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Updated: Jul 6, 2026

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids
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A Practical Guide to Developing and Troubleshooting Patient-Derived "Mini-Gut" Colorectal Organoids for Clinical

Rex Devasahayam Arokia Balaya1,2, Zahra Heydari1, Gobinda Sarkar1

  • 1Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN 55905, USA.

Methods and Protocols
|October 28, 2025
PubMed

Insights

This study details a protocol for generating patient-derived organoids (PDOs) from colorectal tissues. These PDOs are crucial for personalized medicine, drug screening, and understanding cancer mechanisms.

Area of Science:

  • Biotechnology
  • Gastroenterology
  • Oncology

Background:

  • Patient-derived organoids (PDOs) are valuable models for personalized medicine and cancer research.
  • PDOs replicate tumor heterogeneity, aiding drug screening and precision treatment strategies.
  • Understanding disease mechanisms and ex vivo exposures is enhanced by organoid models.

Purpose of the Study:

  • To present a detailed step-by-step protocol for generating organoids from normal, polyp, and tumor colorectal tissues.
  • To provide methods for tissue processing, crypt isolation, culture establishment, and polarity transition.
  • To include immunofluorescence staining and quality control for cellular characterization.

Main Methods:

  • Standardized protocol for colorectal tissue processing and crypt isolation.
  • Organoid culture establishment and basolateral to apical-out polarity transition.
  • Immunofluorescence staining for cellular characterization and quality control.

Main Results:

  • High-efficiency and reproducible generation of organoids from diverse colorectal tissues.
  • Successful implementation of methods for tissue processing, crypt isolation, and culture.
  • Standardized approach addresses technical challenges and improves success rates.

Conclusions:

  • The developed protocol enhances reproducibility in organoid research.
  • Organoid utility is expanded for translational applications in personalized medicine.
  • This resource is particularly valuable for colorectal cancer research and precision treatment.
Keywords:
colorectal cancerpatient-derived organoidsthree-dimensional culture (3D)

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