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SKIN Publication-derived

Modeling of solar UV-induced photodamage on hair follicles in human skin organoids

Source Kim et al., 2024 · Adult Stem Cell Research Center and Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University · 10.1177/20417314241248753journal

👤 Min-Ji Kim, Hee-Jin Ahn, Dasom Kong, Seunghee Lee, Da-Hyun Kim, Kyung-Sun Kang ⏱ 130 days 📋 18 phases 🧫 Human iPSC (line CMC003)

Abstract

This protocol describes the generation of human iPSC-derived skin organoids with hair follicles and their exposure to solar UV radiation to model photodamage. The organoids recapitulate UV-induced skin damage including barrier disruption, extracellular matrix degradation, and inflammatory responses in hair follicles. The model is used to evaluate exosome-based therapeutics.

Cell source
Human iPSC (line CMC003)
Application
Disease modeling (photodamage); Therapeutic efficacy evaluation

Protocol overview

73 steps across 18 phases

Human iPSC Culture and Maintenance Ongoing (passages 1–50–70)
  1. 1 iPSC Culture Setup
  2. 2 Passage iPSCs
Embryoid Body (EB) Generation Day 0
  1. 1 Seed iPSCs for EB Formation
Non-Neural Ectoderm Induction Day 0–4
  1. 1 Monitor EB Size
  2. 2 Transfer EBs to Differentiation Medium
  3. 3 Culture in Non-Neural Ectoderm Induction Medium
Cranial Neural Crest Cell Induction Day 4–6
  1. 1 Add NCC Induction Factors
  2. 2 Add Fresh Medium
Medium Exchange and Organoid Growth Day 8–12
  1. 1 Perform Half Medium Changes
  2. 2 Monitor Organoid Development
Self-Assembly and Maturation (Orbital Shaker Phase) Day 12–95
  1. 1 Transfer Organoids to 6-Well Plates
  2. 2 Continue Culture Until Day 85
  3. 3 Monitor for Hair Follicle Formation
Air–Liquid Interface (ALI) Culture and Maturation Day 85–120
  1. 1 Cut and Seed Organoids onto Transwell Inserts
  2. 2 Culture on Transwell Inserts with Liquid Medium
  3. 3 Transfer to Dry-Conditioned Incubator
Solar UV Exposure and Photodamage Modeling Day 120–130 (or after ALI maturation completion)
  1. 1 Prepare UV Source
  2. 2 Prepare Organoids for UV Exposure
  3. 3 First UV Exposure (Day 0 of Photodamage Protocol)
  4. 4 Second UV Exposure (2 h after First)
  5. 5 Repeat Exposure Cycle (Total 3 Cycles)
Histological Sample Preparation and Staining On demand (post-exposure)
  1. 1 Fix Organoids in Paraformaldehyde
  2. 2 Wash and Cryoprotect
  3. 3 Freeze and Cryosection
  4. 4 H&E Staining
  5. 5 Masson's Trichrome Staining
Immunohistochemistry and Immunofluorescence Analysis On demand (post-fixation)
  1. 1 Permeabilization and Blocking
  2. 2 Primary Antibody Incubation
  3. 3 Wash and Secondary Antibody Incubation
  4. 4 Nuclear Staining and Mounting
  5. 5 Image Acquisition
  6. 6 Quantitative Image Analysis
RNA Extraction and qRT-PCR Analysis On demand (post-exposure or treatment)
  1. 1 RNA Extraction
  2. 2 cDNA Synthesis
  3. 3 qRT-PCR Setup and Reaction
  4. 4 Data Analysis
Isolation and Characterization of Umbilical Cord Blood-Derived Exosomes (UCB-Exos) Variable (exosome preparation)
  1. 1 Culture UCB-MSCs
  2. 2 Collect Conditioned Medium
  3. 3 Tangential Flow Filtration (TFF)
  4. 4 Ultracentrifugation at 3000 g
  5. 5 Ultracentrifugation at 100,000 g
  6. 6 Size Exclusion Chromatography (SEC)
  7. 7 Transmission Electron Microscopy (TEM) Characterization
  8. 8 Nanoparticle Tracking Analysis (NTA)
  9. 9 Western Blot for Exosomal Markers
  10. 10 Flow Cytometry for Exosomal Marker Verification
Exosome Penetration Assay Variable (post-exosome preparation)
  1. 1 Fluorescent Labeling of Exosomes
  2. 2 Incubate Labeled Exosomes with Skin Organoids
  3. 3 Fix and Image Organoids
  4. 4 Quantify Exosome Localization
Exosome Treatment of UV-Damaged Organoids Day 4 (post-UV exposure) – Day 7
  1. 1 Determine Optimal Exosome Concentration
  2. 2 Apply Exosomes Post-UV Exposure
  3. 3 Maintain Culture Conditions
Western Blot Analysis (Protein Level Assessment) On demand (post-treatment)
  1. 1 Prepare Whole Cell Lysate
  2. 2 Prepare Nuclear and Cytoplasmic Fractions
  3. 3 SDS-PAGE Electrophoresis
  4. 4 Transfer to PVDF Membrane
  5. 5 Block and Incubate with Primary Antibodies
  6. 6 Wash and Incubate with Secondary Antibodies
  7. 7 Detection
Enzyme-Linked Immunosorbent Assay (ELISA) for Cytokine Quantification On demand (post-treatment)
  1. 1 Collect Culture Supernatant
  2. 2 Perform ELISA for IL-1β, IL-6, and TNF-α
  3. 3 Read Absorbance and Analyze Results
NF-κB Pathway Inhibition Studies Day 0–3 (parallel to UV exposure and exosome treatment)
  1. 1 Precondition Organoids with NF-κB Inhibitor
  2. 2 UV Exposure During Inhibition
  3. 3 Post-UV Inhibition
  4. 4 Harvest and Analyze
Statistical Analysis On demand (post-data collection)
  1. 1 Data Compilation and Quality Control
  2. 2 Descriptive Statistics
  3. 3 Determine Appropriate Statistical Tests
  4. 4 Perform Statistical Tests
  5. 5 Define Statistical Significance Threshold
  6. 6 Generate Graphs

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Kim et al., 2024. 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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