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RETINA Publication-derived

Retinal Degeneration 2D Cell Cultures

Full SOP with Academic or a plan

Source Zhu et al., 2013 · Center for Regenerative Therapies, Technical University Dresden, Dresden, Germany · 10.1371/journal.pone.0054552

Authors: Yu Zhu, Madalena Carido, Andrea Meinhardt, Thomas Kurth, Mike O. Karl, Marius Ader, Elly M. Tanaka Duration: 60 days 7 phases Cell source: Human ESC (H9, H1), Human iPSC (O27-08)

Abstract

This protocol describes a three-dimensional epithelial cyst culture method using Matrigel and neural induction medium (N2B27) to efficiently differentiate human embryonic stem cells (hESCs) into polarized neuroepithelial cysts within 5 days, followed by directed differentiation to retinal pigment epithelial (RPE) cells within 30 days. The resulting RPE cells display mature morphological and functional characteristics and can be directly transplanted without selection or expansion, with demonstrated capacity to rescue photoreceptor degeneration in a rat model.

Cell source
Human ESC (H9, H1), Human iPSC (O27-08)
Application
Disease modeling and RPE cell production for retinal degeneration treatment

Protocol overview

29 steps across 7 phases

Phase 1 · Neuroepithelial Cyst Formation in Matrigel Day 0-5
  1. a hESC Culture and Preparation
  2. b Preparation of hESC Clumps for Embedding
  3. c Embedding in Matrigel
  4. d Neural Induction Culture
  5. e Optional: IGF-1 Signaling Inhibition Test
Phase 2 · Retinal Progenitor Cell Characterization (Cyst Phase) Day 5-15
  1. a Immunofluorescence Characterization at Day 5
  2. b RT-PCR Analysis of Retinal Identity Genes
  3. c In Situ Hybridization Verification
  4. d Quantitative Assessment at Day 15
Phase 3 · RPE Differentiation on Transwell Filters Day 5-30
  1. a Cyst Recovery and Cell Dissociation
  2. b Plating on Growth Factor-Reduced Matrigel-Coated Transwells
  3. c Medium Switch to RPE-Supporting Conditions (H9 cells with Activin A)
  4. d Alternative: RPE Differentiation from H1 Cells (Endogenous Activin A)
  5. e Characterization of RPE Cells: Morphology and PAX6/MITF Expression
Phase 4 · RPE Maturation and Functional Characterization Day 30-50
  1. a Tight Junction Assessment via ZO-1 Immunostaining
  2. b Transepithelial Resistance (TER) Measurement
  3. c Ultrastructural Analysis via Transmission Electron Microscopy (TEM)
  4. d Mature RPE Marker Expression (RPE65 and BESTROPHIN)
  5. e Cell Cycle Exit Assessment via EdU Incorporation
Phase 5 · Functional Assay: Phagocytosis of Photoreceptor Outer Segments Day 30+
  1. a Co-culture Setup with Transgenic Retinal Explants
  2. b Co-culture Incubation and Fixation
  3. c Detection of Phagocytosed Outer Segments and Early Endosome Marker EEA1
Phase 6 · Alternative Differentiation: Neural Retina from Retinal Progenitor Cysts Day 5-60
  1. a Cyst Dissociation and Plating for Neural Retina Differentiation
  2. b Early Neural Retina Characterization (Days 20-40)
  3. c Late Neural Retina Maturation (Days 40-60)
Phase 7 · In Vivo Transplantation and Rescue Assessment Variable (pre-transplant: Days 1-30)
  1. a RPE Cell Preparation for Transplantation
  2. b Surgical Transplantation into RCS Rat Subretinal Space
  3. c In Vivo Integration and Monolayer Formation Assessment
  4. d Quantification of Outer Nuclear Layer (ONL) Rescue

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Attribution

This SOP was written by Organthis based on the published method in Zhu et al., 2013. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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