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BRAIN Publication-derived

Three-Dimensional Retinal Tissue with Functional Photoreceptors

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

👤 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 ⏱ 28 days 📋 6 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Zhong et al.. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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