Standard Operating Procedure (SOP): Wound Healing Assay

Disclaimer

This SOP is based on personal experience and is intended to serve as a guideline. It is essential for researchers to adapt this protocol to their specific experimental conditions and laboratory settings. Variations might be necessary based on cell type, culture conditions, and specific experimental requirements.

Introduction to the Wound Healing Assay

The wound healing assay, also known as the scratch assay, is a popular method for studying cell migration in vitro. This assay simulates a wound or scratch to create a gap in a confluent cell monolayer and measures the ability of cells to move into this open area. Cell migration is a key process in the healing of wounds in multicellular organisms and is also a fundamental aspect of processes such as cancer metastasis, embryonic development, and tissue regeneration. The purpose of this assay is to assess the rate and regulation of cellular migration under various treatment conditions, such as the application of inducers or inhibitors to modulate the wound healing process.

Materials Required

6-well plates.
1 mL or 200 µL pipette tips.
Phosphate Buffered Saline (PBS), sterile.
Complete culture medium.
Treatments (e.g., inducers or inhibitors) as required by the experimental design.
EVOS Cell Imaging System (Thermo Fisher Scientific) or equivalent.
ImageJ software (NIH) or similar for image analysis.

SOP Steps

Cell Culture Preparation:

Before seeding the cells, use a marker to draw three straight lines on the underside of each well in the 6-well plate. These lines will serve as reference points to ensure that images are consistently taken from the same location throughout the experiment.
Seed cells in a 6-well plate at a density sufficient to reach full confluence. It is crucial that the cell monolayer is completely confluent before the assay starts to avoid gaps that can affect the migration and healing process.

Wound Creation:

When confluence is achieved, use a sterile 1 mL pipette tip to create a straight scratch across the center of the well. For smaller wounds, a 200 µL tip can be utilized. To create the scratch, two pens with tape and placed a plastic piece between them to keep them separated.
Gently wash the cells twice with PBS to remove detached cells and debris that may affect the healing process.

Treatment Application:

Apply the experimental treatments, such as cell migration inducers or inhibitors, to the cells in complete medium to modulate the healing process.

Monitoring and Analysis:

Immediately after creating the scratch and applying treatments (0 hours), take initial photographs of the wound with the EVOS Cell Imaging System.
Take additional images, for example N = 3, of the wound at the specific time points of interest, such as 6, 18, 24, 48, and 96 h, or continue until the wound has completely closed. It is crucial to maintain consistency in the imaging process to enable accurate comparisons.
Use ImageJ software to analyze the images and calculate the wound area. The wound healing rate can be quantified by comparing the wound closure at multiple time points, such as 0, 6, 18, 24, 48, and 96 h, or continue monitoring until the wound has completely closed. This approach allows for a comprehensive assessment of the healing process over time.

In this experiment, fibroblasts were stimulated with TGF-β, which is well established as an inducer of cell migration. The wound was monitored for 24 and 48 h after the scratch was made. The top shadows, as previously explained, ensure that the same area can be compared.

Data Analysis:

Analyze the data by calculating the percentage of wound closure using the initial and final wound areas. Using the formula:

$\text{Migration Rate} (\%) = 100 \times \left(1 - \frac{\text{Area}_{i+1}}{\text{Area}_i}\right)$

This formula calculates the percentage of the wound area that has been closed between two time points, 𝑖 (initial) and 𝑖+1(subsequent).

Statistical analysis can be performed to evaluate the significance of the effects observed between different treatment groups.

3D Printed Wound Maker

I have developed a 3D printed wound maker designed using SolidWorks and fabricated with a Formlabs Form 3 printer. This innovative device is tailored for use with the Greiner 6 Well Multiwell Plate, though its design can be adapted for other plates by modifying their geometry in SolidWorks. The primary advantage of this wound maker is its capacity to produce scratches of consistent size and at the same position across experiments, irrespective of the operator. This repeatability significantly enhances experimental reliability compared to traditional methods. The following figure represents the 3D design from SolidWorks.

This figure shows the physical printed version along with its dimensions.

The wound maker was rigorously tested by three different operators on fibroblast cultures and benchmarked against scratches produced by the CytoSelect™ 24-Well Wound Healing Assay and manual scratching techniques. The results demonstrated that the 3D printed wound maker not only guarantees a straighter and more uniform scratch, but also offers a higher degree of reproducibility and reliability. Additionally, its design ensures that it does not contact cells directly, allowing for repeated use without risk of contamination.

As this is a prototype, I am seeking additional funding to further develop and refine this tool, aiming to enhance its applicability and validate its effectiveness across a broader range of cell culture assays.

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Standard Operating Procedure (SOP): Wound Healing Assay