Skip to content
Back to browse
SKIN Publication-derived

Skin Organoids for Regeneration and Disease Modelling

Full SOP with Academic or a plan

Source Shafiee et al., 2024 · University of Queensland, Herston Biofabrication Institute · 10.1002/smll.202304879

Authors: Abbas Shafiee, Jane Sun, Imaan A. Ahmed, Felicia Phua, Gustavo R. Rossi, Cheng-Yu Lin, Fernando Souza-Fonseca-Guimaraes, Ernst J. Wolvetang, Jason Brown, Kiarash Khosrotehrani Duration: 125 days 8 phases Cell source: Human iPSC (skin fibroblast-derived and placental CD34+-derived)

Abstract

This protocol describes the generation of physiologically relevant skin organoids (SKOs) from human induced pluripotent stem cells (hiPSCs) through optimized embryoid body formation coupled with modulation of TGF-β, FGF, and BMP signaling pathways. The resulting SKOs develop stratified epidermal layers, pigmented hair follicles, sebaceous glands, and eccrine sweat glands, as well as Merkel cells, enabling studies of skin development, disease modeling, and wound healing.

Cell source
Human iPSC (skin fibroblast-derived and placental CD34+-derived)
Application
Disease modeling and regenerative medicine

Protocol overview

36 steps across 8 phases

Phase 1 · iPSC Maintenance and Preparation Before differentiation
  1. a Coat culture plates with Matrigel
  2. b Maintain hiPSCs in culture
  3. c Passage confluent hiPSCs
Phase 2 · Embryoid Body Formation and Initial Differentiation (Days 0-6) Days 0-6
  1. a Prepare hiPSCs for aggregation
  2. b Form embryoid bodies in Ultra-Low Attachment plates
  3. c Incubate for EB formation
  4. d Apply day 0 differentiation medium
  5. e Incubate for 3 days
  6. f Apply day 3 differentiation medium
  7. g Incubate for 3 more days
Phase 3 · Extended Culture and Transfer to Larger Plates (Days 6-18) Days 6-18
  1. a Add fresh E6 medium on day 6
  2. b Perform media changes on days 8 and 10
  3. c Transfer organoids to 24-well plates on day 12
  4. d Initiate half media changes from day 18
Phase 4 · Maturation and Continued Culture (Days 18-120+) Days 18-120+
  1. a Continue half media changes three times per week
  2. b Increase media volume from day 80 onwards
  3. c Continue culture to day 120 or beyond
Phase 5 · Organoid Fixation and Tissue Preparation Days 16, 50, 65, 83, 100, 120
  1. a Harvest and wash organoids
  2. b Fix organoids with paraformaldehyde
  3. c Prepare samples for histology
  4. d Prepare samples for immunofluorescence (cryosections)
Phase 6 · Immunofluorescent Staining and Imaging After fixation and sectioning
  1. a Wash cryosections
  2. b Block and permeabilize
  3. c Apply primary antibodies
  4. d Wash after primary antibodies
  5. e Apply secondary antibodies
  6. f Wash after secondary antibodies
  7. g Apply DAPI staining
  8. h Final washing and mounting
  9. i Image using confocal microscopy
Phase 7 · Immunofluorescent Staining of Paraffin Sections After paraffin embedding
  1. a Dewax and rehydrate paraffin sections
  2. b Perform antigen retrieval
  3. c Proceed with immunostaining as described in Phase 6
Phase 8 · Hair Follicle and Placode Quantification Days 72, 83, 93, 104, 115, 125
  1. a Visualize organoids under light microscope
  2. b Capture images and count hair structures
  3. c Document results and calculate averages

Full SOP

Full SOPs need an account and a plan

Create an account, then get free Academic access (for verified academics) or choose a plan. Plans include step-by-step procedures, reagent concentrations, QC checklists and batch record templates.

Create an account

Already registered? Log in

Attribution

This SOP was written by Organthis based on the published method in Shafiee et al., 2024. Once the originating laboratory has confirmed it, the SOP is marked Verified.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence