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INTESTINAL Publication-derived

Colonic Organoids for Developmental Studies (2013)

Full SOP with Academic or a plan

Source Wang et al., 2013 · Stowers Institute for Medical Research · 10.1053/j.gastro.2013.04.050

Authors: Fengchao Wang, David Scoville, Xi C. He, Maxime M. Mahé, Andrew Box, John M. Perry, Nicholas R. Smith, Nan Ye Lei, Paige S. Davies, Megan K. Fuller, Jeffrey S. Haug, Melainia McClain, Adam D. Gracz, Sheng Ding, Matthias Stelzner, James C. Y. Dunn, Scott T. Magness, Melissa H. Wong, Martin G. Martin, Michael Helmrath, Linheng Li Duration: 100 days 8 phases Cell source: Mouse Lgr5-GFP ISCs; Human small intestinal crypts and isolated IECs

Abstract

This protocol establishes a surface marker-based approach using CD44, CD24, CD166, GRP78, and c-Kit combinations to isolate and characterize functional intestinal stem cells (ISCs) from mouse small intestine and colon, and human intestinal tissue. The method incorporates a robust single-cell culture assay using GSK3β inhibitor (CHIR99021) and E-cadherin stabilizer (Thiazovivin) to achieve 25-30% colony-forming efficiency (CFE) in isolated ISCs, enabling functional validation and applicability to human tissue.

Cell source
Mouse Lgr5-GFP ISCs; Human small intestinal crypts and isolated IECs
Application
Intestinal stem cell (ISC) identification, characterization, and functional assay development

Protocol overview

44 steps across 8 phases

Phase 1 · Tissue Preparation and Crypt Isolation Day 0
  1. a Intestinal tissue washing and preparation
  2. b EDTA incubation for crypt isolation (small intestine)
  3. c Mechanical shaking to release crypts
  4. d Filter crypts and remove debris
  5. e Crypt collection by centrifugation
Phase 2 · Cell Dissociation and Preparation for Flow Cytometry Day 0
  1. a Enzymatic dissociation of crypt cells
  2. b Remove cell clumps and mucus
  3. c Wash dissociated cells
  4. d Prepare cell suspension for flow cytometry
Phase 3 · Antibody Staining and Flow Cytometry Day 0
  1. a Primary antibody staining
  2. b Wash cells after primary staining
  3. c Secondary antibody staining
  4. d Final wash and viability staining
  5. e Flow cytometry and cell sorting
Phase 4 · Matrigel Preparation and Cell Seeding Day 0
  1. a Dissolve and prepare Matrigel with growth factors
  2. b Prepare pre-warmed 96-well plate
  3. c Spin down sorted cells and remove excess medium
  4. d Mix cells with Matrigel
  5. e Seed Matrigel with cells into pre-warmed wells
  6. f Polymerization of Matrigel
  7. g Prepare and add seeding medium
Phase 5 · Growth Factor Addition and Medium Management (Days 2-14) Days 2-14
  1. a Add growth factors on day 2
  2. b First medium change on day 4
  3. c Maintenance medium changes every 4 days (days 8, 12, etc.)
  4. d Monitor colony formation and growth
Phase 6 · Colony-Forming Efficiency (CFE) Measurement Day 0 (parallel to culture setup)
  1. a Prepare cell-counting wells in parallel
  2. b Fix and stain counting wells
  3. c DAPI staining of seeded cells
  4. d Image and count seeded cells
  5. e Count colonies in culture wells
  6. f Calculate colony-forming efficiency
Phase 7 · RNA Extraction and Quantitative Reverse-Transcription PCR (qRT-PCR) Day 0 (for sorted cells) or Day 10+ (for colonoids)
  1. a Collect sorted cells or colonoid material for RNA extraction
  2. b Extract total RNA
  3. c Assess RNA quality
  4. d Reverse transcription to synthesize cDNA
  5. e Pre-amplification (optional but recommended)
  6. f Prepare qPCR plate
  7. g Run quantitative real-time PCR
  8. h Data analysis and normalization
Phase 8 · Passaging and Long-Term Culture Maintenance Days 10-100+ (for colony passaging)
  1. a Prepare for enteroid/colonoid passaging
  2. b Mechanically dissociate enteroids/colonoids from Matrigel
  3. c Transfer dissociated colonies to fresh Matrigel
  4. d Add Thiazovivin and/or Rock inhibitor to passaged culture for first 2 days
  5. e Maintain passaged cultures with regular medium changes

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Attribution

This SOP was written by Organthis based on the published method in Wang et al., 2013. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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