3 Results & Discussion
Typical CNNs experiments in Petri dishes usually concentrate the cell
population in an approximately circular area (radius ~5
mm), which had been previously coated for cell adhesion. However, this
procedure does not allow an accurate selection of the area to be
functionalized, which finally results in a certain degree of cell
dispersion and the reduction of the cell concentration in the area of
interest. The custom design of our chips permits different shapes and
sizes to develop the desired areas to grow cells. The work area
contention is achieved by designing and fabricating just a circular area
connected to the inlet and outlet entries.
We conducted two culture on chips experiments along this study. In our
experiments it was successfully observed that the microflux drag force
does not affect the cells adhesion and their uniform distribution. As
expected, neurons moved slightly to form neuronal aggregates, but they
were heavily attached to the surface, and no cell loss or detachment due
to a failing in the coating or the flow were observed.
Continuous flow can be regulated to supplement the amount of fresh
medium wanted. Although the flow was able to drag small particles and
debris, larger dissociation particles as void ganglia capsules were not
removed. The flow also washed off particles and toxic metabolites
products of cellular activity, as melanin, particularly important in
cell cultures from insects when hemolymph is added to the medium and
whose accumulation can be cytotoxic due to phenol oxidase activity and
can damage the cells. Consequently, the CNNs developed in the chip
survived longer, at least 22 days, than the ones grown on Petri dish (up
to 14-18 days) , where the medium is not changed. In fact the CNN was
still connected in a good conditions until that day.
Another point to analyse was the homogeneity of the flow distribution.
It has not been observed greater process of detachment of the cells in
the most peripheral areas of the chip, and in addition, network
processes have also been formed in these areas of the chip, so it seems
that the flow has been homogeneous in all the culturing volume.
In experiments where the culture status evaluation and measurement are
done by means of optical microscopy, the microfluidic device design must
fulfil several requirements. In addition to be transparent enough, the
chip dimensions must fit the microscope holding plate. Also, the overall
thickness must allow to focus the culturing surface, and therefore must
fit the range imposed by the microscope focal.
Once both requirements are met, the new neuron-on-a-chip culture is
connected to a microfluidic system with hemolymph enriched medium, as
explained above. The chip is monitored for at least 18 days following
the described observation protocol.
The longitudinal observation of the development of the CNN, as reflected
in Fig. 3, seems to follow a similar path as reported in our previous
works. Isolated neurons in the first day in vitro (Fig.2 A1) started to
grow neurites and being linked to their neighbouring neurons around 3-6
days of culture, getting closer among to form clusters. At DIV 7 (Fig.2
A2), connections among farther neurons are observed and junctions
previously formed are more entrenched. In this step we observed an
increase in the number of connections and a larger network is settled.
After this stage the networks grow, forming bigger clusters, until the
CNNs settled as a consolidated and mature network at day 15 of culture
(Fig.2 A3). No significant changes in the network were observed beyond
this point under our observation until day 22.
In order to properly quantify the different network states with the
tools of graph theory, the resulting graphs from the segmented mosaics
(Fig.2 B1-3) were mapped into adjacency matrices (Fig.2 C1-3) whose
elements aij are equal to 1 if nodes i and nodej are connected and 0 otherwise. Results of the evolution and
development of the graph of the two CNNs grown on chips have similar
trends to those grown on Petri dishes with similar initial density. As
shown in Fig.3A, the number of connected nodes, nodes having at least 1
connection, increases until the 6-7 days in vitro, as a result of the
neurite growth and connections being made. After this stage, the network
development is steady, and in fact, there is a slightly decrease of the
number of connected nodes because when connected neurons form clusters,
the segmentation algorithm identifies them as a single node.
Other network properties we focused on were the longitudinal progression
of the averaged clustering coefficient (C) and of the shortest path
length L, normalised by the size of the largest connected component
SGC1, L / SGC (Fig. 3B). Regarding the normalized
shortest path, high values were observed at the start of the experiment.
Around 4-6 days in vitro, this value decreases, as links between nodes
are formed. Beyond that, L/SGC keeps a low constant value in the more
mature stage. Clustering coefficient shows near 0 values at the start,
as expected as there are no connections between neurons. In the critical
stage of network formation, around 3-6 days in vitro, C increases
revealing the new connections between neurons. When the mature network
is settled, the C value keeps constant at a relatively high value
indicating the presence of loop circuits in the network.
Both values, low shortest path values, and the high clustering
coefficient are studied frequently as characteristics of a small world
structure, where the high tendency to form clusters of nodes in highly
interconnected subgroups and short distance between them contribute to
an optimal functionality in the network based in high efficiency. These
results are very similar to the ones obtained in Petri dishes mentioned
above, with no significant alteration, so we can confirm that the use of
our chips to grow optimal functional CNNs with the desired parameters
not only does not affect their development but they promote a longer
network survival.