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SKIN Publication-derived

Generation of Human Induced Pluripotent Stem Cell-derived Planar Hair-bearing Skin Organoids Using an Air-Liquid Interface Culture System

Source De Henau et al., 2025 · Leiden University Medical Center, Department of Anatomy and Embryology · 10.3791/69088

👤 Charlotte M. S. De Henau, Victor Lorrain, Myrthe P. Flesseman, Veronika Ramovs, Karine Raymond ⏱ 90 days 📋 6 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of hiPSC-derived planar skin organoids containing hair follicles using an air-liquid interface culture system. Cystic skin organoids are first developed from hiPSCs over 70–90 days, then mechanically flattened and cultured at an air-liquid interface for an additional 27 days to produce physiologically relevant, stratified skin models with functional hair follicles and sebaceous gland-like structures.

Cell source
Human iPSC
Application
Disease modeling and developmental study of skin with hair follicles

Protocol overview

19 steps across 6 phases

Cell Aggregation and ROCK Inhibitor Priming Day -2 to Day -1
  1. 1 Culture hiPSCs to confluence and dissociate
  2. 2 Form cell aggregates in ultra-low-attachment plate
  3. 3 Dilute ROCK inhibitor
Surface Ectoderm Induction Day 0 to Day 3
  1. 1 Prepare and induce surface ectoderm
  2. 2 Induce non-epithelial cell populations
Early Organoid Development and Nutrient Supply Day 6 to Day 12
  1. 1 Supply nutrients
  2. 2 Medium refreshment
  3. 3 Transition to low-attachment 24-well plates
Cystic Organoid Maturation and Maintenance Day 15 to Day 90
  1. 1 Medium refreshment with basement membrane matrix
  2. 2 Half medium change (Days 18–45)
  3. 3 Half medium change and full medium change (Days 45–90)
Selection and Flattening of Cystic Organoids Day 70 to Day 90
  1. 1 Select cystic organoids for transition
  2. 2 Prepare collagen-coated inserts
  3. 3 Excise byproducts from cystic organoid
  4. 4 Trim and unfold the organoid
  5. 5 Cut and transfer tissue to collagen-coated insert
  6. 6 Initiate air-liquid interface culture
Air-Liquid Interface Culture Maintenance Day 1 to Day 27 post-flattening
  1. 1 Daily/three-times-weekly medium changes (Days 1–21 post-flattening, humidified)
  2. 2 Daily medium changes with reduced humidity (Days 21–27 post-flattening)

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in De Henau et al., 2025. 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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