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RETINA Publication-derived

Retinal Organoids for Developmental Studies under Hypoxia

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Source Du et al., 2023 · Senior Department of Ophthalmology, the Third Medical Center of Chinese PLA General Hospital, Beijing · 10.18240/ijo.2023.10.03

Authors: Jin-Lin Du, Li-Xiong Gao, Tao Wang, Zi Ye, Hong-Yu Li, Wen Li, Quan Zeng, Jia-Fei Xi, Wen Yue, Zhao-Hui Li Duration: 52 days 4 phases Cell source: Human iPSC

Abstract

This protocol investigates the effects of physiological hypoxia (5% O₂) on the development of human iPSC-derived retinal organoids (ROs) using the SFEBq 3D culture method. Hypoxia promotes retinal cell proliferation and stemness during early developmental stages (before day 40) while restricting progenitor cell differentiation, as measured by increased SOX2-positive cells and decreased PAX6/CHX10-positive cells.

Cell source
Human iPSC
Application
Developmental study of hypoxia effects on retinal organoid differentiation and progenitor cell biology

Protocol overview

22 steps across 4 phases

Phase 1 · hiPSC Culture and Embryoid Body Generation Days 0-18
  1. a hiPSC Maintenance Culture
  2. b hiPSC Dissociation and Single-Cell Preparation
  3. c Embryoid Body Reaggregation in V-Bottomed Plates
  4. d BMP4 Supplementation and Sequential Dilution
  5. e Transfer to Neural Retina Differentiation Medium
Phase 2 · Hypoxia Treatment and Culture Maintenance Days 30-52
  1. a Division into Normoxic and Hypoxic Groups
  2. b Continuous Hypoxia Maintenance and Medium Changes
  3. c Photography for Volume Measurement (Days 38, 45, 52)
Phase 3 · Sample Fixation, Sectioning, and Immunofluorescence Days 38-52 (sampling and analysis)
  1. a Selection and Fixation of Retinal Organoids
  2. b Dehydration and Paraffin Embedding
  3. c Drying and Storage of Sections
  4. d Deparaffinization and Rehydration
  5. e Permeabilization and Blocking
  6. f Primary Antibody Incubation
  7. g PBS Washing
  8. h Secondary Antibody Incubation
  9. i DAPI Counterstaining
  10. j Microscopy and Image Acquisition
Phase 4 · Cell Counting, Analysis, and Statistical Evaluation Post-imaging analysis
  1. a Image Processing and Region-of-Interest Selection
  2. b Manual Cell Counting and Density Calculation
  3. c Data Organization and Statistical Testing
  4. d Calculation of Embryoid Body Volume

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Attribution

This SOP was written by Organthis based on the published method in Du et al., 2023. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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