(2020) used the assay to investigate novel hydrogel blends as a potential wound dressing. Additionally, the produced data is relatively easy to analyze with an ample variety of open access software packages. endobj endobj In this regard, in S5 Fig we performed wound area measurements of images taken under different conditions (i.e., light, contrast, type of microscope), to find the range in which each of the parameters should be varied to identify the proper scratch area. <> Wounds with different angles of inclination. Sign up for our newsletter. [17] K. Kobiyama, R. Saigusa, and K. Ley, Vaccination against atherosclerosis, Curr. The study was able to identify certain cytotoxic alkaloids as anti-migrating agents that could be investigated further [19]. [18] A. Alves et al., Xanthan Gum--Konjac Glucomannan Blend Hydrogel for Wound Healing, Polymers (Basel)., vol. The manual extraction of data is very time-consuming, subjective, and prone to error. The wound healing assay can be used in the study of wound dressings. endobj The second one considers that if the scratch is diagonal, with fixed width measurement according to the inclination angle, derived by fitting the selected ROI to an ellipse. endobj Cell migration is the movement of individual cells, cell sheets, and clusters from one location to another, and two main types have been identified, namely single-cell migration and collective cell migration. We analyzed selected regions of interest using a Zeiss inverted microscope on a 10X objective with NA/0.25 every 2 hours for 28 hours. The measures of the wounds width is likely to be impacted by its inclination. Conceptualization, Copyright 2022 CytoSMART Technologies B.V. 5 0 obj In contrast, Fig 5B shows that for gaps with an irregular shape, even though we used the same parameters of the MRI method, our plugin determined an area that is closer to that of MiToBo. Red lines of 0.1 mm represent the edges cut by the laser cutter, while Blacklines of 0.1 mm delineate regions for engraving. [14] T. Gebck, M. M. P. Schulz, P. Koumoutsakos, and M. Detmar, TScratch: a novel and simple software tool for automated analysis of monolayer wound healing assays: Short Technical Reports, Biotechniques, vol. Yes An increase in the threshold value increases the determined area (S3B Fig). This approach is however demanding, tedious, and time-consuming. We performed Paired t-tests to compare the difference between the area and the average length obtained with the Wound_healing_size_tool with respect to the manual measurement and the other available macros for ImageJ/Fiji, considering as significant p-values of less than 0.05. The wrong parameter selection can significantly alter the resulting analyses, for example, a low variance window radius can limit the recognition of the scratch while a higher value leads to underestimation of the area (S3A Fig). <>/F 4/Dest[ 25 0 R/XYZ 68 757 0] /StructParent 10>> Yes 8 0 obj [11] S. Martinotti and E. Ranzato, Scratch Wound Healing Assay, 2019. Even though not comparable with our study, similar results for wound closure were found by Walter et al. This is also the case with respect to our plugin where overestimations approached 6.5% 4.4 and 10.33% 6.9, respectively. A. We determined the scratch area, wound coverage of total area, and average and standard deviation of the scratch width with the aid of our plugin. Cellular behavior regulates wound healing during the phases of proliferation, migration, matrix formation, and contraction. It allows the analysis of bright field, phase contrast, and fluorescence images. We seeded the HaCaT cells in a 24-well plate at a concentration of 1x105 cells /well and we left them until they reached 80% of confluence [10]. Dulbeccos Eagle's medium modified high glucose medium (DMEM) Penicillin/streptomycin (P/S), Fetal bovine serum (FBS), Trypsin-EDTA (1X) were purchased from Biowest (Nuaill, France) and PBS (1X) pH 7.4 was acquired from Sigma-Aldrich (St. Louis, MO, USA). endobj However, since the open wound area usually contains single cells or cell islets, the obtained binary image will not include these regions as part of the wound. The wound healing size tool has the following advantages: https://doi.org/10.1371/journal.pone.0232565.s001, https://doi.org/10.1371/journal.pone.0232565.s002, https://doi.org/10.1371/journal.pone.0232565.s003, https://doi.org/10.1371/journal.pone.0232565.s004. 1, p. 99, 2020. Depending on the cell type used in the assay, cell proliferation can be a confounding factor. conditions of the tissue before wounding such as, an intact epithelium. <>/F 4/Dest[ 25 0 R/XYZ 68 547 0] /StructParent 12>> endobj 14 0 obj Physiol., vol. [13] C. N. Ramirez et al., Validation of a high-content screening assay using whole-well imaging of transformed phenotypes, Assay Drug Dev. Rep., vol. Understanding the processes involved in this migration can provide potential targets for inhibition. Automation also has greater functionality; for example, it can be used to determine the experimental endpoints and generate kinetic, functional, and quantitative measurements of living cells (see Figure 2) [1]. Careful optimization is necessary as any changes to the medium can result in unpredictable time- and cell-type-dependent effects that can interfere in the assay [1]. Decreasing the concentration of serum (serum starvation) is the most common change. broad scope, and wide readership a perfect fit for your research every time. Want to stay up to date? #C? <>/F 4/Dest[ 17 0 R/XYZ 68 547 0] /StructParent 6>> 7987, 2016. The plugin presents all results for each image, area of the wound, wound coverage of total area, and average of the width and its standard deviation in a table with the set scale (Fig 2B and 2C). 19 0 obj A Schematic of the use of mold for scratching wounds on 2D cultures. In such cases, it determines the inclination angle and adjusts each calculated width with trigonometric relations (S4 Fig). The variance window radius represents the radius of the variance filter, which we establish to determine the empty or the occupied zones. endobj The first one applies when the user has multiple images with the same calibration scale. endobj Methodology, Also, both treatments were fitted to linear regressions that show (from the slope) higher wound closure rates in the presence of the conditioned medium. The authors demonstrated that hydrogels could be used to improve the wound healing process by promoting fibroblast migration, adhesion, and proliferation [18]. Methods that are more sophisticated include molds or cell inserts [19, 20], electric currents [21], lasers [4] and magnets [20]. <>/ExtGState<>/XObject<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/MediaBox[ 0 0 595.32 841.92] /Contents 113 0 R/Group<>/Tabs/S/StructParents 25>> This is because the MiToBo tool is a highly reliable plugin based on histogram features and support vector machines (SVMs) [29]. Formal analysis, Four processes occur during wound healing, namely haemostasis, inflammation, migration-proliferation, and maturation-remodeling [1], [2]. 7 0 obj 45 0 obj It is easy to use and can be adapted to analyze both individual images and stacks. We estimated these migration times with the data analysis provided by our plugin. 18 0 obj 13 0 obj Whole-well imaging systems can be applied to the wound-healing assay. https://doi.org/10.1371/journal.pone.0232565.s006. The most frequently used method is to wound the monolayer by mechanical scratching (scratch wound) or stamping. In order to develop robust therapeutic approaches, it is imperative to study these processes in detail [4]. B. 17, no. Formal analysis, As a result, a number of plugins have been developed for these software platforms to accelerate the analysis process by automatically quantifying cell number and wound area [22, 25]. Validation, ------------------------------------------------------. Supervision, Migration is considered the rate-limiting process during healing, and therefore migration assays are a key part of investigating wound healing [2]. Both the MRI wound healing tool (which is also based on a variance-based method [19] and the manual method overestimate the area values found with the MiToBo tool. 5965, 2012. <> 2, pp. Writing review & editing, * E-mail: jc.cruz@uniandes.edu.co (JCC); c.munoz2016@uniandes.edu.co (CMC), Roles <>/F 4/Dest[ 17 0 R/XYZ 68 452 0] /StructParent 7>> Copyright: 2020 Suarez-Arnedo et al. The plugin and detailed information and user manual can be found in the S2 File and in https://github.com/AlejandraArnedo/Wound-healing-size-tool/wiki as an open source wiki. With a cell-free gap prepared, optical microscopy can be used to observe cells migrating into the wound area. Opin. Cell proliferation can compete with cell migration to fill the gap made during the assay. Is the Subject Area "Wound healing" applicable to this article? endobj Moreover, to compare the scratches made using the molds or using only a 200 L sterile pipette tip, we evaluated the width homogeneity of the gap with our plugin by the coefficient of variation (CV = SD/mean) and standard deviation with respect to a straight line. Writing original draft, Affiliation Growth factors and matrix signals that determine the function of cells in regeneration processes orchestrate this behavior [1]. endobj Examples of some of these applications are presented below. 40 0 obj Snapshot of the results in table format in pixels show area of the wound, wound coverage of total area, and average of the width and its standard deviation. endobj The wound healing size tool provides an automatic method of processing images obtained from a scratch/ wound healing assay. The final method measures the relative wound density Circ. cWblsIn+Hwma%?~;Hsu^!G;))p{85ZO ]8b$Yt{UmgNd% *UGpvgpqE]p:qRO}\&=v$;{bEteS"0"s*d4"Ty#C|?.k'#Bi|1e%\Bdsot:N@v$tu9 5(ZI8QQ{HW10qLC?a"s5m5\7k\5z95L\1`Y`%>mwI=^ui[ebMdW,TOynopCSR98~G4uJ\1/zF: X+ ki{?RU]Wum}v 1j74IhxR8 ]1}jC0&i!>Emi}Ds|`?On:"XKRxE{du:(d=1{TOyB^4k=AI1Ck*IT- oxzpkEb8ZZQK#.du^lz_-w sNN There are open-source programs such as imageJ to analyze images of in vitro scratch wound healing assays, but these tools require manual tuning of various parameters, which is time-consuming and limits image throughput. The relative wound density method can be further refined to factor in cell proliferation or pharmacological effects. (2014) [4]. Methodology, For that reason, we developed an optimized plugin for imageJ to automatically recognize the wound healing size, correct the average wound width by considering its inclination, and quantify other important parameters such as: area, wound area fraction, average wound width, and width deviation of the wound images obtained from a scratch/ wound healing assay. Technol., vol. 9, no. In addition to measuring changes in the wound area, the wound healing assay can also be applied to tracking the movement of individual cells at the leading edge of the wound. Moreover, in some regions, the detected edges of the scratch area overlapped between methods. Autom., vol. 24 0 obj By increasing this value, we expect an increase in the contrast. Before running the analysis in all the stack of collected images, we selected three images randomly at different time points to set the right parameters for wound identification. [10] A. P. Looney and M. Bhattacharya, Fibroblast Gap-closure Assay-Microscopy-based in vitro Assay Measuring the Migration of Murine Fibroblasts, Bio-protocol, vol. The second method calculates the change in wound area over time as a percentage of wound closure. Competing interests: The authors have declared that no competing interests exist. Yes Finally, we can observe in Fig 4F that, after 12 hours, the rate of cell migration of cells exposed to hAdMSCs conditioned medium decreases and approaches an average value of 1 4.3 m /hour until the end of the experiment. To validate the quality and efficiency of Wound_healing_size_tool, we compared it with other ImageJ plugins, including MRI_Wound_Healing_Tool (http://dev.mri.cnrs.fr/projects/imagejmacros/wiki/Wound_Healing_Tool) (MRI), the ScratchAssayAnalyzer tool from A microscope image analysis toolbox (MiToBo) (https://mitobo.informatik.uni-halle.de/index.php/Applications/ScratchAssayAnalyzer) [29], as well as manually drawn edges of the scratch (Manual) in 30 different images. 27 0 obj Yes Percentage of coefficient of variation between scratch made with pipette tip of 200 L with or without using the mold [n = 4 images per method]. The plugin features an interface window with two options to adjust the values of all parameters (Fig 2A). This creates a new image with high intensity pixels within the cell monolayer and low intensity pixels within the open wound area. <>/ExtGState<>/XObject<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/MediaBox[ 0 0 595.32 841.92] /Contents 64 0 R/Group<>/Tabs/S/StructParents 20>> The assay is also scalable allowing for high-throughput screening of genes involved in cancer cell migration, small molecule discovery, and drug discovery [1], [2], [16]. We made horizontal reference lines on the bottom of the plate with an ultrafine tip marker to have a grid for alignment to obtain the same field for each image acquisition run. 32 0 obj Immunol., vol. <>/ExtGState<>/XObject<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/MediaBox[ 0 0 595.32 841.92] /Contents 115 0 R/Group<>/Tabs/S/StructParents 26>> Our Wound_healing_size_tool plugin facilitates a high throughput calculation of a number of parameters from several images including scratch area, wound coverage of total area, scratch width average, and standard deviation of the scratch width. The effect of the hydrogel on wound closure was assessed by incorporating it into the cell medium. Three-dimensional collective migration takes place on a tissue scaffold, and it is the movement of cells organized in a multicellular network of strands. We quantified the differences between existing methods regarding our plugin using Eq 3: Then, we added 1 mL of hAdMSCs conditioned medium, which was obtained from cells in the 4-6th passage according to the protocol established by Qazi et al. Accordingly, analyzing the variance within different neighborhoods in the image could be helpful for discriminating between cell monolayer and open wound regions. 18 m/hour) during the first 12 hours of the experiment. e0232565. endobj These results are consistent with previous studies that have established the potential of mesenchymal stem cells secretome in cell regeneration and migration [7, 10, 27, 33, 34]. Total cell count within the wound area can also be used to assess cell migration and wound healing [2]. 17, no. Most wound healing assays still use manual data acquisition and analysis in part. Nonetheless, after this time, we reduced the migration rate to 14.3 m/hour (Fig 4F). The mold was laser cut using a Trotec Speedy 100 CO2 (Marchtrenk, Austria). ?YDTd0 Seeing is believing: label-free, time-lapse microscopy for quantifying cell proliferation, Remote monitoring of iPSC-derived neural crest cells, Wound healing assays: discussing subtypes of cell removal assays, PromoCell and CytoSMART experiment: wound closure assay, Cytotoxicity assays: cell viability analysis using cell growth curves. C. Snapshot of the results in table format in m show area of the wound, wound coverage of total area, and average of the width and its standard deviation. This allows researchers the opportunity to understand the role of genes in the regulation of cell migration [3]. In addition, the multitude of assays available makes comparison between experiments difficult [2]. Fig 4E shows the standard deviation of the scratch width, which is likely an indicative of the heterogeneity of the scratch width at each time point and it suggests differences into how the cells migrate. Furthermore, the mold produced more reproducible and homogeneous scratches in contrast with gaps made using only a pipette tip. Studies have also examined the migration of vascular smooth muscle cells [5], [6]. <>>> Moreover, we also consider the standard deviation of the calculated widths to assess the homogeneity of the scratch and subsequently its closure. 1 0 obj endobj Supervision, 265274, 2009. Validation, Roles It can be adapted to analyze both individual images and stacks. Most often, epithelial and endothelial cells are used to produce the monolayer, and in particular, cell types capable of sheet migration [4]. For in-depth wound healing assay guidelines on parameters to ensure quantitative and reproducible results, please see the review by Jonkman et al. endobj 17 0 obj According to Fig 6, there is no significant difference between the average width calculated with our plugin or calculated manually. Validation, With careful inspection of the gray-scaled images obtained from the wound healing assay, we noticed that the pixels in open wound regions had very similar intensity values, while cell monolayer regions had higher variation due to cell presence. After segmenting the open wound, its area and the coverage with respect to the whole image are easily determined with the regions metadata using the ROI manager.

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