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SKIN Publication-derived

Recessive Dystrophic Epidermolysis Bullosa Epidermal Organ-on-Chip Models

Full SOP with Academic or a plan

Source Garriga-Cerda et al., 2025 · Columbia University Medical Center · 10.1177/20417314251397594

Authors: Laura Garriga-Cerda, Alberto Pappalardo, Charlotte Y. Lee, Jeffrey Kysar, Kristin Myers, Hasan Erbil Abaci Duration: 45 days 8 phases Cell source: Patient-Derived iPSC (RDEB), Wild-type iPSC

Abstract

This protocol describes a method for generating iPSC-derived keratinocytes and fibroblasts from complex skin organoids at day 45 of differentiation, which can be expanded in 2D culture and then engineered into functional 3D skin constructs that recapitulate wild-type and RDEB disease phenotypes. The resulting cells display marker expression comparable to primary counterparts and produce stratified epidermis with significant extracellular matrix remodeling and protein deposition.

Cell source
Patient-Derived iPSC (RDEB), Wild-type iPSC
Application
Disease modeling of recessive dystrophic epidermolysis bullosa (RDEB) and functional 3D engineered skin construct development

Protocol overview

33 steps across 8 phases

Phase 1 · iPSC Culture and Organoid Formation Initiation Day -2 to Day 0
  1. a Prepare iPSC single cell suspension
  2. b Dilute rock inhibitor on Day -1
  3. c Initialize organoid differentiation on Day 0
Phase 2 · Early Organoid Development and Factor Addition Day 3 to Day 12
  1. a Add LDN and bFGF on Day 3
  2. b Dilute factors on Day 6
  3. c Perform half media change on Day 9
  4. d Transfer organoids to 24-well plates on Day 12
Phase 3 · Organoid Maturation and Culture Optimization Day 15 to Day 45
  1. a Perform half media change on Day 15
  2. b Perform half media change on Day 18
  3. c Maintain culture until Day 45 with twice-weekly media changes
Phase 4 · Cell Sorting and Isolation from Organoids Day 45
  1. a Collect and dissociate organoids
  2. b MACS sorting for keratinocytes (iKCs)
  3. c MACS sorting for fibroblasts (iFBs)
  4. d Plate sorted iKCs
  5. e Plate sorted iFBs
Phase 5 · Cell Expansion and Passage Day 45 to Passage 2
  1. a Culture iKCs to 80% confluence and re-sort
  2. b Culture iFBs to 80% confluence
  3. c Freeze cells for banking
Phase 6 · Fibroblast Maturation (Optional Enhancement) 5 days prior to ESC assembly
  1. a Set up transwell co-culture
  2. b Detach and prepare matured iFBs
Phase 7 · 3D Engineered Skin Construct (ESC) Assembly 14-21 days
  1. a Prepare dermal compartment
  2. b Culture dermis in appropriate medium
  3. c Seed keratinocytes
  4. d Submerge constructs in epidermalization medium
  5. e Transition to air-liquid interface (ALI)
  6. f Harvest ESCs for analysis
Phase 8 · Characterization Assays Day 45 organoids and after ESC harvest
  1. a Immunofluorescence staining of 2D cultured cells
  2. b Immunofluorescence staining of 3D ESCs
  3. c Hematoxylin and eosin (H&E) staining
  4. d Flow cytometry analysis
  5. e Mechanical testing by microindentation
  6. f Epidermal thickness quantification
  7. g Contraction quantification

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Attribution

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

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