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BRAIN Publication-derived

Tubular Human Brain Organoids to Model Microglia-Mediated Neuroinflammation

Source Ao et al., 2021 · Indiana University · 10.1039/d1lc00030f

👤 Zheng Ao, Hongwei Cai, Zhuhao Wu, Sunghwa Song, Hande Karahan, Byungwook Kim, Hui-Chen Lu, Jungsu Kim, Ken Mackie, Feng Guo ⏱ 42 days 📋 12 phases 🧫 Human ESC (WA01)

Abstract

This protocol generates tubular human brain organoids using 3D-printed hollow mesh scaffolds integrated into multiwell plates, enabling standardized, scalable production with reduced hypoxia. The method allows perfusion-based incorporation of isogenic microglia to model microglia-mediated neuroinflammation and study neuro-immune interactions, including responses to opioid exposure.

Cell source
Human ESC (WA01)
Application
Disease modeling

Protocol overview

36 steps across 12 phases

Preparation of Human Pluripotent Stem Cells and Embryonic Bodies Day 0–3
  1. 1 Maintain WA01 hPSC culture
  2. 2 Generate embryonic bodies (EBs)
  3. 3 Prepare EBs for loading into tubular device
Tubular Organoid Device Setup and EB Loading Day 3–4
  1. 1 Load EBs into tubular device
  2. 2 Initiate neural induction with dual-SMAD inhibitors
Tubular Structure Formation and Matrigel Embedding Day 7–14
  1. 1 Load Matrigel into inner lumen
  2. 2 Switch to Wnt activation medium
  3. 3 Culture for neural epithelium expansion
Rocking Platform Perfusion and Neuron Maturation Day 14–35
  1. 1 Transfer device to rocking platform
  2. 2 Switch to neuron growth-supporting medium
  3. 3 Monitor organoid maturation
Microglia Differentiation from WA01 hPSC (Parallel Process) Day 0–21 (parallel to organoid culture)
  1. 1 Plate WA01 cells for hematopoietic differentiation
  2. 2 Differentiate HPC into induced microglia (iMG)
  3. 3 Confirm iMG activation capability
Microglia Integration into Tubular Organoids Day 35–38
  1. 1 Introduce iMG into tubular organoid medium reservoir
  2. 2 Allow iMG migration and integration
Drug Treatment and Neuroinflammation Modeling Day 38–40
  1. 1 Treat with LPS and ATP (positive control for microglia activation)
  2. 2 Treat with DAMGO (opioid receptor agonist)
  3. 3 Treat with LY2828360 (CB2 agonist) to reverse activation
Sample Collection and Analysis Day 38–40
  1. 1 Collect conditioned medium for cytokine analysis
  2. 2 Fix organoids for immunofluorescence and histology
Cryosectioning and Immunofluorescence Staining Day 40–41
  1. 1 Prepare organoids for cryosectioning
  2. 2 Freeze and section organoids
  3. 3 Perform immunofluorescence staining
  4. 4 Apply secondary antibodies and mount
Microglia Morphology and Inflammasome Analysis Day 41–42
  1. 1 Image and analyze microglia morphology
  2. 2 Quantify microglia morphological parameters
  3. 3 Perform proximity ligation assay (PLA) for NLRP3/ASC colocalization
  4. 4 Quantify inflammasome activation
Cytokine Quantification by ELISA Day 41–42
  1. 1 Perform ELISA for IL-1β, IL-18, and TNF-α
  2. 2 Read and analyze ELISA plates
  3. 3 Normalize and compare cytokine levels
Gene Expression Analysis by qRT-PCR (Optional) Day 16 and Day 35
  1. 1 Harvest organoids and extract RNA
  2. 2 Reverse transcribe RNA to cDNA
  3. 3 Perform qRT-PCR
  4. 4 Analyze gene expression data

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Ao et al., 2021. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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