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BRAIN Publication-derived

Fungal brain infection modelling in a human neurovascular-unit-on-a-chip with a functional blood–brain barrier

Source Kim et al., 2021 · Department of Biotechnology, 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 📋 10 phases 🧫 Human neural stem cells (NSC), human brain microvascular endothelial cells (EC), human brain vascular pericytes (PC)

Abstract

This protocol describes the development and characterization of a microfluidic human neurovascular unit (hNVU) chip that reconstitutes a functional blood–brain barrier (BBB) with integrated neuronal and vascular components. The chip models fungal brain infection, specifically Cryptococcus neoformans penetration of the BBB, through co-culture of human NSCs, brain ECs, and pericytes in a 3D brain-mimetic extracellular matrix under gravity-driven unidirectional flow.

Cell source
Human neural stem cells (NSC), human brain microvascular endothelial cells (EC), human brain vascular pericytes (PC)
Application
Disease modeling of fungal brain infection and BBB penetration mechanisms

Protocol overview

39 steps across 10 phases

Microfluidic chip fabrication and surface coating Day -7 to Day -1
  1. 1 Fabricate PDMS microfluidic chips
  2. 2 Coat microchannels with poly-L-lysine and collagen I
Preparation of 3D brain extracellular matrix hydrogel (BHEM) Day -2 to Day 0
  1. 1 Prepare catechol-modified hyaluronic acid (HA-CA)
  2. 2 Prepare brain hybrid ECM (BHEM) hydrogel at optimal composition
  3. 3 Validate hydrogel elastic modulus and swelling properties
Cell isolation and preparation Day -3 to Day 0
  1. 1 Isolate and culture human neural stem cells (NSCs)
  2. 2 Isolate and culture human brain microvascular endothelial cells (ECs)
  3. 3 Isolate and culture human brain vascular pericytes (PCs)
hNVU chip assembly and cell seeding Day 0 to Day 1
  1. 1 Seed neural stem cells (NSCs) into central channel with BHEM hydrogel
  2. 2 Allow NSCs to attach (2–4 hours)
  3. 3 Sequentially seed pericytes (PCs) and endothelial cells (ECs) into side channels
  4. 4 Allow EC and PC attachment (4–6 hours)
Optimization of co-culture medium conditions Day 0 to Day 5
  1. 1 Replace NSC medium with optimized NEP co-culture medium
  2. 2 Initiate stepwise gravity-driven unidirectional flow on Day 1
  3. 3 Gradually increase shear stress to physiological range
  4. 4 Monitor NSC differentiation and BBB formation
BBB permeability characterization and functional validation Day 5 onwards
  1. 1 Perform BBB permeability assay using 70 kDa FITC-dextran
  2. 2 Validate barrier function using 4 kDa FITC-dextran probe
  3. 3 Assess P-glycoprotein efflux pump function using calcein AM
  4. 4 Test BBB response to pro-inflammatory stimulus (TNF)
Fungal inoculation and BBB penetration analysis Day 6 onwards
  1. 1 Prepare Cryptococcus neoformans or control fungal strains
  2. 2 Label fungal cells with calcofluor white (CFW)
  3. 3 Inoculate fungal cells into hNVU chip upper channel
  4. 4 Monitor fungal penetration in real time using live imaging
  5. 5 Quantify BBB penetration ability
  6. 6 Perform immunostaining of fungal-infected BBB
  7. 7 Assess direct astrocyte–fungal interactions
Host paracrine signalling analysis upon fungal infection Day 6 to Day 7
  1. 1 Collect culture medium from infected and control hNVU chips
  2. 2 Perform immunoassay to quantify secreted angiogenesis-related proteins
  3. 3 Analyze paracrine factor levels and interpret host response
Multi-organ hNVU chip for neurotropism assessment Day 5 to Day 7
  1. 1 Assemble the hNVU chip containing brain and liver units.
  2. 2 Seed multi-organ chip with NSCs, ECs, PCs, and Huh-7 cells
  3. 3 Inoculate fungal cells into multi-organ chip
  4. 4 Quantify degree of neurotropism
  5. 5 Assess effect of inositol supplementation on neurotropism
Screening fungal mutants for BBB penetration and neurotropism genes Day 5 to Day 7
  1. 1 Prepare C. neoformans gene-deletion mutants
  2. 2 Perform parallel neurotropism and penetration screen
  3. 3 Classify mutants as BBB-penetration-defective, neurotropism-defective, or both
  4. 4 Validate findings and propose mechanistic insights

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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