Focused Ultrasound Neuromodulation of Human In Vitro Neural Cultures in Multi-Well Microelectrode Arrays
May 3rd, 2024
Here, we present a protocol for using a high-throughput system that enables the monitoring and quantification of the neuromodulatory effects of focused ultrasound on human-induced pluripotent stem cell (HiPSC) neurons.
Focused ultrasound neuromodulation. This up and coming exciting field is a topic of ongoing studies In the field of focused ultrasound, the optimal stimulation parameters for a particular application are often unknown. This study aims at a systematic determination of these parameters through empirical in vitro testing on neurons.
This method can explore the neuronal mechanism underlying focus of resound neuromodulation by enabling researcher to analyze the responses of genetically and pharmaceutically modified neurons. To begin aspirate the culture medium to fill a single well in a 24 well neuron culture plate, having embedded micro electrode arrays or MEA. After preparing 300 milliliters of degassed and deionized water, carefully fill it in the focused ultrasound or FUS transducer cone.
Using a customized threaded rod, secure the 3D printed holder to a frame. Position the frame such that the head of the FUS transducer is over the well that will be stimulated. Use a rubber band to secure paraform over the well on the 24 well MEA plate containing the medium and the human induced pluripotent stem cells or HIPSCs.
Set the FUS parameters on the transducer power output or TPO control panel. The values of the various parameters are mentioned on the screen. Next, apply coupling gel on top of the paraform over the well and lower the FUS transducer into the coupling gel ensuring contact with the gel with minimal air bubbles.
Start the FUS sonication by pressing the bottom right button on the TPO and wait at least five minutes between each round of sonication to allow the neurons to return to a baseline state. If the connection is appropriate, the trigger pulse generated by the FUS system will automatically align the FUS stimulation sequence with the MEA recording. Analyze the signal by reading the FUS sonication time with the transfer data based on the change in the firing rate associated with the FUS.
The FUS focal spot was characterized both by visualizing it on thermo chromatic sheets and through water hydrophone scanning. Post-processing steps, including filtering, thresholding, and calculating the firing rate, filtered the noise from the environment to reveal the neuronal activity changes caused by FUS. The raster plots showed the detected spikes in each channel.
The firing rate plot showed that the chosen stimulation parameters increased the neuronal firing rate. The pre FUS firing rate was 140 hertz, while the post FUS firing rate was 786 hertz with continuous wave FUS. Altering the FUS sonication mode also changed the amount of time before the neurons return to their baseline state.
It is crucial to carefully minimize bubble formation when placing the transducer onto the paraform interface. Another consideration is the electrical power delivered to the transducer. The power must be high enough to induce an effect, but must be low enough to not induce transducer or cell damage.
This technique could identify the optimal stimulation parameter to treat Parkinson's disease and other neurological disorders, eliminating risk associated with the invasive surgery, including irreversible damage, lengthy recovery, and rehabilitation time. Further efforts to uncover its mechanism and control preclinical optimization are necessary to ensure safety in future studies, including clinical trials.
Overview
This study presents a protocol for a high-throughput system designed to monitor and quantify the effects of focused ultrasound on human-induced pluripotent stem cell (HiPSC) neurons. The research aims to systematically determine optimal stimulation parameters through in vitro testing, enabling deeper insights into the neuromodulatory mechanisms at play.
Key Study Components
Area of Science
- Neuroscience
- Neuromodulation
- Stem Cell Biology
Background
- Focused ultrasound (FUS) is a growing field of interest.
- The optimal stimulation parameters for FUS applications are frequently unknown.
- This study investigates the neuronal mechanisms involved in FUS neuromodulation.
- It utilizes genetically and pharmaceutically modified neurons for empirical testing.
Purpose of Study
- To establish a systematic approach for determining FUS stimulation parameters.
- To explore neuronal responses to FUS on HiPSC-derived neurons.
- To assess the potential therapeutic applications of this non-invasive technique for neurological disorders.
Methods Used
- The methodology includes culturing neurons in a 24-well plate using microelectrode arrays (MEAs).
- HiPSC-derived neurons are the primary biological model.
- FUS parameters are set on a transducer control panel, with careful monitoring to prevent air bubble formation.
- Key steps involve the application of coupling gel and the analysis of firing rates post-sonication.
Main Results
- Application of continuous wave FUS significantly increased neuronal firing rates from 140 Hz to 786 Hz.
- The study identified optimal stimulation parameters without damaging the neurons.
- Alterations in sonication modes affected the timing of neuronal recovery to baseline states.
Conclusions
- This research demonstrates a non-invasive method to modulate neuronal activity, with implications for treating disorders like Parkinson's disease.
- Future studies will focus on refining the methodology and exploring safety in clinical trials.
- Understanding these mechanisms enhances the potential for effective neuromodulation therapies.
Frequently Asked Questions
Executive Industry Relevance
Focused ultrasound neuromodulation using human iPSC-derived neural cultures on multi-well microelectrode arrays enables systematic, high-throughput interrogation of neuronal response to FUS parameters. This platform addresses a critical gap in mechanistic understanding, supporting predictive confidence for non-invasive neuromodulation strategies in early discovery and translational neuroscience portfolios. The approach facilitates parameter optimization and de-risking for future therapeutic development targeting neurological and psychiatric disorders.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Empirically interrogates neuromodulatory hypotheses by quantifying neuronal firing rate changes in response to FUS.
- Enables mechanistic de-risking through pharmaceutical and genetic manipulation of human neurons.
- Supports functional target validation by isolating FUS effects on disease-relevant neural systems.
Screening & Assay Development
- Establishes a reproducible, high-throughput assay platform using MEA-equipped multi-well plates.
- Delivers quantitative electrophysiological outputs for robust comparison of FUS parameter sets.
- Facilitates assay standardization and scalability for compound or parameter screening workflows.
Translational & Preclinical Research
- Aligns in vitro human neural models with disease-relevant phenotypes for translational continuity.
- Enables risk-adjusted advancement of FUS-based neuromodulation strategies toward preclinical validation.
- Provides a foundation for biomarker discovery linked to electrophysiological readouts.
Pipeline & Workflow Integration
This method integrates into the discovery-to-preclinical continuum by enabling systematic parameter optimization, mechanistic de-risking, and quantitative assessment of neuromodulatory interventions in human neural systems.
- Discovery Biology: Supports hypothesis testing and pathway clarification for FUS neuromodulation.
- Screening: Provides reproducible, quantitative firing rate measurements for parameter or compound evaluation.
- Analytics: Delivers electrophysiological data and statistical outputs for cross-condition comparison.
- Translational Research: Bridges in vitro findings to preclinical models using human-relevant neural cultures.
- Enterprise Reuse: Offers a scalable, adaptable platform for ongoing neuromodulation research and screening.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in neuromodulation research.
- Operational Value: Enables standardized, high-throughput, and reproducible electrophysiological assays.
- Strategic Value: Informs go/no-go decisions and optimizes resource allocation for neuromodulation portfolios.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of FUS-based therapeutic candidates.
Implementation Considerations
- Requires expertise in human iPSC culture, MEA operation, and FUS instrumentation.
- Demands precise control of FUS parameters and careful minimization of air bubble formation.
- Needs robust analytical infrastructure for electrophysiological data processing and statistical analysis.
- Standardization across wells and experiments is critical for reproducibility and cross-team collaboration.
- Power calibration must balance efficacy with avoidance of cellular or transducer damage.
