Name: in vitro scratch assay to demonstrate effects of arsenic on skin cell migration
Uploaded: 2019-02-23T13:00:00
Duration: 9 min 24 s
Description: Watch this Scientific Journal Video about In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration at JoVE.com

Research reported in this publication was supported by the National Institute on Minority Health and Health Disparities of the National Institutes of Health under Award Number U54MD012388. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

<ol>
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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+temporal+variability+of+arsenic+in+groundwater+used+for+drinking+water+supply+in+the+United+States.">Factors affecting temporal variability of arsenic in groundwater used for drinking water supply in the United States.</a> Science of the Total Environment. , (2014).</li><li>Dummer, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geostatistical+modeling+of+arsenic+in+drinking+water+wells+and+related+toenail+arsenic+concentrations+across+Nova+Scotia,+Canada.">Geostatistical modeling of arsenic in drinking water wells and related toenail arsenic concentrations across Nova Scotia, Canada.</a> Science of the Total Environment. , (2014).</li><li>Sorg, T. J., Chen, A. S., Wang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arsenic+species+in+drinking+water+wells+in+the+USA+with+high+arsenic+concentrations.">Arsenic species in drinking water wells in the USA with high arsenic concentrations.</a> Water Research. 48, 156-169 (2014).</li><li>Beamer, P. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+Children's+Urinary+CC16+Levels+with+Arsenic+Concentrations+in+Multiple+Environmental+Media.">Association of Children's Urinary CC16 Levels with Arsenic Concentrations in Multiple Environmental Media.</a> International Journal of Environmental Research and Public Health. 13, (2016).</li><li>Pomari, E., Dalla Valle, L., Pertile, P., Colombo, L., Thornton, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracrine+sex+steroid+synthesis+and+signaling+in+human+epidermal+keratinocytes+and+dermal+fibroblasts.">Intracrine sex steroid synthesis and signaling in human epidermal keratinocytes and dermal fibroblasts.</a> The FASEB Journal. 29 (2), 508-524 (2015).</li><li>Gotsulyak, Y., Kosach, R., Cherednyk, V., Tykhonkova, O., Khoruzhenka, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+cell+motility+evaluation+in+scratch+assay.">Optimization of cell motility evaluation in scratch assay.</a> Biopolymers and Cell. 30, 223-228 (2014).</li><li>Goetsch, K. P., Niesler, C. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+the+scratch+assay+for+in+vitro+skeletal+muscle+wound+healing+analysis.">Optimization of the scratch assay for in vitro skeletal muscle wound healing analysis.</a> Analytical Biochemistry. 411, 158-160 (2011).</li><li>Harishkumar, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revealing+the+mechanism+of+in+vitro+wound+healing+properties+of+citrus+tamurana+extract.">Revealing the mechanism of in vitro wound healing properties of citrus tamurana extract.</a> BioMed Research International. 13, (2013).</li><li>Johnston, S. T., Simpson, M. J., McElwain, D. L. 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The authors have nothing to disclose.

Application of this assay provides a high-throughput and timely evaluation of cellular migration and may directly translate to complex physiological systems5,6,7. For this reason, the proposed bench-top model has been tuned and practiced to evaluate the effects of various therapeutic agents and environmental toxins on wound healing6. The concentrations of arsenic used in the current study were deemed environmentally relevant through extensive literature search of known exposure levels, and from retrospective review of various data bases14,15,16,17,18,19,20,21. Use of this in vitro scratch assay demonstrated that exposure to an arsenic concentration within the high, but environmentally relevant range, delayed wound closure (cellular migration).
This bench top model was also employed to isolate and study an individual cell line (fibroblast) to further understand how fibroblast migration contributes to wound healing outcomes. Furthermore, it is difficult to study the functions of an individual cell line in complex in vivo systems with many other cells and tissues present that can interfere with the analysis. Additionally, the assay provides a means of achieving semi-quantitative percent wound closure data that can be used to target specific cellular mechanisms through which compounds may influence wound healing. For example, cells used in the scratch assay can be collected and stored in a desired reagent and used in gene expression (signal mRNA) or protein expression assays22. This information can be useful to the investigator if they seek to understand what signals or proteins may be largely contributing to changes in cellular migration in the presence of varying treatments (i.e., arsenic)22.
Human neonatal dermal fibroblasts were selected for this work based on their prominent role in wound healing. However, this scratch assay protocol has been implemented using various cell types and for a range of research purposes. For example, the scratch assay has been adopted into the field of oncology for studying migration inhibition of chemotherapy drugs23; for skeletal muscle cell migration, proliferation, and diffusion24; to study the effect of plant extracts on cellular migration25; and to understand how keratinocyte migration and proliferation contribute to wound healing9. Additionally, a previous paper by Pinto et al. evaluated the effects of uranium (U) and platelet-rich plasma (PRP) on hDFn migration using the scratch assay6. The purpose of this work was to understand the extent to which this assay could test the effect of an agent thought to slow cellular migration (U), as well as an agent thought to speed up cellular migration (PRP). As can be seen in the aforementioned work and research by other investigators, application of the scratch assay has high potential for use in various experimental models26. In spite of the detail provided within the methods, variance should be expected between investigators and experiments. For example, cellular migration rates will likely fluctuate based on the cell line in use, in addition to the required micronutrients needed for unique cell types. Many of the important observations to aid in cell yield and viability are listed as NOTEs throughout this protocol. However, it is strongly recommended that investigators follow manufacturer&#39;s recommendations for optimal growth conditions, and to use this method as supplementary instruction for general cell culture work.
Although the scratch assay is extremely versatile for study of cellular activity; unforeseen biases may arise in measuring techniques. For example, when using ImageJ to manually trace cell migration fronts, variation may exist among users, which can limit accuracy and representative data collection. It should also be noted that migration fronts should be read blind to the treatment to minimize user bias. Furthermore, manually identifying migration fronts may result in miscalculation of average distance traveled due to pseudopod formation by cells and their ability to reach out to form focal adhesions, which can be visually misleading. In like manner, the use of fluorescent &#34;cell tracker&#34; dyes on cell surface or nuclear proteins to detect migration may unintentionally fix cells resulting in inaccuracies when measuring cellular migration over time. It is recommended that appropriate positive and/or negative controls be enrolled within the experiment to completely asses the effect of treatment on the cell lines. Practice of this assay must be completed in recognition of its limitations. This assay does not guarantee that cellular migration results obtained from in vitro experimentation will directly translate to in vivo outcomes. Wound healing and cellular migration in a complex living system involves a multitude of cell types and molecular signals; each of which has the potential to influence healing responses.
In summary, the scratch assay has been found to provide high-throughput screening of cellular migration in response to changing environments6,25. We specifically utilized this protocol to successfully detect changes in cellular migration as a result of arsenic exposure at various environmentally relevant doses. These data have provided justification to use the scratch assay to further study and evaluate the mechanistic pathways of cellular migration in this assay and how these pathways are altered by arsenic exposure.

Wound healing consists of a complex series of overlapping steps that involve various cell types, signaling molecules, and extracellular matrix components1. Together, these components work in concert to achieve wound resolution or repair, which ultimately leads to the formation of a protective fibrotic scar depending on the degree of trauma1. To capture the individual processes and functions of wound healing in one experiment is difficult; individual steps within the wound healing process need to be identified and tested separately. Among the many steps of wound healing, the process of cellular migration has been considered critical when evaluating healing rates2. Cellular migration can be observed in all phases of wound healing, and thus necessitates further understanding of how alterations to cell migration can impact wound closure. For example, cellular migration is seen in both early and late phase inflammation, with blood coagulation and platelet aggregation at the wound site3. These events prevent excessive blood loss by forming a temporary blockage to fill wounded areas devoid of tissue3. Platelets in circulation will synthesize and secrete vasoactive mediators along with chemotactic factors, which together signal for inflammatory leukocyte migration (white blood cells) to the wounded tissue for repair4. Re-epithelialization occurs within hours of tissue injury and involves covering the wound site through activity and migration of epithelial keratinocytes to prevent further contamination or invasion of foreign particles to the wound site2. These examples capture only a few of the many cell migration functions associated with wound healing.
The scratch assay has been described and used to better understand cell migration and its many roles in wound healing5,6. This assay is considered simplistic, provides high-throughput, and enables the investigator to collect representative cell migration data. Migration assays have successfully demonstrated that certain compounds may accelerate or slow down the rate of cellular migration6. Furthermore, these assay results provide translational physiologic information that can be used to formulate target treatments, dosing concentrations, and genes of interest prior to in vivo experimentation. The assay requires basic cell culture techniques and supplies, a cell line of choice, and imaging technology to capture movement over time or movement in response to treatments.
The assay can be readily manipulated or tailored to suit the needs of the investigator. However, there are four basic questions to consider prior to experimentation. What general or specific question(s) is/are the investigator(s) trying to answer? This holds true for most hypotheses driven research; however, it is critical to this assay because it will determine many of the parameters needed to accurately and completely answer the question(s). What cell line or cell lines (if multiple) should be used to represent the physiologic system being studied? For example, in a cutaneous wound healing study, a dermal fibroblast5,6,7, stem cell8, or epidermal keratinocyte9 might be considered to represent cell populations that are normally found in abundance in skin tissue10. Alternatively, neutrophils, macrophages or other inflammatory cells could be evaluated in this system to represent cell migration of other components of wound healing within skin tissue10. Additionally, identifying the specific cell line being evaluated will help to identify which reagents are optimal to use to promote cell metabolic activity. How will cell migration be tracked/imaged? There are a number of techniques used to observe cell migration within a plate or flask. For example, dyeing or fluorescently tagging cells and using fluorescence or confocal imaging to track cells provides a strong contrast, which allows the investigator to clearly define cellular vs. non-cellular locations and cellular movement11. Another common technique, described in this study, is the use of inverted light microscopy with phase contrast6,7. This alternative to cell dyes and fluorescence allows the user to live track cells without having to terminally fix cells prior to imaging and also allows for capturing images of the same individual wells containing wounded monolayers of cells across time points. What outcome measures will be used for analysis? Using the images gathered throughout the study, data can be generated in which changes to cellular migration rates can be understood. In our lab, microscopic images captured on the inverted light microscope are uploaded to image analysis software and the percent wound closure and summed area under the curve (AUC) are calculated.
We will highlight the specific use of this assay to elucidate changes to dermal fibroblast migration in the presence of a known environmental contaminant, arsenic. Arsenic is a naturally occurring metalloid found within the earth&#39;s crust. Various locations have been found with elevated levels of arsenic, which poses a risk to individuals who live near these locations. As a result, food and water sources have been shown to have increased levels of arsenic contamination, which increases the likelihood for individuals to become exposed to arsenic. Environmental arsenic exposure has been shown to have long-term health consequences in human populations above or even below the current United States Environmental Protection Agency (USEPA) Max Contaminant Level (MCL) of 10 ppb (10 &#181;g/L)12,13. Although the USEPA has set this MCL and deemed it safe for drinking limits, arsenic concentrations that exceed this limit are not uncommon in water sources worldwide. In the Southwestern United States, numerous ground-water, well water, and springs have been documented to contain concerning levels of arsenic14. For example, in Verde Valley, AZ, Montezuma Well contains 210 &#181;g/L&#160;of arsenic15. Groundwater around the Verde River, AZ contains 16 &#181;g/L&#160;of arsenic on average, with peak values reaching 1.3 mg/L15,16. Notably, arsenic concentrations in many wells in the Verde River water shed exceed 50 &#181;g/L15,16. With this knowledge, environmentally relevant arsenic concentrations were selected that range from below the MCL at 0.1 &#181;M (7.5 ppb), to above the MCL at 10 &#181;M (750 ppb) in the current study14,15,16,17,18,19,20.
The information gathered from these experiments will be used as an early indicator of potential changes to cellular migration rates in an in vivo wound contaminated with arsenic. Afterward, signaling molecules can be selected based on their role in facilitating wound healing and cellular migration, and future quantitative polymerase chain reaction (qPCR) based gene expression experiments may be set up upon completion of the current studies. If migration rates in this assay change in the presence of arsenic, specific gene targets involved in cellular migration may be the target for deleterious effects of arsenic, and thus may be the mechanism of disruption.

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			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Pipette</td>
			<td>Fisherbrand</td>
			<td>13-678-11B</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight Edge Ruler</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Human neonatal dermal fibroblasts (hDFn)</td>
			<td>lot: 1734, 2902</td>
			<td>106-05n</td>
			<td></td>
		</tr>
		<tr>
			<td>Rstudio Statistical Software</td>
			<td></td>
			<td>ver: 3.5.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Image J</td>
			<td>NIH</td>
			<td>ver: 1.8.0_112</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro scratch assay to demonstrate effects of arsenic on skin cell migration

Understanding the physiologic mechanisms of wound healing has been the focus of ongoing research for many years. This research directly translates into changes in clinical standards used for treating wounds and decreasing morbidity and mortality for patients. Wound healing is a complex process that requires strategic cell and tissue interaction and function. One of the many critically important functions of wound healing is individual and collective cellular migration. Upon injury, various cells from the blood, surrounding connective, and epithelial tissues rapidly migrate to the wound site by way of chemical and/or physical stimuli. This migration response can largely dictate the outcomes and success of a healing wound. Understanding this specific cellular function is important for translational medicine that can lead to improved wound healing outcomes. Here, we describe a protocol used to better understand cellular migration as it pertains to wound healing, and how changes to the cellular environment can significantly alter this process. In this example study, dermal fibroblasts were grown in media supplemented with fetal bovine serum (FBS) as monolayer cultures in tissue culture flasks. Cells were aseptically transferred into tissue culture treated 12-well plates and grown to 100% confluence. Upon reaching confluence, the cells in the monolayer were vertically scratched using a p200 pipet tip. Arsenic diluted in culture media supplemented with FBS was added to individual wells at environmentally relevant doses ranging 0.1-10 &#956;M. Images were captured every 4 hours (h) over a 24 h period using an inverted light microscope to observe cellular migration (wound closure). Images were individually analyzed using image analysis software, and percent wound closure was calculated. Results demonstrate that arsenic slows down wound healing. This technique provides a rapid and inexpensive first screen for evaluation of the effects of contaminants on wound healing.

Sample sizes (n) for all treatment groups was n = 28. Cells were successfully cultured following aseptic techniques and lab protocols (Figure 1). Uniform scratches were observed across all treatment groups with the mean scratch width measuring 0.8 mm 0.4 mm (Figure 2). Control groups had an average summed area under the curve value of 1,355.83; 0.01 &#181;M As treatment groups had an average summed area under the curve value of 1,366.21; 0.1 &#181;M As treatment groups had an average summed area under the curve value of 1,326.24, 1.0 &#181;M As treatment group had an average summed area under the curve value of 1,295.10; and finally the 10 &#181;M As treatment group had an average summed area under the curve value of 535.12, which was statistically lower compared to all other groups (p &#60; 0.05) (Figure 3). R-program for statistical analysis was used to run a one-way ANOVA with a Tukey post hoc adjustment to determine statistical differences among treatment groups (p &#60; 0.05).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58838/58838fig2.jpg" /> 
Figure 2: Representative scratch assay images over a 20 h period. Images A-D represent cellular migration of control cells; images E-H represent cellular migration of cells contaminated with 1.0 &#181;M As; images I-L represent cellular migration of cells contaminated with 10 &#181;M As. From these images, it is clear that cellular migration is slowed in the presence of arsenic. The control images show a completely &#34;healed&#34; scratch after 20 h, while the other two As-treatment groups were not &#34;healed&#34;. The 10 &#181;M As treatment inhibited complete closure altogether after 24 h. <a href="https://www.jove.com/files/ftp_upload/58838/58838fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58838/58838fig3.jpg" /> 
Figure 3: Arsenic slows cellular migration in the scratch assay. Images were captured over a 24 h period to observe changes to cellular migration rates in the presence of varying concentrations of arsenic. Raw images were analyzed using an automated software (e.g., in MATLAB), which initially measured wound width, then calculated percent closure and finally area under the curve (AUC). Error bars represent standard error of the mean. The 10 &#181;M As treatment group statistically slowed cellular migration of human dermal fibroblasts neonatal (hDFn), shown by a statistically lower AUC value, compared to all other treatment groups using a one-way ANOVA with a Tukey post hoc adjustment (p&#60;0.05). Statistical differences are represented by the letters &#34;A&#34; and &#34;B&#34;, which were obtained through using the Compact Letter Display method available in R-program. Treatments that do not share a common letter are statistically significant from each other (p&#60;0.05). <a href="https://www.jove.com/files/ftp_upload/58838/58838fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration at JoVE.com

In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration

This study focuses on an in vitro model of wound healing (scratch assay) as a mechanism for determining how environmental contaminants such as arsenic influence cellular migration. The results demonstrate that this in vitro assay provides rapid and early indications of changes to cellular migration prior to in vivo experimentation.

In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration

Understanding the physiologic mechanisms of wound healing has been the focus of ongoing research for many years. This research directly translates into ...

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