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LUNG Publication-derived

Airway Bioprinted and Engineered Tissues for Regenerative Medicine

Full SOP with Academic or a plan

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

Authors: Briana R Dye, Priya H Dedhia, Alyssa J Miller, Melinda S Nagy, Eric S White, Lonnie D Shea, Jason R Spence Duration: 105 days 6 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Dye et al., 2016. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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