Published: December 4th, 2020
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 formation4,5,6. 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)
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).
|Alexa Fluor 546 carboxylic acid, succinimidyl ester
|Cell Medium (DMEM High Glucose)
|Add 10% FBS, 1% PNS, 1% L-Glutamine, 1% Sodium Pyruvate
|Cover Slip #1.5
|DIMETHYL SULPHOXIDE 99.5% GC DMSO
|Dulbecco's Phosphate-Buffered Saline
|EVICEL Fibrin Sealant (Human)
|Fibrinogen: 70 mg/mL, Thrombin: 800-1200 IU/mL
|Recipe for 1L: 7g NaCl, 2.94g trisodium citrate dihydrate, 9g glycine, 20g arginine hydrochloride & 0.15g calcium chloride dihydrate. Bring final volume to 1L with PuW (pH 7.0-7.2)
|Fluorescent micro-beads FluoSpheres (1 µm)
Provided as suspension (2% solids) in water plus 2 mM sodium azide
|High-Temperature Silicone Rubber
E = 1.5 Mpa
Poisson Ratio = 0.48
Tensile Strength = 4.8 MPa
Upper limit of stretch = +300% engineering strain
|HiTrap desalting column 5 mL (Sephadex G-25 packed)
|HIVAC-G High Vacuum Sealing Compound
|Shin-Etsu Chemical Co., Ltd.
|ImageJ FIJI software39
|National Institute of Health, Bethesda, MD
|Microcontroller (Adruino Uno + Adafruit Motorshield v2.3)
|MicroVL 21R Centrifuge
|Primovert Light Microscope
|Carl Zeiss Suzhou Co., Ltd.
|SCyUS CAD (Solidworks)
|Servomotor - TowerPro SG-5010
|SL 16R Centrifuge
|For 50 mL tubes
|Sterile 10 cm non-culture plates
|Recipe for 1L: 20g mannitol, 8.77g NaCl, 2.72g sodium acetate trihydrate, 24 mL 25% Human Serum Albumin, 5.88g calcium chloride. Bring final volume to 1L with PuW (pH 7.0)
|Trypsin EDTA Solution B (0.25%), EDTA (0.05%)
|USB Cable (Type B Male to Type A Male)
|Zeiss LSM 880 Confocal Microscope
|Carl Zeiss AG
|ZEN 2.3 SP1 FP3 (black)
|Carl Zeiss AG
|Release Version 18.104.22.168
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