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SKIN Publication-derived

Skin Organoids for Regenerative Medicine

Full SOP with Academic or a plan

Source Lee et al. · Indiana University School of Medicine / Boston Children's Hospital, Boston, USA · 10.1038/s41586-020-2352-3

Authors: Lee J, Rabbani CC, Gao H, Steinhart MR, Woodruff BM, Pflum ZE, Kim A, Heller S, Liu Y, Shipchandler TZ, Koehler KR Duration: 150 days 7 phases Cell source: Human iPSC / Human ESC

Abstract

Landmark protocol for generating complex hair-bearing skin entirely from human pluripotent stem cells using stepwise TGFβ and FGF modulation. Over 4-5 months, organoids develop stratified epidermis, pigmented hair follicles, sebaceous glands, and sensory neurons recapitulating fetal skin architecture.

Cell source
Human iPSC / Human ESC
Application
Regenerative Medicine & Skin Grafting

Protocol overview

22 steps across 7 phases

Phase 1 · hPSC Culture and Maintenance Day -30 to Day 0
  1. a Coat culture plates with Vitronectin
  2. b Culture hPSCs in Essential 8 Flex medium
  3. c Passage hPSCs at ~80% confluency
Phase 2 · Cell Aggregate Formation and Pre-Differentiation Day -2 to Day 0
  1. a Dissociate hPSCs to single cells
  2. b Prepare high Y-concentration cell suspension
  3. c Seed cells into U-bottom 96-well plates
  4. d Incubate aggregates at 37°C
  5. e Add fresh medium to dilute Y (day –1)
Phase 3 · Initial Differentiation and Epithelial Induction (Day 0-3) Day 0 to Day 3
  1. a Transfer aggregates to differentiation medium (day 0)
  2. b Induce CNCC formation (day 3)
Phase 4 · Expansion and Maturation in Suspension (Day 6-12) Day 6 to Day 12
  1. a Add fresh medium (day 6)
  2. b Perform half-medium changes (days 8 and 10)
  3. c Transfer aggregates to 24-well plates with organoid maturation medium (day 12)
  4. d Place plates on orbital shaker
Phase 5 · Organoid Maturation and Differentiation (Day 15-45) Day 15 to Day 45
  1. a Perform half-medium change (day 15)
  2. b Perform half-medium changes every 3 days (days 18-45)
Phase 6 · Late Maturation and Hair Follicle Development (Day 45-70) Day 45 to Day 70
  1. a Perform half-medium changes every other day (days 45-150)
  2. b Perform full-medium changes once weekly (starting day 45)
  3. c Monitor for hair follicle emergence (days 70 ± 5)
Phase 7 · Continued Maturation and Hair Follicle Growth (Day 70-150) Day 70 to Day 150
  1. a Increase medium volume if necessary (day 80 onwards)
  2. b Maintain half-medium changes every other day and full-medium changes once weekly
  3. c Harvest organoids for analysis or grafting (day 100-150)

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Attribution

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

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