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RETINA Publication-derived

Generation of Versatile Retinal Organoids with Accelerated Ganglion Cell Development (MG/FF Protocol)

Source Wagstaff et al., 2021 · Department of Clinical Genetics, Amsterdam UMC, University of Amsterdam · 10.1038/s41598-020-79651-x

👤 Ellie L. Wagstaff, Anneloor L. M. A. ten Asbroek, Jacoline B. ten Brink, Nomdo M. Jansonius, Arthur A. B. Bergen ⏱ 160 days 📋 5 phases 🧫 Human ESC (H1, WiCell)

Abstract

This protocol combines 3D matrigel (MG) encasement of embryoid bodies with free-floating (FF) culture to generate human retinal organoids with accelerated ganglion cell development within 4 weeks, without external growth factors or small molecules. The organoids develop all major retinal cell types including retinal pigmented epithelium (RPE), horizontal cells, amacrine cells, photoreceptors, and retinal ganglion cells (RGCs), with long-term cultures achieving full lamination and photoreceptor maturation when supplemented with FBS, taurine, retinoic acid, and T3 from day 37 onwards.

Cell source
Human ESC (H1, WiCell)
Application
Disease modeling (glaucoma and ocular neuropathies); retinal tissue engineering and cell replacement therapy

Protocol overview

21 steps across 5 phases

Embryoid Body Formation in Matrigel (MG Phase) 0-4
  1. 1 Dissociate hESCs into small clumps
  2. 2 Suspend clumps in undiluted matrigel
  3. 3 Allow matrigel to gel
  4. 4 Add initial differentiation medium (1:1 mTeSR1:NIM)
  5. 5 Progressive medium transition to full NIM (Day 1-3)
  6. 6 Harvest embryoid bodies from matrigel
Adherent Culture and Neural Enrichment 4-11
  1. 1 Plate embryoid bodies on matrigel-coated adherent surface
  2. 2 Change NIM medium every other day
  3. 3 Mechanically isolate neural centers on day 11
Free-Floating Organoid Development (FF Phase) 11-37 (baseline) or up to 160+ (long-term culture)
  1. 1 Culture isolated neural centers in retinal differentiation medium
  2. 2 Confirm early retinal organoid formation by day 14
  3. 3 Change RDM medium every other day (days 11-36)
  4. 4 Dissociate organoids for retinal ganglion cell isolation (day 23-28 optional)
Long-Term Lamination and Photoreceptor Maturation (Optional) 37-160+
  1. 1 Begin supplementation with FBS and Taurine on day 37
  2. 2 Add Retinoic Acid on day 44
  3. 3 Halve Retinoic Acid concentration and add T3 on day 84
  4. 4 Remove Retinoic Acid on day 120
  5. 5 Harvest organoids for downstream analyses
Retinal Pigmented Epithelium (RPE) Differentiation (Optional) 37-70+ (parallel to organoid development)
  1. 1 Dissociate organoids at day 37 to isolate RPE
  2. 2 Culture RPE on inserts in RDM with Activin-A
  3. 3 Passage RPE monolayers if longer culture is desired

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Wagstaff et al., 2021. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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