Fig. 1. Fabrication of high-throughput bladder cancer-on-a-chip (BCOC) model with microfluidic system. (A) Schematic illustration of the BCOC system. Four BCOC-embedded systems were operated by a single syringe pump. (B) Components of the BCOC system. The system consists of a top/bottom/nut case and top/middle/bottom polydimethylsiloxane (PDMS) layer. The top and bottom layers have a microchannel with a cross-sectional geometry of 1 mm (width)×1 mm (height), while the middle layer has a cylindrical chamber with a geometry of 3 mm (radius)×3 mm (height). The layers were fabricated from PDMS with the advantage of deformability, allowing for easy connection and leak-proof fluidic connection. The three layers were separated by a polycarbonate track-etched membrane (GVS Filter Technology, Sanford, ME, USA) for stable cell tissue positioning and diffusion-based drug supplied on the middle layer. The three layers containing four microfluidic channels/chambers were assembled by acryl case and tightened with bolts and nuts. The whole system was operated by a single syringe pump (Fusion 200; Chemyx Inc., Stafford, TX, USA) with four syringes. (C) Acrylic cases and PDMS layer molds. The cases and PDMS layer molds were made of acrylic plates. (D) PDMS layer fabrication processes. First of all, a 10-mm-thick acrylic plate was engraved using end milling and the desired shape was obtained. A commercial acrylic film was attached to the engraved acrylic molds to flatten the open side. Next the film was pierced with a pin to allow the PDMS to flow through. Uncured PDMS solution, which is a mixture of a prepolymer and a curing agent at a 10:1 ratio (Sylgard® 184; Dow Inc., Midland, MI, USA), was poured onto the film-attached acrylic mold. The degassing process under vacuum conditions was applied for 20 minutes and the vacuum was released very slowly to allow the PDMS to flow in. After the PDMS was perfectly filled inside the acrylic plate covered with the acrylic membrane, it was cured for 2 hours at 80℃ on a hot plate (MSH-30D; DAIHAN Scientific, Wonju, Korea) to create the PDMS microchannel/microchamber layers. The acrylic case parts were fabricated using computer numerical control milling.