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BRAIN Publication-derived

Fungal Brain Infection Modeled in a Human Neurovascular-Unit-on-a-Chip with a Functional Blood–Brain Barrier

Source Kim et al., 2021 · Yonsei University, Seoul, Republic of Korea · 10.1038/s41551-021-00743-8

👤 Jin Kim, Kyung-Tae Lee, Jong Seung Lee, Jisoo Shin, Baofang Cui, Kisuk Yang, Yi Sun Choi, Nakwon Choi, Soo Hyun Lee, Jae-Hyun Lee, Yong-Sun Bahn, Seung-Woo Cho ⏱ 7 days 📋 9 phases 🧫 Human neural stem cells (NSCs), human brain microvascular endothelial cells, human brain vascular pericytes

Abstract

This protocol describes the development and culture of a human neurovascular-unit-on-a-chip (hNVU chip) that reconstitutes a functional blood-brain barrier (BBB) comprising human neural stem cells, brain microvascular endothelial cells, and brain vascular pericytes in a 3D hybrid brain extracellular matrix (BHEM) hydrogel under stepwise gravity-driven unidirectional flow. The hNVU chip is used to model and visualize fungal brain infection, particularly the neurotropic behavior and BBB penetration of Cryptococcus neoformans.

Cell source
Human neural stem cells (NSCs), human brain microvascular endothelial cells, human brain vascular pericytes
Application
Disease modeling; fungal meningitis mechanism study; blood-brain barrier permeability and pathogen penetration assay

Protocol overview

28 steps across 9 phases

Microfluidic Chip Fabrication and NSC Seeding Day 0
  1. 1 Fabricate PDMS microfluidic chip with three parallel channels
  2. 2 Coat microchannel with poly-L-lysine and collagen I
  3. 3 Prepare brain hybrid extracellular matrix (BHEM) hydrogel
  4. 4 Seed neural stem cells (NSCs) into the central hydrogel channel
Endothelial Cell and Pericyte Seeding; Culture Medium Optimization Day 1
  1. 1 Seed brain microvascular endothelial cells (ECs) in the upper channels
  2. 2 Sequentially seed brain vascular pericytes (PCs) with ECs
  3. 3 Use optimized NEP (NSC+EC+PC) culture medium
Initiation of Stepwise Gravity-Driven Unidirectional Flow Day 5
  1. 1 Begin stepwise gravity-driven unidirectional medium flow
  2. 2 Monitor barrier formation under flow
BBB Barrier Characterization and Validation Day 5–6 (post-culture)
  1. 1 Perform permeability test using 70 kDa FITC-dextran
  2. 2 Validate P-glycoprotein efflux transporter function with calcein AM assay
  3. 3 Test BBB response to pro-inflammatory cytokine TNF
Fungal Infection: Cryptococcus neoformans Inoculation and Monitoring Day 6 (post-5–6 days culture); infection monitored for 1–2 days
  1. 1 Prepare and label Cryptococcus neoformans cells
  2. 2 Inoculate C. neoformans into the brain unit (3D BHEM hydrogel channel)
  3. 3 Monitor live fungal penetration via time-lapse fluorescence microscopy
  4. 4 Quantify BBB penetration using image analysis
Multi-Organ Chip Experiments: Brain-Liver Neurotropism Assay Day 6–7 (post-5–6 days culture)
  1. 1 Establish a human neurovascular unit chip that integrates both brain and liver units.
  2. 2 Inoculate C. neoformans into the multi-organ chip
  3. 3 Quantify neurotropism as organ-specific fungal distribution
  4. 4 Optional: Test effect of inositol supplementation on neurotropism
Gene Screening: C. neoformans Mutant Penetration and Neurotropism Analysis Day 6–7 (post-5–6 days culture)
  1. 1 Prepare C. neoformans gene-deletion mutants
  2. 2 Inoculate C. neoformans mutants into multi-organ hNVU chips
  3. 3 Quantify BBB penetration and neurotropism for each mutant
Host Paracrine Signaling Analysis and Immunoassays Day 5–6 (post-culture) or Day 1 post-infection
  1. 1 Collect culture medium from hNVU chips
  2. 2 Perform immunoassay for human angiogenesis-related proteins
Immunofluorescence Staining and Cellular Characterization Day 5–6 (post-culture) or Day 1 post-infection
  1. 1 Fix hNVU chips and perform immunofluorescence staining
  2. 2 Acquire confocal microscopy images
  3. 3 Quantify cell populations by immunofluorescence

Full SOP

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Attribution

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

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