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RETINA Publication-derived

Retinal Organoids for Disease Modelling (No. 2)

Full SOP with Academic or a plan

Source Children's Hospital Los Angeles (CHLA)

Duration: 42 days 9 phases Cell source: 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

Phase 1 · hESC Culture and Maintenance Ongoing (passaged every 4-5 days)
  1. a Maintain hESCs on mouse embryonic fibroblasts
Phase 2 · iPSC Generation and Maintenance Ongoing (passaged every 5-7 days)
  1. a Generate iPSCs from peripheral blood mononuclear cells
  2. b Maintain iPSCs in Essential 8 media or TeSRE8 media
Phase 3 · hESC Retinal Organoid Differentiation (Days 0-42+) Days 0-42+
  1. a Dissociate hESC colonies and form aggregates
  2. b Change media to retinal differentiation media
  3. c Add Growth Factor Reduced Matrigel and IWR1
  4. d Maintain organoid culture
Phase 4 · iPSC Retinal Organoid Differentiation (Days 0-42+) Days 0-42+
  1. a Form cell aggregates with ISL factors
  2. b Change media to gfCDM/KSR with ISL
  3. c Add BMP and additional IWR1
  4. d Omit induction reversal; maintain in NR-differentiation medium
  5. e Begin supplementation with retinoic acid and taurine
Phase 5 · Sample Preparation for Immunofluorescence At desired timepoint
  1. a Fix retinal organoids in 4% PFA
  2. b Cryoprotect in 30% sucrose
  3. c Embed in OCT and prepare cryosections
Phase 6 · Immunofluorescent Staining Same day as imaging
  1. a Heat slides and post-fix sections
  2. b Rinse sections
  3. c Block non-specific binding
  4. d Incubate with primary antibodies
  5. e Wash sections after primary antibodies
  6. f Incubate with secondary antibodies
  7. g Wash and apply DAPI
  8. h Coverslip and mount
Phase 7 · Immunofluorescence Imaging After coverslipping
  1. a Acquire fluorescence images
Phase 8 · Hyperspectral Imaging After preparation
  1. a Acquire spectral images
  2. b Configure excitation and detection parameters
Phase 9 · Fluorescence Lifetime Imaging (FLIM) After preparation
  1. a Set up FLIM instrumentation
  2. b Configure image acquisition parameters
  3. c Acquire FLIM data
  4. d Process FLIM data
  5. e Calibrate FLIM using coumarin 6 standard

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Attribution

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

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