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RETINA Publication-derived

Retinal Organoids for Disease Modelling (2021)

Full SOP with Academic or a plan

Source Isla-Magrané et al., 2021 · Vall d'Hebron Institut de Recerca (VHIR), Vall d'Hebron Barcelona Hospital Campus · 10.1186/s13287-021-02651-9

Authors: Helena Isla-Magrané, Anna Veiga, José García-Arumí, Anna Duarri Duration: 140 days 8 phases Cell source: Human iPSC

Abstract

This protocol describes a 2-step method for generating multiocular organoids from human induced pluripotent stem cells (hiPSCs) that recapitulate the complexity of developing eyes. First, self-organized multizone ocular progenitor cells (mzOPCs) are generated from 2D hiPSC cultures within three weeks. Subsequently, mzOPCs are manually isolated and cultured in suspension to produce 3D multiocular organoids containing retina, cornea, corneal epithelium, and retinal pigment epithelium (RPE), which model important cellular features and interactions of early eye development.

Cell source
Human iPSC
Application
Disease modeling and developmental biology of ocular tissues

Protocol overview

31 steps across 8 phases

Phase 1 · hiPSC Culture and Maintenance Ongoing
  1. a Coat plates with Matrigel
  2. b Culture hiPSCs in mTeSR1 medium
  3. c Passage hiPSCs with EDTA
Phase 2 · Differentiation into Multizone Ocular Progenitor Cells (mzOPCs) — 2D Culture Day 0-30
  1. a Prepare induction medium
  2. b Initiate eye-field induction at 75% confluency
  3. c Change medium every other day for 30 days
  4. d Monitor mzOPC formation via microscopy
Phase 3 · Generation of 3D Multiocular Organoids — Lifting and Suspension Culture Day 30-90
  1. a Manually isolate mzOPC clusters at day 30
  2. b Transfer mzOPCs to low-attachment plates
  3. c Culture organoids in suspension for 60 days (Day 30-90)
  4. d Monitor organoid growth and morphology
Phase 4 · Maturation Phase — Specialized Medium Supplementation (Day 90-140) Day 90-140
  1. a Separate organoid types and apply differential medium
  2. b Continue culture with medium replenishment every other day (Day 90-140)
  3. c Monitor RPE pigmentation and corneal transparency
Phase 5 · Optional: Isolation and Purification of Individual Organoid Types Day 60-140
  1. a Mechanically isolate individual retinal, corneal, or RPE organoids
  2. b Culture individual organoid types separately in low-attachment plates
  3. c Disaggregate RPE organoids to obtain RPE monolayer (optional)
Phase 6 · Histology and Immunohistochemistry Day 90-140
  1. a Fix organoids in paraformaldehyde
  2. b Wash in phosphate-buffered saline
  3. c Embed in paraffin and section
  4. d Deparaffinize and antigen retrieve
  5. e Permeabilize and block non-specific binding
  6. f Incubate with primary antibodies
  7. g Wash and incubate with secondary antibodies
  8. h Stain nuclei with DAPI and mount
  9. i Image acquisition via confocal microscopy
Phase 7 · Gene Expression Analysis by RT-qPCR Day 30-140
  1. a Extract total RNA
  2. b Reverse transcribe mRNA to cDNA
  3. c Perform RT-qPCR
Phase 8 · Standard PCR for Gene Expression Confirmation Day 30-140
  1. a Amplify cDNA by standard PCR
  2. b Visualize PCR products by gel electrophoresis

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Attribution

This SOP was written by Organthis based on the published method in Isla-Magrané et al., 2021. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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