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SKIN Publication-derived

Air-Liquid Interface Culture Enhances Skin Organoid Hair

Source Sun et al. · Frazer Institute, University of Queensland, Brisbane, Australia · 10.1093/burnst/tkae070

👤 Sun J, Ahmed I, Brown J, Khosrotehrani K, Shafiee A ⏱ 130 days 📋 7 phases 🧫 Human iPSC

Abstract

Demonstrates that air-liquid interface culture enhances hair follicle development and epidermal maturation in iPSC-derived skin organoids. Bridges organoid and traditional ALI skin equivalent approaches to improve translational relevance for burn wound therapy.

Cell source
Human iPSC
Application
Hair Follicle Research

Protocol overview

40 steps across 7 phases

hiPSC Culture and Maintenance Prior to Day 0
  1. 1 Coat plates with hESC-qualified Matrigel
  2. 2 Culture hiPSC lines in mTeSR Plus medium
  3. 3 Passage hiPSCs at 80% confluency
hiPSC Reprogramming (for P111 and P112 lines) Prior to Phase 1
  1. 1 Isolate CD34+ cells from human term placenta
  2. 2 Expand CD34+ cells in StemPro34 medium
  3. 3 Reprogram CD34+ cells to iPSCs using Sendai vectors
  4. 4 Identify and manually select hiPSC colonies
SKO Differentiation — Embryoid Body Formation and Early Induction (Day −1 to Day 12) Day −1 to Day 12
  1. 1 Prepare hiPSCs for embryoid body formation
  2. 2 Dissociate hiPSCs to single-cell suspension
  3. 3 Distribute cells into 96-well low-attachment plate
  4. 4 Centrifuge plates to facilitate embryoid body aggregation (Day −1)
  5. 5 Initiate differentiation (Day 0)
  6. 6 Induce cranial neural crest cells (Day 3)
  7. 7 Medium expansion (Day 6)
  8. 8 Medium changes (Days 8 and 10)
  9. 9 Transfer SKOs to respective culture systems (Day 12)
Floating SKO (F-SKO) Culture — Maturation Phase (Day 12 to Day 130) Day 12 to Day 130
  1. 1 Transfer organoids to low-attachment 24-well plate (Day 12)
  2. 2 Half-medium change with GFR-Matrigel (Day 15)
  3. 3 Half-medium change to OMM only (Day 18)
  4. 4 Maintain half-medium changes every other day (Day 18 to Day 130)
  5. 5 Increase medium volume as organoids mature (Day 80 onward)
Transwell SKO (T-SKO) Culture — Air-Liquid Interface (Day 12 to Day 130) Day 12 to Day 130
  1. 1 Transfer organoids to transwell insert (Day 12)
  2. 2 Position medium to establish ALI interface
  3. 3 Replace medium with reduced Matrigel (Day 15)
  4. 4 Transition to full-medium changes without Matrigel (Day 18 onward)
  5. 5 Increase medium volume as organoids grow (Day 80 onward)
Immunofluorescence and Histological Analysis Days 16, 50, 65, 75, 85, 90, 120 (for analysis)
  1. 1 Fix organoids in 4% paraformaldehyde
  2. 2 Embed organoids in OCT cryo-embedding medium
  3. 3 Prepare cryosections
  4. 4 Store sectioned samples
  5. 5 Block cryosections with blocking buffer
  6. 6 Incubate with primary antibodies
  7. 7 Wash and incubate with secondary antibodies
  8. 8 Wash and stain nuclei with DAPI
  9. 9 Image sections with confocal microscopy
  10. 10 Perform whole-mount immunofluorescence (optional)
  11. 11 Perform Hematoxylin and Eosin (H&E) staining
  12. 12 Image H&E sections with slide scanner
Bright-Field Microscopy Imaging Days 65, 75, 85, 95, 115 (analysis timepoints)
  1. 1 Select organoids for bright-field imaging
  2. 2 Image organoids using Olympus IX73 inverted microscope

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Sun et al.. 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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