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GASTRIC Publication-derived

A Comprehensive Human Gastric Cancer Organoid Biobank: Establishment and Characterization

Source Yan et al., 2018 · Department of Pathology, The University of Hong Kong, Queen Mary Hospital · 10.1016/j.stem.2018.09.016

👤 Helen H.N. Yan, Hoi Cheong Siu, Simon Law, Siu Lun Ho, Sarah S.K. Yue, Wai Yin Tsui, Dessy Chan, April S. Chan, Stephanie Ma, Ka On Lam, Sina Bartfeld, Alice H.Y. Man, Bernard C.H. Lee, Annie S.Y. Chan, Jason W.H. Wong, Priscilla S.W. Cheng, Anthony K.W. Chan, Jiangwen Zhang, Jue Shi, Xiaodan Fan, Dora L.W. Kwong, Tak W. Mak, Siu Tsan Yuen, Hans Clevers, Suet Yi Leung ⏱ 90 days 📋 11 phases 🧫 Patient-Derived Primary Gastric Cancer and Normal Gastric Epithelial Tissue

Abstract

This protocol describes the establishment and comprehensive characterization of a biobank of patient-derived gastric cancer organoids (46 cancer lines, 17 normal lines) from 34 patients, capturing diverse molecular subtypes and enabling large-scale drug sensitivity screening for personalized gastric cancer therapy. The organoids maintain genomic and transcriptomic fidelity to original tumors and support identification of therapeutic targets.

Cell source
Patient-Derived Primary Gastric Cancer and Normal Gastric Epithelial Tissue
Application
Disease modeling, Drug screening, Therapeutic development

Protocol overview

33 steps across 11 phases

Tissue Preparation and Sample Processing Day 0–1
  1. 1 Tissue collection and sampling
  2. 2 Normal tissue preprocessing
Normal Gastric Organoid Establishment Day 1–10
  1. 1 Gland isolation via EDTA incubation
  2. 2 Matrigel embedding and plating
  3. 3 Culture medium application and feeding
Tumor Organoid Establishment Day 1–30
  1. 1 Tumor tissue digestion
  2. 2 Cell cluster isolation and straining
  3. 3 Matrigel embedding and initial plating
  4. 4 Manual enrichment for tumor organoids (if normal contamination detected)
  5. 5 TP53 immunostaining and Nutlin-3a enrichment
Organoid Passage and Maintenance Day 14 onwards
  1. 1 Passage of normal organoids
  2. 2 Passage of tumor organoids
  3. 3 Long-term culture and cryopreservation
Preparation of Niche Factors Day 1 (parallel to main culture)
  1. 1 Wnt3a conditioned medium production
  2. 2 R-Spondin-1 conditioned medium production
  3. 3 Noggin conditioned medium production (transient transfection)
DNA and RNA Extraction Day 14–30 (post-culture establishment)
  1. 1 Organoid recovery and cell lysis
  2. 2 Frozen tissue nucleic acid extraction
Whole-Exome and Transcriptome Sequencing Day 30–60 (sample preparation and submission)
  1. 1 Exome library preparation
  2. 2 RNA library preparation
Quality Control and Characterization Day 14–30 (post-establishment)
  1. 1 Proliferation assay
  2. 2 Cryopreservation revival assay
  3. 3 Morphological assessment by H&E staining
  4. 4 Immunohistochemistry for TP53, CAM5.2, HER2, and FGFR2
  5. 5 Dual-ISH for ERBB2 amplification assessment
Molecular Characterization Day 30–90 (parallel to sequencing)
  1. 1 EBV detection by PCR
  2. 2 MLH1 promoter methylation analysis
Drug Sensitivity Screening Day 30–60 (post-establishment; 2-week turnaround per screen)
  1. 1 Organoid preparation and cell counting
  2. 2 Plating for dose-response screening
  3. 3 Cell viability assessment by CellTiter-Glo 2.0
  4. 4 Data analysis and drug sensitivity classification
Specialized Assays (Optional) Day 30–60 (as needed)
  1. 1 WNT withdrawal assay (for APC mutation status assessment)
  2. 2 PARP inhibitor sensitivity assay (for BRCA2 mutation assessment)

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Yan et al., 2018. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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