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RETINA Publication-derived

Generation and Characterization of Retinal Organoids from Pluripotent Stem Cells

Source Children's Hospital Los Angeles (CHLA)

👤 <UNKNOWN> ⏱ 42 days 📋 9 phases 🧫 Human ESC (WA09) and Human iPSC

Abstract

This protocol describes the differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) into retinal organoids in three-dimensional culture. Organoids are subsequently characterized via immunofluorescent staining, hyperspectral imaging, and fluorescence lifetime imaging microscopy (FLIM) to assess retinal differentiation and cellular organization.

Cell source
Human ESC (WA09) and Human iPSC
Application
Developmental study; Disease modeling

Protocol overview

31 steps across 9 phases

hESC Culture and Maintenance Ongoing (passaged every 4-5 days)
  1. 1 Maintain hESCs on mouse embryonic fibroblasts
iPSC Generation and Maintenance Ongoing (passaged every 5-7 days)
  1. 1 Generate iPSCs from peripheral blood mononuclear cells
  2. 2 Maintain iPSCs in Essential 8 media or TeSRE8 media
hESC Retinal Organoid Differentiation (Days 0-42+) Days 0-42+
  1. 1 Dissociate hESC colonies and form aggregates
  2. 2 Change media to retinal differentiation media
  3. 3 Add Growth Factor Reduced Matrigel and IWR1
  4. 4 Maintain organoid culture
iPSC Retinal Organoid Differentiation (Days 0-42+) Days 0-42+
  1. 1 Form cell aggregates with ISL factors
  2. 2 Change media to gfCDM/KSR with ISL
  3. 3 Add BMP and additional IWR1
  4. 4 Omit induction reversal; maintain in NR-differentiation medium
  5. 5 Begin supplementation with retinoic acid and taurine
Sample Preparation for Immunofluorescence At desired timepoint
  1. 1 Fix retinal organoids in 4% PFA
  2. 2 Cryoprotect in 30% sucrose
  3. 3 Embed in OCT and prepare cryosections
Immunofluorescent Staining Same day as imaging
  1. 1 Heat slides and post-fix sections
  2. 2 Rinse sections
  3. 3 Block non-specific binding
  4. 4 Incubate with primary antibodies
  5. 5 Wash sections after primary antibodies
  6. 6 Incubate with secondary antibodies
  7. 7 Wash and apply DAPI
  8. 8 Coverslip and mount
Immunofluorescence Imaging After coverslipping
  1. 1 Acquire fluorescence images
Hyperspectral Imaging After preparation
  1. 1 Acquire spectral images
  2. 2 Configure excitation and detection parameters
Fluorescence Lifetime Imaging (FLIM) After preparation
  1. 1 Set up FLIM instrumentation
  2. 2 Configure image acquisition parameters
  3. 3 Acquire FLIM data
  4. 4 Process FLIM data
  5. 5 Calibrate FLIM using coumarin 6 standard

Full SOP

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Attribution

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

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