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BRAIN Publication-derived

Cell Diversity and Network Dynamics in Photosensitive Human

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

👤 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 ⏱ 15 days 📋 6 phases 🧫 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

Pluripotent stem cell culture and embryoid body formation Day 0–5
  1. 1 Prepare stem cell culture plates
  2. 2 Dissociate pluripotent stem cells
  3. 3 Plate cells for embryoid body formation
  4. 4 Culture embryoid bodies in EBM
Two-step neural induction Day 6–14 (approximately 2 weeks)
  1. 5 Transfer embryoid bodies to intermediate induction medium
  2. 6 Add neural induction medium supplement
  3. 7 Assess neural induction
Embedding in Matrigel and transfer to bioreactor Day 10–15
  1. 8 Embed organoids in Matrigel
  2. 9 Transfer Matrigel-embedded organoids to cerebral differentiation medium
  3. 10 Transfer to spinning bioreactor
Long-term culture and maturation (1–13 months) Month 1–13
  1. 11 Perform media changes
  2. 12 Add brain-derived neurotrophic factor (BDNF) at 1 month
  3. 13 Monitor organoid development and health
  4. 14 Assess culture viability and hypoxia status (optional QC)
  5. 15 Continue culture to desired timepoint
Organoid harvest and characterization At desired timepoint (1–13 months)
  1. 16 Remove organoids from bioreactor
  2. 17 Fix organoids for immunohistochemistry
  3. 18 Cryoprotect and embed in OCT
  4. 19 Section organoids on a cryostat
  5. 20 Perform immunofluorescence staining
  6. 21 Image and analyze stained organoids
Electrophysiological recordings (optional, for mature organoids ≥ 7–9 months) At 7–9 months of culture
  1. 22 Prepare recording chamber and artificial cerebrospinal fluid
  2. 23 Transfer organoid to recording chamber
  3. 24 Equilibrate organoid to recording temperature
  4. 25 Position high-density silicon microelectrode probe
  5. 26 Record spontaneous extracellular activity
  6. 27 Perform spike detection and sorting
  7. 28 Analyze spontaneous activity patterns
  8. 29 Pharmacological validation of activity (optional)
  9. 30 Light stimulation and photosensitivity testing (optional)

Full SOP

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Attribution

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

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