High‐Throughput Generation of Tumor Spheroids via Droplet Microfluidics for siRNA‐Loaded Nanomedicine Assessment

Tumor spheroids, the most widely used model of 3D cell culture, have emerged as a viable platform for assessing drug responses. However, high-throughput validation of novel drugs using tumor spheroids remains hindered by the challenges in generating large-scale, homogeneous, and functionally relevant spheroids. Here, a flow-focusing droplet microfluidic platform is developed for high-throughput generation of uniform tumor spheroids, producing over 50 000 droplets within 5 min, with each microdroplet serving as an individual bioreactor for spheroid formation. The initial size of the tumor spheroids is tuned based on cell concentration and water-to-oil flow rate ratio during microdroplet generation. After being released from the microdroplets, the 3D tumor spheroids continue growing, reaching diameters exceeding 300 µm. The growth and functional characteristics of the spheroids are examined both in a liquid environment and in a 3D collagen matrix. Moreover, these tumor spheroids enable assessment of the therapeutic efficacy of siRNA-based nanomedicine that demonstrates enhanced performance compared to free siRNA treatments. This platform offers a robust and scalable approach for evaluating novel nanomedicines, providing valuable insights into their therapeutic potential and underlying mechanisms of action.