Single Cell-Seeded Human Intestinal Organoids for Organoid Research

Abstract

Recent developments in organoid technology have enabled the creation of patient-derived intestinal organoids (PDIOs) that recapitulate the structural, functional, genetic, and epigenetic features of their original tissues. However, conventional passage-derived organoids inevitably yield heterogeneous populations in size and number, leading to inconsistent results even under identical conditions. To address this, a standardized approach, referred to here as "single cell-seeded PDIOs,"was established. In this method, mature PDIOs were enzymatically dissociated into single cells and seeded at a defined number into individual wells of a 96-well plate. This controlled seeding normalized the size and number of PDIOs. Compared with passage-derived organoids, single cell-seeded PDIOs displayed reduced inter-well variability in organoid numbers and intra-well variability in organoid sizes, which enables the determination of generation efficiency and improves the reproducibility of viability assays. Moreover, this platform is compatible with downstream analysis, including transcriptomic analysis and protein expression profiling. Collectively, this approach may enhance experimental consistency and provide a practical foundation for reproducible PDIO-based studies.