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SKIN Publication-derived

Sweat Gland Organoids Originating from Reprogrammed Epidermal Keratinocytes

Source Sun et al., 2021 · Research Center for Tissue Repair and Regeneration affiliated to the Medical Innovation Research Department and 4th Medical Center PLA General Hospital and PLA Medical College · 10.1002/advs.202103079

👤 Xiaoyan Sun, Jiangbing Xiang, Runkai Chen, Zhijun Geng, Lintao Wang, Yiqiong Liu, Shuaifei Ji, Huating Chen, Yan Li, Cuiping Zhang, Peng Liu, Tao Yue, Lei Dong, Xiaobing Fu ⏱ 60 days 📋 7 phases 🧫 Human iPSC-derived (EDA-reprogrammed HEK)

Abstract

This protocol generates functional human sweat gland organoids (iSwGOs) from reprogrammed epidermal keratinocytes (HEKs) via a three-step strategy: reprogramming HEKs into sweat gland cells (iSwGCs) using EDA overexpression combined with β2-AR activation and sweat gland-specific medium; establishing iSwGO via 3D organoid culture; and transplanting iSwGOs to restore sweat gland function in damaged skin. The regenerated sweat glands exhibit structural, molecular, and functional characteristics of native human sweat glands.

Cell source
Human iPSC-derived (EDA-reprogrammed HEK)
Application
Disease modeling, Wound healing, Sweat gland regeneration

Protocol overview

38 steps across 7 phases

HEK Isolation and Primary Culture Day -1 to Day 0
  1. 1 Tissue Digestion with Dispase II
  2. 2 Epidermal Layer Separation
  3. 3 Trypsin Digestion
  4. 4 Cell Washing and Suspension
  5. 5 Cell Plating on Collagen IV
  6. 6 Standard HEK Culture Initiation
β2-AR Activation and EDA Transduction Day 0 to Day 8
  1. 1 β2-AR Agonist (ISO) Treatment
  2. 2 Lentiviral Transduction with EDA
  3. 3 FACS Sorting of GFP-Positive Cells
  4. 4 Recovery of Sorted Cells
Reprogramming of HEK-EDA into iSwGCs Day 2 to Day 20
  1. 1 Transfer to Sweat Gland Induction Medium (SGM)
  2. 2 Morphological Change Monitoring
  3. 3 Medium Change Schedule
  4. 4 Gene Expression Analysis at Day 8
  5. 5 Immunofluorescence Characterization at Day 20
  6. 6 FACS Analysis of iSwGCs
Functional Characterization of iSwGCs Day 8 to Day 20
  1. 1 Transmission Electron Microscopy (TEM) Analysis
  2. 2 Calcium Imaging Assay
  3. 3 RNA-Sequencing and Transcriptome Analysis
3D Organoid Culture (iSwGO Formation) Day 21 to Day 60
  1. 1 Single-Cell Suspension Preparation
  2. 2 Matrigel Embedding
  3. 3 Initial Organoid Culture (Days 1–14)
  4. 4 Organoid Expansion (Days 14–28)
  5. 5 Immunofluorescence of Organoids
  6. 6 Organoid Passage (Optional)
Microfluidic Culture for Tubular Structure Formation Day 28 to Day 46
  1. 1 Microfluidic Device Preparation
  2. 2 Gradient Channel Setup
  3. 3 Organoid Elongation (Days 1–8)
  4. 4 Tubular Structure Formation (Days 8–18)
  5. 5 Immunofluorescence of Tubular Structures
In Vivo Transplantation and SwG Regeneration Day 1 to Day 30 (post-injury)
  1. 1 Mouse Burn Injury Model
  2. 2 Organoid Preparation for Transplantation
  3. 3 Transplantation of iSwGOs
  4. 4 Starch–Iodine Sweat Testing
  5. 5 Wound Closure Assessment
  6. 6 Tissue Harvest and Histological Analysis
  7. 7 H&E Staining and Morphological Assessment
  8. 8 Immunofluorescence of Regenerated SwGs

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Sun et al., 2021. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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