CO2 laser fabrication of PMMA-based multilayer 3D microchannel networks for tubular organ-on-chip models

Organ-on-chip (OoC) technologies enable in vitro modeling of human tissues, but their adoption is limited by the complexity of fabricating 3D vascularized architectures. Here, we present a rapid, scalable method for fabricating PMMA-based 3D tube-like OoC devices using commercial CO2 laser ablation. Our approach combines drilling, cutting, and bonding to create multilayer microfluidic chips with independent, perfusable channel networks embedded in a central reservoir. We optimized laser parameters to achieve high-aspect ratio holes (∼50:1) with smooth surfaces and minimal spacing. The chips were used to fabricate 3D scaffolds in PDMS or collagen hydrogels, with vessel lumens defined by removable microneedles. Collagen scaffolds were covalently grafted to PMMA via EDC/NHS chemistry. Structural tests confirmed distinct channel layers, with no detectable dye mixing in PDMS scaffolds and fluorescent beads confined to their respective lumens in collagen scaffolds. Biological validation was demonstrated by culturing human Caco-2 epithelial cells, which formed polarized monolayers with intact cell–cell junctions. This robust and high-throughput platform supports long-term cell culture and is compatible with standard protocols, offering a versatile tool for modeling tissue barriers in tissue engineering and preclinical research.