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BRAIN Publication-derived

Fungal Meningitis/Cryptococcal Infection Brain Organ-on-Chip Models

Full SOP with Academic or a plan

Source 10.1038/s41551-021-00743-8

Duration: 7 days 17 phases Cell source: Human fetal NSC (13 weeks gestation), human cerebral microvascular endothelial cells (hCMEC/D3), human brain microvascular pericytes (HBVP)

Abstract

This protocol describes the establishment of a human neurovascular unit (hNVU) microfluidic chip system combining human neural stem cells (NSCs), cerebral microvascular endothelial cells (ECs), and pericytes in 3D hydrogels to model a functional blood–brain barrier. The system recapitulates BBB architecture and function in vitro and enables study of fungal (Cryptococcus neoformans) neurotropism and CNS invasion mechanisms.

Cell source
Human fetal NSC (13 weeks gestation), human cerebral microvascular endothelial cells (hCMEC/D3), human brain microvascular pericytes (HBVP)
Application
Disease modeling; fungal infection model; blood-brain barrier function

Protocol overview

71 steps across 17 phases

Phase 1 · Cell Culture Preparation Ongoing
  1. a Culture human neural stem cells (NSCs)
  2. b Culture human cerebral microvascular endothelial cells (hCMEC/D3)
  3. c Culture human brain microvascular pericytes (HBVP)
Phase 2 · Hydrogel Preparation Day -3 to -1
  1. a Prepare collagen (Col) hydrogel
  2. b Synthesize hyaluronic acid-catechol (HA-CA) conjugate
  3. c Purify HA-CA conjugate by dialysis
  4. d Prepare BHEM hydrogel
Phase 3 · Hydrogel Characterization Day -1
  1. a Measure rheological properties of hydrogels
  2. b Determine swelling and shrinking properties
  3. c Perform FRAP analysis to measure diffusion coefficient
Phase 4 · Decellularized Brain Extracellular Matrix (BEM) Preparation Day -7 to -1
  1. a Prepare porcine brain tissue for decellularization
  2. b Execute decellularization sequential protocol
  3. c Lyophilize and store decellularized brain tissue
  4. d Perform mass spectrometry analysis of BEM components
  5. e Prepare BEM-hydrogel mixture
Phase 5 · Microfluidic Device Fabrication Day -5 to -2
  1. a Prepare PDMS and cast mold
  2. b Cure PDMS
  3. c Prepare PDMS device
  4. d Sterilize and dry components
  5. e Perform oxygen plasma treatment and assembly
  6. f Post-bonding curing
Phase 6 · hNVU Chip Preparation and Cell Seeding Day 0
  1. a Coat microchannels with poly-L-lysine (PLL)
  2. b Prepare and administer NSC-seeded hydrogels to center channel
  3. c Coat upper channel with collagen and administer medium
  4. d Culture NSCs in 3D hydrogel overnight
  5. e Seed pericytes (HBVP) and endothelial cells (hCMEC/D3) to upper channel
Phase 7 · hNVU Chip Culture and Medium Optimization Day 1-5
  1. a Administer optimized triple co-culture medium
  2. b Establish gradual flow with stepwise shear stress increase
  3. c Maintain lower chambers and channel vacant until analysis
  4. d Monitor barrier function during culture period
Phase 8 · Immunohistochemistry Day 5
  1. a Fix cells in hNVU chip
  2. b Permeabilize cell membranes
  3. c Block non-specific antibody binding
  4. d Incubate with primary antibodies
  5. e Wash with PBS
  6. f Incubate with fluorescently-labeled secondary antibodies
  7. g Final washing and nuclear counterstaining
Phase 9 · Quantitative Real-Time PCR (qPCR) Analysis Day 5
  1. a Collect and extract total RNA
  2. b Synthesize complementary DNA (cDNA)
  3. c Perform qPCR with TaqMan assays
  4. d Analyze and calculate relative gene expression
Phase 10 · RNA Sequencing (Optional Advanced Characterization) Day 5
  1. a Isolate total RNA from NSCs in co-culture
  2. b Prepare RNA-seq library
  3. c Perform sequencing
  4. d Process sequencing data
Phase 11 · Barrier Permeability and Efflux Pump Function Assays Day 5
  1. a Validate barrier integrity with FITC-dextran permeability test
  2. b Measure P-glycoprotein (P-gp) efflux pump functionality
Phase 12 · Cell Viability, Live Imaging, and Cell Tracking Day 5
  1. a Perform Live/Dead viability assay
  2. b Label cells for tracking before seeding
  3. c Image entire chip regions using tile scan and z-stack modules
  4. d Quantify cell numbers using IMARIS
Phase 13 · Angiogenesis Protein Secretion Analysis Day 5
  1. a Collect culture medium from hNVU chips
  2. b Quantify total protein concentration
  3. c Perform human angiogenesis antibody array
  4. d Visualize and quantify array signals
Phase 14 · Fungal Strain Preparation Day -1
  1. a Culture fungal strains
  2. b Prepare and standardize fungal cell suspension
  3. c Label fungal cells with Calcofluor White (CFW) for imaging
Phase 15 · Fungal Blood-Brain Barrier Invasion Assay Day 5
  1. a Validate chip barrier integrity prior to fungal inoculation
  2. b Prepare fungal inoculum for chip inoculation
  3. c Conduct 24-hour fungal infection assay
  4. d Image fungal invasion using multiple microscopy modalities
  5. e Quantify fungal penetration degree
Phase 16 · Multi-Organ hNVU Chip Study for Fungal Neurotropism Day 0-5
  1. a Seed multi-organ chip with brain and liver units
  2. b Administer CFW-labeled fungal cells to liver unit
  3. c Add inositol to liver unit lower channel
  4. d Culture multi-organ chip with fungal challenge
  5. e Assess fungal neurotropism using image analysis
Phase 17 · Statistical Analysis and Data Reporting Upon completion of experiments
  1. a Organize and tabulate experimental data
  2. b Perform statistical comparisons
  3. c Generate graphical representations

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Attribution

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

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