A cleanroom-free membrane-integrated organ-on-a-chip platform for rapid intestinal tissue formation.

Polydimethylsiloxane (PDMS)-based organ-on-a-chip (OoC) platforms typically rely on cleanroom photolithography, custom masks, and master molds, which limit their accessibility and impede rapid prototyping efforts. Here, we present a simple, mask-free, cleanroom-free method to fabricate membrane-integrated PDMS microfluidic devices using a low-cost digital craft cutter and plasma-assisted adhesive bonding. The process achieves 150µm feature resolution, and forms PDMS-adhesive interfaces with high mechanical robustness (>370 kPa tensile strength) and hydrolytic stability, supporting flow rates up to 20 ml min-1without leakage. Using this method, we fabricated one-lane and two-lane OoC platforms incorporating commercial polycarbonate membranes and lab-made bacterial nanocellulose membranes without requiring membrane modification for bonding. These membrane-integrated devices enabled rapid formation of intestinal tissues within 72 h, significantly faster than conventional PDMS or transwell-based models, which typically require 1-3 weeks to achieve epithelial differentiation. Under physiological shear stress, epithelial tissues exhibited 2-3 fold increase in expression of differentiation markers (Mucin-2, Villin) and substantial reduction in stemness marker expression compared with static culture. In two-lane co-culture systems, epithelial-endothelial interfaces developed functional barrier properties within the same 72 h window and demonstrated membrane-dependent differences in maturation, with nanocellulose membranes promoting enhanced three-dimensional organization. This cleanroom-free platform reduces fabrication barriers for membrane-integrated PDMS OoC devices. Its ability to support rapid, robust intestinal tissue formation makes it a practical platform for accessible organ-mimetic systems in research settings and translational applications.