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SKIN Publication-derived

Shaped Microfluidic Device with ECM and Morphogen Gradients

Source Quílez et al. · Columbia University Irving Medical Center, New York, USA · 10.1002/adhm.202400405

👤 Quílez C, Jeon EY, Pappalardo A, Pathak P, Abaci HE ⏱ 32 days 📋 9 phases 🧫 Human iPSC

Abstract

Novel approach generating skin organoids in a 3D bioprinted spindle-shaped hydrogel device with defined ECM (Collagen I + Laminin 511). Overcomes the inverted anatomy limitation of conventional skin organoids and achieves 2-fold keratinocyte and 8-fold hair follicle increase compared to free-floating conditions.

Cell source
Human iPSC
Application
Organ-on-Chip Skin Models

Protocol overview

31 steps across 9 phases

mESC Maintenance Ongoing maintenance prior to day 0
  1. 1 Maintain mESCs in feeder-free conditions
Initial Embryoid Body (EB) Formation Day 0–Day 3
  1. 1 Detach and resuspend mESC colonies
  2. 2 Seed cells in ultra-low attachment plate for EB formation
  3. 3 Culture EBs in incubator
  4. 4 Medium supplementation on Day 1
ECM Hydrogel Encapsulation (Day 3) Day 3
  1. 1 Prepare collagen I hydrogel solution
  2. 2 Embed EBs in hydrogel
  3. 3 Crosslink hydrogel
  4. 4 Add differentiation medium with morphogens
Early Differentiation (Day 4 onwards) Day 4–Day 8
  1. 1 Add neural crest differentiation factors on Day 4
  2. 2 Transfer to maturation culture on Day 8
Maturation Phase (Day 8–Day 32) Day 8–Day 32
  1. 1 Maintain maturation culture with regular medium changes
  2. 2 Monitor organoid growth and morphology
3D Bioprinting of Spindle Device (Optional—for enhanced growth) Device fabrication prior to Day 3 seeding
  1. 1 Design and 3D bioprint spindle device
  2. 2 Clean and sterilize printed devices
  3. 3 Prepare devices for cell seeding
PSO Generation in Spindle Device (Alternative to standard plate culture) Day 3–Day 32
  1. 1 Seed EBs in spindle device on Day 3
  2. 2 Culture spindle devices with initial differentiation medium
  3. 3 Add neural crest factors on Day 4
  4. 4 Transition to maturation medium on Day 8
  5. 5 Continue maturation with regular medium changes
Microfluidic Device Fabrication and Setup (Optional—for gradient control) Device fabrication prior to Day 2 seeding
  1. 1 3D bioprint microfluidic device with gradient channels
  2. 2 3D print perfusion chamber in PLA
  3. 3 Assemble microfluidic and perfusion system
  4. 4 Prepare devices for cell seeding
PSO Generation in Microfluidic Device with Morphogen Gradients Day 2–Day 32
  1. 1 Seed EBs in microfluidic device on Day 2
  2. 2 Culture EBs with E6 medium in lateral chambers
  3. 3 Establish epidermal differentiation gradient on Day 4
  4. 4 Establish dermal differentiation gradient on Day 5
  5. 5 Transition to maturation medium on Day 10
  6. 6 Maintain maturation culture through Day 32

Full SOP

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Attribution

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

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