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BRAIN Publication-derived

Generation and Characterization of 3D Neocortical Organoids from Human Pluripotent Stem Cells

Source <UNKNOWN>

👤 <UNKNOWN> ⏱ 66 days 📋 7 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of 3D neocortical organoids from human pluripotent stem cells using dual SMAD and FGF inhibition, followed by detailed characterization methods including immunohistochemistry, electrophysiology, and real-time PCR analysis. The organoids recapitulate cortical development and serve as in vitro models for studying cocaine exposure effects and cytochrome P450 expression during neural differentiation.

Cell source
Human iPSC
Application
Disease modeling, Drug screening, Developmental study

Protocol overview

50 steps across 7 phases

hPSC Maintenance and Propagation Ongoing
  1. 1 Culture hPSCs on MEF feeder cells
  2. 2 Passage hPSC colonies
BrdU Incorporation and Immunostaining Day 45, Day 51–52
  1. 1 Fix and cryoprotect neocortical organoids
  2. 2 Permeabilize organoid sections with methanol
  3. 3 Treat with HCl
  4. 4 Neutralize HCl treatment
  5. 5 Block and stain with anti-BrdU antibody
  6. 6 Apply secondary antibody
CLARITY Clearing and Immunohistochemistry Day 66
  1. 1 Prepare organoids for hydrogel embedding
  2. 2 Incubate in Hydrogel Monomer Solution
  3. 3 Desiccate and polymerize hydrogel
  4. 4 Extract organoids from hydrogel
  5. 5 Initial clearing solution wash
  6. 6 Secondary clearing solution washes
  7. 7 Passive clearing
  8. 8 PBST wash after clearing
  9. 9 Primary antibody incubation
  10. 10 Primary antibody washout
  11. 11 Secondary antibody incubation
  12. 12 Secondary antibody washout
  13. 13 DAPI staining
  14. 14 DAPI washout
  15. 15 Refractive index matching with glycerol
  16. 16 Embed organoids in agarose
  17. 17 Image cleared organoids
Electrophysiology Recording Variable (Post-differentiation)
  1. 1 Suspend organoid with suction pipette
  2. 2 Record in bathing medium
  3. 3 Use patch-clamp amplifier and electrode
Gene Expression Analysis by RT-PCR and RT-qPCR Variable (Post-differentiation)
  1. 1 Extract total RNA
  2. 2 Treat RNA with DNase
  3. 3 Synthesize cDNA
  4. 4 Perform RT-PCR
  5. 5 Perform RT-qPCR with probe-based detection
  6. 6 Perform RT-qPCR with SYBR Green detection
Lentiviral Vector Construction and Transduction Day 28 transduction; analysis day 32+
  1. 1 Select and validate shRNA sequences
  2. 2 Clone shRNA sequences into lentiviral vector
  3. 3 Amplify lentiviral vectors in bacteria
  4. 4 Produce lentiviral particles
  5. 5 Purify and concentrate viral particles
  6. 6 Freeze and store viral particles
  7. 7 Titer viral particles
  8. 8 Prepare organoids for lentiviral infection
  9. 9 Infect organoids with lentivirus
  10. 10 Remove viral particles at medium change
ROS Content Analysis Day 44
  1. 1 Incubate organoids with DCFH-DA
  2. 2 Wash organoids
  3. 3 Lyse organoid tissue
  4. 4 Measure fluorescence
  5. 5 Quantify protein concentration
  6. 6 Calculate ROS content

Full SOP

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Attribution

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

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