InVitroGap: an open-source tool for automated quantification of wound closure in the in vitro scratch assay

Abstract Background and Objective: Scratch assays are widely used to study wound closure in vitro, but quantitative image analysis remains constrained by manual variability, proprietary workflows, and tools requiring programming expertise. We developed InVitroGap, a Python-based application with a browser-accessible interface for automated quantification of scratch assay closure from sequential microscopy images. Methods: RCC-ER and Renca cells were seeded in 96-well ImageLock plates and scratched using a WoundMaker device for uniform linear wounds or a 200 uL pipette tip for crisscross wounds. Phase-contrast time-lapse images acquired at 0, 24, and 48 h with an IncuCyte SX5 system were independently analyzed using IncuCyte 2023A Rev2 and InVitroGap. The InVitroGap pipeline combines Gaussian smoothing, gradient-based texture mapping, adaptive percentile thresholding, and morphological post-processing to quantify wound confluence and relative wound density (RWD). Agreement was evaluated using paired comparisons, Pearson and Spearman correlations, Bland-Altman analysis, and mean absolute error (MAE). Results: InVitroGap measurements closely tracked IncuCyte outputs across both cell lines, with no significant between-method differences (p > 0.05), strong pooled correlations (R square = 0.964 for RWD; R square = 0.983 for wound confluence), and small mean biases (absolute bias [≤] 1.64%). The tool successfully processed crisscross wounds from brightfield image series, and a complete four-timepoint series was analyzed in approximately 10 seconds, with robust performance across distinct cell morphologies and wound geometries. Conclusions: InVitroGap provides a transparent, computationally efficient, and platform-independent alternative for scratch assay analysis, delivering performance comparable to commercial systems while remaining freely accessible at https://invitrogap.vercel.app/.