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RETINA Publication-derived

Differentiation of retinal organoids from human pluripotent stem cells

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

👤 Clarisse M. Fligor, Kang-Chieh Huang, Sailee S. Lavekar, Kirstin B. VanderWall, Jason S. Meyer ⏱ 200 days 📋 3 phases 🧫 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

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

Full SOP

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Attribution

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

This wording is awaiting legal review.

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