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

Abstract

3D bioprinting becomes one of the popular approaches in the tissue engineering. In this emerging application, bioink is crucial for fabrication and functionality of constructed tissue. The use of cell spheroids as bioink can enhance the cell-cell interaction and subsequently the growth and differentiation of cells in the 3D printed construct with the minimum amount of other biomaterials. However, the conventional methods of preparing the cell spheroids have several limitations, such as long culture time, low-throughput, and medium modification. In this study, the formation of cell spheroids by SSAW was evaluated both numerically and experimentally in order to overcome the aforementioned limitations. The effects of excitation frequencies on the cell accumulation time, diameter of the formed cell spheroids, and subsequently, the growth and viability of cell spheroids in the culture medium over time were studied. Using the high-frequency (23.8 MHz) excitation, cell accumulation time to the pressure nodes could be reduced in comparison to that of the low-frequency (10.4 MHz) excitation, but in a smaller spheroid size. SSAW excitation at both frequencies does not affect the cell viability up to 7 days, > 90% with no statistical difference compared with the control group. In summary, SSAW can effectively prepare the cell spheroids as bioink for the future 3D bioprinting and various biotechnology applications (e.g., pharmaceutical drug screening and tissue engineering).

Keywords: bioink; cell spheroid; cell viability; interdigital transducer (IDT); standing surface acoustic wave (SSAW).

Figure 1

(A) Schematic diagram of experimental setup of forming cell spheroids by SSAW and (B) zoomed photo showing two pairs of interdigital transducers (IDTs) and PDMS cavity.

Figure 2

Numerical simulation of (A) 10 μm-cell trajectory excited by low- (10.4 MHz) and (B) high- (23.8 MHz) frequency standing surface acoustic wave across the cavity at the power of 0.5 W from different initial positions to the pressure nodes, and (C) the effect of thediameter of cell (8 μm, 10 μm, and 15 μm) at the excitation power of 1.0 W and (D) the effect of excitation power (0.1 W, 0.5 W, and 1.0 W) on motion of 10-μm diameter cellat thelow and high frequency with the same initial distance to the corresponding pressure node of 42 μm.

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.

Figure 4

Progressive growth of the cell spheroids after the formation by SSAW (A) at 10.4 MHz (solid circle) and 23.8 MHz (hollow circle) over seven days of culture, and representative photo of cell spheroid of 10.4 MHz at (B) hour 0 (immediately after the formation), (C) hour 4, on (D) day 1, and (E) day 3 with a scale bar of 20 μm.

Figure 5

Cells stained with live/dead assay, (A) individual HepG2 cellswithout acoustic excitation in the control group, in the formed cell spheroids by the acoustic excitation on (B) day 0, (C) day 7, and (D) thepercentage of cell viability of cells with and without acoustic excitation on day 1, 3, 5, and 7.