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International Journal of Bioprinting Pregabalin impact on 3D neuronal models

spikes and bursts decreased in all the conditions compared
to the baseline measurements. However, network bursts
increased in the PDL/laminin- and IIZK-cultured neurons
and nearly returned to the baseline value in PDL-cultured
neurons. Collectively, these observations underscore
the modulation of neuronal activity in the presence of
pregabalin (Figure 7).
3.6. High-throughput 3D bioprinting for
neuropharmacology applications
In this study, we aimed to evaluate the potential
of high-throughput 3D bioprinting techniques in
neuropharmacology applications. Our study pioneered
the application of this advanced printing method to the
fabrication of CN models. By employing 3D bioprinting
technology coupled with a coaxial nozzle, as previously
described and applied for neurons by Susapto et al.
29
35
(Figure 8A), we succeeded in bioprinting a 3D matrix
environment using 2 mg/mL IIZK peptide hydrogel within
96-well plates (Video S4, Supporting Information).
The experiment involved bioprinting cortical neurons
treated with 10 μM concentrations of pregabalin and
untreated controls using the 3D peptide matrix. The
gelation of the IIZK-based bioink was sped up using 5×
PBS, ensuring that the cells remained viable and functional
throughout the printing process and subsequent culture
period. Following the bioprinting process, the cultures
were maintained for 3 days before live/dead staining was
conducted. This assay revealed excellent post-printing
cell viability for both treated and untreated groups, as
Figure 5. Changes in gene expression induced by pregabalin exposure evidenced by the predominance of green fluorescence
in embryonic cortical neurons (ECNs). No significant changes were indicating live cells in the fluorescent images (Figure 8B
observed in the expressions of (A) Emx2, (B) Gsx2, (C) Lhx6, and (D)
Nkx2.1 in ECNs. Pregabalin exposure led to significant alterations in and C).
the expression of (E) Dlx2, (F) Olig2, (G) Nhlh2, (H) Otp2, (I) Zic1, and To address concerns regarding the potential impact
(J) Gad67. Data are presented as the mean ± standard error of the mean
(SEM) and were analyzed using a t-test. * p < 0.05; ** p < 0.01. of printing shear stress or other printing-related factors
on neuronal integrity, we performed immunostaining for
firing rates, number of bursts, and their durations were neuronal markers TUJ1 and TBR1 on day 3 post-printing.
observed in 3D mouse ECNs on day 14 of culture on IIZK- Treated and untreated neurons exhibited both markers
based scaffolds (Figure 6B–E). and maintained normal morphology (Figure 8D and E),
affirming that the bioprinting process had not adversely
Neurons that displayed high activity (i.e., spike count affected their fundamental cellular characteristics.
exceeding 1000) were selected for pregabalin treatment. Moreover, the cells were observed to be distributed across
Baseline recording was conducted before treating the different layers within the 3D structures, a complexity
neurons with pregabalin. Neuronal activity was then captured through Z-stack imaging, underscoring the
recorded at different time points up to 20 h after adding technique’s capability to replicate the intricate architecture
pregabalin. Notably, in the 3D-cultured neurons, a surge of neural tissue (Video S5, Supporting Information).
in activity was observed for up to 4 h after the treatment,
evidenced by the increased number of spikes, bursts, and 4. Discussion
network bursts. A significant increase in the network burst
activity was observed after 15 min of exposure to pregabalin In this study, we investigated the mechanisms through
compared to the untreated 3D-cultured ECNs (Videos S2 which the administration of pregabalin during pregnancy
and S3, Supporting Information). After 8 h, the number of impacts the development of primary cortical neurons

Volume 10 Issue 4 (2024) 414 doi: 10.36922/ijb.3010