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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.
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.
Focal adhesion clusters (FAC) are dynamic and complex structures that help cells to sense physicochemical properties of their environment. Research in biomaterials, cell adhesion or cell migration often involves the visualization of FAC by fluorescence staining and microscopy, which necessitates quantitative analysis of FAC and other cell features in microscopy images using image processing. Fluorescence microscopy images of human umbilical vein endothelial cells (HUVEC) obtained at 63x magnification were quantitatively analysed using ImageJ software. A generalised algorithm for selective segmentation and morphological analysis of FAC, nucleus and cell morphology is implemented. Further, a method for discrimination of FACnear the nucleus and around the periphery is implemented using masks. Our algorithm is able to effectively quantify different morphological characteristics of cell components and shows a high sensitivity and specificity while providing a modular software implementation.
Programmable nano-bio interfaces driven by tuneable vertically configured nanostructures have recently emerged as a powerful tool for cellular manipulations and interrogations. Such interfaces have strong potential for ground-breaking advances, particularly in cellular nanobiotechnology and mechanobiology. However, the opaque nature of many nanostructured surfaces makes non-destructive, live-cell characterization of cellular behavior on vertically aligned nanostructures challenging to observe. Here, a new nanofabrication route is proposed that enables harvesting of vertically aligned silicon (Si) nanowires and their subsequent transfer onto an optically transparent substrate, with high efficiency and without artefacts. We demonstrate the potential of this route for efficient live-cell phase contrast imaging and subsequent characterization of cells growing on vertically aligned Si nanowires. This approach provides the first opportunity to understand dynamic cellular responses to a cell-nanowire interface, and thus has the potential to inform the design of future nanoscale cellular manipulation technologies.
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.
Medical implants play a central role in modern medicine and both, naturally derived and synthetic materials have been explored as biomaterials for such devices. However, when implanted into living tissue, most materials initiate a host response. In addition, implants often cause bacterial infections leading to complications. Polyelectrolyte multilayer (PEM) coatings can be used for functionalization of medical implants improving the implant integration and reducing foreign body reactions. Some PEMs are also known to show antibacterial properties. We developed a PEM coating suggesting that it can decrease the risk of bacterial infections occurring after implantation while being highly biocompatible. We applied two different standard tests for evaluating the PEM’s antibacterial properties, the ISO norm (ISO 22196) and one ASTM norm (ASTM E2180) test. We found a reduction of bacterial growth on the PEM but to a different degree depending on the testing method. This result demonstrates the need for defining proper method to evaluate antibacterial properties of surface coatings.
Thermoplastic polycarbonate urethane elastomers (TPCU) are potential implant materials for treating degenerative joint diseases thanks to their adjustable rubber-like properties, their toughness, and their durability. We developed a water-containing high-molecular-weight sulfated hyaluronic acid-coating to improve the interaction of TPCU with the synovial fluid. It is suggested that trapped synovial fluid can act as a lubricant that reduces the friction forces and thus provides an enhanced abrasion resistance of TPCU implants. Aims of this work were (i) the development of a coating method for novel soft TPCU with high-molecular sulfated hyaluronic acid to increase the biocompatibility and (ii) the in vitro validation of the functionalized TPCUs in cell culture experiments.
Endogenous electrical fields play an important role in various physiological and pathological events. Yet the effects of electrical cues on processes such as wound healing, tumor development or metastasis are still rarely investigated, though it is known that direct current electrical fields can alter cell migration or proliferation in vitro. Several 2D experimental models for studying cell responses to direct current electrical fields have been presented and characterized but suitable experimental models for electrotaxis studies in 3D are rare. Here we present a novel, easy-to-produce, multi-well-based galvanotactic-chamber for the use in 2D and 3D cell experiments for investigations on the influence of electrical fields on tumor cell migration and tumor spheroid growth. Our presented system allows the simultaneous application of electrical field to cells in four chambers, either cultured on the bottom of the culture-plate (2D) or embedded in hydrogel filled channels(3D). The set-up is also suitable for, live-cell-imaging. Validation tests show stable electrical fields and high cell viabilities inside the channel. Tumor spheroids of various diameters can be exposed to direct current electrical fields up to one week.
Drug-induced liver toxicity is one of the most common reasons for the failure of drugs in clinical trials and frequent withdrawal from the market. Reasons for such failures include the low predictive power of in vivo studies, that is mainly caused by metabolic differences between humans and animals, and intraspecific variances. In addition to factors such as age and genetic background, changes in drug metabolism can also be caused by disease-related changes in the liver. Such metabolic changes have also been observed in clinical settings, for example, in association with a change in liver stiffness, a major characteristic of an altered fibrotic liver. For mimicking these changes in an in vitro model, this study aimed to develop scaffolds that represent the rigidity of healthy and fibrotic liver tissue. We observed that liver cells plated on scaffolds representing the stiffness of healthy livers showed a higher metabolic activity compared to cells plated on stiffer scaffolds. Additionally, we detected a positive effect of a scaffold pre-coated with fetal calf serum (FCS)-containing media. This pre-incubation resulted in increased cell adherence during cell seeding onto the scaffolds. In summary, we developed a scaffold-based 3D model that mimics liver stiffness-dependent changes in drug metabolism that may more easily predict drug interaction in diseased livers.
Polyurethane-bases block copolymers (TPCUs) are block-copolymers with systematically varied soft and hard segments. They have been suggested to serve as material for chondral implants in joint regeneration. Such applications may require the adhesion of chondrocytes to the implant surface, facilitating cell growth while keeping their phenotype. Thus, aims of this work were (1) to modify the surface of soft biostable polyurethane-based model implants (TPCU and TSiPCU) with high-molecular weight hyaluronic acid (HA) using an optimized multistep strategy of immobilization, and (2) to evaluate bioactivity of the modified TPCUs in vitro. Our results show no cytotoxic potential of the TPCUs. HAbioactive molecules (Mw =700kDa) were immobilized onto the polyurethane surface via polyethylenimine (PEI) spacers, and modifications were confirmed by several characterization methods. Tests with porcine chondrocytes indicated the potential of the TPCU-HA for inducing enhanced cell proliferation.