A. A Scratch identifies with our plugin.
Proliferation inhibitors, such as mitomycin C, can also be added to the medium. The relative wound density method can be further refined to factor in cell proliferation or pharmacological effects. ------------------------------------------------------. The manual extraction of data is very time-consuming, subjective, and prone to error. endobj [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. We would like to acknowledge Natalia Bolaos and Dr. John Mario Gonzlez for providing the HaCaT cells for the study.
endobj
It can be adapted to analyze both individual images and stacks. For more information about PLOS Subject Areas, click By increasing this value, we expect an increase in the contrast. All these biomolecules activate signaling cascades that enhance the migratory and proliferative potential of various cell types [7, 10, 35]. 1524, 2019. Two of the principal attributes to be found in a standardized assay are reproducibility and repeatability. Yes <>
Copyright: 2020 Suarez-Arnedo et al. 247261, 2011. The average runtime per image is another plugin feature we evaluated. We then washed away detached cells with PBS (1X). For more information about PLOS Subject Areas, click In contrast, after the same time, the rate of cell migration for the control cells slightly increases and approaches an average value of 7.9 m/hour until the end of the experiment. Yes Autom., vol. Scale bar = 200 pixels. 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]. <>/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>> endobj Additionally, we convert the image resulting from the variance filter to a mask by applying the given threshold. Some of these molecules include growth factors, proteins, natural compounds, and small pharmacological principles, among many others [2, 8, 9]. All the measurements and parameters were taken for 28 hours [n = 4 replicas per time for HaCaT exposed to hAdMSCs conditioned medium and n = 2 replicas per time for HaCaT exposed to control medium]. endobj 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).
Nonetheless, after this time, we reduced the migration rate to 14.3 m/hour (Fig 4F). The percentage of saturated pixels allows enhancing the contrast of the image by determining the number of pixels that could saturate in the image. over time expressed as a percentage and is most often applied in live-cell imaging software [1], [12]. <>/F 4/Dest[ 17 0 R/XYZ 68 644 0] /StructParent 5>> The most frequently used method is to wound the monolayer by mechanical scratching (scratch wound) or stamping. 4, no. As shown in Fig 4, conditioned medium from hAdMSCs promoted healing in an in vitro epidermal model. Migration is considered the rate-limiting process during healing, and therefore migration assays are a key part of investigating wound healing [2]. [15] K. A. This mini-live cell imaging system operates from within cell culture incubators and allows for side-by-side comparison between cell cultures.
<>/F 4/Dest[ 37 0 R/XYZ 68 771 0] /StructParent 15>> Lab. <>>> endobj <>/F 4/Dest[ 25 0 R/XYZ 68 648 0] /StructParent 11>> endobj The difference between MRI method and our plugin can be explained by the image enhancement used as pre-processing. [17] K. Kobiyama, R. Saigusa, and K. Ley, Vaccination against atherosclerosis, Curr. F. Rate of cell migration in m/hour. Growth factors and matrix signals that determine the function of cells in regeneration processes orchestrate this behavior [1]. Most wound healing assays still use manual data acquisition and analysis in part. An increase in the threshold value increases the determined area (S3B Fig). Main, C. M. Mikelis, and C. L. Doi, In Vitro Wound Healing Assays to Investigate Epidermal Migration, 2019. 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. <>/F 4/Dest[ 12 0 R/XYZ 68 771 0] /StructParent 3>> 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. endobj 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. A microfluidic wound healing assay was developed by Wei et al. 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. The S1 File and S1 Fig show molds for wound scratching for 6, 24, 48 and 96-well plates. LIRa{lxo~w(AEA.e-x\HrCOJ{Dx^AZvT? _39ODM8_*ejAli i{M?7Sr]]SCS6O@"rV:[@$LC@, Wound with manual measurement of width. <>/F 4/Dest[ 22 0 R/XYZ 68 771 0] /StructParent 9>> 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. 43 0 obj This last tool avoids setting up parameters manually because it is a user-trainable plugin based on a support vector machine (SVM) algorithm [29]. https://doi.org/10.1371/journal.pone.0232565.g002. Sign up for our newsletter. This is most likely because, as described by Liang et al. Furthermore, to analyze the results of the Wound healing size tool test we used Two-Way ANOVA and the Bonferroni tests to perform multiple comparisons. 16, 2019. 20, no. The measures of the wounds width is likely to be impacted by its inclination. 4 0 obj Some of the most popular software packages for this application include the open source imageJ/Fiji and licensed ones such as Matlab. W*];2Whh mxWKY>LZ^2%Qs|~0w'fB\(k+vg`v!=^.+uHEqiW_# <>/F 4/Dest[ 29 0 R/XYZ 68 748 0] /StructParent 14>> *4!Mn'1k8:OsM^Y4PxQrG!0:RU@`'^{BW[>&ufK3DWM /7e3xZNS|X?X[=z`Q|(T%+? <>/F 4/Dest[ 17 0 R/XYZ 68 547 0] /StructParent 6>> <>/F 4/Dest[ 41 0 R/XYZ 68 771 0] /StructParent 17>> Three-dimensional collective migration takes place on a tissue scaffold, and it is the movement of cells organized in a multicellular network of strands. [ 33 0 R] Even though not comparable with our study, similar results for wound closure were found by Walter et al. Technol., vol. 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. 101, no. [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. 11 0 obj PLOS ONE promises fair, rigorous peer review, 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. [4] J. E. N. Jonkman et al., An introduction to the wound healing assay using live-cell microscopy, Cell Adh. Fig 5A shows how all evaluated methods distinguish gaps with regular shape similarly. e11--e16, 2017. <> 32 0 obj [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. This width is the average distance between the two margins of the scratch. 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. <>/F 4/Dest[ 29 0 R/XYZ 68 771 0] /StructParent 13>> 5, p. 14049, 2015. 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. No, Is the Subject Area "Pipettes" applicable to this article? 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]. 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]. 112, 2019. This can be achieved by counting cells in subregions found inside and outside the wound area to determine relative cell density [12]. 7987, 2016. The plugin features an interface window with two options to adjust the values of all parameters (Fig 2A). https://doi.org/10.1371/journal.pone.0232565.g006. <> We calculate the Euclidean distance between paired pixels, and we determine their average to describe wound width. Total cell count within the wound area can also be used to assess cell migration and wound healing [2]. 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. 13 0 obj 7 0 obj Scale bar = 27550 m) images of wound healing closure in HaCaT exposed to hAdMSCs conditioned medium. 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]. endobj <>/ExtGState<>/XObject<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/MediaBox[ 0 0 595.32 841.92] /Contents 115 0 R/Group<>/Tabs/S/StructParents 26>> Examples of some of these applications are presented below. Decreasing the concentration of serum (serum starvation) is the most common change. %PDF-1.5 Data Availability: All relevant data are within the manuscript and its Supporting Information files. This value should always be greater than zero. endobj [18] A. Alves et al., Xanthan Gum--Konjac Glucomannan Blend Hydrogel for Wound Healing, Polymers (Basel)., vol. <> 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]. 9, no. endobj Nonetheless, the implementation of the wound healing assay is generally challenging due to the impact of multiple variables such as the tool used to make the scratch, the pressure, the velocity of the scratching, and the experience of the researcher [19, 20, 30]. endobj endobj [19] X. Wang, C. C. Decker, L. Zechner, S. Krstin, and M. Wink, In vitro wound healing of tumor cells: inhibition of cell migration by selected cytotoxic alkaloids, BMC Pharmacol. The variation was only of about 5% (Fig 3C). Click through the PLOS taxonomy to find articles in your field. 26 0 obj % Methods that avoid cell damage altogether, such as physical exclusion, have also been described [4], [10]. 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. This process involves cell migration, proliferation, and differentiation [1]. endobj Once images of the gap closing have been acquired, several methods of analysis can be used to quantify the rate of cell migration. Data curation, Validation, Roles <>/F 4/Dest[ 12 0 R/XYZ 68 218 0] /StructParent 4>> 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]. 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]. 137, no. It can be used with different operating systems and it is easy to install. [6] Y. Wei et al., A tubing-free microfluidic wound healing assay enabling the quantification of vascular smooth muscle cell migration, Sci. (2020) used the assay to investigate novel hydrogel blends as a potential wound dressing. For these reasons, we designed a polymethyl methacrylate mold for a 24-well plate. Roles 36 0 obj Moreover, since there can be small regions within the cell monolayer with low variance, this algorithm can yield more than one region labeled as open wound. [5] A. D. der Meer, K. Vermeul, A. after 28 hours (closer to 95%) [10]. This is because the MiToBo tool is a highly reliable plugin based on histogram features and support vector machines (SVMs) [29]. Collective migration can take on two different forms depending on the extracellular matrix. 14 0 obj Cell proliferation can compete with cell migration to fill the gap made during the assay. Our results indicate that accurate analyses are likely to be found for variance window radii is between 3 to 25, threshold values in the range of 50 to 150 and percentages of saturated pixels between 0.001 to 0.4. 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. The area obtained with our plugin led to no significant differences with respect to the other methods (Fig 5). <>/F 4/Dest[ 43 0 R/XYZ 68 742 0] /StructParent 19>> We grew cells in DMEM supplemented with 10% FBS, 1% P/S at 37 C, 5% CO2 and 75% of humidity. 5, pp. For researchers looking to evaluate single-cell migration patterns, we recommend the CytoSMART Lux2 Duo Kit. A. Scratch with low variation of calculated area between the different methods. [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. The latter is defined by Grada et al. 5vB1M?`9: {N|=MQ*K#hA+ Xo{]#A$`QjX'T TLoq}wusM?W3P*S~]@.Y` A[;LcTXB02Ur2_l0`}v,c"9'&zi7rrVb[n7x9+afkxg?$0 k} ?>&0 Or"6sl0RP[rRh\JeOAeF@hIX<4xTn_dS=x+fB5=0A GJvqUhP We calculated the rate of cell migration (RM) and the percentage of wound closure according to (Eq 1) and (Eq 2), respectively [28]: Additionally, special gratitude to Laura Bocanegra for her collaboration with image acquisition. It is easy to use and can be adapted to analyze both individual images and stacks. Some adherent cell lines used for the assay are endothelial, fibroblast and epithelial [4]. (2015) to investigate vascular smooth muscle cell (VSMC) migration. We estimated these migration times with the data analysis provided by our plugin. <>/F 4/Dest[ 43 0 R/XYZ 68 771 0] /StructParent 18>> 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]. Both of these diseases are a major cause of morbidity and mortality globally [17]. 27 0 obj here. If this occurs, the cell medium can be optimized to reduce cell proliferation. H719--H725, 2010. <> Furthermore, our plugin is 6 times faster than the manual method. The wound healing size tool provides an automatic method of processing images obtained from a scratch/ wound healing assay.
endobj V6!o j3[RxS:3+Xa.BPv>#1%etR0#C>rW~ %W-){bdul?6bFndC9b J1L}*Y/|vHXSxWAu-{[Zn:. No, Is the Subject Area "Magnetic resonance imaging" applicable to this article? The technique reproduces wounding by creating a gap in a confluent cell monolayer and consists of four main steps that will be elaborated upon below (see Figure 1 for an overview) [1]. While this assay does not recapitulate the exact conditions of a wound, it can be successfully applied to model and investigate cell movement in a controlled in vitro environment. endobj Conceptualization, 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. 2016 [2]. https://doi.org/10.1371/journal.pone.0232565.s007. xn8xi &/-ivv2u`ooPE"-nvC9/eY_xXr^|I^]_Wwg70NI/#&==?4L8y%zR$bjDLe"'sg5iB+2 ,OnqTvuLKI+r/r&Q$4ed3? 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]. [7] C. R. Keese, J. Wegener, S. R. Walker, and I. Giaever, Electrical wound-healing assay for cells in vitro, Proc. endobj In the study microfluidic wound healing assay uses five types of VSMCs [6]. 37 0 obj p-value<0.0001****, p-value<0.001***, p-value<0.01** Some error bars are shorter than the size of the symbols. Cellular behavior regulates wound healing during the phases of proliferation, migration, matrix formation, and contraction. 18 m/hour) during the first 12 hours of the experiment. Yes endobj We changed the medium three times per week until the cells reached 70% of confluence. Depending on the cell type used in the assay, cell proliferation can be a confounding factor. 12, pp. 25 0 obj 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. The study was able to identify certain cytotoxic alkaloids as anti-migrating agents that could be investigated further [19]. <>
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 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. 9, no. D. Scratch width in m. Figure 2 | The advantages of automating the wound healing assay. [8] R. Riahi, Y. Yang, D. D. Zhang, and P. K. Wong, Advances in wound-healing assays for probing collective cell migration, J. Department of Biomedical Engineering, Universidad de los Andes, Bogot, Colombia, Roles Sci., vol.
endobj Writing review & editing.
Figure 3 | Methods to quantify cell migration in the wound healing assay. successfully screened cytotoxic alkaloids for the ability to inhibit biological processes related to cell migration and cytoskeleton dynamics. Effect of changing the threshold value while maintaining constant the variance window radius = 20 and the percentage of saturated pixels = 0.01.
15 0 obj 2, pp. It quantifies the area, wound coverage of total area, average wound width and width standard deviation. E. Standard deviation of the scratch width in m. <>/ExtGState<>/XObject<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/MediaBox[ 0 0 595.32 841.92] /Contents 94 0 R/Group<>/Tabs/S/StructParents 22>> (2016) as the coordinated movement of a group of cells that maintains their intercellular connections and collective polarity [1].
The migration images can then be used to collect measurements or be visually scored [4]. 8, no. Lin, K.-Y. 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. Four processes occur during wound healing, namely haemostasis, inflammation, migration-proliferation, and maturation-remodeling [1], [2]. A Schematic of the use of mold for scratching wounds on 2D cultures. Therefore, we can apply a simple threshold to the processed image in order to obtain binary segments with a user-defined input. PLoS ONE 15(7): 1, p. 21, 2004. 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. This reproducibility can be improved with the use of commercial tools for making uniform gaps [1]. Formal analysis, 31 0 obj conditions of the tissue before wounding such as, an intact epithelium.
Automated data analysis software, such as TScratch and ImageJ, has been developed that can significantly accelerate analysis and overcome limitations associated with image quality [14], [15]. Inhibitors of cell invasion and metastasis can be effectively screened using the wound healing assay. Acad. 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]. 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. A. endobj Understanding the processes involved in this migration can provide potential targets for inhibition. The second method calculates the change in wound area over time as a percentage of wound closure. 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). <>/F 4/Dest[ 17 0 R/XYZ 68 452 0] /StructParent 7>> 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. 30 0 obj 265274, 2009. 16 0 obj Time lapse (0, 12, 18 and 24 hours. This result is most likely due to the high rate of cellular migration (aprox. <>/F 4/Dest[ 39 0 R/XYZ 68 471 0] /StructParent 16>> 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. 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. Yes 44 0 obj endobj Yes The cells were isolated following the protocol by Linero et al. 42 0 obj [7]. The first one applies when the user has multiple images with the same calibration scale. endobj The small difference found between the area estimated with the MiToBo plugin and our plugin validates the robustness of our approach. Fig 1 presents an overview of our image-processing algorithm. This modified wound healing assay is a closer representation of the microenvironment present in the vasculature. This live-cell imaging system operates from within cell culture incubators and is fully automated, to easily create time-lapse videos of gap closure. 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.
here. 9, no. <>/ExtGState<>/XObject<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/MediaBox[ 0 0 595.32 841.92] /Contents 64 0 R/Group<>/Tabs/S/StructParents 20>>
endobj
Wounds with different angles of inclination. 8 0 obj Finally, the algorithm also considers cases in which the wound is not vertical but made at a specific angle. <>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/MediaBox[ 0 0 595.32 841.92] /Contents 117 0 R/Group<>/Tabs/S/StructParents 36>> This monolayer represents the in vivo 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. e0232565. https://doi.org/10.1371/journal.pone.0232565.g005. endobj 1, p. 38, 2015. Studies have also examined the migration of vascular smooth muscle cells [5], [6]. To address this issue, we designed an acrylic (polymethyl methacrylate) mold that fits 6, 24, 48, and 96 well plates (S1 File) (Fig 3A). This straightforward technique relies on adherent cells that could vary depending on the particular cellular processes to study. Writing original draft, Affiliation 111, 2019. Wang et al. endobj Some phases that occur during wound healing have also been observed during cancer invasion [2, 3]. 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).
We created the schematic representation of the use of the mold for scratching wounds on 2D cultures using (www.biorender.com).
9, no. Supervision, 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]. 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. It allows the analysis of bright field, phase contrast, and fluorescence images.
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