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INTESTINAL Publication-derived

Colorectal Cancer Colonic Tumouroids (2020)

Full SOP with Academic or a plan

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

Authors: Rebekah M. Engel, Wing Hei Chan, David Nickless, Sara Hlavca, Elizabeth Richards, Genevieve Kerr, Karen Oliva, Paul J. McMurrick, Thierry Jardé, Helen E. Abud Duration: 15 days 7 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Engel et al., 2020. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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