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- ItemGlycerylphytate as an ionic crosslinker for 3D printing of multi-layered scaffolds with improved shape fidelity and biological features(London : Royal Society of Chemistry, 2020) Mora-Boza, A.; Włodarczyk-Biegun, M.K.; Del Campo, A.; Vázquez-Lasa, B.; Román, J.S.The fabrication of intricate and long-term stable 3D polymeric scaffolds by a 3D printing technique is still a challenge. In the biomedical field, hydrogel materials are very frequently used because of their excellent biocompatibility and biodegradability, however the improvement of their processability and mechanical properties is still required. This paper reports the fabrication of dual crosslinked 3D scaffolds using a low concentrated (<10 wt%) ink of gelatin methacryloyl (GelMA)/chitosan and a novel crosslinking agent, glycerylphytate (G1Phy) to overcome the current limitations in the 3D printing field using hydrogels. The applied methodology consisted of a first ultraviolet light (UV) photopolymerization followed by a post-printing ionic crosslinking treatment with G1Phy. This crosslinker provides a robust framework and avoids the necessity of neutralization with strong bases. The blend ink showed shear-thinning behavior and excellent printability in the form of a straight and homogeneous filament. UV curing was undertaken simultaneously to 3D deposition, which enhanced precision and shape fidelity (resolution ≈150 μm), and prevented the collapse of the subsequent printed layers (up to 28 layers). In the second step, the novel G1Phy ionic crosslinker agent provided swelling and long term stability properties to the 3D scaffolds. The multi-layered printed scaffolds were mechanically stable under physiological conditions for at least one month. Preliminary in vitro assays using L929 fibroblasts showed very promising results in terms of adhesion, spreading, and proliferation in comparison to other phosphate-based traditional crosslinkers (i.e. TPP). We envision that the proposed combination of the blend ink and 3D printing approach can have widespread applications in the regeneration of soft tissues.
- ItemCharacterization of immune cell migration using microfabrication(Berlin ; Heidelberg : Springer, 2021) Vesperini, Doriane; Montalvo, Galia; Qu, Bin; Lautenschläger, FranziskaThe immune system provides our defense against pathogens and aberrant cells, including tumorigenic and infected cells. Motility is one of the fundamental characteristics that enable immune cells to find invading pathogens, control tissue damage, and eliminate primary developing tumors, even in the absence of external treatments. These processes are termed “immune surveillance.” Migration disorders of immune cells are related to autoimmune diseases, chronic inflammation, and tumor evasion. It is therefore essential to characterize immune cell motility in different physiologically and pathologically relevant scenarios to understand the regulatory mechanisms of functionality of immune responses. This review is focused on immune cell migration, to define the underlying mechanisms and the corresponding investigative approaches. We highlight the challenges that immune cells encounter in vivo, and the microfabrication methods to mimic particular aspects of their microenvironment. We discuss the advantages and disadvantages of the proposed tools, and provide information on how to access them. Furthermore, we summarize the directional cues that regulate individual immune cell migration, and discuss the behavior of immune cells in a complex environment composed of multiple directional cues.
- ItemSelf-Adhesive Silicone Microstructures for the Treatment of Tympanic Membrane Perforations(Weinheim : Wiley-VCH, 2021) Lana, Gabriela Moreira; Sorg, Katharina; Wenzel, Gentiana Ioana; Hecker, Dietmar; Hensel, René; Schick, Bernhard; Kruttwig, Klaus; Arzt, EduardInspired by the gecko foot, polymeric microstructures have demonstrated reliable dry adhesion to both stiff objects and sensitive surfaces such as skin. Microstructured silicone patches are proposed, herein, for the treatment of tympanic membrane perforations with the aim of serving as an alternative for current surgical procedures that require anesthesia and ear canal packing. Sylgard 184 PDMS micropillars of 20 μm in diameter and 60 μm in length are topped by a Soft Skin Adhesive (SSA) MG7-1010 terminal layer, of about 25 μm thickness. The adhesion is evaluated by specially designed tack tests against explanted murine eardrums and, for comparison, against a rigid substrate. Functional effects are evaluated using auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAE). The adhesion strength of the microstructure and unstructured controls to explanted murine tympanic membranes is comparable (typically 12 kPa), but the microstructured patches are easier to handle by the surgeon. For the first time, partial recovery of hearing performance is measured immediately after patch application. The novel patches adhere without the need for further fixation, removing the need for ear canal packing. The proposed material design holds great promise for improving clinical treatments of tympanic membrane perforations.
- ItemMyxobacteria-Derived Outer Membrane Vesicles: Potential Applicability Against Intracellular Infections(Basel : MDPI, 2020) Goes, Adriely; Lapuhs, Philipp; Kuhn, Thomas; Schulz, Eilien; Richter, Robert; Panter, Fabian; Dahlem, Charlotte; Koch, Marcus; Garcia, Ronald; Kiemer, Alexandra K.; Müller, Rolf; Fuhrmann, GregorIn 2019, it was estimated that 2.5 million people die from lower tract respiratory infections annually. One of the main causes of these infections is Staphylococcus aureus, a bacterium that can invade and survive within mammalian cells. S. aureus intracellular infections are difficult to treat because several classes of antibiotics are unable to permeate through the cell wall and reach the pathogen. This condition increases the need for new therapeutic avenues, able to deliver antibiotics efficiently. In this work, we obtained outer membrane vesicles (OMVs) derived from the myxobacteria Cystobacter velatus strain Cbv34 and Cystobacter ferrugineus strain Cbfe23, that are naturally antimicrobial, to target intracellular infections, and investigated how they can affect the viability of epithelial and macrophage cell lines. We evaluated by cytometric bead array whether they induce the expression of proinflammatory cytokines in blood immune cells. Using confocal laser scanning microscopy and flow cytometry, we also investigated their interaction and uptake into mammalian cells. Finally, we studied the effect of OMVs on planktonic and intracellular S. aureus. We found that while Cbv34 OMVs were not cytotoxic to cells at any concentration tested, Cbfe23 OMVs affected the viability of macrophages, leading to a 50% decrease at a concentration of 125,000 OMVs/cell. We observed only little to moderate stimulation of release of TNF-alpha, IL-8, IL-6 and IL-1beta by both OMVs. Cbfe23 OMVs have better interaction with the cells than Cbv34 OMVs, being taken up faster by them, but both seem to remain mostly on the cell surface after 24 h of incubation. This, however, did not impair their bacteriostatic activity against intracellular S. aureus. In this study, we provide an important basis for implementing OMVs in the treatment of intracellular infections.
- ItemQuantification of EGFR-HER2 Heterodimers in HER2-Overexpressing Breast Cancer Cells Using Liquid-Phase Electron Microscopy(Basel : MDPI, 2021) Peckys, Diana B.; Gaa, Daniel; de Jonge, NielsCurrently, breast cancer patients are classified uniquely according to the expression level of hormone receptors, and human epidermal growth factor receptor 2 (HER2). This coarse classification is insufficient to capture the phenotypic complexity and heterogeneity of the disease. A methodology was developed for absolute quantification of receptor surface density ρR, and molecular interaction (dimerization), as well as the associated heterogeneities, of HER2 and its family member, the epidermal growth factor receptor (EGFR) in the plasma membrane of HER2 overexpressing breast cancer cells. Quantitative, correlative light microscopy (LM) and liquid-phase electron microscopy (LPEM) were combined with quantum dot (QD) labeling. Single-molecule position data of receptors were obtained from scanning transmission electron microscopy (STEM) images of intact cancer cells. Over 280,000 receptor positions were detected and statistically analyzed. An important finding was the subcellular heterogeneity in heterodimer shares with respect to plasma membrane regions with different dynamic properties. Deriving quantitative information about EGFR and HER2 ρR, as well as their dimer percentages, and the heterogeneities thereof, in single cancer cells, is potentially relevant for early identification of patients with HER2 overexpressing tumors comprising an enhanced share of EGFR dimers, likely increasing the risk for drug resistance, and thus requiring additional targeted therapeutic strategies.
- 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.
- ItemTargeting extracellular lectins of Pseudomonas aeruginosa with glycomimetic liposomes(London [u.a.] : RSC, 2021) Metelkina, Olga; Huck, Benedikt; O'Connor, Jonathan S.; Koch, Marcus; Manz, Andreas; Lehr, Claus-Michael; Titz, AlexanderThe antimicrobial resistance crisis requires novel approaches for the therapy of infections especially with Gram-negative pathogens. Pseudomonas aeruginosa is defined as priority 1 pathogen by the WHO and thus of particular interest. Its drug resistance is primarily associated with biofilm formation and essential constituents of its extracellular biofilm matrix are the two lectins, LecA and LecB. Here, we report microbial lectin-specific targeted nanovehicles based on liposomes. LecA- and LecB-targeted phospholipids were synthesized and used for the preparation of liposomes. These liposomes with varying surface ligand density were then analyzed for their competitive and direct lectin binding activity. We have further developed a microfluidic device that allowed the optical detection of the targeting process to the bacterial lectins. Our data showed that the targeted liposomes are specifically binding to their respective lectin and remain firmly attached to surfaces containing these lectins. This synthetic and biophysical study provides the basis for future application in targeted antibiotic delivery to overcome antimicrobial resistance.
- ItemHigh glucose distinctively regulates Ca2+ influx in cytotoxic T lymphocytes upon target recognition and thapsigargin stimulation(Hoboken, NJ : Wiley, 2020) Zou, Huajiao; Yang, Wenjuan; Schwär, Gertrud; Zhao, Renping; Alansary, Dalia; Yin, Deling; Schwarz, Eva C.; Niemeyer, Barbara A.; Qu, BinIn CTLs: High glucose‐culture enhances thapsigargin‐induced SOCE but decreases target recognition‐induced Ca2+ influx. High glucose‐culture regulates expression of ORAIs and STIMs without affecting glucose uptake. More high glucose‐cultured CTLs are prone to necrosis after execution of killing.
- ItemRole of Extracellular Vimentin in Cancer-Cell Functionality and Its Influence on Cell Monolayer Permeability Changes Induced by SARS-CoV-2 Receptor Binding Domain(Basel : Molecular Diversity Preservation International, 2021) Thalla, Divyendu Goud; Jung, Philipp; Bischoff, Markus; Lautenschläger, FranziskaThe cytoskeletal protein vimentin is secreted under various physiological conditions. Extracellular vimentin exists primarily in two forms: attached to the outer cell surface and secreted into the extracellular space. While surface vimentin is involved in processes such as viral infections and cancer progression, secreted vimentin modulates inflammation through reduction of neutrophil infiltration, promotes bacterial elimination in activated macrophages, and supports axonal growth in astrocytes through activation of the IGF-1 receptor. This receptor is overexpressed in cancer cells, and its activation pathway has significant roles in general cellular functions. In this study, we investigated the functional role of extracellular vimentin in non-tumorigenic (MCF-10a) and cancer (MCF-7) cells through the evaluation of its effects on cell migration, proliferation, adhesion, and monolayer permeability. Upon treatment with extracellular recombinant vimentin, MCF-7 cells showed increased migration, proliferation, and adhesion, compared to MCF-10a cells. Further, MCF-7 monolayers showed reduced permeability, compared to MCF-10a monolayers. It has been shown that the receptor binding domain of SARS-CoV-2 spike protein can alter blood–brain barrier integrity. Surface vimentin also acts as a co-receptor between the SARS-CoV-2 spike protein and the cell-surface angiotensin-converting enzyme 2 receptor. Therefore, we also investigated the permeability of MCF-10a and MCF-7 monolayers upon treatment with extracellular recombinant vimentin, and its modulation of the SARS-CoV-2 receptor binding domain. These findings show that binding of extracellular recombinant vimentin to the cell surface enhances the permeability of both MCF-10a and MCF-7 monolayers. However, with SARS-CoV-2 receptor binding domain addition, this effect is lost with MCF-7 monolayers, as the extracellular vimentin binds directly to the viral domain. This defines an influence of extracellular vimentin in SARS-CoV-2 infections.
- ItemGraphene Enclosure of Chemically Fixed Mammalian Cells for Liquid-Phase Electron Microscopy(Cambridge, MA : JoVE, 2020) Blach, Patricia; Keskin, Sercan; de Jonge, NielsA protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using scanning transmission electron microscopy (STEM). Cells of the mammalian breast cancer cell line SKBR3 were grown on silicon microchips with silicon nitride (SiN) windows. Cells were chemically fixed, and HER2 proteins were labeled with quantum dot nanoparticles (QDs), using a two-step biotin-streptavidin binding protocol. The cells were coated with multilayer graphene to maintain a hydrated state, and to protect them from electron beam damage during STEM. To examine the stability of the samples under electron beam irradiation, a dose series experiment was performed. Graphene-coated and non-coated samples were compared. Beam induced damage, in the form of bright artifacts, appeared for some non-coated samples at increased electron dose D, while no artifacts appeared on coated samples.