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International Journal of Bioprinting Laser bioprinting of hiPSC-derived neural stem cells and neurons

be different due to the spatially decreasing effect of the Funding
Matrigel . Matrigel is widely used in 2D and 3D cell The research presented here was funded by European
TM
TM
culture and for bioprinting as well, but its composition is Union’s Horizon 2020 projects MESO-BRAIN, Grant
rather undefined. Therefore, more defined alternatives are 713140, PLATFORMA, Grant 951890, the Ministerio
required for standardized test-systems. Further important de Ciencia e Innovación (Spain, Grant PID2019-
aspects are the impact of different printing patterns on 108842GB-C21), the Generalitat de Catalunya (Spain,
neuronal network formation and the interaction between Grant 2017- SGR-1061), and German Cluster of Excellence
different cells in these patterns, especially when printing Ex62/2 Rebirth.
different cells at specific positions.
5. Conclusion Conflict of interest

NSCs could be laser-printed with high viability of The authors declare no conflicts of interests.
about 95% and without affecting their stemness and Author contributions
differentiation potential. In contrast, the viability of printed
neurons (pre-differentiated NSCs) decreased with duration Conceptualization: Andrea Deiwick, Lothar Koch
of pre-differentiation period (below 70% for 20 days of pre- Formal analysis: Lothar Koch, Jordi Soriano
differentiation). We found significant differences in further Funding acquisition: Boris Chichkov, Jordi Soriano
differentiation of printed NSCs and neurons compared Investigation: Andrea Deiwick, Lothar Koch
to normal cell culture, especially the differentiation to Methodology: Andrea Deiwick, Lothar Koch, Jordi Soriano
astrocytes under neuronal differentiation conditions was Supervision: Boris Chichkov
increased. This can be explained by the higher local cell Visualization: Andrea Deiwick, Lothar Koch, Jordi Soriano
density in the printed droplets—at similar average cell Writing – original draft: Lothar Koch, Andrea Deiwick,
density. Synaptogenesis, neuronal network formation and Jordi Soriano
neuronal activity of connected cells were observed during Writing – review & editing: Lothar Koch, Andrea Deiwick,
differentiation of printed NSCs and neurons. While we did Jordi Soriano, Boris Chichkov
not observe differences in synaptogenesis, we found distinct
differences in network formation and neuronal activity. Ethics approval and consent to participate
Pre-differentiation of NSCs before printing did Not applicable.
accelerate both neuronal network formation after printing
and onset of collective neuronal activity. However, further Consent for publication
development of frequency and intensity of collective Not applicable.
activity was superior when NSCs were applied. Addition of
astrocytes to NSCs further supported network formation
and increase of collective activity, while no comparable Availability of data
effect was observed when astrocytes were added to neurons. The data used in this study are available from the
Therefore, NSCs seems to be the best choice for corresponding author upon reasonable request.
printing of functional neuronal networks. However, if
complex systems containing neurons and other cell types References
shall be printed, differentiation post-printing alone might
not be possible. Moreover, proliferation and migration of 1. WHO, 2006, Neurological Disorders: Public Health Challenges,
World Health Organization, Geneva, Switzerland, 204, table
NSCs were stronger than those of neurons; maintenance of A.4.7.
printed pattern was better when pre-differentiated neurons
were printed. Possibly, these findings would be different 2. Zhuang P, Sun AX, An J, et al., 2018, 3D neural tissue
if other biomaterials were applied, which better support models: From spheroids to bioprinting. Biomaterials, 154:
network formation by pre-differentiated neurons. We aim 113–133.
to investigate this in a subsequent study. https://doi.org/10.1016/j.biomaterials.2017.10.002
3. Turunen S, Joki T, Hiltunen ML, et al., 2017, Direct laser
Acknowledgments writing of tubular microtowers for 3D culture of human
The authors would like to thank Axol Biosciences Ltd. pluripotent stem cell-derived neuronal cells. ACS Appl
(Cambridge, UK) for providing NSCs and culture Mater Interfaces, 9:25717–25730.
materials. https://doi.org/10.1021/acsami.7b05536

Volume 9 Issue 2 (2023) 365 https://doi.org/10.18063/ijb.v9i2.672