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RETINA Publication-derived

Repopulation of Decellularized Retinas with hiPSC-Derived Retinal Pigment Epithelial and Ocular Progenitor Cells

Source Maqueda et al., 2021 · Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), Barcelona, Spain · 10.1016/j.biomaterials.2021.121049

👤 Maria Maqueda, Jose Luis Mosquera, José García-Arumí, Anna Veiga, Anna Duarri ⏱ 140 days 📋 5 phases 🧫 Human iPSC

Abstract

This protocol describes the decellularization of mouse and porcine retinas to create acellular retinal ECM (arECM) scaffolds, followed by repopulation with hiPSC-derived retinal pigment epithelial (RPE) cells or ocular progenitor cells (OPCs). The protocol demonstrates that arECM scaffolds support cell engraftment, proliferation, differentiation, and organization into cell-type-specific clusters, providing a tissue-engineered model for studying retinal development and regeneration.

Cell source
Human iPSC
Application
Tissue engineering and regenerative medicine using acellular ECM scaffolds

Protocol overview

23 steps across 5 phases

Retinal Isolation and Decellularization Day 0
  1. 1 Tissue collection and preparation
  2. 2 Retinal dissection
  3. 3 DNase I treatment
  4. 4 Non-ionic detergent treatment
  5. 5 Ionic detergent treatment
  6. 6 Washing with distilled water
  7. 7 Final PBS wash and storage
Characterization of Acellular Retinal ECM Day 0-1
  1. 1 DNA quantification
  2. 2 Scanning electron microscopy (SEM) preparation
  3. 3 Proteomic analysis by LC-MS/MS
hiPSC Culture and Differentiation Day 0-30
  1. 1 hiPSC maintenance culture
  2. 2 Ocular progenitor cell (OPC) differentiation
  3. 3 RPE differentiation and GFP transduction
Recellularization of Acellular Retinal ECM Day 0-140
  1. 1 ECM equilibration
  2. 2 RPE cell preparation and seeding
  3. 3 OPC cell preparation and seeding
  4. 4 Medium replacement - day 1
  5. 5 Removal of non-attached cells - day 2
  6. 6 Continued culture and medium changes
Characterization of Recellularized ECM Day 30-140
  1. 1 Histology and H&E staining
  2. 2 Immunohistochemistry (IHC)
  3. 3 Real-time quantitative PCR (qPCR)
  4. 4 Photoreceptor outer segment (POS) phagocytosis assay

Full SOP

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Attribution

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

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