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INTESTINAL Publication-derived

Patient-Derived Colorectal Cancer Organoids: Establishment, Drug Sensitivity Testing, and Stem Cell Marker Analysis

Source Engel et al., 2020 · Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia · 10.3390/jcm9010128

👤 Rebekah M. Engel, Wing Hei Chan, David Nickless, Sara Hlavca, Elizabeth Richards, Genevieve Kerr, Karen Oliva, Paul J. McMurrick, Thierry Jardé, Helen E. Abud ⏱ 15 days 📋 7 phases 🧫 Patient-Derived Tumor Tissue (Colorectal Cancer)

Abstract

This protocol describes the establishment and characterization of patient-derived colorectal cancer organoids (PDCOs) from treatment-naïve patients. PDCOs are generated from primary tumor tissue and cultured in Matrigel to enable in vitro modeling of tumor heterogeneity and evaluation of chemotherapeutic response to 5-fluorouracil (5-FU). The protocol includes histological validation, immunohistochemical analysis, quantitative RT-PCR for stem cell marker expression profiling, and drug sensitivity assays.

Cell source
Patient-Derived Tumor Tissue (Colorectal Cancer)
Application
Disease modeling and drug sensitivity testing for chemotherapy response prediction

Protocol overview

44 steps across 7 phases

Tissue Preparation and Cell Isolation Day 0
  1. 1 Tissue Sectioning and Washing
  2. 2 Enzymatic Digestion
  3. 3 Mechanical Dissociation
  4. 4 Gravity Settling and Supernatant Collection
  5. 5 Centrifugation and Pelleting
  6. 6 Cell Straining
  7. 7 Final Centrifugation and Resuspension
Organoid Establishment and Culture Day 0–7
  1. 1 Matrigel Seeding in Culture Plates
  2. 2 Culture Medium Preparation
  3. 3 Overlay with Culture Medium
  4. 4 Addition of Y-27632 Kinase Inhibitor (Initial Culture Period)
  5. 5 Culture Maintenance
Organoid Dissociation and Re-seeding for Drug Testing Day 7–10
  1. 1 Organoid Dissociation
  2. 2 Re-seeding in 48-Well Plates
  3. 3 Overlay and Initial Culture (Pre-Treatment Baseline)
Organoid Drug Sensitivity Testing Day 10–15
  1. 1 Baseline Viability Measurement (Day 0 of Drug Treatment)
  2. 2 5-Fluorouracil (5-FU) Treatment Application
  3. 3 Drug-Induced Viability Measurement (Day 5 of Drug Treatment)
  4. 4 Data Analysis - IC50 and AUC Determination
Histological Processing and Immunohistochemistry Day 0–3 (parallel to organoid culture)
  1. 1 Primary Tissue and Organoid Fixation
  2. 2 Organoid Extraction from Matrigel and Agarose Embedding
  3. 3 Paraffin Embedding
  4. 4 Tissue Section Preparation
  5. 5 Routine Haematoxylin and Eosin (H&E) Staining
  6. 6 Deparaffinization and Rehydration
  7. 7 Antigen Retrieval
  8. 8 Blocking
  9. 9 Primary Antibody Incubation
  10. 10 Secondary Antibody Detection
  11. 11 Peroxidase Activity Detection
  12. 12 Counterstaining and Mounting
  13. 13 Microscopy and Image Acquisition
Quantitative RT-PCR Analysis for Stem Cell Marker Expression Day 0–1 (tissue processing) and Day 5 (gene expression during drug treatment)
  1. 1 Total RNA Isolation
  2. 2 RNA Quantification and Quality Assessment
  3. 3 Complementary DNA (cDNA) Synthesis
  4. 4 Quantitative PCR Setup and Thermal Cycling
  5. 5 qPCR Instrument Operation and Data Acquisition
  6. 6 Relative Gene Expression Quantification
  7. 7 Analysis of Stem Cell Marker Expression
Statistical Analysis and Patient Outcome Correlation Data analysis phase (concurrent with experimental work or post-hoc)
  1. 1 Correlation Analysis: Stem Cell Markers vs. Drug Sensitivity
  2. 2 Gene Expression Response to 5-FU Treatment
  3. 3 TCGA Survival Analysis (CLU Expression)
  4. 4 Cox Survival Analysis (GSE41258 Database)
  5. 5 Integration of Patient Data with In Vitro Results

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Engel et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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