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SKIN Publication-derived

Skin Organoids

Full SOP with Academic or a plan

Source Lee et al. · Boston Children's Hospital / Harvard Medical School, Boston, USA · 10.1038/s41596-022-00681-y

Authors: Lee J, van der Valk WH, Serdy SA, Deakin C, Kim J, Le AP, Koehler KR Duration: 150 days 5 phases Cell source: Human iPSC / Human ESC

Abstract

Definitive detailed protocol for generating hair-bearing skin organoids from human PSCs over 150 days. Includes comprehensive reagent tables, cell line optimization, troubleshooting, and the novel qCe3D clearing technique for whole-mount immunostaining and 3D imaging.

Cell source
Human iPSC / Human ESC
Application
Skin Biology Research

Protocol overview

21 steps across 5 phases

Phase 1 · hPSC Culture and Preparation Pre-differentiation
  1. a Thaw frozen hPSCs
  2. b Maintain hPSC culture
  3. c Passage hPSCs
  4. d Freeze hPSCs for long-term storage
Phase 2 · Cell Aggregation (Days −2 to −1) Days -2 to -1
  1. a Prepare single-cell suspension
  2. b Form cell aggregates in 96-well U-bottom plates
  3. c Dilute Y-27632 on day −1
Phase 3 · Early Differentiation (Days 0-12) Days 0-12
  1. a Transition to E6SFB on day 0
  2. b Apply LDN and bFGF on day 3
  3. c Provide nutrition on day 6
  4. d Half medium change on day 9
  5. e Transition to floating culture on day 12
Phase 4 · Organoid Maturation (Days 15-150) Days 15-150
  1. a Half medium change on day 15
  2. b Dilute Matrigel on day 18
  3. c Perform weekly full and interval half medium changes (days 18-150)
Phase 5 · Downstream Assays and Analysis Days 6-150
  1. a Dissociate organoids for single-cell RNA-seq
  2. b Fix organoids for cryo-embedding
  3. c Cryo-embed organoids in OCT
  4. d Cryo-section organoids at 12 μm
  5. e Immunostain organoid sections
  6. f Whole-mount immunostaining with tissue clearing (qCe3D)

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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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