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RETINA Publication-derived

Retinal 2D Cell Cultures for Disease and Development

Full SOP with Academic or a plan

Source Mellough et al., 2015 · Newcastle University, Institute of Genetic Medicine · 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 8 phases Cell source: Human ESC (line H9, WiCell)

Abstract

This protocol demonstrates the differentiation of human embryonic stem cells (hESCs) into three-dimensional laminated neural retina containing mature photoreceptors with functional phototransduction capacity, along with retinal pigmented epithelium and accessory ocular structures (lens and corneal-like tissue). The addition of insulin-like growth factor 1 (IGF-1) significantly enhances optic vesicle and cup formation, enabling the generation of correctly laminated retinal tissue with photoreceptor cells displaying inner and outer segment-like structures and functional synapses as early as 6.5 weeks of differentiation.

Cell source
Human ESC (line H9, WiCell)
Application
Retinal tissue engineering and photoreceptor generation for disease modeling and cell replacement therapy

Protocol overview

34 steps across 8 phases

Phase 1 · hESC Expansion and Embryoid Body Formation Days 0-5
  1. a Prepare Embryoid Bodies
Phase 2 · Early Neural Induction with IGF-1 Supplementation Days 0-37
  1. a Prepare Ventral Neural Induction Medium (VNIM) with IGF-1
  2. b Add N2 Supplement from Day 37
  3. c Monitor EB Morphology
Phase 3 · Late Differentiation with Elevated IGF-1 and Basal Medium Days 37-90
  1. a Switch to Basal Knockout Serum-Free Medium with Increased IGF-1
  2. b Continue Culturing EBs in Suspension
  3. c Observe Optic Vesicle and Cup Formation
Phase 4 · Immunocytochemistry Analysis and Characterization Days 30-90
  1. a Harvest and Fix EBs
  2. b Prepare Cryostat Sections
  3. c Perform Immunocytochemistry
  4. d Image and Analyze Sections
Phase 5 · Transmission Electron Microscopy (TEM) Analysis Days 30-90
  1. a Fix Tissues for TEM
  2. b Process for Embedding
  3. c Prepare Ultrathin Sections
  4. d Image with Transmission Electron Microscope
Phase 6 · Electrophysiological Analysis of Photoreceptor Function Days 30, 45, 90
  1. a Prepare Cultures for Functional Analysis
  2. b Dark Adaptation
  3. c Load Calcium Indicator Dye
  4. d Perfuse with Oxygenated Artificial Cerebrospinal Fluid (aCSF)
  5. e Apply 8-br-cGMP Stimulation
  6. f Record Calcium Imaging
  7. g Perform Control Recordings
  8. h Analyze Fluorescence Changes
  9. i Immunostain Responding Cells
  10. j Statistical Analysis
Phase 7 · IGF-1 Receptor Signaling Interference Studies Days 0-90
  1. a Prepare IGF-1 Receptor Inhibitor
  2. b Prepare IGF-II Receptor Blocking Antibody
  3. c Differentiate with Inhibitors
  4. d Count Optic Vesicles and Cups
  5. e Analyze Survival and Structure Development
Phase 8 · Statistical Analysis and Quantification Throughout (Days 0-90)
  1. a Count and Record Optic Structures
  2. b Calculate Mean ± SEM
  3. c Perform Statistical Testing
  4. d Quantify Cellular Responses

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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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