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07:36 min
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April 9th, 2021
DOI :
April 9th, 2021
•0:05
Introduction
1:08
Systems for Acoustic Transfection (SAT) Preparation
2:15
SAT2 Preparation
3:47
Data Collection
5:00
Results: Representative Passive Cavitation Detector Responses
6:57
Conclusion
副本
These bench-top system design, data collection and analysis principles can be used as a rational foundation for the in-vitro study of cavitation enhanced therapies. Our techniques enable high throughput testing while preserving the critical experimental attributes of interval cavitation monitoring, repeatable sample alignment and compatibility with common cellular analysis methods. Cavitation enhanced therapy has multiple potential applications, including the treatment of diseases such as cancer and stroke.
By better understanding the underpinning mechanisms, we can develop better therapies. This work provides an easily reproducible system design and implementation framework to allow research into a wide variety of cavitation induced cellular bio effects. Including drug delivery, sonoporation and sonoprinting.
Getting a meaningful results requires ensuring reproducibility and controlling for all variables in the experiment. It is imperative to perform all the relevant controls and measure the electrical noise. For preparation of the systems for acoustic transfection, degas the fill liquid under a pressure of 100kPa for at least two hours to minimize the likelihood of cavitation in the propagation path.
Confirmation with a dissolved oxygen probe that the partial pressure of oxygen is below 10kPa is recommended. Fill the test chamber slowly to minimize the re-introduction of air into the degassed liquid and immediately clear any residual bubbles from the transducer and medium container surfaces. Allow the ultrasound source power amplifier to warm up per the manufacturer's recommendation so that the gain and output are stable with respect to time.
And dilute the cavitation agent while gently and continuously stirring to make a uniform suspension without entrapping macro bubbles or destroying the agent. When working with micro bubbles, make sure to handle them carefully. For SAT2 preparation, before beginning the experiment, use ethanol to sterilize the PDMs lid.
Press fit the sterilized lid to the culture dish to form the cell compartment and load a 10 milliliter syringe equipped with an 18 gauge blunt needle with 10 milliliters of the fill liquid. Insert the needle through one of the PDMs fill holes and slowly fill the chamber while tilting so that macro bubbles can escape through the open fill hole. When the chamber has been filled, insert a four to five millimeter polymer rod into the open hole and position the assembly so that holes are horizontal.
Remove the needle while injecting extra fluid so that air is not drawn into the chamber and close the fill hole with another polymer rod. Visually check the compartment for evidence of entrapped macro bubbles and press fit the cell exposure compartment into the compartment holder. Consider the buoyancy of the particles in suspension and how their buoyancy will affect their contact with cells when deciding the orientation of the cell exposure compartment.
Then lowering the chamber lid at a horizontal angle to discourage macro bubbles from resting on the submerged parts of the device, install the lid onto the top of the chamber. Before performing the experimental measurements, allow the suspension to thermally equilibrate with the chamber temperature. Using a fine needle thermocouple to confirm that the temperature in the chamber has stabilized.
To monitor the experiments in real time, in both time and frequency domains, start the data collection process and turn on the ultrasound source drive signal. Use a high voltage probe to monitor the amplifier output signal that drives the ultrasound source throughout the experiment to ensure that the exposure is proceeding as expected. And make sure that the oscilloscope is set to compensate for the probe attenuation.
Time domain monitoring of the passive cavitation detector reveals whether signals are sized appropriately for the current instrumentation settings and whether the cavitation signals are seen earlier than expected. Frequency domain monitoring allows analysis of the type of bubble behavior and can be used to adjust drive levels as necessary to achieve the desired cell stimulus. In this analysis, at the lowest incident pressure, the passive cavitation detector response, consisted entirely of integer harmonics of the 0.5MHz fundamental ultrasound frequency.
Increasing from 0.2 to 0.3MPa resulted in pronounced ultra harmonics in the spectrum in addition to a further elevated integer harmonics. The time domain wave forms at these two pressures looked similar although the 0.3MPa results demonstrated more variability over the pulse duration. At the highest pressure, the time domain wave form amplitude grew non-linearly relative to the lower pressures as a result of clearly elevated broadband noise, likely due to inertial cavitation caused by micro bubble destruction.
Here, full spectra are shown over a 50 second exposure time during which the source emitted two millisecond pulses every 0.2 seconds. As observed in this graph, illustrating the corresponding total harmonic and broadband powers, large amplitude broadband responses were generated with the initial spike considered to correlate with the destruction of the largest bubbles. After a few seconds, the broadband response rapidly diminish, apparently due to bubble destruction.
In this analysis using a 20 to 1 dilution of micro bubbles and normal PBS, the time and sample average spectra showed that the unfocused passive cavitation detector contained a stronger broadband response than the focus detector. Accompanied by a reduced sample to sample variability in both the harmonic and ultra harmonic powers. Cavitation induced bio effects can be assessed using techniques such as fluorescence microscopy, flow cytometry or biological assays.
This enables the establishment of a robust relationship between cavitation activity and the biological effects. This technique has allowed us to better identify the relationships between bubble behavior and biological effects which have revealed some potential new mechanisms underpinning cavitation mediated drug delivery.
The presented experimental protocol can be used to perform real time measurements of cavitation activity in a cell culture device with the aim of enabling investigation of the conditions required for successful drug delivery and/or other bioeffects.
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