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bioRxiv · 10.1101/2025.09.17.676825

In Vitro Wound Simulation: A High-Throughput Device for ScratchAssays

Labovitz, J. E.·Kulaga, P. J.·DuBois, E. M.·Li, K.·O'Shea, T. M.
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Abstract

Traumatic injury to the healthy central nervous system (CNS) causes mechanical tissue damage that results in localized cell death and blood-brain-barrier (BBB) disruption. CNS tissue damage stimulates a multicellular wound response to limit the extent of damage but fails to reestablish the normal function of injured tissue. There is strong interest in developing new strategies to augment regeneration after CNS injury. To enable therapy development, reliable assays to screen and identify molecular approaches to augment glial-based wound responses over fibrotic scarring are needed. Scratch assays, which involve mechanically removing cells from an in vitro culture, allow for the simulation of wounds with high throughput and tight control over applied treatments to mechanistically study cell migration and proliferation functions that are critical to effective repair. Current methods require researchers to individually scratch each well with a pipette tip, resulting in low throughput as well as inconsistent scratch widths, straightness, and efficacy within and between wells. Here, we describe the design of a quickly assembled (<30min), inexpensive (<$110) scratch assay rig that readily creates uniform scratches that are straight (average tortuosity < 1.1), have tunable widths (730-1100{micro}m), and fully remove damaged cells from the simulated wound region. Designed for a 24-well plate, the rig allows for high-throughput screening of varied experimental conditions or for testing many replicates. Application of the scratch assay device on an in vitro culture of neural progenitor cells (NPC) demonstrates the ability to detect differences in wound closure rate for three unique media conditions. These results support the implementation of this high-throughput scratch assay rig as a method to standardize and improve the efficiency of in vitro wound healing studies.

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Labovitz, J. E., Kulaga, P. J., DuBois, E. M., Li, K., O'Shea, T. M.. 2025-09-20. In Vitro Wound Simulation: A High-Throughput Device for ScratchAssays. https://doi.org/10.1101/2025.09.17.676825

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