Protocol to generate large human intestinal organoids using a rotating bioreactor

Junichi Takahashi; Hady Yuki Sugihara; Shu Kato; Sayaka Nagata; Ryuichi Okamoto; Tomohiro Mizutani

doi:10.1016/j.xpro.2023.102374

Protocol to generate large human intestinal organoids using a rotating bioreactor

STAR Protoc. 2023 Sep 15;4(3):102374. doi: 10.1016/j.xpro.2023.102374. Epub 2023 Jun 22.

Authors

Junichi Takahashi¹, Hady Yuki Sugihara², Shu Kato², Sayaka Nagata², Ryuichi Okamoto², Tomohiro Mizutani³

Affiliations

¹ Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University (TMDU), 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. Electronic address: jtakahashi.gast@tmd.ac.jp.
² Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University (TMDU), 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.
³ Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University (TMDU), 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. Electronic address: tmizutani.gast@tmd.ac.jp.

Abstract

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.

Keywords: Cell Biology; Cell Differentiation; Cell culture; Organoids; Stem Cells; Tissue Engineering.

MeSH terms

Bioreactors
Humans
Induced Pluripotent Stem Cells*
Intestines
Organoids*
Reproducibility of Results