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 in vitro migration analyses while reducing reliance on commercial devices.