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International Journal of Bioprinting Droplet-based bioprinting of tumor spheroids

and is compatible with diverse substrates, including well bioprinted by TIJ is approximately within the range of 75%
plates, microwells, scaffolds, and planar substrates. Apart to 90%. 14-16,18,19
from these general properties, there are also different
features associated with the working mechanism. In 2.2. Piezoelectric inkjet bioprinting
this section, we illustrate the bioprinting principles and Similar to thermal inkjet bioprinting with the deformation
performances of droplet-based bioprinting technologies of bioink by external actuation, piezoelectric inkjet
for spheroid fabrication. Conventional droplet-based bioprinting (PIJ) leverages a piezoelectric actuator, which
bioprinting can be categorized into three types based on is embedded in the printhead surrounding the chamber.
the droplet formation mechanism: inkjet, acoustic, and When supplied with a high-voltage pulse, the piezoelectric
microvalve-based. Inkjet bioprinting is the mostly adapted actuator produces a radial compression, which drives the
technique, and it can be further classified into three: bioink chamber to generate a transitory radial deformation.
continuous inkjet bioprinting, drop-on-demand inkjet The sudden contraction of chamber volume induces a
bioprinting (thermal, piezoelectric, and electrostatic), and pressure wave, resulting in droplet ejection (Figure 2A,
electrohydrodynamic inkjet bioprinting. In this review, middle). During droplet ejection, the back pressure applied
continuous ink-jetting mode is not discussed, owing to in the fluid chamber has a range of 200 to 500 Pa. 20,21 Many
the incapability to control droplets on demand. Notably, factors affect the droplet size and cell viability, such as the
combined with microfluidics, droplet-based microfluidic length of the piezoelectric inkjet nozzle, diameter of the
bioprinting has attracted increasing interests in recent orifice, and amplitude and frequency of the voltage pulse.
years. We summarize and compare these techniques (see The most utilized mode to excite the actuator is the bipolar
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Figure 2 and Table 1) and present a review of them in this excitation waveform. For instance, Xu et al. fabricated
section. scaffold-free complex tubes with an overhang structure
using a bipolar excitation voltage with an amplitude of 45
2.1. Thermal inkjet bioprinting V. The droplet generated by PIJ shows a diameter range
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The initial thermal inkjet bioprinter is derived from a of 25 to 100 μm, 20,21,23 suitable for printing cells with a wide
thermal inkjet 3D printer. During thermal-based inkjet range of sizes. Despite that many studies have attempted
bioprinting (TIJ; Figure 2A, left), the thermal actuator to control the PIJ bioprinting process, challenges remain
locally heats the bioink solution with a voltage pulse, to reduce the number and diameter of satellite droplets
which generates vapor bubbles. Subsequently, the bubbles and to improve the printing efficiency. In addition, the
expand rapidly and explode, leading to a pressure pulse mechanical deformation pressure produces a relatively
that deforms bioink to eject droplets from the nozzle. high shear stress (more than 10 kPa), which may damage
Thermal inkjet bioprinter, which was directly transformed cells ; thus, the cell viability after PIJ is approximately
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from commercial deskjet printer with modifications, can within the range of 70 to 90%. 20,23
produce a very high throughput at frequency of above
10,000 Hz, with the actuation of a high-frequency voltage 2.3. Electrostatic inkjet bioprinting
pulse. 14,15 Other technological improvement includes the The electrostatic inkjet printer was initially developed by
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integration of multiple nozzles, based on which higher Kamisuki et al. in 1998. Similarly, electrostatic inkjet
printing efficiency can be achieved. 15 bioprinting (EIJ) generates droplets through electrostatic
attraction between the electrode and pressure plate;
Droplets generated by TIJ have a diameter range of 30 the pressure plate is deformed when a voltage pulse is
to 60 μm, meeting the requirements of most droplet-based applied, leading to a volume change of fluid chamber
bioprinting. 14,16 TIJ is capable of dispensing a variety of and subsequently droplet ejection (Figure 2A, right). The
biological materials, such as cellular solutions and proteins. frequency of voltage pulse directly affects the throughput
The major concern regarding this technique is whether of droplet ejection, and a maximum throughput of
printed cells and tissues can proliferate and differentiate approximately 2 kHz was demonstrated. Printed droplets
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normally because the heat needed to eject droplets during showed a diameter range of 10 to 60 μm, which meets the
printing is relatively high. Campbell et al. found that the requirement for single-cell positioning. The survival rate
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MCF-7 breast cancer cells had a viability rate of 73% after of cells after EIJ is relatively low, and tested cell viability is
thermal-based bioprinting. When cultured in tamoxifen roughly 70%. 25
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solution, the cells exhibited an increase of viability and
the secretion of chaperone proteins. Overall, biological 2.4. Electrohydrodynamic jet bioprinting
and physiological experiments have demonstrated that Droplet-on-demand inkjet bioprinting utilizes a very high
the heating process and mechanical stress can damage level of heat or pressure to eject droplets from a small
cells and decrease cell viability; thus, the viability of cells nozzle, in which heat or mechanical stress may be harmful

Volume 10 Issue 1 (2024) 109 https://doi.org/10.36922/ijb.1214