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LUNG Publication-derived

A bioengineered niche promotes in vivo engraftment and maturation of pluripotent stem cell derived human lung organoids

Source Dye et al., 2016 · University of Michigan Medical School · 10.7554/elife.19732.001

👤 Briana R Dye, Priya H Dedhia, Alyssa J Miller, Melinda S Nagy, Eric S White, Lonnie D Shea, Jason R Spence ⏱ 105 days 📋 6 phases 🧫 Human ESC (UM63-1, H1, H9)

Abstract

This protocol describes the generation and transplantation of human pluripotent stem cell-derived lung organoids (HLOs) onto microporous poly(lactide-co-glycolide) (PLG) scaffolds into the epididymal fat pad of NSG mice. The method enables long-term survival, engraftment, and maturation of HLO epithelium in vivo, resulting in airway-like structures with enhanced cellular organization and differentiation resembling adult human lung tissue.

Cell source
Human ESC (UM63-1, H1, H9)
Application
In vivo maturation and engraftment of lung organoids; disease modeling and tissue engineering

Protocol overview

25 steps across 6 phases

Maintenance and generation of hESCs and foregut spheroids Day 0 - Day 35+
  1. 1 Maintain hESCs on Matrigel-coated plates
  2. 2 Generate human lung organoids (HLOs) from hESCs
  3. 3 Select HLOs at appropriate developmental stage
Preparation of PLG scaffolds Day -7 to Day -1 (before transplant)
  1. 1 Prepare PLG scaffold base material
  2. 2 Press and foam scaffolds
  3. 3 Leach scaffolds to remove salt
  4. 4 Sterilize and prepare scaffolds for seeding
Seeding HLOs onto PLG scaffolds Day -7 to Day -1
  1. 1 Mix HLOs with Matrigel
  2. 2 Seed HLOs onto scaffold
  3. 3 Incubate seeded scaffolds
  4. 4 Overlay scaffolds with culture media
In vitro culture of HLO-scaffold constructs Day -7 to Day -1
  1. 1 Culture seeded scaffolds in vitro
  2. 2 Prepare scaffolds for transplantation
Scaffold preparation for transplantation and surgical implantation Day 0 (transplant day)
  1. 1 Option A: Dip scaffold in Matrigel/FGF10 before transplantation
  2. 2 Option B: Proceed directly to transplantation
  3. 3 Anesthetize mice
  4. 4 Prepare surgical site
  5. 5 Expose epididymal fat pad
  6. 6 Position and secure scaffold construct
  7. 7 Administer antibiotic and close incision
Post-transplant monitoring and harvest Day 1 - Day 105 (4–15 weeks post-transplant)
  1. 1 Monitor mice post-operatively
  2. 2 Harvest transplanted tissue at 4 weeks
  3. 3 Harvest transplanted tissue at 8 weeks
  4. 4 Optional: Harvest at 15 weeks
  5. 5 Process tissue for histology and immunofluorescence

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Dye et al., 2016. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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