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RETINA Publication-derived

Generation of Retinal Organoids from Human Pluripotent Stem Cells

Source Galindo-Cabello et al., 2025 · University of Valladolid, Institute of Applied Ophthalmobiology (IOBA) · 10.3390/ijms26073263

👤 Nadia Galindo-Cabello, Estefanía Caballano-Infantes, Gregorio Benites, Salvador Pastor-Idoate, Francisco J. Diaz-Corrales, Ricardo Usategui-Martín ⏱ 150 days 📋 6 phases 🧫 Human iPSC, Human ESC

Abstract

This protocol describes the generation of three-dimensional retinal organoids from human pluripotent stem cells (iPSCs and ESCs) that recapitulate the structure and function of the native retina. The organoids develop multiple retinal cell types including photoreceptors, ganglion cells, amacrine cells, bipolar cells, horizontal cells, and Müller cells, enabling disease modeling for inherited retinal diseases and screening of therapeutic interventions.

Cell source
Human iPSC, Human ESC
Application
Disease modeling of retinal degeneration

Protocol overview

26 steps across 6 phases

Neuroectoderm Induction and ESC/iPSC Aggregation Days 0–6
  1. 1 Fragment ESCs or iPSCs into single cells
  2. 2 Reaggregate cells in minimal adhesion 96-well plates
  3. 3 Culture aggregates for 6 days in basal medium
  4. 4 Add BMP4 on day 6
Retinal Specification and Neural Retina Formation Days 6–18
  1. 1 Continue culture with BMP4 and other retinal specification factors
  2. 2 Monitor optic vesicle-like structure formation
  3. 3 Isolate and collect neuroretinal-like structures on day 18
RPE Induction and Retinal Maturation Culture Days 18–30+
  1. 1 Transfer isolated NR structures to RPE-induction medium
  2. 2 Transfer to retinal maturation medium
  3. 3 Culture in long-term suspension or orbital rotation
  4. 4 Monitor retinal ganglion cell (RGC) emergence (around day 30) and photoreceptor maturation (around day 130)
Alternative Protocol: 2D-3D Hybrid Culture (Cowan et al. variant with modifications) Days 0–28+
  1. 1 Culture iPSCs at 70% confluence in essential medium without FGF2 and TGFβ
  2. 2 Add N2 supplement on day 2
  3. 3 Culture for 4 weeks in 2D to allow neural retina-like structure formation
  4. 4 Select and transfer NR-like structures to suspension culture
  5. 5 Continue long-term culture in 3D suspension
Alternative Protocol: Embryoid Body (EB) Method Days 0–28+
  1. 1 Fragment ESCs into tiny clumps
  2. 2 Grow clumps in mTeSR1 with Blebbistatin to form embryoid bodies
  3. 3 Transfer EBs to neural induction medium on day 1
  4. 4 Place EBs on matrigel-coated plates on day 7
  5. 5 Select, dissect, and transfer NR-like structures to long-term orbital culture
  6. 6 Culture in long-term orbital rotation
Advanced Maturation and Photoreceptor Development (Optional Supplementation) Days 50–150+
  1. 1 Add Notch inhibitor (DAPT) if desired to enhance photoreceptor stratification
  2. 2 Optionally supplement with Taurine and 9-cis-Retinal
  3. 3 Consider FGF pathway inhibition (SU5402) if enhanced photoreceptor development is desired
  4. 4 Monitor maturation with stage-specific markers (Day 100–150)

Full SOP

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Attribution

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

This wording is awaiting legal review.

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