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. <>/XObject<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/Annots[ 11 0 R 15 0 R 16 0 R 18 0 R 19 0 R 20 0 R 21 0 R 23 0 R 26 0 R 27 0 R 28 0 R 30 0 R 36 0 R 38 0 R 40 0 R 42 0 R 44 0 R] /MediaBox[ 0 0 595.32 841.92] /Contents 4 0 R/Group<>/Tabs/S/StructParents 0>> lymphatic elm motility lec This approach is however demanding, tedious, and time-consuming. <>/F 4/Dest[ 37 0 R/XYZ 68 771 0] /StructParent 15>> wound healing experiments with these CytoSMART devices: full-plate scanner Omni, This proves the effectiveness of our plugin to calculate the wounds width for different imaging conditions. Furthermore, the mold produced more reproducible and homogeneous scratches in contrast with gaps made using only a pipette tip. p-value<0.0001****, p-value<0.001***, p-value<0.01** Some error bars are shorter than the size of the symbols. 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. 43 0 obj Software, Roles In such cases, it determines the inclination angle and adjusts each calculated width with trigonometric relations (S4 Fig). 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]. [5] A. D. der Meer, K. Vermeul, A. 1, pp. Once the microscope is set up, a series of time-lapse images (snapshot method) can be acquired as cells migrate into the cell-free gap [1]. 111, 2019. 5 0 obj

Accurate measurements can be manually acquired using a digital camera fitted to the microscope; however, this process is time-consuming, and maintaining the same field-of-view along each gap can be difficult [4]. Click through the PLOS taxonomy to find articles in your field. Yes The first method measures the change in the wound width (nanometres) over time. Yes <>/ExtGState<>/XObject<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/MediaBox[ 0 0 595.32 841.92] /Contents 94 0 R/Group<>/Tabs/S/StructParents 22>> A. List of parameters for the collected images. These packages have been previously used to manually count cells (selecting and tallying individual cells) and to assess wound closure (tracing the wound perimeter and calculating the percentage of closure) [23, 24]. 38 0 obj <> To discriminate adequately between cell monolayer and open wound area, we developed a classic computer vision segmentation algorithm focused on assessing neighboring pixel intensity variance (i.e., the morphology and visual characteristics). The measures of the wounds width is likely to be impacted by its inclination. Accordingly, it is advisable to test two or three images with different combinations of the parameters before starting the analysis in order to localize an appropriate area of analysis such that the scratch can be fitted appropriately. https://doi.org/10.1371/journal.pone.0232565.s006. Therefore, the analysis of large datasets remains a bottleneck with many assays. Researchers can perform [12] S. T. Johnston, E. T. Shah, L. K. Chopin, D. L. S. McElwain, and M. J. Simpson, Estimating cell diffusivity and cell proliferation rate by interpreting IncuCyte ZOOMTM assay data using the Fisher-Kolmogorov model, BMC Syst. For researchers interested in such a functionality see the CytomSMART Omni landing page. Autom., vol. endobj Cellular behavior regulates wound healing during the phases of proliferation, migration, matrix formation, and contraction. The area obtained with our plugin led to no significant differences with respect to the other methods (Fig 5). We tested the plugin in the task of analyzing images derived from a scratch assay where Human skin keratinocytes HaCaT were exposed to conditioned media of Human adipose-derived mesenchymal stem cells (hAdMSCs). Most often, epithelial and endothelial cells are used to produce the monolayer, and in particular, cell types capable of sheet migration [4]. We performed mold design using Autodesk Inventor Professional 2020 (Autodesk, Inc., San Rafael, California, USA, www.autodesk.com) to guarantee a completely closed piece with the dimensions of the real object and optimized for laser cutting.

According to Fig 4B and 4C, the wound closure proceeds at a constant rate, without significant changes over the period evaluated (R2 = 0.9851), reaching almost 95% of closure after 28 hours. Alternatives to mechanical damage can include thermal, electrical, and optical wounding [2], [7]. Data curation, ------------------------------------------------------. JFIF Ducky. 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. here. Some phases that occur during wound healing have also been observed during cancer invasion [2, 3]. 32 0 obj 39 0 obj 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. The effect of the hydrogel on wound closure was assessed by incorporating it into the cell medium. This modified wound healing assay is a closer representation of the microenvironment present in the vasculature. Conceptualization, C. Percentage of deviation from the straight line of each scratch made with pipette tip of 200 L with or without using the mold [n = 4 images per method] p_value<0.05*. Scale bar = 1000 pixels B. Scratch with high variation of calculated area between the different methods C. Quantification of the difference in measurements between methods with our plugin [n = 30 images]. For these reasons, we designed a polymethyl methacrylate mold for a 24-well plate. Rep., vol. 1 0 obj Also, both treatments were fitted to linear regressions that show (from the slope) higher wound closure rates in the presence of the conditioned medium. Scale bar = 200 pixels. 7 0 obj The disadvantages experienced with manual acquisition can largely be overcome using automation that allows for live-cell imaging. 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. have been used to clear cells from fluid channels [5], [6], [8], [9]. https://doi.org/10.1371/journal.pone.0232565.g006. <>/F 4/Dest[ 17 0 R/XYZ 68 452 0] /StructParent 7>> 46, no. Funding: This work was financially supported by the Department of Biomedical Engineering at Universidad de los Andes, by the start-up funding to Assistant Professors FAPA (Fondo de Apoyo a Profesores Asistentes of Carolina Muoz), and by an internal call for termination of publications (CI-001-Pblica tus nuevos conocimientos o expone tus nuevas creaciones) from Vice-provost of Research at Universidad de los Andes. Circ. Additionally, the produced data is relatively easy to analyze with an ample variety of open access software packages. (2016) as the coordinated movement of a group of cells that maintains their intercellular connections and collective polarity [1]. This allows researchers the opportunity to understand the role of genes in the regulation of cell migration [3]. In this regard, our plugin is comparable to the MRI wound healing tool as they both take between 6 to 13 seconds to perform the image analysis. This plugin facilitates the quantification of wound characteristics and circumvents their manual acquisition in many images obtained during each experiment. We calculated the rate of cell migration (RM) and the percentage of wound closure according to (Eq 1) and (Eq 2), respectively [28]: Although both methods require the use of a pipette tip, the mold gives the advantage of limited tip movement and fixed inclination for more uniform scratch edges. Discover a faster, simpler path to publishing in a high-quality journal. For in-depth wound healing assay guidelines on parameters to ensure quantitative and reproducible results, please see the review by Jonkman et al. endobj For this reason, we included a function in the plugin that allows correction with respect to an inclination angle. Alves et al. Collective migration can take on two different forms depending on the extracellular matrix. The migration images can then be used to collect measurements or be visually scored [4]. 2016 [2]. <>/ExtGState<>/XObject<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/Annots[ 108 0 R 109 0 R] /MediaBox[ 0 0 595.32 841.92] /Contents 107 0 R/Group<>/Tabs/S/StructParents 24>> <> To remove these structures, we perform a morphological reconstruction by erosion, also known as hole filling, on the area binary labeled as open wound. progression pancreatic malignant promotes invasion panc bxpc scratch With a cell-free gap prepared, optical microscopy can be used to observe cells migrating into the wound area. To eliminate the regions falsely detected as wounds, we select the largest connected component within the detected open wound regions as the true wound area. <> 112, 2019. We calculate the Euclidean distance between paired pixels, and we determine their average to describe wound width. 30 0 obj In microfluidic wound healing assays, chemical and enzymatic treatments 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. 17 0 obj Scale bar = 1000 pixels B. Quantification of the difference in measurements between wound healing size tool (WHST) and scratching manually [n = 30 images, 10 lines per area in the manual method] *p_value<0.01. 9, no. 4, pp. Methodology, endobj No, Is the Subject Area "Image analysis" applicable to this article? No, PLOS is a nonprofit 501(c)(3) corporation, #C2354500, based in San Francisco, California, US, Corrections, Expressions of Concern, and Retractions, https://doi.org/10.1371/journal.pone.0232565, https://github.com/AlejandraArnedo/Wound-healing-size-tool/wiki, http://dev.mri.cnrs.fr/projects/imagejmacros/wiki/Wound_Healing_Tool, https://mitobo.informatik.uni-halle.de/index.php/Applications/ScratchAssayAnalyzer, https://doi.org/10.1016/j.jid.2016.11.020. It can be adapted to analyze both individual images and stacks. V6!o j3[RxS:3+Xa.BPv>#1%etR0#C>rW~ %W-){bdul?6bFndC9b J1L}*Y/|vHXSxWAu-{[Zn:. Depending on the cell type used in the assay, cell proliferation can be a confounding factor. A. endobj Examples of some of these applications are presented below. [27]. The results of the width homogeneity of the scratch (Fig 3B), using the mold, show a reduction in variability of approximately 50% when compared with manually made scratches using a pipette tip. As shown in Fig 4, conditioned medium from hAdMSCs promoted healing in an in vitro epidermal model. Drawbacks with the manual method of wounding include low throughput and well-to-well variation of the gap width [1], [11]. <> Investigation, endobj 7987, 2016. In this context, the in vitro scratch wound healing assay is useful to evaluate the proliferation and migration process when exposing cells to metabolites present in the conditioned media. endobj Dermatol., vol. Methodology, 21 0 obj We created the schematic representation of the use of the mold for scratching wounds on 2D cultures using (www.biorender.com). 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. 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). endobj after 28 hours (closer to 95%) [10]. Total cell count within the wound area can also be used to assess cell migration and wound healing [2]. (Eq 3). <>/ExtGState<>/XObject<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/Annots[ 49 0 R 50 0 R] /MediaBox[ 0 0 595.32 841.92] /Contents 47 0 R/Group<>/Tabs/S/StructParents 1>>

Yes endobj This process involves cell migration, proliferation, and differentiation [1]. Ensuring reproducibility is important for the following data acquisition step. (Eq 2). 34 0 obj As shown in Fig 4, the exposure of keratinocytes to the conditioned medium from hAdMSCs promotes the migration and wound healing in a 2D layer of such cells. Yes endobj

1, p. 38, 2015. A Scratch identifies with our plugin. Methodology, The assay can be used to investigate the effects of the cell-matrix and cell-cell interaction on cell migration and be combined with transfection to determine the effect of the expression of exogenous genes on the migration of individual cells [1], [3]. 2, pp. 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. Furthermore, it is possible to study the response of free cells and those seeded on scaffolds made of both synthetic polymers and natural extracellular matrices [11, 1214].

J. Physiol. The most remarkable advantages of this assay are the low requirements of specialized equipment or expensive reagents, which makes it adaptable to research groups with limited budgets. The difference between MRI method and our plugin can be explained by the image enhancement used as pre-processing. 24 0 obj Yes E. Standard deviation of the scratch width in m.

9, no. The wound healing assay is used to investigate cell migration and wound healing. An analysis of the same wound but with different inclination showed an average width difference of only 1.35 + -0.59% with respect to the original measurement, which was perpendicular to the x-plane of the image (S4 Fig). All these biomolecules activate signaling cascades that enhance the migratory and proliferative potential of various cell types [7, 10, 35]. 5965, 2012. These two methods can be time-consuming when performed manually. <>/ExtGState<>/XObject<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/MediaBox[ 0 0 595.32 841.92] /Contents 113 0 R/Group<>/Tabs/S/StructParents 25>> B. Wang et al. over time expressed as a percentage and is most often applied in live-cell imaging software [1], [12]. Most wound healing assays still use manual data acquisition and analysis in part. successfully screened cytotoxic alkaloids for the ability to inhibit biological processes related to cell migration and cytoskeleton dynamics. 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. 9, no. 3 0 obj

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The wound healing size tool provides an automatic method of processing images obtained from a scratch/ wound healing assay. stream Where Wi is the average of the initial wound width, Wf is the average of the final wound width both in m and t is the time span of the assay in hours.

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