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GASTRIC Publication-derived

Gastric Cancer Organoid Isolation, Culture, and Drug Sensitivity Testing

Source <UNKNOWN> · 10.7150/ijbs.10248

👤 <UNKNOWN> ⏱ 7 days 📋 7 phases 🧫 Patient-Derived Gastric Cancer Tissue

Abstract

This protocol describes the isolation of primary gastric cancer cells from patient tumour tissue via enzymatic digestion and mechanical dissection, followed by organoid culture in a defined medium supplemented with growth factors and ROCK inhibitor. Cultured organoids are exposed to chemotherapeutic agents (5-FU, oxaliplatin, irinotecan, epirubicin, docetaxel) and targeted therapeutics (trastuzumab, palbociclib, imatinib) to assess drug responses and establish patient-specific sensitivity profiles for gastric cancer.

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

Protocol overview

18 steps across 7 phases

Tumour tissue preparation and enzymatic digestion Day 0
  1. 1 Tissue cutting and washing
  2. 2 Enzymatic digestion of tumour tissue
  3. 3 Tumour patch isolation
  4. 4 Centrifugation of tumour patches
Organoid embedding and culture initiation Day 0–1
  1. 5 Resuspension in Matrigel
  2. 6 Overlay with culture medium and ROCK inhibitor
Organoid expansion and maintenance Day 1–7
  1. 7 Culture medium composition
Chemotherapeutic drug treatment Day 2–5
  1. 8 Organoid seeding into multiwell plates
  2. 9 Treatment with chemotherapeutic agents (5-FU, oxaliplatin, irinotecan, epirubicin, docetaxel)
  3. 10 Treatment with targeted therapeutics
Viability and response assessment Day 2–5
  1. 11 Presto Blue cell viability assay
  2. 12 Apoptosis assay by flow cytometry
  3. 13 EdU proliferation assay
Immunohistochemistry and microscopy Day 5–7
  1. 14 Organoid fixation and embedding
  2. 15 Immunohistochemical staining
  3. 16 Imaging
Protein analysis by Western blot Day 5–7
  1. 17 Protein extraction and quantification
  2. 18 Western blot antibody incubation

Full SOP

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Attribution

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

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