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INTESTINAL Publication-derived

Colorectal Cancer Colonic Tumouroids for Regenerative Medicine

Full SOP with Academic or a plan

Source Fumagalli et al., 2017 · Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Center (UMC) Utrecht · 10.1073/pnas.1701219114

Authors: Arianna Fumagalli, Jarno Drost, Saskia J. E. Suijkerbuijk, Ruben van Boxtel, Joep de Ligt, G. Johan Offerhaus, Harry Begthel, Evelyne Beerling, Ee Hong Tan, Owen J. Sansom, Edwin Cuppen, Hans Clevers, Jacco van Rheenen Duration: 112 days 12 phases Cell source: Human intestinal stem cells, normal donor and patient-derived colon tissue

Abstract

This protocol describes the generation of CRISPR/Cas9-engineered human colon organoids harboring combinations of CRC mutations (APC, KRAS, P53, SMAD4) and their orthotopic transplantation into the caecal submucosa of immune-deficient mice to model adenoma-carcinoma sequence progression and metastatic dissemination.

Cell source
Human intestinal stem cells, normal donor and patient-derived colon tissue
Application
Disease modeling - colorectal cancer progression and metastasis

Protocol overview

68 steps across 12 phases

Phase 1 · Human Colon Organoid Derivation and Culture Ongoing (maintenance culture)
  1. a Human colon tissue acquisition
  2. b Prepare complete organoid culture medium
  3. c Establish organoid cultures from tissue
Phase 2 · CRISPR/Cas9 Engineering of Mutant Organoids 2-14 days post-transfection
  1. a Prepare CRISPR/Cas9 constructs
  2. b Dissociate organoids into single cells
  3. c Cotransfect organoid cells with Cas9 and sgRNA plasmids
  4. d Apply functional selection strategy
  5. e Pick and expand single organoid clones
  6. f Confirm mutations by Sanger sequencing and genotyping
Phase 3 · In Vitro Characterization of Engineered Organoids 3-7 days
  1. a Perform morphological assessment
  2. b Perform cell cycle analysis by flow cytometry
  3. c Assess proliferation rates
  4. d Analyze P53 protein expression
Phase 4 · Organoid Preparation for Orthotopic Transplantation Day −1 before surgery
  1. a Harvest and dissociate organoids
  2. b Prepare collagen drops for transplantation
  3. c Allow organoids to recover overnight in collagen
Phase 5 · Orthotopic Transplantation Surgery Day 0
  1. a Prepare recipient mouse
  2. b Perform midline abdominal incision
  3. c Externalize cecum
  4. d Inject collagen drop into caecal submucosa
  5. e Close abdominal incision
  6. f Post-operative recovery
Phase 6 · Post-Transplantation Monitoring and Tumor Assessment Days 1-112 (16 weeks)
  1. a Weekly abdominal palpation for tumor detection
  2. b Measure tumor growth by palpation
  3. c Monitor animal health and welfare
  4. d Euthanize mice and harvest tissues at endpoint
Phase 7 · Immunohistochemistry and Tumor Analysis Upon tissue harvest
  1. a Fix tissue samples
  2. b Dehydrate and embed tissue
  3. c Prepare tissue sections
  4. d Perform hematoxylin and eosin (H&E) staining
  5. e Perform immunohistochemical staining for tumor characterization
  6. f Analyze histopathology and quantify proliferation
Phase 8 · Intravital Imaging for Migration Analysis (Optional) 10-12 weeks post-transplant
  1. a Transduce organoids with Dendra2 fluorescent reporter
  2. b Implant abdominal imaging window on primary tumor
  3. c Photoconvert Dendra2 in tumor region
  4. d Allow recovery between imaging sessions
  5. e Re-image photoconverted region at 24 hours
  6. f Analyze cell displacement
Phase 9 · Mesenteric Vein Injection Experiment (Metastasis Colonization Assay) Variable (same-day procedure)
  1. a Prepare cell suspension for injection
  2. b Prepare recipient mouse and anesthesia
  3. c Expose mesenteric vein
  4. d Inject cells into mesenteric vein
  5. e Close incision and recover mouse
  6. f Monitor for metastasis development
  7. g Immunostain metastatic lesions for Ki-67
Phase 10 · Intrahepatic Injection and Niche Reconstitution Experiment Variable (same-day procedure + 8-14 days monitoring)
  1. a Transduce organoids with luciferase and Noggin
  2. b Prepare cell suspension for intrahepatic injection
  3. c Prepare recipient mouse for intrahepatic injection
  4. d Perform intrahepatic injection
  5. e Close incision and recover mouse
  6. f Administer Noggin for niche reconstitution (if applicable)
  7. g Perform bioluminescence imaging immediately post-injection
  8. h Repeat bioluminescence imaging every 48 hours
Phase 11 · Whole-Genome Sequencing and Mutation Analysis Variable (performed on archived samples)
  1. a Extract genomic DNA from organoid cultures
  2. b Sort Dendra2-positive tumor cells for sequencing
  3. c Expand metastasized tumor cells in vitro
  4. d Prepare DNA libraries for Illumina sequencing
  5. e Sequence samples using Illumina HiSeq X Ten
  6. f Map sequence reads to human reference genome
  7. g Mark duplicates and perform realignment
  8. h Detect copy number variations
  9. i Call and filter point mutations
  10. j Analyze mutation load and functional consequences
Phase 12 · RNA Isolation and Gene Expression Analysis 3-7 days (culture phase)
  1. a Culture organoids in presence or absence of niche factors
  2. b Harvest organoids in lysis buffer
  3. c Isolate total RNA
  4. d Perform reverse transcription
  5. e Perform quantitative RT-PCR

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Attribution

This SOP was written by Organthis based on the published method in Fumagalli et al., 2017. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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