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INTESTINAL Publication-derived

Generating Human Intestinal Tissue from Pluripotent Stem Cells

Source McCracken et al. · Cincinnati Children's Hospital Medical Center, Cincinnati, USA · 10.1038/nprot.2011.410

👤 McCracken KW, Howell JC, Wells JM, Spence JR ⏱ 140 days 📋 5 phases 🧫 Human iPSC / Human ESC

Abstract

The definitive step-by-step protocol for generating human intestinal organoids from pluripotent stem cells. Provides detailed instructions from PSC culture through definitive endoderm induction, hindgut specification, and long-term 3D organoid culture with expansion over 140 days.

Cell source
Human iPSC / Human ESC
Application
HIO Production Protocol

Protocol overview

37 steps across 5 phases

hPSC Passaging and Preparation Days 0–3
  1. 1 Coat 24-well plate with matrigel
  2. 2 Prepare hPSC culture for passaging
  3. 3 Dispase treatment
  4. 4 Wash wells
  5. 5 Resuspend colonies in mTeSR1
  6. 6 Triturate colonies
  7. 7 Passage colonies to 24-well dish
  8. 8 Distribute cells evenly and incubate overnight
  9. 9 Observe cultures and remove floating debris
  10. 10 Feed cells with mTeSR1 and monitor growth
Definitive Endoderm Induction Days 4–6
  1. 11 Day 1 of Activin A treatment
  2. 12 Day 2 of Activin A treatment
  3. 13 Day 3 of Activin A treatment
  4. 14 End of Activin A treatment and assessment
Mid/Hindgut Patterning and Spheroid Formation Days 7–10
  1. 15 Day 1 of FGF4/WNT3a treatment
  2. 16 Days 2–3 of FGF4/WNT3a treatment and spheroid observation
  3. 17 Collect mid/hindgut spheroids
  4. 18 Optional immunostaining of epithelial monolayers
Three-Dimensional Organoid Expansion in Matrigel Days 10–24
  1. 19 Thaw Intestinal Matrigel
  2. 20 Allow spheroids to settle by gravity
  3. 21 Transfer spheroids to cold Matrigel
  4. 22 Plate spheroid-Matrigel suspension
  5. 23 Remove air bubbles and solidify Matrigel
  6. 24 Add Intestine Growth Media
  7. 25 Maintain organoid cultures
Organoid Passage and Further Expansion Days 24–140+
  1. 26 Prepare for organoid splitting at day 14
  2. 27 Prepare cut pipette tips
  3. 28 Dislodge Matrigel bead from plate
  4. 29 Free organoids from Matrigel
  5. 30 Pool Matrigel, organoids, and media
  6. 31 Select and re-embed organoids
  7. 32 Culture for 14 days
  8. 33 Prepare for expansion at day 28 (after second 14-day culture)
  9. 34 Free organoids from Matrigel for expansion
  10. 35 Manually cut organoids
  11. 36 Re-embed cut organoid halves
  12. 37 Continue expansion cycles

Full SOP

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Attribution

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

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