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BRAIN Publication-derived

Simplified Brain Organoids for Rapid and Robust Modeling of Brain Disease

Source Ha et al., 2020 · Stem Cell Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, South Korea · 10.3389/fcell.2020.594090

👤 Jeongmin Ha, Ji Su Kang, Minhyung Lee, Areum Baek, Seongjun Kim, Sun-Ku Chung, Mi-Ok Lee, Janghwan Kim ⏱ 30 days 📋 8 phases 🧫 Human iPSC; Patient-Derived iPSC (Parkinson's disease, LRRK2 G2019S)

Abstract

This protocol establishes simplified brain organoids (simBOs) generated from expandable hPSC-derived primitive neural stem cells (pNSCs) in approximately 2 weeks. The simBOs are structurally simpler but more homogeneous than conventional brain organoids and can be rapidly specified into midbrain-like organoids for Parkinson's disease modeling and drug screening.

Cell source
Human iPSC; Patient-Derived iPSC (Parkinson's disease, LRRK2 G2019S)
Application
Disease modeling and drug screening

Protocol overview

40 steps across 8 phases

hiPSC Maintenance and Differentiation into Primitive Neural Stem Cells (pNSCs) Day 0–7 (initial induction)
  1. 1 Maintain hiPSCs in TeSR-E8 medium
  2. 2 Seed hiPSCs on Geltrex-coated dishes for neural induction
  3. 3 Switch to Neural Induction Medium-I (NIM-I) for 2 days
  4. 4 Switch to Neural Induction Medium-II (NIM-II) for 5 days
  5. 5 Dissociate cells and transfer to Geltrex-coated dishes with Neural Stem Cell Maintenance Medium
Spontaneous Differentiation of pNSCs in 2D Culture (Control/Reference) Day 0–30 (optional, for comparison)
  1. 1 Dissociate pNSCs into single cells
  2. 2 Plate cells on Geltrex-coated 2D culture plates
  3. 3 Switch to differentiation medium and feed every other day
Generation of Simplified Brain Organoids (simBOs) Day 0–20 (organoid differentiation)
  1. 1 Seed pNSCs on ultra-low attachment 96-well plates for self-organization
  2. 2 Culture for 4 days to allow sphere formation
  3. 3 Transfer 96 spheres to bioreactor with differentiation medium
  4. 4 Culture in bioreactor with medium changes every 3 days
  5. 5 Harvest organoids at day 10–20
LRRK2 Inhibitor Treatment (PD Modeling) Day 10–13 (3-day treatment window)
  1. 1 Apply PFE-360, an LRRK2 kinase inhibitor, to midbrain-like simBOs.
  2. 2 Maintain PFE-360 treatment for 3 days
  3. 3 Harvest organoids for analysis
Immunohistochemistry / Immunocytochemistry Variable (performed on harvested organoids or 2D cultures)
  1. 1 Fix organoids with 4% paraformaldehyde overnight
  2. 2 Cryoprotect organoids with sucrose gradient
  3. 3 Embed in OCT compound and freeze
  4. 4 Cryosection organoids and 2D cultures
  5. 5 Block and permeabilize samples
  6. 6 Incubate with primary antibodies
  7. 7 Wash with dilute BSA/DPBS and incubate with secondary antibodies
  8. 8 Acquire fluorescent images
Quantitative Real-Time PCR (qRT-PCR) Variable (performed on harvested organoids)
  1. 1 Lyse organoids and extract total RNA
  2. 2 Synthesize cDNA from total RNA
  3. 3 Perform quantitative real-time PCR
  4. 4 Analyze Ct values and normalize expression data
Western Blotting Variable (performed on harvested organoids)
  1. 1 Prepare whole-cell protein extracts
  2. 2 Quantify protein concentration
  3. 3 Separate proteins on MP TGX Precast Gels
  4. 4 Transfer proteins to PVDF membrane
  5. 5 Block membrane and incubate with primary antibodies
  6. 6 Wash and incubate with HRP-conjugated secondary antibodies
  7. 7 Develop blot and acquire band images
  8. 8 Quantify band intensity
Dopamine Secretion Assay Day 13 post-differentiation (24-hour culture medium collection)
  1. 1 Culture midbrain-like simBOs to day 13
  2. 2 Collect 24-hour culture medium
  3. 3 Perform enzyme-linked immunosorbent assay (ELISA) for dopamine
  4. 4 Quantify dopamine concentration

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Ha et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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