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RETINA Publication-derived

Differentiation Protocol for 3D Retinal Organoids, Immunostaining and Signal Quantitation

Source Döpper et al., 2020 · Institute of Human Genetics, University Hospital Essen, University of Duisburg-Essen · 10.1002/cpsc.120

👤 Hannah Döpper, Julia Menges, Morgane Bozet, Alexandra Brenzel, Dietmar Lohmann, Laura Steenpass, Deniz Kanber ⏱ 152 days 📋 8 phases 🧫 Human ESC (H9 line)

Abstract

This protocol describes the generation of three-dimensional retinal organoids from human embryonic stem cells (H9 line) containing all seven retinal cell types arranged in physiologically relevant layers. The organoids are produced using a Matrigel-free method that prevents fusion and improves differentiation monitoring. The protocol includes immunostaining of cryosections for phenotypic characterization and quantitation of immunofluorescent signals using Fiji image analysis.

Cell source
Human ESC (H9 line)
Application
Disease modeling and developmental study of human retinogenesis

Protocol overview

69 steps across 8 phases

Cell Preparation and Organoid Formation d0–d1
  1. 1 Prepare DMEM/F12-GlutaMAX with ROCK inhibitor
  2. 2 Prepare AggreWell medium with ROCK inhibitor
  3. 3 Prepare medium M0
  4. 4 Wash cells with D-PBS
  5. 5 Dissociate cells with Accutase
  6. 6 Prepare tube with DMEM/F12 during Accutase incubation
  7. 7 Verify cell detachment
  8. 8 Transfer cell suspension to prepared tube
  9. 9 Centrifuge cell suspension
  10. 10 Resuspend cell pellet
  11. 11 Count viable cells using hemocytometer
  12. 12 Prepare cell suspension at correct concentration
  13. 13 Recount cells (optional verification)
  14. 14 Seed cells into 96-well U-bottom plate
Medium M1 Application and Initial Differentiation Signaling d1–d15
  1. 15 First medium change with differentiation factors at d1
  2. 16 Incubate until d6
  3. 17 Add BMP4 and perform medium change at d6
  4. 18 Incubate until d9
  5. 19 Half-medium changes at d9 and d12
  6. 20 Incubate until d12 and d15
Medium M2 Application and Retinal Commitment d15–d21
  1. 21 Prepare medium M2 at d15
  2. 22 Add signaling factors and perform complete medium change at d15
  3. 23 Incubate until d16
  4. 24 Half-medium change at d16 with continued signaling
  5. 25 Incubate until d19
  6. 26 Complete medium change at d19 with signaling factors
  7. 27 Incubate until d21
Transfer to 48-Well Plates and Long-Term Culture Initiation d21–d23
  1. 28 Prepare medium M3 for long-term culture at d21
  2. 29 Coat 48-well plates with agarose and add medium
  3. 30 Complete medium change in 96-well plate at d21
  4. 31 Transfer organoids to 48-well plates
  5. 32 Incubate until d23
Long-Term Culture with Retinoic Acid Feeding d23–d152+
  1. 33 Establish and maintain regular feeding schedule with retinoic acid
  2. 34 Harvest organoids at desired time points for analysis
Organoid Embedding and Cryosectioning (Support Protocol) Harvest day + 1–2 days
  1. 1 Transfer organoid to a tube for fixation
  2. 2 Remove medium and wash with D-PBS
  3. 3 Fix organoid with 4% PFA
  4. 4 Wash fixed organoid
  5. 5 Incubate in 30% sucrose for cryoprotection
  6. 6 First incubation in O.C.T. compound
  7. 7 Transfer organoid to fresh O.C.T. compound
  8. 8 Embed organoid in tissue mold and freeze
  9. 9 Store frozen cryoblock
  10. 10 Section cryoblock using cryotome
Immunostaining of Cryosections 1 day
  1. 1 Thaw slides and prepare humid chamber
  2. 2 Outline staining areas and post-fix
  3. 3 Wash sections with TBS-T
  4. 4 Block non-specific binding
  5. 5 Incubate with primary antibody overnight
  6. 6 Wash sections after primary antibody
  7. 7 Incubate with secondary antibody
  8. 8 Wash after secondary antibody
  9. 9 Stain nuclei with DAPI and add
  10. 10 Treat with TrueBlack to reduce autofluorescence
  11. 11 Final D-PBS washes
  12. 12 Mount slide with VectaShield and seal
  13. 13 Store slides for imaging
Image Analysis and Quantitation Using Fiji After microscopy
  1. 1 Install and prepare Fiji software
  2. 2 Convert and prepare macro for Fiji
  3. 3 Determine optimal fluorescence signal threshold
  4. 4 Verify threshold accuracy using overlay images
  5. 5 Optimize particle size threshold for signal recognition
  6. 6 Create results folder and prepare batch processing
  7. 7 Run macro with optimized parameters on all images
  8. 8 Export and review results
  9. 9 Calculate mean DAPI signal across all images
  10. 10 Calculate mean fluorescent signal per organoid
  11. 11 Calculate percentage of positive cells
  12. 12 Perform statistical analysis on percentage values

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Döpper et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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