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

Summary

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.

Abstract

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 < 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.

Introduction

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 patients^1,2,3. 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'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's operating angle, force, and speed, affecting the method'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'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.

Protocol

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

1. 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%.
2. 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.
3. Investigate the experimentalists' 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 section^14,15.
   ​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 abstract^17,18,19.

2. Design and three-dimensional modeling

1. 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.
   1. Elaborate on the dimensions and layout of each component by applying the Dim, DimRadius, and DimDiameter functions in the modeling software.
   2. 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.
2. Construct a three-dimensional model and render it.
   1. 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.
   2. 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 & 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.
   3. Finally, enhance the image'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.
      1. Use Image > Adjustments, Brightness/Contrast to adjust the contrast of an image. Use Image > Hue/Saturation to modify the saturation level of the image. Use Text Tool (T icon) to add text information to the image .
         ​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).
3. After completing the three-dimensional model, proceed to produce the cell scraper via the grinding and assembly method^20,21,22.

3. Production

1. 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 materials^23,24,25.
   1. Select the colors to be used in the cell scraper from standard color system, including Black 6 C, Cool Gray 6 C, and 11-0601TPG^26,27.
   2. 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.
      ​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.
   3. 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.
   4. Follow this with a fine-grit, 220-grit sandpaper to achieve a smooth surface.
   5. Post-sanding to smooth out the surfaces, ensuring the plug-in or built-in connectors are correctly matched.
   6. 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's strength, apply adhesive (glue) to bond the components further.
   7. Autoclave the scraper at 121.3 °C for 30 min to ensure it retains its original shape and physical properties.

4. Cell culture

1. Grow HOS cells in minimum essential medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Culture them at 37 °C with 5% CO₂.
2. Change the culture medium every 2 days.
3. 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.

5. Assessing cell wounding potential of the cell scraper and tips-based method

1. Prepare all materials for wounding and sterilize them with ultraviolet radiation by exposing for 30 min.
2. 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.
   1. For the tip-based method, use a 100 µL tip and drag it across the cell containing medium with 1 stroke in the horizontal direction and the other in the vertical direction.
   2. 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.
3. Analyze all cell wounds using a digital microscope system and imaging software.

6. Measuring cell viability and cell proliferation

1. Perform all cell viability assays following the protocol provided in the CCK-8 Kit.
2. Incubate the cells with CCK-8 solution for 2 h at 37 °C, then measure their absorbance at 450 nm using a microplate reader to quantify cell viabilities.
3. 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.
4. Stain the cells and image them using a digital microscope system. Ensure that each experiment is carried out in triplicate.

Results

Dissecting user demands for tools to generate cell wounds
The current experimental method to generate cell wounds demands further enhancement to address many issues that compromise biological reproducibility, robustness, economic consumption, and user experience of cell wound healing assay. We utilized the hard laddering method to analyze the requirements of users involved in biological experiments via questionnaires²⁹ (Figure 1A). The information involved in the questionnaire, such as names, was anonymized to protect their privacy. The details of the questionnaire are shown in Table 1. During 6 weeks of survey for 100 individuals, 97 fully completed questionnaires were received, 3 suspicious and invalid questionnaires were excluded; the response rate was 97.00%.

Results demonstrate that the most troubling problems in cell wound healing assay were serrated edge (not straight) and unstable width or areas of wounds, accounting for 55.67% and 29.90%, respectively. These were significantly higher than those of lower cell growth rate, cell contamination and other options with 3.09%, 5.15%, and 6.19%, respectively (p < 0.001, Figure 1B). Regarding the selection of tools to create wounds, the use of pipette tips was much more prevalent than cell culture inserts (Figure 1C). It was mainly ascribed to the fact that cell culture inserts are rather expensive and non-reusable (Figure 1D), while pipette tips are cheap, show experimental reproducibility (6.59%) and cell viability (20.88%, Figure 1E). Importantly, up to 69.23% of users argued that the price of cell culture inserts was over their budget (Figure 1D). In contrast, only 6 subjects chose cell culture inserts, in which only 16.67% thought their price was acceptable (Figure 1F). Finally, as for the innovative tool to generate cell wounds, 86.60% of them were optimistic, while only fewer users were unwilling (4.12%, Figure 1G).

Subsequently, product features that users urgently needed were dissected^30,31,32. These features included straight wounds, stable width and area of wounds, high reproducibility, and acceptable price. Accordingly, subsequent design activities were carried out.

Decision to design elements and materials used in developing cell scraper
The data above shows that laboratory supplies implemented in routine biological research must be strictly sterile to avoid cell contamination³³. Therefore, the proposed cell scraper should be sterile. Although disposable tools could be sterilized by ethylene oxide, the experimental cost will inevitably increase^34,35,36. Therefore, instead of disposable materials, we propose to design the cell scraper with a reusable material with great tolerance to high-pressure steam sterilization (121.3 °C), to reduce the cost and increase the flexibility when applied in different cases³⁷. To meet these requirements, the material used to construct the cell scraper must have certain physical and chemical features, including resistance to heat, high pressure, and water vapor corrosion.

For the intended use of the cell scraper, in addition to meeting the basic functions such as making the edge-straight wounds, it should also have an appealing design and color. In terms of modeling, considering the targeted and application fields, the overall style should be congenial for the laboratories. The appearance should not be too ostentatious to hinder the personnel from observing the experimental progression. Moreover, its size should be suitable for six-well plates, and the surface should have a protrusion for the tweezers to pick it up stably.

CMF in design is a critical aspect of product design influencing aesthetics and user experience. Color choice impacts emotional response, material selection affects functionality and sustainability, and finish determines the tactile feel and visual appeal. CMF designers harmonize these elements, considering market trends and brand identity, to create products that resonate with users.

According to the CMF theory, three critical factors, including color, material, and finishing process, were considered to determine their impacts on function and visual effect²⁴. The color selection was conducted first. In this context, color refers to the intrinsic color, which is the inherent color of an object or substance under standard lighting conditions, devoid of the influence of external light sources or reflections. Given that the six-well plate is made of PP with a transparent surface, dark gray was chosen as the intrinsic color. This choice was made for easy identification and operation, as dark gray provides optimal contrast and visibility when overlaid with other colors. Besides, dark gray is less likely to cause color distortion when combined with other colors, which is also crucial for observation. Essential functional components were further distinguished by black (Figure 2A).

The commercial color matching system was selected as the standard for color selection due to its widespread acceptance and use in various industries. Relying on the company's ability to reproduce consistent colors across different mediums, it was chosen for the study as this is crucial to ensure accurate and consistent color representation.

The material selection was performed based on the aforementioned results of questionnaires. The shell and components of the scraper were made from a heat-resisting plastic, polypropylene (PP), to meet the requirement of sterilization via autoclaving as an example shown in Figure 2B. high-carbon steel is suitable for springs, because it is stable against high- and low-temperature, corrosion-resistant laboratory environments. High-density sponges were used for buffering and placed at the end of the slide.

Finally, the finishing process was developed. As the cell scraper is frequently used and easily contaminated and the surface quality affects sterilization, a smooth and glossy surface is preferable for the cell scraper as it helps the user to quickly identify the source of contamination and deal with it, giving a sense of cleanliness. The tips are partially sanded to avoid damage to the bottom of the culture dish, which can affect the culture environment and result in restriction of cell proliferation³⁸.

Design and prototype of the cell scraper
Three prototypes of cell scraper were designed based on the results mentioned above (Figure 3A). Studies show that the most commonly used cell wounds within cell culture plates were cross-shaped³⁹, which is quite different from the prototype I that employed rectangular wounds; thus prototype I had been waived (Figure 3A). Although prototype II and III could both create cross-shaped cell-free areas, the slide rail of prototype II was difficult to fix in cell culture discs and its infrastructure was rather complicated (Figure 3A). After prudent discussions, prototype III was distinguished as the final model to produce cell scraper.

Notably, given that the shape of tips could directly affect the cell-free area in terms of edges and sizes, three sketches were designed for selection. The sketch I had a sharp top, which could provide very straight edges; however, the culture interface allowing cell proliferation might be impaired if the tips are too sharp (Figure 3B). Sketch II offered a square top, which might avoid impairment of the cell culture plates. However, as the contact surface between the tip and the bottom of the culture plate is square, it is unstable when using it (Figure 3B). Sketch III provided a blunt rounded tip, effectively avoiding the above problems (Figure 3C). Therefore, it was selected as the final design.

The design was optimized in three aspects: the fixer fixing method, the built-in cushion sponge, and the ease of gripping, respectively. First, the expected product fixing methods included buckling⁴⁰, mortise and tenon⁴¹, and pivoting⁴². Considering the actual production cost and operability, a claw was designed under the cross-shaped fixer to facilitate clamping the edge of the well. Additionally, in the cell scratch healing test, we observed that the tips bounced back after touching the wall near the end due to the fixed length and hardness of the slides, which affected the scratch state. Therefore, we added a cushioning sponge to the end of the slide to stabilize the motion of the tips. (Figure 3A,C). Finally, a surface depression was added to the protrusion to facilitate tweezer gripping.

A three-dimensional model was further constructed using the 3D design software. Its rendering is shown in Figure 4A. The explosive plot elaborates the detailed inner structures of the cell scraper, which shows its necessary characteristics discussed above (Figure 4B). Additionally, to achieve the goal of smooth sliding of the tips and straight edges, the internal mechanical power transmission structure is adopted. The whole functional unit of the cell scraper is composed of the control part. In actual work, the linkage lever inside the cell scraper is connected with the top button, and the end moves downward after applying pressure to make the end buckle. The spring inside the slide is released to push the tips to move. Finally, it stops at the sponge with a narrow slit to finish the scraping and then toggles to reset when used again (Figure 5A). Moreover, the ease of learning and convenient operation are the criteria for a biologist to measure product quality. Therefore, for the appearance design of the product, the surface layout followed the principle of symmetry and neatness^43,44, distinguishing and highlighting different components through different shades of black, white, and gray colors (Figure 5A).

Cell scraper is superior to the tips scratching method in practical use
The physical product of the cell scraper was prepared using the grinding and assembly method (Figure 5B). As expected, the cell scraper integrated excellently with the six-well plate, with the upper cross-shaped holder fitting closely to the edge of the plate, providing a strong fixation (Figure 5C). Moreover, the tips fitted precisely to the bottom of the six-well plate, facilitating the smooth generation of cell-free areas. Finally, the cell scraper was autoclaved to test whether it could be reusable. As results shown in Figure 5D, the present cell scraper held the original shape and physical performances even after autoclaving under 121.3 °C for 30 min.

The cell scraper outperformed the existing cell wound creation method in the benchmark test. After repeated tests, the cell scratcher showed high stability and reproducibility in the preparation of the wound, presented a low fluctuation in the area of each wound and nearly straight edges, with a standard deviation of cell-free area of 483.73 and deviation to straight edge of 5041.33 ± 2420.00. In contrast, the area of each wound prepared by the pipette tips showed a higher fluctuation, with a standard deviation of cell-free area of 3905.80 and deviation to straight edge of 19562.33 ± 1068.39 (Figure 6A-C).

Finally, the effect on cell viability after cell wounds was evaluated (Figure 6D). The results demonstrated that 12 h after the cell wound completion, the cell viability was slightly but significantly higher in the cell scraper group than in the tips group. EdU assay was conducted to confirm these results further and clarify the impact of different cell wound preparation methods on cell proliferation, i.e., DNA replication. The results of the EdU assay showed that the percentage of proliferating cells in the cell scraper group (24.41% ± 0.48) was significantly higher than that in the tips method (18.79% ± 1.46) after 12 h (Figure 6E,F).

These data strongly attested that the present cell wound generator outperformed the current method to create cell wounds in terms of stability and biological reproducibility. It had a low impact on cell viability and proliferation. All these remarkable advantages of the cell wound generator confirmed its potential to improve the current cell wound assays by ensuring the reliability and repeatability of the resultant data.

Figure 1: User demands analyses. (A) Sankey plot presents the workflow of questionnaires-based investigation for experimenters. (B) to (G) exhibit the results of questionnaires, including Questions 1 to 6. Please click here to view a larger version of this figure.

Figure 2: Product design with colors and materials used. (A) Colors selection. (B) Autoclave used in biological experiments. Please click here to view a larger version of this figure.

Figure 3: Product design of detailed structures. (A) Three prototype candidates of cell scraper. (B) Three sketches of tip design. (C) Three-view drawing of selected prototype III presenting its overall design and the physical and spatial parameters. Please click here to view a larger version of this figure.

Figure 4: Rendered three-dimensional model and inner structures display. (A) Render of three-dimensional model. (B) Explosive plot demonstrating the inner structures of the proposed cell scraper. Please click here to view a larger version of this figure.

Figure 5: Working mechanisms of cell scraper. (A) The sketches show how cell scraper works. (B) Physical product of cell scraper. (C) The cell scraper fits in a 6-well plate. Please click here to view a larger version of this figure.

Figure 6: Experimental validation of cell scraper. (A) Cell wounds generated by different methods and their grouped comparison results were shown in (B) and (C), the scale bar is 100 µm. (D) Cell viability results. (E) and (F) Edu assay results, the scale bar is 50 µm. For the statistical evaluation, t-test was conducted for grouped comparison, and the original p-value was adjusted by Holm-Bonferroni test to avoid false positive results. Please click here to view a larger version of this figure.

I) Which of the following is the most troubling issue for you in the cell scratching experiment?

A) No straight edges

B) Unstable width

C) Slow proliferation

D) Cell contamination

E) Other

II) Which tools do you use for generating cell wounds

A) Pipette tips (turn to question III)

B) Cell culture inserts (turn to question IV-V)

III) Why don’t you choose cell culture inserts?

A) Cell viability

B) Not reusable

C) Cost

D) Other

IV) Why did you choose cell culture inserts?

A) Cost

B) Repeatability

C) Other

V) What do you think are the most severe problems with cell culture inserts?

A) Cell status

B) Not reusable

C) Expensive

D) Other

E) Satisfied

VI) If there was an inexpensive, reusable (autoclavable), highly reproducible scratching tool, how willing would you be to buy and use it?

A) Very willing

B) Willing

C) Not sure

D) Unwilling

Table 1: The questionnaire used in this study comprises six questions intended to understand situations biologists encounter during cell wound healing assays.

Discussion

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 > 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.

Materials

List of materials used in this article

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