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In vitro, hydrogel-based ECMs for functionalizing surfaces of various material have played an essential role in mimicking native tissue matrix. Polydimethylsiloxane (PDMS) is widely used to build microfluidic or organ-on-chip devices compatible with cells due to its easy handling in cast replication. Despite such advantages, the limitation of PDMS is its hydrophobic surface property. To improve wettability of PDMS-based devices, alginate, a naturally derived polysaccharide, was covalently bound to the PDMS surface. This alginate then crosslinked further hydrogel onto the PDMS surface in desired layer thickness. Hydrogel-modified PDMS was used for coating a topography chip system and in vitro investigation of cell growth on the surfaces. Moreover, such hydrophilic hydrogel-coated PDMS is utilized in a microfluidic device to prevent unspecific absorption of organic solutions. Hence, in both exemplary studies, PDMS surface properties were modified leading to improved devices.
Analysis of multicellular patterns is required to understand tissue organizational processes. By using a multi-scale object oriented image processing method, the spatial information of cells can be extracted automatically. Instead of manual segmentation or indirect measurements, such as general distribution of contrast or flow, the orientation and distribution of individual cells is extracted for quantitative analysis. Relevant objects are identified by feature queries and no low-level knowledge of image processing is required.
Characterisation of porous knitted titanium for replacement of intervertebral disc nucleus pulposus
(2017)
Effective restoration of human intervertebral disc degeneration is challenged by numerous limitations of the currently available spinal fusion and arthroplasty treatment strategies. Consequently, use of artificial biomaterial implant is gaining attention as a potential therapeutic strategy. Our study is aimed at investigating and characterizing a novel knitted titanium (Ti6Al4V) implant for the replacement of nucleus pulposus to treat early stages of chronic intervertebral disc degeneration. Specific knitted geometry of the scaffold with a porosity of 67.67 ± 0.824% was used to overcome tissue integration failures. Furthermore, to improve the wear resistance without impairing original mechanical strength, electro-polishing step was employed. Electro-polishing treatment changed a surface roughness from 15.22 ± 3.28 to 4.35 ± 0.87 μm without affecting its wettability which remained at 81.03 ± 8.5°. Subsequently, cellular responses of human mesenchymal stem cells (SCP1 cell line) and human primary chondrocytes were investigated which showed positive responses in terms of adherence and viability. Surface wettability was further enhanced to super hydrophilic nature by oxygen plasma treatment, which eventually caused substantial increase in the proliferation of SCP1 cells and primary chondrocytes. Our study implies that owing to scaffolds physicochemical and biocompatible properties, it could improve the clinical performance of nucleus pulposus replacement.
Increasing number of studies are focused on how adherent cells respond, in vitro, to different properties of a material. Typical properties are the surface chemistry, topographical cues (at the nano- and micro-scale) of the surface, and the substrate stiffness. Cell Response studies are of importance for designing new biomaterials with applications in cell culture technologies, regenerative medicine, or for medical implants. However, only very few studies take the cell age factor, respectively the donor age, into account. In this work, we tested two types of human vascular cells (smooth muscle and endothelial cells) from old and young donors on (a) micro-structured surfaces made of pol (dimethylsiloxane) or on (b) flat polyacrylamide hydrogels with varying stiffnesses. These experiments reveal age-dependent and cell typedependent differences in the cell response to the topography and stiffness, and may establish the Basis for further studies focusing on cell age-dependent responses.
Age-dependent migratory behavior of human endothelial cells revealed by substrate microtopography
(2019)
Cell migration is part of many important in vivo biological processes and is influenced by chemical and physical factors such as substrate topography. Although the migratory behavior of different cell types on structured substrates has already been investigated, up to date it is largely unknown if specimen's age affects cell migration on structures. In this work, we investigated age-dependent migratory behavior of human endothelial cells from young (≤ 31 years old) and old (≥ 60 years old) donors on poly(dimethylsiloxane) microstructured substrates consisting of well-defined parallel grooves. We observed a decrease in cell migration velocity in all substrate conditions and in persistence length perpendicular to the grooves in cells from old donors. Nevertheless, in comparison to young cells, old cells exhibited a higher cell directionality along grooves of certain depths and a higher persistence time. We also found a systematic decrease of donor age dependent responses of cell protrusions in orientation, velocity and length, all of them decreased in old cells. These observations lead us to hypothesize a possible impairment of actin cytoskeleton network and affected actin polymerization and steering systems, caused by aging.
A wide variety of cell types exhibit substrate topography-based behavior, also known as contact guidance. However, the precise cellular mechanisms underlying this process are still unknown. In this study, we investigated contact guidance by studying the reaction of human endothelial cells (ECs) to well-defined microgroove topographies, both during and after initial cell spreading. As the cytoskeleton plays a major role in cellular adaptation to topographical features, two methods were used to perturb cytoskeletal structures. Inhibition of actomyosin contractility with the chemical inhibitor blebbistatatin demonstrated that initial contact guidance events are independent of traction force generation. However, cell alignment to the grooved substrate was altered at later time points, suggesting an initial ‘passive’ phase of contact guidance, followed by a contractility-dependent ‘active’ phase that relies on mechanosensitive feedback. The actin cytoskeleton was also perturbed in an indirect manner by culturing cells upside down, resulting in decreased levels of contact guidance and suggesting that a possible loss of contact between the actin cytoskeleton and the substrate could lead to cytoskeleton impairment. The process of contact guidance at the microscale was found to be primarily lamellipodia driven, as no bias in filopodia extension was observed on micron-scale grooves.
Although integrins are responsible for the interaction of cells with their environment, e.g., the extracellular matrix or artificial substrates, there is still a lack of knowledge about their role in cell adhesion and migration on protein-coated substrates with microtopography. Understanding such interactions could lead to new applications in e.g., medical implants as well as shed light on processes such as embryonic development, angiogenesis, wound healing, and tumor progression. In this work, the influence of surface topography and chemistry on αvβ3 and α5β1 integrin-mediated cell adhesion and migration of healthy and malignant human cell types (human coronary artery endothelial cells, human osteosarcoma cells, and human skin fibroblasts cells) was studied, using microgrooved and flat substrates covered by two different extracellular proteins, fibronectin and vitronectin. Although some general behaviors can be observed, cell migration (speed, directionality, and persistence time) and morphological adaptation (cell area, aspect ratio, and circularity) of cells on protein coated microgrooved substrates are mainly dependent on the cell type and its specific integrin expression.
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.
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.
The spreading area of cells has been shown to play a central role in the determination of cell fate and tissue morphogenesis; however, a clear understanding of how spread cell area is determined is still lacking. The observation that cell area and force generally increase with substrate rigidity suggests that cell area is dictated mechanically, by means of a force-balance between the cell and the substrate. A simple mechanical model, corroborated by experimental measurements of cell area and force is presented to analyze the temporal force balance between the cell and the substrate during spreading. The cell is modeled as a thin elastic disc that is actively pulled by lamellipodia protrusions at the cell front. The essential molecular mechanisms of the motor activity at the cell front, including, actin polymerization, adhesion kinetics, and the actin retrograde flow, are accounted for and used to predict the dynamics of cell spreading on elastic substrates; simple, closed-form expressions for the evolution of cell size and force are derived. Time-resolved, traction force microscopy, combined with measurements of cell area are performed to investigate the simultaneous variations of cell size and force. We find that cell area and force increase simultaneously during spreading but the force develops with an apparent delay relative to the increase in cell area. We demonstrate that this may reflect the strain-stiffening property of the cytoskeleton. We further demonstrate that the radial cell force is a concave function of spreading speed and that this may reflect the strengthening of cell–substrate adhesions during spreading.
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.
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.
Continuum physics modeling of tumor growth is a rich topic with room for rather sophisticated models of reaction-transport and mechanics. It also has the attraction of being able to pose and examine solutions to certain questions on tumor growth that are difficult to access using experimental methods alone. However, the imperative of experimental biophysical investigations cannot by understated.
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.