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Materials Science in Additive Manufacturing SLA 3D printed triaxial nozzle

USA) supplemented with 5% human platelet lysate 2.7.2. Cytoskeleton staining
(StemCell Technologies, USA), 2 mM L-glutamine, and 1% The cell-laden constructs were first fixed with 4%
penicillin/streptomycin (GIBCO, ThermoFisher, USA). formaldehyde solution for 30 min. Subsequently, the
BMECs were cultured at seeding of 2 × 10 cells/cm in cells were permeabilized using a cold cytoskeleton buffer
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T175 tissue culture flasks and subcultured when cultures (consisting of 3 mM MgCl , 300 mM sucrose, and 0.5%
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reached 80% confluence. The cells were maintained in Triton X-100 in PBS) for 5 min. Then, the permeabilized
complete growth media consisting of MCDB-131 media cells were incubated in a blocking buffer solution
supplemented with 10% fetal bovine serum (FBS), 2 mM (comprising 5% FBS, 0.1% Tween-20, and 0.02% sodium
L-glutamine, and 1% penicillin/streptomycin (all from azide in PBS) for 30 min at 37°C. To detect F-actin,
GIBCO, ThermoFisher, USA). Cells in passage 3 or 4 were rhodamine-phalloidin (1:300) was added to the cells for 1 h.
used for bioprinting experiments. Subsequently, the cells were treated with DAPI for 5 min to
counterstain the nucleus. The cells were then observed and
2.6.2. Bioprinting imaged using a laser scanning confocal microscope (Zeiss
For bioprinting, the cells were mixed with 1× PBS solution LSM 710 Inverted Confocal Microscope, Germany), and
to prepare cell suspensions with a final concentration z-stack images of the samples were captured.
of 10 × 10 cells/mL or 20 × 10 cells/mL, depending on
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whether hBM-MSCs or BMEC were used. Those cell 3. Results and discussion
concentrations were identified as the optimal density This study highlights the benefits of using AM to produce
in terms of cell viability and cell-cell interaction after customized 3D-printed nozzles for 3D bioprinting. Using
bioprinting. Subsequently, the microfluidic pumps of the resin-based 3D printing as a fabrication method for
robotic arm bioprinter were loaded with the cell solution. nozzles has several advantages, including precision and
During the printing process, flow rates of 55 μL/min, reproducibility. This new design for a 3D-printed nozzle
15 μL/min, and 15 μL/min were maintained for peptide addresses some of the limitations observed in previous
solution, 5× PBS, and cells, respectively. Cuboid cell- designs , such as the complexities of the assembly process
[29]
laden structures with 10 mm edges and 2.6 mm height and the potential for backflow. Moreover, this nozzle design
were printed. Following the printing process, the cell- features a cell inlet that extrudes cells inside the nozzle,
laden constructs were incubated in the CO incubator for allowing for the introduction of cells into the bioink after
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5 min, after which complete growth media were added. gelation and before the final extrusion.
The printed cell-laden constructs were placed in standard
conditions (37°C, 5% CO , and 95% relative humidity), 3.1. Design and fabrication of the nozzle
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and the media were changed every 3 days. This nozzle was designed with three inlets and one outlet. The
top two inlets are meant for the addition of the material to
2.7. Assessment of cell-laden constructs
be mixed and gelled inside the nozzle, while the third inlet
2.7.1. Cell viability assessment is used to extrude the cells just before the final extrusion of
The viability of 3D bioprinted cells was assessed using the the materials (Figure 2A). This allows for the addition of
LIVE/DEAD Viability/Cytotoxicity Kit (ThermoFisher, cells to the gel once gelation occurs within the nozzle mixing
USA), in which calcein acetoxymethyl ester (Calcein-AM) region. In addition, the nozzle is compatible with the Luer-
is used to detect viable cells, and ethidium homodimer-I Lok for ease of use despite the larger diameter of the Luer-Lok
(EthD-I) is used to detect dead cells. The 3D cell-laden compared to the desired interior diameter of our materials.
bioprinted constructs were washed twice with Dulbecco’s To maintain optimal flow, the nozzle channels are kept at a
phosphate-buffered saline (D-PBS). Then, a staining consistent 1 mm inner diameter throughout the entire nozzle.
solution of 2 μM Calcein-AM and 4 μM of EthD-1 was A backflow prevention feature was also incorporated to prevent
added to the 3D cell-laden bioprinted constructs and any backflow before the materials were mixed. The backflow
incubated for 45 min in the CO incubator. Subsequently, issue was particularly prevalent in previous experiments we
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the staining solution was removed, and the 3D-bioprinted performed with other nozzles, where the different flow rates
constructs were washed with 1× DPBS. The stained printed between peptide and PBS led to a difference in pressure. As
cell-laden constructs were imaged using an inverted the peptide flowed toward the PBS inlet, premature mixing
immunofluorescent microscope (Evos, Invitrogen, occurred before reaching the intended mixing region, leading
ThermoFischer, USA). The assessment of cell viability was to clog formation and disrupting gel continuity.
performed at days 3, 7, and 10 for hBM-MSCs and at days After designing using NX CAD software, the 3D model
5 and 10 for BMECs. was extruded as an STL file. The file was then uploaded to a

Volume 2 Issue 3 (2023) 5 https://doi.org/10.36922/msam.1786