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RETINA Publication-derived

Retinal Organoids for Drug Screening and Disease Modelling

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 8 phases Cell source: 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

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

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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.

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