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RETINA Publication-derived

Retinal Organoids for Disease and Development (2020)

Full SOP with Academic or a plan

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

Authors: Florian Regent, Holly Y. Chen, Ryan A. Kelley, Zepeng Qu, Anand Swaroop, Tiansen Li Duration: 200 days 4 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Regent et al., 2020. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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