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RETINA Publication-derived

Optimized Adeno-Associated Virus Vectors for Efficient Transduction of Human Retinal Organoids

Source Völkner et al., 2021 · German Center for Neurodegenerative Diseases (DZNE), Dresden, Germany · 10.1089/hum.2020.321

👤 Manuela Völkner, Marina Pavlou, Hildegard Büning, Stylianos Michalakis, Mike O. Karl ⏱ 225 days 📋 9 phases 🧫 Human iPSC

Abstract

This protocol describes the differentiation of human retinal organoids (HROs) from induced pluripotent stem cells and their transduction with novel adeno-associated virus (AAV) vectors carrying a self-complementary CMV-eGFP cassette. The HRO system serves as a human testing platform to evaluate the tropism and transduction efficiency of engineered AAV capsid variants, specifically AAV9.GL, AAV9.NN, AAV2.GL, and AAV2.NN, in a three-dimensional tissue containing all major retinal neuronal and glial cell types in a laminated structure.

Cell source
Human iPSC
Application
Gene therapy vector screening and optimization

Protocol overview

34 steps across 9 phases

hiPSC Maintenance and Retinal Organoid Differentiation Initiation D0-D5
  1. 1 hiPSC Maintenance
  2. 2 Preparation of HRO Initiation: Cell Cluster Formation
  3. 3 Initial Floating Culture and Neuroepithelial Cyst Formation
  4. 4 Transition to Adherent Culture
Epithelial Sheet Isolation and Floating Culture D13-D25
  1. 5 Epithelial Sheet Detachment
  2. 6 Transfer to Floating Culture in B27 Medium
  3. 7 Retinal Epithelial Domain Isolation
  4. 8 Addition of FBS and Synthetic Retinoid
Retinal Organoid Maturation D25-D200
  1. 9 Medium Optimization at Day 100
  2. 10 Continued Culture and Maturation to Day 200
AAV Vector Production Concurrent with organoid differentiation
  1. 11 Synthesis of Peptide Insert Oligonucleotides
  2. 12 Hybridization and Cloning of AAV Capsid Genes
  3. 13 Preparation of Vector Plasmid
  4. 14 rAAV Vector Production
AAV Transduction of Day 200 Retinal Organoids D200-D210
  1. 15 Preparation of Organoids for Transduction
  2. 16 Vector Transduction at Multiple Doses
  3. 17 Removal of Excess Vector Particles
  4. 18 Post-Transduction Culture and Monitoring
  5. 19 AAV2 Transduction (Separate Experiment)
Tissue Fixation and Cryoprotection D210 (or D225 for AAV2)
  1. 20 Fixation of Organoids
  2. 21 Cryoprotection with Sucrose
  3. 22 Embedding in O.C.T. Compound
Cryosectioning and Immunohistochemistry After embedding (can be performed immediately or after storage)
  1. 23 Cryosectioning
  2. 24 Rehydration of Sections
  3. 25 Heat-Mediated Antigen Retrieval (If Necessary)
  4. 26 Blocking and Primary Antibody Incubation
  5. 27 Secondary Antibody Incubation
  6. 28 Nuclear Counterstaining with DAPI
  7. 29 Final Washing and Mounting
Cell Death Assessment (Optional) At 10 DPT (concurrent with immunohistochemistry)
  1. 30 TUNEL Staining for Cell Death Detection
Image Acquisition and Analysis After immunostaining is complete
  1. 31 Overview Image Acquisition for Quantitative Analysis
  2. 32 Image Processing and Quantification
  3. 33 Qualitative Colocalization Analysis
  4. 34 TUNEL Analysis (If Performed)

Full SOP

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Attribution

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

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