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BRAIN Publication-derived

Retinal Organoids

Full SOP with Academic or a plan

Source Quadrato et al. · Harvard University, Cambridge, MA, USA · 10.1038/nature22047

Authors: Quadrato G, Nguyen T, Macosko EZ, Sherwood JL, Min Yang S, Berger DR, Maria N, Scholvin J, Goldman M, Kinney JP, Boyden ES, Lichtman JW, Williams ZM, McCarroll SA, Arlotta P Duration: 15 days 6 phases Cell source: Human iPSC

Abstract

Quadrato et al. characterized 8-month-old cerebral organoids using Drop-seq single-cell transcriptomics, revealing remarkable cell diversity including cortical neurons, interneurons, astrocytes, and unexpectedly photosensitive retinal-like cells capable of responding to light stimulation. This Nature 2017 study provided the most comprehensive cell type atlas of long-term brain organoids.

Cell source
Human iPSC
Application
Long-Term Brain Organoid Characterization

Protocol overview

30 steps across 6 phases

Phase 1 · Pluripotent stem cell culture and embryoid body formation Day 0-5
  1. a Prepare stem cell culture plates
  2. b Dissociate pluripotent stem cells
  3. c Plate cells for embryoid body formation
  4. d Culture embryoid bodies in EBM
Phase 2 · Two-step neural induction Day 6-14 (approximately 2 weeks)
  1. a Transfer embryoid bodies to intermediate induction medium
  2. b Add neural induction medium supplement
  3. c Assess neural induction
Phase 3 · Embedding in Matrigel and transfer to bioreactor Day 10-15
  1. a Embed organoids in Matrigel
  2. b Transfer Matrigel-embedded organoids to cerebral differentiation medium
  3. c Transfer to spinning bioreactor
Phase 4 · Long-term culture and maturation (1-13 months) Month 1-13
  1. a Perform media changes
  2. b Add brain-derived neurotrophic factor (BDNF) at 1 month
  3. c Monitor organoid development and health
  4. d Assess culture viability and hypoxia status (optional QC)
  5. e Continue culture to desired timepoint
Phase 5 · Organoid harvest and characterization At desired timepoint (1-13 months)
  1. a Remove organoids from bioreactor
  2. b Fix organoids for immunohistochemistry
  3. c Cryoprotect and embed in OCT
  4. d Section organoids on a cryostat
  5. e Perform immunofluorescence staining
  6. f Image and analyze stained organoids
Phase 6 · Electrophysiological recordings (optional, for mature organoids ≥ 7-9 months) At 7-9 months of culture
  1. a Prepare recording chamber and artificial cerebrospinal fluid
  2. b Transfer organoid to recording chamber
  3. c Equilibrate organoid to recording temperature
  4. d Position high-density silicon microelectrode probe
  5. e Record spontaneous extracellular activity
  6. f Perform spike detection and sorting
  7. g Analyze spontaneous activity patterns
  8. h Pharmacological validation of activity (optional)
  9. i Light stimulation and photosensitivity testing (optional)

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Attribution

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

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