Name: optimization of the wound scratch assay to detect changes in murine mesenchymal stromal cell migration after damage by soluble cigarette smoke extract
Uploaded: 2015-12-03T10:15:00
Description: Watch this Scientific Journal Video about Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract at JoVE

We are grateful to the other members of the lab for their technical assistance in developing the CSE damage assay, and their support and advice during the development of this assay. In addition, we are grateful to the Holy Cross Fenwick Scholar Committee for their support of Nicholas Cormier, and the Holy Cross College Honors program for their support of Alexander Yeo.

Note: For this study, lung mesenchymal stromal cells (LR-MSCs) from distal lung tissue were isolated either based on their expression of cell surface markers (CD45neg, CD31neg, Sca-1high, Epcamneg) 14,15, using explant out-growth 16, or using enzymatic digestion 17. Adherent LR-MSCs were cultured in DMEM with high glucose and no glutamine, 15% FBS, 1x antibiotic/antimycotic, and 2 mM glutamine (henceforth complete media) and incubated at 37 &#1...

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The protocol described here provides a quantitatively robust, standardized method to perform and analyze scratch assays. Simple scratch assays are routinely used in many different areas of study to examine cellular migration. However, traditionally the scratch assay has lacked a standardized set-up and quantification protocol, which has led to issues with reproducibility 7,10. Many of the modifications and optimizations for improving the scratch assay have reduced these issues, including individual live-cell i...

Cell migration is highly coordinated and vital to many physiological processes such as tissue development, repair, and regeneration, as well as pathological processes such as cancer metastasis and arteriosclerosis 1. An understanding of cell migration is particularly relevant to emerging therapies used to repair damaged tissues and treat pathological conditions, including cell transplantation technologies and artificial tissue grafting 2. Given the current interest in the role of mesenchymal stromal cells (MSCs) in mediating tissue repair 3, quantifying the migratory capacity of these cells using an assay that is rigorously quantifiable, adaptable, and cost-effective is of interest. Importantly, such an assay must be sufficiently sensitive to detect relatively subtle changes in cellular migratory capacity after damage. Current methods for quantifying cell migration, including scratch assays, trans-well migration assays (Boyden chambers), micropillar arrays, and cell exclusion zone assays possess a range of limitations in reproducibility, customizability, quantification, and cost-effectiveness 1,4,5. The optimized scratch assay described here demonstrates robust outcomes, quantifiable and image-based analysis capabilities, cost-effectiveness, and adaptability to other applications.
Scratch assays have been used in multiple capacities to assess cell migration and proliferation under different experimental conditions 5. The assay entails seeding the designated cells, growing them to complete or near confluence, and scratching the resultant monolayer with a sterile needle or pipet tip 6. Analysis is most commonly performed by comparing the width of the scratch over multiple time points at randomly selected locations 7-9. Despite its prominent use, the scratch assay is confounded by issues with reproducibility and quantification. Variability in generation of the scratch not only alters the microenvironment of the cells, but can also impede cell migration by damaging the plate surface and underlying extracellular-matrix 5. Assays are frequently conducted over 7 to 12 hr, however for cell lines displaying slower migration and longer assay times, proliferation becomes a confounding variable 7,10. Lastly, senescent cells generated by the scratching process can release factors that interfere with the extracellular signaling required to close the gap in the monolayer 1. Optimizing the scratch assay requires creation of a consistent gap that does not interfere with surface properties, minimizing assay time length, and preventing unwanted cell death during the manipulation. The stopper based assay is an optimization of a cell exclusion zone assay. This assay utilizes a stopper placed in the middle of the well that excludes cell growth, but allows cells to be plated around the central exclusion zone. To assess migration, the stopper is removed, and the resultant exclusion zone provides a surface for migration to occur. However, this assay is difficult to customize or adapt 10 and for some applications, this technique can also be cost prohibitive.
In contrast to scratch assays and their derivatives, trans-well migration assays (or Boyden chamber assays) assess migration by quantifying the number of cells that move from one chamber, through a microporous filter membrane, into a chamber containing chemotactic agents 8,11,12. This technique has limited utility for adherent cells like MSCs because following migration through the porous membrane, cells adhere to the membrane side exposed to the chemotactic agents, and can be hard to accurately quantify. While the assay is able to examine some three-dimensional migration patterns, the restricted cell types for which it is able to accurately quantify cell migration limit its utility 10. Another alternative to scratch assays uses a micro-pillar array, which measures cellular motility through a three-dimensional space using the ability of cells to deform and migrate into the array as a surrogate. Polydimethlysiloxane (PDMS) elastomers cured over a precise mold and treated with ozone and fibronectin produces a homogenous and non-degradable microenvironment. Micro-pillar spacing can also be varied as needed to gauge the ability of cells to enter the array 4. The mold is created through deep reactive ion etching of silicon wafers to create a negative version of the high aspect-ratio array 13. While the assay is strengthened by its customizability, ability to model three-dimensional migration, and analysis through direct visualization of migrating cells, difficulty in creating micro-pillar arrays economically impedes its widespread use.
The optimized scratch assay described in this protocol provides an efficient, cost-effective method for producing consistent scratches that can be analyzed using freely available software. Instead of simple width measurements made across the scratch before and after cell migration, the software enables the user to determine total scratch areas before and after migration. This advancement limits the issue of trying to determine where the scratch the width measurements should be taken, and whether the width of the scratch is uniform along it&#39;s length. In addition, careful optimization of cell numbers, cell confluence and the type and degree of damage inflicted on the cells is discussed in order to further optimize the assay.

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	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">DMEM (high glucose, no added glutamine)</td>
			<td style="width:221px;">Life Technologies</td>
			<td style="width:88px;">31053-036</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Fetal Bovine Serum, Thermo Scientific HyClone</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td style="width:88px;">SH3091002</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Antibiotic/antimycotic, Thermo Scientific HyClone&#160;</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td style="width:88px;">SV3007901</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:527px;">Glutamine, 200mM, Thermo Scientific HyClone</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td style="width:88px;">SH3003401</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">TypeLE cell dissociation reagent</td>
			<td style="width:221px;">Life Technologies</td>
			<td style="width:88px;">12563-01</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Dulbecco&#39;s Phosphate Buffered Saline, Thermo Scientific HyClone&#160;</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td style="width:88px;">SH3002802</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Falcon standard 60mm tissue culture dishes</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td style="width:88px;">08-772B</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Fisherbrand Sterile 200ul pipet tips</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td>02-707-502</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Inverted light microscope with camera attachment</td>
			<td style="width:221px;">Variable</td>
			<td style="width:88px;"></td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:527px;">ImageJ software&#160;</td>
			<td style="width:221px;">http://imagej.nih.gov/ij/</td>
			<td style="width:88px;"></td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:527px;">MRI_Wound_Healting plugin</td>
			<td style="width:221px;"><a href="http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Wound_Healing_Tool">http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Wound_Healing_Tool</a></td>
			<td style="width:88px;"></td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Statistical analysis software</td>
			<td style="width:221px;">Microsoft Excel</td>
			<td style="width:88px;"></td>
			<td style="width:307px;"></td>
		</tr>
	</tbody>
</table>

optimization of the wound scratch assay to detect changes in murine mesenchymal stromal cell migration after damage by soluble cigarette smoke extract

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and inflammatory responses. Given the current interest in the role of mesenchymal stromal cells in mediating tissue repair, we are interested in quantifying the migratory capacity of these cells, and understanding how migratory capacity may be altered after damage. Optimization of a rigorously quantitative migration assay that is both easy to customize and cost-effective to perform is key to answering questions concerning alterations in cell migration in response to various stimuli. Current methods for quantifying cell migration, including scratch assays, trans-well migration assays (Boyden chambers), micropillar arrays, and cell exclusion zone assays, possess a range of limitations in reproducibility, customizability, quantification, and cost-effectiveness. Despite its prominent use, the scratch assay is confounded by issues with reproducibility related to damage of the cell microenvironment, impediments to cell migration, influence of neighboring senescent cells, and cell proliferation, as well as lack of rigorous quantification. The optimized scratch assay described here demonstrates robust outcomes, quantifiable and image-based analysis capabilities, cost-effectiveness, and adaptability to other applications.

The scratch assays presented here were performed using murine lung resident mesenchymal stromal cells (LR-MSCs) and isolated as referenced in the protocol notes. The LR-MSCs were seeded at a density of 500,000 cells in a 60 mm tissue culture dish, and grown to 90% confluence in 48 hr. To generate damage, 4% CSE (described above) was incubated with the cells for 24 hr following the initial seeding period but before the scratch assay. For each assay, the scratch was generated using a 200 &#181;l pipet tip. Initial images w...

Watch this Scientific Journal Video about Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract at JoVE

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

The capacity to migrate is a key function of many different cell types, including mesenchymal stromal cells (MSCs). However, quantifying alterations in migratory capacity after damage is challenging. This protocol describes an easily adaptable migration assay that uses rigorous statistics to quantify changes in MSC migratory capacity after damage.

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and ...

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