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INTESTINAL Publication-derived

Intestinal Organoids for Infection Studies

Full SOP with Academic or a plan

Source Forbester et al. · Wellcome Sanger Institute and University of Cambridge, UK · 10.1128/iai.00161-15

Authors: Forbester JL, Goulding D, Vallier L, Hanber N, Hale C, Pickard D, Mukhopadhyay S, Dougan G Duration: 9 days 14 phases Cell source: iPSC-Derived (Human iPSC-Derived Intestinal Organoids)

Abstract

Modeled Salmonella Typhimurium invasion using microinjection into iPSC-derived intestinal organoid lumens. Bacteria invaded epithelial cells with intracellular replication and triggered NF-κB-mediated innate immune responses including IL-8 and TNF-α secretion, recapitulating key features of in vivo Salmonella infection.

Cell source
iPSC-Derived (Human iPSC-Derived Intestinal Organoids)
Application
Bacterial Infection Modeling

Protocol overview

78 steps across 14 phases

Phase 1 · hIPSC Culture and Maintenance Ongoing (refresh every 5 days)
  1. a Precoat tissue culture plastic with porcine gelatin
  2. b Prepare MEF medium
  3. c Culture hIPSCs in feeder-free conditions
  4. d Passage hIPSCs every 5 days
Phase 2 · Preparation for Differentiation Days 0-1
  1. a Passage hIPSCs and grow under maintenance conditions
Phase 3 · Endoderm Differentiation Days 2-3
  1. a Initiate endoderm differentiation (Days 2-3)
  2. b Complete endoderm differentiation without CHIR99021
Phase 4 · Hindgut Patterning Days 4-9
  1. a Pattern definitive endoderm to hindgut (Days 4-5)
  2. b Hindgut specification (Days 6-9)
Phase 5 · Three-Dimensional Intestinal Organoid Formation Days 9+
  1. a Harvest hindgut tissue
  2. b Prepare tissue for embedding in Matrigel
  3. c Plate organoids in Matrigel droplets
  4. d Add iHO base growth medium
  5. e Change medium every 2-3 days
  6. f Passage iHOs every 4-6 days
Phase 6 · Bacterial Culture and Preparation Day of experiment
  1. a Grow S. Typhimurium overnight
  2. b Prepare bacterial inoculum for infection
  3. c Prepare bacterial suspension for microinjection
Phase 7 · Stimulation Experiments (Apical or Basal) After 6 weeks in culture
  1. a Culture iHOs for at least 6 weeks
  2. b Basal stimulation: Add bacteria to culture medium
  3. c Incubate stimulated organoids
  4. d Harvest RNA from stimulated organoids
Phase 8 · Microinjection of iHOs Day of experiment (4 days after passaging)
  1. a Prepare iHOs for microinjection
  2. b Plate organoids in glass-bottom microinjection dishes
  3. c Perform microinjection
  4. d Incubate injected organoids
  5. e Harvest phenol red-marked organoids for RNA sequencing
  6. f Fix organoids for microscopy
Phase 9 · RT-qPCR Analysis Post-infection
  1. a Reverse transcription of isolated RNA
  2. b Perform RT-qPCR
  3. c Analyze RT-qPCR data
Phase 10 · RNA Sequencing and Analysis Post-infection
  1. a Prepare RNA for sequencing
  2. b Prepare mRNA libraries
  3. c Sequence libraries
  4. d Assess sequence quality
  5. e Align RNA-Seq reads
  6. f Generate read counts
  7. g Normalize and detect differentially expressed genes
  8. h Construct heat maps and PCA plots
  9. i Perform gene ontology enrichment analysis
Phase 11 · Cytokine and Chemokine Multiplex Assays Post-infection
  1. a Collect culture supernatants
  2. b Prepare samples for analysis
  3. c Perform Luminex multiplex assay
  4. d Capture cytokines overnight
  5. e Wash and add detection reagents
  6. f Final washing and data acquisition
  7. g Analyze and report data
Phase 12 · Invasion Assay (Modified Gentamicin Protection) Day of experiment
  1. a Prepare iHOs for invasion assay
  2. b Incubate injected organoids
  3. c Isolate organoids from Matrigel
  4. d Disaggregate organoid ultrastructure
  5. e Treat with gentamicin
  6. f Wash and lyse cells
  7. g Enumerate bacteria via CFU counting
  8. h Incubate plates and count colonies
  9. i Analyze invasion assay data
Phase 13 · Immunohistochemistry and Immunofluorescence Post-infection (optional for morphological characterization)
  1. a Fix organoids in paraformaldehyde and glutaraldehyde
  2. b Cool and dehydrate samples
  3. c Infiltrate and polymerize samples
  4. d Store embedded samples
  5. e Prepare thin sections
  6. f Block nonspecific binding (immunoperoxidase)
  7. g Wash slides with TBS buffer
  8. h Incubate with primary antibodies
  9. i Detect bound primary antibodies
  10. j Counterstain nuclei
  11. k Mount and set slides
  12. l Immunofluorescence staining for LAMP-1 and CSA-1
  13. m Mount immunofluorescence samples
Phase 14 · Transmission Electron Microscopy (TEM) Post-infection (optional for ultrastructural analysis)
  1. a Fix organoids for electron microscopy
  2. b Post-fix in osmium tetroxide
  3. c Dehydrate with ethanol series
  4. d Polymerize embedded samples
  5. e Cut semithin sections
  6. f Cut and prepare ultrathin sections
  7. g Contrast ultrathin sections
  8. h View via transmission electron microscopy
  9. i Capture and record images

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Attribution

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

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