A process engineering approach to increase organoid yield. Author Natasha Arora, Jasmin Alsous, Jacob Guggenheim, Michael Mak, Jorge Munera, James Wells, Roger Kamm, H Harry Asada, Stanislav Shvartsman, Linda Griffith Publication Year 2017 Type Journal Article Abstract Temporal manipulation of the environment and growth factors can direct differentiation of human pluripotent stem cells into organoids - aggregates with multiple tissue-specific cell types and three-dimensional structure mimicking native organs. A mechanistic understanding of early organoid formation is essential for improving the robustness of these methods, which is necessary prior to use in drug development and regenerative medicine. We investigated intestinal organoid emergence, focusing on measurable parameters of hindgut spheroids, the intermediate step between definitive endoderm and mature organoids. We found that 13% of spheroids were pre-organoids that matured into intestinal organoids. Spheroids varied by several structural parameters: cell number, diameter and morphology. Hypothesizing that diameter and the morphological feature of an inner mass were key parameters for spheroid maturation, we sorted spheroids using an automated micropipette aspiration and release system and monitored the cultures for organoid formation. We discovered that populations of spheroids with a diameter greater than 75 μm and an inner mass are enriched 1.5- and 3.8-fold for pre-organoids, respectively, thus providing rational guidelines towards establishing a robust protocol for high quality intestinal organoids. Keywords Humans, Flow Cytometry, Cell Count, Cells, Cultured, Cell Size, Spheroids, Cellular, Tissue Engineering, Digestive System, Organoids Journal Development Volume 144 Issue 6 Pages 1128-1136 Date Published 2017 Mar 15 ISSN Number 1477-9129 DOI 10.1242/dev.142919 Alternate Journal Development PMCID PMC5358111 PMID 28174251 PubMedPubMed CentralGoogle ScholarBibTeXEndNote X3 XML