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INTESTINAL Publication-derived

Clostridium difficile Toxin Effects on Human Intestinal Organoids

Source Leslie et al. · University of Virginia School of Medicine, Charlottesville, USA · 10.1128/iai.02561-14

👤 Leslie JL, Huang S, Opp JS, Nagy MS, Kobayashi M, Young VB, Spence JR ⏱ 90 days 📋 10 phases 🧫 Primary (Human Colonic Biopsy-Derived Organoids)

Abstract

Characterized dose-dependent damage of C. difficile toxins A and B on human colonic organoids including tight junction disruption and cytoskeletal collapse. Toxin B was 10-fold more potent than Toxin A, and the anti-toxin antibody bezlotoxumab rescued organoid damage, validating the clinical mechanism of action.

Cell source
Primary (Human Colonic Biopsy-Derived Organoids)
Application
C. difficile Infection Modeling

Protocol overview

40 steps across 10 phases

HIO Generation and Differentiation 0–90
  1. 1 Endoderm Induction
  2. 2 Intestinal Specification
  3. 3 Spheroid Collection and Matrigel Embedding
  4. 4 HIO Expansion in Intestinal Growth Medium
C. difficile Strain Preparation and Growth As needed before experiments
  1. 1 Anaerobic Culture Setup
  2. 2 Bacterial Growth for Microinjection
  3. 3 Bacterial Supernatant Preparation
Microinjection and HIO Colonization Experimental day
  1. 1 HIO Preparation and Re-embedding
  2. 2 Glass Capillary Preparation
  3. 3 Capillary Loading with Bacteria and FD4
  4. 4 Microinjection of HIOs
  5. 5 Post-injection Culture and Imaging
Quantification of Viable C. difficile in HIOs 0–12 h postinjection
  1. 1 HIO Harvesting
  2. 2 HIO Disruption and Bacterial Recovery
  3. 3 Serial Dilution and Plating
  4. 4 Colony Enumeration
Vero Cell Cytotoxicity Assay for Toxin Activity As needed for toxin validation
  1. 1 Vero Cell Culture
  2. 2 Vero Cell Harvesting and Seeding
  3. 3 Toxin Preparation and Serial Dilution
  4. 4 Toxin-Serum Control Incubation
  5. 5 Toxin Exposure and Cell Rounding Assessment
Purified Toxin Injection into HIOs Experimental day
  1. 1 Toxin Procurement and Preparation
  2. 2 HIO Preparation
  3. 3 Toxin Microinjection
  4. 4 Post-injection Culture and Monitoring
FD4 Pixel Intensity Quantification During and after barrier function assays
  1. 1 Image Acquisition
  2. 2 ImageJ Analysis Setup
  3. 3 HIO Perimeter Delineation and Measurement
  4. 4 Pixel Intensity Calculation
Basolateral Barrier Function Assay Experimental day
  1. 1 HIO Placement and Toxin Preparation
  2. 2 Toxin Exposure (First 2 Hours)
  3. 3 FD4 Exposure (Final 1 Hour)
  4. 4 PBS Wash and Imaging
Immunofluorescence and Confocal Microscopy Following barrier function assays
  1. 1 HIO Fixation
  2. 2 Cryoprotection and Freezing
  3. 3 Sectioning
  4. 4 Immunofluorescence Staining
  5. 5 Confocal Microscopy Imaging
Optional: Apoptosis Induction and Detection As needed
  1. 1 TNF-α and IFN-γ Treatment
  2. 2 Sample Fixation and Preparation

Full SOP

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Attribution

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

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