A Bioinert Hydrogel Framework for Precision 3D Cell Cultures: Advancing Automated High‐Content and High‐Throughput Drug Screening

Enhanced drug testing efficiency has driven the prominence of high-content and high-throughput screening (HCHTS) in drug discovery and development. However, traditional HCHTS in well-plates often lack complexity of in vivo conditions. 3D cell cultures, like cellular spheroids/organoids, offer a promising alternative by replicating in vivo conditions and improving the reliability of drug responses. Integrating spheroids/organoids into HCHTS requires strategies to ensure uniform formation, systemic function, and compatibility with analysis techniques. This study introduces an easy-to-fabricate, low-cost, safe, and scalable approach to create a bioinert hydrogel-based inverted colloidal crystal (BhiCC) framework for uniform and high-yield spheroid cultivation. Highly uniform alginate microgels are fabricated and assembled into a colloidal crystal template with controllable contact area, creating engineered void spaces and interconnecting channels within agarose-based BhiCC through the template degradation by alginate lyase and buffer. This results in a multi-layered iCC domain, enabling the generation of in-vitro 3D culture models with over 1000 spheroids per well in a 96-well plate. The unique hexagonal-close-packed geometry of iCC structure enables HCHTS through conventional plate reader analysis and fluorescent microscopy assisted by house-developed automated data processing algorithm. This advancement offers promising applications in tissue engineering, disease modeling, and drug development in biomedical research.