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INTESTINAL Publication-derived

Salmonella Typhimurium Invasion of Human Intestinal Organoids

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

👤 Forbester JL, Goulding D, Vallier L, Hanber N, Hale C, Pickard D, Mukhopadhyay S, Dougan G ⏱ 9 days 📋 14 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Forbester et al.. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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