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Formation of cell spheroids using Standing Surface Acoustic Wave (SSAW)

differentiation [22,23] because they represent more similar in may produce significant biological effects, such as damages
vivo biological behaviors. Therefore, cell spheroids could to the cell membrane [37] , apoptosis, and necrosis for the
be an alternative format of the bioink. More importantly, reduced cell viability. Furthermore, the fluid medium may
such novel bioink enhances cell-cell interaction, growth, also be heated up by the acoustic exposure due to the
differentiation, and resistance to the environment because energy absorption, especially in a small cavity at high power
of the high cell density in the construct. As a result, the output and high acoustic frequency, which may harm
printed vascular construct shows a better cell-cell interaction biological cells [38,39] .
and differentiation [24,25] . Additionally, tissue construct In this study, the effects of excitation frequency on the
printed using the cell spheroids could minimize the inclusion formation of cell spheroids (accumulation time and size)
of biomaterials [26 ] , enhance the growth in the natural and their biological characteristics (growth and cell viability)
condition, and reduce the potential biodegradation which in the culturing afterward were studied. The motion of
may release the toxic or unnatural byproducts [25] . cells by SSAW for the formation of cell spheroids was
The current methods of forming cell spheroids, such simulated and then compared with the experimental results. It
as using the U-bottom plate, cell hanging drop [27] , is hypothesized that the high-frequency excitation could
dielectrophoresis [28] and magnetic-assisted assembly [29] , reduce the accumulation time, but size of cell spheroids
require additional chemicals to modify the cell culture as well. The potential damage of acoustic exposure to the
medium or the use of a complex device or complicated formed cell spheroids was evaluated up to 7 days after
fabrication process, but in low throughput. Although rotating the production. Our study may be able to provide the
cell culture [30] , using non-adhesive surface [31] , and cell guideline for the preparation of cell spheroids by SSAW
culturing in scaffold [32] can improve the throughput, they are as bioink for the future biotechnical applications.
still time-consuming and tedious with inconsistent production 2. Materials and Methods
of cell spheroids in size. Microvalve-based printer is another
high-throughput method to form cell spheroids, but low cell 2.1 Governing Equation
viability and inhomogeneity were found [33,34] . In comparison,
microparticle manipulation by the acoustic wave has been In the fluid, the motion of cells in the acoustic field depends
utilized in the field of lab-on-a-chip because of its advantages on the resultant forces from acoustophoresis and Stroke
of non-invasiveness, low power consumption, free labeling, drag. The Stokes drag force applied to the cells is due to
[40]
biocompatibility, and high throughput. Standing wave the velocity differences between fluid and cells .
generated from the bulk acoustic wave (BAW) could trap (1)

the individual cells loaded into a certain device to the
[35]
pressure nodes and then form cell spheroids . However, where r is the radius of cell in the shape of a sphere, and

excitation frequency for BAW is quite low (mostly below 4 are the velocities of fluid and cells, respectively, is

MHz), resulting in weak acoustic radiation force, low the dynamic viscosity. As cells have different physical
throughput, and domination of acoustic streaming and properties from fluid media, the propagation of an acoustic
temperature instability at the high power. In the recent wave causes the cells to oscillate and pulsate, which leads to
year, surface acoustic wave (SAW) was introduced in the monopole and dipole scattering expressed in the resultant
microparticle manipulation [36] . In comparison to BAW, acoustic radiation force [41] .
SAW has the advantages of high excitation frequency, high

throughput, low power consumption, less excessive heat and

disturbance of acoustic streaming, simple manufacture of
device in arbitrary design, and large range of operating , (2)
parameters. However, the effect of excitation frequency
on the formation of cell spheroids by standing surface where and are the density of cell and fluid, and

acoustic wave (SSAW) and their biological characteristics are the compressibility of particle and fluid, and

has not been explored. As the distance between pressure are the dimensionless scattering coefficients for the

nodes in the standing acoustic field is half of the wavelength, monopole and dipole, respectively, and is the acoustic

which is inversely proportional to the excitation frequency, wave number. In the acoustic standing wave field, the
and the acoustic radiation force applied to the microparticles acoustic radiation force acting on the cell is simplified as
is proportional to the frequency, the preparation time and

size of cell spheroids is highly dependent on the excitation (3)

frequency. In addition, acoustic exposure at high intensity
2 International Journal of Bioprinting (2018)–Volume 4, Issue 1