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

Figure 3. High-throughput fabrication of aggregates. (a) The number of aggregates fabricated by the 3D acoustic device were controllable with the
adjustment on the height (H) of the solution. Scale bar: 1 mm. (b) Both the number of aggregate layer (L) and the yield show an increasing relationship
with the H. In this acoustic device, more than 13,000 aggregates were fabricated.
linearly proportional to the H, having a good agreement cells or cell aggregates [49,50] . In this experiment, once the
with the simulation results. Accordingly, the total number cell aggregates were acoustically assembled, blue light was
of aggregates was also increased. At the H value of 5.5 mm, turned on for 30 s to initiate the photopolymerization
the number of aggregates was more than 13,000. of GelMA to form a hydrogel scaffold. Consequently,
cell aggregates encapsulated in GelMA hydrogel can be
3.4. Acoustic assembly of cell spheroids easily and non-disruptively transferred into Petri dish
Having demonstrated the 3D acoustic assembly device’s supplemented with corresponding medium for a week
ability to produce high-throughput particle aggregates in a and cultured in a humidified incubator. To sustain the
3D-dot array pattern, we then applied them to fabricate cell supply of oxygen and nutrients for encapsulated cells, the
spheroids. Hepatocellular carcinoma cell line, C3A cells, GelMA hydrogel scaffold was divided into smaller pieces
were evenly suspended in the 6% (w/v) GelMA solution (2 × 2 × 3 mm). Figure 4c shows the dynamic growth of
(2 × 10 /mL) containing 0.5% (w/v) LAP, and added into the cell aggregates within the GelMA scaffold. At day 1,
6
the acoustic chamber. The height of the solution was these loosely assembled aggregates were still composed
controlled at 3 mm. Once applying RF signals, a 3D-dot of individual cells. Interestingly, at day 3 they formed
array of cell aggregates was generated, as shown in a serial compact spheroids with smooth contours. At day 7,
view (Videoclip S4, with description in Supplementary the spheroids further matured and become darker. We
File). It was observed that the acoustically assembled determined the formation efficiency of spheroid, which
cell aggregates alternately appeared and disappeared as depends on the incubation time, by calculating the ratio
the microscope’s focal layer continued to move upward of the number of compact spheroids to the total number of
from the bottom. Figure 4a shows the distribution of cell initial cell aggregates.
aggregates at three different layers. It clearly demonstrates
that cell aggregates in each layer were arranged in a dot- Figure 4d shows that the efficiency of spheroid formation
array pattern, and had the same periodical distribution had reached more than 90% at day 3. Furthermore, we
characteristic (Figure 4b). In this experimental setup, 7500 tested the viability of cells during the acoustic assembly
(25 × 25 × 12) cell aggregates were fabricated per operation. and culture process (Figure 4e and f). The results revealed
good cell viability (>90%) and no significant difference as
To further mature the cell aggregates, these loosely compared to cells without acoustic treatment. Together,
formed aggregates need to be transferred into an incubator. these results demonstrated that the novel 3D acoustic
However, the transfer process would inevitably disrupt their assembly device enabled to fabricate cell spheroids in a
aggregation structure, resulting in low production efficiency high-efficiency, high-throughput, and low-cell damage
and low yield of spheroids. To prevent that, we employed the manner.
GelMA material to constitute the cell suspension. It has two
major characteristics: (i) rapid photopolymerization into a 3.5. Cell spheroid retrieval
crosslinked hydrogel to maintain and protect the structure To flexibly apply the cell spheroids for diverse biological
of acoustically assembled cell aggregates, and (2) superior studies, such as high-throughput drug screening and tissue
biocompatibility to enable the growth and maturation of engineering, the acoustically fabricated cell spheroids need

Volume 9 Issue 4 (2023) 266 https://doi.org/10.18063/ijb.733