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

making the total cost of COTS materials and 3D-printed (FBS), 1% penicillin–streptomycin solution, and 0.5%
parts to be approximately US$ 297. This represents a neomycin. The cells were then stored in a humidified
very low price point for a quad-extrusion 3D bioprinting 5% CO incubator at 37°C. Before mixing the cells with
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system compared to what has thus far been reported in the GelMA, cells were stained with green fluorescence using
literature. A detailed table of components can be found CFDA-SE (CFDA-SE, Invitrogen, CA, USA) to track
in the GitHub repository mentioned in the Availability of their proliferation and migration within the final printed
data section. constructs using a wide-field fluorescence microscope
(IX83P1ZX, Olympus, TYO, JP).
2.2. QEH design and QEB development
The QEH was designed and developed in-house using
Autodesk Fusion 360, where all motions and tolerances 2.3.2. Viability and invasion assays
were simulated to verify the proper functioning of the The steps done by Zhu et al. were followed exactly for
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system. With the compactness of this minimalistic low- conducting viability assays. LIVE/DEAD staining kits
profile design, the maximization of printing volumes with (LIVE/DEAD Viability/Cytotoxicity Kit, Invitrogen, CA,
functionally biomimetic characteristics was achieved. USA) were used. Tissue constructs were washed with
Dulbecco’s phosphate-buffered saline (DPBS) and stained
The fabrication of the main body of the QEH was with 2 μM calcein blue AM (green stain for live cells) and
done in-house using an FDM 3D printer with PLA+ 4 μM ethidium homodimer-1 (red stain for dead cells)
filament (DURAMIC 3D, Amazon, USA). This allows the solution and then incubated for 30 min. After incubation,
maintenance of the tight tolerances desired and minimizes the stained constructs were washed and imaged under
any warping within the model. The gears and syringe the wide-field microscope with fluorescein isothiocyanate
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plunger holders were printed with acrylonitrile butadiene (FITC; green stain for live cells) and tetramethylrhodamine
styrene (ABS) to ensure high strength within those parts (TRITC; red stain for dead cells) channels. The images were
since they are moving parts with high torque exerted on then analyzed using ImageJ for cell counting and viability
them. results.
To properly control the QEH, the appropriate firmware To characterize the function of cells in bioprinted
configurations need to be developed. For that purpose, tissue constructs, a trophoblast (HTR-8) invasion assay in
the open-source Marlin 2.0.3 firmware (MarlinFirmware/ a simplified 3D-bioprinted placenta model was performed.
Marlin, GitHub) was adopted and modified to suit the The placenta model was printed with four different bioinks
physical properties and requirements of our bioprinting using IAP for ease of culturing and the lack of necessity
system (GitHub link can be found in the Availability of data of post-processing. Figure 6A shows a top-view design
section). Namely, the firmware was made to accommodate of the placenta model. This model is composed of two
four extruders and allow cold extrusion by setting a dummy main modules, the epidermal growth factor (EGF) (EGF/
temperature sensor to compensate for the removal of the Alexa-555 conjugate, ThermoFisher, USA) and the HTR-
existing temperature sensor. The modified firmware was 8 modules. All sections of the model are composed of
then uploaded to the upgraded motherboard, and the step GelMA bioinks. The two modules were separated by two
motors were calibrated to output the correct number of main channels that allow us to study the invasion of the
turns and speeds as needed. This allowed the control of the HTR-8 cells from the cell module to the EGF module.
QEB through any open-source control software, namely The two main channels are the control channel, which is
Repetier Host (Repetier-Host, Repetier) used herein. composed only of GelMA, and the experimental model,

The g-code generated needed to be customized based which comprises GelMA and a low concentration of EGF.
on the machine and the model being printed. Starting and To ensure that the cells invade only through the control
ending g-codes as well as the extruder change g-code were and experimental channels at the same conditions, GelMA
added into the slicer to modify the generated g-code based separation channels, made of 10% GelMA, were added
on the type of printing paradigm being adopted (IAP and highly crosslinked. This would make those separation
versus SBP). The starting Z-level was also modified to meet channels stiffer and harder for cells to invade through.
the desired starting height based on each type of substrate. To ensure that the cells do not migrate to the surface or
bottom of the bioprinted modules, highly crosslinked
2.3. Biological studies GelMA bed and cap layers were added below and above
2.3.1. Cell culture and fluorescence staining the modules, respectively, as shown in Figure 6B. With
HTR-8/SVneo (HTR-8) derived from the SV40-transfected this model configuration, cells would only invade through
first trimester EVT cell line (ATCC, USA) were cultured the designated channels where the effect of EGF on cell
in DMEM/F12 medium containing 5% fetal bovine serum invasion can be studied. FITC and TRITC fluorescence

Volume 10 Issue 1 (2024) 296 https://doi.org/10.36922/ijb.0159