The CellRaft AIR® System: A novel system enabling organoid imaging, identification, and isolation

 Three-dimensional (3D) culture systems have been developed that can re-capitulate organ level responses, simulate compound diffusion through complex structures, and assess cellular heterogeneity of tissues, making them attractive models for advanced in vitro research and discovery. Organoids are a unique subtype of 3D cell cul- ture that are grown from stem cells, are self-organizing, and closely replicate in vivo pathophysiology. Organoids have been used to understand tissue development, model diseases, test drug sensitivity and toxicity, and advance regenerative medicine. However, traditional organoid culture methods are inadequate because they are low throughput and ill-suited for single organoid imaging, phenotypic assessment, and isolation from heterogenous organoid populations. To address these bottlenecks, we have adapted our tissue culture consumable and instrumentation to enable automated imaging, identification, and isolation of individual organoids. Organoids grown on the 3D CytoSort® Array can be reliably tracked, imaged, and phenotypically analyzed using brightfield and fluorescent microscopy as they grow over time, then released and transferred fully intact for use in downstream applications. Using mouse hepatic and pancreatic organoids, we have demonstrated the use of this technology for single-organoid imaging, clonal organoid generation, parent organoid subcloning, and single- organoid RNA extraction for downstream gene expression or transcriptomic analysis. The results validate the ability of the CellRaft AIR® System to facilitate efficient, user-friendly, and automated workflows broadly applicable to organoid research by overcoming several pain points: 1) single organoid time-course imaging and phenotypic assessment, 2) establishment of single cell-derived organoids, and 3) isolation and retrieval of single organoids for downstream applications.