Abstract

We have developed hetero-cellular 3D liver, islet, and tumor spheroid models, and an accompanying 384 microplate technology that supports microtissue production, cultivation, and high-resolution 3D imaging. The combined platform enables the cell-level analysis of spheroids with added spatial/architectural context. The platform is scalable and automation-compatible making it ideal for studying tissue-level responses at early stages in the drug discovery pipeline. Using pancreatic islet microtissues we examined the compatibility of the Akura™ 384 spheroid plate with high-resolution confocal imaging. Human islet microtissues, reconstituted from dissociated native islets, were labeled with three nuclear markers, DAPI, anti-NKX6.1 (beta cell-specific marker) and EdU (cell proliferation marker). Using a 3D nuclear colocalization assay requiring single cell resolution, we compared the images acquired from 3D microtissues in Akura™ 384 plates to those acquired in u-bottom spheroid plates. Our results demonstrate that a continuous, flat, ultra-thin, transparent bottom significantly minimizes optical aberrations observed with u-bottom plates, dramatically improving the image quality, and enabling single cell resolution throughout the entire spheroid volume. By combining islet microtissues with the Akura™ 384 plate technology and high-resolution imaging capabilities of the Yokogawa CQ1 we were able to develop an exquisitely sensitive assay to quantify beta cell proliferation in hetero-cellular islets.As the dependence on 3D models and high content imaging continues to expand, maximizing 3D image quality is imperative to the development of accurate and comprehensive spheroid measurements. Innovations, such as the development of standardized 3D models and 3D-optimized microplates, may soon overcome the remaining obstacles limiting the broader utilization 3D cell models in early drug discovery.