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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.

Evaluation of arrhythmogenic drugs is required by regulatory agencies before any new compound can obtain market approval. Despite rigorous review, cardiac disorders remain the second most common cause for safety-related market withdrawal. On the other hand, false-positive preclinical findings prohibit potentially beneficial candidates from moving forward in the development pipeline. Complex

Multipotent mesenchymal stromal cells (MSC) and MSC-derived products have emerged as promising therapeutic tools. To fully exploit their potential, further mechanistic studies are still necessary and bioprocessing needs to be optimized, which requires an abundant supply of functional MSC for basic research. To address this need, here we used a novel technology to establish a human adipose-derived MSC line with functional characteristics representative of primary MSC. Primary MSC were isolated and subjected to lentiviral transduction with a library of expansion genes. Clonal cell lines were generated and evaluated on the basis of their morphology, immunophenotype, and proliferation potential. One clone (K5 iMSC) was then selected for further characterization. This clone had integrated a specific transgene combination including genes involved in stemness and maintenance of adult stem cells. Favorably, the K5 iMSC showed cell characteristics resembling juvenile MSC, as they displayed a shorter cell length and enhanced migration and proliferation compared with the non-immortalized original primary MSC (p < 0.05). Still, their immunophenotype and differentiation potential corresponded to the original primary MSC and the MSC definition criteria, and cytogenetic analyses revealed no clonal aberrations. We conclude that the technology used is applicable to generate functional MSC lines for basic research and possible future bioprocessing applications.

We have developed a highly integrated lab-on-a-chip containing embedded electrical microsensors, μdegassers and pneumatically-actuated micropumps to monitor allergic hypersensitivity. Rapid antigen-mediated histamine release (e.g. s to min) and resulting muscle contraction (

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., Powell-Palm et al. demonstrate sub-zero centigrade preservation and revival of autonomously beating, 3D human induced pluripotent stem cell (hiPSC)-derived cardiac microtissues via isochoric supercooling, without the use of chemical cryoprotectants. Their study suggests that functional 3D engineered tissues may provide a 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.

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.

Microphysiological systems (MPSs) mimicking human organ function in vitro are an emerging alternative to conventional cell culture and animal models for drug development. Human induced pluripotent stem cells (hiPSCs) have the potential to capture the diversity of human genetics and provide an unlimited supply of cells. Combining hiPSCs with microfluidics technology in MPSs offers new perspectives for drug development. Here, the integration of a newly developed liver MPS with a cardiac MPS—both built with the same hiPSC line—to study drug-drug interaction (DDI) is reported. As a prominent example of clinically relevant DDI, the interaction of the arrhythmogenic gastroprokinetic cisapride with the fungicide ketoconazole was investigated. As seen in patients, metabolic conversion of cisapride to non-arrhythmogenic norcisapride in the liver MPS by the cytochrome P450 enzyme CYP3A4 was inhibited by ketoconazole, leading to arrhythmia in the cardiac MPS. These results establish functional integration of isogenic hiPSC-based liver and cardiac MPSs, which allows screening for DDI, and thus drug efficacy and toxicity, in the same genetic background.

The immature physiology of cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) limits their utility for drug screening and disease modelling. Here we show that suitable combinations of mechanical stimuli and metabolic cues can enhance the maturation of hiPSC-derived cardiomyocytes, and that the maturation-inducing cues have phenotype-dependent effects on the cells' action-potential morphology and calcium handling. By using microfluidic chips that enhanced the alignment and extracellular-matrix production of cardiac microtissues derived from genetically distinct sources of hiPSC-derived cardiomyocytes, we identified fatty-acid-enriched maturation media that improved the cells' mitochondrial structure and calcium handling, and observed divergent cell-source-dependent effects on action-potential duration (APD). Specifically, in the presence of maturation media, tissues with abnormally prolonged APDs exhibited shorter APDs, and tissues with aberrantly short APDs displayed prolonged APDs. Regardless of cell source, tissue maturation reduced variabilities in spontaneous beat rate and in APD, and led to converging cell phenotypes (with APDs within the 300-450 ms range characteristic of human left ventricular cardiomyocytes) that improved the modelling of the effects of pro-arrhythmic drugs on cardiac tissue.

Targeted nanomedicine builds on the concept that nanoparticles can be directed to specific tissues while remaining inert to others organs. Many studies have been performed on the synthesis and cellular interactions of core-shell nanoparticles, in which a functional inorganic core is coated with a biocompatible polymer layer that should reduce nonspecific uptake and cytotoxicity. However, work is lacking that relates structural parameters of the core-shell structure and colloidal properties directly to interactions with cell membranes and further correlates these interactions to cell uptake. We have synthesized monodisperse (SD10%), single-crystalline, and superparamagnetic iron oxide nanoparticles (SPION) of different core size (3-8 nm) that are densely grafted with nitrodopamine-poly(ethylene glycol) (NDA-PEG(5 kDa)) brushes. We investigated the interactions of the PEGylated SPION with biomimetic membranes and cancer and kidney cells. It is shown that a dense homogeneous PEG shell suppresses membrane interactions and cell uptake but that nanoparticle curvature can influence membrane interactions for similarly grafted nanoparticles. Weak adsorption to anionic lipid membranes is shown to correlate with eukaryote cell uptake and is attributed to double-layer interactions without direct membrane penetration. This attraction is strongly suppressed during physiological conditions and leads to unprecedented low cell uptake and full cell viability when compared to those of traditional dextran-coated SPION. Less curved (larger core) PEGylated SPION show weaker membrane adsorption and lower cell uptake due to effectively denser shells. These results provide a better understanding of design criteria for core-shell nanoparticles in terms of avoiding nonspecific uptake by cells, reducing toxicity, and increasing circulation time.

The p-value is the most prominent established metric for statistical significance in non-targeted metabolomics. However, its adequacy has repeatedly been the subject of discussion criticizing its uncertainty and its dependence on sample size and statistical power. These issues compromise non-targeted metabolomics in model systems, where studies typically investigate 5–10 samples per group. In this paper we propose a different approach for assessing the relevance of fold change (FC) data, where the FC is treated as a quantitative value and is validated by uncertainty budgeting. For the purpose of large-scale application in non-targeted metabolomics, we present a simplified approach for uncertainty propagation using experimental standard deviations of metabolite intensities as type A-summarized standard uncertainties. The resulting expanded FC uncertainty can be used to derive a minimum relevant FC as a complementary criterion in metabolomics data evaluation. This concept overcomes the need for a uniform p-value cut-off for all metabolites by considering the experimental uncertainty for each metabolite individually. The proposed procedure is part of analytical method validation, however the concept has not previously been applied to non-targeted metabolomics. A case study on mesenchymal stem cells cultured in normoxia and hypoxia demonstrates the practical value of this approach, in particular for studies with a small sample size. An online two-dimensional LC method coupled to mass spectrometry was crucial in providing both broad metabolome coverage and excellent experimental precision (

It has long been a central tenet of biomedical research that coating of nanoparticles with hydrated polymers can improve their performance in biomedical applications. However, the efficacy of the approach in vivo is still debated. In vitro model systems to test the performance of engineered nanoparticles for in vivo applications often use nonrepresentative cell lines and conditions for uptake and toxicity tests. We use our platform of monodisperse iron oxide nanoparticles densely grafted with nitrodopamide-poly(ethylene glycol) (PEG) to probe cell interactions with a set of cell types and culture conditions that are relevant for applications in which nanoparticles are injected into the bloodstream. In the past, these particles have proved to have excellent stability and negligible interaction with proteins and membranes under physiological conditions. We test the influence of flow on the uptake of nanoparticles. We also investigate the transport through endothelial barrier cell layers, as well as the effect that PEG-grafted iron oxide nanoparticles have on cell layers relevant for nanoparticles injected into the bloodstream. Our results show that the dense PEG brush and resulting lack of nonspecific protein and membrane interaction lead to negligible cell uptake, toxicity, and transport across barrier layers. These results contrast with far less well-defined polymer-coated nanoparticles that tend to aggregate and consequently strongly interact with cells, for example, by endocytosis.

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) are a promising in vitro tool for drug development and disease modeling, but their immature electrophysiology limits diagnostic utility. Tissue engineering approaches involving aligned 3D cultures enhance hiPSC-CM structural maturation but are insufficient to induce mature electrophysiology. We hypothesized that mimicking post-natal switching of the heart’s primary ATP source from glycolysis to fatty acid oxidation could enhance electrophysiological maturation of hiPSC-CM. We combined hiPSC-CM with microfabricated culture chambers to form 3D cardiac microphysiological systems (MPS) that enhanced immediate microtissue alignment and tissue specific extracellular matrix (ECM) production. Using Robust Experimental design, we identified a maturation media that improved calcium handling in MPS derived from two genetically distinct hiPSC sources. Although calcium handling and metabolic maturation were improved in both genotypes, there was a divergent effect on action potential duration (APD): MPS that started with abnormally prolonged APD exhibited shorter APD in response to maturation media, whereas the same media prolonged the APD in MPS that started with aberrantly short APD. Importantly, the APD of both genotypes was brought near the range of 270-300ms observed in human left ventricular cardiomyocytes. Mathematical modeling explained these divergent phenotypes, and further predicted the response of matured MPS to drugs with known pro-arrhythmic effects. These results suggest that systematic combination of biophysical stimuli and metabolic cues can enhance the electrophysiological maturation of hiPSC-derived cardiomyocytes. However, they also reveal that maturation-inducing cues can have differential effects on electrophysiology depending on the baseline phenotype of hiPSC-CM. In silico models provide a valuable tool for predicting how changes in cellular maturation will manifest in drug responsiveness.

Background Co-cultures of endothelial cells with mesenchymal stem cells currently represent one of the most promising approaches in providing oxygen and nutrient supply for microvascular tissue engineering. Still, to translate this model into clinics several in vitro parameters including growth medium and scaffold degradation need to be fine-tuned. Methods We recently described the co-culture of adipose-derived stem cells with endothelial cells in fibrin, resulting in capillary formation in vitro as well as their perfusion in vivo. Here, we aimed to further characterise microvascular tube formation in fibrin by determining the role of scaffold degradation, thrombin concentration and culture conditions on vascularisation. Results We observed that inhibition of cell-mediated fibrin degradation by the commonly used inhibitor aprotinin resulted in impaired vascular network formation. Aprotinin had no effect on laminin and collagen type IV deposition or formation of tube-like structures in scaffold-free co-culture, indicating that poor vascularisation of fibrin clots is primarily caused by inhibition of plasminogen-driven fibrinolysis. Co-culture in plasminogen- and factor XIII-depleted fibrin did not result in different vascular network density compared to controls. Furthermore, we demonstrate that thrombin negatively affects vascular network density at high concentrations. However, only transient activation of incorporated endothelial cells by thrombin could be observed, thus excluding a long-term inflammatory response in tissue-engineered micro-capillaries. Finally, we show that vascularisation of fibrin scaffolds in basal medium is undermined because of increased fibrinolytic activity leading to scaffold destabilisation without aprotinin. Conclusions Taken together, our data reveal a critical role of fibrinolysis inhibition in in vitro cell-mediated vascularisation of fibrin scaffolds. Electronic supplementary material The online version of this article (10.1186/s13287-017-0764-2) contains supplementary material, which is available to authorized users.

To advance existing in vitro cell impedance spectroscopy and improve reliability of impedimetric sensors for cell analysis, we have electrically insulated interdigitated microelectrodes using the high-k biomaterial zirconium dioxide (ZrO2). We report the smallest passivation thickness to electrode distance ratio of 10−3 using atomic layer deposition resulting in electrically insulated metal oxide films of 15 nm thickness. For the first time the influence of the insulation on sensor performance is experimentally and theoretically analyzed using numerical simulations. In addition an equivalent electrical circuit model was established and validated using non-linear least square fitting. Results of the computational simulations revealed improved electrical current distribution across the electrically insulated interdigitated electrode structures in comparison to open (not passivated) electrodes. Furthermore, we found linear decrease of current density in z-direction within 5 μm distance from the sensor surface in the presence of ZrO2 nanocoatings is ideally suited to assess confluent cell layers. Final practical application of the ZrO2 nanolayer passivated impedimetric sensors is demonstrated for nanotoxicological investigations, where sensitivity and repeatability are crucial parameters for cell analysis. Results of our study show that the reproducible and standardizable deposition of a uniform metal oxide nanocoating improves current density distributions, has no performance drawbacks compared to open sensors and enables sensitive detection of protein-coating effects on cytotoxic silica nanoparticles. The presented novel sensor design allows for the integration of alternative electrode materials such as aluminum enabling cost-effective fabrication of large-volume sensor arrays.

Mesenchymal stem cells (MSC) exhibit a high self-renewal capacity, multilineage differentiation potential and immunomodulatory properties. This set of exceptional features makes them an attractive tool for research and clinical application. However, MSC are far from being a uniform cell type, which makes standardization difficult. The exact properties of human MSC (hMSC) can vary greatly depending on multiple parameters including tissue source, isolation method and medium composition. In this review we address the most important influence factors. We highlight variations in the differentiation potential of MSC from different tissue sources. Furthermore, we compare enzymatic isolation strategies with explants cultures focusing on adipose tissue and umbilical cords as two relevant examples. Additionally, we address effects of medium composition and serum supplementation on MSC expansion and differentiation. The lack of standardized methods for hMSC isolation and cultivation mandates careful evaluation of different protocols regarding efficiency and cell quality. MSC characterization based on a set of minimal criteria defined by the International Society for Cellular Therapy is a widely accepted practice, and additional testing for MSC functionality can provide valuable supplementary information. The MSC secretome has been identified as an important signaling mechanism to affect other cells. In this context, extracellular vesicles (EVs) are attracting increasing interest. The thorough characterization of MSC-derived EVs and their interaction with target cells is a crucial step toward a more complete understanding of MSC-derived EV functionality. Here, we focus on flow cytometric approaches to characterize free as well as cell bound EVs and address potential differences in the bioactivity of EVs derived from stem cells from different sources. © 2017 International Society for Advancement of Cytometry.

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.

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.

Intramyocardial hydrogel injection is an innovative and promising treatment for myocardial infarction (MI) and has recently entered clinical trials. By providing mechanical support to the ventricular wall, hydrogel injectate may act to preserve cardiac function and slow the remodeling process that leads to heart failure. However, improved outcomes will likely depend on the use of hydrogels specifically designed for this unique application, and better understanding of the mechanisms affected by the intervention. In this work, we present the first large animal study achieving functional and geometrical improvements in treating MI using a relatively stiff, fully synthetic hydrogel designed for intramyocardial injection. In addition, the renin-angiotensin system coincided with the mechanical effects of hydrogel injection and attenuated left ventricular remodeling, even after significant hydrogel degradation had occurred in vivo. These results may inspire further optimization of hydrogel materials used in intramyocardial hydrogel injection therapy and a better description of physiologic pathways affected by its implementation to facilitate successful clinical translation.

Nanotechnology provides the tools to develop novel biosensors with improved performance, including sensitivity and response time that can be readily integrated into diagnostic devices. We have developed a miniaturized cell analysis platform to advance microfluidic cell cultures by combining two complementary, label-free and non-invasive cell analysis methods for the long-term monitoring of dynamic cell behavior. The novel dual-parameter cell-on-a-chip detects light scattering from adherent cells to provide information on cell numbers and intracellular granularity, while simultaneously performing impedance spectroscopy to monitor cell adhesion and cell-cell interaction. In the present work we have integrated spray-coated organic photodiode arrays with a lab-on-a-chip containing embedded interdigitated electrode structures to improve assay reproducibility, reliability and accuracy. We successfully demonstrate that the complementary cell chip technology can accurately detect cell numbers, clarify misleading results during cell-substance interaction assays, as well as the cytotoxicity screening of drug substances. The ability to precisely determine cell numbers within minutes constitutes a major step towards standardization.

In this chapter the state of the art of live cell microarrays for high-throughput biological assays are reviewed. The fabrication of novel microarrays with respect to material science and cell patterning methods is included. A main focus of the chapter is on various aspects of the application of cell microarrays by providing selected examples in research fields such as biomaterials, stem cell biology and neuroscience. Additionally, the importance of microfluidic technologies for high-throughput on-chip live-cell microarrays is highlighted for single-cell and multi-cell assays as well as for 3D tissue constructs.

The uptake of large particles by cells (phagocytosis) is an important factor in cell biology and also plays a major role in biomedical applications. So far, most methods for determining the phagocytic properties rely on cell-culture incubation and end-point detection schemes. Here, we present a lab-on-a-chip system for real-time monitoring of magnetic particle uptake by human fibroblast (NHDF) cells. It is based on recording the time evolution of the average position and distribution of magnetic particles during phagocytosis by giant-magnetoresistive (GMR) type sensors. We employ particles with a mean diameter of 1.2 mu m and characterize their phagocytosis-relevant properties. Our experiments at physiological conditions reveal a cellular uptake rate of 45 particles per hour and show that phagocytosis reaches saturation after an average uptake time of 27.7 h. Moreover, reference phagocytosis experiments at 4 degrees C are carried out to mimic environmental or disease related inhibition of the phagocytic behavior, and our measurements clearly show that we are able to distinguish between cell-membrane adherent and phagocytosed magnetic particles. Besides the demonstrated real-time monitoring of phagocytosis mechanisms, additional nano-biointerface studies can be realized, including on-chip cell adhesion/spreading as well as cell migration, attachment and detachment dynamics. This versatility shows the potential of our approach for providing a multifunctional platform for on-chip cell analysis. (C) 2012 Elsevier B.V. All rights reserved.

Luminescent sensors with improved performance for monitoring dissolved oxygen in microfluidic devices were developed. Our brightness enhanced sensing films with reduced film thickness showed higher signals than standard sensors and responded in real-time. Sensor layers were integrated into microfluidic chips and readout off-chip on a fluorescence microscope with lifetime imaging or ratiometric imaging using the color channels of a CCD camera. Cell respirometry of model culture systems was monitored with this sensor setup.

In this chapter the state of the art of live cell microarrays for high-throughput biological assays are reviewed. The fabrication of novel microarrays with respect to material science and cell patterning methods is included. A main focus of the chapter is on various aspects of the application of cell microarrays by providing selected examples in research fields such as biomaterials, stem cell biology and neuroscience. Additionally, the importance of microfluidic technologies for high-throughput on-chip live-cell microarrays is highlighted for single-cell and multi-cell assays as well as for 3D tissue constructs.

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.

Today highly complex 3D cell culture formats that closely mimic the in vivo situation are increasingly available. Despite their wide use, the development of analytical methods and tools that can work within the depth of 3D-tissue constructs lags behind. In order to get the most information from a 3D cell sample, adequate and reliable assays are required. However, the majority of tools and methods used today have been originally designed for 2D cell cultures and translation to a 3D environment is in general not trivial. Ideally, an analytical method should be non-invasive and allow for repeated observation of living cells in order to detect dynamic changes in individual cells within the 3D cell culture. Although well-established laser confocal microscopy can be used for these purposes, this technique has serious limitations including penetration depth and availability. Focusing on two relevant analytical methods for live-cell monitoring, we discuss the current challenges of analyzing living 3D samples: microscopy, which is the most widely used technology to observe and examine cell cultures, has been successfully adapted for 3D samples by recording of so-called “z-stacks”. However the required equipment is generally very expensive and therefore access is often limited. Consequently alternative and less advanced approaches are often applied that cannot capture the full structural complexity of a 3D sample. Similarly, image analysis tools for quantification of microscopic images range from highly specialized and costly to simplified and inexpensive. Depending on the actual sample composition and scientific question the best approach needs to be assessed individually. Another more recently introduced technology for non-invasive cell analysis is Raman micro-spectroscopy. It enables label-free identification of cellular metabolic changes with high sensitivity and has already been successful applied to 2D and 3D cell cultures. However, its future significance for cell analysis will strongly depend on the availability of application oriented and user-friendly systems including specific tools for easy analysis and interpretation of spectral data focusing on biological relevant information.

We have developed a lab-on-a-chip system for continuous and non-invasive monitoring of microfluidic cell cultures using integrated high-frequency contactless impedance spectroscopy. Electrically insulated microfabricated interdigitated electrode structures were embedded into four individually addressable microchambers to reliably and reproducibly detect cell-substrate interactions, cell viability and metabolic activity. While silicon nitride passivated sensor substrates provided a homogeneous cell culture surface that minimized cell orientation along interdigitated electrode structures, the application of high-frequency AC fields reduced the impact of the 300 nm thick passivation layer on sensor sensitivity. The additional implementation of multivariate data analysis methods such as partial least square (PLS) for high-frequency impedance spectra provided unambiguous information on intracellular pathway activation, up and down-regulation of protein synthesis as well as global cellular stress responses. A comparative cell analysis using connective tissue fibroblasts showed that high-frequency contactless impedance spectroscopy and time-resolved quantification of IL-6 secretion using ELISA provided similar results following stimulation with circulating pro-inflammatory cytokines IL-1β and TNFα. The combination of microfluidics with contactless impedance sensing and time-resolved quantification of stress factor release will provide biologist with a new tool to (a) establish a variety of uniform cell culture surfaces that feature complex biochemistries, micro- and nanopatterns; and (b) to simultaneously characterize cell responses under physiologically relevant conditions using a complementary non-invasive cell analysis method.

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.

The recent advances on a monolithically integrated sensor platform based on ring-shaped organic photo detectors are presented. Various sensing chemistries based on luminescence for the detection of a number of parameters such as oxygen, carbon dioxide, humidity and pH in gaseous and/or liquid phase were investigated and optimized to the requirements of the sensor platform. Aiming on practical application, the need and methods to reference luminescence signals are evaluated including two wavelength rationing and lifetime measurements. Finally, we will discuss potential applications of the platform and present a micro-fluidic chip containing an array of integrated sensor spots and organic photodiodes.

Double layer interactions between charged particles and surfaces play a vital role in a variety of technical and biological systems because they determine the stability of, e.g., protein-membrane biointerfaces. The underlying theoretical principle is based on the overlap of two different double layers that induce surface charges to be shifted to a new equilibrium distribution, which can be approximated by the Poisson-Boltzmann equation. In the present work we show theoretical and experimental results involving double layer capacitance of surfaces that exhibit charge regulation behavior. Charge regulation is an important parameter to consider when investigating protein-membrane interactions because it defines surface properties between ideal constant charge and constant potential behavior. In this work we introduce a novel theoretical model that also includes charge regulation behavior and can assess changes of double layer disruptions at TiO(2) and supported lipid-bilayers (SLB). The selected surfaces represent important biointerfaces that can be found on implants or cell membranes. We also demonstrate that contactless impedance spectroscopy is well suited to measure double layer capacitance interactions using differently charged silica beads. The combination of a theoretical model with experimental data allowed us further to identify charge regulation effects during protein adsorption (BSA and Annexin V) events at supported lipid-bilayers (SLB) used as a simple cell membrane model. Finally, the first indications of changed charge regulation behavior during protein surface crystallization events were also documented.

As nanotechnology moves towards widespread commercialization, new technologies are needed to adequately address the potential health impact of nanoparticles (NPs). Assessing the safety of over 30,000 NPs through animal testing would not only be expensive, but it would also raise a number of ethical considerations. Furthermore, existing in vitro cell-based assays are not sufficient in scope to adequately address the complexity of cell-nanoparticle interactions including NP translocation, accumulation and co-transport of e.g. allergens. In particular, classical optical/fluorescent endpoint detection methods are known to provide irreproducible, inaccurate and unreliable results since these labels can directly react with the highly catalytic surfaces of NP. To bridge this technological gap we have developed a lab-on-a-chip capable of continuously and non-invasively monitoring the collagen production of primary human fibroblast cells (NHDF) using contactless dielectric microsensors. Human dermal fibroblast cells are responsible for the maintenance of soft tissue integrity, are found throughout the human body and their primary function is collagen expression. We show that cellular collagen production can be readily detected and used to assess cellular stress responses to a variety of external stimuli, including exposure to nanoparticles. Results of the study showed a 20% and 95% reduction of collagen production following 4 hour exposure to 10 μg mL(-1) gold and silver nanoparticles (dia.10 nm), respectively. Furthermore a prolonged perfusion of sub-toxic concentrations (0.1 μg mL(-1)) of silver NP reduced NHDF collagen production by 40% after 10 h indicating increased NP take up and accumulation. We demonstrate that the application of microfluidics for the tailored administration of different NP treatments constitutes a powerful new tool to study cell-nanoparticle interactions and nanoparticle accumulation effects in small cell populations.

We have developed a μ-fluidic biochip capable of monitoring in real-time cellular phenotype dynamics by non-invasively assessing cell viability and reproduction over long periods of time. The μ-fluidic biochip contains integrated organic photodiodes that continuously perform light scattering measurements of adherent cell populations. In the presented work, we experimentally show that light scattering measurements are an effective label-free method for accurately assessing cell number variations. Furthermore, results obtained from 3D FDTD simulations indicate that side scatter characteristics of cells allow in principle predictions on cellular health status.

Background The synovium is primarily built by fibroblast-like synoviocytes (FLS) that are connected to one another via cellular processes, thus forming a continuous network of cells. Its multicellularity requires precise coordination between cells to bring forth specialised tissue functions critical to joint homeostasis. Cell-to-cell transfer of cytoplasmic cargo may provide a means for intercellular communication, thereby facilitating the concerted behaviour of FLS. Using a 3D in-vitro model of the synovium, we analysed FLS capacity for exchange of cytoplasmic content. Methods Human FLS were prepared from synovial tissues obtained as discarded specimens following joint arthroplasty. Cells were cultured in spherical matrigel micromasses with an average size of 2 mm O. For confocal live cell imaging and transmission electron microscopy, FLS were loaded with fluorescent non-degradable microspheres and labelled with fluorescent membrane dyes. Analysis of the resulting 4D movies was done with Imaris® software. Results To examine intercellular cytoplasmatic transfer, we labelled 50% of FLS with red cell tracker dye and loaded the other 50% with green microspheres. In a time series (8 days), we found that microspheres do indeed appear in red labelled cells. First evidence was found on Day 1 and over the course of the following days microspheres accumulated in red labelled cells with a transfer rate of 10% of newly affected cells/day. A similar experiment in 2D demonstrated microsphere movement within interconnecting membrane nanotubes. Transfer rates for microspheres into red cells were identical. Transmission Electron Microscopy (TEM) identified extracellular vesicles (exosomes) in discrete regions of intimate cell-to-cell contact but also open interconnecting tubes, suggesting distinct modes for transfer. Conclusions These studies reveal transfer of cytoplasmic cargo between FLS and may provide insight into how the synovial tissue operates. Further studies will demonstrate the significance of directed cargo exchange for cellular cooperation and the function of the normal as well as the diseased synovium.

In this work we present a high resolution oxygen imaging approach, which can be used to study 2D oxygen distribution inside microfluidic environments. The presented setup comprises a fabrication process of microfluidic chips with integrated luminescent sensing films combined with referenced oxygen imaging applying a color CCD-camera. Enhancement of the sensor performance was achieved by applying the principle of light harvesting. This principle enabled ratiometric imaging employing the red and the green channel of a color CCD-camera. The oxygen sensitive emission of platinum(ii)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorphenyl)-porphyrin (PtTFPP) was detected by the red channel, while the emission of a reference dye was detected by the green channel. This measurement setup allowed for accurate real-time 2D oxygen imaging with superior quality compared to intensity imaging. The sensor films were subsequently used to measure the respiratory activity of human cell cultures (HeLa carcinoma cells and normal human dermal fibroblasts) in a microfluidic system. The sensor setup is well suited for different applications from spatially and temporally resolving oxygen concentration inside microfluidic channels to parallelization of oxygen measurements and paves the way to novel cell based assays, e.g. in tissue engineering, tumor biology and hypoxia reperfusion phenomena.