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International Journal of Bioprinting Pregabalin impact on 3D neuronal models

were then placed in a CO incubator for 24 h. Following equivalent volume of CellTiter-Glo® Reagent (Promega,
2
incubation, PDL was discarded, and the wells were USA) to that of the cell culture medium was added and
washed twice with 1× PBS before the introduction of cells. thoroughly mixed by pipetting 10 times. Following a 25-
Laminin-521 was then diluted to 10 µg/mL in PBS and min incubation at room temperature, the luminescence
applied to the PDL-prepped plates to create a PDL/laminin of the plates was recorded using the PHERAstar FS plate
mixture for coating. The plates were incubated overnight reader (BMG LabTech, Germany). These procedures were
at 4°C or in the incubator. Before their subsequent repeated across specific sample sizes (n = 7 for viability
application, the plates were cleansed twice with PBS. 31 and n = 3 for ATP). The mean and standard error of the
mean (SEM) were computed, and a t-test was applied to
The advantage of 3D in vitro cultures is that they naturally
resemble in vivo development. Neuronal viability, ATP determine statistical significance.
release, morphogenesis, and quantitative polymerase chain 2.4. Immunocytochemistry
reaction (qPCR) assays were conducted on the 3D cultures Untreated ECN cultures were utilized as control groups.
developed for this investigation. For 3D culture, freshly For each experimental condition, including both control
extracted primary mouse ECNs (E12.5) were seeded at a and treated groups, a total of three biological replicates
density of 6 × 10 cells/well in 96-well cell culture plates. We were examined. Moreover, to ensure the reliability and
4
used a previously described short peptide IIZK, prepared reproducibility of our findings, each biological replicate
in Dulbecco’s PBS (DPBS), to prepare 3D hydrogels. Half was subjected to three technical replicates. After culturing
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of the necessary final volume of nuclease-free sterile water for 3 days, the ECNs were fixed using 4% paraformaldehyde
was used to suspend the weighed IIZK peptides. A total of (Santa Cruz Biotechnology, USA) and stored at 4°C in 1×
20 µL of peptide in water was added to each well of the 96- PBS before staining. The detection of tyrosine hydroxylase
well cell culture plate, followed by the addition of 2× PBS (TH) and class III beta-tubulin (TUJ1), markers specific
at an equivalent volume. To ensure complete gelation, the to mouse neurons, involved the use of primary antibodies
plates were incubated at 37°C for approximately 5 min. The G7121 (Promega, USA) and ab112 (Abcam, United
required number of cells was added to the previously made Kingdom [UK]), respectively. The fixed cultures were then
hydrogels. Before adding the cell culture medium, the plates incubated overnight at room temperature with primary
were incubated again for 2–3 min. The culture plates were antibodies against TUJ1 (at a dilution of 1:1500) and TH
filled with N2 medium and cultured for 72 h at 37°C and (at a dilution of 1:500) in a blocking buffer composed of
5% CO . Pregabalin (Sigma-Aldrich, USA) was dissolved 5% goat serum, 0.3% Triton-X, and 0.2% sodium azide.
2
in sterile 1× PBS following the manufacturer’s guidelines. Following the primary antibody application, the cultures
Upon cell seeding, the group designated for pregabalin were exposed to this buffer for an additional hour at room
treatment was administered a 10 µM concentration of the temperature. Secondary antibody treatment involved goat
drug, while the control groups were given a comparable anti-rabbit IgG H&L (Alexa Fluor® 555) and anti-mouse
volume of sterile 1× PBS. Alexa Fluor 488 (ab150078; Abcam, UK), both diluted in
blocking buffer at a ratio of 1:200 and incubated for 2 h at
2.3. Viability of embryonic cortical neurons and room temperature. Subsequently, the wells were rinsed and
assessment of adenosine triphosphate release maintained in 1× PBS. Cell nuclei were stained with DAPI
To elucidate the impact of pregabalin administration on (D1306; Thermo Fisher Scientific, USA) diluted in 1× PBS
the metabolic activity and survival of ECNs, untreated for 5 min, followed by visualization using DMi8 inverted
cultures were utilized as a control for viability and ATP fluorescence microscopy (Leica Microsystems, Germany),
measurements compared to ECNs treated with 10 µM of from which images were captured.
pregabalin on day 3 of cell culture. ECNs were plated at a
concentration of 60,000 cells per well in 96-well cell culture 2.5. Morphogenetic analysis
plates. To evaluate the viability of ECNs in both control In this experiment, we aimed to study the impact of
and pregabalin-exposed samples, AlamarBlue™ reagent pregabalin on the development of central neurons (CNs).
(Thermo Fisher Scientific, USA) was utilized, adhering The effect of pregabalin on neuron development was
to the instructions provided by the manufacturer. The evaluated in ECNs immunolabeled with TUJ1 and Tbr1,
fluorescence was measured using a PHERAstar FS plate as described in the previous section. ECNs were seeded
reader (BMG LabTech, Germany) after preparing the well at a density of 60,000 cells per well in 96-well cell culture
plates. Furthermore, to assess cellular metabolic activity, plates. The developmental parameters under scrutiny
the release of ATP was evaluated using the CellTiter- included the number of neurites, their total length, the
Glo® 3D Cell Viability Assay (Promega, USA). To dissolve length of dominant neurites, and the count of branches,
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the 3D structure formed by the cells and hydrogel, an all of which were assessed using LAS X software (Leica,

Volume 10 Issue 4 (2024) 408 doi: 10.36922/ijb.3010