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RETINA Publication-derived

Retinal Organoids for Regeneration and Transplantation

Full SOP with Academic or a plan

Source Lopez et al., 2022 · Department of Ophthalmology & Vision Science, School of Medicine, U.C. Davis · 10.1111/cpr.13198

Authors: Antonio Jacobo Lopez, Sangbae Kim, Xinye Qian, Jeffrey Rogers, J. Timothy Stout, Sara M. Thomasy, Anna La Torre, Rui Chen, Ala Moshiri Duration: 100 days 4 phases Cell source: Rhesus macaque iPSC (Macaca mulatta)

Abstract

This protocol generates three-dimensional retinal organoids from rhesus macaque induced pluripotent stem cells (rhiPSCs) through a stepwise neural induction and retinal differentiation process. The resulting organoids develop laminated retinal tissue containing photoreceptors, Müller glia, bipolar cells, and other retinal cell types, with differentiation occurring approximately 30-40% faster than human iPSC-derived retinal organoids due to the shorter gestational period of rhesus macaques.

Cell source
Rhesus macaque iPSC (Macaca mulatta)
Application
Disease modeling and developmental biology; preclinical retinal allograft transplantation studies

Protocol overview

22 steps across 4 phases

Phase 1 · rhiPSC2431 Derivation and Stem Cell Culture Day 0-5+
  1. a Generate rhesus macaque fibroblasts from skin punches
  2. b Transfect fibroblasts with reprogramming plasmids using Neon system
  3. c Culture transfected cells for 4-5 days
  4. d Maintain rhiPSC2431 colonies in defined xeno-free medium
  5. e Maintain rhiPSC89 and rhiPSC90 on MEF feeders
  6. f Maintain human H9 ESCs for comparison
Phase 2 · Embryoid Body Formation and Neural Induction Day 0-7
  1. a Prepare iPSC colonies for differentiation
  2. b Treat colonies with dispase
  3. c Prepare neural induction media
  4. d Plate fragmented colonies in non-adherent conditions
  5. e Sequential weaning of EBs into neural induction media
  6. f Treat EBs with BMP4 on day 6
  7. g Plate EBs on Matrigel on day 7
  8. h Media changes on days 9 and 12
Phase 3 · 2D Retinal Differentiation and Optic Vesicle Formation Day 7-30
  1. a Media change to retinal differentiation media on day 15
  2. b Continue RDM media changes through day 30
  3. c Observe optic vesicle-like structures
  4. d Pick retinal organoids on day 30
Phase 4 · 3D Retinal Organoid Culture and Maturation Day 30-100+
  1. a Resuspend organoids in 3D retinal differentiation media
  2. b Culture organoids with RA supplementation until day 100
  3. c Culture organoids beyond day 100 without RA
  4. d Monitor morphological stages

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Attribution

This SOP was written by Organthis based on the published method in Lopez et al., 2022. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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