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RETINA Publication-derived

An alternative approach to produce versatile retinal organoids with accelerated ganglion cell development

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 📋 8 phases 🧫 Human ESC (H1, WiCell)

Abstract

This protocol describes a method for generating human retinal organoids with accelerated retinal ganglion cell (RGC) development within four weeks using a combination of 3D matrigel encasement (MG) and free-floating (FF) culture. The MG/FF approach produces organized embryoid bodies and retinal organoids with laminated structure containing multiple retinal cell types (RGCs, RPE, photoreceptors, horizontal cells, bipolar cells, amacrine cells) suitable for disease modeling and therapeutic studies.

Cell source
Human ESC (H1, WiCell)
Application
Disease modeling (glaucoma, ocular neuropathies); retinal organoid generation for therapeutic screening

Protocol overview

38 steps across 8 phases

Embryoid body formation in matrigel 0-4
  1. 1 Prepare stem cell clumps
  2. 2 Suspend stem cells in matrigel
  3. 3 Plate matrigel drops
  4. 4 Gel matrigel
  5. 5 Add gradual medium transition (day 0)
  6. 6 Medium change day 1
  7. 7 Medium change day 2
  8. 8 Medium change day 3
Embryoid body expansion in adherent culture 4-11
  1. 1 Recover embryoid bodies from matrigel
  2. 2 Plate embryoid bodies on matrigel-coated wells
  3. 3 Maintain with NIM medium
Neural center isolation and retinal organoid initiation 11-14
  1. 1 Dislodge and isolate neural centres
  2. 2 Transfer to retinal differentiation medium
  3. 3 Culture as free-floating organoids
  4. 4 Observe organoid formation
Retinal organoid maintenance and accelerated RGC development 14-37
  1. 1 Continue organoid culture in RDM
  2. 2 Monitor for ganglion cell markers
  3. 3 Continue culture until day 37 or until desired cell type is achieved
Long-term organoid lamination with external factors (optional) 37-160
  1. 1 Initiate treatment at day 37 with FBS and Taurine
  2. 2 Add Retinoic Acid at day 44
  3. 3 Add T3 and halve Retinoic Acid at day 84
  4. 4 Maintain FBS, Taurine, RA, and T3 until day 120
  5. 5 Remove Retinoic Acid at day 120
Dissociation and plating for retinal ganglion cells 23-28
  1. 1 Prepare organoids for dissociation
  2. 2 Dissociate organoids to single cells
  3. 3 Prepare coated coverslips
  4. 4 Plate dissociated cells
  5. 5 Maintain plated RGC cultures
Dissociation and plating for retinal pigmented epithelium 37
  1. 1 Select pigmented organoid sections
  2. 2 Dissociate organoids to single cells
  3. 3 Prepare growth factor-reduced matrigel-coated inserts
  4. 4 Seed cells on inserts
  5. 5 Culture RPE in RDM with Activin A
  6. 6 Passage RPE cells (optional)
Dissociation and plating for photoreceptors 160
  1. 1 Prepare mature organoids for dissociation
  2. 2 Dissociate organoids to single cells
  3. 3 Plate on prepared coverslips
  4. 4 Maintain cultures

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.

This wording is awaiting legal review.

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