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RETINA Publication-derived

Retinal Embryo Models for Disease Modelling

Full SOP with Academic or a plan

Source Mellough et al., 2019 · Institute of Genetic Medicine, Newcastle University, Newcastle, United Kingdom · 10.1002/sctm.18-0267

Authors: Carla B. Mellough, Joseph Collin, Rachel Queen, Gerrit Hilgen, Birthe Dorgau, Darin Zerti, Majed Felemban, Kathryn White, Evelyne Sernagor, Majlinda Lako Duration: 150 days 7 phases Cell source: Human ESC (H9, WiCell); Human iPSC (Neo1 neonatal, AD3 adult)

Abstract

This protocol compares three methods of embryoid body (EB) formation—mechanical, enzymatic, and dissociation–reaggregation—in combination with ROCK inhibition and static versus shaking culture conditions for generating laminated retinal organoids from human pluripotent stem cells. The most efficient approach combines mechanical EB generation with Y-27632 supplementation for the first 48 hours under static culture conditions, consistently producing laminated retinal tissue with mature, light-responsive photoreceptors.

Cell source
Human ESC (H9, WiCell); Human iPSC (Neo1 neonatal, AD3 adult)
Application
Disease modeling; Developmental study; Light-responsive retinal tissue generation

Protocol overview

53 steps across 7 phases

Phase 1 · Cell Culture and Feeder-Free Adaptation Maintenance phase (ongoing before differentiation)
  1. a Prepare culture plates with growth factor-reduced Matrigel
  2. b Culture pluripotent stem cells in feeder-free conditions
  3. c Passage cells using versene EDTA
Phase 2 · Embryoid Body Formation—Mechanical Method (M) Day 0-2
  1. a Grow cells to 90% confluence
  2. b Make cross hatches to fragment colonies
  3. c Collect and transfer colony fragments
  4. d Refresh media and seed into petri dishes
  5. e Culture EBs under static or shaking conditions (day 0-2)
  6. f Change media to retinal differentiation medium at 48 hours
Phase 3 · Embryoid Body Formation—Enzymatic Method (E) Day 0-2
  1. a Grow cells to 90% confluence
  2. b Wash cells with phosphate-buffered saline
  3. c Apply collagenase/dispase detachment solution
  4. d Collect detached colonies
  5. e Dilute detachment solution and settle colonies
  6. f Remove supernatant and wash colonies
  7. g Resuspend colonies and transfer to petri dish
  8. h Culture EBs under static or shaking conditions (day 0-2)
  9. i Change media to retinal differentiation medium at 48 hours
Phase 4 · Embryoid Body Formation—Dissociation–Reaggregation Method (V9) Day 0-12
  1. a Harvest cells at 90% confluence
  2. b Dissociate cells with Accumax
  3. c Add TeSR1 medium and collect cells
  4. d Spin down and count cells
  5. e Reaggregate cells in V-bottomed 96-well plates
  6. f Culture EBs under static or shaking conditions (day 0-2)
  7. g Change media to retinal differentiation medium at 48 hours
  8. h Change media every 3 days
  9. i Transfer EBs to larger culture vessels (day 12)
  10. j Continue culture under assigned conditions
Phase 5 · Long-Term Retinal Organoid Differentiation and Maturation Day 2-150 (weeks 1-21)
  1. a Culture in retinal differentiation medium with IGF-1 and B27
  2. b Add N2 supplement from day 37
  3. c Reduce serum supplementation to 0% by day 37
  4. d Perform media changes every 3 days
  5. e Monitor organoid morphology throughout differentiation
  6. f Maintain static or shaking culture conditions throughout
Phase 6 · Organoid Harvest and Analysis Days 35-150 (weeks 5-21) and beyond
  1. a Fix organoids for immunocytochemistry
  2. b Perform immunohistochemistry on cryostat sections
  3. c Image immunostained sections
  4. d Prepare tissue for transmission electron microscopy
  5. e Extract RNA for gene expression analysis
  6. f Synthesize cDNA and perform preamplification
  7. g Perform qPCR on Fluidigm BioMark system
Phase 7 · Electrophysiological Characterization (Weeks 20-24) Day 140 onwards (weeks 20-24)
  1. a Add 9-cis-retinal to culture medium
  2. b Transfer organoids to artificial cerebrospinal fluid
  3. c Open organoid and mount on multielectrode array
  4. d Allow organoid to settle on MEA
  5. e Record spontaneous activity in darkness
  6. f Apply white light pulse stimulation
  7. g Apply sustained blue light stimulation
  8. h Apply pharmacological agents—cGMP puff
  9. i Apply pharmacological agents—GABA puff
  10. j Extract spikes and perform spike sorting
  11. k Analyze firing rate responses
  12. l Classify ipRGCs and photoreceptor-driven RGCs

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Attribution

This SOP was written by Organthis based on the published method in Mellough et al., 2019. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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