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Optimisation of cell culture protocol for neuronal populations coupled to microtransducer arrays

Embryonic day 18 rats were harvested by caesarean section from anaesthetised pregnant dam (animal care followed standard procedures that are in accordance with institutional guidelines).

Cerebral cortices were isolated and chopped into small pieces and exposed to a trypsin (0.125%) digestion and mechanical dissociation through fine-tipped pipette. The resulting tissue was resuspended in Neurobasal medium (Invitrogen) supplemented with 2% B27 and 1% glutamax (both Invitrogen), no antimitotic agent was used to control glial cells proliferation. Dissociated cells were counted, diluted and plated, in a drop of serum-free medium from 7µl up to 150µl, both at high density and low density and, finally, placed in a humidified incubator at 37° C, 5% CO2 for about one hour. When cells adhered to the substrate, 1 ml of Neurobasal supplemented medium was added to the culture.

Once a week 50% of old medium was replaced with fresh pre-warmed medium. Culturing was usually carried on from 5 up to 6 weeks, but spontaneous cortical network activity already arises at the end of the first week. Low-density culture (600cells/mm2) was used onto glass coverslips 20mm, pre-treated only with poly-lysine solution, to characterize neuronal system by immunofluorescence technique. We evaluate from DIV7 to DIV 28 a panel of neuronal markers as indicators of the synaptogenesis maturation( synaptophysin ,synapsin , V-Glut 1, V-GAT, or cytoskeleton neuron protein tubulin βIII and astrocytes fibrillary acidic protein GFAP.

On the other hand, high-density culture was used onto microelectrode arrays, pre-coated with adhesion promoting molecules: Poly-lysine and Laminin, in order to monitoring the electrophysiological activity of neuron network over a long period. During these last two years we used two different types of micro-electrodes MEAS and IMT clustering chip. On the first commercial type we plate around 1000 - 1200cells/mm2, while on the IMT chip we plate on each of the five clusters 2000cells/mm2.

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