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Development. 2017 Mar 15;144(6):1128-1136. doi: 10.1242/dev.142919. Epub 2017 Feb 7.

A process engineering approach to increase organoid yield.

Author information

1
Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139, USA.
2
Department of Chemical and Biological Engineering, Princeton University, Washington Road, Princeton, NJ 08540, USA.
3
Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139, USA.
4
Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229, USA.
5
Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139, USA griff@mit.edu.

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.

KEYWORDS:

Aggregates; Intestinal; Organoids; Process engineering; Sorting; Yield

PMID:
28174251
PMCID:
PMC5358111
DOI:
10.1242/dev.142919
[Indexed for MEDLINE]
Free PMC Article

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