In vitro CNS tissue analogues formed by self‐organisation of reaggregated post‐natal brain tissue

Abstract

Abbreviations used: CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; D-APV, D-2-amino-5-phosphonovalerate; DIV, days in vitro; eGFP, enhanced green fluorescent protein; GFAP, glial fibrillary acidic protein; MEA, multielectrode array; NR1, NMDA receptor subunit 1; PI, propidium iodide; PSD-95, post-synaptic density protein 95 kDa; TEM, transmission electron microscopy; VGAT, vesicular GABA transporter; VGLUT1, vesicular glutamate transporter. AbstractIn this paper, we report the characterization of 'Hi-Spot' cultures formed by the re-aggregation of dissociated postnatal CNS tissue grown at an air-liquid interface. This produces a self-organised, dense, organotypic cellular network. Western blot, immunohistochemical, viral transfection and electron microscopy analyses reveal neuronal and glial populations, and the development of a synaptic network. Multi-electrode array recordings show synaptically driven network activity that develops through time from single unit spiking activity to global network bursting events. This activity is blocked by tetanus toxin and modified by antagonists of glutamatergic and GABAergic receptors suggesting tonic activity of excitatory and inhibitory synaptic signaling. The tissue-like properties of these cultures has been further demonstrated by their relative insensitivity to glutamate toxicity. Exposure to millimolar concentrations of glutamate for hours is necessary to produce significant excitotoxic neuronal death, as in vivo. We conclude that 'Hi-Spots' are biological analogues of CNS tissue at a level of complexity that allows for detailed functional analyses of emergent neuronal network properties.

Dissociated Neurons and Glial Cells Derived from Rat Inferior Colliculi after Digestion with Papain

“…We were able to cultivate the obtained cells for 2–7 days in complemented MACS® Neuro Medium. A neuromedium specifically designed to promote cultivation of cells of the central [37]–[41] and peripheral [42], [43] nervous system seems to be more suitable for the cultivation of the IC than the also tested Panserin 401.…”

IL-1β and HMGB1 are anti-neurogenic to endogenous neural stem cells in the sclerotic epileptic human hippocampus

“…As we hypothesised that neurogenesis is altered via a pro-in ammatory microenvironmental effect, we developed a novel 3D primary culture system based on a technique using rodent tissue [31] in an attempt to model the environmental effects on neurogenesis in human mTLE. The primary cell suspension was diluted to 50,000 viable cells per µl and plated in 5µL drops onto 6mm polytetra uoroethylene membrane discs placed onto the membranes of Millicell inserts in 6-well plates with cortical medium (sTable 2; Supplementary materials) and incubated at 37°C in humidi ed air with 5% CO 2 , with 3-day medium changes (Figure 1A).…”