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GASTRIC Publication-derived

Human Gastric Cancer Organoid Culture and Drug Sensitivity Testing

Source Seidlitz et al., 2019 · University Hospital Carl Gustav Carus, Technische Universität Dresden · 10.1136/gutjnl-2017-314549

👤 Therese Seidlitz, Sebastian R. Merker, Alexander Rothe, Falk Zakrzewski, Clàre 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 ⏱ 35 days 📋 12 phases 🧫 Human gastric cancer tissue (patient-derived)

Abstract

This protocol describes the establishment and culture of human gastric cancer organoids from surgical resection specimens, followed by characterization via immunohistochemistry, whole genome sequencing, and drug sensitivity testing to conventional chemotherapeutics and targeted therapies. The organoids maintain primary tumor characteristics and enable personalized therapy response assessment.

Cell source
Human gastric cancer tissue (patient-derived)
Application
Disease modeling and drug screening

Protocol overview

41 steps across 12 phases

Human Gastric Tissue Acquisition and Processing Day 0
  1. 1 Surgical tissue collection
  2. 2 Tissue optimization for organoid initiation
Human Organoid Culture Initiation and Maintenance Day 1 onwards
  1. 1 Seed organoid culture in standard organoid medium
  2. 2 Passage organoid cultures
  3. 3 Cryopreserve organoid lines
Growth Factor Dependency Analysis Day 1–35
  1. 1 Deplete media components individually
  2. 2 Assess phenotypical response to media modifications
Two-Dimensional Culture Assessment Day 1
  1. 1 Attempt 2D culture in plastic flasks
Xenograft Transplantation Day 0–variable (follow tumor growth curve)
  1. 1 Prepare organoids for subcutaneous injection
  2. 2 Inject organoids subcutaneously into mouse hind legs
  3. 3 Harvest and verify xenograft tumors
Immunohistochemical Characterization Day 1–3 (tissue processing)
  1. 1 Prepare organoid samples for immunohistochemistry
  2. 2 Perform immunohistochemical staining
  3. 3 Evaluate immunohistochemical results
Conventional Chemotherapy Drug Sensitivity Testing Day 1–4
  1. 1 Prepare chemotherapeutic agents
  2. 2 Seed organoids into multi-well assay plates
  3. 3 Treat organoids with chemotherapeutic agents (24 hours post-seeding)
  4. 4 Assess cell viability via viability assays
  5. 5 Perform apoptosis detection (optional confirmation)
  6. 6 Calculate IC50 values and define active drugs
Genomic Characterization - Whole Genome Sequencing Day 1–5
  1. 1 Extract genomic DNA from organoids and normal tissue
  2. 2 Prepare DNA library and perform whole genome sequencing
  3. 3 Sequence primary tumor tissue
  4. 4 Perform bioinformatic processing
Transcriptomic Characterization - RNA Sequencing Day 1–5
  1. 1 Extract RNA from organoid cultures
  2. 2 Prepare stranded RNA-Seq library
  3. 3 Perform RNA-Seq on Illumina NextSeq500
  4. 4 Perform bioinformatic analysis of RNA-Seq data
Pathway Analysis and Molecular Classification Day 1–7
  1. 1 Perform gene set enrichment analysis (GSEA)
  2. 2 Visualize pathway alterations
  3. 3 Classify organoid lines according to TCGA gastric cancer subtypes
Targeted Therapy Testing Day 1–5
  1. 1 Identify druggable mutations and activated pathways
  2. 2 Test Anti-HER2 Therapy (Trastuzumab)
  3. 3 Assess downstream signaling in HER2-targeted organoids
  4. 4 Test combination therapy (Trastuzumab + Chemotherapy)
  5. 5 Test CDK4/6 Inhibition (Palbociclib)
  6. 6 Test Tyrosine Kinase Inhibition (Imatinib)
Statistical Analysis and Data Reporting Day 1–7
  1. 1 Perform statistical testing
  2. 2 Generate graphs and visualizations
  3. 3 Calculate and report IC50 values
  4. 4 Quantify and analyze Western blot results

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Seidlitz et al., 2019. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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