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RETINA Publication-derived

Retinal Organoids for Drug Screening and Infection

Full SOP with Academic or a plan

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

Authors: Manuela Völkner, Marina Pavlou, Hildegard Büning, Stylianos Michalakis, Mike O. Karl Duration: 225 days 9 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Völkner et al., 2021. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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