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INTESTINAL Publication-derived

Intestinal Organoids for Regenerative Medicine with Enhanced Maturation

Full SOP with Academic or a plan

Source Watson et al. · Cincinnati Children's Hospital Medical Center, Cincinnati, USA · 10.1038/nm.3737

Authors: Watson CL, Mahe MM, Munera J, Howell JC, Sundaram N, Poling HM, Schweitzer JI, Vallance JE, Mayhew CN, Sun Y, Liang G, Rapber R, Shroyer NF, Wells JM, Helmrath MA Duration: 42 days 9 phases Cell source: iPSC-Derived (Human iPSC-Derived HIOs)

Abstract

Demonstrated that iPSC-derived intestinal organoids transplanted into mouse kidney capsule mature into functional adult-like intestinal tissue with crypt-villus architecture, smooth muscle layers, and peptide absorption capacity, with 100-fold size increase after transplantation.

Cell source
iPSC-Derived (Human iPSC-Derived HIOs)
Application
Regenerative Medicine

Protocol overview

50 steps across 9 phases

Phase 1 · hPSC Maintenance and Culture Preparation Days 0-1
  1. a Prepare cell culture plates
  2. b Maintain hPSCs in feeder-free conditions
  3. c Passage hPSCs for endoderm differentiation
  4. d Culture until confluence
Phase 2 · Definitive Endoderm (DE) Induction Days 2-4
  1. a Treat cells with Activin A
  2. b Change media daily during Activin A treatment
Phase 3 · Mid-Hindgut Spheroid Formation Days 5-8
  1. a Prepare hindgut induction medium
  2. b Culture for 4 days to form mid-hindgut spheroids
  3. c Harvest and replicate spheroids in Matrigel
Phase 4 · Human Intestinal Organoid (HIO) Generation and In Vitro Maturation Days 9-35
  1. a Culture spheroids in intestinal growth medium
  2. b Change media at day 3
  3. c Change media twice weekly
  4. d Replicate HIOs every 14 days
  5. e Assess HIO morphology at day 35
Phase 5 · HIO Preparation for Transplantation Day 35 (Day -1 to Day 0 relative to surgery)
  1. a Remove HIO from Matrigel
  2. b Embed HIO in type I collagen
  3. c Incubate collagen-embedded HIO in growth medium overnight
Phase 6 · Surgical Transplantation of HIO under Kidney Capsule Day 0
  1. a Anesthetize mouse
  2. b Prepare surgical site
  3. c Make left-posterior subcostal incision
  4. d Expose the left kidney
  5. e Create subcapsular pocket
  6. f Insert collagen-embedded HIO into subcapsular pocket
  7. g Return kidney to peritoneal cavity
  8. h Administer intraperitoneal antibiotic flush
  9. i Close surgical incision
  10. j Administer subcutaneous analgesic
Phase 7 · Post-Operative Care and HIO Engraftment in Vivo Days 1-42 post-transplant
  1. a Maintain post-operative housing and monitoring
  2. b Maintain antibiotic supplementation
  3. c Allow HIO engraftment and maturation for 6 weeks
  4. d Assess engraftment success (optional, before further experimentation)
Phase 8 · Optional: Ileocecal Resection (ICR) and Intestinal Adaptation Study 6 weeks post-transplant, plus 7 days post-ICR
  1. a Switch mice to liquid diet before ICR surgery
  2. b Switch to liquid antibiotics
  3. c Randomize mice to sham or ICR groups
  4. d Anesthetize mouse for ICR or sham surgery
  5. e Prepare surgical site for ICR
  6. f Open abdomen via anterior incision
  7. g Identify and isolate ileocecal region
  8. h Perform ileocecal resection (ICR group only)
  9. i Perform sham operation (Sham group only)
  10. j Perform reanastomosis (ICR group only)
  11. k Close abdominal incision
  12. l Administer post-operative analgesia and antibiotics
  13. m Post-operative care: warmth and monitoring
  14. n Euthanize and harvest tissues at post-operative day 7
Phase 9 · HIO Harvest and Tissue Processing for Analysis 6 weeks post-transplant (or 7 days post-ICR)
  1. a Euthanize transplant recipient mouse
  2. b Expose engrafted HIO in kidney
  3. c Harvest engrafted HIO
  4. d Fix engraftment for histology or immunofluorescence
  5. e Prepare tissue for embedding

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Attribution

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

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