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International Journal of Bioprinting Multi-scale vascularization strategy for 3D-bioprinted tissue

2.8. Fluorescence staining of bioprinted tissue based bioprinting method for fabricating vascularized
For assessing cell viability, live and dead staining was tissue constructs requires two bioink supply equipment,
[15]
performed on the bioprinted tissue. The culture medium i.e., pneumatic dispenser and syringe pump. On the other
was removed from the bioprinted tissue and rinsed three hand, only a pneumatic dispenser is required for pre-set
times with prewarmed DPBS. Cells were then stained extrusion bioprinting with coaxial core–shell precursor
with calcein-AM (C1430, Thermo Fisher Scientific) and cartridge to fabricate mid-scale vasculature-embedded
ethidium homodimer-1 (E1169, Thermo Fisher Scientific) tissue. Therefore, fabricating mid-scale vasculature-
solution for 40 min at 25°C. embedded tissue using pre-set extrusion bioprinting is
more cost-effective than coaxial nozzle-assisted bioprinting
In addition, the bioprinted tissue was stained with
CD31 and alpha-smooth muscle actin for visualizing technique.
the vasculatures and the surrounding CCD-986Sk cells. Furthermore, pre-set extrusion bioprinting can be used
Briefly, the culture medium was removed from the samples for controlling the diameter of the vasculature by adjusting
and rinsed with DPBS. The sample was then fixed with 4% the concentration of the bioink and the geometry of the
paraformaldehyde solution for 5 min and washed with precursor cartridge. In this technique, the similarity in
DPBS five times. Then, the sample was soaked in a 0.25% the shear viscosity of bioinks in a precursor cartridge is
Triton X-100 solution for 1 min for permeabilization. For important because it determines the co-printability of the
[9]
blocking, the permeabilized sample was soaked in a 1% bioinks . Within the co-printable range (Figure S3), the
bovine serum albumin solution for 1 h. Subsequently, the areal proportion of cross section of the printed construct,
samples were immunostained with CD31 (ab9498, Abcam, fabricated by the pre-set extrusion bioprinting technique,
UK) and α-SMA (ab124964, Abcam) primary antibodies is controlled by adjusting the concentration of each bioink
for 12 h. Alexa Fluor 488 anti-mouse goat (ab150113, because of the difference in shear viscosity. Considering
Abcam) and Alexa Fluor 594 anti-rabbit goat (ab150080, this possibility, the lumen size in the printed construct was
Abcam) secondary antibodies were stained for 2 h. Finally, controllable (Figure 2A). When the concentration of the
the nuclei of cells in the bioprinted construct were stained sacrificial bioink (alginate) was diluted, a larger lumen was
with Hoechst 33342 (62249, Thermo Fisher Scientific). generated, because the viscosity of the alginate solution
All fluorescently stained tissues were subjected to laser decreased with dilution (Figure S4). Another method for
scanning confocal microscopy (FV1200; Olympus). changing the lumen size in a printed construct is changing
the geometry of the precursor cartridge (Figure 2B). The
3. Results and discussion results showed that a small change in the area of the core
region of a precursor cartridge caused large differences
Figure 1A shows the vascularization strategy used in in the area of the lumen in the bioprinted construct. This
this study for fabricating bioprinted tissue. The mid- result can be attributed to the geometry of the core–shell
scale vasculatures were created in bioprinted tissue using precursor cartridge and syringe.
a pre-set extrusion bioprinting technique (Figure 1B).
Subsequently, the bioprinted tissue was cultured for the The printed tissue construct had high initial cell
attachment of cells. Next, we designed an experimental viability (Figure 2C). Coaxial nozzle-assisted bioprinting
setup for generating a biochemical gradient in bioprinted sometimes requires a small-diameter nozzle in the core
tissue (Figure 1C). As a result, the endothelial cells formed region for fabrication of small vasculature-embedded tissue.
perfusable capillary branches. When cells in the bioink are extruded through a small-
diameter nozzle, they can be damaged by shear stress .
[16]
Pre-set extrusion bioprinting is a derivative technique However, in the pre-set extrusion bioprinting process,
from extrusion-based bioprinting method . The small-vasculature-embedded tissue can be fabricated by
[12]
resolution of conventional extrusion bioprinting is lower adjusting the concentration of bioink and the geometry of
than other bioprinting method such as jetting-based the precursor cartridge without a small-diameter nozzle.
[13]
and polymerization-based bioprinting method. For this Thus, the pre-set extrusion bioprinting technique has a high
[14]
reason, conventional extrusion bioprinting is not effective potential for fabricating tissue constructs with embedded
for building mid-scale lumen demonstrated in this paper. small-diameter vasculatures with high cell viability. In
On the other hand, pre-set extrusion bioprinting has high addition, endothelial cells were well attached to the inner
resolution that is able to build lumen with a diameter of surface of the lumen (Figure 2D) and proliferated to form
185–445 μm in bioprinted tissue (Figure 2A and B) by mid-scale vasculature in the culture (Figure 2E). Although
simply using a coaxial core–shell precursor cartridge, which the pre-set extrusion bioprinting technique is capable of
is a major advantage of pre-set extrusion bioprinting. Also, fabricating mid-scale vasculature-embedded tissue, it is
coaxial nozzle-assisted bioprinting, a common extrusion- not effective for capillary branches in the native tissue.

Volume 9 Issue 4 (2023) 168 https://doi.org/10.18063/ijb.726