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International Journal of Bioprinting Low-cost quad-extrusion 3D bioprinting system

images were taken after 30 h of incubation to visualize crosslinking, no CaCl was added to the solution as would
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the CFDA-SE-stained HTR-8 cells in green and the EGF/ be the case with alginate. The Laponite B concentration
Alexa-555 conjugate in red, respectively. adopted was 1.5% (w/v).
2.4. Bioink preparation 2.6. 3D bioprinting, ultraviolet crosslinking, and
GelMA bioinks were prepared akin to the method post-processing of printed bioinks
described in Ding et al. with minor differences based To prepare the toolpath g-codes of the computer-
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on the recommendations of the biomaterial vendor. First, aided design (CAD) models to be printed, Cura (Cura,
lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP; Ultimaker, USA) was used to slice the CAD models and
Allevi Inc., PA, USA) was fully dissolved in DPBS at obtain the toolpath for printing. However, to accommodate
60°C for 15–30 min with a 0.5% w/v concentration of the the physical characteristics of our QEB, custom starting
final bioink solution. It is important to keep the solution and ending g-code modifications are needed to avoid
in an amber container or covered with aluminum foil to physical interference. These modifications also need to be
prevent contact with light and premature photoinitiator customized based on the type of substrate used to print on,
activation. When the LAP was fully dissolved in the DPBS in the case of IAP, or within, in the case of SBP.
solution, lyophilized gelMA foam (GelMA, Allevi Inc.,
PA, USA) was added to the solution with a 10% w/v final Before bioprinting, warm (room temperature) cell-free
concentration. The final mixture was thoroughly mixed at or cell-laden bioinks were filled into 3 mL syringes. The
60°C for over an hour in the dark. It is noteworthy that syringes were equipped with 2-inch 23G nozzles (Nordson
the material preparation was performed under a sterile EFD, RI, USA) and were covered with aluminum foil to
biohood to minimize contamination. When the as- prevent premature crosslinking. The prepared bioink
prepared GelMA bioink mixture was well dissolved and syringes were then mounted onto the QEH. To ensure the
homogenized, it was stored overnight in the dark at 4°C. alignment of the four nozzle tips along the z-direction,
For cell-free bioprinting, GelMA was melted down in a a specific point on the print bed grid was identified, and
warm water bath (37°C), diluted in DPBS to the desired the four nozzles were zeroed along the z-levels by simply
concentration, and mounted in a 3 mL syringe that was turning the needle tips along with the Luer locks. This
covered with aluminum foil to maintain the prevention of is necessary since the four nozzles are in-line and may
any premature crosslinking before and during printing. interfere with the printed structures.
For the cell-laden bioink formulation, melted GelMA To crosslink the bioprinted gelMA bioinks, a handheld
was sterile-filtered in two stages using sterile filters with 8 Watt, 365 nm ultraviolet (UV) light (UVP, UVL-18
pores sizes of 0.45 µm and 0.20 µm, sequentially. The 365 nm UV light, Analytik Jena US, CA, USA) was used
sterile GelMA was then mixed with a cell pellet mixed in in the case of IAP. The UV lamp was placed on top of
cell media in volumes that would render a certain desired the petri dish, covering the whole area of the bioprinted
final bioink concentration. For the advanced case study sample. Since the size and shape of the UV lamp are
herein, due to their availability, HTR-8/SVneo cells were rectangular, wide, and long enough to cover the whole
used after being centrifuged at 300 rpm for 5 min. The cell substrate being printed on, it is safe to assume uniform
pellet was mixed with cell media, which was then mixed crosslinking has occurred within the samples. However,
by gently pipetting with the melted sterile GelMA to reach for the case of SBP, the beaker carrying the support bath,
a final concentration of 5% cell-laden GelMA bioink with along with the printed construct, was placed in a UV
approximately 2 × 10 cells mixed therein. curing station (Sovol 3D SL1 Curing Station, Amazon,
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USA) that has UV light strips on the sides and the top
2.5. Support bath material preparation while being completely mirror-polished on the inside with
The support bath material (SBM) is prepared from a turntable in the center of the bottom plate. This ensures
Laponite nanoclay (Na Si Mg Li O (OH) ) that creates a uniform omnidirectional UV light dispersion within
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0.7
20
8
0.3
5.5
a yield stress bath that can self-support the printed bioink the mirrored curing box allowing uniform crosslinking of
inside. The Laponite nanoclay was prepared following the bioprinted samples in the support bath. The distance
the manufacturer’s recommendations as well as the steps would vary depending on the size and geometry of the
undertaken by Ding et al. The SBM tested was Laponite bioprinted sample. Crosslinking times were varied based
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B (BYK Additives Inc., Gonzales, TX, USA). The nanoclay on the desired final stiffness of the bioprinted constructs.
powder was slowly and continuously added to a beaker The crosslinking process was either done simultaneously
with deionized water, and it was then vigorously stirred for throughout the printing process or at a single instance
24 h to ensure proper dispersion in the final solution. Since during post-processing, depending on the printing
the printed bioink is GelMA and does not require chemical paradigm used. For SBP, the final bioprinted structures
Volume 10 Issue 1 (2024) 297 https://doi.org/10.36922/ijb.0159