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Method Article

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

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DOI:

10.3791/65794

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October 4th, 2024

 ,  ,  , 
1Faculty of Art and Communication, Kunming University of Science and Technology, 2The School of Integrated Chinese and Western Clinical Medicine, Anhui University of Chinese Medicine, 3School of Industrial Design, Hubei University of Technology

* These authors contributed equally

In This Article

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 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'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.

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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 section14,15.
    ​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 abstract17,18,19.

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 method20,21,22.

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 materials23,24,25.
    1. Select the colors to be used in the cell scraper from standard color system, including Black 6 C, Cool Gray 6 C, and 11-0601TPG26,27.
    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.

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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...

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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 e...

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Disclosures

The authors declare that they have no conflict of interest.

Acknowledgements

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).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
CCK-8 KitBeyotime Company, ChinaC0037
digital microscope systemOlympusIX81
fetal bovine serumGibco, USA16000044
HOS Procell Life Science & Technology Co., LtdCL-0360
Image-Pro PlusMedia Cyberneticsversion 6.0
KeyShotLuxionversion 11.03D rendering software
microplate readerBioTek, GermanELX808
Minimum Essential MediumGibco, USA11095080
Pantone matching systemPantonecommercial color matching
penicillin-streptomycinBeyotime Company, ChinaST488
PhotoshopAdobephoto and design software
Rhinoceros 3DRobert McNeel & Associatesversion 7.03D design software
TC20 Automated Cell CounterBio-RadTC20
TrypsinCytiva HyClone, United StateSH30042.01

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