Page 415 - IJB-10-4

International Journal of Bioprinting Pregabalin impact on 3D neuronal models

committees of King Abdulaziz University (KAAU), King if there was observation of a vaginal plug that morning.
Abdullah University of Science and Technology, and Taif On E12.5, mouse ECNs were harvested and placed in an
University (7-CEGMR-Bioeth-2018 and 21IBEC023). ice-cold L15 medium (Thermo Fisher Scientific, United
Animals exhibiting any signs of distress, as per the rigorous States of America [USA]). The boundaries of the isthmic
guidelines outlined by the Institutional Animal Care and organizer, telencephalon, and mesencephalon were cut to
Use Committee (IACUC), were promptly excluded and, separate the midbrain and cortical tissues. Tissue from the
when necessary, humanely euthanized. The experimental posterior third of the midbrain was excised to augment
technique used in this study is illustrated in Figure 1. the population of ECNs in the culture. Trypsin (0.05%)
Swiss mice were mated to produce embryos for use in (Thermo Fisher Scientific, USA) and DNase (0.1%) (Stem
the experiment at the Animal Experiment Facility, King Cell Technologies, USA) were used to treat isolated cortices.
2+
2+
Fahad Medical Research Center, King Abdulaziz University, These were diluted in Hanks’ Ca /Mg -free balanced salt
Jeddah, Saudi Arabia. Pregnancy was confirmed based solution (HBSS) (Thermo Fisher Scientific, USA) at 37°C
on estrus cycle monitoring and careful observation of for 15 min. The tissues were washed three times in HBSS
weight gain in the female mice, ensuring a more accurate medium before further incubation in an N2 medium (F12
determination of pregnancy initiation. A vaginal smear medium + minimum essential medium [MEM] with 1
was taken from the female mice to determine the oval mM glutamine + 1% N2 supplement + 1 mg/mL bovine
estrus phase, which allowed for precise identification of serum albumin + 15 mM HEPES with 6 mg/mL glucose
the optimal mating time. The presence of cornified cells + 1% penicillin/streptomycin) (Thermo Fisher Scientific,
indicated the pre-estrus or estrus stage, ensuring a high USA). Primary neurons were extracted within the lab
conception rate of 95%. The animals that were considered 3 days prior to the study, depending on the experiment.
ready for mating were weighed, and this weight was Neurons were isolated from the embryos of five pregnant
registered as a baseline weight. Mating occurred following mice. Each litter yielded 10–12 embryos, contributing to
this confirmation by introducing male mice into the cage an approximate total of 5 million cortical neurons utilized
at 6 a.m. The male mouse was removed the next day at 6 for the experimental procedures.
a.m., and the day after mating was considered embryonic 2.2. 2D and 3D cortical neuron cultures and
day 0.5 (E0.5). The female mice were weighed again after treatment with pregabalin
3 days, and the weight was compared to the baseline The effects of pregabalin were investigated on primary
weight and the weight of a control mouse from the same ECNs cultivated in both 2D and 3D environments. For the
species that was not exposed to this procedure to confirm untreated cultures, 2D-cultured ECNs on poly-D-lysine
pregnancy. The female mice were weighed again after (PDL)-coated cell culture plates were used as controls,
4–5 days to further confirm pregnancy. The day after the providing a baseline for comparison. For cultures treated
animals had mated during the night was recorded as E0.5 with pregabalin, the untreated cultures served as controls,
allowing for a thorough assessment of pregabalin’s
effect. The number of experimental replicates (N) was
3–6, depending on the specific endpoint experiments.
Any cultures displaying abnormal growth patterns,
contamination, or irregularities during the experimental
period were excluded from subsequent experiments and
data analysis. By employing both 2D and 3D culture
techniques, we aimed to explore the intricate responses
of ECNs, shedding light on their behavior and molecular
dynamics in various culture conditions, which will be vital
for advancing our understanding of neuronal physiology
and drug interactions. Primary ECNs, extracted from the
cortex at E12.5, were cultured in 96-well plates coated with
PDL (Sigma Aldrich, USA) at a density of 60,000 cells per
well. The PDL treatment involved dissolving 5 mg of PDL
in 5 mL of 1× phosphate-buffered saline (PBS) (Gibco,
USA), with this solution then being stored at −20°C. For
the coating process, PDL was diluted to a concentration
Figure 1. The overall experimental procedure used in the research.
Abbreviations: ATP, adenosine triphosphate; qPCR, quantitative of 10 µg/mL (1:100 dilution) in 1× PBS and applied to the
polymerase chain reaction. plates in amounts adjusted for the surface area. These plates

Volume 10 Issue 4 (2024) 407 doi: 10.36922/ijb.3010