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INTESTINAL Publication-derived

Colorectal Cancer Colonic Tumouroids for Disease Modelling (2016)

Full SOP with Academic or a plan

Source Fessler et al., 2016 · Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine (CEMM), Academic Medical Center (AMC), University of Amsterdam · 10.15252/emmm.201606184

Authors: Evelyn Fessler, Jarno Drost, Sander R van Hooff, Janneke F Linnekamp, Xin Wang, Marnix Jansen, Felipe De Sousa E Melo, Pramudita R Prasetyanti, Joep EG IJspeert, Marek Franitza, Peter Nürnberg, Carel JM van Noesel, Evelien Dekker, Louis Vermeulen, Hans Clevers, Jan Paul Medema Duration: 14 days 10 phases Cell source: Human iPSC (genetically engineered BRAF[V600E]); Patient-derived tubuler adenoma and normal colon organoids

Abstract

This protocol investigates the role of TGFβ signaling in directing colorectal cancer precursor lesions toward distinct molecular subtypes. Human organoid cultures from normal colon, tubular adenomas, and a genetically engineered BRAF[V600E]-mutant model are treated with TGFβ to assess differential responses including apoptosis, epithelial-mesenchymal transition, and subtype-specific gene expression changes relevant to colorectal cancer prognosis.

Cell source
Human iPSC (genetically engineered BRAF[V600E]); Patient-derived tubuler adenoma and normal colon organoids
Application
Disease modeling

Protocol overview

72 steps across 10 phases

Phase 1 · Organoid Isolation and Establishment Days 1-14
  1. a Obtain tissue samples
  2. b Cut tissue into small pieces
  3. c Wash tissue pieces
  4. d Plate in Matrigel
  5. e Add organoid culture medium
  6. f Culture organoids until expansion
Phase 2 · BRAF[V600E] Mutation Introduction (Optional for Serrated Adenoma Modeling) Days 1-14
  1. a Prepare CRISPR-Cas9 transfection complex
  2. b Recover transfected organoids
  3. c Apply puromycin selection
  4. d Pick and expand single clones
  5. e Genotype clones
  6. f Culture BRAF[V600E]-mutant organoids
Phase 3 · TGFβ Treatment Days 1-5
  1. a Prepare organoid cultures for treatment
  2. b Prepare TGFβ treatment medium
  3. c Prepare control medium
  4. d Replace medium with treatment or control
  5. e First medium refresh (Day 3)
  6. f Continue culture until day 5
Phase 4 · Analysis - Sample Harvesting and Preparation Day 5 onward
  1. a Harvest organoids for RNA extraction
  2. b Extract total RNA
  3. c Quantify and assess RNA quality
  4. d Harvest organoids for protein extraction
  5. e Lyse cells for protein extraction
  6. f Quantify protein concentration
  7. g Harvest organoids for immunohistochemistry
Phase 5 · Downstream Analysis - qRT-PCR Day 1-2 (after RNA extraction)
  1. a Synthesize cDNA
  2. b Perform qRT-PCR
  3. c Calculate relative gene expression
Phase 6 · Downstream Analysis - Western Blotting Day 1-2 (after protein extraction)
  1. a Prepare polyacrylamide gel
  2. b Load samples
  3. c Perform electrophoresis
  4. d Transfer proteins to membrane
  5. e Block membrane
  6. f Incubate with primary antibodies
  7. g Wash membrane
  8. h Incubate with secondary antibodies
  9. i Wash membrane again
  10. j Develop blot
  11. k Capture signal
Phase 7 · Downstream Analysis - Immunohistochemistry Day 1-3 (after fixation)
  1. a Dehydrate and embed fixed organoids
  2. b Cut sections
  3. c Rehydrate sections (CDX2, SMAD4, FN1)
  4. d Perform antigen retrieval (CDX2, SMAD4, FN1)
  5. e Block endogenous peroxidase (CDX2, SMAD4, FN1)
  6. f Apply ultraV block
  7. g Incubate with primary antibodies (CDX2, SMAD4, FN1)
  8. h Incubate with post-antibody blocking
  9. i Incubate with secondary antibody
  10. j Develop with DAB
  11. k Counterstain with hematoxylin
  12. l Dehydrate and mount
  13. m Acquire images
  14. n ZEB1 staining - alternative antigen retrieval
  15. o Cleaved Caspase-3 staining - modified protocol
Phase 8 · Downstream Analysis - Flow Cytometry (Nicoletti Assay) Day 1 (after organoid harvest)
  1. a Harvest organoids
  2. b Prepare Nicoletti buffer
  3. c Stain cells
  4. d Perform flow cytometry
  5. e Quantify apoptosis
Phase 9 · Gene Expression Analysis - Microarray Day 1-3 (after RNA extraction and QC)
  1. a Verify RNA integrity and quantity
  2. b Prepare samples for microarray
  3. c Hybridize samples to GeneChip arrays
  4. d Scan arrays
  5. e Normalize and summarize data
  6. f Annotate probe sets
  7. g Identify differentially expressed genes
  8. h Perform gene set enrichment analysis
Phase 10 · Quality Control and Troubleshooting Throughout experiment
  1. a Check for mycoplasma contamination
  2. b Monitor organoid morphology
  3. c Verify TGFβ bioactivity
  4. d Validate key findings with orthogonal methods
  5. e Document all procedures and results

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Attribution

This SOP was written by Organthis based on the published method in Fessler et al., 2016. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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