TY - JOUR A1 - Gehre, Christian A1 - Flechner, Marie A1 - Kammerer, Sarah A1 - Küpper, Jan-Heiner A1 - Coleman, Charles Dominic A1 - Püschel, Gerhard Paul A1 - Uhlig, Katja A1 - Duschl, Claus T1 - Real time monitoring of oxygen uptake of hepatocytes in a microreactor using optical microsensors JF - Scientific reports N2 - Most in vitro test systems for the assessment of toxicity are based on endpoint measurements and cannot contribute much to the establishment of mechanistic models, which are crucially important for further progress in this field. Hence, in recent years, much effort has been put into the development of methods that generate kinetic data. Real time measurements of the metabolic activity of cells based on the use of oxygen sensitive microsensor beads have been shown to provide access to the mode of action of compounds in hepatocytes. However, for fully exploiting this approach a detailed knowledge of the microenvironment of the cells is required. In this work, we investigate the cellular behaviour of three types of hepatocytes, HepG2 cells, HepG2-3A4 cells and primary mouse hepatocytes, towards their exposure to acetaminophen when the availability of oxygen for the cell is systematically varied. We show that the relative emergence of two modes of action, one NAPQI dependent and the other one transient and NAPQI independent, scale with expression level of CYP3A4. The transient cellular response associated to mitochondrial respiration is used to characterise the influence of the initial oxygen concentration in the wells before exposure to acetaminophen on the cell behaviour. A simple model is presented to describe the behaviour of the cells in this scenario. It demonstrates the level of control over the role of oxygen supply in these experiments. This is crucial for establishing this approach into a reliable and powerful method for the assessment of toxicity. Y1 - 2020 U6 - https://doi.org/10.1038/s41598-020-70785-6 SN - 2045-2322 VL - 10 IS - 1 PB - Macmillan Publishers Limited, part of Springer Nature CY - [London] ER - TY - JOUR A1 - Velk, Natalia A1 - Uhlig, Katja A1 - Vikulina, Anna A1 - Duschl, Claus A1 - Volodkin, Dmitry T1 - Mobility of lysozyme in poly(L-lysine)/hyaluronic acid multilayer films JF - Colloids and surfaces : an international journal devoted to fundamental and applied research on colloid and interfacial phenomena in relation to systems of biological origin ; B, Biointerfaces N2 - The spatial and temporal control over presentation of protein-based biomolecules such as growth factors and hormones is crucial for in vitro applications to mimic the complex in vivo environment. We investigated the interaction of a model protein lysozyme (Lys) with poly(L-lysine)/hyaluronic acid (PLL/HA) multilayer films. We focused on Lys diffusion as well as adsorption and retention within the film as a function of the film deposition conditions and post-treatment. Additionally, an effect of Lys concentration on its mobility was probed. A combination of confocal fluorescence microscopy, fluorescence recovery after photobleaching, and microfluidics was employed for this investigation. Our main finding is that adsorption of PLL and HA after protein loading induces acceleration and reduction of Lys mobility, respectively. These results suggest that a charge balance in the film to a high extent governs the protein-film interaction. We believe that control over protein mobility is a key to reach the full potential of the PLL/HA films as reservoirs for biomolecules depending on the application demand. (C) 2016 The Authors. Published by Elsevier B.V. KW - Layer-by-layer KW - Protein KW - Diffusion KW - Release KW - FRAP Y1 - 2016 U6 - https://doi.org/10.1016/j.colsurfb.2016.07.055 SN - 0927-7765 SN - 1873-4367 VL - 147 SP - 343 EP - 350 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Uhlig, Katja A1 - Madaboosi, Narayanan A1 - Schmidt, Stephan A1 - Jäger, Magnus S. A1 - Rose, Jürgen A1 - Duschl, Claus A1 - Volodkin, Dmitry V. T1 - 3d localization and diffusion of proteins in polyelectrolyte multilayers JF - Soft matter N2 - The interaction of diverse biomaterials with surfaces is more crucial than ever for biomedical applications to ensure efficiency and reproducibility. Very interesting surface materials are micrometer-thick polyelectrolyte multilayers. Not only their surface but also the bulk can be loaded with biomaterials like proteins or DNA for various purposes. Therefore, we established a method to analyze the lateral and vertical distribution of fluorescently labelled proteins of various size and charge in polyelectrolyte films composed of poly(L-lysine) and hyaluronic acid by confocal laser scanning microscopy. This approach enables us to measure the diffusion coefficients of the proteins via fluorescence recovery after photobleaching as a function of their vertical position in the film and facilitates the understanding of molecular interactions in the film with a high resolution in both space and time. As a result, we confirm that protein loading in the film is driven by electrostatic interactions - uncharged dextran molecules of 10 and 500 kDa do not diffuse into the film. Proteins of different sizes (3-11 nm) can diffuse relatively fast (D = 2-4 mm(2) s(-1)) independent of their net charge, indicating complex interpolymer interactions. This approach is a new powerful experimental tool to design the polyelectrolyte multilayers for bio-applications by finding a relationship between intermolecular interactions and mobility and availability of biomolecules to biological samples (e.g. cells) or detection units (e.g. biosensors). Y1 - 2012 U6 - https://doi.org/10.1039/c2sm26500a SN - 1744-683X VL - 8 IS - 47 SP - 11786 EP - 11789 PB - Royal Society of Chemistry CY - Cambridge ER - TY - JOUR A1 - Wischerhoff, Erik A1 - Glatzel, Stefan A1 - Uhlig, Katja A1 - Lankenau, Andreas A1 - Lutz, Jean-Francois A1 - Laschewsky, André T1 - Tuning the thickness of polymer brushes grafted from nonlinearly growing multilayer assemblies N2 - A new versatile method for tuning the thickness of surface-tethered polymer brushes is introduced. It is based on the combination of polyelectrolyte multilayer deposition and surface-initiated atom transfer radical polymerization. To control the thickness of the brushes, the nonlinear growth of certain polyelectrolyte multilayer systems is exploited. The method is demonstrated to work with different polyelectrolytes and different monomers. The relevance for applications is demonstrated by cell adhesion experiments oil grafted thermoresponsive polymer layers with varying thickness. Y1 - 2009 UR - http://pubs.acs.org/journal/langd5 U6 - https://doi.org/10.1021/La804197j SN - 0743-7463 ER - TY - JOUR A1 - Uhlig, Katja A1 - Wischerhoff, Erik A1 - Lutz, Jean-Francois A1 - Laschewsky, André A1 - Jäger, Magnus S. A1 - Lankenau, Andreas A1 - Duschl, Claus T1 - Monitoring cell detachment on PEG-based thermoresponsive surfaces using TIRF microscopy N2 - Recently, we introduced a thermoresponsive copolymer that consists of oligo(ethylene glycol) methacrylate (OEGMA) and 2-(2- methoxyethoxy) ethyl methacrylate (MEO(2)MA). The polymer exhibited an LCST at 35 degrees C in PBS buffer and was anchored onto gold substrates using disulfide polymerisation initiators. It allows the noninvasive detachment of adherent cells from their substrate. As the mechanisms that determine the interaction of cells with such polymers are not well understood, we employed Total Internal Reflection Fluorescence (TIRF) microscopy in order to monitor the detachment process of cells of two different types. We identified contact area and average cell-substrate distance as crucial parameters for the evaluation of the detachment process. The sensitivity of TIRF microscopy allowed us to correlate the specific adhesion pattern of MCF-7 breast cancer cells with the morphology of cell deposits that may serve as fingerprints for a nondestructive characterisation of live cells. Y1 - 2010 UR - http://www.rsc.org/Publishing/Journals/sm/index.asp U6 - https://doi.org/10.1039/C0sm00010h SN - 1744-683X ER -