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BRAIN Publication-derived

Cerebral Organoids for Infection Studies with Microglia

Full SOP with Academic or a plan

Source Xu et al., 2021 · Rutgers University · 10.1016/j.stemcr.2021.06.011

Authors: Ranjie Xu, Andrew J. Boreland, Xiaoxi Li, Caroline Erickson, Mengmeng Jin, Colm Atkins, Zhiping P. Pang, Brian P. Daniels, Peng Jiang Duration: 40 days 5 phases Cell source: Human iPSC, Human ESC

Abstract

This protocol generates microglia-containing cerebral organoids by co-culturing hPSC-derived primitive neural progenitor cells (pNPCs) and primitive macrophage progenitors (PMPs) in 3D culture. The resulting organoids contain ~8% microglia (mimicking in vivo ratios), develop functional neurons and astrocytes, and enable study of microglial functions including phagocytosis, synaptic pruning, and responses to viral infection such as Zika virus.

Cell source
Human iPSC, Human ESC
Application
Disease modeling (Zika virus infection), developmental neurobiology, pathology studies

Protocol overview

23 steps across 5 phases

Phase 1 · Neural progenitor cell (pNPC) generation and expansion Day 0-21
  1. a Dual SMAD inhibition for neural induction
  2. b Plate EBs on Matrigel-coated dishes
  3. c Manual isolation of neural rosettes
  4. d Expand pNPCs in proliferation medium
  5. e Dissociate pNPCs into single cells
Phase 2 · Primitive macrophage progenitor (PMP) generation and collection Day 0-35
  1. a Generate yolk sac embryoid bodies (YS-EBs)
  2. b Plate YS-EBs and initiate myeloid differentiation
  3. c Collect PMPs from supernatant
  4. d Assess PMP identity and proliferation
Phase 3 · Co-culture of pNPCs and PMPs: organoid initiation and proliferation stage Day 24-35
  1. a Co-plate pNPCs and PMPs in ultra-low-attachment plates
  2. b Culture in 96-well plates for initial aggregation
  3. c Transfer organoids to 6-well plates
  4. d Initiate orbital shaking and patterning (optional)
  5. e Maintain proliferation stage through day 35
Phase 4 · Neural differentiation and microglia maturation stage Day 35 onwards
  1. a Switch to neural differentiation medium
  2. b Maintain organoids in differentiation medium
  3. c Optional: Enhance neuronal maturation with BrainPhys medium
  4. d Assess microglia and neural markers
Phase 5 · Functional assessment and downstream applications Day 40 onwards (dependent on application)
  1. a Assess microglia phagocytic function
  2. b Assess synaptic pruning by microglia
  3. c Perform whole-cell patch-clamp recordings
  4. d Perform Zika virus infection (optional disease model)
  5. e Measure gene expression by qRT-PCR

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Attribution

This SOP was written by Organthis based on the published method in Xu et al., 2021. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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