Page 108 - IJB-9-6

International Journal of Bioprinting Affordable temperature-controlled bioprinter

the possibility of sample contamination and any potential For evaluation of the postprinting cell viability, the
damage to the system. A scheme of the complete cells were stained using a Live/Dead kit (Thermo Fisher,
bioprinter setup is shown in Figure 1A. USA). The staining solution was directly pipetted over the

A syringe pump flow rate of 0.1 mL/min was used bioprinted structures, and the samples were incubated for
to extrude the ink/bioink down to the tip of the cooling 30 min at 37°C at 5% CO and 100% humidity. After two
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nozzle. The flow rate was then changed and maintained washes with DPBS, the samples were imaged using an Axio
at 0.05 mL/min to avoid freezing the bioink and clogging M2 Observer fluorescence microscope (ZEISS, Germany).
the cooling/heating nozzle. We ascertained that a stream The images were processed and the dimensions of the
of filament of constant thickness was observed for at least structures with cells were analyzed using open-source Fiji
20 s to ensure good bioprinting quality. In cases of clogging software (Image J). For dimension analyses, at least 12
due to unexpected freezing of the bioink, the tubing on the measurements per dimension were taken to calculate the
top was disconnected and hot water at 70°C was injected means and standard deviations.
into the printhead through the top fitting. Subsequently, In a different set of experiments, a C2C12-laden bioink
the bioprinting protocol was restarted. was bioprinted into three multilayered 1 × 1-cm squares
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and cultured for 7 days under the same conditions (37°C,
2.7. Printability assays 5% CO , and 100% humidity) to observe long-term survival
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A series of experiments was conducted to study the effect of and elongation. After the culture period, the cells were
the nozzle feed rate on the thickness of the printed GelMA stained with phalloidin/4′,6-diamidino-2-phenylindole
or Pluronic lines and to determine the optimal printing (DAPI) to observe the cellular structures. Micrographs
parameters. We analyzed three different feed rates at a were obtained with a Zeiss Axio M2 Observer fluorescence
fixed flow rate in a single test to generate a custom G-code. microscope.
This G-code enabled the sequential printing of three
different squares (5 × 5 mm ), in which the linear velocity 2.9. Statistical analysis
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decreased from the first to the third. The printability was Data are presented as the mean ± standard deviation from
quantitatively analyzed by measuring the dimensions (i.e., at least three repetitions (n = 3). Significant differences
line thickness and square area) of the printed squares by (p < 0.05) were found by analysis of variance (ANOVA).
image analysis with ImageJ [34,35] . Different 2D structures
were also printed utilizing our printhead, with and without 3. Results and discussion
temperature control. 3.1. Bioprinter development
We developed a cost-effective bioprinter capable of
2.8. Cell culture, cell-laden bioink preparation, and bioprinting simple cell-laden GelMA constructs. We used
cell viability assessment the Anet A8 3D printer, a commercially available fused-
C2C12 myoblasts (CRL 1772, ATCC, USA) were cultured deposition model, as our mechanical backbone. This 3D
to about 80% confluency in Dulbecco’s modified Eagle’s printer has been used before as a frame to develop cost-
medium (DMEM, Sigma-Aldrich, USA) supplemented effective bioprinters [18,36] , due to its relatively low cost
with 10% fetal bovine serum (Thermo Fisher, USA) and (currently between 200 and 300 USD), easy assembly, and
1% penicillin-streptomycin (Sigma-Aldrich, USA) at 37°C, off-the-shelf availability. In addition, the use of this 3D
5% CO , and 100% humidity. For bioink preparation, printer is supported by a vast community of users globally,
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the cultured C2C12 cells were trypsinized, collected, and making it widely useful for customizing—a quality that
resuspended in the GelMA pregel solution at a concentration is tremendously lacking in commercial bioprinters. The
of 1 × 10 cells/mL. This bioink was charged into a 10-mL (bio)printer was assembled by closely following the
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syringe (BD Plastics, USA) and refrigerated at 4°C for 20 manufacturer’s directions and subsequently subjected to
min before loading into the syringe pump for bioprinting. in-house modifications using common maker tools. As
an additional resource, many tutorials are available on the
The GelMA-based bioink was bioprinted in a grid
pattern and in the shape of the logo of Tecnologico de internet describing the procedures for assembly of the Anet
A8 3D printer. Figure 1A shows the overall architecture of
Monterrey using a flow rate of 0.05 mL/min, a feed rate our Anet A8-derived bioprinter.
(i.e., nozzle linear speed) of 300 mm/min, and a water
circulation temperature of −5°C. The bioprinted patterns In this project, we used the concept of “a printer that
were subsequently UV-crosslinked using an Omnicure prints itself.” Before conducting any modifications, we
2000 (Excelitas Technologies, USA) apparatus for 60 s at used the as-supplied 3D printer to print the modifications
800 mJ/s. DMEM was added to the bioprinted structures needed to increase the stability and robustness of our
until further assessment. bioprinting machine. The X-axis carriage and printhead

Volume 9 Issue 6 (2023) 100 https://doi.org/10.36922/ijb.0244