Microfluidic-based gelatin methacrylate microgel as a scaffold to create reverse-polarity HT29 spheroids.

The in vitro model for the human gut is an important tool for predicting drug absorption and toxicity, as well as for understanding infectious diseases due to the interactions between pathogens and hosts. We replicate the tops of intestinal villi (convex surfaces) using gelatin methacrylate (GelMa) microgels to culture HT29 on the surface and investigate the reverse-polarity spheroids. Microgels were utilized at different concentrations (7 and 10 % w/v) and stiffness, with Young's modulus of approximately 0.01 kPa produced via flow-focusing droplet microfluidics. The flow rate ratio was optimized to achieve microgels approximately 80 μm in size, enhancing cell adhesion. The HT29 cell line was cultured on the microgel surface, maintaining 95 % viability. Immunostaining indicated that the cells polarized in response to the curvature of the microgel, with cell nuclei oriented toward the gel and high expression of F-actin on the apical side. Furthermore, the HT29 cells cultured on the GelMa7 microgel exhibited higher expression of tight junctions than those on the GelMa microgels. We refer to this cell-laden microstructure, characterized by reverse-polarity spheroids formed by cell growth around microgels, as the microgut. The potential applications of the microgut method include drug absorption, toxicity assessments, and infectious disease research, underscoring its significance as a promising tool for the scientific community.