2.5 Biological sample obtention
The CNNs grown inside the microfluidic chip are primary cultures of
dissociated neurons from the frontal ganglia of Schistocerca
gregaria locusts. This model is widely used as it shares neuronal basic
features with vertebrates while having larger neurons. This last feature
makes our model an advantageous alternative to mammals for the study of
structural details of the neuronal network. Locusts are stored in a cage
at 30°C with 12:12 night-day cycle and daily fed with grass.
CNNs are obtained following the protocol described in Ref.. Each adult
locust is dissected to extract its frontal ganglion, which contains
approximately 100 neurons. The connectivity tissue of the ganglia is
removed by enzymatic and mechanical procedures to obtain a suspension of
dissociated neuron somas in Leibovitz’s L-15 medium supplemented with
L-glutamine (Sigma Aldrich/Merck) and 0.01 % penicillin-streptomycin
(Biological Industries, Israel). The medium is furthermore enriched with
5% locust hemolymph as a growth factor for the neurite development. To
inhibit the damaging melanin production, the hemolymph is heated at 60°C
for 1 h, then frozen at -20°C, and finally filtered and centrifugated
before being added to the medium.