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BRAIN Publication-derived

Alzheimer's Disease Cerebral Organoids with Electrophysiology

Full SOP with Academic or a plan

Source Ghatak et al., 2019 · The Scripps Research Institute · 10.7554/elife.50333

Authors: Swagata Ghatak, Nima Dolatabadi, Dorit Trudler, XiaoTong Zhang, Yin Wu, Madhav Mohata, Rajesh Ambasudhan, Maria Talantova, Stuart A Lipton Duration: 56 days 23 phases Cell source: Patient-Derived iPSC (Alzheimer's Disease - Familial)

Abstract

This protocol generates cerebrocortical neurons and cerebral organoids from hiPSC lines bearing Alzheimer's disease-related mutations (PS1 ΔE9, PS1 M146V, or APP Swedish). The resulting neurons and organoids recapitulate early AD pathophysiology, including hyperexcitability, altered ion channel properties, aberrant synaptic function, and shortened neurites, serving as a human cellular model for investigating AD mechanisms.

Cell source
Patient-Derived iPSC (Alzheimer's Disease - Familial)
Application
Disease modeling

Protocol overview

64 steps across 23 phases

Phase 1 · hiPSC Maintenance and Pluripotent State Days 1-5 (continuous cycling)
  1. a Culture feeder-free hiPSCs on Matrigel
Phase 2 · Initiation of Neural Differentiation Days 1-6 of differentiation
  1. a Replace medium with differentiation cocktail
Phase 3 · Neural Progenitor Specification and Expansion Days 6-20 of differentiation
  1. a Manual scraping to form PAX6+ neurospheres
  2. b Plate neurospheres on p-ornithine/laminin-coated dishes
Phase 4 · Terminal Neuronal Differentiation - Protocol A (Co-culture with Astrocytes) Days 20-35 (5-6 weeks total differentiation)
  1. a Seed hNPCs with neonatal mouse astrocytes
Phase 5 · Terminal Neuronal Differentiation - Protocol B (Compound E Treatment) Days 20-35 (5-6 weeks total differentiation)
  1. a Treat hNPCs with γ-secretase inhibitor Compound E
  2. b Transition to BrainPhys medium at week 3
Phase 6 · Cerebral Organoid Generation Days 0-56 (approximately 8 weeks)
  1. a Seed hiPSCs in embryoid body (EB) formation media
  2. b Transfer EBs to expansion phase
  3. c Embed EBs in Matrigel and transfer to expansion medium
  4. d Transition to maturation medium for cortical layer formation
  5. e Media changes during organoid maturation
Phase 7 · Characterization: Amyloid-Beta Quantification Week 6 of 2D culture; Weeks 4-8 of organoid culture
  1. a Collect culture medium for ELISA
  2. b Run an ELISA to quantify Aβ peptide forms Aβ1-38, Aβ1-40, and Aβ1-42.
Phase 8 · Electrophysiology: Whole-Cell Patch-Clamp Recording Setup Week 5-6 of 2D culture; Week 8+ of organoid culture
  1. a Prepare patch pipettes and solutions
  2. b Set up patch-clamp rig and amplifier
Phase 9 · Electrophysiology: Spontaneous and Evoked Action Potential Recording Week 5-6 of 2D culture
  1. a Record spontaneous action potentials in gap-free mode
  2. b Record evoked action potentials
  3. c Quantify action potential properties
Phase 10 · Electrophysiology: Voltage-Clamp Sodium and Potassium Currents Week 5-6 of 2D culture
  1. a Record whole-cell sodium and potassium currents
  2. b Measure current density and kinetics
Phase 11 · Electrophysiology: Spontaneous Postsynaptic Currents (sPSCs) Week 5-6 of 2D culture
  1. a Record spontaneous excitatory postsynaptic currents (sEPSCs)
  2. b Record spontaneous inhibitory postsynaptic currents (sIPSCs)
  3. c Analyze postsynaptic current parameters
Phase 12 · Electrophysiology: Miniature Postsynaptic Currents (mPSCs) Week 5-6 of 2D culture
  1. a Block action potentials with tetrodotoxin (TTX)
  2. b Record miniature excitatory postsynaptic currents (mEPSCs)
  3. c Record miniature inhibitory postsynaptic currents (mIPSCs)
  4. d Analyze miniature current parameters
Phase 13 · Electrophysiology: Readily Releasable Pool (RRP) Measurement Week 5-6 of 2D culture
  1. a Prepare hypertonic sucrose solution
  2. b Record sucrose-evoked currents
  3. c Quantify RRP size
Phase 14 · Pharmacology: γ-Secretase and BACE1 Inhibitor Treatment Week 5-6 of 2D culture (pre-incubation) and recording
  1. a Incubate PS1 mutant neurons with γ-secretase inhibitor (Compound E)
  2. b Incubate APP Swedish mutant neurons with BACE1 inhibitor
  3. c Record electrophysiology in treated vs. control neurons
Phase 15 · Immunocytochemistry: Neuronal Morphology and Markers Week 5-6 of 2D culture
  1. a Fix neuronal cultures with paraformaldehyde (PFA)
  2. b Permeabilization and blocking
  3. c Incubate with primary antibodies
  4. d Incubate with secondary antibodies and DAPI counterstain
  5. e Imaging and quantification of neuronal morphology
Phase 16 · Immunocytochemistry: GFP Transfection and Detailed Neurite Tracing Week 4-6 of 2D culture (transfection at ~day 25)
  1. a Transfect neurons with GFP expression vector
  2. b Image GFP-labeled neurons
  3. c Trace neurites using ImageJ Neurite Tracer
Phase 17 · Immunocytochemistry: Synaptic Markers (Synapsin, Homer, VGLUT1, VGAT) Week 5-6 of 2D culture
  1. a Fix and stain for synaptic markers
  2. b Secondary antibodies, imaging, and quantification
Phase 18 · Immunocytochemistry: GABAergic and Parvalbumin-Positive Neurons Week 5-6 of 2D culture
  1. a Stain for inhibitory neuronal markers
  2. b Secondary antibodies and cell counting
Phase 19 · Immunocytochemistry: Cerebral Organoids - Layer Formation and Aβ Week 8 (2 months) of organoid culture
  1. a Fix and section organoids
  2. b Stain for cortical layer markers
  3. c Stain for amyloid-beta deposition
  4. d Secondary antibodies and imaging
Phase 20 · Immunohistochemistry: Golgi Staining of Organoid Dendrites Week 8 (2 months) of organoid culture
  1. a Fix organoids and prepare vibratome sections
  2. b Golgi impregnation procedure
  3. c Visualize Golgi-stained neurons and trace dendrites
  4. d Quantify dendrite length
Phase 21 · Multielectrode Array (MEA) Recording of Organoid Network Activity Week 8+ of organoid culture (2 months and beyond)
  1. a Plate organoids on MEA-compatible culture plates
  2. b Record spontaneous network electrical activity
  3. c Analyze network firing patterns
Phase 22 · Western Blotting: VGLUT1 and VGAT Quantification Week 4-5 of 2D culture
  1. a Harvest and lyse neuronal cultures
  2. b Prepare protein samples and run SDS-PAGE
  3. c Transfer to PVDF membrane and probe with antibodies
  4. d Detect labeled proteins and quantify
Phase 23 · Data Analysis and Statistical Testing Upon completion of experiments
  1. a Organize and summarize data
  2. b Perform statistical tests
  3. c Generate figures and summary statistics

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Attribution

This SOP was written by Organthis based on the published method in Ghatak et al., 2019. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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