A process engineering approach to increase organoid yield

TLDR

Temporal manipulation of the in vitro environment and growth factors directs human pluripotent stem cells into organoids, and understanding early organoid formation is essential for improving robustness for drug development and regenerative medicine. The study tests whether spheroid diameter and inner‑mass morphology predict maturation into intestinal organoids by sorting and monitoring them with an automated micropipette aspiration system. We investigated intestinal organoid emergence by measuring hindgut spheroid parameters and sorted spheroids by diameter and inner‑mass morphology using an automated micropipette aspiration and release system. Thirteen percent of spheroids were pre‑organoids, and spheroids larger than 75 µm with an inner mass were 1.5‑ and 3.8‑fold enriched for pre‑organoids, offering rational guidelines for a robust intestinal organoid protocol.

Abstract

Temporal manipulation of the in vitro 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.