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The tissue microenvironment is a major contributor to cellular functions, such as cell adhesion, migration and invasion. A critical physical parameter for determining the effect of the microenvironment on cellular functions is the average pore-size of filamentous scaffolds, such as 3D collagen fiber matrices, which are assembled by the polymerization of biopolymers. The scaffolds of these matrices can be analyzed easily by using state-of-the-art laser scanning confocal imaging. However, the generation of a quantitative estimate of the pore-size in a 3D microenvironment is not trivial. In this study, we present a reliable and fast analytical method, which relies on a two-step 3D pore-size analysis utilizing several state-of-the-art image analysis methods, such as total variation (TV) denoising and adaptive local thresholds, and another crucial parameter, such as pore-coverage. We propose an iterative approach of pore-size analysis to determine even the smallest and obscure pores in a collagen scaffold. Additionally, we propose a novel parameter, the pseudo-pore-size, which describes a virtual scaffold porosity. In order to validate the advanced two-step pore-size analysis different types of artificial collagens, such as a rat and bovine mixture with two different collagen concentrations have been utilized. Additionally, we compare a traditional approach with our method using an artificially generated network with predefined pore-size distributions. Indeed, our analytical method provides a precise, fast and parameter-free, user-independent and automatic analysis of 3D pore topology, such as pore-sizes and pore-coverage. Additionally, we are able to determine non-physiological network topologies by taking the pore-coverage as a goodness-of-fit parameter.

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The magnetic tweezer technique has become a versatile tool for unfolding or folding of individual molecules, mainly DNA. In addition to single molecule analysis, the magnetic tweezer can be used to analyze the mechanical properties of cells and extracellular matrices. We have established a magnetic tweezer that is capable of measuring the linear and non-linear viscoelastic behavior of a wide range of soft matter in precisely controlled environmental conditions, such as temperature, CO 2 and humidity. The magnetic tweezer presented in this study is suitable to detect specific differences in the mechanical properties of different cell lines, such as human breast cancer cells and mouse embryonic fibroblasts, as well as collagen matrices of distinct concentrations in the presence and absence of fibronectin crosslinks. The precise calibration and control mechanism employed in the presented magnetic tweezer setup provides the ability to apply physiological force up to 5 nN on 4.5 µm superparamagnetic beads coated with fibronectin and coupled to the cells or collagen matrices. These measurements reveal specific local linear and non-linear viscoelastic behavior of the investigated samples. The viscoelastic response of cells and collagen matrices to the force application is best described by a weak power law behavior. Our results demonstrate that the stress stiffening response and the fluidization of cells is cell type specific and varies largely between differently invasive and aggressive cancer cells. Finally, we showed that the viscoelastic behavior of collagen matrices with and without fibronectin crosslinks measured by the magnetic tweezer can be related to the microstructure of these matrices.

The extracellular matrix (ECM) is a three-dimensional, acellular scaffold of living tissues. Incorporating the ECM into cell culture models is a goal of cell biology studies and requires biocompatible materials that can mimic the ECM. Among such materials are hydrogels: polymeric networks that derive most of their mass from water. With the tuning of their properties, these polymer networks can resemble living tissues. The microarchitectural properties of hydrogels, such as porosity, pore size, fiber length, and surface topology can determine cell plasticity. The adequate characterization of these parameters requires reliable and reproducible methods. However, most methods were historically standardized using other biological specimens, such as 2D cell cultures, biopsies, or even animal models. Therefore, their translation comes with technical limitations when applied to hydrogel-based cell culture systems. In our current work, we have reviewed the most common techniques employed in the characterization of hydrogel microarchitectures. Our review provides a concise description of the underlying principles of each method and summarizes the collective data obtained from cell-free and cell-loaded hydrogels. The advantages and limitations of each technique are discussed, and comparisons are made. The information presented in our current work will be of interest to researchers who employ hydrogels as platforms for cell culture, 3D bioprinting, and other fields within hydrogel-based research.

The knowledge of cell mechanics is required to understand cellular processes and functions, such as the movement of cells, and the development of tissue engineering in cancer therapy. Cell mechanical properties depend on a variety of factors, such as cellular environments, and may also rely on external factors, such as the ambient temperature. The impact of temperature on cell mechanics is not clearly understood. To explore the effect of temperature on cell mechanics, we employed magnetic tweezers to apply a force of 1 nN to 4.5 µm superparamagnetic beads. The beads were coated with fibronectin and coupled to human epithelial breast cancer cells, in particular MCF-7 and MDA-MB-231 cells. Cells were measured in a temperature range between 25 and 45 °C. The creep response of both cell types followed a weak power law. At all temperatures, the MDA-MB-231 cells were pronouncedly softer compared to the MCF-7 cells, whereas their fluidity was increased. However, with increasing temperature, the cells became significantly softer and more fluid. Since mechanical properties are manifested in the cell’s cytoskeletal structure and the paramagnetic beads are coupled through cell surface receptors linked to cytoskeletal structures, such as actin and myosin filaments as well as microtubules, the cells were probed with pharmacological drugs impacting the actin filament polymerization, such as Latrunculin A, the myosin filaments, such as Blebbistatin, and the microtubules, such as Demecolcine, during the magnetic tweezer measurements in the specific temperature range. Irrespective of pharmacological interventions, the creep response of cells followed a weak power law at all temperatures. Inhibition of the actin polymerization resulted in increased softness in both cell types and decreased fluidity exclusively in MDA-MB-231 cells. Blebbistatin had an effect on the compliance of MDA-MB-231 cells at lower temperatures, which was minor on the compliance MCF-7 cells. Microtubule inhibition affected the fluidity of MCF-7 cells but did not have a significant effect on the compliance of MCF-7 and MDA-MB-231 cells. In summary, with increasing temperature, the cells became significant softer with specific differences between the investigated drugs and cell lines.

Cell migration performs a critical function in numerous physiological processes, including tissue homeostasis or wound healing after tissue injury, as well as pathological processes that include malignant progression of cancer. The efficiency of cell migration and invasion appears to be based on the mechano-phenotype of the cytoskeleton. The properties of the cytoskeleton depend on internal cytoskeletal and external environmental factors. A reason for this are connections between the cell and its local matrix microenvironment, which are established by cell-matrix adhesion receptors. Upon activation, focal adhesion proteins such as PINCH1 are recruited to sites where focal adhesions form. PINCH1 specifically couples through interactions with ILK, which binds to cell matrix receptors and the actomyosin cytoskeleton. However, the role of PINCH1 in cell mechanics regulating cellular motility in 3D collagen matrices is still unclear. PINCH1 is thought to facilitate 3D motility by regulating cellular mechanical properties, such as stiffness. In this study, PINCH1 wild-type and knock-out cells were examined for their ability to migrate in dense extracellular 3D matrices. Indeed, PINCH1 wild-type cells migrated more numerously and deeper in 3D matrices, compared to knock-out cells. Moreover, cellular deformability was determined, e.g., elastic modulus (stiffness). PINCH1 knock-out cells are more deformable (compliable) than PINCH1 wild-type cells. Migration of both PINCH1−/− cells and PINCH1fl/fl cells was decreased by Latrunculin A inhibition of actin polymerization, suggesting that actin cytoskeletal differences are not responsible for the discrepancy in invasiveness of the two cell types. However, the mechanical phenotype of PINCH1−/− cells may be reflected by Latrunculin A treatment of PINCH1fl/fl cells, as they exhibit resembling deformability to untreated PINCH1−/− cells. Moreover, an apparent mismatch exists between the elongation of the long axis and the contraction of the short axis between PINCH1fl/fl cells and PINCH1−/− cells following Latrunculin A treatment. There is evidence of this indicating a shift in the proxy values for Poisson’s ratio in PINCH1−/− cells compared with PINCH1fl/fl cells. This is probably attributable to modifications in cytoskeletal architecture. The non-muscle myosin II inhibitor Blebbistatin also reduced the cell invasiveness in 3D extracellular matrices but instead caused a stiffening of the cells. Finally, PINCH1 is apparently essential for providing cellular mechanical stiffness through the actin cytoskeleton, which regulates 3D motility.