Page 408 - IJB-9-4

International Journal of Bioprinting Development and characterization of AAMP for hydrogel bioink preparation

Despite tremendous breakthroughs made in hydrogel In an active mixer, two syringes loaded with hydrogel
bioprinting, challenges remain, with achievement components are placed against each other and connected
of hydrogel bioink homogeneity being one of them. by a female-to-female syringe connector. By pumping
Syringe extrusion is the most used hydrogel bioprinting the two-syringe system back and forth, the components
approach, where the hydrogel is first prepared by mixing would start mixing in the sealed chamber formed by
[25]
of the monomer, crosslinker, and other additives, and then the two syringes and the connector . As the back-
incubated to form the hydrogel, followed by the loading of and-forth pumping could have infinite number of
hydrogel into a syringe for subsequent printing . During cycles, homogeneity could eventually be reached if the
[15]
this process, homogeneous mixing and proper loading components are non-reacting. However, the gelation
are crucial to the homogeneity of the final product to be of hydrogel pre-polymer components adds more
printed . As the hydrogel pre-polymer solutions are complexity to the process, and if not mixed fast enough,
[16]
usually highly viscous and difficult to mix, traditional gelation would affect the final homogeneity. Therefore,
[17]
vortexing usually works poorly for hydrogel pre-polymer the pumping is expected to be fast with high pumping
mixing. The fast gelation kinetics also result in the change frequency and pumping speed. So far, this approach is
of viscosity and diffusion coefficients during the mixing, still carried out by manually pumping the two syringes,
[15]
which adds more complexity to the mixing process . which is not only laborious, but also limited in its
Moreover, during all the mixing and loading steps, it is pumping speed and frequency. With the pumping of very
difficult to avoid formation or trapping of air bubbles . viscous fluids repeatedly with only thumbs in the span
[15]
These factors can greatly hinder the bioink preparation of a few minutes, one may even be at risk for long-term
process, resulting in heterogeneous bioink, which, in turn, ergonomics-based diseases. Moreover, manual operation
affects the print quality. induces human error as the pumping speed can hardly be
kept constant. All these factors add up to the complexity
Various efforts have then emerged to address these of the mixing and gelation process during the hydrogel
issues so as to achieve a homogeneous bioink. The most bioink preparation, making it difficult to analyze and
straightforward approach is to apply powerful mixing standardize. Therefore, many hydrogel preparation
apparatus. Different types of mixing apparatus, including approaches still suffer from batch-to-batch variations [26,27] .
blenders [18,19] or agitators, [20,21] can be used. These powerful Such variation not only limits the applications of hydrogel
mixers can achieve fast mixing homogeneity even before bioinks, but also hinders the theoretical understanding of
the sol-gel transition begins, so the change in viscosity the intrinsic gelation process of hydrogels.
and diffusivity can be negligible. They are also capable of
batch-processing large quantity of gels due to their large In this work, we propose an automated active mixing
form factors. However, these methods are still subject to platform (AAMP). The AAMP is an active mixer that has
formation or trapping of air bubbles during the blending the potential to relieve this variation obstacle. On one
or on the loading of bioink into the printing syringe hand, the AAMP frees human labor from the laborious
barrel. Once the gel is formed, it is relatively difficult to pumping operation. On the other hand, it allows for
be transferred from one container to another without controllable mixing settings, including pumping cycles
trapping air. To address this, approaches have been and speed. Such capabilities can help minimize the batch-
developed to perform the mixing in the format of syringes. to-batch variation of hydrogel bioinks, as was validated by
For example, a static syringe mixer was developed with consecutive successful rounds of homogenous mixing. We
the brandmark of CELL-MIXER [22-24] . In such static mixer, also systematically characterized the AAMP performance
different components to be mixed are loaded in separate under different parameter settings, including cycle and
2+
syringes placed in parallel and pushed together through a speed, using a representative alginate/Ca hydrogel with
mixing tip whose outlet is connected to the target syringe. high-viscosity additives. All experiments were validated
using colorimetric analysis . We believe, the high
[25]
The mixing tip is designed with swirling blades, so that repeatability provided by AAMP could greatly facilitate
the components can be thoroughly mixed when passing the fabrication of hydrogel bioink with high quality, as
through. As the mixer is static and the mixing is a one- well as provide more predictable data for theoretical
time operation, the homogeneity of the mixing depends study of the gelation process. Furthermore, to validate
on the length and geometry of the mixing tip. For very our experimental results and provide more insights
viscous components, the tip might never be long enough into the hydrogel gelation study, we also carried out
to achieve homogeneous mixing.
COMSOL simulation for the AAMP mixing process. We
In these cases, another type of syringe-based mixer, varied mixing cycle and speed in the simulation and the
referred to as active mixer herein, would be preferred. outcome matched with experimental results. In addition,

Volume 9 Issue 4 (2023) 400 https://doi.org/10.18063/ijb.705