Inference of long-range cell-cell force transmission from ECM remodeling fluctuations.

Cells sense, manipulate and respond to their mechanical microenvironment in a plethora of physiological processes, yet the understanding of how cells transmit, receive and interpret environmental cues to communicate with distant cells is severely limited due to lack of tools to quantitatively infer the complex tangle of dynamic cell-cell interactions in complicated environments. We present a computational method to systematically infer and quantify long-range cell-cell force transmission through the extracellular matrix (cell-ECM-cell communication) by correlating ECM remodeling fluctuations in between communicating cells and demonstrating that these fluctuations contain sufficient information to define unique signatures that robustly distinguish between different pairs of communicating cells. We demonstrate our method with finite element simulations and live 3D imaging of fibroblasts and cancer cells embedded in fibrin gels. While previous studies relied on the formation of a visible fibrous 'band' extending between cells to inform on mechanical communication, our method detected mechanical propagation even in cases where visible bands never formed. We revealed that while contractility is required, band formation is not necessary, for cell-ECM-cell communication, and that mechanical signals propagate from one cell to another even upon massive reduction in their contractility. Our method sets the stage to measure the fundamental aspects of intercellular long-range mechanical communication in physiological contexts and may provide a new functional readout for high content 3D image-based screening. The ability to infer cell-ECM-cell communication using standard confocal microscopy holds the promise for wide use and democratizing the method.

Strain Gradient Programming in 3D Fibrous Hydrogels to Direct Graded Cell Alignment.

Biological tissues experience various stretch gradients which act as mechanical signaling from the extracellular environment to cells. These mechanical stimuli are sensed by cells, triggering essential signaling cascades regulating cell migration, differentiation, and tissue remodeling. In most previous studies, a simple, uniform stretch to 2D elastic substrates has been applied to analyze the response of living cells. However, induction of nonuniform strains in controlled gradients, particularly in biomimetic 3D hydrogels, has proven challenging. In this study, 3D fibrin hydrogels of manipulated geometry are stretched by a silicone carrier to impose programmable strain gradients along different chosen axes. The resulting strain gradients are analyzed and compared to finite element simulations. Experimentally, the programmed strain gradients result in similar gradient patterns in fiber alignment within the gels. Additionally, temporal changes in the orientation of fibroblast cells embedded in the stretched fibrin gels correlate to the strain and fiber alignment gradients. The experimental and simulation data demonstrate the ability to custom-design mechanical gradients in 3D biological hydrogels and to control cell alignment patterns. It provides a new technology for mechanobiology and tissue engineering studies.

FITC-Dextran Release from Cell-Embedded Fibrin Hydrogels.

Fibrin hydrogel is a central biological material in tissue engineering and drug delivery applications. As such, fibrin is typically combined with cells and biomolecules targeted to the regenerated tissue. Previous studies have analyzed the release of different molecules from fibrin hydrogels; however, the effect of embedded cells on the release profile has yet to be quantitatively explored. This study focused on the release of Fluorescein isothiocyanate (FITC)-dextran (FD) 250 kDa from fibrin hydrogels, populated with different concentrations of fibroblast or endothelial cells, during a 48-h observation period. The addition of cells to fibrin gels decreased the overall release by a small percentage (by 7-15% for fibroblasts and 6-8% for endothelial cells) relative to acellular gels. The release profile was shown to be modulated by various cellular activities, including gel degradation and physical obstruction to diffusion. Cell-generated forces and matrix deformation (i.e., densification and fiber alignment) were not found to significantly influence the release profiles. This knowledge is expected to improve fibrin integration in tissue engineering and drug delivery applications by enabling predictions and ways to modulate the release profiles of various biomolecules.

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging.

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (>100 µm) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel's 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user's needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

Motion magnification analysis of microscopy videos of biological cells.

It is well recognized that isolated cardiac muscle cells beat in a periodic manner. Recently, evidence indicates that other, non-muscle cells, also perform periodic motions that are either imperceptible under conventional lab microscope lens or practically not easily amenable for analysis of oscillation amplitude, frequency, phase of movement and its direction. Here, we create a real-time video analysis tool to visually magnify and explore sub-micron rhythmic movements performed by biological cells and the induced movements in their surroundings. Using this tool, we suggest that fibroblast cells perform small fluctuating movements with a dominant frequency that is dependent on their surrounding substrate and its stiffness.

Straining 3D Hydrogels with Uniform Z-Axis Strains While Enabling Live Microscopy Imaging.

External forces play an important role in the development and regulation of many tissues. Such effects are often studied using specialized stretchers-standardized commercial and novel laboratory-designed. While designs for 2D stretchers are abundant, the range of available 3D stretcher designs is more limited, especially when live imaging is required. This work presents a novel method and a stretching device that allow straining of 3D hydrogels from their circumference, using a punctured elastic silicone strip as the sample carrier. The system was primarily constructed from 3D-printed parts and low-cost electronics, rendering it simple and cost-efficient to reproduce in other labs. To demonstrate the system functionality, > 100 µm thick soft fibrin gels (< 1 KPa) were stretched, while performing live confocal imaging. The subsequent strains and fiber alignment were analyzed and found to be relatively homogenous throughout the gel's thickness (Z axis). The uniform Z-response enabled by our approach was found to be in contrast to a previously reported approach that utilizes an underlying elastic substrate to convey strain to a 3D thick sample. This work advances the ability to study the role of external forces on biological processes under more physiological 3D conditions, and can contribute to the field of tissue engineering.

Good to the bone: microbial community thrives within bone cavities of a bison carcass at Yellowstone National Park.

The discovery of unanticipated microbial diversity in remote, often hostile environments has led to a greater appreciation of the complexity and richness of the natural world. Yellowstone National Park (YNP) has long been a focus of work on taxa that inhabit extreme environments. Here we report the finding of microbial flora that inhabit an unexpected niche: the cavities of bone remnants from a bison carcass in Norris Geyser Basin in YNP. Although bleached white on the surface, the bone cavities are bright green due to the presence of Stichococcus-like trebouxiophyte green algae. The cavities also harbour different fungi and bacteria. Stichococcus species are common lichen photobionts and the Thelebolales fungi present in the bone cavities have previously been found in association with animal remains. Scanning electron microscope analysis suggests the fungi and algae do not form lichen-like associations in the bone. Rather these taxa and the bacteria appear to be opportunists that have colonized an isolated oasis that provides nutrients and protection from desiccation and UV radiation.