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RETINA Publication-derived

Retinal Organoids for Drug Screening and Development

Full SOP with Academic or a plan

Source Fligor et al., 2020 · Indiana University–Purdue University Indianapolis / Indiana University School of Medicine · 10.1016/bs.mcb.2020.02.005

Authors: Clarisse M. Fligor, Kang-Chieh Huang, Sailee S. Lavekar, Kirstin B. VanderWall, Jason S. Meyer Duration: 200 days 3 phases Cell source: Human hPSC (hESC and hiPSC)

Abstract

A comprehensive protocol for the differentiation of three-dimensional retinal organoids from human pluripotent stem cells (hPSCs), which recapitulate human retinogenesis in vitro. The protocol guides hPSCs through progressive developmental stages—from neural induction through formation of optic vesicle-like structures to fully laminated retinal organoids containing retinal ganglion cells, photoreceptors, and other retinal cell types.

Cell source
Human hPSC (hESC and hiPSC)
Application
Developmental study; Disease modeling; Drug screening; Cell replacement therapies

Protocol overview

25 steps across 3 phases

Phase 1 · Maintenance and expansion of human pluripotent stem cells Ongoing (4-6 days per passage)
  1. a Coat culture plates with Matrigel
  2. b Aspirate excess Matrigel and add culture medium
  3. c Thaw frozen hPSC vial (if starting with frozen cells)
  4. d Centrifuge and resuspend cell pellet
  5. e Distribute cells evenly in well
  6. f Monitor colony growth for passaging readiness
  7. g Treat colonies with Dispase to initiate enzymatic passaging
  8. h Wash colonies and dislodge with mechanical pipetting
  9. i Transfer and settle cell aggregates
  10. j Resuspend and distribute aggregates to new wells
  11. k Distribute cells in new wells and maintain until next passage
Phase 2 · Induction to a primitive retinal fate (Formation of hPSC aggregates and neural induction) Days 0-16
  1. a Generate hPSC aggregates by enzymatic lifting and transition to neural medium
  2. b Gradually transition aggregates to neural induction medium (Days 0-3)
  3. c Maintain aggregates in neural induction medium and add BMP4 (Days 3-6)
  4. d Perform half-medium changes to dilute BMP4 (Days 6-12)
  5. e Plate hPSC aggregates onto 6-well dishes with FBS (Day 8)
  6. f Gradually wean BMP4 and transition to pure NIM (Days 9-15)
Phase 3 · Generation and long-term maintenance of retinal organoids (3D culture and maturation) Days 16-200+
  1. a Lift primitive retinal aggregates and transition to low-adherence 3D culture (Day 16)
  2. b Maintain organoids with regular media changes (Days 16-20)
  3. c Supplement RDM with fetal bovine serum in a stepwise fashion (Days 20-26)
  4. d Add Taurine to support photoreceptor maturation (Day 35)
  5. e Add retinoic acid to promote photoreceptor differentiation (Day 60)
  6. f Switch to retinal maturation medium (RMM) at Day 90
  7. g Maintain regular feeding schedule throughout long-term culture
  8. h Monitor organoid development and morphology through long-term culture

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Attribution

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

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