Verena Charwat

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.

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.

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.

Isolation, cultivation, and characterization of human mesenchymal stem cells

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.

Cell Microarrays for Biomedical Applications.

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

BackgroundCo-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.MethodsWe 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.ResultsWe 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.ConclusionsTaken together, our data reveal a critical role of fibrinolysis inhibition in in vitro cell-mediated vascularisation of fibrin scaffolds.

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.

Uncertainty budgeting in fold change determination and implications for non-targeted metabolomics studies in model systems

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 (<8% CV for peak areas, on average 0.5% CV for retention times) that was required for sensitive differential analysis as low as FC 1.1.

Characterization of double layer alterations induced by charged particles and protein-membrane interactions using contactless impedance spectroscopy.

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.