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SKIN Publication-derived

Skin Organoids for Disease Modelling with Hair Follicles

Full SOP with Academic or a plan

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

Authors: Charlotte M. S. De Henau, Victor Lorrain, Myrthe P. Flesseman, Veronika Ramovs, Karine Raymond Duration: 90 days 6 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in De Henau et al., 2025. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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