A Protocol Guide to Micro Milling for Bio-Microfluidics.

Micro milling is a subtractive manufacturing method for fabricating micro-scale three-dimensional features from hard substrates like acrylic, wood, or metal. It enables rapid prototyping of biomicrofluidic devices and master molds, offering advantages over traditional fabrication methods like photolithography. Micro milling is seldom applied in the fabrication of organs-on-a-chip, in part due to its requirement for knowledge of computer numerical machining techniques that are required to program and operate micro mills. This protocol provides practical guidelines for micro milling-based fabrication of organs-on-a-chip, including toolpath optimization, SolidWorks and Fusion workflows, and troubleshooting tips. A case study demonstrates the design and fabrication of master molds for a human airway-on-a-chip, validated in a recent publication. This resource supports the expansion of micro milling techniques into organs-on-a-chip, which will enhance capacity for rapid device prototyping and design of more complex 3D features that better recapitulate human physiology. Key features • Stepwise guide to lung-on-a-chip design and fabrication via micro milling, a specialized type of computer numerical control (CNC) machining. • Demonstration of model design, tool path optimization, micro milling, device assembly, and cell culture. • Example SolidWorks and Fusion documents illustrating best practices for model design and toolpath generation.