Droplet microfluidic fabrication of stiffness-tunable alginate-Matrigel microspheres with innovative external gelation for high-throughput tumor organoid assays

Abstract Matrigel-based hydrogel microspheres fabricated using microfluidic technology have emerged as promising carriers for tumor organoid modeling. However, their intrinsic low stiffness limits their ability to mimic the mechanical properties of the native tumor microenvironment. To overcome this limitation, we developed an interpenetrating polymer network by incorporating alginate into Matrigel, yielding alginate-Matrigel (AM) composite hydrogel microspheres with tunable stiffness via droplet microfluidics technique. Additionally, to eliminate the cytotoxic effects associated with acidic conditions in alginate-based droplet microfluidics process by acid-driven gelation methods, a novel external gelation strategy was designed. A Ca ²⁺ preloaded gelatin substrate (contact angle > 80°) was employed to receive AM droplets, enabling rapid in situ crosslinking and solidification. Notably, cells encapsulated within AM microspheres exhibited high viability throughout the gelation process, and the resulting microspheres displayed excellent sphericity and structural uniformity. The entire workflow—spanning droplet formation, allocation, gelation, culture, and drug testing—was integrated into a streamlined single-step process optimized for high-throughput screening. The stiffness significantly increased over 7-fold, elevating from 0.6 kPa in Matrigel microsphere to 5.0 kPa in AM microsphere. Compared to Matrigel-only microspheres, both patient-derived tumor organoids and cell line spheroids in AM microspheres demonstrate enhanced chemoresistance, as indicated by elevated IC ₅₀ values. Taken together, this simple, biocompatible, and reproducible fabrication strategy offers a powerful platform for organoid modeling, drug screening, and patient-relevant drug testing.