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INTESTINAL Publication-derived

Microvillus Inclusion Disease Intestinal Organoids

Full SOP with Academic or a plan

Source Wigerninck et al., 2014 · University Medical Center Utrecht, Department of Pediatric Gastroenterology, Wilhelmina Children's Hospital · 10.1053/j.gastro.2014.04.002

Authors: Caroline L. Wigerninck, Andreas R. Janecke, Kerstin Schneeberger, Georg F. Vogel, Johanna C. Escher, Thomas Müller, Sabine Middendorp Duration: 230 days 9 phases Cell source: Patient-Derived iPSC (Microvillus Inclusion Disease)

Abstract

This protocol describes the establishment and culture of human intestinal organoids derived from patient duodenal biopsy tissue, as well as lentiviral-mediated expression of wild-type and truncated syntaxin 3 (STX3) in Caco-2 intestinal epithelial cells. The methods enable recapitulation of variant microvillus inclusion disease (MVID) phenotypes in vitro to validate loss-of-function mechanisms of STX3 mutations.

Cell source
Patient-Derived iPSC (Microvillus Inclusion Disease)
Application
Disease modeling and cellular mechanism investigation

Protocol overview

48 steps across 9 phases

Phase 1 · Patient Material and Genetic Characterization Day 1-14
  1. a Obtain Duodenal Biopsy Specimens
  2. b Perform Whole-Exome Sequencing on Patient 1
  3. c Perform Whole-Exome Sequencing on Patient 2
  4. d Comparative Variant Analysis
  5. e Confirm STX3 Mutations by Sanger Sequencing
  6. f Screen Additional MYO5B-Negative Samples
Phase 2 · Organoid Culture Establishment Day 15-70
  1. a Isolate Crypts from Duodenal Biopsy Specimens
  2. b Culture Organoids in Expansion Medium (Weeks 1-5 to 10)
  3. c Induce Differentiation
Phase 3 · Plasmid Construction and Lentiviral Preparation Day 71-100
  1. a Amplify STX3 cDNA
  2. b Ligate STX3 into pENTR4-mCitrine Vector
  3. c Clone Fusion Constructs into Lentiviral Expression Vector
  4. d Transfect HEK293LTV Cells for Lentiviral Production
  5. e Harvest Lentiviral Supernatant
Phase 4 · Caco-2 Cell Culture and Lentiviral Transduction Day 101-120
  1. a Culture Caco-2 Cells Under Standard Conditions
  2. b Infect Caco-2 Cells with Lentiviral Particles
  3. c Select Transduced Caco-2 Cells
  4. d Culture Transduced Cells on Transwell Filters
Phase 5 · Histological Analysis and Sample Preparation Day 121-140
  1. a Fix Biopsy Tissue for Histology
  2. b Prepare Paraffin Sections for Light Microscopy
  3. c Prepare Semithin Resin Sections for Toluidine Blue Staining
  4. d Prepare Tissue for Transmission Electron Microscopy
  5. e Prepare Organoids for Histological Analysis
Phase 6 · Immunofluorescence and Immunohistochemistry Day 141-165
  1. a Prepare Duodenal Biopsy Sections for Immunofluorescence
  2. b Block Nonspecific Binding in Biopsy Sections
  3. c Incubate Biopsy Sections with Primary Antibodies
  4. d Apply Secondary Antibodies and Phalloidin in Biopsy Sections
  5. e Prepare Caco-2 Cells on Filters for Immunofluorescence
  6. f Incubate Caco-2 Cells with Primary Antibodies
  7. g Apply Secondary Antibodies and Phalloidin to Caco-2 Cells
  8. h Nuclear Counterstaining and Mounting
  9. i Image Sections using Epifluorescence Microscopy
  10. j Image Organoids using Confocal Microscopy
  11. k Image Caco-2 Cells using Confocal Microscopy
Phase 7 · Electron Microscopy Analysis Day 166-190
  1. a Prepare Biopsy Tissue for Transmission Electron Microscopy
  2. b Prepare Organoids for Transmission Electron Microscopy
  3. c Prepare Caco-2 Cells for Transmission Electron Microscopy
  4. d Perform Scanning Electron Microscopy of Caco-2 Cells
Phase 8 · Protein Analysis and Quantification Day 191-210
  1. a Lyse Caco-2 Cells for Western Blotting
  2. b Perform Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
  3. c Incubate Membranes with Primary Antibodies
  4. d Apply Secondary Antibodies and Develop Blots
  5. e Lyse Organoids for Western Blotting
  6. f Detect Organoid Proteins using Odyssey System
Phase 9 · Quantitative Morphometry and Statistical Analysis Day 211-230
  1. a Quantify Microvillus Inclusions and Basolateral Microvilli
  2. b Count Ring- and Dot-like Actin and CD26/DPPIV Structures
  3. c Use Transmission Electron Microscopy to Distinguish Microvillus Inclusions from Basolateral Microvilli
  4. d Perform Statistical Analysis

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Attribution

This SOP was written by Organthis based on the published method in Wigerninck et al., 2014. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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