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.

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.