Page 353 - IJB-9-2

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

1. Introduction laser-based bioprinting applied in this study, results on
potential cell impairment are not directly transferable. We
Three-dimensional (3D)-bioprinted organ models are prefer laser-based bioprinting due to its versatility, to print
promising alternatives for testing of pharmaceuticals, droplets of cell-containing sols (non-gelled precursors
chemicals, or cosmetics on animals or simple cell cultures. of gels) with low or high cell density (>10 mL ) and
8
-1
They could also provide better understanding into cellular low or high viscosity (1 mPa·s up to more than 1 Pa·s for
mechanisms of specific diseases and, by applying patient- printing of cells) with volumes from a few picoliters up to
derived cells, could enable generation of patient-specific the nanoliter range. For instance, Chrisey et al. published
disease models for personalized medicine.
two studies of printing rat neuronal cells with laser-based
For neural disorders, like epilepsy, schizophrenia, bioprinting, applying a neuronal blastoma cell line and
[42]
bipolar disorder, Parkinson’s disease, or Alzheimer’s dorsal root ganglion neurons . They applied different
[23]
disease, which affect more than one out of seven people , biomaterials in each study and investigated cell survival
[1]
different cell-based brain models are being investigated , after printing. To the best of our knowledge, however, a
[2]
which usually are based on simple cell culture systems, direct comparison of the behavior of different neural cell
3D cell cultures in hydrogels, cell-seeded scaffolds, or cell types upon laser-based bioprinting has not been explored
aggregates (organoids, spheroids) [3-11] . 3D bioprinting could yet, and has only been investigated to a very limited extent
advance such models toward 3D brain tissue constructs in studies applying other printing technologies [43,44] .
with a higher level of complexity and functionality.
Having all the different options for cells raises the
For personalized brain models, patient-derived neural following questions: (i) Which cellular differentiation
cells can be generated by reprogramming cells from blood or stages are optimal for printing brain tissue models? (ii) For
skin to human induced-pluripotent stem cells (hiPSCs) [12,13] these differentiation stages, does co-printing of other cell
and differentiating them to multipotent neural stem cells types like glial cells support neuronal network formation?
(NSCs) and further down the neuronal differentiation In this study, we investigated laser printing of NSCs
[14]
pathway. Thus, different types of required cells like neurons, and pre-differentiated neurons, partially together with
glial cells such as astrocytes or oligodendrocytes, and astrocytes, which were all derived from hiPSCs.
endothelial cells can be generated [15-17] and 3D-bioprinted In a previous study, we compared the application
with determined brain-tissue-like patterning.
of different biomaterials for printing of hiPSCs and
[45]
Published studies demonstrating the printing of found that the best approach was to print with a bioink
neural cells applied mouse, rat, or human cells. These composed of hyaluronic acid and cell culture medium onto
were primary neurons (rat [18-24] and mouse ), primary a layer of Matrigel . Given these findings, we used the
TM
[25]
neural stem and progenitor cells or neural stem cell lines same biomaterials in our experiments on hiPSC-derived
(mouse [26,27] ), neural progenitor cell lines (human ), or NSCs presented here. Matrigel was also used by other
TM
[18]
neurons and neural stem and progenitor cells derived from groups [28,30,42] for printing of neural cells.
iPSCs (mouse and human [28-33] ). Gu et al. printed hiPSCs Our present study includes assessment of post-printing
[28]
and differentiated them to neurons post-printing and viability and proliferation of NSCs and pre-differentiated
[34]
also used immortalized human neural progenitor cells . neurons (all derived from hiPSCs), maintenance of NSCs
[35]
Aside from neural stem cells and neurons, glial cells such stemness, differentiation of printed NSCs toward neurons
as astrocytes (rat ), oligodendrocytes (mouse iPSC- and glia cells, formation of dendrites and synapses by
[19]
derived ), and Schwann cells (rat [36-40] , porcine ) were printed NSCs during differentiation, as well as statistical
[28]
[41]
printed.
evaluation of the functionality of neuronal networks formed
For different printing technologies and bioinks, by printed post-differentiated NSCs and neurons. We used
these studies demonstrated printing of neural cells in fluorescence calcium imaging to monitor and evaluate the
controlled patterns, achieving high viability post-printing, behavior of the printed neuronal networks. Thereby, the
maintenance of neuronal phenotypes, morphology, and frequency and intensity of collective neuronal activity, i.e.,
basic electrophysiological functions [18,21-23,25,29,30,32,37-39,41] ; events in which many neurons are active simultaneously, are
neurite outgrowth, formation of synaptic contacts and considered a measure of the functionality of these networks.
networks, as well as spontaneous and stimulated neuronal NSCs were compared with NSCs cultivated under
activity were shown [19,20,22,24,25,28,32,35] .
neuronal differentiation conditions (pre-differentiated) for 5,
For these printing experiments, mostly extrusion 10, or 20 days to identify the optimal differentiation stage for
and inkjet printers were used. Since their nozzle-based generating neuronal networks by laser printing. Furthermore,
printing techniques differ fundamentally from nozzle-free we also studied whether co-printing of astrocytes increases

Volume 9 Issue 2 (2023) 345 https://doi.org/10.18063/ijb.v9i2.672