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.