Skip to content
Back to browse
BRAIN Publication-derived

Synucleinopathy Cerebral Organoids

Full SOP with Academic or a plan

Source Kim et al., 2025 · Department of Molecular Physiology, College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University · 10.3390/cells14181436

Authors: So Jin Kim, Won Hee Jung, Mu Seog Choe, Ye Seong Jeon, Min Young Lee Duration: 150 days 9 phases Cell source: Human ESC (hESC SNUhES31 line)

Abstract

This protocol describes the generation of cerebral organoids (COs) from hESCs carrying the SNCA[A53T] mutation to model synucleinopathy. The A53T COs exhibit elevated α-synuclein expression, phosphorylation, Lewy body-like aggregations, and tau pathology, making them suitable for studying synucleinopathy mechanisms and screening aggregation inhibitors.

Cell source
Human ESC (hESC SNUhES31 line)
Application
Disease modeling and drug screening for synucleinopathy

Protocol overview

29 steps across 9 phases

Phase 1 · hESC Culture and Maintenance Continuous maintenance (prior to modification)
  1. a Feeder-dependent hESC culture
  2. b Transition to feeder-free culture
Phase 2 · Generation of SNCA[A53T] Mutant hESC Line Variable (typically 2-4 weeks from transfection to colony isolation)
  1. a Lentiviral vector construction
  2. b Virus production via HEK293T transfection
  3. c hESC transduction with lentiviral particles
  4. d Single-cell cloning and colony selection
  5. e Characterization of A53T hESC line
Phase 3 · Cerebral Organoid Generation (Days 0-150) Day 0 to Day 150
  1. a Embryoid body (EB) formation (Days 0-6)
  2. b Neural induction (Days 6-10)
  3. c Matrigel culture phase (Days 10-14)
  4. d Long-term spinner flask culture (Days 14-150)
Phase 4 · Immunocytochemistry (Characterization of hESCs and early differentiation) As needed for pluripotency assessment
  1. a Cell fixation and permeabilization
  2. b Primary antibody incubation
  3. c Secondary antibody incubation and imaging
Phase 5 · Immunohistochemistry (Organoid characterization and pathology assessment) At day 150 (mature organoids)
  1. a Organoid cryoprotection and sectioning
  2. b Antibody staining (general immunofluorescence)
  3. c DAB staining for phospho-α-synuclein
Phase 6 · Western Blot Analysis At day 150 (mature organoids)
  1. a Protein extraction by sonication
  2. b SDS-PAGE and membrane transfer
  3. c Membrane blocking and primary antibody incubation
  4. d Secondary antibody and detection
Phase 7 · Enzyme-Linked ImmunoSorbent Assay (ELISA) At day 150 (mature organoids)
  1. a Organoid lysis and protein quantification
  2. b ELISA for phospho-α-Syn and Aβ
Phase 8 · Bielschowsky's Silver Staining for Neurofibrillary Tangles At day 150 (mature organoids)
  1. a Silver nitrate incubation
  2. b Ammonium silver incubation
  3. c Developer and fixation
  4. d Dehydration and mounting
Phase 9 · Drug Screening: SynuClean D Treatment Day 70 to Day 150
  1. a SynuClean D preparation and treatment
  2. b Assessment of drug efficacy

Full SOP

Full SOPs need an account and a plan

Create an account, then get free Academic access (for verified academics) or choose a plan. Plans include step-by-step procedures, reagent concentrations, QC checklists and batch record templates.

Create an account

Already registered? Log in

Attribution

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

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence