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RETINA Publication-derived

Retinal Organoid Cultivation, Differentiation, and Cell Transplantation

Source Li et al., 2021 · Department of Ophthalmology, West China Hospital, Sichuan University · 10.3389/fncel.2021.638439

👤 Xuying Li, Li Zhang, Fei Tang, Xin Wei ⏱ 100 days 📋 8 phases 🧫 Human iPSC, Human ESC, Mouse ESC

Abstract

This protocol describes the cultivation and differentiation of retinal organoids from pluripotent stem cells into mature three-dimensional structures containing all retinal cell types. The protocol includes strategies to promote differentiation, increase yield, reduce heterogeneity, and enable extraction and transplantation of photoreceptor and retinal ganglion cells for treating retinal degenerative diseases.

Cell source
Human iPSC, Human ESC, Mouse ESC
Application
Disease modeling and replacement therapy for retinal degeneration

Protocol overview

35 steps across 8 phases

Stem Cell Aggregate Formation and Embryoid Body Induction Days 0-7
  1. 1 Prepare pluripotent stem cells in suspension
  2. 2 Add extracellular matrix to culture medium
  3. 3 Culture aggregates in neural induction medium
  4. 4 Monitor PAX6 expression
Optic Vesicle and Optic Cup Formation Days 7-30
  1. 5 Transfer to retinal differentiation medium
  2. 6 Promote optic cup self-organization
  3. 7 Manually separate aggregates if needed
  4. 8 Monitor optic cup morphology
Retinal Organoid Maturation and Cell Differentiation Days 30-100+
  1. 9 Culture maturing organoids in differentiation medium
  2. 10 Promote differentiation with growth factors
  3. 11 Optional: Culture on electrospun scaffolds to enhance ganglion cell development
  4. 12 Monitor metabolic status and differentiation in real-time
  5. 13 Assess cell type differentiation by immunocytochemistry
  6. 14 Verify photoreceptor maturation
Photoreceptor Cell Extraction and Enrichment Days 50-100+
  1. 15 Promote photoreceptor cell differentiation with Notch pathway inhibition
  2. 16 Optional: Add COCO to promote photoreceptor precursor generation
  3. 17 Label photoreceptor cells with CRX reporter gene
  4. 18 Separate photoreceptor cells using CD73 and CRX markers
  5. 19 Optional: Use High Efficiency Hypoxia Induced Generation of Photoreceptors (HEGP) protocol
Retinal Ganglion Cell Extraction and Enhancement Days 50-100+
  1. 20 Convert organoids to two-dimensional culture to amplify ganglion cells
  2. 21 Screen mature RGCs using CD184 and CD171 markers
  3. 22 Screen RGCs using THY1 marker
  4. 23 Co-culture RGCs with Muller cells to enhance survival
Organoid Quality Control and Staging Throughout culture (especially days 30-100+)
  1. 24 Stage organoids using morphological classification
  2. 25 Confirm layered structure formation by OCT
  3. 26 Perform immunohistochemistry to verify cell type distribution
  4. 27 Monitor metabolic status using fluorescence lifetime imaging microscopy
Photoreceptor Cell Transplantation Variable (typically after day 30-70 culture equivalent to 8-14 week embryo)
  1. 28 Prepare photoreceptor cell grafts from stage 2-3 organoids
  2. 29 Optional: Purify photoreceptor precursor cells before transplantation
  3. 30 Optionally add neurons to transplant preparation
  4. 31 Perform subretinal transplantation into recipient retina
  5. 32 Verify synaptic integration post-transplantation
Retinal Ganglion Cell Transplantation Variable (after RGC extraction)
  1. 33 Prepare retinal progenitor cells or mature RGCs for ganglion cell layer transplantation
  2. 34 Transplant RGCs or RPCs into ganglion cell layer
  3. 35 Optional: Co-transplant Muller glial cells to enhance RGC survival

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Li et al., 2021. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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