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RETINA Publication-derived

Retinal 2D Cell Cultures for Disease Modelling (2015)

Full SOP with Academic or a plan

Source Mellough et al., 2015 · Institute of Genetic Medicine, Newcastle University · 10.1002/stem.2023

Authors: Carla B. Mellough, Joseph Collin, Mahmoud Khazim, Kathryn White, Evelyne Sernagor, David H. W. Steel, Majlinda Lako Duration: 90 days 7 phases Cell source: Human ESC (H9, WiCell)

Abstract

A protocol for differentiating human embryonic stem cells (hESCs) into three-dimensional laminated neural retina with photoreceptor cells displaying advanced maturation features including inner and outer segment-like structures and phototransduction capacity by 6.5 weeks of differentiation. The protocol employs IGF-1 supplementation in minimal serum-free media and also generates accessory ocular structures including retinal pigmented epithelium, lens, and corneal-like epithelium.

Cell source
Human ESC (H9, WiCell)
Application
Disease modeling; Developmental study; Cell replacement therapy

Protocol overview

24 steps across 7 phases

Phase 1 · Embryoid Body Formation and Initial Neural Induction Days 0-37
  1. a hESC Expansion and Embryoid Body Formation
  2. b Ventral Neural Induction Media Supplementation
  3. c Time-Lapse Morphological Documentation
Phase 2 · Neural Retinal Differentiation and Maturation Days 37-90
  1. a Transition to Basal Knockout Serum-Free Media with IGF-1 Continuation
  2. b Extended Maturation Culture
  3. c Media Changes
Phase 3 · Tissue Harvest and Immunocytochemistry Analysis Days 30-90
  1. a Embryoid Body Fixation
  2. b Cryostat Sectioning
  3. c Immunocytochemical Staining
  4. d Microscopy and Image Acquisition
Phase 4 · Transmission Electron Microscopy (TEM) Analysis Days 30-90
  1. a Tissue Preparation for Electron Microscopy
  2. b TEM Imaging and Analysis
Phase 5 · Functional Electrophysiology Studies Days 30, 45, 90
  1. a Embryoid Body Dark Adaptation
  2. b Fura-2-AM Loading
  3. c Perfusion with Oxygenated Artificial Cerebrospinal Fluid (aCSF)
  4. d cGMP Stimulation and Control Recordings
  5. e Calcium Imaging Acquisition
  6. f Fluorescence Data Analysis
  7. g Confirmation of Photoreceptor Identity by Immunocytochemistry
Phase 6 · IGF-1 Receptor Signaling Interference Studies Days 0-90
  1. a IGF-IR Inhibition Experiments
  2. b IGF-IIR Neutralization Experiments
  3. c Optic Vesicle and Cup Counting
Phase 7 · Statistical Analysis and Data Reporting Throughout differentiation (days 0-90)
  1. a Data Collection and Presentation
  2. b Statistical Significance Testing

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Attribution

This SOP was written by Organthis based on the published method in Mellough et al., 2015. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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