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RETINA Publication-derived

Decellularised extracellular matrix-derived peptides from neural retina and retinal pigment epithelium enhance the expression of synaptic markers and light responsiveness of human pluripotent stem cell derived retinal organoids

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

👤 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 ⏱ 150 days 📋 7 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Dorgau et al., 2019. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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