Fabrication and installation of a flexible polydimethylsiloxane (PDMS) porous membrane as a substitute for a rigid, conventional, track-etched polyethylene terephthalate (PET) membrane on cell culture inserts for gut barrier co-culture.

We report a simple, clean-room-free strategy for fabricating an ultrathin (≈ 5 µm) and highly flexible polydimethylsiloxane (PDMS) porous membrane that functions as a mechanically dynamic scaffold for an intestine-on-a-chip platform capable of co-culturing human epithelial cells and gut bacteria. The membrane is produced by a two-dimensional phase separation process in which polystyrene (PS) of defined molecular weight (MW = 1300-170,000) acts as the sacrificial layer in a PDMS/toluene matrix. Systematic variation of phase separation time (3-24 h), PS molecular weight, and PS:PDMS:toluene mixing ratio revealed that PS of MW = 5780 at a 1:7.5:7.5 weight ratio generated through pores 10-100 µm in diameter, whereas PS of MW = 1300 yielded sub-2 µm pores but required optimization to secure pore continuity. Scanning electron microscopy images confirmed homogeneous lateral distribution of the pores and negligible surface collapse after sacrificial removal of PS by overnight toluene extraction and subsequent thermal curing. Fluorescein permeability assays demonstrated that membranes fabricated with PS of MW = 5780 or 1300 displayed solute transport rates that showed a similar trend to 1.0 µm track-etched polyethylene terephthalate (PET) inserts at 12 h, underscoring successful formation of through pores despite an order of magnitude reduction in overall thickness. Importantly, Caco-2 colon epithelial cells adhered, proliferated, and formed confluent monolayers within 7 days on collagen-coated PDMS membranes, whereas confluent cultures on PET required 10 days. Long-term culture experiments (up to 18 days in our setting) highlighted contrasting behaviors: cells cultured on the larger-pore (10-100 µm) membranes detached after ≈13 days, likely due to accumulation of autocrine inhibitors on the impermeable basal side; in contrast, cells on the smaller pore (≈2 µm) PDMS membranes remained viable, although some cells migrated through the membrane to the lower chamber, illustrating the need to match pore size distribution to cell diameter. The present methodology eliminates photolithography and plasma bonding steps commonly associated with PDMS microfabrication and can be completed with benchtop spin coating and solvent-casting equipment in <48 h. Because the resulting membrane combines (i) compliance suitable for pneumatically driven peristaltic deformation, (ii) molecular permeability that showed a similar trend to conventional PET inserts, and (iii) optical transparency for real-time microscopy, it provides a versatile foundation for constructing gut-on-a-chip systems that more faithfully recapitulate the biochemical and mechanical microenvironment of the intestinal epithelium. Beyond microbiome-epithelium interaction studies, the platform is readily adaptable to barrier-function assays, drug-transport screening, and host-pathogen investigations involving mechanically active mucosal tissues.