Page 359 - IJB-9-2

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

proliferation marker Ki67 combined with general cell were put into the printing setup but were not transferred,
nuclei marker Hoechst 33342 are shown in Figure S1; while control cells were those that were not put into the
the ratios of cells positive for these markers, counted in setup but stored in a vial during the printing procedure.
between 12 and 18 of such images from 3 independent In general, we observed that the survival rate of printed
experiments, are presented in Figure 1C. neurons depended on duration of pre-differentiation
For neuronal differentiation, the percentage of MAP2- period. Control, printed, and donor (c/p/d) cells’ viability
positive cells increased from 18.4 ± 0.7% (day 0 / NSCs) decreased with the duration of pre-differentiation.
to 43.8 ± 1.7% (day 5), 56.4 ± 2.0% (day 10) and 81.9 ± While the viability of d5 neurons (control: 89.9 ± 0.4%;
1.2% (day 20), while the percentage of GFAP-positive cells printed: 93.0 ± 0.6%; donor: 91.0 ± 0.5%) was similar (no
remained low (day 0: 0.01 ± 0.01%; day 5: 0.05 ± 0.02%; significant difference for c/p/d; see P values in Figure 2C)
day 10: 0.04 ± 0.02%; day 20: 0.4 ± 0.1%). For astrocytic to that of NSCs (control: 91.1 ± 0.5%; printed: 94.5 ± 0.6%;
differentiation, the percentage of GFAP-positive cells donor: 89.1 ± 0.5%), the viability of d10 neurons (control:
increased from 0.01 ± 0.01% (day 0 / NSCs) to 93.4 ± 0.8% 76.5 ± 0.9%; printed: 79.1 ± 0.5%; donor: 75.3 ± 0.5%) was
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(astrocytes), while here the percentage of MAP2-positive significantly (P < 10 for c/p/d) lower and, when compared
cells decreased from 18.4 ± 0.7% (NSCs) to 2.4 ± 0.6% with d10 neurons, d20 neurons viability (control: 68.4 ±
(astrocytes). 0.8%; printed: 68.4 ± 1.3%; donor: 67.5 ± 1.0%) was again
significantly lower (P < 10 for c/p/d).
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The percentage of cells positive for proliferation marker
It is noticeable that the viability of printed cells was always
Ki67 decreased during neuronal differentiation from 40.1 ± higher or equal (d20 neurons) than that of donor or control
2.1% (NSCs) to 34.5 ± 1.7% (day 5), 19.7 ± 1.3% (day 10), cells. This difference was significant in five out of eight cases
and 13.6 ± 1.0% (day 20), while it decreased to 31.4 ± 2.1% (three out of four cases for printed/donor and two out of
during astrocytic differentiation. We observed astrocytes four for printed/control); no significant difference could be
positive for GFAP and Ki67, which showed that these seen for d20 neurons. We also observed significantly higher
astrocytes were not yet fully matured. However, neurons, viability of control NSCs when compared with donor NSCs;
positive for MAP2, were not found to be positive for Ki67.
however, this might have been an artifact.
Protein expression levels of neuronal markers β3-
tubulin, doublecortin, MAP2, synaptophysin, synapsin-1, 3.3. Effect of cell density on formation of neuronal
PSD95, and GFAP, investigated by Western blot, are shown dendrites
in Figure 1 for NSCs, d5, d10, d20 neurons and astrocytes as When printing cell-containing droplets, the effect of
images (Figure 1D) as well as in a quantitative representation droplet size and cell number per droplet on cells behavior
(Figure 1E). For the quantitative representation, all these post-printing is of interest. Figure 2D and 2E show the
levels were normalized by dividing by the β-actin expression formation of neuronal dendrites of printed NSCs at day 7
level as a house-keeping protein. Except for GFAP, all these of differentiation post-printing (day 9 pp, diff 0/7), which
protein expression levels increase from NSCs via d5 and were printed in smaller (Figure 2D) and larger (Figure 2E)
d10 neurons to d20 neurons. As expected, astrocytes are droplets with lower and higher cell number per droplet
GFAP-positive, while all NSCs and cell cultures under (bioink: hyaluronic acid and culture media; substrate:
TM
neuronal differentiation are almost all GFAP-negative. Matrigel ). Phase contrast images taken at day 1 post-
Astrocytes also expressed β3-tubulin and doublecortin printing illustrate different droplet sizes, while neuron-
(weak), which is in accordance with literature [58,59] . specific cytoskeletal marker β3-tubulin and Hoechst 33342
cell nuclei staining at day 7 post-printing (day 9 pp, diff
3.2. Cell viability after printing 0/7) show that the number of neuronal dendrites relative
Figure 2A and 2B depict fluorescence microscopy images to the number of neuronal cells is lower in smaller droplets
of live/dead-staining 24 hours after printing for NSCs, d5, and higher in larger droplets.
d10, and d20 neurons with two different magnifications. 3.4. Proliferation and migration after printing
Live/dead staining revealed apparent agglomeration of Figure 3A–C compare the proliferation and migration of
printed d10 and d20 neurons compared to NSCs and d5 NSCs and d20 neurons. They are shown at 2 days (NSCs
neurons, an effect which increased with duration of pre- only; Figure 3A) and 12 days after printing under neuronal
differentiation. Dead cells could be seen within these differentiation conditions (day 12 pp, diff 0/10 for NSCs,
agglomerations in high-magnification images (Figure 2B). Figure 3B; day 12 pp, diff 20/12 for neurons, Figure 3C).
Figure 2C compares the viability of printed cells, i.e., Panels show staining of proliferating cells (red, Ki67) alone
NSCs and neurons (pre-differentiated for 5, 10, and 20 and together with cell nuclei staining (blue, Hoechst 33342),
days), with non-printed donor and control cells. Donor cells as well as for the same cells, neuron-specific marker β3-

Volume 9 Issue 2 (2023) 351 https://doi.org/10.18063/ijb.v9i2.672