This study is supported by the grant of National Social Science Foundation (22FYSB023), Hubei Industrial Design Center Research Foundation (08hqt201412046), and Humanities and Social Science Foundation of Hubei Provincial Education Department (15Y054).

Full written informed consent was provided by all participants. Ethics approval was not applicable since no animal or human tissue samples were included in the present study.
1. Investigating the requirements of the user community
<ol>
	<li>Deliver questionnaires to the biologists/experimenter working on cell wound healing assays. Collect the completed questionnaires and use them for the study. For this study, from 12 Oct 2021 to 3 Feb 2022, 100 questionnaires were delivered to 100 biologists. The response percent was 97%.</li>
	<li>Ask respondents to answer questions like, which of the cell scratching experiments bothered you the most? and which tool was used to perform the cell scratching? Follow these questions with sub-questions to trace the causes.</li>
	<li>Investigate the experimentalists&#39; perceptions of tools such as pipette tips and cell culture inserts in cell wound healing experiments. Understand and gather their reasons for selecting one tool over another, using the Means-end-chain theory based on laddering techniques in the questionnaires section14,15. 
	&#8203;NOTE: The ladder method is divided into two types: the soft ladder method with in-depth interviews and the hard ladder method with questionnaires or paper-and-pencil quizzes16. The hard laddering method was the primary technique utilized in this study. The Means-end-chain theory suggests that the explicit knowledge held by the target users is superficial and concrete, while the tacit knowledge is deep and abstract17,18,19.</li>
</ol>
2. Design and three-dimensional modeling
<ol>
	<li>Draw a sketch from the insights gathered from the previous questionnaire survey and initiate the design process. Use this preliminary sketch as the foundational blueprint.
	<ol>
		<li>Elaborate on the dimensions and layout of each component by applying the Dim, DimRadius, and DimDiameter functions in the modeling software.</li>
		<li>Perform measurements by the vernier caliper with a precision of 0.02 mm on actual 6-well plates to ascertain the final dimensions of the cell scraper. Confirm these to be 42.1 mm in length, 42.1 mm in width, and 18.5 mm in height. Pay attention to the details in the design phase, especially the product height and fitness in the well, to facilitate a smoother assembly.</li>
	</ol>
	</li>
	<li>Construct a three-dimensional model and render it.
	<ol>
		<li>Begin 3D designing in a software by creating a basic model. Click buttons like ExtrudeCrv for stretching and Loft for shaping the design. Refine the model and then click FilletEdge for smooth edges. 
		NOTE: Other used function buttons include, Lines - To draw straight line segments, Polylines - To create continuous lines composed of multiple segments, Rectangles - To draw rectangular shapes, Circles - To draw circular shapes, Arcs - To draw arc or elliptical shapes, Points - To place single point markers, Text - To add text labels, Dimensions - To add measured dimensions to models, Array - To create copied patterns of objects, Rotate - To rotate objects to desired angles, Move - To move objects to new locations, Scale - To resize objects bigger or smaller, Trim/Extend - To trim or extend objects to meet other geometry, Boolean Union/Difference/Intersection - To combine, subtract from, or find the intersection of objects, Layers - To organize objects on different layers, Render - To generate rendered views with materials and lighting and Export - To export model geometry to other file formats.</li>
		<li>Import the model into a 3D rendering software. Apply materials including plastic, sponge, and steel, then adjust the lighting for realistic rendering. 
		NOTE: A generalized list of function buttons include, Drag &#38; Drop - To apply materials directly to parts or models by dragging a material from the library and dropping it onto the desired component in the real-time view, Right-click - When right-clicking on a material in the library, you will typically see options to: Apply to Selection - Apply the material to a selected part in the real-time view. Edit Material - Adjust the material properties. Material Properties (after selecting a material) - To access and modify specific properties of the material, such as color, roughness, refractive index, and other attributes. Search Bar - To quickly find a material in the library by typing its name or associated keywords. Categories/Folders - To navigate through different categories or folders of materials like metals, plastics, glass, etc. Add to Favorites - To mark certain materials as favorites for easy access during future sessions. Hotkeys - Some operations can be accessed via hotkeys. For instance, M is usually the hotkey to quickly bring up the material properties of a selected part.</li>
		<li>Finally, enhance the image&#39;s contrast (+56) and saturation (Vibrance +19, Saturation +7) in photo and design software, and add background elements (text information and color from white to light gray gradient background) for context to ensure a high-quality, realistic product representation.
		<ol>
			<li>Use Image &#62; Adjustments, Brightness/Contrast to adjust the contrast of an image. Use Image &#62; Hue/Saturation to modify the saturation level of the image. Use Text Tool (T icon) to add text information to the image . 
			&#8203;NOTE: A generalized list of function buttons include Ellipse Tool (U) - To draw dots/circles. Set the tool to draw a shape, choose the fill color you want, and then click and drag (holding Shift to maintain a perfect circle) to create a dot. Line Tool (U) - To draw straight lines on the image. Select the tool, set the desired line width, and then click and drag to draw a line. Brush Tool (B) - An alternative way to draw dots and lines. Select the brush size and shape, then click (for dots) or click and drag (for lines).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>After completing the three-dimensional model, proceed to produce the cell scraper via the grinding and assembly method20,21,22.</li>
</ol>
3. Production
<ol>
	<li>Employ the color, materials, finish (CMF) theory in the current study to design the prototypes of the cell scraper. CMF theory serves as a design methodology in scientific research. It enhances product usability, shapes user perception, and directs the selection of materials23,24,25.
	<ol>
		<li>Select the colors to be used in the cell scraper from standard color system, including Black 6 C, Cool Gray 6 C, and 11-0601TPG26,27.</li>
		<li>To build the cell scraper that meets lab standards, choose appropriate materials including polypropylene (PP), sponge, and high-carbon steel. For the fabrication of physical prototypes, initiate by employing a 3D printer to produce the structural framework. Subsequently, utilize connectors or adhesive agents to complete assembling the scraper. 
		&#8203;NOTE: To prepare the physical product of the cell scraper, grind and assemble the necessary materials. Safety protocols must be followed throughout the entire process to ensure a safe and effective final product.</li>
		<li>Move on to the finishing process, which may involve cutting, grinding, polishing, stamping, or other techniques. Start by sanding the model using a medium-grit, 120-grit sandpaper to remove any rough edges.</li>
		<li>Follow this with a fine-grit, 220-grit sandpaper to achieve a smooth surface.</li>
		<li>Post-sanding to smooth out the surfaces, ensuring the plug-in or built-in connectors are correctly matched.</li>
		<li>Join slide I and II securely using the connectors (fixed rods) to ensure a robust and durable assembly of the cell scraper. Achieve the final fixed form of the cell scraper through a combination of mechanical fastening and adhesive bonding. Use fasteners, specifically a mortise and tenon structure, to secure parts together through mechanical force. Additionally, to enhance the assembly&#39;s strength, apply adhesive (glue) to bond the components further.</li>
		<li>Autoclave the scraper at 121.3 &#176;C for 30 min to ensure it retains its original shape and physical properties.</li>
	</ol>
	</li>
</ol>
4. Cell culture
<ol>
	<li>Grow HOS cells in minimum essential medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Culture them at 37 &#176;C with 5% CO2.</li>
	<li>Change the culture medium every 2 days.</li>
	<li>When cell growth surpasses 80% confluency, add 1 mL of trypsin with 0.25% EDTA and digest for 60 s. Afterwards, centrifuge the cells at 300 x g for 5 min for subculture. Remove supernatant and add culture medium to get a final concentration of 5 x 104 cells per well. Use an automated cell counter for counting cells.</li>
</ol>
5. Assessing cell wounding potential of the cell scraper and tips-based method
<ol>
	<li>Prepare all materials for wounding and sterilize them with ultraviolet radiation by exposing for 30 min.</li>
	<li>After sterilization, wound 100% confluent HOS cells in 6-well plates at a concentration of 5 x 104 cells per well by using either the cell scraper method or the tips-based method.
	<ol>
		<li>For the tip-based method, use a 100 &#181;L tip and drag it across the cell containing medium with 1 stroke in the horizontal direction and the other in the vertical direction.</li>
		<li>For the cell scraper method, place the scraper prototype in one of the wells and with the help of the tweezer gently press once. The cells are wounded. Carry out each wounding experiment in triplicate.</li>
	</ol>
	</li>
	<li>Analyze all cell wounds using a digital microscope system and imaging software.</li>
</ol>
6. Measuring cell viability and cell proliferation
<ol>
	<li>Perform all cell viability assays following the protocol provided in the CCK-8 Kit.</li>
	<li>Incubate the cells with CCK-8 solution for 2 h at 37 &#176;C, then measure their absorbance at 450 nm using a microplate reader to quantify cell viabilities.</li>
	<li>Design the 5-ethynyl-20-deoxyuridine (EdU) incorporation assay to accurately quantify DNA duplication and directly quantify the cell proliferation ratio. Determine the influence of different methods to generate cell wounds upon cell proliferation using the EdU assay, following the protocol outlined in previous publication28.</li>
	<li>Stain the cells and image them using a digital microscope system. Ensure that each experiment is carried out in triplicate.</li>
</ol>

Innovating an Improved Cell Scraper for Cell Wounding by User Requirements Analysis

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<li>Cottle, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+principal+pivoting+method+revisited%5BTitle%5D)+AND+%22Mathematical+Prog%22%5BJournal%5D)">The principal pivoting method revisited.</a> Mathematical Prog. 48, 369-385 (1990).</li>
<li>Cross, N. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aCross%2C+N+%22Engineering+design+methods%3A+strategies+for+product+design%22">Engineering design methods: strategies for product design</a>. , John Wiley & Sons. (2021).</li>
<li>Otto, K., Wood, K. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aOtto%2C+K+%22Product+design%3A+techniques+in+reverse+engineering+and+new+product+development%22">Product design: techniques in reverse engineering and new product development</a>. , Pearson. (2001).</li>
<li>Tariq, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gefitinib+inhibits+M2-like+polarization+of+tumor-associated+macrophages+in+Lewis+lung+cancer+by+targeting+the+STAT6+signaling+pathway%5BTitle%5D)+AND+%22Acta+Pharmacol+Sin%22%5BJournal%5D)">Gefitinib inhibits M2-like polarization of tumor-associated macrophages in Lewis lung cancer by targeting the STAT6 signaling pathway.</a> Acta Pharmacol Sin. 38 (11), 1501-1511 (2017).</li>
<li>Rahimi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPR%2FCas9-mediated+knockout+of+Lcn2+effectively+enhanced+CDDP-induced+apoptosis+and+reduced+cell+migration+capacity+of+PC3+cells%5BTitle%5D)+AND+%22Life+Sci%22%5BJournal%5D)">CRISPR/Cas9-mediated knockout of Lcn2 effectively enhanced CDDP-induced apoptosis and reduced cell migration capacity of PC3 cells.</a> Life Sci. 231, 116586(2019).</li>
<li>Cormier, N., Yeo, A., Fiorentino, E., Paxson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+the+wound+scratch+assay+to+detect+changes+in+murine+mesenchymal+stromal+cell+migration+after+damage+by+soluble+cigarette+smoke+extract%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Optimization of the wound scratch assay to detect changes in murine mesenchymal stromal cell migration after damage by soluble cigarette smoke extract.</a> J Vis Exp. (106), e53414(2015).</li>
<li>Pinto, B. I., Cruz, N. D., Lujan, O. R., Propper, C. R., Kellar, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+scratch+assay+to+demonstrate+effects+of+arsenic+on+skin+cell+migration%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">In vitro scratch assay to demonstrate effects of arsenic on skin cell migration.</a> J Vis Exp. (144), e58838(2019).</li>
<li>Rodriguez, L. G., Wu, X., Guan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Wound+healing+assay%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Wound healing assay.</a> Methods Mol Biol. 294, 23-29 (2005).</li>
</ol>

The authors declare that they have no conflict of interest.

The present study aimed to develop an automatic mechanized tool for cell wound healing assay. To the best of our knowledge, it represents the first attempt to apply a mechanized driven structure to create cell wounds in a one-click way automatically. Through this, we aim to address the shortcomings of the traditional tips-based method, such as low reproducibility and unstable scratch state. Benefiting from the positive results and the encouraging feedback from the user community, the cell scraper is expected to provide experimenters with an efficient and stable alternative for creating cell wounds, thus significantly improving the confidence level of the experimental analysis results.
For the usage of present cell scraper, the user should 1) prepare cells pending to create cell-free gap similar to traditional methods like tip-based method, i.e., seeding cells onto six-well plate at a given density; 2) wait for cellular convergence rate to reach &gt; 90%; 3) put sterile cell scraper in a well of plate, click the button as illustrated in Figure 5, and the cell-free gap will be automatically created within 1-2 s. 
The cell wound healing assay has been widely employed to measure cell migration capacity. Typically, experimenters use pipette tips to create cell wounds. For example, Tariq et al. used a pipette tip to create cell wounds to assess cell migration capacity45; not coincidentally, Rahimi et al. used a pipette tip in an assay to observe the migratory capacity of PC3 cells46; in the mouse mesenchymal cell migration assay Cormier et al. also used a pipette gun47; in demonstrating the effect of arsenic on skin cell migration, Pinto et al. used a pipette tip for vertical scraping48, among others.
However, as previously mentioned, using a tip to create cellular wounds has several problems, such as variable cell-free area, jagged edges, and dependence on the operator's force and scratch angle, resulting in low reproducibility49. The present results of questionnaires also proved that the tip-based method has more apparent advantages over cell culture inserts, specifically in terms of price, cell survival rate, and tool reusability. Alternatively, cell inserts are too costly and are difficult to reuse, while either tool has defects. Therefore, the user community is generally optimistic and willing to try innovative tools. This study designed and developed a new cell-free area preparation tool to facilitate and stabilize cell wound creation.
The most important contribution of the present study lies in the development and experimental validation of cell scraper. Compared with the previous studies, the improvements of the present cell scraper include the following three main points. 1) stable and convenient fabrication of cell wounds. This study showed that compared with the traditional tips-based method, the cell wounds manufactured by the present cell scraper had straight edges and could create similar wounds in repeated experiments, which greatly improved the reproducibility and reliability of the cell wound healing experiments. 2) The present cell scraper had less effect on the cell status. It did not affect the cell viability and proliferation (DNA amplification), which was significantly better than the tip-based method. Therefore, this cell scraper is expected to further reduce the influence of external factors on the cell wound healing assay in routine experiments. 3) This cell scraper is in good agreement with the demands of the user community. The results of the previous questionnaire survey showed that the users' requirements for the new cell wounds tool mainly include uniform stroke width, high reproducibility, simple operation procedure, and moderate price, which have been achieved in the present cell scraper. In addition, the cell scraper is designed in neutral colors to convey this product's simple and practical design concept. At the same time, the strong black and white contrast effectively highlights its functional areas. The overall style is a cross shape formed by the rounded articulation of the curved surface, referencing the conventional wound shape, and suggesting the product's role. The operation's difficulty is reduced by simplifying the operation and manual reset function. In summary, the results indicate a promising alternative tool for experimenters to prepare cell scratches in a one-click, automated, and robust manner, thus improving the reproducibility of scientific results.
The limitations of this study must be acknowledged. The sample size of the questionnaire interviewees limits the generalizability of this study; therefore, we will further expand the sample size in a follow-up study to improve the cell scraper. Secondly, the cell scratcher needs to be included in a well-planned study with a larger population of users so that the performance of this cell scraper can be evaluated more comprehensively.

Tumors are characterized by distinct hallmarks such as selective growth advantages, metabolic rewiring, and immune modulation, all of which intriguingly contribute to enhanced cell migration, a critical malignant behavior of tumor cells. This feature directly affects the distant metastasis of the primary tumor, compromising the long-term survival of patients1,2,3. Selective growth advantages enable cancer cells to outcompete normal cells, while metabolic rewiring supports this rapid proliferation by altering energy pathways. Concurrently, immune modulation allows tumors to evade the body&#39;s defenses. Studies underscore the severity of this issue, showing that lung metastasis, often a result of enhanced cell migration, is a terminal event leading to the death of patients with various cancers4. For instance, breast cancers5, cervical carcinoma4, and osteosarcoma6 account for 20%, 9%, and 30% of such cases, respectively. Therefore, assessing tumor cell migration has become integral to current oncology research, further highlighting the multifaceted nature of tumor progression.
Cell wound healing assay is an easy-to-use method for measuring in vitro cellular migration, often employed in oncological studies7. Most experimenters use pipette tips to create cell wounds manually8. Although such a method could form cell wounds rapidly and conveniently, it still has many limitations that affect reproducibility and accuracy for evaluating cell migration. Firstly, using pipette tips to create scratches manually is highly influenced by the operator&#39;s operating angle, force, and speed, affecting the method&#39;s repeatability8. Secondly, tip-generated cell defects usually have jagged edges rather than straight edges because pipette tips are plastic products with certain elasticity9. Some studies generate wounds by placing prefabricated culture inserts directly into the cell culture plate to restrict the range of cell proliferation10. This approach circumvents the limitation of the tips-based method, such as jagged edges and reproducibility. However, even with biocompatible materials, the long-term coexistence of the embedding with the cells still impacts cell growth11. Moreover, the embedding may also cause cellular epigenetic changes in the marginal zone due to contact restriction12. Also, contact-insert produced from biocompatible materials are expensive and difficult to reuse, limiting their feasibility13. Therefore, a novel, reproducible, and practical tool is needed to easily quantify in vitro cellular migration.
The primary goal of this method is to introduce an innovative tool for quantifying in vitro cellular migration in oncological studies, addressing the limitations of existing techniques and enhancing reproducibility and accuracy in assessing cellular migration.
The rationale behind this technique&#39;s development lies in the critical importance of evaluating tumor cell migration in oncology. Tumors exhibit distinctive hallmarks, including selective growth advantages, metabolic rewiring, and immune modulation, all contributing to enhanced cell migration, a fundamental aspect of cancer malignancy. This method aims to provide a more dependable means of studying cellular migration, contributing to a deeper understanding of tumor behavior.
This method offers substantial advantages over existing techniques. Manual scratch assays can suffer from operator-dependent interference, while culture inserts may impact cell growth and gene expression. In contrast, this method offers improved repeatability, accuracy, and practicality, presenting a cost-effective solution for measuring in vitro cellular migration in oncology research. It addresses a crucial need for a reliable and accessible tool to study cell migration in various cancer types, making it a valuable addition to the field.

<table><tbody><tr><td>CCK-8 Kit</td><td>Beyotime Company, China</td><td>C0037</td><td></td></tr><tr><td>digital microscope system</td><td>Olympus</td><td>IX81</td><td></td></tr><tr><td>fetal bovine serum</td><td>Gibco, USA</td><td>16000044</td><td></td></tr><tr><td>HOS&amp;nbsp;</td><td>Procell Life Science &amp;amp; Technology Co., Ltd</td><td>CL-0360</td><td></td></tr><tr><td>Image-Pro Plus</td><td>Media Cybernetics</td><td>version 6.0</td><td></td></tr><tr><td>KeyShot</td><td>Luxion</td><td>version 11.0</td><td>3D rendering software</td></tr><tr><td>microplate reader</td><td>BioTek, German</td><td>ELX808</td><td></td></tr><tr><td>Minimum Essential Medium</td><td>Gibco, USA</td><td>11095080</td><td></td></tr><tr><td>Pantone matching system</td><td>Pantone</td><td></td><td>commercial color matching</td></tr><tr><td>penicillin-streptomycin</td><td>Beyotime Company, China</td><td>ST488</td><td></td></tr><tr><td>Photoshop</td><td>Adobe</td><td></td><td>photo and design software</td></tr><tr><td>Rhinoceros 3D</td><td>Robert McNeel &amp;amp; Associates</td><td>version 7.0</td><td>3D design software</td></tr><tr><td>TC20 Automated Cell Counter</td><td>Bio-Rad</td><td>TC20</td><td></td></tr><tr><td>Trypsin</td><td>Cytiva&amp;nbsp;HyClone, United State</td><td>SH30042.01</td><td></td></tr></tbody></table>

a tool to automatically create stable and reproducible cell-free gaps for improving the reliability of cell wound healing assay

The reliability of cellular migration measurement in wound healing assays is frequently undermined by the prevalent methodological instability, i.e., tip-based method. We introduce an innovative instrument designed to address these limitations. Our novel cell scraper surpasses the current approach, generating a more consistent and stable cell-free gap. Repeated biological experiments reveal that the cell-free gap produced by the cell scraper exhibits straighter edges and uniform size and shape compared to the tip-based technique (p &lt; 0.05). In terms of product design, the cell scraper boasts a refined color scheme suited for laboratory environments, enhancing the monitoring of experimental outcomes, and permits sterilization through autoclaving for reuse. Notably, after treatment, the cell scraper demonstrates a negligible effect on cell viability and proliferation (97.31% and 24.41%, respectively). Conversely, the tip-based method yields lower cell viability (91.37%) and proliferation (18.79%). This investigation presents the cell scraper as a novel, reusable device capable of generating reproducible cell-free gaps while preserving cellular viability, thereby augmenting the reliability of wound healing assays in comparison to existing techniques.

A Tool to Automatically Create Stable and Reproducible Cell-free Gaps for Improving the Reliability of Cell Wound Healing Assay

Through the incorporation of interaction experience design and user requirements analysis, we introduce an innovative cell scraper that enhances cellular wound healing assays in terms of reproducibility, dependability, practicality, cellular integrity, and user experience.

The reliability of cellular migration measurement in wound healing assays is frequently undermined by the prevalent methodological instability, i.e., ...

a-tool-to-automatically-create-stable-reproducible-cell-free-gaps-for

Universidad San Sebastian

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Research

JoVE Journal

Biology

440 Views.  Kunming University of Science and Technology. Through the incorporation of interaction experience design and user requirements analysis, we introduce an innovative cell scraper that enhances cellular wound healing assays in terms of reproducibility, dependability, practicality, cellular integrity, and user experience.

Video: A Tool to Automatically Create Stable and Reproducible Cell-free Gaps for Improving the Reliability of Cell Wound Healing Assay

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration and invasion abilities to understand how cancer cells disseminate, with the aim of developing new treatment strategies. Analysis of the cellular and molecular basis of these abilities has led to the characterization of cell mobility and the physicochemical properties of the cytoskeleton and cellular microenvironment. For many years, the Boyden chamber assay and the scratch wound assay have been the standard techniques to study cell invasion and migration. However, these two techniques have limitations. The Boyden chamber assay is difficult and time consuming, and the scratch wound assay has low reproducibility. Development of modern technologies, especially in microscopy, has increased the reproducibility of the scratch wound assay. Using powerful analysis systems, an &#34;in-incubator&#34; video microscope can be used to provide automatic and real-time analysis of cell migration and invasion. The aim of this paper is to report and compare the two assays used to study cell invasion and migration: the Boyden chamber assay and an optimized in vitro video microscope-based scratch wound assay.

This study reports two different methods for the analysis of cell invasion and migration: the Boyden chamber assay and the in vitro video microscope-based wound-healing assay. The protocols for these two experiments are described, and their benefits and disadvantages are compared.

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration ...

Cancer Research

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of cyclic nucleotides. By modulating intracellular cyclic nucleotide concentration, MRP4 can regulate multiple cyclic nucleotide-dependent cellular events including cell migration. Previously, we demonstrated that in the absence of MRP4, fibroblast cells contain higher levels of intracellular cyclic nucleotides and can migrate faster. To understand the underlying mechanisms of this finding, we adopted a direct yet multifaceted approach. First, we isolated potential interacting protein complexes of MRP4 from a MRP4 over-expression cell system using immunoprecipitation followed by mass-spectrometry. After identifying unique proteins in the MRP4 interactome, we utilized Ingenuity Pathway Analysis (IPA) to explore the role of these protein-protein interactions in the context of signal transduction. We elucidated the potential role of the MRP4 protein complex in cell migration and identified F-actin as a major mediator of the effect of MRP4 on cell migration. This study also emphasized the role of cAMP and cGMP as key players in the migratory phenomena. Using high-content microscopy, we performed cell-migration assays and observed that the effect of MRP4 on fibroblast migration is completely abolished by disruption of the actin cytoskeleton or inhibition of cAMP-dependent kinase A (PKA). To visualize signaling modulations in a migrating cell in real time, we utilized a FRET-based sensor for measuring PKA activity and found, the presence of more polarized PKA activity near the leading edge of migrating Mrp4-/- fibroblast, compared to Mrp4+/+fibroblasts. This in turn increased cortical actin formation and augmented the process of migration. Our approach enables identification of the proteins acting downstream to MRP4 and provides us with an overview of the mechanism involved in MRP4-dependent regulation of fibroblast migration.

MRP4 regulates various cyclic nucleotide-dependent signaling events including a recently elucidated role in cell migration. We describe a direct, but multifaceted approach to unravel the downstream molecular targets of MRP4 resulting in identification of a unique MRP4 interactome that plays key roles in the fine-tuned regulation of fibroblast migration.

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of ...

Developmental Biology

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor metastasis. In this assay, cells are grown to form a confluent monolayer and a mechanical wound is created by scratching with a device. Then the migration rate of the cells towards the denuded area can be monitored by imaging. Our 8-channel mechanical wounder is designed to tackle most of the problems associated with the cell migration assay. Firstly, our wounder can be easily sterilized by autoclaving or with common disinfectants. Secondly, the individual adjustable pins allow even contact with the cell culture plate so that sharp and reproducible wounds can be created. Thirdly, the guiding bars on both sides of the wounder ensure consistent wounding position in each well. The use of disposable plastic pipette tips for wounding can further provide better handling of the wounder as well as to minimize cross-contamination. In conclusion, our cell wounder can provide researchers with a user friendly and reproducible device for performing the cell migration assay using the standard 96-well culture plate.

Here, we present a protocol to perform a semi-high throughput cell migration assay on a 96-well cell culture plate. This protocol is a fast, simple and economical method to create consistent scratch wounds on a cell monolayer.

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Bioengineering

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

Immunology and Infection

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

This manuscript describes how incision-like lesions made on cultured epithelial cell monolayers conveniently model wound healing in vitro, allowing for imaging by confocal or laser scanning microscopy, and which can provide high-quality quantitative and qualitative data for studying both cell behavior and the mechanisms involved in migration.

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

Medicine

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...

Wound Assay to Evaluate the Migratory Capacity of Murine Myoblasts in Co-Culture

Source: Toubat, O. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/63247">Modeling Paracrine Noncanonical Wnt Signaling In Vitro.</a> J. Vis. Exp. (2021).
This video describes a non-contact co-culture wound healing assay to evaluate the migratory capacity of murine myoblasts. This helps to understand signal-sending and signal-receiving components of the paracrine non-canonical Wnt signaling interactions in vitro.

Source: Toubat, O. et al., Modeling Paracrine Noncanonical Wnt Signaling In Vitro. J. Vis. Exp. (2021).
This video describes a non-contact co-culture ...

A Tool to Automatically Create Stable and Reproducible Cell-free Gaps for Improving the Reliability of Cell Wound Healing Assay

Summary

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Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

Sandwich-like Microenvironments to Harness Cell/Material Interactions

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

In vitro Cell Migration and Invasion Assays

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Wound Assay to Evaluate the Migratory Capacity of Murine Myoblasts in Co-Culture