Moshksayan, K.; Khanna, R.; Camli, B.; Yakay, Z. N.; Harihara, A.; Zdyrski, C.; Mondal, S.; Hegarty, E.; Mochel, J. P.; Allenspach, K.; Ben-Yakar, A.

High-content imaging (HCI) and analysis are the keys for advancing our understanding of the science behind organogenesis. To this end, culturing adult stem cell-derived organoids (ASOs) in a platform that also enables live imaging, staining, immobilization, and fast high-resolution imaging is crucial. However, existing platforms only partially satisfy these requirements. In this study, we present the OrganoidChip+, an all-in-one microfluidic device designed to integrate both culturing and HCI of ASOs all within one platform. We previously developed the OrganoidChip as a robust imaging tool. Now, the OrganoidChip+ incorporates several additional features for culturing organoids in addition to fluorescence staining and imaging without the need for sample transfer. The organoids grown within a culture chamber are stained and then transferred to immobilization chambers for blur-free, high-resolution imaging at predetermined locations. We cultured adult stem cell-derived intestinal organoids in the chip for 7 days and tracked growth rates of each organoid using intermittent brightfield images, followed by multiple image-based assays, including viability assay using widefield fluorescence imaging, a redox ratio assay using label-free, two-color, two-photon microscopy, and immunofluorescence assays using confocal microscopy. These assays serve as proof-of-concept to showcase the chip\'s capabilities in HCI of ASOs. Organoids cultured in the chip exhibited superior average growth rates over those in traditional Matrigel dome cultures, off-chip. Viability and redox ratio measurements of on-chip organoids were comparable or slightly better than their off-chip counterparts. Confocal imaging further confirmed that the OrganoidChip+ supports robust organoid culture while enabling detailed, high-resolution analysis. This all-in-one platform holds great potential for advancing ASO-based research, offering a scalable and cost-effective solution for HCI and analysis in organogenesis, drug screening, and disease modeling.