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- ItemIntegrating Biophysics in Toxicology(Basel : MDPI, 2020) Del Favero, G.; Kraegeloh, A.Integration of biophysical stimulation in test systems is established in diverse branches of biomedical sciences including toxicology. This is largely motivated by the need to create novel experimental setups capable of reproducing more closely in vivo physiological conditions. Indeed, we face the need to increase predictive power and experimental output, albeit reducing the use of animals in toxicity testing. In vivo, mechanical stimulation is essential for cellular homeostasis. In vitro, diverse strategies can be used to model this crucial component. The compliance of the extracellular matrix can be tuned by modifying the stiffness or through the deformation of substrates hosting the cells via static or dynamic strain. Moreover, cells can be cultivated under shear stress deriving from the movement of the extracellular fluids. In turn, introduction of physical cues in the cell culture environment modulates differentiation, functional properties, and metabolic competence, thus influencing cellular capability to cope with toxic insults. This review summarizes the state of the art of integration of biophysical stimuli in model systems for toxicity testing, discusses future challenges, and provides perspectives for the further advancement of in vitro cytotoxicity studies.
- ItemTargeted delivery of functionalized PLGA nanoparticles to macrophages by complexation with the yeast Saccharomyces cerevisiae(Chichester : John Wiley and Sons Ltd, 2020) Kiefer, R.; Jurisic, M.; Dahlem, C.; Koch, M.; Schmitt, M.J.; Kiemer, A.K.; Schneider, M.; Breinig, F.Nanoparticles (NPs) are able to deliver a variety of substances into eukaryotic cells. However, their usage is often hampered by a lack of specificity, leading to the undesired uptake of NPs by virtually all cell types. In contrast to this, yeast is known to be specifically taken up into immune cells after entering the body. Therefore, we investigated the interaction of biodegradable surface-modified poly(lactic-co-glycolic acid) (PLGA) particles with yeast cells to overcome the unspecificity of the particulate carriers. Cells of different Saccharomyces cerevisiae strains were characterized regarding their interaction with PLGA-NPs under isotonic and hypotonic conditions. The particles were shown to efficiently interact with yeast cells leading to stable NP/yeast-complexes allowing to associate or even internalize compounds. Notably, applying those complexes to a coculture model of HeLa cells and macrophages, the macrophages were specifically targeted. This novel nano-in-micro carrier system suggests itself as a promising tool for the delivery of biologically active agents into phagocytic cells combining specificity and efficiency.
- ItemGelation kinetics of thiol-methylsulfone (MS) hydrogel formulations for 3D cell culture(Washington, D.C. : American Chemical Society, 2022) de Miguel-Jiménez, Adrián; Ebeling, Bastian; Paez, Julieta I.; Fink-Straube, Claudia; Pearson, Samuel; del Campo, AranzazuCrosslinking chemistries that allow hydrogel formation within minutes are essential to achieve homogeneous networks and cell distributions in 3D cell culture. Thiol-methylsulfone (MS) crosslinking chemistry offers minutes-scale gelation under near-physiological conditions showing many desirable attributes for 3D cell encapsulation. Here we investigate the gelation kinetics and mechanical properties of PEG-based hydrogels formed by thiol-tetrazole methylsulfone (TzMS) crosslinking as a function of buffer, crosslinker structure, and degree of TzMS functionalization. Appropriate buffer selection ensured constant pH throughout crosslinking. The formulation containing cell adhesive ligand RGD and enzymatically-degradable peptide VPM gelled in ca. 4 min at pH 7.5, and stiffness could be increased from hundreds of Pascals to > 1 kPa by using excess VPM. The gelation times and stiffnesses for these hydrogels are highly suitable for 3D cell encapsulations, and pave the way for reliable 3D cell culture workflows in pipetting robots.
- ItemScanning electron microscopy preparation of the cellular actin cortex: A quantitative comparison between critical point drying and hexamethyldisilazane drying(San Francisco, California, US : PLOS, 2021) Schu, Moritz; Terriac, Emmanuel; Koch, Marcus; Paschke, Stephan; Lautenschläger, Franziska; Flormann, Daniel A.D.The cellular cortex is an approximately 200-nm-thick actin network that lies just beneath the cell membrane. It is responsible for the mechanical properties of cells, and as such, it is involved in many cellular processes, including cell migration and cellular interactions with the environment. To develop a clear view of this dense structure, high-resolution imaging is essential. As one such technique, electron microscopy, involves complex sample preparation procedures. The final drying of these samples has significant influence on potential artifacts, like cell shrinkage and the formation of artifactual holes in the actin cortex. In this study, we compared the three most used final sample drying procedures: critical-point drying (CPD), CPD with lens tissue (CPD-LT), and hexamethyldisilazane drying. We show that both hexamethyldisilazane and CPD-LT lead to fewer artifactual mesh holes within the actin cortex than CPD. Moreover, CPD-LT leads to significant reduction in cell height compared to hexamethyldisilazane and CPD. We conclude that the final drying procedure should be chosen according to the reduction in cell height, and so CPD-LT, or according to the spatial separation of the single layers of the actin cortex, and so hexamethyldisilazane.
- ItemExogenous supply of Hsp47 triggers fibrillar collagen deposition in skin cell cultures in vitro(London : BioMed Central, 2020) Khan, E.S.; Sankaran, S.; Llontop, L.; Del Campo, A.Background: Collagen is a structural protein that provides mechanical stability and defined architectures to skin. In collagen-based skin disorders this stability is lost, either due to mutations in collagens or in the chaperones involved in collagen assembly. This leads to chronic wounds, skin fragility, and blistering. Existing approaches to treat such conditions rely on administration of small molecules to simulate collagen production, like 4-phenylbutyrate (4-PBA) or growth factors like TGF-β. However, these molecules are not specific for collagen synthesis, and result in unsolicited side effects. Hsp47 is a collagen-specific chaperone with a major role in collagen biosynthesis. Expression levels of Hsp47 correlate with collagen deposition. This article explores the stimulation of collagen deposition by exogenously supplied Hsp47 (collagen specific chaperone) to skin cells, including specific collagen subtypes quantification. Results: Here we quantify the collagen deposition level and the types of deposited collagens after Hsp47 stimulation in different in vitro cultures of cells from human skin tissue (fibroblasts NHDF, keratinocytes HaCat and endothelial cells HDMEC) and mouse fibroblasts (L929 and MEF). We find upregulated deposition of fibrillar collagen subtypes I, III and V after Hsp47 delivery. Network collagen IV deposition was enhanced in HaCat and HDMECs, while fibril-associated collagen XII was not affected by the increased intracellular Hsp47 levels. The deposition levels of fibrillar collagen were cell-dependent i.e. Hsp47-stimulated fibroblasts deposited significantly higher amount of fibrillar collagen than Hsp47-stimulated HaCat and HDMECs. Conclusions: A 3-fold enhancement of collagen deposition was observed in fibroblasts upon repeated dosage of Hsp47 within the first 6 days of culture. Our results provide fundamental understanding towards the idea of using Hsp47 as therapeutic protein to treat collagen disorders.
- ItemSynthesis of biphasic Al/Al2O3 nanostructures under microgravity and laser structuring on Al/Al2O3 surfaces for selective cell guidance(Saarbrücken : Universität des Saarlandes, 2013) Lee, JuseokThe first part of this thesis is dealing with gravity effect on the synthesis of biphasic core/shell Al/Al2O3 composites. By chemical vapor deposition of the precursor [tBuOAlH2]2 at 400°C, only spherical nanoparticles were observed on the substrate surface. The formation of nanowires was observed at 600°C. It is a good agreement with our previous results on earth condition and there is no gravity impact on the chemical reaction. At increased gravity levels, the nanoparticles formed large clusters and the nanowires showed bundle formation while the nanowires at microgravity have predominantly linear structures. It is proposed that the chaotic nature of nanowires and cluster formation of nanoparticles were caused by a dominance of gravity over the thermal creep. In the second part the use of Al/Al2O3 nanowire layers for bio applications is considered. Contact cell guidance and alignment were studied to understand how cells recognize and respond to certain surface patterns. Linear micro channels were created on Al/Al2O3 layer by direct laser writing and laser interference patterning. Although surface topography was altered, the surface chemistry was always identical (Al2O3) due to the unique core/shell nature of Al/Al2O3 nanowires. Human osteoblast, normal human dermal fibroblast and neuronal cells were cultured and investigated. The results indicate that different cell types show diverse responses to the topography independent from the surface chemistry of the material.