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International Journal of Bioprinting Droplet-based bioprinting of tumor spheroids

to cells. In contrast, electrohydrodynamic jet bioprinting waves, which generate acoustic radiation pressure. When
(EHDJ) pulls the droplets out of the orifice with an electric the force exerted at the focal point exceeds the surface
field instead of mechanical deformation of bioink. The tension, the bioink is elongated to eject droplets. Because
EHDJ was initially rooted in the electrohydrodynamic acoustic bioprinting works without a nozzle, cells are not
atomization mechanism, and the first bioprinting was exposed to high shear stress during bioprinting process,
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performed by Eagles et al. During bioprinting (Figure and clogging in the nozzle is avoided as well. 45,46 The
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2B), a high electric field with a voltage of 0.5–20 kV between droplet size is determined by the amplitude and frequency
the nozzle and the substrate is applied, which induces the of acoustic waves. Jentsch et al. verified that the droplet
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ejection of droplets. During bioprinting, the accuracy size was negatively correlated with acoustic frequency. By
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and cell survival rate are affected by the voltage, flow rate, tuning the acoustic frequency, the droplet diameter could
and distance between the nozzle and substrate, 28,29 among be controlled within the range of 10 to 200 μm. Besides,
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which the applied voltage significantly influences droplet Demirci et al. demonstrated that the throughput could be
formation and droplet size. Gasperini et al. confirmed increased to ~1000 Hz. Because cells are not exposed
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that droplet size decreased with the increase of voltage to harmful external stressors (heat, pressure, and electric
from 14 to 20 kV, above which the droplet formation field), cell survival rate after acoustic bioprinting can be kept
was disordered. Droplet diameter could be tuned from at more than 90%. Owing to superior biocompatibility,
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10 to 1000 μm by varying the voltage. 28,30 Because the acoustic bioprinting exhibits good potential in tissue
droplet generation is dependent on the electric force engineering.
and is not limited by the orifice size, EHDJ is suitable for
bioprinting applications requiring high-concentration 2.7. Microfluidic bioprinting
cells. Moreover, the low shear stress reduces the cell It is difficult to manipulate cells or droplets during
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damage during printing and thus results in a relatively conventional droplet-based bioprinting. However,
high cell survival rate of above 90%. However, there integrating microfluidics into conventional bioprinting
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are some shortcomings that restrict the application of platform can enhance the droplet printing performances.
EHDJ. For instance, during droplet printing, it works in Microfluidics has seen considerable advances since its
a continuous manner, and several droplets are generated rapid growth from the 1990s, and a myriad of microfluidic
at a time, making it a great challenge to realize the precise modules have been developed. The manipulation of fluids,
placement of single droplets. 32-34 droplets, and cells of microfluidics improves the precision
and complexity of bioprinting. For instance, multiple
2.5. Microvalve-based bioprinting bioink fluids can be controllably mixed, cells can be
Microvalve-based bioprinting generates droplets with encapsulated and sorted, and droplet composition can be
an electromechanical valve (Figure 2C). In general, the manipulated. In the early stage, microfluidic bioprinting,
electromagnetic force, actuated by a voltage pulse, is which was directly modified from the capillary
applied on one plunger that gates the orifice to generate microfluidics, can generate droplets as building blocks
droplets. The droplet formation is influenced by properties to fabricate biological constructs (Figure 2E, left). 51-53 The
of bioink, pneumatic pressure, valve gating time, nozzle droplet size can be tuned with varying fluid flow rates and
length, and nozzle geometry. 32,35-37 In addition, Chen wettability of capillaries.
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et al. found that stable droplet formation worked with
a Z number (the inverse of the Ohnesorge number, Droplet-based microfluidic bioprinting possesses
2<Z<15) of the bioink, and the increase of concentration the similar properties of the conventional technologies
of bioink solution had a negligible impact on the droplet and manifests new capabilities. For instance, Zhang and
diameter and cell viability. Overall, for microvalve-based Abate conducted single-cell printing using a new type of
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bioprinting, the survival rate of cells can be maintained at droplet-based microfluidic bioprinting technique they
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85–95%. 39-41 Besides, microvalve-based bioprinting has a developed (Figure 2E, right). In their method, cell-
throughput of up to 1 kHz with a droplet diameter range of containing droplets, formed by shear of bioink with co-
100 to 600 μm. 39,42-44 Limited by the droplet diameter, the flowed air streams, can be sorted in air, based on which
tissues fabricated by microvalve-based bioprinting show a deterministic single-cell printing is achieved. They
lower resolution than that by TIJ, PIJ, or EHDJ. demonstrated that the survival rate of different types of
cells was more than 95%. They fabricated multicellular
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2.6. Acoustic bioprinting liver spheroids with single-cell printing and showed
Acoustic bioprinting leverages focused acoustic waves. As that the size and composition of these spheroids can
shown in Figure 2D, two focal interdigital transducers are be controlled, which is conducive to the accuracy of
placed on the piezoelectric substrates to generate acoustic drug screening.

Volume 10 Issue 1 (2024) 111 https://doi.org/10.36922/ijb.1214