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

Summary

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.

Abstract

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 μM. 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.

Introduction

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 healing^5,6. 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 fibroblast^5,6,7, 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 contrast^6,7. 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'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 µg/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 µg/L of arsenic¹⁵. Groundwater around the Verde River, AZ contains 16 µg/L of arsenic on average, with peak values reaching 1.3 mg/L^15,16. Notably, arsenic concentrations in many wells in the Verde River water shed exceed 50 µg/L^15,16. With this knowledge, environmentally relevant arsenic concentrations were selected that range from below the MCL at 0.1 µM (7.5 ppb), to above the MCL at 10 µM (750 ppb) in the current study^{14,15,16,17,18,19,20}_.

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.

Protocol

1. Preparation of a Biosafety II Cabinet (BSC)

NOTE: The methods described in this protocol should be conducted with personal protective equipment, while using good laboratory practices to achieve aseptic technique.

1. Lift the BSC protective glass to operating height and turn on laminar flow fans.
   1. Use 70% isopropanol aqueous solution to perform a sanitation wipe of the working area. Wipe BSC from the back wall and side walls and work down to the base plate.
   2. Wipe all materials that will be used in the assay with 70% alcohol (isopropanol or EtOH) and place them inside the BSC.

2. Human Dermal Fibroblasts Seeding in a T75 Flask

1. Obtain vials with the desired cryopreserved cell line (neonatal human dermal fibroblasts [hDFn], at cell concentration of 500,000 cells/vial, and at passage p3–p5).
   1. Place a vial with cells in a 1.0-cm-deep water bath at 37 °C to allow thawing. Thaw vial until approximately 70% of cell solution is liquid. Wipe down vial with 70% alcohol and place vial in vial rack inside BSC.
      NOTE: Ensure water does not come in contact with threads or cap of vial to prevent potential contamination during cell seeding. Prevent complete thawing of cells in water bath as failure to do so may cause unintentional cell death.
2. Draw out 10 mL of media supplemented with 10% Fetal Bovine Serum (FBS). Use an automatic pipettor and 10 mL pipette tip to aspirate into T75 tissue culture flask. Allow media to fully saturate the base of the flask by gently rocking the flask back and forth.
   1. Open vial with cell stock and aspirate solution from the vial, being mindful of pipetting speed, as cell membranes will be vulnerable from cryopreservation.
   2. Transfer cells to a T75 tissue flask with media. Eject thawed cell into tissue flask, again being mindful of pipetting speed.
      NOTE: Seeding density of cells in tissue flask is approximated to be 6,600 cells/cm². Seeding density can be altered to accommodate the user and desired timing of experimentation.
   3. Measure out 1 mL of media from T75 tissue flask and transfer volume back into the vial. Transfer volume from the vial back into the T75 flask.
      NOTE: Performing this step helps maximize cell yield from the vial.
   4. Tighten the tissue culture flask cap and gently rock to uniformly disperse cells in suspension across the entire surface. Check for even distribution of cells using an inverted microscope.
3. Label flask with cell type, date, initials, lineage, and purpose (e.g., hDFn, 07/11/2018, NDC, Passage 4, Arsenic Scratch Assay). Place tissue culture flask in a humidified incubator at 37 °C and 5.0% CO₂ for 72 h.
   NOTE: Growth medium should be changed 12-24 h after initial seeding to remove trace amounts of dimethyl sulfoxide (DMSO) cryopreservative, and then changed every 48 h after that to ensure cells are properly nourished. The incubation and growth period will depend on the calculated number of cells needed for the scratch assay. The doubling time for hDFn cells is typically 16-24 h under normal conditions. However, doubling rate may change due to elevation, pH, temperature, humidity, medium type, etc., and must be accounted for when planning experiments.

3. Cell Counting and Subculture into 12-Well Tissue Culture Plate

1. Retrieve the T75 tissue culture flask from incubator. Observe adhered cells using an inverted microscope at 10X objective magnification (100X total magnification) and determine the approximate cell confluence.
   1. Scan the majority of the tissue culture flask when observing confluence to ensure the most representative percentage is approximated. Evaluate individual cell morphology for any abnormalities, or for bacterial and/or fungal contamination.
2. Assemble the automatic pipettor with a 10 mL pipette tip. Use pipettor to aspirate the full volume of media from the T75 and transfer solution to a waste container. Prevent the pipet tip from contacting the cell adhering surface. Discard pipette tip in a biohazard bin.
   1. Assemble the automatic pipettor with a 5 mL pipette tip. Aspirate 5 mL of the balanced salt solution, dispense in the flask, and gently rock the flask to rinse excess media, dead cells, and waste product.
   2. Draw the volume from the T75 and dispense into the waste container. Discard the pipette tip into the biohazard bin.
   3. Assemble the automatic pipettor with a 5 mL pipette adapter. Aspirate 2 mL of trypsin from stock, dispense in the flask, and gently rock the flask to disperse the enzyme over the adhered cells. Ensure complete saturation of the adherent surface. Place the tissue flask in the incubator for 2-3 minutes (min).
      NOTE: The maximum enzyme-substrate reaction should not exceed 10 min.
   4. Observe the tissue flask under an inverted microscope at a 10X objective lens magnification (100X total magnification). Apply gentle vibrations to facilitate cell detachment.
   5. Wipe the T75 flask with 70% alcohol and place inside the BSC.
   6. Assemble the automatic pipettor with a 5 mL pipette tip. Aspirate 5 mL of media from the stock and add to the T75 tissue flask to stop the enzyme-substrate reaction. The media:trypsin ratio should be 3:1. Pipet up and down the base of the flask 2-3 times to rinse and ensure that the reaction has stopped. Aspirate the complete volume of fluid from the flask and transfer into a sterile 15 mL conical tube.
   7. Fill a second 15 mL conical tube with an identical volume of media.
   8. Place both 15 mL conical tubes in an antipodal position to one another. Apply 200 g of centrifugal force for 5 min at 25° C by setting equipment’s running parameters.
   9. Wipe the 15 mL conical tube with pelleted cells with 70% alcohol and place inside the BSC.
   10. Use the automatic pipettor with a 5 mL attachment to pipet off media, leaving behind approximately 250 µL of fluid and cell pellet. Pay attention to the location of the pipet tip, as the cell pellet should remain undisturbed. Dispense the volume collected into a waste container and discard the tip in a biohazard bin.
   11. Use a microcentrifuge tube rack to run the bottom of the conical tube across the vial slots, creating a swift vibration, to disturb and re-suspend the cell pellet (sometimes called “rack raking”).
       NOTE: Avoid using a vortex mixer to perform this step. Gravity forces created by vortex mixers exceed cell g-force thresholds and can cause lysing. When completed correctly, the new cell solution will appear as a cloudy mixture with no remaining pellet. Diligence performing this step will oftentimes create a single cell suspension, leading to increased accuracy when counting cells.
   12. Add 2 mL of media to the cell stock or resuspension. Pipet the cells gently down the side of the tube 20 times to break up any clumps. Record the total cell suspension volume and set aside briefly.
3. Pipet 20 µL of Trypan Blue stock (0.4%) into an empty well of a 96-well plate using a sterile pipet.
   1. Mix the stock cell solution to generate a uniform cell suspension before transfer. Use a sterile pipet tip to remove 20 µL of cell stock (avoiding touching the tube walls). Add to the 20 µL of Trypan Blue solution in the 96-well plate.
   2. Pipet gently to mix contents. Pipet 10 µL of the mixture between the coverslip and the counting chamber on the hemocytometer.
   3. Observe the cells and the grid under a 10X objective. Observe nine large squares within the field of view. Count cells only in the center and 4 corner squares (total of 5 squares).
      NOTE: Look for a uniform distribution of cells across the entire grid to ensure an accurate count.
   4. Tally all cells (transparent and blue) in the 5 identified squares of the grid. Separately, tally only the nonviable (blue) cells in the same 5 squares.
   5. Calculate viable cell count using: 
      where x = viable cell count, a = total cells, b = non-viable cells
   6. Calculate the viable cell density using: 
      where x = viable cell density and y = total sum of viable cells.
   7. Calculate total viable cells: 
      where x = total viable cells, y = viable cell density, f = cell suspension volume (mL).
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Results

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 µM As treatment groups had an average summed area under the curve value of 1,366.21; 0.1 µM As treatment groups had an average summed area under the curve value of 1,326.24, 1.0 µM As treatment group had an average summed area under the curve value of 1,295.10; and finally the 10 µM 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 < 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 < 0.05).

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 µM As; images I-L represent cellular migration of cells contaminated with 10 µM As. From these images, it is clear that cellular migration is slowed in the presence of arsenic. The control images show a completely "healed" scratch after 20 h, while the other two As-treatment groups were not "healed". The 10 µM As treatment inhibited complete closure altogether after 24 h. Please click here to view a larger version of this figure.

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 µM 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<0.05). Statistical differences are represented by the letters "A" and "B", 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<0.05). Please click here to view a larger version of this figure.

Discussion

Application of this assay provides a high-throughput and timely evaluation of cellular migration and may directly translate to complex physiological systems^5,6,7. 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 bases^{14,15,16,17,18,19,20,21}. 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'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 "cell tracker" 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 environments^6,25. 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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Class II A/B3 Biological Safety Cabinet	Forma Scientific	15201-816
Nitrile Gloves	Kimberly-Clark	55082
Water Bath	Precision	Model 182
Inverted Microscope	Leica	Q080318
CPR Centrifuge	Beckman	349702
Analytical Scale	Ohaus	I093 1125062111P
Weighting paper	VWR	H351125781212A
Biohazard bags	Fisherbran	01-830A
Water Jacketed CO2 Incubator	Thermo Scientific	309739-31053
Hanks Balanced Salt Solution	Gibco	14175-095
Dulbecco's Modified Eagle Medium	Gibco	10566-016
Fetal Bovine Serum	Gibco	26140-079
Automatic Pipettor	Drummond	140685
p200 Pipette	Gilson
p20 Pipette	Gilson
Denominator	Fisher Scientific	D59707
Trypan Blue Stain	Gibco	15250-061
T75 tissue flask	Corning	430725U
15 mL Conical Tube	Fisherbrand	05-539-5
50 mL Conical Tube	VWR	21008-178
5 mL Pipette	Fisherbrand	13-678-11D
10 mL Pipette	Fisherbrand	13-678-11E
Tissue Marker	Securline	92167
Wipes	Kintech	34155
70% Ethanol	Fisher	63-67-0
Non-Filter Pipet Tips	Fisherbrand	16160210
Bleach	Great Value	220-04
96-well	Falcon	353915
Cryovial Rack	Nalgene	5030-0505
Hemacytometer	Gizmo Supply	B00SCOGY56
TrypLE Express	Gibco	12605-028
Conical Tube Rack	n/a	n/a
12-well plate	Corning	3598
Waste Beaker	n/a	n/a
1 mL Pipette	Fisherbrand	13-678-11B
Straight Edge Ruler	n/a	n/a
Human neonatal dermal fibroblasts (hDFn)	lot: 1734, 2902	106-05n
Rstudio Statistical Software		ver: 3.5.1
Image J	NIH	ver: 1.8.0_112