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RETINA Publication-derived

A simple and efficient method for generating human retinal organoids

Source Regent et al., 2020 · Neurobiology, Neurodegeneration & Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, MD

👤 Florian Regent, Holly Y. Chen, Ryan A. Kelley, Zepeng Qu, Anand Swaroop, Tiansen Li ⏱ 200 days 📋 4 phases 🧫 Human iPSC, Human ESC

Abstract

This protocol describes an efficient method for generating human retinal organoids (ROs) from pluripotent stem cells by scraping adherent cultures and culturing in free-floating conditions, avoiding labor-intensive dissection. The approach yields 2.5–4.6-fold higher RO production with enhanced pigmented epithelium cogeneration, facilitating large-scale production for disease modeling and drug screening.

Cell source
Human iPSC, Human ESC
Application
Retinal organoid differentiation, disease modeling, developmental study

Protocol overview

29 steps across 4 phases

Generation and maintenance of hPSCs Pre-differentiation
  1. 1 hPSC line selection and characterization
  2. 2 Plate hPSCs on Matrigel-coated surfaces
  3. 3 Culture hPSCs in maintenance medium
  4. 4 Passage hPSCs using EDTA
Embryoid body (EB) formation and optic vesicle induction in adherent culture D0–D16
  1. 1 Initiate differentiation with EDTA dissociation
  2. 2 Transfer to polyHEMA-coated plates for EB formation
  3. 3 Apply neural induction medium (NIM) at D1–D2
  4. 4 Switch to 100% NIM at D3
  5. 5 Plate EBs onto BD GFR Matrigel at D7
  6. 6 Transition to retinal induction medium (RIM) at D16
  7. 7 Monitor and harvest optic vesicles D20–D30
Generation and maintenance of retinal organoids (ROs) via scraping method D20–D200
  1. 1 Scrape adherent culture into clumps (D20–D30)
  2. 2 Transfer to polyHEMA-coated dishes for free-floating culture
  3. 3 Perform full media change the next day
  4. 4 Harvest and separate optic vesicles (D21 onward)
  5. 5 Maintain ROs in 3:1 RIM + IGF-1 (D21–D34)
  6. 6 Supplement with FBS and Taurine starting D35
  7. 7 Add 9-cis retinal starting D63
  8. 8 Reduce 9-cis retinal concentration at D91
  9. 9 Switch from B27 to N2 supplement at D91
  10. 10 Continue maintenance through D200
Immunofluorescence analysis Harvest at desired timepoint (D60, D200, etc.)
  1. 1 Collect and fix organoids
  2. 2 Cryoprotect with sucrose gradient
  3. 3 Embed in M1 matrix and section
  4. 4 Dry and store sections
  5. 5 Block nonspecific binding
  6. 6 Incubate with primary antibodies
  7. 7 Wash and apply secondary antibodies
  8. 8 Mount and image

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Regent et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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