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RETINA Publication-derived

Accelerated and Improved Differentiation of Retinal Organoids from Pluripotent Stem Cells in Rotating-Wall Vessel Bioreactors

Source DiStefano et al., 2018 · National Eye Institute (NEI), National Institutes of Health · 10.1016/j.stemcr.2017.11.001

👤 Tyler DiStefano, Holly Yu Chen, Christopher Panebianco, Koray Dogan Kaya, Matthew J. Brooks, Linn Gieser, Nicole Y. Morgan, Tom Pohida, Anand Swaroop ⏱ 32 days 📋 5 phases 🧫 Mouse ESC (Nrl-GFP)

Abstract

This protocol describes a bioprocess for differentiating mouse pluripotent stem cells into 3D retinal organoids using rotating-wall vessel (RWV) bioreactors. The RWV culture environment promotes enhanced proliferation, improved morphology, and accelerated differentiation of retinal neurons including ganglion cells and photoreceptors. Organoids cultured in RWV at day 25 achieve similar maturation profiles as static culture controls at day 32, closely recapitulating spatiotemporal development of postnatal retina in vivo.

Cell source
Mouse ESC (Nrl-GFP)
Application
Disease modeling and developmental study of retinal organoids

Protocol overview

16 steps across 5 phases

ESC Maintenance and Preparation Pre-differentiation
  1. 1 Culture ESCs on feeders with LIF
  2. 2 Passage ESCs every three days
Retinal Organoid Initiation (Days 0–7) D0–D7
  1. 1 Plate dissociated ESCs in low-adhesion plates
  2. 2 Add Matrigel on Day 1
  3. 3 Transfer organoids to culture dish on Day 7
Optic Cup Dissection and Culture Initiation (Days 10–17) D10–D17
  1. 1 Dissect optic cups on Day 10
  2. 2 Establish dissected neural retina cultures (D10–D17)
Retinal Maturation and Cell Type Development (Days 18–32) D18–D32
  1. 1 Supplement culture medium from Day 18
  2. 2 Maintain intact organoids (SSCi) from Day 26
Immunohistochemistry Analysis At harvest timepoints
  1. 1 Fix organoids in paraformaldehyde
  2. 2 Cryo-protect in sucrose gradients
  3. 3 Embed and section organoids
  4. 4 Hydrate and block sections
  5. 5 Incubate with primary antibodies
  6. 6 Wash and apply secondary antibodies
  7. 7 Counterstain nuclei with DAPI and image

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in DiStefano et al., 2018. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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