Toxicol., vol. W*];2Whh mxWKY>LZ^2%Qs|~0w'fB\(k+vg`v!=^.+uHEqiW_# while a higher percentage of saturated pixels value leads to detecting smaller areas (S3C Fig). We evaluated the performance of our plugin by analyzing 60 wound images. Besides analyzing specific points in time, the plugin can be useful for time-lapse analysis and even the variability and heterogeneity of the cellular migration. endobj 6 0 obj here. 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. 36 0 obj 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. e0232565. 12, pp. endobj This technique is suitable to study the paracrine signals (conditioned media) produced by stem [10] or other types of cells under either mechanical [7] or electrical stimuli [11]. <>/F 4/Dest[ 29 0 R/XYZ 68 771 0] /StructParent 13>> [18] A. Alves et al., Xanthan Gum--Konjac Glucomannan Blend Hydrogel for Wound Healing, Polymers (Basel)., vol. #C? 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. Accordingly, analyzing the variance within different neighborhoods in the image could be helpful for discriminating between cell monolayer and open wound regions. Formal analysis, 9, no. Methodology, endobj <>/F 4/Dest[ 37 0 R/XYZ 68 771 0] /StructParent 15>> 1, pp. endobj In vitro scratch wound healing assay, a simple and low-cost technique that works along with other image analysis tools, is one of the most widely used 2D methods to determine the cellular migration and proliferation in processes such as regeneration and disease. This reproducibility can be improved with the use of commercial tools for making uniform gaps [1]. Drawbacks with the manual method of wounding include low throughput and well-to-well variation of the gap width [1], [11]. This approach is however demanding, tedious, and time-consuming. [4] J. E. N. Jonkman et al., An introduction to the wound healing assay using live-cell microscopy, Cell Adh. The wound healing assay is a simple and inexpensive method. <> Supervision, A numerical example of the variance window radius value is shown in S2 Fig and S3A Fig. This method calculates the ratio of the occupied area in the gap to the total area of the initial gap. 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. endobj The difference between MRI method and our plugin can be explained by the image enhancement used as pre-processing. This can be achieved by counting cells in subregions found inside and outside the wound area to determine relative cell density [12]. 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. As collective cell migration is linked to many physiological and pathological processes related to embryogenesis, wound repair, and cancer metastasis, the wound healing assay is broadly applicable [4], [8]. 4, pp. Fig 5A shows how all evaluated methods distinguish gaps with regular shape similarly. 112, 2019. This result is most likely due to the high rate of cellular migration (aprox. <>/F 4/Dest[ 17 0 R/XYZ 68 644 0] /StructParent 5>> Growth factors and matrix signals that determine the function of cells in regeneration processes orchestrate this behavior [1]. <> The study was able to identify certain cytotoxic alkaloids as anti-migrating agents that could be investigated further [19]. Roles Investigation, Scale bar = 27550 m) images of wound healing closure in HaCaT exposed to hAdMSCs conditioned medium. The percentage of saturated pixels allows enhancing the contrast of the image by determining the number of pixels that could saturate in the image. 14 0 obj <>/F 4/Dest[ 25 0 R/XYZ 68 757 0] /StructParent 10>> 5, p. 14049, 2015. Autom., vol. Understanding the processes involved in this migration can provide potential targets for inhibition. The image analysis allowed us to validate that the plugin can be useful in images collected in a wide variety of instruments in contrast to some macros that only work to identify the scratch from images collected with a limited number of instruments [25]. 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. No, Is the Subject Area "Cell migration" applicable to this article? This allows researchers the opportunity to understand the role of genes in the regulation of cell migration [3]. endobj 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. The average runtime per image is another plugin feature we evaluated. 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. <> 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. It quantifies the area, wound coverage of total area, average wound width and width standard deviation. endobj This proves the effectiveness of our plugin to calculate the wounds width for different imaging conditions. 29 0 obj To remove these structures, we perform a morphological reconstruction by erosion, also known as hole filling, on the area binary labeled as open wound. [17] K. Kobiyama, R. Saigusa, and K. Ley, Vaccination against atherosclerosis, Curr. Rep., vol. This process involves cell migration, proliferation, and differentiation [1]. Also, both treatments were fitted to linear regressions that show (from the slope) higher wound closure rates in the presence of the conditioned medium. This is because this approach provides an environment that mimics that of a wound healing process in vivo [1517]. <>/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>> Additionally, our plugin allowed us to compare easily the two methods evaluated to make the wounds. 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. PLoS ONE 15(7): 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). 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. B. endobj *4!Mn'1k8:OsM^Y4PxQrG!0:RU@`'^{BW[>&ufK3DWM /7e3xZNS|X?X[=z`Q|(T%+? Yes B. Width measurements with (blue) and without correction (white). 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. 42 0 obj Yes Time lapse (0, 12, 18 and 24 hours. Learn about all CytoSMART imaging solutions here. Methodology, Competing interests: The authors have declared that no competing interests exist. <>/F 4/Dest[ 25 0 R/XYZ 68 547 0] /StructParent 12>> 31 0 obj Lin, K.-Y. <>/F 4/Dest[ 17 0 R/XYZ 68 316 0] /StructParent 8>> For in-depth wound healing assay guidelines on parameters to ensure quantitative and reproducible results, please see the review by Jonkman et al. Cellular behavior regulates wound healing during the phases of proliferation, migration, matrix formation, and contraction. Alternatives to mechanical damage can include thermal, electrical, and optical wounding [2], [7]. Want to stay up to date? For researchers interested in such a functionality see the CytomSMART Omni landing page. Importantly, the time for analysis for 60 images was only 5 to 8 min, which represents less than 10% of the time it takes to do it manually. 34 0 obj [8] R. Riahi, Y. Yang, D. D. Zhang, and P. K. Wong, Advances in wound-healing assays for probing collective cell migration, J. Studies have also examined the migration of vascular smooth muscle cells [5], [6]. A. Interface window to adjust parameters B. 1, p. 38, 2015. 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. 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. [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. As it was expected, deviation of the width appears not a function of the evaluated times or used medium. This is most likely because, as described by Liang et al. 5, pp. This plugin facilitates the quantification of wound characteristics and circumvents their manual acquisition in many images obtained during each experiment. 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]. conditions of the tissue before wounding such as, an intact epithelium. 4 0 obj Citation: Suarez-Arnedo A, Torres Figueroa F, Clavijo C, Arbelez P, Cruz JC, Muoz-Camargo C (2020) An image J plugin for the high throughput image analysis of in vitro scratch wound healing assays. The wound healing assay can be used in the study of wound dressings. 137, no. 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. https://doi.org/10.1371/journal.pone.0232565.g005. We changed the medium three times per week until the cells reached 70% of confluence. %PDF-1.5 (2015) to investigate vascular smooth muscle cell (VSMC) migration. Department of Biomedical Engineering, Universidad de los Andes, Bogot, Colombia, Roles Supervision, 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). The preparation of gaps can be performed manually or be automated. We created the schematic representation of the use of the mold for scratching wounds on 2D cultures using (www.biorender.com). <> 16 0 obj Validation, We then washed away detached cells with PBS (1X). endobj 1, pp. 32 0 obj For more information about PLOS Subject Areas, click 6, pp. endobj broad scope, and wide readership a perfect fit for your research every time. <>/XObject<>/Pattern<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/MediaBox[ 0 0 595.32 841.92] /Contents 72 0 R/Group<>/Tabs/S/StructParents 21>> A. Scratch with low variation of calculated area between the different methods. 9, no. endobj p-value<0.0001****, p-value<0.001***, p-value<0.01** Some error bars are shorter than the size of the symbols. Additionally, At = 0 is the initial wound area, At = t is the wound area after n hours of the initial scratch, both in m2. No, Is the Subject Area "Image analysis" applicable to this article? We would like to acknowledge Natalia Bolaos and Dr. John Mario Gonzlez for providing the HaCaT cells for the study. 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. endobj 111, 2019. 25 0 obj 15 0 obj We analyzed and plotted The results using GraphPad Prism software 7 (GraphPad Software, San Diego, California, USA, www.graphpad.com).
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