Scratching in style: 3D printers as plotters for automated and complex wound-healing assays

Scratch wound healing assays remain widely used 2D migration assays to study the behavior of cultured cells. They are easy and cost-effective, yet poorly standardized in geometric dimensions (e.g., scratch width and line straightness), especially when performed manually. Conventional assays typically apply a single scratch, restricting analysis to a subset of the cells in a well rather than the full population. Commercial "scratch-makers" exist but are expensive and lack support for complex patterns. Complex scratch formations would enable assessment across wells and evaluation of migration-associated protein expression. Accordingly, we developed an open-source, cost-effective method that repurposes widely available 3D printers as 2D plotters to generate repeatable scratch dimensions with different pipette tips. The system operates under culture-hood conditions across common plate formats from multiple manufacturers. We validate reproducible and consistent scratch parameters and demonstrate how programmable patterns can broaden <i>in vitro</i> migration analyses while reducing reliance on commercial devices.