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BRAIN Publication-derived

Schizophrenia Brain Spheroids

Full SOP with Academic or a plan

Source Farkas et al., 2025 · HUN-REN Research Centre for Natural Sciences, Budapest, Hungary · 10.3390/ijms26094444

Authors: Kiara Gitta Farkas, Katalin Vincze, Csongor Tordai, Ece Ilay Özgen, Derin Gürler, Vera Deli, Julianna Lilienberg, Zsuzsa Erdei, Balázs Sarkadi, János Miklós Réthelyi, Ágota Apáti Duration: 30 days 7 phases Cell source: Human iPSC (4 lines: patient-derived with ZMYND11 mutation, patient-derived corrected, control wild-type, control with introduced mutation)

Abstract

This protocol describes the differentiation of human iPSCs into hippocampal neural progenitor cells (NPCs) and their characterization in both 2D and 3D spheroid models to study schizophrenia-associated ZMYND11 mutations and antipsychotic drug effects. The model enables functional assessment of cell proliferation, migration, adhesion, and differentiation in response to first-, second-, and third-generation antipsychotic treatments.

Cell source
Human iPSC (4 lines: patient-derived with ZMYND11 mutation, patient-derived corrected, control wild-type, control with introduced mutation)
Application
Disease modeling and drug screening

Protocol overview

28 steps across 7 phases

Phase 1 · iPSC Culture and NPC Differentiation Days 1-30 (protocol duration varies; differentiation typically ~30 days based on referenced protocol)
  1. a Prepare iPSC lines
  2. b Differentiate iPSCs into hippocampal NPCs
  3. c Culture NPCs in standard medium
Phase 2 · 2D NPC Characterization (Proliferation and Migration Assays) Days 1-3 (proliferation endpoint); 1 day (scratch assay)
  1. a Seed cells for proliferation assay
  2. b Initiate 2D proliferation assay
  3. c Perform 2D scratch assay setup
  4. d Acquire scratch assay images
Phase 3 · 3D Spheroid Formation and Culture Days 1-3 (spheroid aggregation)
  1. a Seed NPCs on ultra-low attachment plates for spheroid formation
  2. b Allow spheroid aggregation
  3. c Measure spheroid diameter
  4. d Transfer spheroids to adherent plates
Phase 4 · Antipsychotic Treatment of 2D NPCs Days 1-3 (from treatment initiation to endpoint)
  1. a Prepare antipsychotic stock solutions
  2. b Prepare DMSO control medium
  3. c Treat NPCs in 2D culture
  4. d Image treated 2D cultures
Phase 5 · Antipsychotic Treatment of 3D Spheroids - Migration Assay Days 3-6 (post-spheroid formation; 3 days of treatment post-attachment)
  1. a Prepare treated medium for spheroid migration assay
  2. b Treat attached spheroids with antipsychotics
  3. c Stain and image treated spheroids for migration assessment
Phase 6 · Antipsychotic Treatment of 3D Spheroids - Differentiation Assay Days 3-7 (post-spheroid formation; 4 days of treatment in differentiation medium)
  1. a Prepare differentiation medium with antipsychotics
  2. b Transfer attached spheroids to differentiation medium with antipsychotics
  3. c Stain differentiating spheroids
  4. d Acquire images of differentiating spheroids
Phase 7 · Image Analysis and Quantification Post-imaging (analysis period variable)
  1. a Analyze 2D proliferation data
  2. b Analyze 2D scratch assay data
  3. c Analyze 3D spheroid size data
  4. d Analyze 3D spheroid outgrowth (cell number)
  5. e Analyze 3D spheroid outgrowth (migration distance)
  6. f Analyze neurite outgrowth in differentiating spheroids

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Attribution

This SOP was written by Organthis based on the published method in Farkas et al., 2025. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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