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SKIN Publication-derived

Establishment of a complex skin structure via layered co-culture of keratinocytes and fibroblasts derived from induced pluripotent stem cells

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

👤 Yena Kim, Narae Park, Yeri Alice Rim, Yoojun Nam, Hyerin Jung, Kijun Lee, Ji Hyeon Ju ⏱ 30 days 📋 4 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Kim et al., 2018. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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