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INTESTINAL Publication-derived

Intestinal Organoids for Developmental Studies (2014)

Full SOP with Academic or a plan

Source Yin et al., 2014 · David H. Koch Institute for Integrative Cancer Research, MIT · 10.1038/nmeth.2737

Authors: Xiaolei Yin, Henner F Farin, Johan H van Es, Hans Clevers, Robert Langer, Jeffrey M Karp Duration: 11 days 7 phases Cell source: Mouse Lgr5-EGFP intestinal stem cells

Abstract

This protocol describes the culture of Lgr5+ intestinal stem cells using CHIR99021 and valproic acid (VPA) to achieve nearly homogeneous stem cell cultures with ~100-fold higher colony-forming efficiency than conventional methods. The protocol maintains self-renewal while preserving multilineage differentiation capability into enterocytes, goblet cells, and Paneth cells.

Cell source
Mouse Lgr5-EGFP intestinal stem cells
Application
Maintenance and differentiation of intestinal stem cells

Protocol overview

30 steps across 7 phases

Phase 1 · Crypt and Single-Cell Isolation Day 0
  1. a Tissue harvesting and preparation
  2. b Tissue fragmentation
  3. c EDTA incubation for crypt release
  4. d Crypt collection and purification
  5. e Single-cell isolation
  6. f FACS sorting of stem cells
Phase 2 · Matrigel embedding and culture setup Day 0
  1. a Matrigel preparation
  2. b Cell embedding in Matrigel
  3. c Matrigel polymerization
  4. d Culture medium addition
Phase 3 · Stem cell expansion (CV condition) Day 0-Day 6 and beyond
  1. a Medium change
  2. b Incubation
  3. c Cell passage as single cells (every 6 days)
  4. d Monitoring stem cell characteristics
Phase 4 · Directed differentiation Day 6-Day 11
  1. a Preparation of homogeneous stem cell starting population
  2. b Transfer to differentiation condition – Enterocyte differentiation
  3. c Goblet cell differentiation
  4. d Paneth cell differentiation
  5. e Alternative – Spontaneous multilineage differentiation
Phase 5 · Flow cytometry analysis As needed
  1. a Cell dissociation
  2. b Cell counting and preparation
  3. c Flow cytometry
Phase 6 · Immunocytochemistry and microscopy As needed
  1. a Fixation
  2. b Matrigel disruption and cell isolation
  3. c Permeabilization
  4. d Antibody staining
  5. e EdU incorporation assay
  6. f Microscopy imaging
Phase 7 · Molecular analysis – qPCR As needed
  1. a RNA extraction
  2. b Quantitative real-time PCR

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Attribution

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

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