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RETINA Publication-derived

Retinal Organoids from Induced Pluripotent Stem Cells: Systematic Review of Methods

Source Lee et al., 2025 · Seoul National University College of Medicine · 10.1007/s12015-024-10802-7

👤 Yoo Jin Lee, Dong Hyun Jo ⏱ 30 days 📋 6 phases 🧫 Patient-Derived iPSC (Inherited Retinal Diseases)

Abstract

This systematic review consolidates diverse protocols for generating retinal organoids (ROs) from patient-derived induced pluripotent stem cells (iPSCs) with inherited retinal diseases (IRDs). ROs are three-dimensional structures that recapitulate retinal architecture and function, enabling disease phenotype reproduction and serving as platforms for therapeutic development including genome editing, gene augmentation, and cell replacement therapy.

Cell source
Patient-Derived iPSC (Inherited Retinal Diseases)
Application
Disease modeling, Drug screening, Developmental study

Protocol overview

29 steps across 6 phases

SFEBq Embryoid Body Formation (Nakano et al. protocol) Days 0-18
  1. 1 Prepare aggregates in V-bottomed 96-well plates
  2. 2 Sequential addition of signaling cues
  3. 3 Apply Wnt agonist for optic vesicle formation
  4. 4 Optic cup invagination and stratification
Kuwahara Induction-Reversal Protocol for Enhanced NR Production Days 0-30+
  1. 1 SFEBq aggregate preparation and BMP4 application
  2. 2 Induction-reversal phase
  3. 3 Reverse with NR induction medium
2D/3D Hybrid Protocol (Zhong et al.) Days 0-week 12+
  1. 1 2D neural induction
  2. 2 Transition to modified retinal differentiation medium
  3. 3 Manual dissection of optic cups
  4. 4 3D suspension culture of dissected organoids
  5. 5 Medium supplementation with FBS and taurine
  6. 6 RA reduction and B27/N2 switching for photoreceptor maturation
2D/3D Hybrid Protocol (Gonzalez-Cordero et al.) Days 0-week 12+
  1. 1 Early differentiation with E6 medium
  2. 2 Neural induction up to week 7
  3. 3 Manual excision and retinal differentiation medium transition
  4. 4 FBS, taurine, and GlutaMAX supplementation
  5. 5 Retinoic acid introduction and N2 addition
2D/3D Hybrid Protocol (Reichman et al.) Days 0-week 12+
  1. 1 E6 medium initiation with N2 supplement
  2. 2 Manual excision and transition to proB27 medium
  3. 3 FGF2 supplementation
  4. 4 FGF2 withdrawal and photoreceptor maturation
  5. 5 Long-term maturation with connecting cilia and outer segment formation
Protocol Refinements and Optimization Strategies Throughout culture (variable)
  1. 1 IGF-1 supplementation for lamination
  2. 2 Photoreceptor acceleration with 9-cis retinal
  3. 3 Notch inhibition for photoreceptor fate specification
  4. 4 Cone-rich RO generation without RA
  5. 5 Ganglion cell acceleration with Matrigel encapsulation
  6. 6 Co-culture with RPE, microglia, or endothelial cells

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Lee et al., 2025. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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