Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

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.

Tissue response to mechanical forces is an integral part of a wide range of biological functions, including gene expression¹, cell differentiation², and tissue remodeling³. Moreover, force-induced changes in the extracellular matrix (ECM) such as fiber alignment and densification can impact cell behavior and tissue formation^4,5,6. The ECM’s fibrous mesh structure has intriguing mechanical properties, such as non-linear elasticity, non-affine deformation and plastic deformations⁷

1. Solution preparation (to be performed in advance)

1. Fibrinogen labeling
   NOTE: The labeling step is required only if analyzing the deformation of the fibrin gel is desired. For cellular experiments, it is possible to use an unlabeled gel.
   1. Add 38 μL of 10 mg/mL succinimidyl ester fluorescent dye (dissolved in DMSO) to 1.5 mL of 15 mg/mL fibrinogen solution (molar ratio of 5:1) in a 50 mL centrifuge tube and place on a shaker for 1 hour at room temperature. Afterwards, place the tube in .......

Representative data from static stretch of increasing magnitudes applied to the silicone strip carrying a 3D fibrin hydrogel, embedded with 1 μm fluorescent beads, is shown in Figure 9. The analysis demonstrates the effect of silicone stretch on geometric changes of the cut-out as well as the developed strains within the gel. Z-stack images of the entire gel are used to evaluate the deformation of the original circle shaped cut-out to the elliptical geometry (

The method and protocol presented herein are largely based on our previous study by Roitblat Riba et al.⁴¹ We include here the full computer-aided design (CAD), Python and microcontroller codes of the SCyUS device.

The major advantages of the presented method over existing approaches include the possibility to strain very soft 3D hydrogels (Elastic Modulus of ~100 Pa) from their circumference, and under live confocal imaging. Other methods are usually .......

Some figures included here have been adapted by permission from the Copyright Clearance Center: Springer Nature, Annals of Biomedical Engineering. Straining 3D hydrogels with uniform z-axis strains while enabling live microscopy imaging, A. Roitblat Riba, S. Natan, A. Kolel, H. Rushkin, O. Tchaicheeyan, A. Lesman, Copyright© (2019).

https://doi.org/10.1007/s10439-019-02426-7