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SKIN Publication-derived

Skin Organoids for Disease Modelling (2024)

Full SOP with Academic or a plan

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

Authors: Min-Ji Kim, Hee-Jin Ahn, Dasom Kong, Seunghee Lee, Da-Hyun Kim, Kyung-Sun Kang Duration: 130 days 18 phases Cell source: 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

Phase 1 · Human iPSC Culture and Maintenance Ongoing (passages 1-50-70)
  1. a iPSC Culture Setup
  2. b Passage iPSCs
Phase 2 · Embryoid Body (EB) Generation Day 0
  1. a Seed iPSCs for EB Formation
Phase 3 · Non-Neural Ectoderm Induction Day 0-4
  1. a Monitor EB Size
  2. b Transfer EBs to Differentiation Medium
  3. c Culture in Non-Neural Ectoderm Induction Medium
Phase 4 · Cranial Neural Crest Cell Induction Day 4-6
  1. a Add NCC Induction Factors
  2. b Add Fresh Medium
Phase 5 · Medium Exchange and Organoid Growth Day 8-12
  1. a Perform Half Medium Changes
  2. b Monitor Organoid Development
Phase 6 · Self-Assembly and Maturation (Orbital Shaker Phase) Day 12-95
  1. a Transfer Organoids to 6-Well Plates
  2. b Continue Culture Until Day 85
  3. c Monitor for Hair Follicle Formation
Phase 7 · Air–Liquid Interface (ALI) Culture and Maturation Day 85-120
  1. a Cut and Seed Organoids onto Transwell Inserts
  2. b Culture on Transwell Inserts with Liquid Medium
  3. c Transfer to Dry-Conditioned Incubator
Phase 8 · Solar UV Exposure and Photodamage Modeling Day 120-130 (or after ALI maturation completion)
  1. a Prepare UV Source
  2. b Prepare Organoids for UV Exposure
  3. c First UV Exposure (Day 0 of Photodamage Protocol)
  4. d Second UV Exposure (2 h after First)
  5. e Repeat Exposure Cycle (Total 3 Cycles)
Phase 9 · Histological Sample Preparation and Staining On demand (post-exposure)
  1. a Fix Organoids in Paraformaldehyde
  2. b Wash and Cryoprotect
  3. c Freeze and Cryosection
  4. d H&E Staining
  5. e Masson's Trichrome Staining
Phase 10 · Immunohistochemistry and Immunofluorescence Analysis On demand (post-fixation)
  1. a Permeabilization and Blocking
  2. b Primary Antibody Incubation
  3. c Wash and Secondary Antibody Incubation
  4. d Nuclear Staining and Mounting
  5. e Image Acquisition
  6. f Quantitative Image Analysis
Phase 11 · RNA Extraction and qRT-PCR Analysis On demand (post-exposure or treatment)
  1. a RNA Extraction
  2. b cDNA Synthesis
  3. c qRT-PCR Setup and Reaction
  4. d Data Analysis
Phase 12 · Isolation and Characterization of Umbilical Cord Blood-Derived Exosomes (UCB-Exos) Variable (exosome preparation)
  1. a Culture UCB-MSCs
  2. b Collect Conditioned Medium
  3. c Tangential Flow Filtration (TFF)
  4. d Ultracentrifugation at 3000 g
  5. e Ultracentrifugation at 100,000 g
  6. f Size Exclusion Chromatography (SEC)
  7. g Transmission Electron Microscopy (TEM) Characterization
  8. h Nanoparticle Tracking Analysis (NTA)
  9. i Western Blot for Exosomal Markers
  10. j Flow Cytometry for Exosomal Marker Verification
Phase 13 · Exosome Penetration Assay Variable (post-exosome preparation)
  1. a Fluorescent Labeling of Exosomes
  2. b Incubate Labeled Exosomes with Skin Organoids
  3. c Fix and Image Organoids
  4. d Quantify Exosome Localization
Phase 14 · Exosome Treatment of UV-Damaged Organoids Day 4 (post-UV exposure) – Day 7
  1. a Determine Optimal Exosome Concentration
  2. b Apply Exosomes Post-UV Exposure
  3. c Maintain Culture Conditions
Phase 15 · Western Blot Analysis (Protein Level Assessment) On demand (post-treatment)
  1. a Prepare Whole Cell Lysate
  2. b Prepare Nuclear and Cytoplasmic Fractions
  3. c SDS-PAGE Electrophoresis
  4. d Transfer to PVDF Membrane
  5. e Block and Incubate with Primary Antibodies
  6. f Wash and Incubate with Secondary Antibodies
  7. g Detection
Phase 16 · Enzyme-Linked Immunosorbent Assay (ELISA) for Cytokine Quantification On demand (post-treatment)
  1. a Collect Culture Supernatant
  2. b Perform ELISA for IL-1β, IL-6, and TNF-α
  3. c Read Absorbance and Analyze Results
Phase 17 · NF-κB Pathway Inhibition Studies Day 0-3 (parallel to UV exposure and exosome treatment)
  1. a Precondition Organoids with NF-κB Inhibitor
  2. b UV Exposure During Inhibition
  3. c Post-UV Inhibition
  4. d Harvest and Analyze
Phase 18 · Statistical Analysis On demand (post-data collection)
  1. a Data Compilation and Quality Control
  2. b Descriptive Statistics
  3. c Determine Appropriate Statistical Tests
  4. d Perform Statistical Tests
  5. e Define Statistical Significance Threshold
  6. f Generate Graphs

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Attribution

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

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