Novel microfluidic protein patterning method for human-organs-on-chip: liver-tumor application

INTRODUCTION: Micropatterned co-cultures (MPCCs) of hepatocytes and 3T3 fibroblasts can maintain higher hepatic functions for several days in vitro, compared to standard culture techniques. Despite the micrometer resolution of protein patterns, so far MPCCs have been used in standard multi-well plates or glass substrates only. Indeed, no methods were described to combine this technology with microfluidic systems. Possible advantages in coupling MPCCs with microfluidic designs include: higher throughput, lower reagent consumption, compatibility with organotypic or multi-organ designs. Here we present a novel technique that allows culture and analysis of MPCCs within a microdevice specifically designed for liver-tumor compartmentalized cultures. The platform is of use in studying drug effects on tumor tissue in the presence of liver metabolism. METHODS: Microfluidic devices were fabricated following standard photo (SU-8) and soft (PDMS) -lithographies. The liver and tumor chambers were separated by an array of microchannels (5µm high, 3µm wide, 500µm long) to impose a diffusion-based exchange between the compartments. Collagen islands (500µm diameter, 1.2mm center-to-center) were obtained inside microfluidic devices by means of consecutive operations: protein coating, stamp positioning, plasma ablation and bonding of the device. HepG2 cells were seeded at 20·106 cells/mL and allowed to adhere for 20 minutes before a washing step. The following day, 3T3 fibroblasts were seeded at 5·106 cells/mL forming MPCCs. HCT-116 colon cancer cells were seeded at 10·106 cells/mL in the tumor compartment and medium was changed every 24h. Medium supplemented with 100µM Tegafur was added in the liver compartment and the viability of the tumor cells was then evaluated. RESULTS: Microfluidic devices were fabricated and evaluated by dye diffusion to assess the integrity of the microchannels. The patterning and bonding technique resulted efficient with collagen islands of defined circularity successfully obtained inside the liver compartment of the microdevice. MPCCs resulted viable and stable in size. MPCCs were assessed with LIVE/DEAD assay and showed high viability (82%) even after 8 days of culture. Morphologically, HepG2 islands were well maintained during the culture period even though a small loss of circularity was observed after the third day of culture. Ongoing experiments are aimed at validating the platform for pharmacokinetic-based drug screenings. Indeed, Tegafur is a known model drug that increases tumor cytotoxicity after being metabolized by the liver. DISCUSSION & CONCLUSIONS: Owing to the fine control of microfluidics, the device described is particularly suitable to study the effect of liver metabolism on anti-tumor drugs. However, our new technology can be applied to potentially all combinations of protein patterns and microfluidic layouts, enabling for new human in vitro studies.