Page 355 - IJB-9-2

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

(Axol Biosciences)-coated 6-well plates and cultured for period, were dissociated into single cells with Accutase™
24 hours at 37°C in a 5% CO environment. The plating for 5 minutes at 37°C. After gentle pipetting, the cells
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medium was then replaced with expansion medium were collected by centrifugation at 200 ×g for 5 minutes
consisting of KnockOut™ DMEM/F-12 supplemented and carefully resuspended in their respective cell culture
with 2% StemPro™ Neural Supplement, 1% Glutamax™, medium supplemented with 1% RevitaCell™. The majority
basic fibroblast growth factor (bFGF, 20 ng mL ; Axol of these cells were used for the printing experiments, and
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Bioscience), epidermal growth factor (EGF, 20 ng mL ; a smaller portion of the cells were analyzed with respect
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Axol Bioscience), and 50 µg mL gentamycin. For to their differentiation stage and composition. The latter
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passaging, NSCs were washed once with phosphate- were seeded at a cell concentration 2.5 × 10 cells cm on
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buffered saline (PBS) and then dissociated into single Matrigel -coated glass coverslips in their respective cell
TM
cells with Accutase™ for 5 minutes at 37°C. After gentle culture medium and allowed to attach for 4 hours in an
pipetting, the cells were collected by centrifugation at incubator at 37°C in a 5% CO environment.
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400 ×g for 5 minutes. NSCs were seeded at a density of
5 × 10 cells cm and cultured onto Geltrex-ESC™-coated 2.3.2. Cell printing system
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6-well plates in expansion medium supplemented with The printing system applied in this study was described
1% RevitaCell™ during initial plating. Medium was fully in our previous publication . Briefly, for laser printing,
[46]
exchanged to expansion medium without RevitaCell™ after cells are suspended in a sol (referred to as bioink), usually
24 hours. NSCs were maintained in expansion medium consisting of several components; a sol is the non-gelled
with medium changes twice a week. NSCs from passages precursor of a gel. A glass slide (donor) is coated with a
3–5 were used for the cell printing experiments. 60 nm thin laser-absorbing gold layer and a thicker (50–
70 µm) layer of the cell containing bioink. The donor is
2.2.2. Neuronal differentiation mounted upside-down (Figure 1A) above a second glass
For the experiments with pre-differentiated neurons, NSCs slide (collector), which is coated with a layer of hydrogel
were plated at a density of 1 × 10 cells cm onto Geltrex- (referred to as substrate) to provide a humid environment
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ESC™-coated plates in expansion medium supplemented and to prevent printed cells from dehydration and drying
with 1% RevitaCell™. Medium was changed the next day out due to the extremely small droplet volumes. For some
to expansion medium without further supplementation. cell types, the hydrogel layer is also necessary to enable cell
At a confluence of 40%–60%, the expansion medium adhesion after printing.
was switched to neuronal differentiation medium
(Neurobasal medium, 2% B27™ Serum-Free Supplement, For printing bioink droplets, laser pulses are focused
1% Glutamax™, 200 µm ascorbic acid-2-phosphate, and through the donor glass slide into the absorbing layer
50 µg mL gentamycin). Half of the medium volume was which is evaporated in the laser focus. An expanding
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exchanged every second day. vapor bubble is generated, which propels the subjacent
bioink downward. Due to the vapor bubble collapsing
2.2.3. Astrocytic differentiation after a few microseconds, a bioink jet is formed, lasting
For the printing experiments with astrocytes, NSCs were for a few hundred microseconds . The bioink deposits
[47]
plated at a density of 1 × 10 cells cm onto Geltrex-ESC™- as a droplet on the collector substrate. Arbitrary patterns
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coated plates in expansion medium supplemented with 1% can be generated by moving laser focus and donor slide;
RevitaCell™. Medium was switched the next day to astrocyte 3D structures and tissues are printable layer-by-layer. The
differentiation medium (DMEM, 1% N2™ Supplement, 1% printed droplet volume depends on laser pulse energy and
fetal bovine serum (FBS), 1% Glutamax™, and 50 µg mL focused laser spot size, as well as on thickness and viscosity
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gentamycin). Medium was thereafter fully exchanged of laser-absorbing layer and bioink layer. In this study,
twice a week. Cells were split (1:4 ratio) at a confluence of droplet volumes in the range of 0.1–1 nL were printed.
80%–90% by using Accutase . Astrocytes from passages Former studies demonstrated that no heat shock was
TM
5–7 were used for the cell printing experiments. Astrocytic induced to the printed cells .
[48]
differentiation was confirmed by the staining of glial
fibrillary acidic protein (GFAP), a general astrocyte marker A Nd:YAG laser (DIVA II; Thales Laser, Orsay, France)
that does not require fully mature astrocytes. with 1064 nm wavelength, approximately 10 ns pulse
duration (FWHM) and 20 Hz repetition rate was used. The
2.3. Cell printing laser pulses were focused with a 50 mm achromatic lens
2.3.1. Cell preparation for printing and into an ablation spot with a 40 µm diameter. Depending on
immunostaining the bioink layer thickness (approx. 60 µm) and viscosity,
For printing and immunostaining, NSCs, astrocytes the laser pulse energy was set between 10 and 15 µJ,
and neurons after 5, 10, and 20 days pre-differentiation corresponding to laser fluences around 1–2 J cm .
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Volume 9 Issue 2 (2023) 347 https://doi.org/10.18063/ijb.v9i2.672