Page 356 - IJB-9-2

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

appropriate for the cells to be printed at a ratio of 15%–
85% (v/v) was applied as bioink.
For printing, 1 or 2 million cells were suspended in
50 µl of bioink. 26 × 26 mm² donor glass slides were blade-
coated with 50 µl bioink (74 µm layer thickness) and 23 ×
23 mm² collector glass slides were coated with 60 µl of
substrate hydrogel (113 µm layer thickness). Subsequently,
droplets of bioink were printed onto the substrate layer
on the collector slide in x/y-arrays of distinct dots (see
Figure 1B) with 1 mm dot distance.
2.3.4. Cell handling after printing
After printing, the collector slide with hydrogel
substrate and printed cells was stored submerged in cell
culture medium in an incubator at 37°C in a 5% CO
2
environment. Culture medium was expansion medium
with RevitaCell for printed NSCs and neuronal
TM
differentiation medium with RevitaCell for pre-
TM
differentiated neurons. The culture medium of printed
NSCs was changed after 2 days to neuronal differentiation
medium (without RevitaCell™).
For cell viability assay (Figure 1C and 1D), cells were
printed with hyaluronic acid and culture media as bioink
on Matrigel substrate. Non-printed cells residing on
TM
the donor glass slides (referred to as donor cells) were
rinsed after printing, collected, centrifuged, resuspended
in expansion or differentiation medium with RevitaCell™,
and 50,000 of them (similar to the number of printed
cells) were seeded on a glass slides coated with Matrigel .
TM
Figure 1. Printing process and cell culture homogeneity. (A) Schemat-
ic sketch of the laser-assisted bioprinting technique. (B) Example of bi- Control cells were stored with culture medium in vials
oprinted neuronal stem cells (NSCs): Fluorescence microscopic image (while other cells were printed) and were seeded like donor
of NSCs printed in a dot-pattern with a dot-to-dot distance of 600 µm, cells when printing was finished.
immunostained with nestin antibody (green fluorescence) for neuronal
stem cells 2 days after printing. (C) Quantitative investigation of NSC’s 2.4. Cell evaluation
differentiation toward neurons and astrocytes (without printing); count- 2.4.1. Analysis of cell viability
ing of positive cells for mature neuronal (MAP2), proliferation (Ki67) and
astrocyte (GFAP) markers among NSCs, d5, d10, d20 neurons and as- Cell viability of printed, donor, and control cells was
trocytes from fluorescent microscopic images; representative images are determined via calcein AM (2 µM, green-live cells) and
depicted in Figure S2 (in Supplementary File). (D) Western blot (left) ethidium-homodimer-1 (4 µM, red-dead cells) staining
analysis of neuronal proteins and its quantitative representation (right) of 24 hours after printing. The samples were analyzed by
the expression level of β3-tubulin, doublecortin (DCX), MAP2, synapto- fluorescence microscopy using an AxioImager A1.m
physin (SYN), synapsin-1 (SYP1), PSD95, and GFAP of cells in NSC, d5,
d10, d20 neuron and astrocyte culture. Data are normalized relative to microscope (Carl Zeiss, Oberkochen, Germany) equipped
internal standard β-actin band density. with AxioCam ICc1 camera and AxioVision Rel. 4.8
software. Live/dead cells were counted from 10 fields
of 3 independent experiments of printed, donor, and
2.3.3. Bioink preparation for printing control cells. Percentage of viability is reported as mean
The preparation for printing followed the method used in with standard error of mean. Significance of differences
our previous study with hiPSCs . The following materials is calculated by applying Student’s unpaired two-sample
[45]
were used as bioinks and culture substrates: 66.67% (v/v) t-test.
Corning™ Matrigel , mixed with 33.33% (v/v) EBM-2
TM
medium (Lonza, Basel, Switzerland), was applied as culture 2.4.2. Immunostaining
substrate; 1% (w/w) hyaluronic acid from Streptococcus For immunofluorescence analysis, cells printed in patterns
equi (mol wt ∼1.5–1.8 × 10 Da) in 0.1 M Tris-buffered as well as those prepared as described in section 2.4.1.,
6
saline (TBS), pH 7.4, mixed with the culture medium were fixed in 4% paraformaldehyde for 30 minutes and

Volume 9 Issue 2 (2023) 348 https://doi.org/10.18063/ijb.v9i2.672