Page 371 - IJB-9-2

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

pre-differentiated neurons. In the latter case, it is also of eight cases, than the viability of donor and control cells.
important to ascertain which stage of differentiation is We hypothesize that cell viability is cell density-dependent,
optimal. Essential aspects were viability, maintenance of and that the higher viability of printed cells is caused by
stemness and differentiation potential, as well as ability to the high density of cells concentrated in printed droplets
form functional neuronal networks. compared to the density of donor and control cells seeded
An aspect for future research will be the identification homogeneously by pipetting with the same total amount of
of other biomaterials for printing, bioinks and culture cells per area. In contrast to neurons, the viability of NSCs
substrates, which probably could even better support is much higher and about 91.1 ± 0.5% for control cells and
formation of functional neuronal networks; however, we 94.5 ± 0.6% for printed cells, which is in good accordance to
[32]
applied Matrigel as substrate, and hyaluronic acid and 92 ± 3% reported by Sharma et al. for printing of hiPSC-
TM
cell culture media as bioink in the present study, since one derived NSCs, while other groups reported lower viability
[28,35]
[30]
of our previous studies found that these materials are the of 75%–80% for printing of NSCs. Salaris et al.
best for printing hiPSCs , from which all cells applied in reported viability of 78 ± 4% for hiPSC-derived neurons.
[45]
the present study were generated. However, in these studies a different extrusion-based
bioprinting technique was applied; therefore, the results
These generated cell types were not present as were not directly comparable. By applying a similar laser-
homogeneous cultures. NSCs at passage 3–5 that were based bioprinting technique and rat dorsal root ganglion
printed already contained some TBR1- and MAP2-positive neurons, Curley et al. demonstrated a viability of 84.9
[23]
cortical neurons due to spontaneous differentiation, and ± 4.7% for printed cells, 86.4 ± 3.3% for donor cells, and
a few cells (≈ 0.1%) differentiated toward astrocytes at 89.3 ± 2.0% for control cells. Compared with these results,
passage 5. However, no astrocytes were observed in NSCs we achieved higher viability for NSCs and d5 neurons,
at passage 3, and no oligodendrocytes were observed at but lower viability for d10 and d20 neurons. Furthermore,
any of these passages. On the other hand, d20 neurons Curley et al. did not observe higher but lower viability
[23]
still contained undifferentiated NSCs and some astrocytes for printed cells.
(≈ 0.4%), but over 80% of cells were MAP2-positive. We In our study on printing of hiPSCs with the same
found that there were more than 90% GFAP-positive biomaterials, we achieved generally lower viability of
cells within astrocytes. Furthermore, it can be noted printed (82 ± 1%), donor (84 ± 1%), and control cells (87 ±
that very few astrocytes were generated under neuronal 1%); here the viability of printed cells was significantly
differentiation and very few neurons developed under lower than the control cell viability. However, pluripotent
astrocytic differentiation conditions in conventional hiPSCs are known to be sensitive to environmental
2D cell culture; in contrast, many more astrocytes were parameters, such as culture media components, paracrine
generated under neuronal post-differentiation in printed factors, and matrices. These factors have an impact on cell
3D cell samples. Especially for long-term cultivation post- viability and proliferation; when dissociated into single
printing, the compositions of different cell types need to be cells, programmed cell death (apoptosis) of hiPSCs is
taken into consideration.
immediately induced. The present study confirmed that, in
Initially, the viability post-printing was studied, which comparison to hiPSCs, hiPSC-derived multipotent NSCs
is affected not only by the printing process itself and the are less delicate.
applied biomaterials; harvesting neurons from culture
plates caused dissociation of neuronal networks that Obviously, NSCs proliferate much more than pre-
were spontaneously formed in cell culture and can reduce differentiated neurons. Figure 3C suggests that proliferation
viability. Figure 2C demonstrates this effect. Even control of neurons, in opposite to NSCs, depends on cell density,
neurons that were not printed and were not in contact with which is in line with the fact discussed above that neurons,
any of the biomaterials, bioink, or culture substrate showed like NSCs, concentrated in printed droplets showed higher
a reduced viability that decreased with the duration of viability than cells homogeneously seeded by pipetting.
pre-differentiation to 68.4 ± 0.8% for d20 neurons. We Printed NSCs maintained their multipotency, if
assume that neurons were harmed due to chemical and cultivated under expansion culture conditions, as
mechanical dissociation by pipetting required to dissolve demonstrated by stem cell markers nestin, SOX2, and
networks formed under differentiation culture conditions PAX6 (Figure S2B and 2C). On the other hand, if NSCs
(depicted in Figure S1). On the other hand, the printing were cultivated under neuronal differentiation conditions
process and the applied biomaterials, hyaluronic acid and after printing for 20 days, many TBR1- and MAP2-positive
Matrigel , did not reduce viability. On the contrary, the cortical neurons could be observed (Figure 3D), while
TM
viability of printed cells was higher, significantly in five out among printed d5 neurons cultivated for further 22 days

Volume 9 Issue 2 (2023) 363 https://doi.org/10.18063/ijb.v9i2.672