Suspension culture in a rotating bioreactor for efficient generation of human intestinal organoids

Cell Rep Methods. 2022 Nov 15;2(11):100337. doi: 10.1016/j.crmeth.2022.100337. eCollection 2022 Nov 21.


Human intestinal organoids (HIOs) derived from human pluripotent stem cells (hPSCs) hold great promise for translational medical applications. A common method to obtain HIOs has been to harvest floating hindgut spheroids arising from hPSCs. As this technique is elegant but burdensome due to the complex protocol and line-to-line variability, a more feasible method is desired. Here, we establish a robust differentiation method into suspension-cultured HIOs (s-HIOs) by seeding dissociated cells on a spheroid-forming plate. This protocol realizes the reliable generation of size-controllable spheroids. Under optimized conditions in a rotating bioreactor, the generated spheroids quickly grow and mature into large s-HIOs with supporting mesenchyme. Upon mesenteric transplantation, s-HIOs further mature and develop complex tissue architecture in vivo. This method demonstrates that intestinal tissue can be generated from iPSC-derived HIOs via suspension induction and bioreactor maturation, establishing a reliable culture platform with wide applications in regenerative medicine.

Keywords: hindgut spheroids; human intestinal organoids; human pluripotent stem cells; induced pluripotent stem cells; regenerative medicine; rotating bioreactor; suspension culture; transplantation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bioreactors
  • Digestive System
  • Humans
  • Intestines*
  • Organoids
  • Pluripotent Stem Cells*