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International Journal of Bioprinting 3D cell culture model for neural cell analysis

3.2. Optimization of 3D bioprinting process observed for NG108-15 cells; however, a slight decline in
and cell viability viability was noted on Day 5 compared to Day 3, suggesting
In this study, extrusion pressure was the primary variable a potential sensitivity to prolonged culture conditions or
parameter, while bed temperature, print head temperature, particle exposure (Figure 4b).
and print speed were maintained at constant values of 10°C, Our statistical analysis, conducted using separate
24°C, and 11 mm/s, respectively. Our findings indicate that one-way ANOVA tests, indicated that the viability of
an extrusion pressure of 6 kPa facilitated the generation C6 astrocyte-like cells showed no significant differences
of consistent GelMA droplets with satisfactory structural between the control group (cells-only embedded in
integrity (Figure 3a and b). Furthermore, the effect of four 3D-bioprinted hydrogels) and our embedded model
distinct nozzle gauge sizes—20G, 22G, 25G, and 27G— particles in 3D-bioprinted hydrogels: CrCoMo (p = 0.65),
was evaluated for the droplet printing of GelMA hydrogels ZTA (p = 0.34), PEEK-OPTIMA™ (p = 0.058), and Ceridust ®
mixed with particles. The print speed and extrusion 3615 (p = 0.94). Comparable results were observed for
pressure were fixed at 11 mm/s and 6 kPa, respectively. NG108-15 cells. Our statistical analysis revealed no
Nozzles with gauge sizes of 20G (0.58 mm inner diameter) significant differences in NG108-15 cell viability between
and 22G (0.41 mm inner diameter) successfully extruded the control group (cells-only in 3D-printed hydrogels) and
the GelMA hydrogel–particle mixture. Conversely, nozzles cell exposed to model particles in 3D-bioprinted hydrogels:
with gauge sizes of 25G (0.25 mm inner diameter) and CrCoMo (p = 0.32), ZTA (p = 0.83), PEEK-OPTIMA™
27G (0.20 mm inner diameter) failed to achieve consistent (p = 0.64), and Ceridust 3615 (p = 0.56). Additionally,
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extrusion of the bioink–particle mixture. Based on there were no significant variations in the viability of C6
these observations, the 22G nozzle was selected for the astrocyte-like and NG108-15 cells across different particle
fabrication of 3D model particle constructs, both with and volumes (0.5, 5, and 50 μm³ per cell) within the CrCoMo
without the incorporation of neural cells (Figure 3c). model particle (p < 0.46). However, for both cell types, a
To assess the biocompatibility and proliferation significant revduction in viability (p < 0.05) was observed in
potential of neural cells within the 3D-bioprinted the positive control (cells exposed to DMSO) at each time
environment, cell viability was evaluated at Days 1, 3, point when compared to the cell-only negative control.
and 7 within 5% (w/v) GelMA hydrogels and compared We performed a two-way ANOVA statistical analysis
to 2D cell culture models as a control (Figure 3d and e). to assess the impact of culture time, biomaterial type,
Both C6 Astrocyte-like and NG108-15 cells were seeded and their interaction on the viability of C6 astrocyte-like
at an initial concentration of 1 × 10⁴ cells/mL across both cells. The assessment was based on estimated marginal
2D and 3D models (Figure 3f). A progressive increase in mean luminescent values (±95% confidence interval [CI])
cell viability was observed from Day 1 to Day 7 in both obtained from an ATP assay, which served as an indicator
environments. Notably, the proliferation rates of cells of cell viability (Figure 5). These values were measured for
within the GelMA hydrogels were comparable to those C6 astrocyte-like cells exposed to different model particles
in the 2D control (Figure 3d and e). Qualitative analysis or at different dose concentrations over time (Table 2).
further confirmed enhanced proliferation over the 7 days
of cell culture. By Day 7, NG108-15 cells exhibited neurite Our statistical analysis demonstrated a significant
outgrowth, indicating that the bioprinted 3D droplets interaction between culture time and biomaterial type
supported the growth of these cells. The incidence of cell (p < 0.001, Figure 5a). Additionally, both culture time and
death remained minimal throughout the 7-day culture biomaterial type had a significant impact on the viability
period, as evidenced by the sparse presence of red dots of C6 astrocyte-like cells (p < 0.001, Figure 5b and c). The
from propidium iodine staining. post hoc analysis further revealed significant differences in
cell viability across the culture time points (Days 1, 3, and
3.3. Biological assessments 5), with a progressive increase from Day 1 to Day 3, which
continued through Day 5 (Figure 5b).
3.3.1. Cell viability
The viability of C6 astrocyte-like and NG108-15 cells We observed no significant difference in cell viability
was assessed over a 5-day culture period in both (p < 0.9) for CoCrMo model particles across varying
experimental groups (cells embedded with model particles volumes (0.5, 5, and 50 μm³ per cell, Tables 1 and 2).
in 3D-bioprinted hydrogels) and control groups (cells However, the overall cell viability was significantly higher for
embedded without particles) (Figure 4). Luminescence CoCrMo (50 μm³ per cell) compared to PEEK-OPTIMA™,
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readings, indicative of cellular metabolic activity, showed Ceridust 3615, and ZTA model particles (p < 0.001). No
a continuous increase in viability for C6 astrocyte-like significant differences in cell viability were found between
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cells across the 5 days (Figure 4a). A comparable trend was PEEK-OPTIMA™ and Ceridust (p = 0.6) or between

Volume 11 Issue 4 (2025) 263 doi: 10.36922/IJB025180174