Organoid-in-Bead (OrB): vortex-based compartmentalization enables scalable, high-density intestinal organoid culture

Intestinal organoids are powerful in vitro models, but their use in large-scale analyses remains constrained by the low throughput, labor-intensive handling, and high reagent consumption of conventional Matrigel dome culture. Here, we present Organoid-in-Bead (OrB), a vortex-based compartmentalization workflow that partitions organoid fragments into thousands of discrete Matrigel microbeads, enabling scalable, high-density culture from a single batch preparation. OrB maintains dome-comparable organoid growth and epithelial polarity, supports passaging-based culture expansion, yields more than 5,000 organoids in the final 10 cm dish format, and reduces Matrigel and medium consumption by approximately 70% on a per-organoid basis. OrB therefore provides a practical and scalable upstream workflow for generating screening-scale intestinal organoids. HighlightsO_LIOrB generates Matrigel microcompartments by vortexing without microfluidics C_LIO_LIOrB enables scalable, high-density intestinal organoid culture in one batch C_LIO_LIOrB maintains dome-comparable growth and epithelial polarity and supports passaging C_LIO_LIOrB yields >5,000 organoids per batch with [~]70% less Matrigel/medium per organoid C_LI