Protocol to generate large human intestinal organoids using a rotating bioreactor

Abstract

Summary: Current techniques for producing induced-pluripotent-stem-cell-derived mid/hindgut spheroids have faced major hurdles in consistency and reproducibility. Here, we present a protocol that uses mid/hindgut cells to generate homogeneous spheroids that subsequently mature into human intestinal organoids (HIOs). We describe steps for stepwise differentiation and spheroid formation using a 96-well plate. We then detail cell maturation in a suspended state and the implementation of a rotational bioreactor platform to maximize the culture efficiency of larger HIOs.For complete details on the use and execution of this protocol, please refer to Takahashi et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell Biology
• Cell culture
• Stem Cells
• Cell Differentiation
• Organoids
• Tissue Engineering