A Novel In Vitro Mouse Model to Study Mycobacterium tuberculosis Dissemination Across Brain Vessels: A Combination Granuloma and Blood‐Brain Barrier Mouse Model

Abstract

In vitro culture models of the blood-brain barrier (BBB) provide a useful platform to test the mechanisms of cellular infiltration and pathogen dissemination into the central nervous system (CNS). We present an in vitro mouse model of the BBB to test Mycobacterium tuberculosis (Mtb) dissemination across brain endothelial cells. One-third of the global population is infected with Mtb, and in 1%-2% of cases bacteria invade the CNS through a largely unknown process. The "Trojan horse" theory supports the role of a cellular carrier that engulfs bacteria and carries them to the brain without being recognized. We present for the first time a protocol for an in vitro BBB-granuloma model that supports the Trojan horse mechanism of Mtb dissemination into the CNS. Handling of bacterial cultures, in vivo and in vitro infections, isolation of primary astroglial and endothelial cells, and assembly of the in vitro BBB model is presented. These techniques can be used to analyze the interaction of adaptive and innate immune system cells with brain endothelial cells, cellular transmigration, BBB morphological and functional changes, and methods of bacterial dissemination.

Blood–brain barrier dysfunction in l-ornithine induced acute pancreatitis in rats and the direct effect of l-ornithine on cultured brain endothelial cells

“…Primary rat brain endothelial cells and primary brain microvessel pericytes along with primary brain astroglia cells were isolated according to our well established and characterized method [ 27 , 29 , 30 ]. Reagents for the isolation are listed in our previous paper [ 31 ]. Primary cell based culture models are widely used and suitable systems to investigate the pathology, pharmacology and physiology of the BBB in vitro [ 32 ].…”

“…Cell culture medium during all experiments’ growth period contained 10% plasma derived serum (PDS, First Link UK Ltd., Birmingham, UK), 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml sodium selenite (ITS, Thermo Fisher Scientific Inc.), 1 ng/ml basic fibroblast growth factor, 10 mM HEPES, 100 µg/ml heparin and 50 µg/ml gentamycin in DMEM/F12 (Thermo Fisher Scientific Inc.). In all experiments 24 h before treatment cells received 550 nM hydrocortisone to tighten barrier properties [ 31 , 32 ].…”

Mycobacterium bovis Bacillus Calmette–Guérin–Infected Dendritic Cells Induce TNF-α–Dependent Cell Cluster Formation That Promotes Bacterial Dissemination through an In Vitro Model of the Blood–Brain Barrier

“…The in vitro coculture BBB model was established as described previously (20). For each experiment performed using the Transwell inserts, 1 Â 10 5 uninfected or infected DCs were cultured on the top of brain ECs with or without the addition of 1 Â 10 5 P25 PBMCs.…”

“…For each experiment performed using the Transwell inserts, 1 Â 10 5 uninfected or infected DCs were cultured on the top of brain ECs with or without the addition of 1 Â 10 5 P25 PBMCs. All groups received 20% liver granuloma supernatant to enhance in vitro granuloma formation (20). The cell culture medium consisted of DMEM/F12, 10% FBS (Corning, Corning, NY), heparin (100 mg/ml; Sigma-Aldrich, St. Louis, MO), insulin (5 mg/ml), transferrin (5 mg/ml), sodium selenite (5 ng/ml; Sigma-Aldrich, St. Louis, MO), and gentamicin (10 mg/ml; Sigma-Aldrich, St. Louis, MO).…”

“…These models are suitable for analyzing intercellular interactions within an artificial granuloma but lack the possibility of studying M. tuberculosis dissemination in situ or across a vessel wall. To address this, we developed a novel, to our knowledge, combination model of an in vitro granuloma and BBB coculture system (20). This model allows us to study the interaction of Mycobacteriuminfected DCs, Mycobacterium Ag-specific P25 T cells, PBMCs, and the BBB.…”