Three-dimensional organoid culture using the CellRaft microwell technology

Project Summary Organoids are mini-­??tissue structures that are revolutionizing in vitro studies due to the fact that they are derived from normal or diseased tissues from human donors, induced pluripotent stem cells (iPSCs), and nearly every model organism. The use of organoids in disease modeling has become a powerful method to replicate pathophysiology using straightforward cell culture conditions. Virtually every tissue type now has an in vitro organoid correlate. As the name implies, organoids are representations of tissue layers (typically epithelium) that have a specific function in an organism. They are typically spherical, and are stem cell driven, given them potential to produce all the differentiated cell types found in any given tissue. This has led to inaccurate results, failure in predicting drug efficacy, and loss of millions of dollars and man hours following lines of research based on artifact. Challenges associated with rapidly establishing organoid cultures are largely associated with initiating a clonal colony from a single cell, tracking colony growth and inducing differentiation processes. In addition, systems supporting organoid culture must allow time-­??course based analysis of physiological phenotypes. Cell Microsystems has developed the CellRaft Technology, a microwell array-­??based platform where single cells can be seeded in small culture chambers, grown into clonal colonies and tracked over time using virtually any imaging modality. These capabilities have shown initial promise in addressing many of the challenges associated with organoid culture. Scott Magness, PhD of the University of North Carolina at Chapel Hill has published methods using CellRaft Arrays for establishing, growing and analyzing organoids derived from various enteric stem cells. The research proposed here builds on both innovative array designs and materials by Cell Microsystems, as well as the workflows established by Dr. Magness. Under this collaborative project, we will develop a novel CellRaft Array, the 3D-­??CytoSort Array, with larger microwells than are currently manufactured (500 microns and 1 millimeter square) with greater depth (300 microns instead of the standard 80 microns) to facilitate the growth, differentiation and analysis of various types of organoid. These arrays will be validated for use with Cell Microsystems AIR? System, an automated platform for the imaging, sorting and isolation of cells or colonies from the CellRaft Array. This instrument will allow not only the imaging of organoids for temporal analysis, but also isolation of organoids for downstream molecular analysis via next generation sequencing or other molecular analysis modality. Dr. Magness? team will translate their current organoid culture methods to the new, larger 3D-­??CellRaft Array. Successful recapitulation of their previously published data using the new array and the AIR? System will serve as an initial validation of the product?s performance as a platform for broad organoid culture methods. Pending completion of the Specific Aims proposed here, a Phase II project will be proposed that will expand the range of organoid types with the goal of expanding the utility of the AIR System software for organoid-­??specific analysis.