## 4 Discussion  
Previous studies have extensively explored the use of a 3D environment to create neural tissue by using natural or synthetic polymers. Such strategies are focused on the creation of hydrogel  
Figure 4: Flow cytometry of 3D bioprinted NPCs after 15 days of culture in vitro (**a**) \(\beta\)T-III; (**b**) Olig2; (**c**) HB9. n = 3 for all groups. One-way ANOVA and Tukey post-hoc analysis was performed for statistical analysis using a confidence level of 95% (\(p\) < 0.05) and 99.9% (\(p\) < 0.001). * represents \(p\) < 0.05 and ** 99.9% \(p\) < 0.001. The same data visualized as a scatterplot can be seen in Supplementary Figure S2. Figure 5: Immunocytochemistry after 30 days of culture in vitro. (**a**–**e**) N+; (**f**–**j**) P+; (**k**–**o**) R+; (**p**–**t**) PR+; (**u**–**y**) PR\(-\). Scale bar for (**a**–**d**,**f**–**i**,**k**–**n**,**p**–**s**,**u**–**x**) represents 100 \(\upmu\)m. Scale bar for (**e**,**j**,**o**,**t**,**y**) represent 50 \(\upmu\)m.  
constructs, nerve guidance channels, or electrospinningning [16; 30; 31; 32]. However, those approaches require a large amount of labor and raw materials while their reproducibility remains challenging. Bioprinting technologies offer great advantages over conventional tissue engineering methods as they require less labor and materials and can be automated, producing high-throughput and reproducible processes [18; 19]. In addition, patterned-specific structures with micro-architectures at the micrometer size resolution can be created to mimic the native tissues [18; 19]. Recent studies have used 3D bioprinting technologies where multichannel scaffolds were 3D printed and NPCs within a bioink were extruded in a layer-by-layer fashion [33]. A different study performed by [PERSON] et al. created functional mini tissues by bioprinting human neural stem cells (hNSCs) using a 3D-biophotted system where the bionk is extruded, followed by incubation in crosslinking solution [34]. In this study, we successfully bioprinted NPCs using the microfluidic LOPTM and differentiated these cells into ChaT expressing MNs after 30 days. With this LOPTM technology, all the components of the bioink are mixed prior to the printing process and crosslinked at the 200 \(\upmu\)m \"Y\" junction prior to ejection from the single nozzle. This special feature, where the bioink is surrounded by two crosslinking channels at the Y junction, initiates the crosslinking process from both sides and creates a sheath surrounding the bioink that protects the cells from shear-stress while being extruded. In addition, the 700 \(\upmu\)m extrusion nozzle contained in this LOPTM is made out of PDMS and is therefore flexible and less stiff in comparison with other expression needles used in other studies [33; 34; 35]. Using a pneumatic pressure driven flow, our fibers are extruded at 25 mbar for the bioink, and an increased pressure of 40 mbar for the crosslinker, leading to the creation of printed fiber diameters of (176 \(\pm\) 16 \(\upmu\)m), with all the bioink components homogenously distributed. These diameters are similar to those reported previously when bioprinting NPCs using a point dispensing printing method [33]. On day 7, cell viability was above 81% for all groups, where P+ and R+ showed the highest levels of cell viability at 93% and 91%, respectively. These percentages are higher than those reported by [PERSON] et al., where cell viability is reported to be \(>\)75% after four days of printing iPSC-derived spinal neural progenitor cells (sNPCs) and oligodendrocyte progenitor cells (OPCs) in a 50% Matrigel bioink [33]. Flow cytometry analysis was performed to assess the differentiation of the bioprinted NPCs cultured with combinations of small molecules that have shown to promote MN differentiation after 15 days of culture [25; 26]. At this time point, P+ and PR+ showed the highest levels of the neuronal marker, \(\beta\)T-III, expression, with no statistically significant differences between groups.