GHz Ultrasound and Electrode Chip-Scale Arrays Stimulate and Influence Morphology of Human Neural Cells
Priya S. Balasubramanian, Amit Lal
IEEE Transactions on Ultrasonics, Ferroelectrics, and Frequency Control 2022 · 10.1109/tuffc.2022.3152427
Abstract
This study describes the effects of chip-scale gigahertz (GHz) ultrasound (US) and electrical stimulus on the morphology, functionality, and viability of neural cells in vitro. The GHz frequency stimulation is achieved using aluminum nitride piezoelectric transducers fabricated on a silicon wafer, operating at 1.47 GHz, corresponding to the film's thickness mode resonance. These devices are used to stimulate SH-SY5Y neural cells in vitro and observe effects on the morphology and viability of the stimulated cells. It is possible to use these devices to deliver either ultrasonic stimulus alone or US stimulus in conjunction with electrical stimulus. Viability tests demonstrated that the neurons retained structural integrity and viability across a wide range of GHz US stimulus intensities (0-1.2 W/cm 2 ), validating that measurements occur at nontoxic doses of US. Neural stimulation is validated with these devices following the outputs of a previous study, with the normalized fluorescence intensity of activated cells between 1.9 and 2.4. The 300-s ultrasonic stimulation at 1.47 GHz and 0.05 W/cm 2 peak intensity led to a decrease in nuclear elongation by 17.5% and a cross-sectional area decrease by 17.8% across three independent trials of over 150 cells per category ( ). The F-actin governed cellular elongation increased in length by up to 16.3% in cells exposed to an ultrasonic stimulus or costimulus ( ). Neurite length increased following ultrasonic stimulation compared with control by 75.8% ( ). This article demonstrates new GHz US and electrical chip-scale arrays with apparent effects in both neural excitation and cell morphology.
Abstract via europepmc.
Exposures
Exposure 1: GHz (1.47 GHz) chip-scale ultrasound stimulation of SH-SY5Y neural cells (Devices A/B/C)
Target: cultured neurons — “SH-SY5Y human neural cells (differentiated) in culture”
Device: custom-built · Institute of Microelectronics (IME), A*STAR, Singapore · chip-scale AlN thin-film piezoelectric GHz ultrasound transducer (Devices A, B and C) ✓
| Waveform | continuous | |
|---|---|---|
| Fundamental frequency (kHz) | 1,470,000 | ✓✓✓ |
| Pulse duration (ms) | not applicable | |
| Pulse repetition frequency (Hz) | not applicable | |
| Duty cycle (%) | not applicable | |
| Sonication duration (s) | 20, 30, 300swept | ✓✓✓ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | not reported | |
| In-situ estimate | not applicable | |
| In-situ pressure (kPa) | not applicable | |
| In-situ Isppa (W/cm²) | not applicable | |
| In-situ Ispta (W/cm²) | not applicable | |
| Isppa, domain unspecified (W/cm²) | 0.05, 1.2swept | ✓?⚑ |
GHz ultrasound was delivered as continuous-wave stimulation. For ion-channel/calcium-imaging experiments, 10 s of prestimulus data was followed by 20 s of continuous-wave GHz ultrasound (Devices A/B) or 30 s of electrical stimulation followed by 30 s of US+electrical costimulation (Device C), then poststimulus recording. For morphology (nuclear/F-actin) experiments, cells were exposed to US for 300 s (three independent trials per condition, >150 cells per category) and assessed 24 h later. For the viability dose-response, ultrasound was applied for 20 s and cells assayed 120 s after exposure across an intensity range of 0-1.2 W/cm2.
Consistency checks: f0 out of range.
Exposure 2: Kilohertz-ultrasound positive control (apoptosis/necrosis assay)
Target: cultured neurons — “SH-SY5Y human neural cells in culture”
Device: custom-built · 92-kHz resonance piezoelectric ceramic disk transducer, 22 mm ✓
| Waveform | not reported | |
|---|---|---|
| Fundamental frequency (kHz) | 92 | ✓✓✓ |
| Pulse duration (ms) | not reported | |
| Pulse repetition frequency (Hz) | not reported | |
| Duty cycle (%) | not reported | |
| Sonication duration (s) | 300 | ✓✓✓ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | not reported | |
| In-situ estimate | not applicable | |
| In-situ pressure (kPa) | not applicable | |
| In-situ Isppa (W/cm²) | not applicable | |
| In-situ Ispta (W/cm²) | not applicable |
Used only as a high-intensity positive control for apoptosis/necrosis staining, applied for 300 s followed by 1 h incubation; intensity is stated only as an inequality ('>5 W/cm2 acts as a positive control'), not a specific value.
Flags from extraction
n_subjects— Different assays report different independent-trial counts (e.g. 'three independent trials', 'n>50 cells per condition', 'over 150 cells per category') with no single stated total number of independent cultures/replicates; left not_reported.sham_type— Main US-free control cells were still interfaced with the (inactive) transducer for the same duration, matching inactive_transducer; a second negative control used off-resonance (300 MHz vs 1.47 GHz) drive voltage plus a pharmacological (gentamicin) channel blocker, which does not match a listed sham mechanism and is recorded as 'other'.exposures[0].unspecified_domain.isppa_w_cm2— 0.05 W/cm2 is the intensity used for the main stimulation experiments and 0-1.2 W/cm2 is the range swept in the viability dose-response; both are reported as spatial-peak/'peak' intensity without a stated measurement domain (water bath vs. cell interface), so both are placed in unspecified_domain.exposures[1].unspecified_domain.isppa_w_cm2— Intensity for the kilohertz positive control is given only as an inequality ('>5 W/cm2'), not a specific value, so it is left not_reported.exposures[1].timing.waveform— Waveform (continuous vs pulsed) of the 92-kHz positive-control transducer is not described in the text.