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RETINA Publication-derived

Retinal Organoids for Regenerative Medicine with Cryopreservation

Full SOP with Academic or a plan

Source Reichman et al., 2017 · Institut de la Vision, Sorbonne Universités, INSERM, CNRS, UPMC, Paris, France · 10.1002/stem.2586

Authors: Sacha Reichman, Amélie Slembrouck, Giuliana Gagliardi, Antoine Chaffiol, Angélique Terray, Céline Nanteau, Anaïs Potey, Morgane Belle, Oriane Rabesandratana, Jens Duebel, Gael Orieux, Emeline F. Nandrot, José-Alain Sahel, Olivier Goureau Duration: 100 days 6 phases Cell source: Human iPSC

Abstract

This protocol describes a two-step xeno-free/feeder-free differentiation system for generating retinal organoids and retinal pigmented epithelial (RPE) cells from adherent human iPSCs. The method bypasses embryoid body formation, enabling efficient production of photoreceptor precursors (CD73+), mature photoreceptors (rods and cones), and functional RPE cells within ~100 days. Both retinal organoids and dissociated retinal cells can be cryopreserved while retaining phenotypic characteristics, supporting large-scale GMP-compliant manufacturing for cellular therapy applications.

Cell source
Human iPSC
Application
Retinal cell therapy and disease modeling

Protocol overview

37 steps across 6 phases

Phase 1 · Adaptation of hiPSCs to Feeder-Free Culture Passages 16-20 prior to differentiation
  1. a Dissociate iPS colonies from feeder layer
  2. b Plate on vitronectin-coated dishes
  3. c Passage feederfree-adapted hiPSCs weekly
  4. d Continue adaptation and characterization
Phase 2 · Retinal Differentiation (Adherent Phase) Day 0 to Day 28
  1. a Prepare hiPSCs at 70-80% confluence
  2. b Switch to Essential 6 medium (Day 0)
  3. c Culture in Essential 6 for 2 days
  4. d Switch to E6N2 medium (Day 2)
  5. e Maintain in E6N2 with regular medium changes
  6. f Monitor formation of neuroepithelial-like structures
Phase 3 · Retinal Organoid Isolation and Floating Culture Day 28 to Day 56
  1. a Isolate retinal organoids at Day 28
  2. b Culture organoids as floating structures
  3. c Perform half medium changes with FGF2
  4. d Remove FGF2 at Day 35
  5. e Continue floating culture maintenance
Phase 4 · RPE Cell Generation and Amplification Day 28 to Day 42 (isolation), then ongoing amplification
  1. a Identify pigmented patches (Day 28-42)
  2. b Cut and isolate pigmented patches at Day 42
  3. c Culture hiRPE cells in ProN2 medium
  4. d Expand RPE cells to confluence
  5. e Dissociate and passage RPE cells
  6. f Continue amplification and banking
Phase 5 · Cryopreservation of Retinal Cells Day 84-100 (retinal organoids) or P1 (RPE cells)
  1. a Prepare retinal organoids for freezing
  2. b Suspend organoids in cryopreservation medium
  3. c Controlled-rate freezing
  4. d Transfer to long-term storage
  5. e Freeze dissociated retinal cells (alternative method)
  6. f Freeze RPE cells for banking
  7. g Thaw frozen cells/organoids
Phase 6 · Immunostaining and 3D Imaging of Retinal Organoids (Optional Characterization) Any timepoint after Day 35
  1. a Incubate organoids in PBSGT permeabilization solution
  2. b Primary antibody incubation
  3. c Wash organoids
  4. d Secondary antibody incubation
  5. e Final wash and storage
  6. f Tissue clearing (3DISCO protocol)
  7. g Delipidation
  8. h Final clearing
  9. i 3D imaging

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Attribution

This SOP was written by Organthis based on the published method in Reichman et al., 2017. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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