Page 112 - IJB-4-1

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

6
pressure node have the low initial acceleration, especially at cell density (2×10 cells/mL), 32.8 ± 4.3 μm vs. 18.8 ± 3.0
the high-frequency excitation and low output power in μm. Low-frequency could gather more cells from a wide
comparison to that at the low-frequency excitation and region due to its large wavelength for the generation of
the same output power input (see 0.1 W in Figure 2D). larger cell spheroids. Overall, it is a tradeoff between the
It is noted that if the height of PDMS cavity is larger size and accumulation speed of cell spheroids. New strategies
than the half wavelength of SSAW excitation multiple or techniques are desired to produce large uniform cell
pressure nodes will be generated in the vertical direction. spheroids in a short time. It is noted that low power output
The magnitude and distribution of these pressure nodes (e.g., 0.1 W) was applied after the cell spheroid formation
in the central region of the cavity are quite uniform, but not for 30–90 min in order to allow sufficient ECM being
at the edge [53] . Those cells accumulated at the middle region secreted to hold them in place and avoid the sedimentation.
of pressure nodes are in the suspension and may have No mergence of cell spheroids was found inside the PDMS
low possibility of attachment to the cavity. However, higher cavity at the cell density used in this study. Afterwards, the
PDMS cavity may not allow the accurate measurement of spheroids are stable enough for handling and transferring.
the size of cell spheroids aligned vertically, but increase During the excitation, most cells initially move toward
the production, which will be evaluated later. and gather with the others at the nearby pressure nodes.
However, it is found that some cells locate slightly away
3.2 Formation of Cell Spheroids by SSAW from the pressure nodes, which may be due to attachment
Cell spheroids were gathered and formed from suspended of cells on the surface of PDMS cavity or LiNbO 3 wafer
individual cells under acoustic excitation, usually within and curved wave front of SSAW inside the cavity. Cells
30 sec. The diameter of spheroids relies on the number of attachment on the surface of microchannel is a common
cells in the adjacent region of pressure nodes. The distance issue due to complicated surface properties [59,60] . Briefly,
between pressure nodes in the PDMS cavity, which is the the attractive forces from the surface are stronger than the
half wavelength and inverse proportional to the excitation combination of electrostatic repulsion force and acoustic
frequency, is one of the important factors for the size of streaming forces applied to the cell [61] . Subsequently, cells
produced cell spheroids. In this experiment, both low- could not move and follow the acoustic radiation force.
and high-frequency excitations could accumulate cells at When the waves travel through PDMS and fluid medium
the pressure nodes successfully (see Figure 3). However, at a long distance, acoustic attenuates particularly at the
the size of cell spheroids and number of accumulated cells high frequency is not spatially uniform because of the
at each pressure nodes are not exactly same. The main heterogeneous properties distribution of each medium. The
reason may be non-uniform cell distribution in the PDMS diffraction waves generated from the flat IDTs lead to
cavity [54] with low Reynold number, <20 (see Figure 3C). the slightly curved wave front, but the flat grid in the
Accumulation of cells and subsequently, the formation of numerical simulation. In addition, cell density may also
cell spheroids is a quite complicated phenomenon involving determine the magnitude of force required to tightly pack
several factors, such as cell aggregation [55] , lateral shear cells into spheroid. In the recent study, during of cells
force [56] , and culture medium (e.g., nutrients [57] , growth High cell density results in the formation of large cell were
[57]
factor [58] , and waste ). Initial average size of cell spheroids found located away from the pressure node at high cells
[36]
generated by the low-frequency excitation is slightly larger density . Furthermore, cell density is an important issue.
[36]
than the reported value in the previous study at the same spheroids, but also high possibility of cell attachment and
(A) (B) (C)

Figure 3. Accumulation of HepG2 cells by SSAW at the frequency of (A) 10.4 MHz, (B) 23.8 MHz, and (C) distribution of
suspended cells without excitation, scale bar of 50 μm.

6 International Journal of Bioprinting (2018)–Volume 4, Issue 1