Overview of the steps of the automated image analysis pipeline.

<p>Representative immunofluorescent image of Human Vena Saphena Cells (HVSCs), (A) stained for the actin cytoskeleton (green), nucleus (blue), and focal adhesions (magenta). To automatically detect and analyze cells (B), nuclei (C) and focal adhesions (D), corresponding grey-scale images were processed using the automated image analysis pipeline. Scale bars: 50 μm.</p

An automated quantitative analysis of cell, nucleus and focal adhesion morphology

<div><p>Adherent cells sense the physical properties of their environment via focal adhesions. Improved understanding of how cells sense and response to their physical surroundings is aided by quantitative evaluation of focal adhesion size, number, orientation, and distribution in conjunction with the morphology of single cells and the corresponding nuclei. We developed a fast, user-friendly and automated image analysis algorithm capable of capturing and characterizing these individual components with a high level of accuracy. We demonstrate the robustness and applicability of the algorithm by quantifying morphological changes in response to a variety of environmental changes as well as manipulations of cellular components of mechanotransductions. Finally, as a proof-of-concept we use our algorithm to quantify the effect of Rho-associated kinase inhibitor Y-27632 on focal adhesion maturation. We show that a decrease in cell contractility leads to a decrease in focal adhesion size and aspect ratio.</p></div

Schematic overview of the morphometric features of the cell, nucleus, and focal adhesions (FAs), providing information about the effects of cell type, physical environment, and pharmacological drugs on cell response.

<p>The cell type, physical properties of the environment, and pharmacological drugs are known to affect cellular, nuclear and FA morphology. With the developed algorithm we were able to detect these changes and translate them into quantifiable parameters.</p

Automatic cell and nucleus segmentation.

<p>Representative immunofluorescence images of the actin cytosketeleton and nuclei and the corresponding segmentation results, with each identified cell and nucleus shown in a different color. Scale bar: 50 μm.</p

The effect of the Rho-associated kinase (ROCK) inhibitor Y-27632 on the morphological features of focal adhesions (FAs) in Human Vena Saphena Cells (HVSCs).

<p>A: Representative immunofluorescence images of the actin cytoskeleton (green), nucleus (blue), FAs (magenta) and zoom-in images of FAs of HVSCs treated with different doses (0-20 μM) of ROCK inhibitor or DMSO (control). Scale bar: 50 μm. Quantitative analysis of FA area (B), FA aspect ratio (C), and fraction of FAs with a defined length (D) reveals that Y-27632 affects FA morphology. At least 20 cells were analyzed per each condition and the results are expressed as the mean ± standard error of the mean (SEM). To assess differences between the different concentrations of ROCK inhibitor on the morphological features of the FAs, the One-Way ANOVA with a Bonferroni post-hoc test was used. ***: p < 0.001.</p

Detection of a single cell, nucleus and focal adhesions (FAs).

<p>Representative grey-scale images of the actin cytoskeleton, nucleus, and FAs of HVSCs on a substrate homogeneously coated with fibronectin. The detected outlines are shown in green, blue, and magenta, respectively, and the orange rectangles marked areas show zoom-in images of the cell, nucleus and FAs. The white arrows indicate some small actin-rich membrane protrusions that were not detected.</p

Heading in the Right Direction: Understanding Cellular Orientation Responses to Complex Biophysical Environments

The aim of cardiovascular regeneration is to mimic the biological and mechanical functioning of tissues. For this it is crucial to recapitulate the in vivo cellular organization, which is the result of controlled cellular orientation. Cellular orientation response stems from the interaction between the cell and its complex biophysical environment. Environmental biophysical cues are continuously detected and transduced to the nucleus through entwined mechanotransduction pathways. Next to the biochemical cascades invoked by the mechanical stimuli, the structural mechanotransduction pathway made of focal adhesions and the actin cytoskeleton can quickly transduce the biophysical signals directly to the nucleus. Observations linking cellular orientation response to biophysical cues have pointed out that the anisotropy and cyclic straining of the substrate influence cellular orientation. Yet, little is known about the mechanisms governing cellular orientation responses in case of cues applied separately and in combination. This review provides the state-of-the-art knowledge on the structural mechanotransduction pathway of adhesive cells, followed by an overview of the current understanding of cellular orientation responses to substrate anisotropy and uniaxial cyclic strain. Finally, we argue that comprehensive understanding of cellular orientation in complex biophysical environments requires systematic approaches based on the dissection of (sub)cellular responses to the individual cues composing the biophysical niche

Protein Micropatterning in 2.5D: An Approach to Investigate Cellular Responses in Multi-Cue Environments

[Image: see text] The extracellular microenvironment is an important regulator of cell functions. Numerous structural cues present in the cellular microenvironment, such as ligand distribution and substrate topography, have been shown to influence cell behavior. However, the roles of these cues are often studied individually using simplified, single-cue platforms that lack the complexity of the three-dimensional, multi-cue environment cells encounter in vivo. Developing ways to bridge this gap, while still allowing mechanistic investigation into the cellular response, represents a critical step to advance the field. Here, we present a new approach to address this need by combining optics-based protein patterning and lithography-based substrate microfabrication, which enables high-throughput investigation of complex cellular environments. Using a contactless and maskless UV-projection system, we created patterns of extracellular proteins (resembling contact-guidance cues) on a two-and-a-half-dimensional (2.5D) cell culture chip containing a library of well-defined microstructures (resembling topographical cues). As a first step, we optimized experimental parameters of the patterning protocol for the patterning of protein matrixes on planar and non-planar (2.5D cell culture chip) substrates and tested the technique with adherent cells (human bone marrow stromal cells). Next, we fine-tuned protein incubation conditions for two different vascular-derived human cell types (myofibroblasts and umbilical vein endothelial cells) and quantified the orientation response of these cells on the 2.5D, physiologically relevant multi-cue environments. On concave, patterned structures (curvatures between κ = 1/2500 and κ = 1/125 μm(–1)), both cell types predominantly oriented in the direction of the contact-guidance pattern. In contrast, for human myofibroblasts on micropatterned convex substrates with higher curvatures (κ ≥ 1/1000 μm(–1)), the majority of cells aligned along the longitudinal direction of the 2.5D features, indicating that these cells followed the structural cues from the substrate curvature instead. These findings exemplify the potential of this approach for systematic investigation of cellular responses to multiple microenvironmental cues