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INTESTINAL Publication-derived

TTC7A Mutations and Intestinal Epithelial Polarity: Organoid Culture and Analysis Protocol

Source Bigorgne et al., 2014 · INSERM U768, Hôpital Necker Enfants-Malades, Paris, France · 10.1172/jci71471

👤 Amélie E. Bigorgne, Henner F. Farin, Roxane Lemoine, Nizar Mahlaoui, Nathalie Lambert, Marine Gil, Ansgar Schulz, Pierre Philippet, Patrick Schlesser, Tore G. Abrahamsen, Knut Oymar, E. Graham Davies, Christian Lycke Ellingsen, Emmanuelle Leteurtre, Brigitte Moreau-Massart, Dominique Berrebi, Christine Bole-Feysot, Patrick Nischke, Nicole Brousse, Alain Fischer, Hans Clevers, Geneviève de Saint Basile ⏱ 20 days 📋 8 phases 🧫 Human Patient-Derived iPSC (Multiple Intestinal Atresia with Combined Immunodeficiency)

Abstract

This protocol establishes 3-dimensional intestinal organoid cultures from patient biopsies to study the effects of TTC7A mutations on epithelial polarity, proliferation, and differentiation. The protocol demonstrates that TTC7A deficiency results in increased Rho kinase activity and disrupted apicobasal polarity, which is reversible by pharmacological ROCK inhibition with Y-27632.

Cell source
Human Patient-Derived iPSC (Multiple Intestinal Atresia with Combined Immunodeficiency)
Application
Disease modeling

Protocol overview

38 steps across 8 phases

Intestinal Biopsy Collection and Preparation Day 0
  1. 1 Collect and dissect small intestine biopsy
  2. 2 Cut tissue into small pieces
  3. 3 Wash tissue pieces
Organoid Embedding and Establishment Day 0–1
  1. 4 Embed tissue in Matrigel
  2. 5 Add culture medium to embedded tissue
  3. 6 Culture for organoid outgrowth
Organoid Passaging and Expansion Day 5–7 onwards
  1. 7 Prepare organoids for passaging
  2. 8 Reseed organoid fragments in fresh Matrigel
Immunofluorescence Analysis of Polarity and Proliferation Day 12–20 (following organoid establishment)
  1. 9 Prepare organoids for immunostaining
  2. 10 Fix organoids for whole-mount immunostaining
  3. 11 Perform F-actin staining for apical brush border visualization
  4. 12 Perform immunostaining for α6 integrin (basolateral marker)
  5. 13 Perform immunostaining for tight junction marker ZO-1
  6. 14 Counterstain nuclei with DAPI
  7. 15 Analyze organoids by confocal microscopy
Proliferation and Differentiation Analysis by Paraffin Section Immunostaining Day 12–20 (concurrent with phase 4)
  1. 16 Harvest organoids and prepare paraffin sections
  2. 17 Cut organoid sections on microtome
  3. 18 Perform Ki67 immunostaining for proliferation assessment
  4. 19 Perform CK20 immunostaining for differentiation assessment
  5. 20 Develop immunostaining with HRP detection system
  6. 21 Mount slides and analyze by light microscopy
Western Blot Analysis of ROCK Pathway Activation Day 12–20 (concurrent with phases 4–5)
  1. 22 Harvest organoids for protein extraction
  2. 23 Lyse organoids in radioimmunoprecipitation (RIPA) buffer
  3. 24 Quantify protein concentration
  4. 25 Perform SDS-PAGE separation
  5. 26 Transfer proteins to PVDF or nitrocellulose membrane
  6. 27 Block membrane and incubate with primary antibodies
  7. 28 Incubate with secondary antibody and detect signal
  8. 29 Quantify band intensities and compare
ROCK Inhibitor Y-27632 Treatment and Assessment Day 5 onwards (parallel to organoid expansion)
  1. 30 Prepare Y-27632 stock solution
  2. 31 Add Y-27632 to patient-derived organoid cultures
  3. 32 Establish matched control cultures (with and without Y-27632)
  4. 33 Quantify organoid morphology and growth
Optional: Complementation Studies with Wild-Type TTC7A in Fibroblasts Day 1–3 post-transfection
  1. 34 Culture patient-derived fibroblasts from skin biopsy
  2. 35 Prepare wild-type TTC7A expression construct
  3. 36 Transfect patient fibroblasts with WT-TTC7A or control plasmid
  4. 37 Analyze ROCK effector phosphorylation in transfected fibroblasts
  5. 38 Assess cell adhesion and proliferation in transfected fibroblasts

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Bigorgne et al., 2014. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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