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INTESTINAL Publication-derived

Intestinal Spheroids

Full SOP with Academic or a plan

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

Authors: McCracken KW, Howell JC, Wells JM, Spence JR Duration: 140 days 5 phases Cell source: 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

Phase 1 · hPSC Passaging and Preparation Days 0-3
  1. a Coat 24-well plate with matrigel
  2. b Prepare hPSC culture for passaging
  3. c Dispase treatment
  4. d Wash wells
  5. e Resuspend colonies in mTeSR1
  6. f Triturate colonies
  7. g Passage colonies to 24-well dish
  8. h Distribute cells evenly and incubate overnight
  9. i Observe cultures and remove floating debris
  10. j Feed cells with mTeSR1 and monitor growth
Phase 2 · Definitive Endoderm Induction Days 4-6
  1. a Day 1 of Activin A treatment
  2. b Day 2 of Activin A treatment
  3. c Day 3 of Activin A treatment
  4. d End of Activin A treatment and assessment
Phase 3 · Mid/Hindgut Patterning and Spheroid Formation Days 7-10
  1. a Day 1 of FGF4/WNT3a treatment
  2. b Days 2-3 of FGF4/WNT3a treatment and spheroid observation
  3. c Collect mid/hindgut spheroids
  4. d Optional immunostaining of epithelial monolayers
Phase 4 · Three-Dimensional Organoid Expansion in Matrigel Days 10-24
  1. a Thaw Intestinal Matrigel
  2. b Allow spheroids to settle by gravity
  3. c Transfer spheroids to cold Matrigel
  4. d Plate spheroid-Matrigel suspension
  5. e Remove air bubbles and solidify Matrigel
  6. f Add Intestine Growth Media
  7. g Maintain organoid cultures
Phase 5 · Organoid Passage and Further Expansion Days 24-140+
  1. a Prepare for organoid splitting at day 14
  2. b Prepare cut pipette tips
  3. c Dislodge Matrigel bead from plate
  4. d Free organoids from Matrigel
  5. e Pool Matrigel, organoids, and media
  6. f Select and re-embed organoids
  7. g Culture for 14 days
  8. h Prepare for expansion at day 28 (after second 14-day culture)
  9. i Free organoids from Matrigel for expansion
  10. j Manually cut organoids
  11. k Re-embed cut organoid halves
  12. l Continue expansion cycles

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Attribution

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

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