Page 109 - IJB-9-6

International Journal of Bioprinting Affordable temperature-controlled bioprinter

mount were also printed utilizing the same 3D printer Yamato water circulator with a water reservoir capacity of
(Figure S3C and D) using PLA as the printing filament. 4 L. However, any conventional laboratory water circulator
We have provided all the STL files (Supplementary File) would be suitable. The printhead design that we present
required to 3D-print the pieces needed for successful here has not been engineered for optimal mass transfer;
conversion of the Anet A8 into a high-performance 3D however, its performance was satisfactory, as we will show,
bioprinter. to assure the printability of our hydrogel-based bioinks
While the Anet A8 has been used by others as a frame to under conventional laboratory settings and experimental
develop low-cost bioprinters [18,36] , we embedded additional conditions (i.e., room temperature between 20°C and 25°C,
features that greatly enhance the capabilities and versatility 10% in-house-made or commercial GelMA bioinks, etc.)
of our bioprinter. For instance, the mechanical driving In our described setup, we continuously measured
system of the Anet A8 printer was adapted for control by a and controlled the temperature in the water bath and
RAMPS controller based on the Arduino Mega and Marlin circulating water, which is significantly different than
firmware. This enabled full access to user modifications, the actual temperature of the extruded bioink. This
thereby greatly enhancing the flexibility of this bioprinting temperature difference (i.e., 7°C ± 2.2°C) is due to the low
system. For the purposes of this project, we used original thermal conductivity of the resin and water and to the
Marlin firmware and the Anet A8 configuration files. Once internal wall thickness between the water jacket and the
the motherboard had a firmware installed, and all of the bioink chamber (~2 mm in thickness) (Figure 1C).
electrical connections are established, we connected the
printer to a host software to allow for manual control as 3.3. Printability assays
well as G-code uploads. Several guides describing the In principle, this bioprinter and printhead can be used
installation of an open-source 3D printer firmware, such to ensure printability of any bioink matrix that requires
as Marlin, on an Arduino Mega board have been developed specific temperature control during extrusion to attain
and are widely available. a desired viscosity. For demonstration purposes, we first
chose GelMA, one of the most widely used hydrogels
This printer/bioprinter is also extremely flexible in terms in tissue engineering and bioprinting research, as our
of the needles that can be used, the range of temperatures bioink matrix. GelMA is known to have a printability
controlled by the printhead jacket, the flow rate of the ink/ that highly depends on its rheology [25,29,31] , and several
bioink used, and the feed rate of the printhead. In this recent papers have described the rheological behavior of
work, we used a standard 21G needle as the printhead tip, GelMA hydrogels in the range of temperatures of interest
a range of feed rates (i.e., linear speeds) between 100 and here [29,31] . We also ran rheological determinations ourselves
300 mm/min, and a spectrum of flow rates between 0.05 for the GelMA formulations utilized in the present study
and 0.1 mL/min produced by the syringe pump. (Figure 2A). The GelMA viscosity increased in the
temperature range between 15°C and 5°C and showed
3.2. Performance of the cooling/heating system viscosity variations of more than one order of magnitude
As previously mentioned, the most differentiating attribute in this range (Figure 2Ai). In general, similar trends can
of our DiY bioprinter with respect to existing DiY models be observed in alternative GelMA-based formulations
is its ability to control temperature during extrusion. commonly used in bioprinting applications (i.e., GelMA-
To achieve this control, we substituted the original alginate blends; Figure 2A). The intersection between the
printing nozzle with a 3D-printed printhead engineered storage and loss moduli, which determines the gel–liquid
for temperature control by water recirculation during transition, occurred in the range of 20°C–25°C, for typical
extrusion (Figure 1B; see illustrative videos [Movie S1; GelMA-based inks (Figure 2Aii). Therefore, temperature
Movie S2] with link given in Supplementary File). The control is clearly crucial to ensure printability and
CAD model and corresponding STL file are included in reproducible bioprinting results.
the Supplementary File to enable its reproduction and
use. This printhead consisted of a straight cylindrical We conducted printing experiments using a 5% (w/v)
ink chamber housing an inlet that accepts conventional GelMA formulation to assess the printability at different
plastic tubing (0.25 cm in I.D.). The cylindrical ink feed rates , ink extrusion rates (i.e., flow rates), and
[34]
reservoir was enclosed in a chamber (or jacket) that allows temperatures of the recirculating water. For that, we
recirculation of water for temperature control. The sides printed a series of squares with an intended dimension of 5
of the recirculating printhead chamber are designed with × 5 mm and connected by lines using a 5% (w/v) GelMA
2
an inlet and outlet to accept conventional rubber hoses bioink with fluorescent microparticles (Figure 2B). During
coming from and returning to the water-recirculating a single printing trace, the bioprinter was challenged to
system. In the experiments reported here, we used a extrude at three different and increasing feed rates while

Volume 9 Issue 6 (2023) 101 https://doi.org/10.36922/ijb.0244