Page 389 - IJB-10-2

International Journal of Bioprinting Automated bioink mixer improves bioprinting

By enabling the direct spatial deposition of living cells and enable rapid bioink mixing. These kits, however, have
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biomaterials, EBB offers great potential for advancing design limitations such as constraining the volume ratio
tissue engineering and regenerative medicine. A critical between the cell suspension and bioink. Additionally,
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component of the EBB process is the bioink, which is these devices suffer from an inherently big loss of material
extruded as a continuous strand and can be stacked to due to the dead volume of the mixing chamber. In some
fabricate 3D objects. Consequently, the properties of laboratories, a planetary mixer is utilized to prepare viscous
the bioink significantly influence printability, structural mixtures. 13,14 This method is good for reproducibility, but
stability, cell growth, and differentiation by acting as it still presents difficulties in cartridge loading for EBB
essential physical support. However, current bioinks still purposes. One widely used approach is the syringe coupler
face numerous limitations, with one of the major challenges method, which involves mixing the materials using two
being the preparation of the bioinks in a reproducible and syringes by repeatedly pushing the plungers back and
cell-friendly manner. Achieving consistency would ensure forth. This method has gained significant popularity
a precise and consistent bioprinting process that maintains in EBB bioink preparation due to its cost-effectiveness,
high cell biocompatibility. 8 reduced waste, avoidance of air entrapment, and low
The preparation of bioinks dictates the success of EBB risk of contamination. 15-19 When using a syringe coupler
technology, as it directly impacts the quality of the printed to mix bioinks, two crucial parameters are the speed at
constructs and the biological properties of the encapsulated which the plungers are pushed and the number of pushes
cells. Inhomogeneous bioink can lead to nozzle clogging, performed, referred to as mixing speed and mixing
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structural collapse, uneven cell distribution, and variations exchanges, respectively.
at different stages of bioprinting. The inconsistency of the Like other mixing methods, the syringe coupler
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matrix components can cause an unstable viscosity of the method also faces the challenge of balancing between
bioink, while the uneven distribution of cells can result in bioink homogeneity and cell viability, particularly when
cell aggregation and cell vacancy. This can negatively affect dealing with viscous bioinks. To address this issue, Dani et
the printability and resolution of the bioprinted constructs al. made improvements by exploring customized couplers
and hamper the accuracy of the experimental outcomes. with different inner geometries. They discovered that a
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For the sake of clarity, we define homogeneity as the screw-like mixing coupler yielded a homogeneous bioink
uniformity of the bioink mixture, which significantly with high cell viability after a lower number of exchanges.
influences the printability, structural integrity, and cell However, the relationship between mixing parameters
distribution of the printed constructs. Conversely, cell (such as mixing speed and mixing exchanges) and bioink
viability refers to the ability of the encapsulated cells to homogeneity/cell viability remains unclear. Further
survive and maintain their biological function during and research is needed to better understand this relationship
after the printing process. However, these two factors, and achieve a balance between bioink homogeneity and
homogeneity and cell viability, often pose a challenge cell viability, for ultimately improving the physical and
during bioink preparation, and one factor generally works biological quality of the fabricated constructs. This is
against the other. Thorough and unstinted mixing is a crucial for enhancing the reliability of the bioprinting
common approach to obtain a homogeneous mixture, but process. Additionally, it is worth noting that in most
it can potentially harm the encapsulated cells. A high rate of the current studies, the researchers’ handling and
of mixing and/or too long a duration may even irreversibly experience in the bioink mixing process can lead to
disrupt the interactions among hydrogel molecules, such significant differences between individuals. This factor
as entangled structures, as well as covalent or non-covalent decreases the reproducibility of the bioprinting process,
bonds. Compared to other bioprinting methods such thereby influencing the subsequent experimental results.
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as stereolithography and inkjet bioprinting, this issue is Thus, standardized protocols in bioink mixing to ensure
particularly pronounced in EBB, which requires a higher consistent and reproducible outcomes in bioprinting are
viscosity bioink than the other techniques. highly desirable.

To achieve a homogeneous bioink for EBB, several In light of these challenges, the primary objective of
techniques have been developed for blending the this study is to establish a standardized approach for the
components. The pipetting method and spatula mixing bioink mixing process, thereby enabling precise control
are simple approaches that involve placing the materials over bioink properties for EBB (Figure 1). This research
in a container and either pipetting them up and down comprehensively investigated the influence of various
or stirring them manually. However, both methods mixing parameters on bioink homogeneity and cell
introduce air bubbles during the mixing process. Many viability. A dedicated mixing device was developed to
companies provide two-in-one-out mixing kits, which ensure reliable and reproducible operations of the desired

Volume 10 Issue 2 (2024) 381 doi: 10.36922/ijb.1974