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INTESTINAL Publication-derived

Inflammatory Bowel Disease Intestinal Organoids for Developmental Studies

Full SOP with Academic or a plan

Source Kraiczy et al., 2017 · 10.1038/mi.2015.88

Authors: Kraiczy J, Nayak K, Ross A, Howell K Duration: 14 days 5 phases Cell source: Patient-Derived iPSC (Pediatric and Foetal samples)

Abstract

This protocol describes the culture and maintenance of human intestinal epithelial organoids (IEOs) derived from pediatric, foetal, and adult patient samples, including media composition for expansion and differentiation, organoid harvesting and nucleic acid extraction, and molecular characterization via DNA methylation, RNA sequencing, immunofluorescence, and genome editing. The organoids are cultured for studies of intestinal development and inflammatory bowel disease modeling.

Cell source
Patient-Derived iPSC (Pediatric and Foetal samples)
Application
Disease modeling and developmental study of intestinal epithelium

Protocol overview

21 steps across 5 phases

Phase 1 · Organoid Culture Setup and Maintenance Days 0-10 (ongoing)
  1. a Prepare Complete Medium for Intestinal Epithelial Organoids
  2. b Seed Newly Isolated Crypts
  3. c Perform Media Changes
  4. d Split Organoids by Mechanical Disruption
Phase 2 · Organoid Harvesting and RNA/DNA Extraction At harvest timepoint
  1. a Dissolve Matrigel
  2. b Wash Samples
  3. c Lyse in RLT Plus Buffer
Phase 3 · Immunofluorescence and Imaging Same day or after fixation storage
  1. a Fix Organoids
  2. b Prepare Blocking Buffer and Block Organoids
  3. c Apply Primary Antibody
  4. d Apply Secondary Antibody and Nuclear Dye
  5. e Final Wash and Imaging
Phase 4 · Vector Construction and Genome Editing Days 1-14 (electroporation to selection)
  1. a Generate Targeting Vector by PCR and Golden Gate Assembly
  2. b Verify Targeting Vector
  3. c Generate sgRNA Vector
  4. d Prepare Organoids in Conditioned-Medium-Free Medium
  5. e Electroporate Organoids
  6. f Selection with Puromycin
  7. g Genotype Individual Organoid Clones
Phase 5 · RNA Sequencing Library Preparation and Sequencing After RNA extraction
  1. a Prepare RNA Sequencing Libraries
  2. b Perform Paired-End RNA Sequencing

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Attribution

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

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