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RETINA Publication-derived

Retinal Organoids for Disease Modelling with Enhanced Maturation

Full SOP with Academic or a plan

Source Dorgau et al., 2019 · Institute of Genetic Medicine, Newcastle University, UK · 10.1016/j.biomaterials.2019.01.028

Authors: Birthe Dorgau, Majed Felemban, Gerrit Hilgen, Martin Kiening, Darin Zerti, Nicola Claire Hunt, Mary Doherty, Phil Whitfield, Dean Hallam, Kathryn White, Yuchun Ding, Natalio Krasnogor, Jumana Al-Aama, Hani Z. Asfour, Evelyne Sernagor, Majlinda Lako Duration: 150 days 7 phases Cell source: Human ESC (H9, Wicell Inc.), Human iPSC (SB-Ad3)

Abstract

This protocol describes the generation of decellularised extracellular matrix-derived peptides from bovine neural retina and retinal pigment epithelium, and their application to enhance the differentiation and functional maturation of human pluripotent stem cell-derived retinal organoids. The supplementation with these decellularised matrix components improves rod photoreceptor generation, synaptic marker expression, and light-driven responses of retinal organoids.

Cell source
Human ESC (H9, Wicell Inc.), Human iPSC (SB-Ad3)
Application
Disease modeling; Retinal organoid development and functional maturation

Protocol overview

50 steps across 7 phases

Phase 1 · Preparation of RPE conditioned medium (CM RPE) Variable; collected daily
  1. a Culture mature RPE cells
  2. b Collect conditioned medium daily
  3. c Centrifuge and dilute CM RPE
  4. d Dilute and filter CM RPE
Phase 2 · Preparation of decellularised ECM from NR and RPE Day 0-2 (tissue isolation and decellularisation); Day 2-4 (drying); Day 4-5 (digestion)
  1. a Isolate RPE and NR tissues
  2. b Initial PBS washes
  3. c Decellularisation of RPE (Standard protocol)
  4. d Decellularisation of NR (Milder protocol)
  5. e Snap-freeze and freeze-dry decellularised tissues
  6. f Store dried decellularised tissues
  7. g Prepare tissue powder for digestion
  8. h Pepsin digestion of ECM powder
  9. i Centrifuge and filter digested ECM
  10. j Store digested ECM peptides
Phase 3 · Retinal organoid differentiation with ECM supplementation Day 0-18 (base differentiation); Day 18-150 (ECM supplementation and culture)
  1. a Expand hPSC lines
  2. b Initiate retinal differentiation
  3. c Add supplementary factors from day 18
  4. d Establish four experimental culture conditions from day 18
  5. e Maintain organoid cultures to day 35, 90, and 150
  6. f Harvest retinal organoids for qRT-PCR at day 35, 90, 150
  7. g Prepare retinal organoids for immunohistochemistry at day 35, 90, 150
  8. h Cut cryostat sections from embedded organoids
Phase 4 · Immunohistochemistry and image analysis Variable; performed at day 35, 90, and 150 sampling
  1. a Prepare cryostat sections for staining
  2. b Block non-specific binding
  3. c Prepare antibody diluent
  4. d Incubate with primary antibodies
  5. e Wash and incubate with secondary antibodies
  6. f Final washes and mounting
  7. g Perform specificity controls
  8. h Image capture using fluorescence microscopy
  9. i Image processing and analysis
  10. j Quantify synaptic markers using ImageJ
Phase 5 · Gene expression analysis by qRT-PCR Variable; performed at day 35, 90, and 150 sampling
  1. a Extract RNA from retinal organoids
  2. b Reverse transcription
  3. c Set up qRT-PCR reactions
  4. d Perform qRT-PCR amplification
  5. e Calculate relative gene expression
Phase 6 · Electrophysiological recordings and light response analysis Day 149-150 (one day before and on recording day)
  1. a Prepare organoids with visual pigment precursor
  2. b Transfer organoids to recording chamber
  3. c Prepare organoids for MEA recording
  4. d Record baseline spontaneous activity in darkness
  5. e Apply white light pulse stimuli
  6. f Apply pharmacological agent (cGMP)
  7. g Extract and sort spike data
  8. h Analyze light responsiveness of RGCs
Phase 7 · Transmission electron microscopy (TEM) analysis Day 150 (terminal timepoint)
  1. a Fix retinal organoid samples
  2. b Prepare ultrathin TEM sections
  3. c Stain ultrathin sections
  4. d Acquire TEM images
  5. e Analyze TEM ultrastructure

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Attribution

This SOP was written by Organthis based on the published method in Dorgau et al., 2019. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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