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BRAIN Publication-derived

Schizophrenia Brain Organoids with Vasculature

Full SOP with Academic or a plan

Source Stankovic et al., 2024 · Weill Cornell Medicine, Cornell University · 10.1038/s41398-024-02740-2

Authors: Isidora Stankovic, Michael Notaras, Paul Wolujewicz, Tyler Lu, Raphael Lis, M. Elizabeth Ross, Dilek Colak Duration: 35 days 6 phases Cell source: Patient-Derived iPSC (Schizophrenia)

Abstract

This protocol describes the generation of 3D cerebral organoids from patient-derived and healthy control iPSCs to model endothelial cell dysfunction in schizophrenia. The protocol combines a morphogen-free organoid culture system with single-cell RNA sequencing, immunohistochemistry, and functional permeability assays to identify altered endothelial cell signatures and blood-brain barrier dysfunction in schizophrenia brain tissue.

Cell source
Patient-Derived iPSC (Schizophrenia)
Application
Disease modeling

Protocol overview

38 steps across 6 phases

Phase 1 · iPSC Maintenance and Preparation Prior to organoid generation
  1. a Obtain and characterize iPSC lines
  2. b Culture iPSCs on Vitronectin-coated plates
Phase 2 · Three-Dimensional Cerebral Organoid Generation Days 0-35
  1. a Generate embryoid bodies (EB stage)
  2. b Neural fate specification
  3. c Embed organoids in Matrigel droplets
  4. d Culture organoids in terminal organoid media without agitation
  5. e Continue culture with agitation
  6. f Apply quality control criteria at multiple stages
Phase 3 · Single-Cell RNA Sequencing Day 30 DIV
  1. a Dissociate organoids to single-cell suspension
  2. b Load cells into 10x Chromium microfluidic devices
  3. c Prepare cDNA libraries
  4. d Perform next-generation sequencing
  5. e Align reads and perform quality control
  6. f Normalize and integrate data
  7. g Perform clustering and cell type annotation
Phase 4 · Immunohistochemistry and Microscopy Analysis Day 30 DIV
  1. a Prepare organoids for cryosectioning
  2. b Serially cryosection organoids
  3. c Perform antigen retrieval
  4. d Incubate primary antibodies
  5. e Incubate secondary antibodies
  6. f Acquire laser-scanning confocal microscopy images
  7. g Quantify endothelial cells and vessel-like structures
Phase 5 · Differentiation of iPSCs to Brain Microvascular Endothelial Cells (iBMECs) Days 0-26
  1. a Expand iPSCs to confluence
  2. b Reduce ROCK inhibitor concentration
  3. c Switch to unconditioned medium
  4. d Transduce cells with lentiviral vectors
  5. e Dissociate and plate cells on coated surfaces
  6. f Expand iBMECs to confluency
  7. g FACS isolation of pure endothelial populations
  8. h Expand sorted iBMECs
Phase 6 · FITC-Dextran Paracellular Permeability Assay Day 26 of iBMEC differentiation
  1. a Prepare Transwell plates with endothelial cells
  2. b Culture cells to confluency
  3. c Add anti-VE-cadherin antibody
  4. d Add FITC-labeled dextran
  5. e Incubate FITC-dextran
  6. f Collect media from lower compartment
  7. g Measure fluorescence intensity
  8. h Calculate FITC-dextran passage

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Attribution

This SOP was written by Organthis based on the published method in Stankovic et al., 2024. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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