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International Journal of Bioprinting 3D acoustically assembled cell spheroids with high-throughput

Figure 1. Working mechanism of the 3D acoustic assembly device for the high-throughput fabrication of cell spheroids. In 3D acoustic fields, the randomly
suspended C3A cells in gelatin methacrylamide (GelMA) solution were acoustically levitated and assembled into cell aggregates with an 3D array pattern,
once three orthogonal bulk acoustic waves (BAWs) generated by piezoelectric transducers (PZTs) were introduced. Then, blue light was turned on to
photocrosslink the GelMA solution to maintain the structure of cell aggregates. After 3 days of culture, cell spheroids were formed and then retrieved for
drug testing.

2.7. Numerical simulation meant that more cell aggregates could be fabricated by
To simulate acoustic field in our MLANs device, a 3D our acoustic devices. In this case, the suspended cells in
finite element analysis (FEA) model was developed using GelMA solution were moved to the nearby LANs under
COMSOL Multiphysics 5.4a. To reduce the computational the action of acoustic radiation force, and finally assembled
amount, we only considered the fluidic domain. The three into a 3D cell aggregate array. To facilitate the maturation
PZTs were considered plane wave radiation boundary of cell aggregates, aggregates need to be transferred into
conditions at the X, Y, Z sides of the fluidic domain incubators. However, the process of transferring them will
(Figure S1b in Supplementary File). A frequency domain inevitably disrupt their aggregation structure, resulting in
solver was used to calculate the 3D acoustic field. low maturation efficiency of spheroids . To overcome
[38]
this problem, we adopted biocompatible GelMA hydrogels
2.8. Statistical analysis that were photocured as supporting scaffolds , enabling
[49]
All data presented are quantified from at least three to maintain and preserve the structure of cell aggregates.
independent experiments. All values are presented as After that, the acoustic fields were withdrawn and these
mean ± standard error of the mean. Significant differences acoustically assembled cell aggregates within the GelMA
between experiment groups were evaluated by Student’s hydrogel can be easily and non-disruptively transferred
t-test, and P < 0.05 was considered statistically significant. into Petri dishes for further incubation. During the 3 days
of culture, most of cell aggregates (>90%) matured into
3. Results and discussion cell spheroids, and were ready to be retrieved from the
GelMA hydrogel dissociated by a GelMA lysis buffer for
3.1. Device design and working principle subsequent drug testing.
To improve the yield of spheroids using acoustic cell
assembly devices, the presence of more acoustic nodes 3.2. Characterization of 3D dot-array of LANs
is a prerequisite for fabricating more spheroid. Here, we In order to verify that the 3D dot-array of LANs was
developed a 3D acoustic cell assembly device for high- created, a numerical model was first used to predict
throughput fabrication of cell spheroids (Figure 1). The the acoustic pressure field of the 3D acoustic assembly
device was made of three PZTs (20 × 10 mm; frequency, device (Figure 2a). The results showed that there are
3 MHz) located at the three orthogonal walls of the square periodic distribution of levitated acoustic nodes in all
chamber. After applying the RF signals, the PZTs excited three directions, as a result of a 3D dot-array of LANs
three orthogonal BAWs that propagated into the bulk created in the acoustic field. In general, suspended cells or
of the solution and coherently interfered with their own particles are pushed toward the LANs under the action of
reflected waves from the opposed chamber wall to form acoustic radiation force (Figure 2b), and assembled into
three orthogonal standing BAWs. As a result, a 3D dot- aggregates [42-44] . We used 10-μm fluorescent polystyrene
array (25 × 25 × 22) of LANs was formed in the acoustic particles to investigate it. To obtain good assembly pattern,
chamber. The number of LANs (>13,000) was higher than the two PZTs in the horizontal direction were first applied
those obtained with currently used BAWs-based (>30) with RF signals (frequency, 3.12 MHz, 3.10 MHz; 2–5 Vpp)
and SAWs-based (>12,000) acoustic devices [43,47] , which to obtain a clear pattern of 2D array (Videoclip S1, with

Volume 9 Issue 4 (2023) 264 https://doi.org/10.18063/ijb.733