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Soft lithography, a tool widely applied in biology and life sciences with numerous applications, uses the soft molding of photolithography-generated master structures by polymers. The central part of a photolithography set-up is a mask-aligner mostly based on a high-pressure mercury lamp as an ultraviolet (UV) light source. This type of light source requires a high level of maintenance and shows a decreasing intensity over its lifetime, influencing the lithography outcome. In this paper, we present a low-cost, bench-top photolithography tool based on ninety-eight 375 nm light-emitting diodes (LEDs). With approx. 10 W, our presented lithography set-up requires only a fraction of the energy of a conventional lamp, the LEDs have a guaranteed lifetime of 1000 h, which becomes noticeable by at least 2.5 to 15 times more exposure cycles compared to a standard light source and with costs less than 850 C it is very affordable. Such a set-up is not only attractive to small academic and industrial fabrication facilities who want to enable work with the technology of photolithography and cannot afford a conventional set-up, but also microfluidic teaching laboratories and microfluidic research and development laboratories, in general, could benefit from this cost-effective alternative. With our self-built photolithography system, we were able to produce structures from 6 μm to 50 μm in height and 10 μm to 200 μm in width. As an optional feature, we present a scaled-down laminar flow hood to enable a dust-free working environment for the photolithography process.
It is well established that the mechanical environment influences cell functions in health and disease. Here, we address how the mechanical environment influences tumor growth, in particular, the shape of solid tumors. In an in vitro tumor model, which isolates mechanical interactions between cancer tumor cells and a hydrogel, we find that tumors grow as ellipsoids, resembling the same, oft-reported observation of in vivo tumors. Specifically, an oblate ellipsoidal tumor shape robustly occurs when the tumors grow in hydrogels that are stiffer than the tumors, but when they grow in more compliant hydrogels they remain closer to spherical in shape. Using large scale, nonlinear elasticity computations we Show that the oblate ellipsoidal shape minimizes the elastic free energy of the tumor-hydrogel system. Having eliminated a number of other candidate explanations, we hypothesize that minimization of the elastic free energy is the reason for predominance of the experimentally observed ellipsoidal shape. This result may hold significance for explaining the shape progressio.
In Neurofibromatosis 1 (NF1) germ line loss of function mutations result in reduction of cellular neurofibromin content (NF1+/−, NF1 haploinsufficiency). The Ras-GAP neurofibromin is a very large cytoplasmic protein (2818 AA, 319 kDa) involved in the RAS-MAPK pathway. Aside from regulation of proliferation, it is involved in mechanosensoric of cells. We investigated neurofibromin replacement in cultured human fibroblasts showing reduced amount of neurofibromin. Full length neurofibromin was produced recombinantly in insect cells and purified. Protein transduction into cultured fibroblasts was performed employing cell penetrating peptides along with photochemical internalization. This combination of transduction strategies ensures the intracellular uptake and the translocation to the cytoplasm of neurofibromin. The transduced neurofibromin is functional, indicated by functional rescue of reduced mechanosensoric blindness and reduced RasGAP activity in cultured fibroblasts of NF1 patients or normal fibroblasts treated by NF1 siRNA. Our study shows that recombinant neurofibromin is able to revert cellular effects of NF1 haploinsuffiency in vitro, indicating a use of protein transduction into cells as a potential treatment strategy for the monogenic disease NF1.
A series of novel biomedical TPCUs with different percentages of hard segment and a silicone component in the soft segment were synthesized in a multi stage one-pot method. The kinetic profiles of the urethane formation in TPCU-based copolymer systems were monitored by rheological, in line FTIR spectroscopic (React IR) and real-time calorimetric (RC1) methods. This process-analytically monitored multi step synthesis was successfully used to optimize the production of medical-grade TPCU elastomers on preparative scale (in lots of several kg) with controlled molecular structure and mechanical properties. Various surface and bulk analytical methods as well as systematic studies of the mechanic response of the elastomer end-products towards compression and tensile loading were used to estimate the bio-stability of the prepared TPCUs in vitro after 3 months. The tests suggested that high bio-stability of all polyurethane formulations using accelerating in vitro test can be attributed to the synthetic design as well as to the specific techniques used for specimen preparation, namely: (1) the annealing for reducing residual polymer surface stress and preventing IES, (2) stabilization of the morphology by long time storage of the specimens after processing before being immersed in the test liquids, (3) purification by extraction to remove the shot chain oligomers which are the most susceptible to degradation. All mechanical tests were performed on cylindrical and circular disc specimens for modelling the thickness of the meniscus implants under application-relevant stress conditions.
Cell migration plays an essential role in wound healing and inflammatory processes inside the human body. Peripheral blood neutrophils, a type of polymorphonuclear leukocyte (PMN), are the first cells to be activated during inflammation and subsequently migrate toward an injured tissue or infection site. This response is dependent on both biochemical signaling and the extracellular environment, one aspect of which includes increased temperature in the tissues surrounding the inflammation site. In our study, we analyzed temperature-dependent neutrophil migration using differentiated HL-60 cells. The migration speed of differentiated HL-60 cells was found to correlate positively with temperature from 30 to 42 °C, with higher temperatures inducing a concomitant increase in cell detachment. The migration persistence time of differentiated HL-60 cells was higher at lower temperatures (30–33 °C), while the migration persistence length stayed constant throughout the temperature range. Coupled with the increased speed observed at high temperatures, this suggests that neutrophils are primed to migrate more effectively at the elevated temperatures characteristic of inflammation. Temperature gradients exist on both cell and tissue scales. Taking this into consideration, we also investigated the ability of differentiated HL-60 cells to sense and react to the presence of temperature gradients, a process known as thermotaxis. Using a two-dimensional temperature gradient chamber with a range of 27–43 °C, we observed a migration bias parallel to the gradient, resulting in both positive and negative thermotaxis. To better mimic the extracellular matrix (ECM) environment in vivo, a three-dimensional collagen temperature gradient chamber was constructed, allowing observation of biased neutrophil-like differentiated HL-60 migration toward the heat source.
Recently described rhizolutin and collinolactone isolated from Streptomyces Gç 40/10 share the same novel carbon scaffold. Analyses by NMR and X-Ray crystallography verify the structure of collinolactone and propose a revision of rhizolutins stereochemistry. Isotope-labeled precursor feeding shows that collinolactone is biosynthesized via type I polyketide synthase with Baeyer–Villiger oxidation. CRISPR-based genetic strategies led to the identification of the biosynthetic gene cluster and a high-production strain. Chemical semisyntheses yielded collinolactone analogues with inhibitory effects on L929 cell line. Fluorescence microscopy revealed that only particular analogues induce monopolar spindles impairing cell division in mitosis. Inspired by the Alzheimerprotective activity of rhizolutin, we investigated the neuroprotective effects of collinolactone and its analogues on glutamate-sensitive cells (HT22) and indeed, natural collinolactone displays distinct neuroprotection from intracellular oxidative stress.
Tumorzellen on the move : mikrosystem-basierter Assay zur Untersuchung der Tumorzellen-Migration
(2016)
Die Invasion von Tumorzellen in umliegendes Gewebe und die Bildung von Metastasen transformieren einen lokal wachsenden Tumor in eine systemische und lebensbedrohliche Krankheit mit schlechter Prognose. Dabei spielt die aktive Migration der Tumorzellen eine entscheidende Rolle. Tumorzellen gelangen durch die aktive Zellbewegung in das Lymph- oder Blutsystem und breiten sich im Körper aus. Bei der Invasion in ein neues Organ migrieren die Zellen ebenfalls wieder in komplexer Weise durch das Gewebe und können schließlich dort Metastasen bilden. Auf Grund der enormen medizinischen Relevanz der Tumorzell-Invasion, wird die Bewegung von Tumorzellen seit Jahrzehnten unter Laborbedingungen umfassend untersucht und ist ein wichtiger Marker für die Aggressivität der Tumorzellen. Zur Bewegungsanalyse gibt es mehrere experimentelle und auch kommerziell erhältliche in-vitro Untersuchungsmethoden. Ziel des interdisziplinären Projektes „MigChip“ ist die Entwicklung, Herstellung und experimentelle Validierung eines Mikrofludik-Chips zur verbesserten, detailgenauen in-vitro Untersuchung der Tumorzellen-Migration.
Human adipose-derived mesenchymal stem/stromal cells (Ad-MSCs) have great potential for bone tissue engineering. Cryogels, mimicking the three-dimensional structure of spongy bone, represent ideal carriers for these cells. We developed poly(2-hydroxyethyl methacrylate) cryogels, containing hydroxyapatite to mimic inorganic bone matrix. Cryogels were additionally supplemented with different types of proteins, namely collagen (Coll), platelet rich plasma (PRP), immune cells-conditioned medium (CM), and RGD peptides (RGD). The different protein components did not affect scaffolds’ porosity or water-uptake capacity, but altered pore size and stiffness. Stiffness was highest in scaffolds with PRP (82.3 kPa), followed by Coll (55.3 kPa), CM (45.6 kPa), and RGD (32.8 kPa). Scaffolds with PRP, CM, and Coll had the largest pore diameters (~60 µm). Ad MSCs were osteogenically differentiated on these scafffolds for 14 days. Cell attachment and survival rates were comparable for all four scaffolds. Runx2 and osteocalcin levels only increased in Ad-MSCs on Coll, PRP and CM cryogels. Osterix levels increased slightly in Ad-MSCs differentiated on Coll and PRP cryogels. With differentiation alkaline phosphatase activity decreased under all four conditions. In summary, besides Coll cryogel our PRP cryogel constitutes as an especially suitable carrier for bone tissue engineering. This is of special interest, as this scaffold can be generated with patients’ PRP.
Perivascular stromal cells, including mesenchymal stem/stromal cells (MSCs), secrete paracrine factor in response to exercise training that can facilitate improvements in muscle remodeling. This study was designed to test the capacity for muscle-resident MSCs (mMSCs) isolated from young mice to release regenerative proteins in response to mechanical strain in vitro, and subsequently determine the extent to which strain-stimulated mMSCs can enhance skeletal muscle and cognitive performance in a mouse model of uncomplicated aging. Protein arrays confirmed a robust increase in protein release at 24 h following an acute bout of mechanical strain in vitro (10%, 1 Hz, 5 h) compared to non-strain controls. Aged (24 month old), C57BL/6 mice were provided bilateral intramuscular injection of saline, non strain control mMSCs, or mMSCs subjected to a single bout of mechanical strain in vitro (4 ×104). No significant changes were observed in muscle weight, myofiber size, maximal force, or satellite cell quantity at 1 or 4 wks between groups. Peripheral perfusion was significantly increased in muscle at 4 wks post-mMSC injection (p < 0.05), yet no difference was noted between control and preconditioned mMSCs. Intramuscular injection of preconditioned mMSCs increased the number of new neurons and astrocytes in the dentate gyrus of the hippocampus compared to both control groups (p < 0.05), with a trend toward an increase in water maze performance noted (p=0.07). Results from this study demonstrate that acute injection of exogenously stimulated muscle-resident stromal cells do not robustly impact aged muscle structure and function, yet increase the survival of new neurons in the hippocampus.
Intermediate filament reorganization dynamically influences cancer cell alignment and migration
(2017)
The interactions between a cancer cell and its extracellular matrix (ECM) have been the focus of an increasing amount of investigation. The role of the intermediate filament keratin in cancer has also been coming into focus of late, but more research is needed to understand how this piece fits in the puzzle of cytoskeleton-mediated invasion and metastasis. In Panc-1 invasive pancreatic cancer cells, keratin phosphorylation in conjunction with actin inhibition was found to be sufficient to reduce cell area below either treatment alone. We then analyzed intersecting keratin and actin fibers in the cytoskeleton of cyclically stretched cells and found no directional correlation. The role of keratin organization in Panc-1 cellular morphological adaptation and directed migration was then analyzed by culturing cells on cyclically stretched polydimethylsiloxane (PDMS) substrates, nanoscale grates, and rigid pillars. In general, the reorganization of the keratin cytoskeleton allows the cell to become more ‘mobile’- exhibiting faster and more directed migration and orientation in response to external stimuli. By combining keratin network perturbation with a variety of physical ECM signals, we demonstrate the interconnected nature of the architecture inside the cell and the scaffolding outside of it, and highlight the key elements facilitating cancer cell-ECM interactions.