Abstract 35: Hydrogel-based microwell chip for improved organoid culture and on-chip imaging

Abstract Organoids have become essential tools for modeling complex biological systems, driving significant advancements in drug screening, cancer research, and regenerative medicine. However, traditional dome-shaped organoid culture methods come with notable limitations. These include variability in organoid size, structural heterogeneity, and difficulties in achieving high-resolution imaging due to overlapping positions and artifacts from the surrounding extracellular matrix (ECM). Such issues often hinder the reproducibility and precision of experimental outcomes, especially in long-term culture settings. To overcome these challenges, we developed a hydrogel-based microwell chip designed to standardize organoid culture and enhance imaging quality. The chip contains a grid of 100 microwells, each optimized to support the formation of up to 100 uniform spheroids or organoids. It is designed with a main microwell channel along with two inlet and outlet channels for hydrogel injection, which can be connected to a pump to enable dynamic culture conditions. Using this system, we successfully generated uniform spheroids from various cancer cell lines and consistent mouse intestinal organoids that retained characteristics comparable to those produced with the dome-shaped method. Importantly, the optimized size of the microwells reduces the volume of culture medium and cell suspension required, offering a more cost-effective and resource-efficient solution. This efficiency is particularly advantageous for drug screening, large-scale studies, or research involving precious samples, such as patient-derived organoids. Another notable advantage of our microwell chip is its compatibility with high-quality, live on-chip imaging throughout the culture period. This capability is essential for real-time studies of dynamic processes in organoid models, such as tissue morphogenesis, drug responses, and disease progression. Furthermore, the chip’s glass-bottom design, combined with a thin hydrogel layer, facilitates confocal imaging either with the hydrogel in place or after it has been dissolved. Overall, our hydrogel-based microwell chip represents a robust and versatile platform that not only standardizes organoid culture but also improves the quality of imaging and data collection. By enhancing reproducibility, efficiency, and throughput, this technology holds promise for applications in precision medicine, drug testing, cancer research, and beyond. Citation Format: Thi Kim Ngan Ngo, Ting-Yuan Tu. Hydrogel-based microwell chip for improved organoid culture and on-chip imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 35.