video: The movies represent confocal reconstructions of hippocampal neurons grown on 3-D-Scaffold. The cultures were first immunolabelled for β-tubulin III (in red) in order to visualize the neurons; Glial Fibrillary Acidic Protein (GFAP, in green) to visualize glial cells and DAPI (in blue) for nuclei. The images were acquired using a sophisticated Laser-Scanning Confocal Microscope for viewing samples in 3-D, which is not possible in standard microscopes. The 3D renderings demonstrate that the cells grown on such substrates are capable of migrating and forming connections in all spatial configurations, thus overcoming the constraints imposed by culturing on flat surfaces. The reconstructions are of neurons and glial cells spreading through a thickness of 60-100 micron (consider that the size of neuronal soma is around 10 micron). Using this highly technological approach we were able to appreciate that the cells and their processes were not simply anchored to the scaffold but were in fact navigating the pores present in the 3-D-structure. view more
Credit: SISSA
Knowledge of the brain (as well as neural engineering technologies) benefits greatly from the possibility of growing networks of live and functioning neurons. Today, neuronal cultures are essentially two-dimensional (they develop on a flat surface like a classic "Petri dish") even though, as is intuitive, the most "natural" condition for a neuron and neural network would be to grow and live in a three-dimensional space. All attempts to date to create 3D cultures have basically been multiple layers of 2D cultures. The structure created by a team coordinated by Laura Ballerini from SISSA is the first to be genuinely three-dimensional, with functioning neurons and astrocytes (for "several weeks").
"We used a elastomeric scaffold, a kind of sponge, on which we then cultured the neurons". Ballerini and her SISSA team (Rossana Rauti and Denis Scaini) worked in close collaboration with the group headed by Maurizio Prato of the University of Trieste (in particular Susanna Bosi, co-first author of the study). Ballerini and Prato have been working together on the study of the interfaces between neurons and nanomaterials for several years.
Recordings of neuronal activity - measured indirectly by imaging calcium variations in the cells' cytoplasm and not directly by recording electrical activity with electrodes, which would be complex for this type of structure - showed that the neurons cultured on the 3D sponge were alive and functional. And more than that, the technique adopted allowed for a direct comparison between the function of the 3D culture and a 2D counterpart, showing that the former was much more complex. "Our technique differs from other attempts made so far, which basically did no more than stack many flat-surface cultures one on top of the other," explains Rauti. "This 'layered' approach has the disadvantage of multiplying the number of neurons in the culture, adding ambiguity to any direct comparison between 3D and conventional cultures, which normally contain a smaller number of cells". "By contrast, this comparison becomes possible with the new technique" explains Scaini, "and that's how we were able to observe that a 3D architecture improves the functional (synaptic) organisation of small clusters of neurons".
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"Proof that the greater functional complexity is a consequence of the 3D structure came from a series of computer simulations and theoretical studies carried out at IIT in Genova which faithfully reproduced our experimental data" explains Ballerini.
Another thing that makes the method adopted in this study unique is the use of nanotubes made of carbon, a material that Ballerini and Prato have been working on for years. "We coated the cavities of the elastomeric scaffold with carbon nanotubes which promote synapse formation among the cultured neurons, thereby further enhancing cell function" comments Ballerini. "The advantage of our method lies in its extreme simplicity. We think that in the future our technique can be adopted by laboratories that grow this type of culture and perhaps become a standard".
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Journal
Scientific Reports