An In Vitro Model for Studying Pathogen Crossing of the Blood-Brain Barrier and Brain Cell Infection

In This Article

Abstract

Source: da Costa, A., et al. A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain. J. Vis. Exp. (2019).

This video demonstrates an in vitro model to study pathogens crossing the blood-brain barrier (BBB). Microvascular endothelial cells, which express tight junctions, form an endothelial layer in the upper chamber, while neurons, astrocytes, and microglia represent the brain parenchyma in the lower chamber, separated by a porous membrane. Virus infection in the BBB model mimics the pathogen crossing from blood to the brain. Upon introducing a neurotropic virus, it crosses the BBB via transcytosis, infects neural cells, replicates, and releases viral progeny, demonstrating successful neuroinvasion.

Protocol

1. Construction and quality control of the BBB-Minibrain (Blood-brain barrier - Minibrain) (Figure 1)

Setting up a BBB-Minibrain Polyester membrane culture insert device (Figure 1B)
1. Grow the human brain endothelial cell line (hCMEC/D3 cells) on 12 well Polyester membrane culture insert filters for 6 days on endothelial cell medium before using them for the experiment.
2. Coat a 12 well plate with poly-D-lysine (1 mL/well, 10 µg/mL, 4 h at RT) and then laminin (1 mL/well, 1 µg/mL, overnight at RT).
3. Remove laminin and add 1 mL of endothelial cell medium supplemented with 5% fetal bovi....

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Results

Brain-blood barrier study; hCMEC/D3 cells in vitro experiment, diagram, and mRNA expression graphs.
Figure 1: the BBB-Minibrain model. A. Time schedule of the hCMEC/D3 cultures and photograph of the BBB-Minibrain device: a polyester membrane culture inserts-filter with hCMEC/D3 endothelial barrier is held with a forcep before to be inserted in to one w.......