiert. [1] El-Ali, J., P.K. Sorger, and K.F. Jensen, Cells on chips. Nature, 2006. 442(7101): p. 403-411. [2] Gómez-Sjöberg, R., et al., Versatile, fully automated, microfluidic cell culture system. Analytical Chemistry, 2007. 79(22): p. 8557-8563.Microfluidics is an emerging technology that provides the means to develop physiologically relevant cell-based assays.[1] While numerous sophisticated and specialized microfluidic systems for cell cultivation have been developed, even today assay read out is often limited to microscopic cell observation.[2] While microscopic images provide valuable qualitative information, quantification is usually difficult. By contrast, meaningful analysis of microfluidic cell cultures should enable quantitative, continuous readings with good temporal resolution and be amenable for automation without requiring multiple handling steps. Here label-free approaches are ideally suited, because they allow for long term non-invasive monitoring of cell population dynamics. Therefore the goal of this thesis was the development of label-free biosensors that can be integrated into microfluidic devices. Three different integrated sensor systems have been fabricated, optimized, characterized and applied for cell culture monitoring. First, an optical sensor for oxygen imaging is presented. A fluorescent sensor layer containing an oxygen sensitive fluorescent dye and a reference dye enables ratiometric oxygen measurements. Spatial and temporal oxygen gradients could be followed with high accuracy which provides crucial information especially for 3D cell cultures, where oxygen supply can become a limiting factor. Next, optical light scattering detection was combined with electric cell impedance measurements in a single device. While light scattering measurements provides important information on cell numbers and morphology, impedance sensors are sensitive towards cell-substrate interaction. Together the complementary sensors enabled improved assay reliability and helped to clarify otherwise ambiguous assay results. Finally, the dual-parameter cell-chip was applied to monitor dynamic cell-cell interactions in microfluidic co-cultures. The third presented system is a lab-on-a-chip station for high frequency impedance monitoring that enables measurements up to 20 MHz. Application of the system to successfully identify various cell responses including cell death, serum starvation, [beta] -adrenergic receptor activation and inflammatory responses are presented. Finally, the general advances and also limitations of the newly developed microfluidic systems with integrated label-free biosensors are presented. [1]El-Ali, J., P.K. Sorger, and K.F. Jensen, Cells on chips. Nature, 2006. 442(7101): p. 403-411. [2]Gómez-Sjöberg, R., et al., Versatile, fully automated, microfluidic cell culture system. Analytical Chemistry, 2007. 79(22): p. 8557-8563.eingereicht von Verena CharwatAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Med. Univ., Diss., 2013OeBB(VLID)171453

Impact of Source and Manufacturing of Collagen Matrices on Fibroblast Cell Growth and Platelet Aggregation

Collagen is a main component of the extracellular matrix. It is often used in medical applications to support tissue regeneration, hemostasis, or wound healing. Due to different sources of collagen, the properties and performance of available products can vary significantly. In this in vitro study, a comparison of seven different collagen matrices derived from bovine, equine, and porcine sources was performed. As performance indicators, the scaffold function for fibroblasts and platelet aggregation were used. We found strong variation in platelet aggregation and fibroblast growth on the different collagen materials. The observed variations could not be attributed to species differences alone, but were highly dependent on differences in the manufacturing process

Do calcium channel blockers applied to cardiomyocytes cause increased channel expression resulting in reduced efficacy?

Abstract In the initial hours following the application of the calcium channel blocker (CCB) nifedipine to microtissues consisting of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we observe notable variations in the drug’s efficacy. Here, we investigate the possibility that these temporal changes in CCB effects are associated with adaptations in the expression of calcium ion channels in cardiomyocyte membranes. To explore this, we employ a recently developed mathematical model that delineates the regulation of calcium ion channel expression by intracellular calcium concentrations. According to the model, a decline in intracellular calcium levels below a certain target level triggers an upregulation of calcium ion channels. Such an upregulation, if instigated by a CCB, would then counteract the drug’s inhibitory effect on calcium currents. We assess this hypothesis using time-dependent measurements of hiPSC-CMs dynamics and by refining an existing mathematical model of myocyte action potentials incorporating the dynamic nature of the number of calcium ion channels. The revised model forecasts that the CCB-induced reduction in intracellular calcium concentrations leads to a subsequent increase in calcium ion channel expression, thereby attenuating the drug’s overall efficacy. The data and fit models suggest that dynamic changes in cardiac cells in the presence of CCBs may be explainable by induced changes in protein expression, and that this may lead to challenges in understanding calcium based drug effects on the heart unless timings of applications are carefully considered

Isochoric supercooled preservation and revival of human cardiac microtissues.

Low-temperature biopreservation and 3D tissue engineering present two differing routes towards eventual on-demand access to transplantable biologics, but recent advances in both fields present critical new opportunities for crossover between them. In this work, we demonstrate sub-zero centigrade preservation and revival of autonomously beating three-dimensional human induced pluripotent stem cell (hiPSC)-derived cardiac microtissues via isochoric supercooling, without the use of chemical cryoprotectants. We show that these tissues can cease autonomous beating during preservation and resume it after warming, that the supercooling process does not affect sarcomere structural integrity, and that the tissues maintain responsiveness to drug exposure following revival. Our work suggests both that functional three dimensional (3D) engineered tissues may provide an excellent high-content, low-risk testbed to study complex tissue biopreservation in a genetically human context, and that isochoric supercooling may provide a robust method for preserving and reviving engineered tissues themselves

In vitro safety “clinical trial” of the cardiac liability of drug polytherapy

Abstract Only a handful of US Food and Drug Administration (FDA) Emergency Use Authorizations exist for drug and biologic therapeutics that treat severe acute respiratory syndrome‐coronavirus 2 (SARS‐CoV‐2) infection. Potential therapeutics include repurposed drugs, some with cardiac liabilities. We report on a chronic preclinical drug screening platform, a cardiac microphysiological system (MPS), to assess cardiotoxicity associated with repurposed hydroxychloroquine (HCQ) and azithromycin (AZM) polytherapy in a mock phase I safety clinical trial. The MPS contained human heart muscle derived from induced pluripotent stem cells. The effect of drug response was measured using outputs that correlate with clinical measurements, such as QT interval (action potential duration) and drug‐biomarker pairing. Chronic exposure (10 days) of heart muscle to HCQ alone elicited early afterdepolarizations and increased QT interval past 5 days. AZM alone elicited an increase in QT interval from day 7 onward, and arrhythmias were observed at days 8 and 10. Monotherapy results mimicked clinical trial outcomes. Upon chronic exposure to HCQ and AZM polytherapy, we observed an increase in QT interval on days 4–8. Interestingly, a decrease in arrhythmias and instabilities was observed in polytherapy relative to monotherapy, in concordance with published clinical trials. Biomarkers, most of them measurable in patients’ serum, were identified for negative effects of monotherapy or polytherapy on tissue contractile function, morphology, and antioxidant protection. The cardiac MPS correctly predicted clinical arrhythmias associated with QT prolongation and rhythm instabilities. This high content system can help clinicians design their trials, rapidly project cardiac outcomes, and define new monitoring biomarkers to accelerate access of patients to safe coronavirus disease 2019 (COVID‐19) therapeutics

Combinatorial in Vitro and in Silico Approach To Describe Shear-Force Dependent Uptake of Nanoparticles in Microfluidic Vascular Models

This document is the unedited Author’s version of a Submitted Work that was subsequently accepted for publication in Analytical Chemistry copyright © American Chemical Society after peer review. To access the final edited and published work see https://doi.org/10.1021/acs.analchem.7b04788.European Union’s Horizon 202

Correction to: The role of fibrinolysis inhibition in engineered vascular networks derived from endothelial cells and adipose-derived stem cells

The original article [1] contains numerous value errors in the graphs in Fig. 2b regarding the markers describing the values for total tubule length and mean tubule length without aprotinin at 2.5 mg/ml concentration of fibrinogen. The corrected version of this figure can be viewed ahead