Page 372 - IJB-9-2

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

under neuronal differentiation conditions, only a few were addition of other cell types, i.e., astrocytes to NSCs and
TBR1-positive but most of them were MAP2-positive neurons or 20% NSCs to neurons, did not lead to stronger
(Figure 3F). Printed neurons lost the early-born cortical activity and even reduced activity in the case of neurons
neuron marker TBR1 during further differentiation. (not significant). The observed neuronal activity of printed
Under neuronal differentiation conditions for 30 NSCs or NSC-based cell mixtures was always higher than
days, printed NSCs already express glutamatergic and the activity of printed neurons or neuron-based mixtures
GABAminergic neuron lineage markers vGLUT1 and (significant when compared with neuron-based mixtures).
GABA; thus, within this period, printed NSCs undergo all When focusing on collective neuronal activity,
differentiation stages from stem cell to specific neuronal statistical evaluation revealed a distinct difference between
lineage. Aside from that, spontaneous differentiation toward NSCs and neurons, even when d5 neurons (NSCs, pre-
glial cells also occurred under neuronal differentiation differentiated for 5 days) were used. When NSCs were
conditions, and even mature oligodendrocytes (Figure 3F) printed and cultivated under neuronal culture condition,
and astrocytes (Figure 3E) were observed. Similar findings the activity increased with time (Figure 8). Conversely, we
were also reported by other groups [29,30,34,35] . observed collective activity earlier in neurons, even in d5
neurons; however, this collective activity did not increase
As shown in Figure 4, the spontaneous differentiation
to astrocytes under neuronal differentiation conditions any further with extended cultivation under neuronal
differentiation condition. Interestingly, we did not observe
was much higher for printed NSCs and d20 neurons than considerable differences in the synaptogenesis of NSCs and
for normal cell culture (≈ 0.1% for passage-5 NSCs, ≈ d20 neurons (Figure 5); therefore, the distinct difference in
0.4% for d20 neurons; Figure 1C). Also noticeable was the collective activity was not expected.
proportion of astrocytes after prolonged cultivation under
neuronal culture conditions of printed samples with and The addition of 50% astrocytes (Figure 10) increased
without added astrocytes (Figure 4); this does not seem burst size and global efficiency of collective neuronal
to have been dependent on the proportion of astrocytes at activity of printed NSCs significantly, while the modularity
the time of printing. We assume that both might be due (community statistic Q) decreased; 20% astrocytes did
to the high cell concentration in the 3D-printed droplets not have a significant effect. In opposite, the spontaneous
compared to 2D cell culture [34,35] . activity was not significantly affected by addition
Figure 5 demonstrates synaptogenesis and formation of astrocytes (Figure 6). Unexpectedly, neither the
of neuronal networks by printed NSCs and d20 neurons spontaneous (Figure 6) nor the collective (data not shown)
cultivated under neuronal differentiation conditions for activity of d20 neurons was significantly affected by added
20 days post-printing. We did not observe difference in astrocytes; actually, we had expected that the astrocytes
[64,65]
synaptogenesis between NSCs and d20 neurons; however, would help the neuronal network develop better .
the formation of dendrites and thus the neuronal networks Therefore, in terms of maximal collective neuronal activity
during differentiation of printed NSCs depended on cell and functionality of neuronal networks, NSCs with 50%
density. Better results were achieved in bigger droplets added astrocytes were the best in our study. Moreover, the
with more cells (Figure 2D and 2E). Formation of synapses presence of astrocytes resulted in reduced migration (NSCs
by printed hiPSCs, differentiated 30–37 days post-printing, only) and the formation of more compact cell aggregates
was also reported by Gu et al. [34,35] . (NSCs and neurons) as well as supported printing pattern
maintenance (NSCs and neurons).
An important part of the experiments within this study
To the best of our knowledge, this is the first study
was the calcium imaging analysis of printed NSCs, pre- that directly compares printing of hiPSC-derived NSCs
differentiated neurons, astrocytes and compositions of and neurons generated from these NSCs at different
this cell. Calcium imaging demonstrates functionality of differentiation stages as well as investigates the impact
neuronal networks by visualizing spontaneous activity and of co-printed astrocytes. Future studies will investigate
collective activity of several neurons (bursting events).
the effect of other biomaterials applied as bioink and
Our study revealed that there was no clear relationship substrate. In this study, Matrigel , a matrix extracted
TM
between strength of spontaneous neuronal activity and from Engelbreth-Holm-Swarm mouse sarcoma, was
cell composition, except from the fact that the neuronal used as a substrate only. We did not mix Matrigel into
TM
activity of astrocytes alone was strongly reduced (Figure 6). the bioink, but printed the bioink with cells onto the
When comparing the neuronal activity of printed NSCs Matrigel substrate. For printing thick neural tissue, the
TM
and pre-differentiated neurons with succeeding neuronal effect of such a substrate is rather limited to the bottom
differentiation via calcium imaging, we found that the layers. When printing thick neural tissue, results could

Volume 9 Issue 2 (2023) 364 https://doi.org/10.18063/ijb.v9i2.672