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SKIN Publication-derived

Epidermal Organoids for Regenerative Medicine

Full SOP with Academic or a plan

Source Kim et al., 2018 · Catholic iPSC Research Center, College of Medicine, The Catholic University of Korea · 10.1186/s13287-018-0958-2

Authors: Yena Kim, Narae Park, Yeri Alice Rim, Yoojun Nam, Hyerin Jung, Kijun Lee, Ji Hyeon Ju Duration: 30 days 4 phases Cell source: Cord Blood Mononuclear Cell (CBMC)-derived Human iPSC

Abstract

This protocol describes the generation of a complex 3D skin organoid from CBMC-derived iPSCs by differentiating them into fibroblasts and keratinocytes, layering them in a Transwell system, and establishing a stratified epidermal-dermal structure. The organoid can be transplanted into mice to create a humanized skin model for wound healing and dermatologic research.

Cell source
Cord Blood Mononuclear Cell (CBMC)-derived Human iPSC
Application
Skin regenerative medicine and in vitro disease modeling

Protocol overview

23 steps across 4 phases

Phase 1 · Fibroblast Differentiation from CBMC-iPSCs Day 0-21
  1. a Embryonic body attachment
  2. b Culture in fibroblast differentiation medium 1 (FDM1)
  3. c Addition of BMP4 to FDM1
  4. d Switch to fibroblast differentiation medium 2 (FDM2)
  5. e Passage onto noncoated dish
  6. f Passage onto type I collagen-coated dish
Phase 2 · Keratinocyte Differentiation from CBMC-iPSCs Day 0-30
  1. a Formation of aggregated embryonic bodies
  2. b Transfer aggregated EBs to TeSR-E8 medium
  3. c EB attachment to type IV collagen-coated plate
  4. d Culture in keratinocyte differentiation medium 1 (KDM1)
  5. e Culture in keratinocyte differentiation medium 2 (KDM2)
  6. f Culture in keratinocyte differentiation medium 3 (KDM3)
Phase 3 · 3D iPSC-Derived Skin Organoid (iSO) Construction Day 0-30
  1. a Preparation of fibroblast-collagen suspension
  2. b Addition of fibroblast-collagen suspension to Transwell insert
  3. c Seeding keratinocytes onto fibroblast layer
  4. d Culture in low-calcium medium (EP1)
  5. e Transition to normal-calcium medium (EP2)
  6. f Establish air-liquid interface (ALI)
Phase 4 · Transplantation of 3D iSO into SCID Mice Day 0-14
  1. a Surgical preparation and skin defect creation
  2. b Preparation of iSO for transplantation
  3. c Placement of iSO into defect site
  4. d Fixation using tie-over dressing method
  5. e Post-transplant monitoring and sacrifice

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Attribution

This SOP was written by Organthis based on the published method in Kim et al., 2018. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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