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SKIN Publication-derived

Epidermal Organoids for Regeneration and Disease Modelling

Full SOP with Academic or a plan

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

Authors: Yena Kim, Ji Hyeon Ju Duration: 30 days 6 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Kim et al., 2019. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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