Introduction: Cellular wound healing assay is an important experimental technique for detecting cell migration in vitro. Scratching on monolayer cells using a pipette tip is commonly used. However, it is difficult to guarantee the scratch with the same width, and the initial scratch width has a large impact on the experimental results for different treatment factors or different cell types. To optimize this assay for diverse experimental requirements, we developed an experimental device capable of generating scratches with variable widths. Methods: Our device offers the flexibility of selecting among four widths to create cell scratches, enabling the choice of an optimal initial scratch width for specific cell types and experimental conditions. Results: This device produced straight, clean wounds with precise widths. Comparing cell growth in the four width wounds, Hepa1-6 and HUMSCs showed the greatest difference in 0.6 cm wound, 143B at 0.9 cm wound and urine-derived stem cells at 1.2 cm wound were significantly different, which suggests that the width of the wounds has a huge impact on the experimental results. Compared to other wound inserts on the market, our device is more efficient and economical. Conclusion: This versatile and practical device provides a valuable solution for studying cell migration, facilitating a deeper understanding of cellular behaviors and the development of therapeutic strategies.

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