Observation of Cell Behavior by Waveform Measurement using Lithium Niobate-Based Surface Acoustic Wave Device

Introduction: Highly matured cells and tissues are essential to improve the quality of regenerative medicine and drug discovery research. Fluorescence observation by immunostaining with antibody labeling of maturation makers is one of the gold standards for evaluating such cells and tissues. To deliver antibodies to antigens in the cell membrane, fixation and permeabilization of cells are necessary for fluorescence observation. Recently, living cells can be observed by gene transfer (Kasahara et al., <b><i>Sci. Adv.</i></b>, 2023), but with the risk of altering the cellular morphology. That is, observation of living cells is difficult without damaging cells.<br/>Here, we propose a non-invasive evaluation method of cultured cells using surface acoustic waves (SAW) for sensing cell behavior without administering any invasive chemicals to the cells or altering their genetic traits. For cell behavior observation, a cell evaluation system with surface acoustic waves transmitted from a SAW device to the cells was fabricated. The SAW device consists of lithium niobate (LN) substrates having a high mechanical quality factor. Thus, LN is suitable for the material of the SAW device because of its low elastic loss and low attenuation of surface acoustic waves.<br/>Materials and methods: The waveform information from the transmitted and received waves was measured to observe cell behavior by this cell evaluation system. The cell evaluation system was composed of the SAW device and energizing probes. The SAW device was fabricated by a lift-off method using an LN substrate with chromium and aluminum deposited to form IDT fingers. To micropattern the cells to the measurement spot, the seeding area was limited by the dimethylpolysiloxane (PDMS) chamber sandwiched between acrylic covers to adhere the chamber to the SAW device. The slit surface was then cleaned using peracetic acid and plasma. The cells were incubated for 6 hours after modifying fibronectin on the surface of the SAW device. After incubation, the acrylic cover and cell-seeding chamber were removed from the cell evaluation system. After seeding cells, an alternating current of around 40 MHz band was applied to the IDT to measure the received wave with an oscilloscope.<br/>Results and discussion: The resonance frequency of the SAW device on the transmitter side of the fabricated cell evaluation system was measured with an impedance analyzer at 37.8 MHz. The SAW devices freely control the frequency band of irradiating SAW by changing the design of the distance between the interdigital transducer (IDT) fingers. In this study, by setting the distance between the IDT fingers to 25 µm, the target resonance frequency of the SAW device was 40 MHz. From the measurement of the impedance analyzer, the SAW device with a resonance frequency close to the target resonance frequency was successfully fabricated. This reproduces a wavelength (= ~100 µm) that provides sufficient resolution for observing cell behavior. The cell seeding area was a 1 mm × 5 mm slit area considering that the attenuation length of the SAW surface displacement at the LN-water interface is 1.24 mm. The SAW was irradiated by applying an alternating current at around 40 MHz band to the IDT fingers of the SAW device on the transmitting side. Waveforms were measured at two different cell counts. The ratio of the amplitude of the received wave to the amplitude of the transmitted wave was 0.88 when the cell density was 627 cells/mm², and 0.97 when the cell density was 83 cells/mm², respectively. Comparing them, the surface acoustic waves were attenuated as the number of cells increased. These results suggest that this system is effective in measuring cell counts. Since ultrasound is capable of reaching deep into tissues, measuring waveforms using this system could be applied to the development of more effective biomaterials including 3D cellular tissues.