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BRAIN Publication-derived

Retinal Organoids for Disease Modelling

Full SOP with Academic or a plan

Source Zhong et al. · Johns Hopkins University, Baltimore, MD, USA · 10.1038/ncomms5047

Authors: Zhong X, Gutierrez C, Xue T, Hampton C, Vergara MN, Cao LH, Peters A, Park TS, Zambidis ET, Meyer JS, Gamm DM, Yau KW, Canto-Soler MV Duration: 28 days 6 phases Cell source: Human iPSC

Abstract

Zhong et al. generated three-dimensional retinal tissue from human iPSCs that developed all major retinal cell types in the correct laminar organization, including photoreceptors with outer segment-like structures capable of light responsiveness. This 200-day culture protocol from Canto-Soler lab achieved functional maturation comparable to native retinal tissue.

Cell source
Human iPSC
Application
Retinal Disease Modeling

Protocol overview

24 steps across 6 phases

Phase 1 · hiPSC Maintenance and Aggregate Formation Day 0 - Day 7
  1. a Prepare hiPSC cultures
  2. b Initiate aggregate formation (Day 0)
  3. c Transition to neural induction medium (Days 1-3)
  4. d Aggregate seeding on Matrigel (Day 7)
Phase 2 · Eye Field Formation and Neural Retina/RPE Differentiation Day 8 - Day 28
  1. a Monitor eye field (EF) domain formation (Days 8-12)
  2. b Identify and track neural retina (NR) and RPE domain segregation (Days 12-25)
  3. c Assess NR domain formation efficiency (Day 20)
Phase 3 · 3D Retinal Cup Formation and Suspension Culture Day 21 - Week 5
  1. a Manually detach and collect NR domains (Days 21-28)
  2. b Culture NR domains in suspension (Day 21 onward)
  3. c Initiate long-term supplementation (Day 42 onward)
  4. d Monitor retinal cup growth and morphology (Days 45-90)
Phase 4 · Spontaneous Retinal Differentiation and Cell Layer Formation Week 5 - Week 22
  1. a Support early retinal differentiation and layer formation (Weeks 5-7)
  2. b Maintain culture conditions through Week 17 (Weeks 7-17)
  3. c Transition to photoreceptor maturation medium (Week 17 onward)
  4. d Monitor layer organization and cell maturation (Weeks 12-22)
Phase 5 · Optimized Photoreceptor Maturation with Retinoic Acid Titration Week 7 - Week 25+
  1. a Test retinoic acid treatment windows for photoreceptor maturation (Weeks 7-17 vs. 10-14)
  2. b Administer optimized retinoic acid treatment (Weeks 10-14)
  3. c Confirm rod and cone opsin expression (Weeks 17-21)
  4. d Observe advanced photoreceptor morphology and outer segment precursors (Weeks 21-25)
Phase 6 · Ultrastructural Analysis and Functional Assessment of Photoreceptors Week 25 - Week 28
  1. a Prepare samples for transmission electron microscopy (TEM) (Weeks 27-28)
  2. b Identify ultrastructural features of developing photoreceptors
  3. c Assess phototransduction protein expression (Weeks 25-28)
  4. d Perform perforated-patch electrophysiology to assess light response (Weeks 25-27)
  5. e Analyze light response data

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Attribution

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

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