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Protocol to generate large human intestinal organoids using a rotating bioreactor.

Junichi Takahashi1, Hady Yuki Sugihara1, Shu Kato1

  • 1Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University (TMDU), 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.

STAR Protocols
|June 23, 2023
PubMed
Summary

This study introduces a new protocol for generating consistent human intestinal organoids (HIOs) from induced pluripotent stem cells. The method improves spheroid formation and maturation for reproducible organoid development.

Keywords:
Cell BiologyCell DifferentiationCell cultureOrganoidsStem CellsTissue Engineering

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Area of Science:

  • Stem cell biology
  • Gastrointestinal research
  • Tissue engineering

Background:

  • Producing consistent induced pluripotent stem cell-derived mid/hindgut spheroids is challenging.
  • Existing methods lack reproducibility and face scalability issues.

Purpose of the Study:

  • To present a novel protocol for generating homogeneous mid/hindgut spheroids.
  • To mature these spheroids into human intestinal organoids (HIOs) with improved consistency.

Main Methods:

  • Stepwise differentiation and spheroid formation in a 96-well plate.
  • Cell maturation in suspension culture.
  • Utilizing a rotational bioreactor for enhanced culture efficiency.

Main Results:

  • Generation of homogeneous spheroids from mid/hindgut cells.
  • Successful maturation into human intestinal organoids (HIOs).
  • Scalable culture of larger HIOs using a rotational bioreactor.

Conclusions:

  • The developed protocol addresses limitations in current HIO generation techniques.
  • This method offers a reproducible and efficient approach for creating human intestinal organoids.
  • The rotational bioreactor platform enhances the culture efficiency of larger organoids.