A process engineering approach to increase organoid yield.
Temporal manipulation of thein vitroenvironment 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.
Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139, USA.
Natasha
N
Arora
Department of Chemical and Biological Engineering, Princeton University, Washington Road, Princeton, NJ 08540, USA.
Jasmin
J
Imran Alsous
Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139, USA.
Jacob W
JW
Guggenheim
Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139, USA.
Michael
M
Mak
Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229, USA.
Jorge
J
Munera
Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229, USA.
James M
JM
Wells
Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139, USA.|Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139, USA.
Roger D
RD
Kamm
Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139, USA.
H Harry
HH
Asada
Department of Chemical and Biological Engineering, Princeton University, Washington Road, Princeton, NJ 08540, USA.
Stanislav Y
SY
Shvartsman
Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139, USA griff@mit.edu.|Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139, USA.
Linda G
0000-0002-1801-5548
LG
Griffith
D002452:Cell Count; D048429:Cell Size; D002478:Cells, Cultured; D004064:Digestive System / Q000166:cytology; D005434:Flow Cytometry; D006801:Humans; D009940:Organoids / Q000166:cytology / Q000254:growth & development*; D018874:Spheroids, Cellular / Q000166:cytology; D023822:Tissue Engineering / Q000379:methods*
Aggregates; Intestinal; Organoids; Process engineering; Sorting; Yield