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GASTRIC Publication-derived

Gastric Cancer Tumouroids with Gene Editing

Full SOP with Academic or a plan

Source Nanki et al., 2018 · Department of Gastroenterology, Keio University School of Medicine · 10.1016/j.cell.2018.07.027

Authors: Kosaku Nanki, Kohta Toshimitsu, Ai Takano, Masayuki Fujii, Mariko Shimokawa, Yuki Ohta, Mami Matano, Takashi Seino, Shingo Nishikori, Keiko Ishikawa, Kenta Kawasaki, Kazuhiro Togasaki, Sirirat Takahashi, Yasutaka Sukawa, Hiroki Ishida, Shinya Sugimoto, Hirofumi Kawakubo, Jihoon Kim, Yuko Kitagawa, Shigeki Sekine, Bon-Kyoung Koo, Takanori Kanai, Toshiro Sato Duration: 60 days 7 phases Cell source: Patient-Derived Gastric Cancer Tissue

Abstract

A protocol for establishing and characterizing patient-derived gastric cancer organoids from clinical specimens. The method generates a tumor organoid library (GTOL) comprising 37 gastric cancer organoid lines covering diverse histological and molecular subtypes, enabling genotype-phenotype correlations and functional validation of driver mutations through CRISPR-based engineering and xenotransplantation studies.

Cell source
Patient-Derived Gastric Cancer Tissue
Application
Disease modeling and drug screening

Protocol overview

28 steps across 7 phases

Phase 1 · Tissue preparation and enzymatic digestion Day 0
  1. a Obtain clinical samples
  2. b Wash and mince tissue
  3. c Primary enzymatic digestion with Liberase TH
  4. d Secondary enzymatic digestion with TrypLE Express
  5. e Enzyme inactivation and washing
Phase 2 · Organoid plating and initial culture Day 0-3
  1. a Prepare basal culture medium
  2. b Prepare complete culture medium with niche factors
  3. c Embed epithelial cells in Matrigel
  4. d Overlay with complete culture medium
  5. e Initiate culture incubation
Phase 3 · Media changes and initial organoid assessment Day 3-7
  1. a Perform first medium change
  2. b Assess organoid morphology and growth
Phase 4 · Niche factor-based enrichment for gastric cancer organoids Day 7-14
  1. a Nutlin-3 selection for TP53 mutations
  2. b ROCK inhibitor withdrawal for RHO-dysregulated GC enrichment
  3. c TGF-β selection for TGF-β insensitivity
  4. d EGF and FGF10 withdrawal for RTK-independent selection
Phase 5 · Organoid expansion and niche factor dependency assessment Day 14-30
  1. a Passage organoids for expansion
  2. b Test individual niche factor dependencies
  3. c Document minimal essential niche conditions
Phase 6 · Organoid characterization and validation Day 30-60
  1. a Verify tumor origin by whole-exome sequencing (WES)
  2. b Perform copy number and MSI analysis
  3. c Assess gene expression and methylation profiles
  4. d Confirm genetic stability over passages
Phase 7 · Cryopreservation and long-term storage Day 60+
  1. a Prepare organoids for cryopreservation
  2. b Mechanically divide organoids and Matrigel
  3. c Freeze organoids in a controlled-rate freezer
  4. d Transfer cryovials to liquid nitrogen storage
  5. e Maintain organoid library records

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Attribution

This SOP was written by Organthis based on the published method in Nanki et al., 2018. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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