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

with multiple materials is achieving more popularity and cell–cell interaction studies, are crucial in promoting
and pertinence in several fields of use, including in vitro more realistic in vitro tissue models that better mimic in
meat development as an alternate food source that helps vivo tissue models and, ultimately, closing the scalability
in reducing methane gas release into the atmosphere gap toward fully functional organ printing. 35
resulting from animal carnage. These engineered meats In this work, a commercial off-the-shelf (COTS) fused
are composed of multiple tissue types embedded within deposition modeling (FDM) 3D printer was systematically
a single construct to mimic the natural architecture of a mass-modified with a novel quad-extrusion head (QEH)
Wagyu beef steak which is essential in developing the that is compact with a low profile, thereby minimizing its
proper taste and quality of meat. 18,19 moment of inertia when in motion. The quad-extrusion
Available bioprinting technologies that enable multi- bioprinter (QEB) was rendered at a relatively low overall
material printing for diverse biological studies are very cost of US$ 297, proving to be a reliable low-cost bioprinter
complex and rarely allow more than two or three materials with versatile capabilities. With that, it becomes possible
to be printed in a single construct. However, with multi- to fabricate multi-material biological constructs, thereby
material bioprinter designs, the cost remains prohibitively widening the scope and accessibility of in vitro studies.
high, rendering the scalability of such technologies to be This is enabled by the large printing volumes that the
a significant challenge. 20-23 Of all the available bioprinting QEB affords, which provides the capability to create tissue
technologies, extrusion-based systems are the most constructs at scale. Moreover, with its developed compact
prominent for multi-material printing due to the ease of and low-profile design, the QEH module is transferable to
development and modularity. 24,25 For example, Shen et al. other FDM 3D printers typified by similar frame designs,
developed a Computer Numerical Control (CNC)-based with minor modifications to the extruder carrier. To
bioprinter that has four separate extruders and allows demonstrate the capabilities of the developed QEB, gelatin
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multi-material bioprinting. However, it costs around US$ methacrylate (GelMA) bioink was used as a biomaterial
6000, which is still considered along the higher end of model to print complex multi-material constructs using two
prices with the current technologies available. Several printing paradigms. One paradigm is the more traditional
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efforts have been made to design and develop low-cost in-air printing (IAP), where the bioink is printed on glass
and ultra-low-cost bioprinters based on reliable process slides or petri dishes suspended only in air. Alternatively,
designs at costs not exceeding US$ 200–250. 27-34 For a more the second paradigm is support or suspension bath
detailed overview, Table S1 (Supplementary File) compiles printing (SBP), where the bioink is printed in a bath of
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an overview of both commercial bioprinters currently nanoclay that can self-support the bioprinted material.
available on the market and relevant research endeavors, The nanoclay bath used in this work was Laponite B. HTR-
specifically focusing on those positioned within the more 8 SV/neo trophoblasts were implemented as a biological
affordable cost range. Although with extant low-cost model to validate the functionality of the bioprinted
process designs available, limited functionality is achieved. hydrogel structures in providing a suitable environment for
Notably, the characteristic bulky and complex nozzle the cells to perform their intended function. Finally, post-
designs that typify existing low-cost bioprinters effectively process structural and biological characterizations of the
limit the reach and range of tissue applications. This bioprinted constructs were performed by way of assessing
fundamental limitation can be attributed to the significant geometric fidelity and cell proliferation and viability by
loss or reduction in printing volumes, even with single or way of microscopic imaging of the fluorescently labeled
dual nozzle configurations. This limits the range of tissue construct.
types and sizes that can be printed with such small printing 2. Materials and methods
volumes and a limited number of material nozzles. On
the one hand, the present design configurations limit the 2.1. Bioprinter components
ability to advance a low-cost bioprinting system capable To develop the in-house bioprinter with the QEH, several
of fabricating more complex tissue constructs with multi- components were directly bought as COTS. As a start, a
materials at arbitrary scales. On the other hand, by endowing Creality Ender 3 Pro 3D printer was purchased off Amazon
a bioprinter with large-scale multi-material capability, the for US$ 209. However, with the stock controller, there is
application range can be extended to include investigations only one pin for a single extruder. To be able to control
into the interactions between multiple bioinks with four extruders at the same time, the Zonestar ZRIB V6
different cellularized matrix content and organization control board was purchased to replace the stock control
that define the tissue mechanical properties. Specifically, board and allow the control of four independent extruder
investigations into the bioink material–material interfaces stepper motors. The Zonestar controller board costs US$
that join disparate target tissues, along with cell–material 49, and the additional three stepper motors cost US$ 19,

Volume 10 Issue 1 (2024) 295 https://doi.org/10.36922/ijb.0159