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RETINA Publication-derived

Intercellular Adhesion-Dependent Cell Survival and ROCK-Regulated Actomyosin-Driven Forces Mediate Self-Formation of a Retinal Organoid

Source Lowe et al., 2016 · Albert Einstein College of Medicine, Department of Genetics and Department of Ophthalmology and Visual Sciences, Bronx, NY 10461, USA · 10.1016/j.stemcr.2016.03.011

👤 Albert Lowe, Raven Harris, Punita Bhansali, Ales Cvekl, Wei Liu ⏱ 187 days 📋 5 phases 🧫 Human ESC (H1 hESC, hiPSC)

Abstract

This protocol establishes an efficient method for generating retinal organoids from human embryonic stem cells via Matrigel-aided cyst formation, spontaneous attachment and spreading, and Dispase-mediated detachment followed by floating culture. The self-organization of retinal organoids requires intercellular adhesion-dependent cell survival and ROCK-regulated actomyosin-driven forces, and produces stratified retinal tissue comprising neuroretina, ciliary margin, and retinal pigment epithelium with mature photoreceptors displaying outer segments.

Cell source
Human ESC (H1 hESC, hiPSC)
Application
Retinal organoid development and disease modeling

Protocol overview

17 steps across 5 phases

Cyst Formation via Matrigel-Aided Floating Culture Day 0–5
  1. 1 Harvest and prepare hESC sheets
  2. 2 Suspend cell pellet in ice-cold Matrigel
  3. 3 Gently disperse Matrigel/hESC clumps
  4. 4 Plate dispersed clumps into 24-well plate
  5. 5 Transfer cysts to new plates for expansion
Spontaneous Attachment, Spreading, and Formation of Patterned Monolayer Sheets Day 5–17
  1. 1 Culture cysts in adherent plates with medium changes
  2. 2 Verify patterning by microscopy
Dispase-Mediated Cell Sheet Detachment and Characterization Day 12–17 (harvest day)
  1. 1 Prepare medium and reagents for detachment
  2. 2 Add Dispase and optional inhibitors to adherent cultures
  3. 3 Retrieve detached cell sheets and rinse
  4. 4 Perform immunostaining of detached sheets (optional)
Floating Culture and Retinal Organoid Self-Formation Day 13 to Day 26+
  1. 1 Initiate floating culture of detached cell sheets
  2. 2 Monitor organoid formation and morphology
  3. 3 Transition to long-term culture medium
  4. 4 Immunostaining of organoids at intermediate timepoints
Extended Culture and Photoreceptor Maturation (Optional) Day 26 to Day 187+
  1. 1 Maintain long-term floating culture
  2. 2 Electron microscopy analysis of outer segments (optional)

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Lowe et al., 2016. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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