Page 364 - IJB-9-2

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

There was only slight migration of d20 neurons after 20 days of neuronal pre-differentiation in both NSCs
(Figure 4E) compared to NSCs (Figure 4A); however, as well as d20 neurons, and synaptic compartments can be
d20 neurons (which also contained NSCs and a few seen on the right panels of (B) and (D) in juxtaposition
spontaneously differentiated astrocytes) still proliferated. and in colocalization. This abundance demonstrated
Several connections between printed droplets developed. neuronal maturation, extensive formation of synapses,
Nevertheless, the printed pattern was clearly visible 23 and connection of neurons via synapses to form neuronal
days after printing. When 20% astrocytes were added networks.
to neurons before printing, the pattern (Figure 4F) was
compact 23 days after printing compared to neurons alone 3.10. Dependence of neuronal activity on printed
(Figure 4E), although there were more MAP2-positive cell type
neurons in the space between these droplets. Similar to Collective neuronal activity always involves an increase of
2+
neurons alone, several connections had formed between the level of calcium ions (Ca ). The intracellular calcium
printed droplets. Furthermore, addition of 20% of NSCs concentration of neurons at rest is about 50–100 nM, but
to neurons before printing was investigated. In this case, transiently rises to ten- or hundred-times higher levels
much more cells were observed after 23 days (Figure 4G), during electrical activity [62,63] . Therefore, electrical activity
with higher proliferation and migration. There were also of neuronal circuits was monitored by imaging of calcium
several connections between the printed droplets; however, levels using the calcium-binding fluorochrome Fluo-8 AM
the space between these droplets was filled with so many in combination with a wide-field fluorescence microscope
migrated cells that the printing pattern was barely visible with video camera.
in some places. The neuronal activity of cells depended on cell type and
Remarkably, the percentage of astrocytes 23 days after duration of pre-differentiation. However, there were strong
printing does not seem to considerably depend on the variations in activity between samples handled equally
percentage of astrocytes at the time of printing, except, with the same printing parameters and biomaterials.
of course, for the case of printing of astrocytes alone The cells’ activity was visualized by calcium imaging and
(Figure 4D). Furthermore, the percentage of astrocytes in roughly rated by applying a grading system ranging from
NSCs cultured for 23 days under neuronal differentiation 0 (no activity) to 24 (abundant and intensive activity
condition post-printing was much higher than the with bursting events). Figure 6A depicts the mean of
percentage of astrocytes in d20 neurons (pre-differentiated these ratings for different cell types (NSCs, d20 neurons,
in 2D culture) at the day of printing (≈ 0.4%). astrocytes, and mixtures of these). Data were averaged
over different durations of post-differentiation. Figure 6B
3.9. Formation of synapses during differentiation of lists the statistical P values, which are highlighted in red
printed NSCs and neurons whenever they are below the significance level of 0.05.
Neurons develop their full functionality with the
establishment of spontaneously active neuronal networks. Noticeably, NSCs show higher levels of activity than
For communication within these networks, the formation pre-differentiated neurons. The activity of mixtures of
of synapses (synaptogenesis) is crucial. Synapses consist of NSCs with 20% (activity 8.6 ± 0.5) or 50% (9.3 ± 1.0)
pre- and post-synaptic compartments of two connecting astrocytes was similar to the activity of NSCs only (8.9 ±
neurons, at which neurotransmitters are transmitted 0.3; no significance), while the activity of mixtures of
from the pre-synaptic cell to the post-synaptic one. The neurons with 20% astrocytes (6.2 ± 0.6) or 20% NSCs
pre- and post-synaptic compartments consist of different (6.4 ± 0.8) was lower than that of neurons only (7.7 ±
proteins and are positioned with a small gap (synaptic 0.5; no significance). The activities of these neuron-based
cleft) in between. These compartments can be observed in mixtures were also significantly lower than those of NSCs
juxtaposition or as colocalization (overlapping). or NSC-based mixtures, while there was no significant
difference between neurons only and NSCs. Additionally,
In Figure 5, synaptic compartments of printed NSCs the activity of astrocytes only (4.2 ± 0.9), which is the
(Figure 5A and 5B) and d20 neurons (Figure 5C and lowest, is depicted for comparison.
5D), neuronal post-differentiated for 20 days (day 22 pp,
diff 0/20 for NSCs; day 20 pp, diff 20/20 for neurons), are 3.11. Development of neuronal activity in long-term
depicted by pre-synaptic synaptophysin staining (red) cultivation
and post-synaptic PSD95 staining (green) together with Samples of NSCs printed with bioink on Matrigel started
TM
Hoechst 33342 cell nuclei staining (blue). The yellow color to show spontaneous activity at day 10 of differentiation
in the right column panels is an overlap of red and green (day 12 pp, diff 0/10; Videoclip S1). Activity became
staining. Both synaptic markers were found in abundance widespread from day 25 onward (day 27 pp, diff 0/25;

Volume 9 Issue 2 (2023) 356 https://doi.org/10.18063/ijb.v9i2.672