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INTESTINAL Publication-derived

Single Cell Analysis of Crohn's Disease Patient-Derived Small Intestinal Organoids

Source Suzuki et al., 2018 · Tokyo Medical and Dental University · 10.1007/s00535-018-1437-3

👤 Kohei Suzuki, Tatsuro Murano, Hiromichi Shimizu, Go Ito, Toru Nakata, Satoru Fujii, Fumiaki Ishibashi, Ami Kawamoto, Sho Anzai, Reiko Kuno, Konomi Kuwabara, Junichi Takahashi, Minami Hama, Sayaka Nagata, Yui Hiraguri, Kento Takenaka, Shiro Yui, Kiichiro Tsuchiya, Tetsuya Nakamura, Kazuo Ohtsuka, Mamoru Watanabe, Ryuichi Okamoto ⏱ 23 days 📋 7 phases 🧫 Patient-Derived iPSC (Crohn's Disease)

Abstract

This protocol establishes small intestinal organoids from enteroscopic biopsy specimens of Crohn's disease patients in active and remission states, and analyzes intestinal stem cell properties using single-cell gene expression profiling and organoid reformation assays. The protocol reveals disease activity-dependent modifications in stem cell marker expression and organoid-reforming capacity.

Cell source
Patient-Derived iPSC (Crohn's Disease)
Application
Disease modeling

Protocol overview

30 steps across 7 phases

Tissue Collection and Immunohistochemistry Day 1–5
  1. 1 Obtain small intestinal biopsy specimens
  2. 2 Perform immunohistochemistry on surgical specimens
Crypt Isolation and Organoid Establishment Day 1–13
  1. 1 Isolate crypts from biopsy specimens
  2. 2 Embed crypts in Matrigel
  3. 3 Prepare WENR culture medium
  4. 4 Add culture medium and Y-27632 to crypts
  5. 5 Culture organoids to passage 3
Immunohistochemistry of Organoids Day 13–15
  1. 1 Fix and prepare passage 3 organoids for staining
  2. 2 Perform immunocytochemistry on organoids
  3. 3 Image organoids and compare marker expression
Single-Cell Dissociation and Capture Day 13–14
  1. 1 Dissociate passage 3 organoids into single cells
  2. 2 Prepare single-cell suspension
  3. 3 Load single cells onto microfluidic chip
Single-Cell Gene Expression Analysis Day 14–15
  1. 1 Perform pre-amplification of target genes
  2. 2 Perform quantitative multiplex PCR
  3. 3 Acquire gene expression data
Data Processing and Statistical Analysis Day 15–20
  1. 1 Process single-cell gene expression data
  2. 2 Perform hierarchical cluster analysis
  3. 3 Perform principal component analysis (PCA)
  4. 4 Perform t-distributed stochastic neighbor embedding (tSNE) analysis
  5. 5 Analyze gene expression correlation within clusters
  6. 6 Perform multivariate analysis of variance (MANOVA)
Single-Cell Organoid Reformation Assay Day 13–23
  1. 1 Dissociate passage 3 organoids for reformation assay
  2. 2 Optimize cell seeding density
  3. 3 Seed single cells at optimized density
  4. 4 Add culture medium to seeded cells
  5. 5 Culture reformed organoids for 10 days
  6. 6 Quantify organoid reformation using 3D scanner
  7. 7 Calculate organoid reformation efficiency
  8. 8 Perform statistical comparison of reformation efficiency

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Suzuki et al., 2018. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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