1 Introduction
One of the remaining questions in Neuroscience is how the Central Nervous system self-organizes into an extremely complex system. Among the several perspectives and approaches undertaken to understand this process, in vitro neural cultures have become a powerful tool which allows a very controlled environment, in contrast to in vivo animal models. Although cultured neuronal networks (CNNs) have their own limitations and are very simplified models, they still retain fundamental structural and functional features that can be extrapolated to in vivo models, as memory or connectivity, and are a significant tool in drug testing.
The importance of this last task triggers the need of producing large amounts of equivalent cultures that can be created in the same conditions and simultaneously controlled. For this purpose, recent advances in integration of microfluidics and micro/nanoengineering have allowed the development of a new concept in cell culturing: the organ-on-a-chip. The aim of this microfluidic device is to contain and replicate an organ or tissue structure with its physiology, dynamics and functionality. In recent years, several organ-on-a-chip devices have been reported with different organs as a target, such as lung, liver, heart, kidney, intestine, among others.
In the case of the neuroscience field, microfluidic devices have been used to improve neuronal cultures especially in the Blood-Brain Barrier models. Microfluidic techniques allow precise control of the cell environment, creation of concentration gradients, the delimitation of the cell seeding area, accurate control of the cell density and the possibility of stimulating specific regions of the culture. Besides, the use of microfluidic based devices enables a cost reduction compared with traditional culturing methods because the reagent volumes (culture medium, cell density, drugs, etc.) are significantly smaller.
This technology has been employed in neuronal cultures as well for synapse studies, designing of predefined neuronal connectivities, analysis of neuronal connectivity, neuron polarity or the study of intracellular dynamics in neuronal networks.
The microfluidics chip designs of these previous CNNs usually incorporate a similar fabrication process and material selection based on soft lithography using polydimethylsiloxane (PDMS) for the microfluidic chambers, often combined with a substrate of microelectrode arrays (MEAs). PDMS offers several advantages for microchip manufacturing: it is transparent, flexible, gas permeable, biocompatible, autoclavable, cost-effective and it can protect electronic components. However, soft lithography PDMS process has the mayor inconvenience that any change in the microfluidic design needs a new mould and a new pattern, which slows down the optimization process and testing stage of the microfluidic chip. For this reason, it is needed to search and test new materials in the fabrication of microfluidic chips. In this work we present for the first time a new microfluidic device fabricated from PDMS, vinyl and glass for the study of neuronal circuits development using optical microscopic techniques in a microfluidic device and compare the resulting networks with those grown in a Petri dish as previously reported in Refs.. Besides the abovementioned micro-nano fabrication benefits, in this article we report the behaviour of this novel CNN on a chip to monitor biological networks in a most effective way, offering excellent key performance indicators such as avoidance of contamination, efficient culture medium renewal, higher life expectancy of neurons, increasing longer the period of observation, portability and easy handling and monitoring of the neuronal network formation process; which make this new technology for cells and tissue culture engineering a promising alternative to the conventional methods reported in the scientific literature.