An In Vitro Brain Endothelial Model for Studies of Cryptococcal Transmigration into the Central Nervous System

Abstract

Cryptococcus neoformans is an environmental yeast found worldwide that causes lethal brain infections, particularly in immunocompromised hosts. In 2016, there were 280,000 cases of cryptococcal meningitis in the HIV+ population, two-thirds of them fatal; other immunocompromised patients are also affected. The burden of cryptococcal disease and the limits of current chemotherapy create a pressing need for improved treatment. One hindrance to the development of new therapies is lack of understanding of how this pathogen breaches the barriers protecting the brain. Here we describe a tool for investigating this process. This simple in vitro blood-brain-barrier (BBB) model, based on a human brain endothelial cell line grown on a permeable membrane, may be used to assay the BBB transmigration of C. neoformans or other neurotropic pathogens. © 2019 by John Wiley & Sons, Inc.

Keywords: Cryptococcus neoformans; blood-brain barrier; cryptococcosis; fungal meningitis; hCMEC/D3; transwell permeable supports.

Figure 1:. Layout for a typical experiment testing two experimental conditions with experimental and control wells in triplicate.

1^st column, empty insert controls used to measure background values for TEER calculations. 2^nd column, inserts with endothelial cells alone, to assess baseline TEER (in the absence of fungi) that can be used to correct all other TEER values. 3^rd and 4^th columns, inserts with both endothelial and fungal cells. The last two columns may be used to assess any alteration in TEER induced by the fungi (by comparing TEER values to those for the 2nd column) and also to measure fungal traversal under the desired experimental conditions, for example opsonized (column 3) versus non-opsonized fungal cells (column 4).