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INTESTINAL Publication-derived

Colorectal Tumor Organoid Establishment and Culture with Optimized Niche Factor Screening

Source Fujii et al., 2016 · Keio University School of Medicine, Department of Gastroenterology · 10.1016/j.stem.2016.04.003

👤 Masayuki Fujii, Mariko Shimokawa, Shoichi Date, Ai Takano, Mami Matano, Kosaku Nanki, Yuki Ohta, Kohta Toshimitsu, Yoshihiro Nakazato, Kenta Kawasaki, Toshio Uraoka, Toshiaki Watanabe, Takanori Kanai, Toshiro Sato ⏱ 120 days 📋 5 phases 🧫 Patient-Derived Tissue (Colorectal Adenoma and Adenocarcinoma)

Abstract

A refined culture protocol for establishing colorectal tumor organoids from patient samples with improved efficiency by screening eight combinatorial niche factor conditions (WENRA, WENRAS, ENA, ENAS) and two oxygen concentrations (normoxia and 1% hypoxia). The protocol enables establishment of organoids from diverse histological subtypes including rare tumors, and characterizes niche factor dependency during adenoma-carcinoma transition.

Cell source
Patient-Derived Tissue (Colorectal Adenoma and Adenocarcinoma)
Application
Disease modeling and tumor characterization

Protocol overview

20 steps across 5 phases

Tissue Preparation and Epithelial Cell Isolation Day 1
  1. 1 Tissue collection and washing
  2. 2 Primary enzymatic digestion with Liberase
  3. 3 Secondary enzymatic digestion with TrypLE Express
  4. 4 Cell collection and centrifugation
Matrigel Embedding and Culture Initiation with Niche Factor Screening Day 1
  1. 5 Matrigel preparation and plating
  2. 6 Basal culture medium preparation
  3. 7 Niche factor addition and media combination screening
  4. 8 Initial organoid growth monitoring
Organoid Expansion and Minimal Niche Factor Determination Days 14–42
  1. 9 First passage and media expansion
  2. 10 Niche factor withdrawal testing
  3. 11 Minimal essential niche factor identification
  4. 12 Sustained culture in minimal medium
Organoid Characterization and Validation Days 42–120
  1. 13 Morphology and histology assessment
  2. 14 Immunofluorescence staining of organoids
  3. 15 Gene expression analysis
  4. 16 Genetic mutation profiling
Xenotransplantation and In Vivo Validation Days 60–120 (after animal procedures)
  1. 17 Organoid preparation for transplantation
  2. 18 Subrenal capsule transplantation
  3. 19 Splenic injection for metastasis assay
  4. 20 Tumor analysis and histology

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Fujii et al., 2016. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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