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GASTRIC Publication-derived

Gastric Cancer Tumouroids for Disease Modelling

Full SOP with Academic or a plan

Source Seidlitz et al., 2019 · Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus, Technische Universität Dresden · 10.1136/gutjnl-2017-314549

Authors: Therese Seidlitz, Sebastian R Merker, Alexander Rothe, Falk Zakrzewski, Cläre von Neubeck, Konrad Grützmann, Ulrich Sommer, Christine Schweitzer, Sebastian Schölch, Heike Uhlemann, Anne-Marlene Gaebler, Kristin Werner, Mechthild Krause, Gustavo B Baretton, Thilo Welsch, Bon-Kyoung Koo, Daniela E Aust, Barbara Klink, Jürgen Weitz, Daniel E Stange Duration: 72 days 9 phases Cell source: Human gastric cancer and normal gastric tissue from surgical resection specimens

Abstract

This protocol describes the establishment and culture of human gastric cancer organoids from surgical resection specimens, their molecular characterization via whole genome sequencing, and testing of chemotherapeutic and targeted drug responses. The organoids maintain characteristics of primary tumors and serve as living biomarkers for therapy response prediction.

Cell source
Human gastric cancer and normal gastric tissue from surgical resection specimens
Application
Disease modeling and drug screening for personalized gastric cancer therapy

Protocol overview

57 steps across 9 phases

Phase 1 · Tissue Collection and Organoid Initiation Day 0-7
  1. a Tissue Collection
  2. b Tissue Digestion
  3. c Isolated Tumor Patch Processing
  4. d Culture Initiation
  5. e Initial Culture Monitoring
Phase 2 · Organoid Expansion and Passage Day 7 onwards
  1. a Routine Passaging
  2. b Media Replacement
  3. c Long-term Culture Maintenance
  4. d Freeze-Thaw Cycles
Phase 3 · Organoid Characterization – Morphology and Growth After 2-4 weeks of culture
  1. a Morphological Assessment
  2. b Proliferation Rate Assessment
  3. c Growth Factor Dependency Testing
  4. d 2D Culture Attempt
Phase 4 · Organoid Characterization – Immunohistochemistry After 4-6 weeks of culture or at selection for detailed analysis
  1. a Organoid Preparation for IHC
  2. b Sectioning and Mounting
  3. c Cytokeratin 7 Immunostaining
  4. d Cadherin 17 Immunostaining
  5. e Carcinoembryonal Antigen Staining
  6. f Periodic Acid-Schiff Staining
  7. g ERBB2 Immunostaining (if indicated)
Phase 5 · Chemotherapy Response Testing At 4-6 weeks of culture; 24-72 hours drug exposure
  1. a Organoid Seeding for Drug Testing
  2. b 5-Fluorouracil (5-FU) Treatment
  3. c Oxaliplatin Treatment
  4. d Irinotecan Treatment
  5. e Epirubicin Treatment
  6. f Docetaxel Treatment
  7. g Cell Viability Assay
  8. h Apoptosis Assay (Annexin V/PI Flow Cytometry)
  9. i Morphological Response Documentation
Phase 6 · Targeted Therapy Testing At 4-8 weeks of culture; 24-72 hours drug exposure
  1. a Trastuzumab Treatment – HER2 Targeting
  2. b Trastuzumab Plus 5-FU Combination Treatment
  3. c Palbociclib Treatment – CDK4/6 Inhibition
  4. d EdU Proliferation Assay for CDK4/6 Inhibition
  5. e Imatinib Treatment – c-KIT Inhibition
  6. f Western Blot – ERK1/2 Phosphorylation Analysis
  7. g Western Blot – Phospho-c-KIT Analysis
  8. h Cell Viability Assay for Targeted Drugs
Phase 7 · Genomic and Transcriptomic Characterization At 4-8 weeks of culture
  1. a Genomic DNA Extraction
  2. b Normal Tissue DNA Extraction
  3. c Whole Genome Sequencing Library Preparation
  4. d Whole Genome Sequencing
  5. e RNA Extraction
  6. f RNA Sequencing Library Preparation
  7. g RNA Sequencing
  8. h Bioinformatic Processing
  9. i Mutation Annotation
Phase 8 · Pathway Analysis and Statistical Analysis During and after genomic characterization
  1. a Gene Set Enrichment Analysis (GSEA)
  2. b KEGG Pathway Visualization
  3. c Statistical Analysis – Drug Response
  4. d IC50 Calculation
  5. e Western Blot Quantification
  6. f Graph Generation and Data Visualization
Phase 9 · Xenograft Transplantation and In Vivo Validation At 6-10 weeks of culture
  1. a Organoid Preparation for Transplantation
  2. b Xenograft Injection
  3. c Tumor Growth Monitoring
  4. d Human Origin Verification
  5. e Xenograft Immunohistochemistry

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Attribution

This SOP was written by Organthis based on the published method in Seidlitz et al., 2019. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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