Abstract

In recent years, 3D organoids have emerged as a new tool for disease modeling and for pre-clinical toxicity screening applications.  Organoids are self-organizing three-dimensional structures derived from either pluripotent stem cells or from primary tissues with the ability to recapitulate some of the spatial architecture and in vivo function of organs.  However, a key hurdle for widespread use of organoids in screening applications is the difficulty in controlling the numbers, sizes and shapes of the organoids.  Here we describe optimization of experimental parameters that allowed us to generate more consistent numbers of similarly sized organoids in a multi-well plate format.  In response to forskolin, hiPSC derived and normal tissue-derived colon organoids swelled in size, indicating that the cystic fibrosis transmembrane conductance regulator (CFTR) was functional. Bulk RNAseq indicates that hiPSC-derived and normal tissue-derived colon organoids express colon-specific gene signatures and share similar transcriptional profiles. The increased reproducibility of organoids between wells should facilitate screening for agents that exert effects on the intestinal epithelium.