Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

Summary

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.

Abstract

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.

Introduction

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

Protocol

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 .......

Discussion

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 .......

Materials

Name	Company	Catalog Number	Comments
Alexa Fluor 546 carboxylic acid, succinimidyl ester	Invitrogen	A20002
Cell Medium (DMEM High Glucose)	Biological Industries	01-052-1A	Add 10% FBS, 1% PNS, 1% L-Glutamine, 1% Sodium Pyruvate
Cover Slip #1.5	Bar-Naor Ltd.	BN72204-30	22×40 mm
DIMETHYL SULPHOXIDE 99.5% GC DMSO	Sigma-Aldrich Inc.	D-5879-500 ML
Dulbecco's Phosphate-Buffered Saline	Biological Industries	02-023-1A
EVICEL Fibrin Sealant (Human)	Omrix Biopharmaceuticals	3902	Fibrinogen: 70 mg/mL, Thrombin: 800-1200 IU/mL
Fibrinogen Buffer	N/A		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)	Invitrogen	F8820	Orange (540/560) Provided as suspension (2% solids) in water plus 2 mM sodium azide
High-Temperature Silicone Rubber	McMaster-Carr	3788T41	580 µm-thick 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)	GE Healthcare	17-1408-01
HIVAC-G High Vacuum Sealing Compound	Shin-Etsu Chemical Co., Ltd.	HIVAC-G 100
ImageJ FIJI software39	National Institute of Health, Bethesda, MD	Version 1.8.0_112
Microcontroller (Adruino Uno + Adafruit Motorshield v2.3)	Arduino/Adafruit	Arduino-DK001/Adafruit-1438
MicroVL 21R Centrifuge	Thermo Scientific	75002470
Parafilm	Bemis	PM-996
Primovert Light Microscope	Carl Zeiss Suzhou Co., Ltd.	491206-0011-000
SCyUS CAD (Solidworks)	Dassault Systèmes	N/A
SCyUS Code37	N/A	N/A
Servomotor - TowerPro SG-5010	Adafruit	155
SL 16R Centrifuge	Thermo Scientific	75004030	For 50 mL tubes
Sterile 10 cm non-culture plates	Corning	430167
Thrombin buffer	N/A		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%)	Biological Industries	03-052-1B
USB Cable (Type B Male to Type A Male)	N/A	N/A
Zeiss LSM 880 Confocal Microscope	Carl Zeiss AG	2811000417
ZEN 2.3 SP1 FP3 (black)	Carl Zeiss AG	Release Version 14.0.0.0