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 &#176;C, 5%CO2. LR-MSCs were passaged 3-4 times to ensure a population of cells with relatively homogenous growth characteristics for the migration assay.
1. Preparing a Confluent Monolayer
<ol>
	<li>To detach MSCs from standard 100 mm culture dishes, wash cells with 10 ml of 37 &#176;C pre-warmed PBS, aspirate the PBS and add 4 ml of trypsin (0.25%). Incubate 3-5 min in a 37 &#176;C incubator. Aspirate the cells into a conical tube, add equal volume of complete media and centrifuge at 300 g for 5 min to pellet down the cells. 
	Note: Longer incubation in trypsin can alter the cell surface receptors and potentially affect migration.</li>
	<li>Aspirate media and resuspend cells in fresh complete media. Count cells, excluding dead cells using trypan blue exclusion and plate 500,000 mesenchymal stromal cells in 4 ml of complete media on a 60 mm cell culture dish. 
	Note: Depending on the source of the cells, tests may be necessary to determine the optimal number of cells needed.</li>
	<li>Incubate cells until plates are 90% confluent for optimal cell attachment and proliferation, usually 48-72 hr for MSCs. 
	Note: 100% confluence is not recommended, since many anchorage-dependent cells experience contact inhibition, which may alter their migratory capacity. Plates with less than 80% confluence will result in unusable photos because the image analysis software will interpret gaps between cells as part of the perimeter of the scratch (see Figure 1D).</li>
	<li>After the cells have reached 90% confluence, damage the cells as appropriate for the application. To assess cell damage after exposure to soluble cigarette smoke extract (CSE), generate 100% CSE by drawing the smoke from 1 University of Kentucky research cigarette (3R4F) through 25 ml of complete media in a B&#252;chner flask over 2 min 18.</li>
	<li>Incubate the cells for 24 hr in 4 ml 1-4% CSE diluted in complete media. After incubation, wash the cells twice with 4 ml of warm sterile PBS, before replacing with 4 ml fresh complete media. 
	Note: Damaging cells with high concentrations of cigarette smoke extract (&#62;5%) may substantially reduce confluence.</li>
</ol>
2. Creating the Scratch
<ol>
	<li>In a sterile environment, remove the lid from the 60 mm plate of damaged MSCs, and lay a straight edge (for example a sterilized plastic ruler) across the top rim of the plate. Without removing the media, use a sterile 200 &#181;l pipet tip guided by the sterile straight-edge to make one to four parallel scratches across the plate approximately 5 mm apart and 50 mm long. 
	Note: A straight edge minimizes the amount of rotational adjustments needed to the plate when it is on the microscope stage. It is imperative that the scratches appear completely vertical (or horizontal) on the screen. Generating multiple scratches on a 60 mm plate does not alter the microenvironment in a way that affects cell migration (data not shown), but may be a consideration for other cell types. The use of smaller wells is not recommended since the variability of cells growing close to the edge of the well becomes more pronounced and may affect uniform generation of scratches.</li>
	<li>Aspirate the media and gently wash the plates with 2 ml of pre-warmed complete media to prevent cells from adhering to the center of the scratch, and to remove cells that have been loosened by the scratching. Replace with 4 ml of fresh complete media.</li>
	<li>Using a sterile permanent marker, label each scratch from 1-4 on the underside of the plate at location that will not interfere with imaging the scratches. 
	Note: It is important that images from the same scratch are compared before and after migration, since there can be variation in scratch width.</li>
</ol>
3. Taking Initial Images of the Scratch
Note: If the scratch assay is performed on multiple plates, it is recommended that initial images of all the scratches on a single plate are taken before scratches are made on the next plate.
<ol>
	<li>Use an inverted phase contrast microscope with a camera to generate the images needed for analysis.</li>
	<li>Position the culture plate on the microscope, and use the low power objective (10X) to locate scratch #1. If condensation on the lid of the culture plate is impeding a clear image of the cells, use a heated stage set to 37 &#176;C.</li>
	<li>Keeping the scratch completely horizontal and in the center of the field of view, obtain as many images as necessary to encompass 90% of the scratch&#39;s length. Obtain images of distinct portions of the scratch with no overlap between images. 
	Note: The refraction of light at the edges of the culture dish overexposes the images, making them impossible to analyze. Therefore, do not take images from the first and last 5% of the scratch&#39;s length.</li>
	<li>Save images from each scratch in a separate folder.</li>
	<li>Repeat steps 3.2-3.4 for scratches 2, 3, and 4.</li>
	<li>Return plates to the incubator immediately after initial imaging. Let cells migrate for 4-7 hr. 
	Note: 7 hr is optimal when assessing MSCs damaged with CSE. Incubating cells for greater than 7 hr is not recommended because cell proliferation can act as a confounding variable for cell migration (see Optional Note 8.2 below).</li>
</ol>
4. Analyzing Initial Scratch Images
<ol>
	<li>Download ImageJ and install the Wound Healing Tool plugin (refer to the Table of Materials/Equipment).</li>
	<li>Open the image file in ImageJ, then click &#34;Process&#34; and &#34;Find Edges&#34; to highlight the cells surrounding the scratch for the Wound Healing Tool to calculate the scratch area (Figure 2B). Next, click &#34;Image&#34;, &#34;Adjust&#34;, &#34;Color Threshold&#34; and in the dropdown menu labeled &#34;Threshold Color&#34; change the value to &#34;B&#38;W.&#34;</li>
	<li>Within the &#34;Color Threshold&#34; menu, adjust both brightness threshold sliders until optimal contrast between the scratch and cell front is achieved. To allow for the greatest range when trying to create contrast in the image, move the bottom slider completely to the right (completely black image), and then adjust the top slider to generate maximum contrast.</li>
	<li>Slowly adjust the top brightness slider from left to right until the image cleanly displays the contour of the scratch (Figure 2C). Once the edges of the scratch have been identified, click &#34;More Tools&#34; in the ImageJ toolbar, select &#34;MRI_Wound_Healing_Tool&#34; from the dropdown menu, and press the &#34;m&#34; button (that will appear in the ImageJ toolbar) to highlight the scratch area and output the computed area in a results pop-out window (Figure 2D).</li>
	<li>Copy and paste all numbers from the results window (Figure 2E) into an excel spreadsheet. Make sure to separate data from individual scratches.</li>
</ol>
5. Taking Final Images of the Scratch
<ol>
	<li>After the cells have migrated (4-7 hr), repeat steps 3.2-3.4. Image the plates in the same order they were scratched so that cells on each plate have had equal time to migrate. 
	Note: The cells can be imaged at multiple time points, depending on their sensitivity to both temperature and pH changes. Alternatively, live cell imaging can used to follow the cells in real time as they migrate.</li>
</ol>
6. Analyzing Final Scratch Images
<ol>
	<li>Repeat steps 4.2-4.5 for final scratch images.</li>
</ol>
7. Calculating the Scratch Area to Quantify Migration
<ol>
	<li>Compute the mean pre-migration area by averaging the initial area values of the respective scratches.</li>
	<li>Compute the total amount of migration by subtracting the final migration area for each image, calculated in step 6, from the average pre-migration area (step 7.1). Generate a list of values for each scratch representing the change in migration.</li>
	<li>Pool the data from multiple scratches within the same test group (i.e. all plates and all scratches on cells exposed to 0% CSE). Use an Analysis of Variance test to analyze the differences between experimental groups.</li>
	<li>If the cells are slow to migrate and require a longer incubation time after scratching (greater than 10-12 hr), perform a BrDU or EdU incorporation assay 19 to determine whether cells are undergoing any proliferation during the period in which the migration assay is occurring. Choose a migration assay time to avoid significant cell proliferation.</li>
</ol>
8. Optional
<ol>
	<li>To assess whether significant cell senescence is occurring as a result of the scratching procedure, perform a senescence-associated Beta-Galactosidase assay on a test plate at predetermined times after scratching 19.</li>
</ol>

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The authors declare that they have no competing financial interests.

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.

<table border="0" cellpadding="0" cellspacing="0" width="1143">
	<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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JoVE Journal

Developmental Biology

16.9K Views.  College of the Holy Cross. 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.

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

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell migration. However, when explants are established, these cells also move collectively, maintaining many of the features which make individual keratocytes an attractive model to study migration: rapid rates of motility, extensive actin-rich lamellae with a perpendicular actin cable, and relatively constant speed and direction of migration. In early explants, the rapid interconversion of cells migrating individually with those migrating collectively allows the study of the role of cell-cell adhesions in determining the mode of migration, and emphasizes the molecular links between the two modes of migration. Cells in later explants lose their ability to migrate rapidly and collectively as an epithelial to mesenchymal transition occurs and genes associated with wound healing and inflammation are differentially expressed. Thus, keratocyte explants can serve as an in vitro model for the reepithelialization that occurs during cutaneous wound healing and can represent a unique system to study mechanisms of collective cell migration in the context of a defined program of gene expression changes. A variety of mutant and transgenic zebrafish lines are available, which allows explants to be established from fish with different genetic backgrounds. This allows the role of different proteins within these processes to be uniquely addressed. The protocols outlined here describe an easy and effective method for establishing these explant cultures for use in a variety of assays related to collective cell migration.

Zebrafish keratocytes migrate in cell sheets from explants and provide an in vitro model for the study of the mechanisms of collective cell migration in the context of epithelial wound healing. These protocols detail an effective way to establish primary explant cultures for use in collective cell migration assays.

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell ...

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms including wound healing disorder. The physiological process of wound healing is highly complex. The formation of granulation tissue is a key step in this process resulting in a preliminary wound closure and providing a network of new capillary blood vessels &#8211; either through vasculogenesis (novel formation) or angiogenesis (sprouting of existing vessels). Both vasculo- and angiogenesis require functional, directed migration of endothelial cells. Thus, investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of chemical induced wound healing disorders and to potentially identify novel strategies for therapeutic intervention.
We assessed impaired wound healing after alkylating agent exposure and tested potential candidate compounds for treatment. We used a set of techniques outlined in this protocol. A modified Boyden chamber to quantitatively investigate chemokinesis of EEC is described. Moreover, the use of the wound healing assay in combination with track analysis to qualitatively assess migration is illustrated. Finally, we demonstrate the use of the fluorescent dye TMRM for the investigation of mitochondrial membrane potential to identify underlying mechanisms of disturbed cell migration. The following protocol describes basic techniques that have been adapted for the investigation of EEC.

Investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of certain illnesses and to potentially identify novel strategies for therapeutic intervention. The following protocol describes techniques to assess cell migration that have been adapted for the investigation of EEC.

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms ...

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via their strong adherence to tissue culture plastic and then further expanded in vitro, most commonly using fetal bovine serum (FBS). Since FBS can cause MSCs to become immunogenic, its presence in MSC cultures limits both clinical and experimental applications of the cells. Therefore, studies employing chemically defined xeno-free (XF) media for MSC cultures are extremely valuable. Many beneficial effects of MSCs have been attributed to their ability to regulate inflammation and immunity, mainly through secretion of immunomodulatory factors such as tumor necrosis factor-stimulated gene 6 (TSG6) and prostaglandin E2 (PGE2). However, MSCs require activation to produce these factors and since the effect of MSCs is often transient, great interest has emerged to discover ways of pre-activating the cells prior to their use, thus eliminating the lag time for activation in vivo. Here we present protocols to efficiently activate or prime MSCs in three-dimensional (3D) cultures under chemically defined XF conditions and to administer these pre-activated MSCs in vivo. Specifically, we first describe methods to generate spherical MSC micro-tissues or &#39;spheroids&#39; in hanging drops using XF medium and demonstrate how the spheres and conditioned medium (CM) can be harvested for various applications. Second, we describe gene expression screens and in vitro functional assays to rapidly assess the level of MSC activation in spheroids, emphasizing the anti-inflammatory and anti-cancer potential of the cells. Third, we describe a novel method to inject intact MSC spheroids into the mouse peritoneal cavity for in vivo efficacy testing. Overall, the protocols herein overcome major challenges of obtaining pre-activated MSCs under chemically defined XF conditions and provide a flexible system to administer MSC spheroids for therapies.

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or 'spheroids' of MSCs primed under xeno-free conditions for experimental and clinical applications.

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor metastasis. In this assay, cells are grown to form a confluent monolayer and a mechanical wound is created by scratching with a device. Then the migration rate of the cells towards the denuded area can be monitored by imaging. Our 8-channel mechanical wounder is designed to tackle most of the problems associated with the cell migration assay. Firstly, our wounder can be easily sterilized by autoclaving or with common disinfectants. Secondly, the individual adjustable pins allow even contact with the cell culture plate so that sharp and reproducible wounds can be created. Thirdly, the guiding bars on both sides of the wounder ensure consistent wounding position in each well. The use of disposable plastic pipette tips for wounding can further provide better handling of the wounder as well as to minimize cross-contamination. In conclusion, our cell wounder can provide researchers with a user friendly and reproducible device for performing the cell migration assay using the standard 96-well culture plate.

Here, we present a protocol to perform a semi-high throughput cell migration assay on a 96-well cell culture plate. This protocol is a fast, simple and economical method to create consistent scratch wounds on a cell monolayer.

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.
Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC&#39;s wider application.
In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N+ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture can be recorded under video microscopy. For analyzing cell migration in the developing brain, slice culture is commonly used to observe cell migration parallel to the slice section, such as radial cell migration. However, limited information can be obtained from the slice culture method to analyze cell migration perpendicular to the slice section, such as tangential cell migration. Here, we present the protocols for time-lapse imaging to visualize tangential cell migration in the developing chick optic tectum. A combination of cell labeling by electroporation in ovo and a subsequent flat-mount culture on the cell culture insert enables detection of migrating cell movement in the horizontal plane. Moreover, our method facilitates detection of both individual cell behavior and the collective action of a group of cells in the long term. This method can potentially be applied to detect the sequential change of the fluorescent-labeled micro-structure, including the axonal elongation in the neural tissue or cell displacement in the non-neural tissue.

We describe the methods for fluorescent labeling of tangentially migrating cells by electroporation, and for time-lapse imaging of the labeled cell movement in a flat-mount culture in order to visualize migrating cell behavior in the developing chick optic tectum.

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...