Page 107 - IJB-9-6

International Journal of Bioprinting Affordable temperature-controlled bioprinter

A mixture of 40% ethanol and 60% tap water (v/v) was solution was combined with 0.056% (w/v) lithium phenyl-
used in the water circulator reservoir to prevent freezing. 2,4,6-trimethylbenzoylphosphinate (LAP) (Cellink,
For cooling applications, prior to printing, the Yamato Sweden) used as a photoinitiator. The GelMA ink was fed
CF301 circulator was set to a temperature of −5°C to into a syringe, allowed to cool down to room temperature,
−10°C and was left for 30 min to ensure the stabilization of and then stored in a refrigerator at 4°C until used for
the circulating water temperature. The room temperature bioprinting. An aqueous solution of 25% Pluronic F-127
was set to 20°C and left to stabilize for 30 min as well. (w/v) for additional printing experiments was prepared by
The ink/bioink-loaded syringe was purged to remove air stirring in a water bath precooled with ice. The powder was
bubbles and then placed for 20 min in a refrigerator set at added and agitated for 40 min until complete dissolution.
a temperature of 4°C. The circulator hose was connected to The rheology of the GelMA and Pluronic hydrogels
the side fittings of the cooling nozzle (Figure 1B) to enable was evaluated using an MCR 500 rheometer (Anton-
a counter-flow between the water and the ink/bioink (i.e., Paar Rheoplus, Austria) equipped with a Peltier cooler
the ink traveled in the downward direction while the water for temperature control. Parallel plate geometry (50 mm)
circulated in the upward direction) to maintain the water was used with a gap of 0.5 mm to determine rheological
jacket full and maximize heat transfer (Figure 1C). properties. The GelMA and Pluronic inks were tempered

2.4. Cooling/heating nozzle and mount fabrication at 37°C before rheology testing; their viscosity was
The temperature-controlled printhead (i.e., the cooling/ measured with a temperature ramping from 40°C to 10°C
−1
heating printhead) is a distinctive feature of the bioprinter at a cooling rate of 1°C min and a constant shear rate of
−1
described here (Figure 1B and C). This printhead was 1 s . Shear stress was evaluated by varying the shear rate
−1
3D-printed at a resolution/layer height of 80 μm from white range between 0.01 and 1000 s in a rotational test at 14°C.
Peopoly professional ultraviolet (UV) resin (Moai Peopoly, The storage (G′) and loss (G″) moduli were measured as a
USA) using a stereolithography apparatus (SLA) printer function of temperature at a constant frequency of 1 Hz
obtained from the same supplier. The cooling/heating nozzle and a constant strain of 0.1% in a temperature range of
−1
had internal volumes of 0.197 cm and 4.35 cm for the ink 4°C–40°C at a cooling rate of 1°C min .
3
3
reservoir and the external circulating water, respectively. The
bottom tip of the cooling/heating nozzle had a male Luer lock 2.6. Printing/bioprinting protocols
fitting that enabled the use of different commercial needles Our printing/bioprinting workflow consisted of preparing
with varying diameters. The sides of the printhead had two a 3D model of a desired structure using computer-aided
fittings for connection to a cooling/heating water circulator. design (CAD) software (SolidWorks, Dassault Systèmes,
The top had a small fitting for insertion of the plastic tubing France, in our case), exporting the model as a STL file,
that serves as the inlet for the inks/bioinks. and then slicing the file to obtain the G-code of the print.
The original extruder of the 3D printer was replaced Repetier-Host (Repetier, Germany) was used as the host
[2]
with a custom-made X-axis extruder carriage and mounted software for manual control of the bioprinter, for slicing
to hold the cooling nozzle (Figure S3C and D). These STL files into G-code, and for running printing routines.
accessories were also 3D-printed using PLA (Hatchbox, Standard petri dishes were used as printing platforms, and
USA) at a layer height of 0.28 mm. The corresponding a laser-cut piece of polymethyl methacrylate (PMMA)
designs are provided in the Supplementary File. The and binder clips were used to anchor the petri dish to
custom carriage used the same screws that held the stock the printer bed to maximize stability (Figure S3E).
carriage, whereas the new mount required four M3 screws We attained the correct height of the first layer for a
with their respective hex nuts to secure the cooling/ particular construct by dampening a piece of paper with
heating nozzle. We reduced the number of screws needed ethanol and placing it on the surface of the petri dish
by employing a coupling design inspired by traditional (Figure S3E). The printhead was then manually moved
Japanese joinery to attach the custom carriage mount. over the petri dish and lowered progressively until slight
[33]
friction was observed between the paper and the needle.
2.5. Inks preparation and rheological After removal of the syringe containing the ink/bioink
characterization from the refrigerator, the syringe outlet was carefully
GelMA with 10% methacryloyl substitution was prepared connected to the inlet of the printhead using plastic
in-house according to previously published protocols . tubing, and the syringe was placed on the syringe pump,
[28]
The GelMA ink was prepared by dissolving GelMA in which was located beside the bioprinter. Approximately
Dulbecco’s phosphate-buffered saline (DPBS, Sigma- 30 cm of space was cleared around the bioprinter, and
Aldrich, USA) at 5% (w/v) at 70°C for 20 min. This GelMA all surfaces were cleaned with 70% ethanol to minimize

Volume 9 Issue 6 (2023) 99 https://doi.org/10.36922/ijb.0244