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SKIN Publication-derived

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

Source Kim et al., 2019 · CiSTEM Laboratory, Catholic Induced Pluripotent Stem Cell (iPSC) Research Center, College of Medicine, The Catholic University of Korea · 10.3791/59297

👤 Yena Kim, Ji Hyeon Ju ⏱ 30 days 📋 6 phases 🧫 Cord blood mononuclear cell-derived induced pluripotent stem cells (CBMC-iPSCs)

Abstract

This protocol describes the differentiation of cord blood-derived induced pluripotent stem cells (CBMC-iPSCs) into keratinocytes and fibroblasts, the main cell types of human skin. The differentiated cells are then used to generate a stratified 3D skin organoid via an air-liquid interface culture that mimics natural skin structure. The resulting organoid can be transplanted onto mice to create a humanized skin graft model for dermatological research.

Cell source
Cord blood mononuclear cell-derived induced pluripotent stem cells (CBMC-iPSCs)
Application
Disease modeling and regenerative medicine

Protocol overview

51 steps across 6 phases

Medium Preparation Day -1 to Day 0 (prior to cell work)
  1. 1 Prepare KDM1 (Keratinocyte Differentiation Medium 1)
  2. 2 Prepare KDM2 (Keratinocyte Differentiation Medium 2)
  3. 3 Prepare KDM3 (Keratinocyte Differentiation Medium 3)
  4. 4 Prepare FDM1 (Fibroblast Differentiation Medium 1)
  5. 5 Prepare FDM2 (Fibroblast Differentiation Medium 2)
  6. 6 Prepare EP1 (Epithelial Medium 1)
  7. 7 Prepare EP2 (Epithelial Medium 2)
  8. 8 Prepare EP3 (Epithelial Medium 3, Cornification Medium)
Embryonic Body Generation Day 0 to Day 1
  1. 1 Generate CBMC-iPSCs
  2. 2 Coat culture dish with vitronectin
  3. 3 Maintain CBMC-derived iPSCs on vitronectin-coated plates
  4. 4 Expand iPSCs to 80% confluence
  5. 5 Dissociate iPSCs with EDTA
  6. 6 Count and resuspend cells for EB formation
  7. 7 Form embryonic bodies using hanging drop method
  8. 8 Incubate hanging droplets
  9. 9 Harvest embryonic bodies
Keratinocyte Differentiation from CBMC-iPSCs Day 1 to Day 30
  1. 1 Treat EBs with BMP4
  2. 2 Coat culture dish with type IV collagen
  3. 3 Attach EBs to collagen-coated plates
  4. 4 Begin keratinocyte differentiation phase 1 (Days 0–8)
  5. 5 Continue keratinocyte differentiation phase 2 (Days 9–12)
  6. 6 Complete keratinocyte differentiation phase 3 (Days 13–30)
Fibroblast Differentiation from CBMC-iPSCs Day 1 to Day 28+
  1. 1 Coat culture dish with basement membrane matrix
  2. 2 Seed EBs onto basement membrane matrix
  3. 3 Attach EBs to basement membrane matrix-coated plates
  4. 4 Add BMP4 (Days 4–6)
  5. 5 Switch to FDM2 (Day 7 onwards)
  6. 6 Harvest and passage cells (Day 14)
  7. 7 Culture on noncoated dish (Days 14–21)
  8. 8 Coat culture dish with type I collagen
  9. 9 Passage cells onto type I collagen (Day 21)
  10. 10 Final passage onto noncoated dish (Day 28)
Generation of 3D Skin Organoid Day 28+ to Day 14 in 3D culture
  1. 1 Prepare neutralized type I collagen solution
  2. 2 Prepare fibroblast-collagen matrix
  3. 3 Form fibroblast-collagen gel in membrane insert
  4. 4 Culture fibroblast-collagen matrix until gelation complete
  5. 5 Harvest keratinocytes and prepare for seeding
  6. 6 Resuspend keratinocytes in low-calcium medium
  7. 7 Seed keratinocytes onto fibroblast layer
  8. 8 Begin low-calcium epithelial culture (Days 1–2)
  9. 9 Switch to normal-calcium epithelial medium (Days 3–4)
  10. 10 Establish air-liquid interface and begin cornification (Day 5 onwards)
  11. 11 Maintain 3D skin organoid in air-liquid interface culture (Days 5–14)
  12. 12 Harvest 3D skin organoid
Skin Graft onto Humanized Mouse Model Post-organoid generation
  1. 1 Anesthetize NOD/scid mice
  2. 2 Prepare skin graft site
  3. 3 Create skin defect
  4. 4 Graft 3D skin organoid onto skin defect
  5. 5 Post-transplant observation
  6. 6 Euthanize mice and harvest tissue for histological analysis

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Kim et al., 2019. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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