A reliable primary human CNS culture protocol for morphological studies of dendritic and synaptic elements

An in vitro model for investigating human autologous neuronal–astrocyte and immune cell interactions underlying neurodegenerative and immunosuppressive processes in neuropathy

“…Cytoskeletal characteristics of neurons and astrocytes in culture were studied with indirect immunofluorescence staining performed on primary cultures (n¼ 8) at sequential time-points of their in vitro lifespan (0-15 DIV). Differentiated neuron-like cells were identified with cytoskeletal proteins MAP2a&b and Tau, both associated with somatodendritic and axonal regions of mature neurons while astrocytes were characterized with Glial fibrilliary acidic protein (GFAP), a protein associated with their cytoskeletal fibrilliary tangles (Hammond et al, 2002). Neuronal cells cultured on poly-λ-lysine coated glass coverslips were fixed with acetic acid/ethanol (1:9 v/v; 15 min at À20 1C) and permeabilized with 0.5% Triton-X100 (5 min).…”

“…Neuronal cultures from embryonic and neonatal neuronal tissue from several animal models have been reported (Corner et al, 2005;Maslov et al, 2004;Caldwell et al, 2001). Human neuronal cell culture research has focused on the usage of either multipotent embryonic and neonatal stem cells that can be induced to differentiate in vitro (Hammond et al, 2002;Song et al, 2002) or post-mortem brain tissue (Verwer et al, 2002;Brewer et al, 2001;De Groot et al, 2000). Viability and dedifferentiation of adult neurons in vitro have so far been the main disadvantages deterring application of the accomplishments from various animal species to humans.…”

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“…The tissue was dissected in fresh Dulbecco's Modified Eagle Medium, centrifuged and resuspended in a serum-free and pyruvate-free medium as previously described [38,39]. Suspension cultures were initiated at a density of 5 × 10 6 cells/cm 3 in T-75 flasks (resulting in free-floating neuroglial aggregates).…”

“…At four weeks in vitro, cultures were exposed to 1 nM purified recombinant gp120 SF2 (Austral Biologicals, San Ramon, CA) via half media exchange as previously described [38]. Cultures were incubated with gp120 for 72 hours (or less, as in the case of the time series study of apoptosis, necrosis and proliferation).…”

“…Immunofluorescence and confocal imaging followed previously published protocols [38,40]. Briefly, seventy-two hours post-gp120 exposure, cultures were rinsed twice with phosphate buffered saline (PBS) and fixed for 30 minutes with 4% paraformaldehyde.…”