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

the 3D acoustic assembly device was designed composed light (405 nm, 60 mW/cm , 30 s) to form a 3D hydrogel
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of three PZTs (PZT-41, Yantai Xingzhiwen Trading Co., scaffold. Subsequently, the scaffold encapsulating particles
Ltd.), a square acrylic chamber (21 × 21 × 10 mm), and or cell aggregates were transferred to a Petri dish (35 mm,
a manual Z-axis moving apparatus (minimum step, 10 Biosharp) for confocal imaging or culture, respectively.
μm). The spacing between the two opposite walls of the For the cell assembly culture, the hydrogel scaffold was
chamber was an integer multiple of the half wavelength further divided into smaller pieces (2 × 2 × 3 mm) using a
(λ = 500 μm). Two PZTs (20 × 10 mm; frequency, 3 MHz) sterile surgical blade to sustain the supply for oxygen and
were glued to the X and Y outer chamber with epoxy, nutrients for encapsulated cells.
respectively, and another PZT above the chamber was
affixed to acrylic substrate (20 × 20 ×10 mm) that was 2.4. Spheroid retrieval
mounted on the Z-axis moving apparatus (Figure S1a in After 3 days of culture, cell aggregates were grown in the
Supplementary File). Each PZT was driven independently GelMA scaffold and mostly fused into spheroids. Then,
by a radio frequency (RF) signal generator (DG5352, these spheroids were released by dissociating the GelMA
Rigol) and a power amplifier (LCY-22 +, Minicircuit). To scaffold using GelMA lysis buffer (EFL-GM-LS-001, EFL)
hold the cell suspension, a 1-mm thick acrylic sheet was according to the kit instructions. The scaffold was washed
glued to the chamber bottom. with phosphate-buffered saline (PBS) three times and
then incubated with GelMA lysis buffer at a ratio of 1:200
2.2. Cell culture dilution with cell culture medium for 30 min at 5% CO
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Human hepatocellular carcinoma cell line C3A cells were and 37°C. The spheroids were released, centrifuged, and
cultured in Dulbecco’s modified Eagle medium (Gibco) resuspended, and then evenly transferred into an ultra-
supplemented with 10% fetal bovine serum (Gibco) and low attachment 96-well plate (Corning) with fresh cell
1% penicillin–streptomycin (Invitrogen, PA). The cells culture medium in an incubator set at 37°C and 5% CO
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were cultivated in a humidified incubator at 5% CO and for subsequent drug testing.
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37°C in a T75 flask. At 80% confluence, cells were harvested
with 0.25% trypsin-EDTA (Gibco) for sub-culture or cell 2.5. Cell viability assay
suspension preparation. The cell viability was assessed with Live-Dead kit
(Beyotime) following manufacturer’s instructions. Briefly,
2.3. Acoustic particle/cell assembly assay cell aggregates in GelMA scaffold or cell spheroids in ultra-
The acoustic assembly device was placed on the fluorescence low attachment 96-well plate were washed with PBS thrice
microscope stage, and a yellow light source was placed on and then immersed in fresh Calcein-AM/propidium iodide
the side of the acoustic device to supplement the upper (PI) staining solution (2 μM Calcein-AM and 4 μM PI in
bright light blocked by the vertical PZT, for observation of PBS). After 30 min of incubation at 5% CO and 37°C, cell
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the assembly process. Then, fluorescent particles or fresh aggregates or spheroids were washed with PBS thrice and
cell suspension (2 × 10 cells/mL) in 6% (w/v) GelMA subsequently observed under a fluorescence microscope
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(Regenovo) solution supplemented with 0.5% (w/v) (Nikon, Ti-U, Japan). The cell viability was calculated as
lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) the ratio of the fluorescent area (green) to the total area
(Sigma-Aldrich) were introduced to the acoustic chamber. using ImageJ software.
Before acoustic assembly assays, a ring-shaped polyimide
heating film placed underneath the device was used to 2.6. Drug testing
maintain the surrounding temperature around 37°C by The retrieved C3A spheroids were tested for drug treatment
a proportional-integral-derivative (PID) controller, in using sorafenib, a targeted drug for advanced hepatocellular
order to keep the GelMA’s solution state. After that, the carcinoma, while the 2D culture was set up as control. To
PZT located above the chamber slowly descended to the prepare the 2D culture, 2 × 10 cells were seeded in each
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solution interface via Z-axis moving apparatus. To obtain well of a tissue culture-treated 96-well plate (Corning,
good assembly pattern, the two PZTs in the horizontal 3916), while the retrieved spheroids were transferred to
direction were first applied with RF signals (frequency, an ultra-low attachment 96-well plate (Corning, 3474).
3.12 MHz, 3.10 MHz; 2–5 Vpp) to get a clear pattern of After 24 h of culture, both the spheroids and 2D cultures
2D array. Then, the vertical PZT was applied with another were treated with different concentrations of sorafenib
RF signal (2–5 Vpp) and the frequency of the signal solution (0, 0.1, 1, 5, 10, 25, 50 μM) in triplicate wells for
(3.15 ± 0.2 MHz) was slowly adjusted until a periodical 48 h of incubation at 5% CO and 37°C. Then, the sorafenib
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pattern of 3D dot-array appeared in the Z direction. Once solution was carefully removed, and fresh Calcein-AM/PI
particles or cells were assembled by desired patterns, staining solution (2 μM Calcein-AM and 4 μM PI in PBS)
the GelMA solution was crosslinked rapidly by a blue was added to access cell viability.

Volume 9 Issue 4 (2023) 263 https://doi.org/10.18063/ijb.733