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INTESTINAL Publication-derived

Colorectal Cancer Colonic Organoids (2016)

Full SOP with Academic or a plan

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

Authors: 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 Duration: 120 days 5 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Fujii et al., 2016. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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