Name: controlled strain of 3d hydrogels under live microscopy imaging
Uploaded: 2020-12-04T13:00:00
Description: Watch this Scientific Journal Video about Controlled Strain of 3D Hydrogels under Live Microscopy Imaging at JoVE.com

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&#169; (2019).
https://doi.org/10.1007/s10439-019-02426-7

1. Solution preparation (to be performed in advance)
<ol>
	<li>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.
	<ol>
		<li>Add 38 &#956;L 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 ...

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 ...

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&#8217;s fibrous mesh structure has intriguing mechanical properties, such as non-linear elasticity, non-affine deformation and plastic deformations7,8,9,10,11,12. These properties impact how cells and their surrounding microenvironment respond to external mechanical forces13,14. Understanding how the ECM and tissues respond to mechanical forces will enable progress in the field of tissue engineering and in the development of more accurate computational and theoretical models.
Most common methods to mechanically stretch samples have focused on cell-laden 2D substrates to explore the effects on cell behavior. These include, for example, applying strain to polydimethylsiloxane (PDMS) substrates and analyzing cell reorientation angles in relation to the stretch direction15,16,17,18,19. Yet, methods investigating the response of 3D cell-embedded hydrogels to external stretch, a situation that more closely mimics tissue microenvironment, are more limited. Advances toward 3D stretching methods are of particular importance because cells behave differently on 2D substrates when compared to 3D matrices20. These behaviors include cellular realignment, protein expression levels, and migration patterns21,22,23.
Methods and devices that allow for 3D sample stretching include both commercially available24,25,26,27,28 and those developed for laboratory research29. These methods use distensible silicone tubes30, multi-well chambers31, clamps26,32, bioreactors11,33, cantilevers34,35,36, and magnets37,38. Some techniques generate stretch that locally deforms 3D hydrogels, for example by pulling needles from two single points in the gel5, while others allow for deformation of the entire bulk of the gel16. Moreover, most of these systems focus on analysis of the strain field in the X-Y plane, with limited information on the strain field in the Z-direction. Additionally, only a handful of these devices are capable of microscopic in situ imaging. The main challenge with in situ high-magnification imaging (e.g., confocal microscope) is the limited working distance of a few hundred microns from the objective lens to the sample. Devices that do allow live imaging during stretch sacrifice the uniformity of strain in the Z-axis or are relatively complex and difficult to reproduce in other laboratories39,40.
This approach to stretch 3D hydrogels allows for static or cyclical uniaxial strain during live confocal microscopy. The stretching device (referred to as &#8216;Smart Cyclic Uniaxial Stretcher &#8211; SCyUS&#8217;) is constructed using 3D printed parts and low-cost hardware, allowing easy reproduction in other labs. Attached to the device is a commercially available silicone rubber with a geometric cut-out in its center. Hydrogel components are polymerized to fill the cut-out. During polymerization, biological hydrogels, such as fibrin or collagen, naturally adhere to the interior walls of the cut-out. Using the SCyUS, the silicone strip is uniaxially stretched, transferring controlled strains to the embedded 3D hydrogel41.
This system allows for a unique combination of features and advantages compared to other existing methods. First, the system allows uniaxial stretching of thick 3D soft hydrogels (&#62;100 &#181;m thick, &#60;1 kPa stiffness) from their periphery, with Z-homogenous deformation throughout the hydrogel. These hydrogels are too soft to be gripped and stretched by conventional tensile techniques. Second, the stretching device can be easily replicated in other labs since 3D printing is readily available to researchers and the electronics used in the design are low-cost. Third, and perhaps the most attractive feature, the geometry and the size of the cut-out in the silicone strip can be easily manipulated, allowing for tunable strain gradients and boundary conditions as well as the use of a variety of sample volumes, down to a few microliters.
The presented protocol consists of molding fibrin gel into ~2 mm diameter disks in 0.5 mm thick silicone rubber strips proceeded by uniaxial stretch under live confocal microscopy. The following discusses in detail the experimental procedures for measuring and analyzing the strains acting on the geometric cut-out, the internal strains developed in the hydrogel, as well as resulting fiber alignment after various stretch manipulations. Finally, the possibility of embedding cells in the hydrogel and exposing them to controlled external stretch is discussed.

<table>
	<tbody>
		<tr>
			<td>Alexa Fluor 546 carboxylic acid, succinimidyl ester</td>
			<td>Invitrogen</td>
			<td>A20002</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Medium (DMEM High Glucose)</td>
			<td>Biological Industries</td>
			<td>01-052-1A</td>
			<td>Add 10% FBS, 1% PNS, 1% L-Glutamine, 1% Sodium Pyruvate</td>
		</tr>
		<tr>
			<td>Cover Slip #1.5</td>
			<td>Bar-Naor Ltd.</td>
			<td>BN72204-30</td>
			<td>22&#215;40 mm</td>
		</tr>
		<tr>
			<td>DIMETHYL SULPHOXIDE 99.5% GC DMSO</td>
			<td>Sigma-Aldrich Inc.</td>
			<td>D-5879-500 ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td>Biological Industries</td>
			<td>02-023-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>EVICEL Fibrin Sealant (Human)</td>
			<td>Omrix Biopharmaceuticals</td>
			<td>3902</td>
			<td>Fibrinogen: 70 mg/mL, Thrombin: 800-1200 IU/mL</td>
		</tr>
		<tr>
			<td>Fibrinogen Buffer</td>
			<td>N/A</td>
			<td></td>
			<td>Recipe for 1L: 7g NaCl, 2.94g trisodium citrate dihydrate, 9g glycine, 20g arginine hydrochloride &#38; 0.15g calcium chloride dihydrate. Bring final volume to 1L with PuW (pH 7.0-7.2)</td>
		</tr>
		<tr>
			<td>Fluorescent micro-beads FluoSpheres (1 &#181;m)</td>
			<td>Invitrogen</td>
			<td>F8820</td>
			<td>Orange (540/560) 
			Provided as suspension (2% solids) in water plus 2 mM sodium azide</td>
		</tr>
		<tr>
			<td>High-Temperature Silicone Rubber</td>
			<td>McMaster-Carr</td>
			<td>3788T41</td>
			<td>580 &#181;m-thick 
			E = 1.5 Mpa 
			Poisson Ratio = 0.48 
			Tensile Strength = 4.8 MPa 
			Upper limit of stretch = +300% engineering strain</td>
		</tr>
		<tr>
			<td>HiTrap desalting column 5 mL (Sephadex G-25 packed)</td>
			<td>GE Healthcare</td>
			<td>17-1408-01</td>
			<td></td>
		</tr>
		<tr>
			<td>HIVAC-G High Vacuum Sealing Compound</td>
			<td>Shin-Etsu Chemical Co., Ltd.</td>
			<td>HIVAC-G 100</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ FIJI software39</td>
			<td>National Institute of Health, Bethesda, MD</td>
			<td>Version 1.8.0_112</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcontroller (Adruino Uno + Adafruit Motorshield v2.3)</td>
			<td>Arduino/Adafruit</td>
			<td>Arduino-DK001/Adafruit-1438</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroVL 21R Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75002470</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>Primovert Light Microscope</td>
			<td>Carl Zeiss Suzhou Co., Ltd.</td>
			<td>491206-0011-000</td>
			<td></td>
		</tr>
		<tr>
			<td>SCyUS CAD (Solidworks)</td>
			<td>Dassault Syst&#232;mes</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>SCyUS Code37</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Servomotor - TowerPro SG-5010</td>
			<td>Adafruit</td>
			<td>155</td>
			<td></td>
		</tr>
		<tr>
			<td>SL 16R Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75004030</td>
			<td>For 50 mL tubes</td>
		</tr>
		<tr>
			<td>Sterile 10 cm non-culture plates</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin buffer</td>
			<td>N/A</td>
			<td></td>
			<td>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)</td>
		</tr>
		<tr>
			<td>Trypsin EDTA Solution B (0.25%), EDTA (0.05%)</td>
			<td>Biological Industries</td>
			<td>03-052-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>USB Cable (Type B Male to Type A Male)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM 880 Confocal Microscope</td>
			<td>Carl Zeiss AG</td>
			<td>2811000417</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEN 2.3 SP1 FP3 (black)</td>
			<td>Carl Zeiss AG</td>
			<td>Release Version 14.0.0.0</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 (&#62;100 &#956;m) 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&#8217;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&#8217;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.

Representative data from static stretch of increasing magnitudes applied to the silicone strip carrying a 3D fibrin hydrogel, embedded with 1 &#956;m 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 (<strong c...

Watch this Scientific Journal Video about Controlled Strain of 3D Hydrogels under Live Microscopy Imaging at JoVE.com

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 ...

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