Product Applications Scientist Cell Microsystems, North Carolina, United States
Two-dimensional (2D) cell-based assays have been crucial tools in research and drug discovery for decades; however, monolayer cell cultures lack complexity and physiological relevance and poorly mimic the in vivo microenvironment. The limitations of 2D cultures have powered the need for three-dimensional (3D) cell culture technologies that mimic the cellular landscape of tissues. Organoids are self-organizing 3D cell culture models that are derived from stem cells isolated from a variety of tissues and species. Their ability to closely replicate the pathophysiology of their original organs, in contrast to traditional 2D monolayer cultures, provides opportunity for use in medical research, pharmaceutical development, and toxicological studies. Organoids are currently being used to examine tissue development, in disease modeling, in testing for drug sensitivity and toxicity, and in regenerative medicine. Despite the utility of organoid models, traditional organoid culture methods are inadequate because they are low-throughput, insufficient for single organoid imaging and phenotypic assessment, and present challenges in evaluating organoid heterogeneity. To overcome the bottlenecks and data gaps in standard organoid culture methods, Cell Microsystems has adapted our CytoSort Array consumable, CellRaft AIR instrument, software, and workflows to provide automated imaging, identification, and isolation of individual organoids. The CellRaft AIR® System relies on the 3D CytoSort® Array, a cell culture consumable made of elastomeric microwells containing releasable 500x500µm polystyrene CellRafts, for establishing compartmentalized organoid culture. Individual organoids on the 3D CytoSort Array can be reliably tracked, imaged, and phenotypically analyzed by the AIR System in brightfield and fluorescence as they grow over time. Single organoids of interest, with sizes ranging from small ( < 250µm) to large (500µm – 1mm) in diameter, can be subsequently released and isolated from the array into standard 96-well tissue culture plates for continued organoid growth and clonal propagation, or into PCR plates or strip tubes for ‘omics or other destructive endpoints. Using mouse hepatic and pancreatic organoids on the CellRaft AIR System, we demonstrated the use of our technology for imaging organoids, establishing clonal organoids, subcloning organoids, and single-organoid RNA extraction for use in downstream gene expression or transcriptomic analysis. The results validate the ability of the CellRaft AIR system to yield more efficient, user-friendly, and automated workflows that are broadly applicable to organoid research by overcoming several common bottlenecks: 1) single organoid time-course imaging and phenotypic assessment, 2) establishment of single cell-derived organoids, and 3) isolation and retrieval of intact single organoids for downstream applications.