21 0 obj 1, pp. We then washed

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21 0 obj 1, pp. We then washed away detached cells with PBS (1X). xn8xi &/-ivv2u`ooPE"-nvC9/eY_xXr^|I^]_Wwg70NI/#&==?4L8y%zR$bjDLe"'sg5iB+2 ,OnqTvuLKI+r/r&Q$4ed3? According to Fig 6, there is no significant difference between the average width calculated with our plugin or calculated manually. endobj 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]. 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. By increasing this value, we expect an increase in the contrast. The first method measures the change in the wound width (nanometres) over time. In such cases, it determines the inclination angle and adjusts each calculated width with trigonometric relations (S4 Fig). [7] C. R. Keese, J. Wegener, S. R. Walker, and I. Giaever, Electrical wound-healing assay for cells in vitro, Proc. The cells were isolated following the protocol by Linero et al. 101, no.

Figure 2 | The advantages of automating the wound healing assay. Fig 5A shows how all evaluated methods distinguish gaps with regular shape similarly. endobj 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. [15] K. A. 8, no. It quantifies the area, wound coverage of total area, average wound width and width standard deviation. stream 1, pp. 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. 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). It is easy to use and can be adapted to analyze both individual images and stacks. In this regard, the scratch or migration assay is a widely used tool for in vitro studies of the rates of migration [5], angiogenesis [6], movement [7], proliferation [2], and healing in response to different novel drug candidates. To remove these structures, we perform a morphological reconstruction by erosion, also known as hole filling, on the area binary labeled as open wound. We grew cells in DMEM supplemented with 10% FBS, 1% P/S at 37 C, 5% CO2 and 75% of humidity. 20 0 obj Immunol., vol. <> The first step is to create manually a scratch in a cell monolayer, using pipette tips, razors [17], cell scrapers [18], or any object with a sharp tip. This is also the case with respect to our plugin where overestimations approached 6.5% 4.4 and 10.33% 6.9, respectively. endobj C. Effect of changing the percentage of saturated pixels while maintaining constant the variance window radius = 20 and the threshold value = 100. https://doi.org/10.1371/journal.pone.0232565.s005. [11] S. Martinotti and E. Ranzato, Scratch Wound Healing Assay, 2019. conditions of the tissue before wounding such as, an intact epithelium. 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.

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. <>/F 4/Dest[ 29 0 R/XYZ 68 771 0] /StructParent 13>>

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 46, no. 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. A. ROI selection in images analyzed with different set parameters B. 298, no. successfully screened cytotoxic alkaloids for the ability to inhibit biological processes related to cell migration and cytoskeleton dynamics. Depending on the cell type used in the assay, cell proliferation can be a confounding factor. endobj <> Main, C. M. Mikelis, and C. L. Doi, In Vitro Wound Healing Assays to Investigate Epidermal Migration, 2019. Upon injury and under the influence of growth factors and cytokines, keratinocytes at the rear of the wound margin begin to proliferate and migrate onto the wound bed. The final method measures the relative wound density Conceptualization, We estimated these migration times with the data analysis provided by our plugin. The preparation of gaps can be performed manually or be automated. Wound with manual measurement of width. 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). These changes in the migration rate of keratinocytes exposed to both media at 12 hours point to the interplay of additional variables such as the intrinsic growth rate of the cells (proliferation) and complex effects elicit by serum starvation [28]. Additionally, we convert the image resulting from the variance filter to a mask by applying the given threshold. [2] A. Stamm, K. Reimers, S. Strau, P. Vogt, T. Scheper, and I. Pepelanova, In vitro wound healing assays--state of the art, BioNanoMaterials, vol. Nonetheless, there is a significant difference (of about 36%) in the standard deviation between the two methods. Accordingly, we identify edge pixels within the segmented region, and we pair them according to their y-coordinate. Opin. 29 0 obj 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. To do this, we initially enhanced the contrast of the image to increase the variance within the cell monolayer and facilitate its posterior binarization. 112, 2019. PLOS ONE promises fair, rigorous peer review, 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. Acad. It corrects the average wound width by considering its inclination. Aspects of acquisition that can be automated include image capturing, point visiting, and environmental control [2], [4]. 30 0 obj For researchers interested in such a functionality see the CytomSMART Omni landing page. Toxicol., vol. F. Rate of cell migration in m/hour. endobj The radius must be big enough, so that the noise variance has no impact on tissue variance. Yes

This plugin is useful for analyzing wound images since it relies on user-defined input values for the neighborhood radius of the variance filter, the threshold value for binarization, and the saturation percentage in the contrast enhancement, which can vary depending on the analyzed image. Physiol., vol. 2 0 obj It allows the analysis of bright field, phase contrast, and fluorescence images. 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]. Similarly, we can observe that after 6 hours the scratch width in Fig 4D was also significantly reduced in the presence of the hAdMSCs conditioned medium. incubator microscopy culture [7]. The first one applies when the user has multiple images with the same calibration scale. Natl. https://doi.org/10.1371/journal.pone.0232565.g005. Four processes occur during wound healing, namely haemostasis, inflammation, migration-proliferation, and maturation-remodeling [1], [2]. The manual extraction of data is very time-consuming, subjective, and prone to error. 10 0 obj 44 0 obj 5, pp. Three-dimensional collective migration takes place on a tissue scaffold, and it is the movement of cells organized in a multicellular network of strands. Two-dimensional (2D) collective migration (sheet migration) takes place across a tissue surface, and it is the movement of flat monolayer sheets. 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. We changed the medium three times per week until the cells reached 70% of confluence. endobj A microfluidic wound healing assay was developed by Wei et al. e0232565. <>/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>> We calculated the rate of cell migration (RM) and the percentage of wound closure according to (Eq 1) and (Eq 2), respectively [28]: Most often, epithelial and endothelial cells are used to produce the monolayer, and in particular, cell types capable of sheet migration [4]. In microfluidic wound healing assays, chemical and enzymatic treatments <> <>/F 4/Dest[ 37 0 R/XYZ 68 771 0] /StructParent 15>> [1] A. Grada, M. Otero-Vinas, F. Prieto-Castrillo, Z. Obagi, and V. Falanga, Research techniques made simple: analysis of collective cell migration using the wound healing assay, J. Writing original draft, Affiliation <>/F 4/Dest[ 17 0 R/XYZ 68 452 0] /StructParent 7>> JFIF Ducky. 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].

Reproducibility in the scratches is imperative, as it has been reported to significantly affect cell migration rates [17, 28]. https://doi.org/10.1371/journal.pone.0232565, Editor: Giuseppe Chirico, Universita degli Studi di Milano-Bicocca, ITALY, Received: April 16, 2020; Accepted: July 11, 2020; Published: July 28, 2020. In conclusion, this new imageJ plugin is a robust tool to automatically standardize and facilitate quantification of different in vitro wound parameters with high accuracy compared with other tools and manual identification. A. Poot, J. Feijen, and I. Vermes, A microfluidic wound-healing assay for quantifying endothelial cell migration, Am. <> 2, pp. Roles Drawbacks with the manual method of wounding include low throughput and well-to-well variation of the gap width [1], [11]. 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). Cell migration is best viewed using phase-contrast imaging rather than fluorescence, and the wound area in the field-of-view should be maximized using the objective lens [12], [13]. Three-dimensional (3D) wound healing assays are beyond the scope of this article and have been succinctly covered by Stamm et al. <>/F 4/Dest[ 17 0 R/XYZ 68 316 0] /StructParent 8>> Percentage of closure area. endobj B. We show images from each of the main steps to illustrate how the wound area is separated from the cell monolayer area. This result is most likely due to the high rate of cellular migration (aprox. endobj 4, no. endobj Migration is considered the rate-limiting process during healing, and therefore migration assays are a key part of investigating wound healing [2]. This is because the MiToBo tool is a highly reliable plugin based on histogram features and support vector machines (SVMs) [29]. This method calculates the ratio of the occupied area in the gap to the total area of the initial gap. <>/F 4/Dest[ 25 0 R/XYZ 68 547 0] /StructParent 12>> To overcome these limitations, we designed, implemented, compared, and tested a user-friendly plugin for ImageJ/Fiji. Additionally, it allows the analysis of images obtained from bright field, phase contrast, and fluorescence microscopes. 25 0 obj Both of these diseases are a major cause of morbidity and mortality globally [17]. 19 0 obj Methods that avoid cell damage altogether, such as physical exclusion, have also been described [4], [10]. 27 0 obj This width is the average distance between the two margins of the scratch. Scale bar = 200 pixels. This can be achieved by counting cells in subregions found inside and outside the wound area to determine relative cell density [12]. Additionally, our plugin allowed us to compare easily the two methods evaluated to make the wounds. The plugin features an interface window with two options to adjust the values of all parameters (Fig 2A). The area obtained with our plugin led to no significant differences with respect to the other methods (Fig 5). This mold is reusable and can be sterilized using different strategies such as supercritical carbon dioxide, hydrogen peroxide gas plasma, ethylene oxide, and even -irradiation [31, 32]. https://doi.org/10.1371/journal.pone.0232565.s007. 1, pp. 23 0 obj This process involves cell migration, proliferation, and differentiation [1]. https://doi.org/10.1371/journal.pone.0232565.g002. https://doi.org/10.1371/journal.pone.0232565.g001. <> Methodology, Sign up for our newsletter. 14 0 obj 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. 9, no. <> <> endobj <> endobj 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. 9, no. 26 0 obj The last step is to monitor the migration path of cells in the leading edge of the scratch by tracking it with the aid of time-lapse microscopy (4) and image analysis software [4, 22]. endstream In contrast, since the average execution time of the ScratchAssayAnalyzer in MiToBo is about 2 minutes our plugin is 94% faster. 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 next step is exposure to the treatment and image acquisition at the beginning and at regular intervals during cell migration as the scratch closes.

This approach is however demanding, tedious, and time-consuming. 18 0 obj <>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/MediaBox[ 0 0 595.32 841.92] /Contents 117 0 R/Group<>/Tabs/S/StructParents 36>> The percentage of saturated pixels allows enhancing the contrast of the image by determining the number of pixels that could saturate in the image.

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. No, Is the Subject Area "Radii" applicable to this article? Nonetheless, it is important to clarify that in our tool it is necessary to adjust, the input parameters to fit accurately the detected area (S3 Fig). endobj Whole-well imaging systems can be applied to the wound-healing assay. endobj Cellular behavior regulates wound healing during the phases of proliferation, migration, matrix formation, and contraction. have been used to clear cells from fluid channels [5], [6], [8], [9]. Furthermore, our plugin is 6 times faster than the manual method. These two methods can be time-consuming when performed manually. The HaCaT [26] cell line was provided by the Basic Medical Sciences Laboratory of the Faculty of Medicine of the Universidad de los Andes. 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. Therefore, we can apply a simple threshold to the processed image in order to obtain binary segments with a user-defined input. Lo, and Y.-S. Sun, A microfluidics-based wound-healing assay for studying the effects of shear stresses, wound widths, and chemicals on the wound-healing process, Sci. 3, pp. endobj 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]. endobj Data curation, endobj To validate the accuracy of our plugin to locate the scratch in each image, 30 images were analyzed and compared with the MRI wound healing tool, the ScratchAssayAnalyzer in MiToBo and with manually drawing the scratch. In the study microfluidic wound healing assay uses five types of VSMCs [6]. 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). Wounds with different angles of inclination. 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. 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. [27]. The most frequently used method is to wound the monolayer by mechanical scratching (scratch wound) or stamping. Fig 1 presents an overview of our image-processing algorithm. 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. 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].

247261, 2011. Once images of the gap closing have been acquired, several methods of analysis can be used to quantify the rate of cell migration. 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]. here. Ensuring reproducibility is important for the following data acquisition step. 12 0 obj In order to develop robust therapeutic approaches, it is imperative to study these processes in detail [4]. 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*. Studies have also examined the migration of vascular smooth muscle cells [5], [6]. stream Inhibitors of cell invasion and metastasis can be effectively screened using the wound healing assay. 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. The wound-healing assay, also known as the scratch assay, is an established two-dimensional (2D) technique that can be used to investigate collective migration and wound healing in vitro [1], [2]. A. Scratch with low variation of calculated area between the different methods. Data curation, In this regard, although the MRI method and our plugin are based on a variance-based method, the preliminary step of contrast enhancement in our plugin helps to identify the edges of the scratch more precisely. <> This live-cell imaging system operates from within cell culture incubators and is fully automated, to easily create time-lapse videos of gap closure. Collective migration can take on two different forms depending on the extracellular matrix.

Even though not comparable with our study, similar results for wound closure were found by Walter et al. We scratched cell cultures with a 200 L sterile pipette tip [7, 22] using the designed mold (see above for details) (S1 File). Additionally, At = 0 is the initial wound area, At = t is the wound area after n hours of the initial scratch, both in m2. 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. Methods that are more sophisticated include molds or cell inserts [19, 20], electric currents [21], lasers [4] and magnets [20]. Also, both treatments were fitted to linear regressions that show (from the slope) higher wound closure rates in the presence of the conditioned medium. endobj endobj [13] C. N. Ramirez et al., Validation of a high-content screening assay using whole-well imaging of transformed phenotypes, Assay Drug Dev. <>/XObject<>/Pattern<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/MediaBox[ 0 0 595.32 841.92] /Contents 72 0 R/Group<>/Tabs/S/StructParents 21>> Our plugin is easy to install and can be used with different operating systems. Yes Sci., vol. after 28 hours (closer to 95%) [10]. 1 0 obj The effect of the hydrogel on wound closure was assessed by incorporating it into the cell medium. <>/ExtGState<>/XObject<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/Annots[ 99 0 R 100 0 R 101 0 R 102 0 R 103 0 R] /MediaBox[ 0 0 595.32 841.92] /Contents 98 0 R/Group<>/Tabs/S/StructParents 23>> 111, 2019. 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. Rep., vol. 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. 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. 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. The wound healing assay is a convenient and economical method to investigate collective cell migration under different experimental conditions.