Additionally, our plugin allowed us to compare easily the two methods evaluated to make the wounds. Also, both treatments were fitted to linear regressions that show (from the slope) higher wound closure rates in the presence of the conditioned medium. 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
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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 Wounds with different angles of inclination. %PDF-1.5
Alves et al. This creates a new image with high intensity pixels within the cell monolayer and low intensity pixels within the open wound area. ?YDTd0 We calculated the rate of cell migration (RM) and the percentage of wound closure according to (Eq 1) and (Eq 2), respectively [28]: We evaluated the performance of our plugin by analyzing 60 wound images. Once we made the reference lines (approximately 3000 m of distance), the plate was placed under a phase-contrast microscope using as guide the reference marks. 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 wound healing assay is a convenient and economical method to investigate collective cell migration under different experimental conditions. The migration of these cells after endothelial injury is an inherent factor in the progression of atherosclerosis and the complications associated with intimal hyperplasia [6]. <>/ExtGState<>/XObject<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/MediaBox[ 0 0 595.32 841.92] /Contents 64 0 R/Group<>/Tabs/S/StructParents 20>>
23 0 obj
Methods that are more sophisticated include molds or cell inserts [19, 20], electric currents [21], lasers [4] and magnets [20]. endobj
As it was expected, deviation of the width appears not a function of the evaluated times or used medium. Supervision, 5965, 2012.
We obtained the hAdMSCs of abdominoplasties realized at Santa Barbara Surgical Center by Dr. Santiago Merchan with previous approval and signing of informed consent and with the approval of the ethics committee of the Vice-presidency of research at the Universidad de Los Andes (Act No 942, 2018). Invest. 18 m/hour) during the first 12 hours of the experiment. (2016) as the coordinated movement of a group of cells that maintains their intercellular connections and collective polarity [1]. 4, pp. Wound with manual measurement of width. 44 0 obj
1, pp. endobj 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. The preparation of gaps can be performed manually or be automated. 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]. endobj
https://doi.org/10.1371/journal.pone.0232565.g004. 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. All these biomolecules activate signaling cascades that enhance the migratory and proliferative potential of various cell types [7, 10, 35]. Methodology, Department of Biomedical Engineering, Universidad de los Andes, Bogot, Colombia, Roles 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. Finally, the algorithm also considers cases in which the wound is not vertical but made at a specific angle. 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]. [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 average runtime per image is another plugin feature we evaluated.
Furthermore, the mold produced more reproducible and homogeneous scratches in contrast with gaps made using only a pipette tip. 9, no. This is also the case with respect to our plugin where overestimations approached 6.5% 4.4 and 10.33% 6.9, respectively. 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 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]. https://doi.org/10.1371/journal.pone.0232565.g006. Even though not comparable with our study, similar results for wound closure were found by Walter et al. <>/F 4/Dest[ 17 0 R/XYZ 68 547 0] /StructParent 6>> Decreasing the concentration of serum (serum starvation) is the most common change. Three-dimensional collective migration takes place on a tissue scaffold, and it is the movement of cells organized in a multicellular network of strands. 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. Understanding the processes involved in this migration can provide potential targets for inhibition. Immunol., vol. 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]. Some of these molecules include growth factors, proteins, natural compounds, and small pharmacological principles, among many others [2, 8, 9]. Yes Therefore, the analysis of large datasets remains a bottleneck with many assays. It can be used with different operating systems and it is easy to install. [13] C. N. Ramirez et al., Validation of a high-content screening assay using whole-well imaging of transformed phenotypes, Assay Drug Dev. Want to stay up to date? Validation, [18] A. Alves et al., Xanthan Gum--Konjac Glucomannan Blend Hydrogel for Wound Healing, Polymers (Basel)., vol. 112, 2019.
<>/F 4/Dest[ 17 0 R/XYZ 68 452 0] /StructParent 7>>
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. Investigation, A. This monolayer represents the in vivo Aspects of acquisition that can be automated include image capturing, point visiting, and environmental control [2], [4]. p-value<0.0001****, p-value<0.001***, p-value<0.01** Some error bars are shorter than the size of the symbols. Fig 1 presents an overview of our image-processing algorithm. 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*. [ 33 0 R]
[9] J.-Y. <>/ExtGState<>/XObject<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/MediaBox[ 0 0 595.32 841.92] /Contents 115 0 R/Group<>/Tabs/S/StructParents 26>>
This proves the effectiveness of our plugin to calculate the wounds width for different imaging conditions. Figure 2 | The advantages of automating the wound healing assay. endobj
111, 2019. These two methods can be time-consuming when performed manually. <>/XObject<>/Pattern<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/MediaBox[ 0 0 595.32 841.92] /Contents 72 0 R/Group<>/Tabs/S/StructParents 21>>
[16] J. C. Yarrow, Z. E. Perlman, N. J. Westwood, and T. J. Mitchison, A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods, BMC Biotechnol., vol. To address this issue, we designed an acrylic (polymethyl methacrylate) mold that fits 6, 24, 48, and 96 well plates (S1 File) (Fig 3A). The first method measures the change in the wound width (nanometres) over time. 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]. endobj
here. The manual extraction of data is very time-consuming, subjective, and prone to error. 17, no. endobj
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. 37 0 obj
9, no. The radius must be big enough, so that the noise variance has no impact on tissue variance. This mini-live cell imaging system operates from within cell culture incubators and allows for side-by-side comparison between cell cultures. Supervision, Our plugin is easy to install and can be used with different operating systems. 1, pp. For in-depth wound healing assay guidelines on parameters to ensure quantitative and reproducible results, please see the review by Jonkman et al. <>/F 4/Dest[ 43 0 R/XYZ 68 771 0] /StructParent 18>>
endobj
https://doi.org/10.1371/journal.pone.0232565.g005. Additionally, special gratitude to Laura Bocanegra for her collaboration with image acquisition. Time lapse (0, 12, 18 and 24 hours. endobj
B. endobj
Some adherent cell lines used for the assay are endothelial, fibroblast and epithelial [4]. Manual data (average = 1149 pixels, standard deviation = 22.82 pixels) WHST data (average = 1165 pixels, standard deviation 102.35 pixels). 2016 [2]. This operator detects all connected components enclosed by the wound area and it includes them as part of the wound. This has been attributed to the components of the medium, which include multiple growth factors such as Transforming growth factor-beta one (TGF-1), Epidermal growth factor (EGF), Fibroblast growth factor (FGF), Hepatocyte growth factor (HGF), Interleukin 6 and 8 (IL-6 and IL8), as well as components of the extracellular matrix such as type I collagen and fibronectin [10]. The small difference found between the area estimated with the MiToBo plugin and our plugin validates the robustness of our approach. 26 0 obj
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]. https://doi.org/10.1371/journal.pone.0232565.g002. The mold was laser cut using a Trotec Speedy 100 CO2 (Marchtrenk, Austria). A. The wound healing size tool provides an automatic method of processing images obtained from a scratch/ wound healing assay. None of the available plugins for ImageJ calculates the width of the wound, and therefore the comparison was only made with respect to the manual method. 2, pp. Main, C. M. Mikelis, and C. L. Doi, In Vitro Wound Healing Assays to Investigate Epidermal Migration, 2019. 298, no. A. 2329. Growth factors and matrix signals that determine the function of cells in regeneration processes orchestrate this behavior [1]. 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 variation was only of about 5% (Fig 3C).
This can be achieved by counting cells in subregions found inside and outside the wound area to determine relative cell density [12]. We grew cells in DMEM supplemented with 10% FBS, 1% P/S at 37 C, 5% CO2 and 75% of humidity. 28 0 obj Nonetheless, after this time, we reduced the migration rate to 14.3 m/hour (Fig 4F). 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. <>/F 4/Dest[ 12 0 R/XYZ 68 218 0] /StructParent 4>> This method was one of the first to be developed for the study of cell migration and measures the rate at which cells, in a cell monolayer, migrate to fill a cell-free gap [1], [3]. 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.
We obtained the hAdMSCs of abdominoplasties realized at Santa Barbara Surgical Center by Dr. Santiago Merchan with previous approval and signing of informed consent and with the approval of the ethics committee of the Vice-presidency of research at the Universidad de Los Andes (Act No 942, 2018). Invest. 18 m/hour) during the first 12 hours of the experiment. (2016) as the coordinated movement of a group of cells that maintains their intercellular connections and collective polarity [1]. 4, pp. Wound with manual measurement of width. 44 0 obj
1, pp. endobj 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. The preparation of gaps can be performed manually or be automated. 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]. endobj
https://doi.org/10.1371/journal.pone.0232565.g004. 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. All these biomolecules activate signaling cascades that enhance the migratory and proliferative potential of various cell types [7, 10, 35]. Methodology, Department of Biomedical Engineering, Universidad de los Andes, Bogot, Colombia, Roles 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. Finally, the algorithm also considers cases in which the wound is not vertical but made at a specific angle. 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]. [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 average runtime per image is another plugin feature we evaluated.
Furthermore, the mold produced more reproducible and homogeneous scratches in contrast with gaps made using only a pipette tip. 9, no. This is also the case with respect to our plugin where overestimations approached 6.5% 4.4 and 10.33% 6.9, respectively. 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 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]. https://doi.org/10.1371/journal.pone.0232565.g006. Even though not comparable with our study, similar results for wound closure were found by Walter et al. <>/F 4/Dest[ 17 0 R/XYZ 68 547 0] /StructParent 6>> Decreasing the concentration of serum (serum starvation) is the most common change. Three-dimensional collective migration takes place on a tissue scaffold, and it is the movement of cells organized in a multicellular network of strands. 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. Understanding the processes involved in this migration can provide potential targets for inhibition. Immunol., vol. 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]. Some of these molecules include growth factors, proteins, natural compounds, and small pharmacological principles, among many others [2, 8, 9]. Yes Therefore, the analysis of large datasets remains a bottleneck with many assays. It can be used with different operating systems and it is easy to install. [13] C. N. Ramirez et al., Validation of a high-content screening assay using whole-well imaging of transformed phenotypes, Assay Drug Dev. Want to stay up to date? Validation, [18] A. Alves et al., Xanthan Gum--Konjac Glucomannan Blend Hydrogel for Wound Healing, Polymers (Basel)., vol. 112, 2019.
<>/F 4/Dest[ 17 0 R/XYZ 68 452 0] /StructParent 7>>
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. Investigation, A. This monolayer represents the in vivo Aspects of acquisition that can be automated include image capturing, point visiting, and environmental control [2], [4]. p-value<0.0001****, p-value<0.001***, p-value<0.01** Some error bars are shorter than the size of the symbols. Fig 1 presents an overview of our image-processing algorithm. 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*. [ 33 0 R]
[9] J.-Y. <>/ExtGState<>/XObject<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/MediaBox[ 0 0 595.32 841.92] /Contents 115 0 R/Group<>/Tabs/S/StructParents 26>>
This proves the effectiveness of our plugin to calculate the wounds width for different imaging conditions. Figure 2 | The advantages of automating the wound healing assay. endobj
111, 2019. These two methods can be time-consuming when performed manually. <>/XObject<>/Pattern<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/MediaBox[ 0 0 595.32 841.92] /Contents 72 0 R/Group<>/Tabs/S/StructParents 21>>
[16] J. C. Yarrow, Z. E. Perlman, N. J. Westwood, and T. J. Mitchison, A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods, BMC Biotechnol., vol. To address this issue, we designed an acrylic (polymethyl methacrylate) mold that fits 6, 24, 48, and 96 well plates (S1 File) (Fig 3A). The first method measures the change in the wound width (nanometres) over time. 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]. endobj
here. The manual extraction of data is very time-consuming, subjective, and prone to error. 17, no. endobj
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. 37 0 obj
9, no. The radius must be big enough, so that the noise variance has no impact on tissue variance. This mini-live cell imaging system operates from within cell culture incubators and allows for side-by-side comparison between cell cultures. Supervision, Our plugin is easy to install and can be used with different operating systems. 1, pp. For in-depth wound healing assay guidelines on parameters to ensure quantitative and reproducible results, please see the review by Jonkman et al. <>/F 4/Dest[ 43 0 R/XYZ 68 771 0] /StructParent 18>>
endobj
https://doi.org/10.1371/journal.pone.0232565.g005. Additionally, special gratitude to Laura Bocanegra for her collaboration with image acquisition. Time lapse (0, 12, 18 and 24 hours. endobj
B. endobj
Some adherent cell lines used for the assay are endothelial, fibroblast and epithelial [4]. Manual data (average = 1149 pixels, standard deviation = 22.82 pixels) WHST data (average = 1165 pixels, standard deviation 102.35 pixels). 2016 [2]. This operator detects all connected components enclosed by the wound area and it includes them as part of the wound. This has been attributed to the components of the medium, which include multiple growth factors such as Transforming growth factor-beta one (TGF-1), Epidermal growth factor (EGF), Fibroblast growth factor (FGF), Hepatocyte growth factor (HGF), Interleukin 6 and 8 (IL-6 and IL8), as well as components of the extracellular matrix such as type I collagen and fibronectin [10]. The small difference found between the area estimated with the MiToBo plugin and our plugin validates the robustness of our approach. 26 0 obj
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]. https://doi.org/10.1371/journal.pone.0232565.g002. The mold was laser cut using a Trotec Speedy 100 CO2 (Marchtrenk, Austria). A. The wound healing size tool provides an automatic method of processing images obtained from a scratch/ wound healing assay. None of the available plugins for ImageJ calculates the width of the wound, and therefore the comparison was only made with respect to the manual method. 2, pp. Main, C. M. Mikelis, and C. L. Doi, In Vitro Wound Healing Assays to Investigate Epidermal Migration, 2019. 298, no. A. 2329. Growth factors and matrix signals that determine the function of cells in regeneration processes orchestrate this behavior [1]. 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 variation was only of about 5% (Fig 3C). This can be achieved by counting cells in subregions found inside and outside the wound area to determine relative cell density [12]. We grew cells in DMEM supplemented with 10% FBS, 1% P/S at 37 C, 5% CO2 and 75% of humidity. 28 0 obj Nonetheless, after this time, we reduced the migration rate to 14.3 m/hour (Fig 4F). 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. <>/F 4/Dest[ 12 0 R/XYZ 68 218 0] /StructParent 4>> This method was one of the first to be developed for the study of cell migration and measures the rate at which cells, in a cell monolayer, migrate to fill a cell-free gap [1], [3]. 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.