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RETINA Publication-derived

Generation of Storable Retinal Organoids and Retinal Pigmented Epithelium from Adherent Human iPS Cells in Xeno-Free and Feeder-Free Conditions

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

👤 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 ⏱ 100 days 📋 6 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Reichman et al., 2017. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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