TY - JOUR
T1 - A two-culture method for exposure of human brain organotypic slice cultures to replicating human immunodeficiency virus type 1
AU - Martinez, Ricardo
AU - Eraso, Daniel
AU - Geffin, Rebeca
AU - McCarthy, Micheline
N1 - Funding Information:
This study was supported by the Department of Veteran Affairs Merit Review Program , and by a developmental grant from the University of Miami Developmental Center for AIDS Research (P30AI073961). We thank Dr. Minh Tran's Lab at VAMC, Miami, FL for the use of their fluorescent microscopes. We are grateful to Alina Fernandez for her excellent technical assistance.
PY - 2011/8/30
Y1 - 2011/8/30
N2 - 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.
AB - 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.
KW - Cell differentiation
KW - HIV
KW - Neuroepithelial progenitor cells
KW - Organotypic slice culture
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U2 - 10.1016/j.jneumeth.2011.06.016
DO - 10.1016/j.jneumeth.2011.06.016
M3 - Article
C2 - 21726582
AN - SCOPUS:79960891715
VL - 200
SP - 74
EP - 79
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
SN - 0165-0270
IS - 1
ER -