A two-culture method for exposure of human brain organotypic slice cultures to replicating human immunodeficiency virus type 1

To evaluate the effect of HIV-1 virus on neural cells, we have developed a method to culture human fetal organotypic brain slices in the presence of live virus. Brain slices were placed on semipermeable hydrophilic membrane inserts, resting on top of wells that contain cultured H9 T-cells chronically producing HIV-1. This system allows free exposure of the brain slices to HIV-1, HIV-1 proteins, and other molecules released by the infected T-cells. After specific lengths of time in culture, slices were stained for viability with Calcein-AM and propidium iodide, for neural cell markers such as GFAP, nestin and β-III-tubulin, tested for cell proliferation, and analyzed by fluorescent and confocal microscopy. When cultured in the presence of neural progenitor medium lacking serum, slices were viable and maintained active cell replication for at least 3 weeks in culture, without significant cell death. By comparison with slices co-cultured with uninfected T-cells or with medium alone, slices cultured in the presence of HIV-1 showed increased nestin and GFAP. Moreover, in slices exposed to HIV-1-producing H9 cells, regions of nestin stain were, over time in culture, replaced with GFAP stain. This suggested the process of gliosis often found in brains of HIV-1 infected individuals. This co-culture method can be used to model the dynamics and the microenvironment of brain tissue exposed to HIV-1 and can potentially be used to test therapies directed at preventing HIV-1-induced neural damage.