Skip to content
Back to browse
RETINA Publication-derived

Glaucoma and Ocular Neuropathies Retinal Organoids

Full SOP with Academic or a plan

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

Authors: Ellie L. Wagstaff, Anneloor L. M. A. ten Asbroek, Jacoline B. ten Brink, Nomdo M. Jansonius, Arthur A. B. Bergen Duration: 160 days 5 phases Cell source: 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

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

Full SOP

Full SOPs need an account and a plan

Create an account, then get free Academic access (for verified academics) or choose a plan. Plans include step-by-step procedures, reagent concentrations, QC checklists and batch record templates.

Create an account

Already registered? Log in

Attribution

This SOP was written by Organthis based on the published method in Wagstaff et al., 2021. Once the originating laboratory has confirmed it, the SOP is marked Verified.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence