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INTESTINAL Publication-derived

Intestinal Organoids for Disease Modelling (No. 2)

Full SOP with Academic or a plan
Duration: 14 days 5 phases Cell source: Human intestinal crypts / Mouse intestinal crypts

Abstract

This protocol describes the establishment, maintenance, and characterization of human and mouse intestinal organoids from isolated crypts. Organoids are cultured in defined media containing growth factors and small molecule modulators, passaged weekly, and analyzed for gene expression, protein markers, and histological features to model normal and diseased intestinal epithelium.

Cell source
Human intestinal crypts / Mouse intestinal crypts
Application
Organoid culture, disease modeling, and developmental study

Protocol overview

34 steps across 5 phases

Phase 1 · Organoid Isolation and Initial Culture Preparation Day 0
  1. a Tissue dissociation with enzymatic digestion
  2. b Crypt collection and washing
  3. c Matrigel embedding and plating
  4. d Initial culture medium application
Phase 2 · Maintenance of Established Organoids Day 1 to Day 7+
  1. a Medium change schedule
  2. b Weekly mechanical dissociation and passaging (1:5 ratio)
  3. c Further mechanical dissociation with fire-polished pasteur pipette
  4. d Washing and centrifugation
  5. e Resuspension in fresh Matrigel and medium
Phase 3 · Long-Term Storage and Recovery Day 7+ (storage phase)
  1. a Preparation for cryopreservation
  2. b Mechanical dissociation in freezing medium
  3. c Transfer to cryotubes
  4. d Slow freezing at -80°C
  5. e Transfer to liquid nitrogen storage
  6. f Thawing frozen organoids
  7. g Recovery culture
Phase 4 · Immunohistochemistry and Imaging Variable (post-culture)
  1. a Organoid fixation
  2. b Paraffin section processing
  3. c Whole-mount immunostaining preparation
  4. d Permeabilization
  5. e Primary antibody staining
  6. f Secondary antibody staining
  7. g EdU proliferation staining
  8. h DNA counterstaining with DAPI
  9. i Confocal microscopy and image acquisition
  10. j 3D image reconstruction
  11. k Karyotyping (optional)
Phase 5 · Gene Expression Analysis Post-culture (Day 3-14)
  1. a Sample preparation for RNA isolation
  2. b RNA isolation
  3. c Microarray analysis (optional)
  4. d Heat map generation
  5. e cDNA synthesis for real-time PCR
  6. f Quantitative real-time PCR
  7. g Data analysis and relative quantification

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Attribution

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

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