Therapeutic ultrasound experiments in vitro: Review of factors influencing outcomes and reproducibility
Introduction
During the past decades, ultrasound has become an indispensable non-invasive medical tool which provides clinicians with a powerful, easily accessible and relatively cheap diagnostic imaging technique [1]. The potential uses of diagnostic ultrasound have still not been fully explored and new methods, encompassing modalities such as 3D and 4D imaging [1], elastography [2], [3], [4], new ultrasound contrast agents [5], [6], speckle tracking technique [7], [8], ultrasonic microscopy [9], [10], [11] and endoscopic [12], [13] and intravascular [14], [15] ultrasound imaging, are still undergoing rapid development [16].
Ultrasound has not only been used as a powerful diagnostic imaging tool, but considerable research has also been conducted on its therapeutic effects. The use of ultrasound for the treatment of diseased organs or structures is referred to as therapeutic ultrasound [17]. In contrast to diagnostic ultrasound, where biological effects are minimized by keeping up to the ALARA (As Low As Reasonably Achievable) principle [18], when using ultrasound for therapeutic purposes the parameters of ultrasound field are intentionally set to the levels which provoke some kind of biological response [19]. Some of these applications have already found their way into clinical use and have been approved by the FDA (Food and Drug Administration) and the EU (European Union). These include physiotherapy procedures, HIFU (High-Intensity Focused Ultrasound), intracorporeal lithotripsy, extracorporeal shock wave therapy, phacoemulsification, liposuction, tissue cutting and vessel sealing, sonothrombolysis, sonophoresis and bone fracture healing [20]. Table 1 gives a very nice shortened overview of these applications which was originally presented in work by Miller et al. [20]. Other possible therapeutic modalities are still being investigated. These include gene therapy, sonodynamic therapy, sonoporation, drug delivery systems and blood-brain barrier opening [21], [22], [23], [24], [25], [26], [27], [28].
A usual first research step is to perform experiments in vitro. Current ultrasound exposure experiments in vitro show considerable variability in methods, experimental geometries and ways of studying biological effects including sonication of ex vivo tissues, tissue mimicking materials, tissue based methods, cultured cells, non-biological-based methods and biological scaffolds. Each of these methods has characteristics that are well suited for a variety of well-defined investigative goals [29]. Great variability of methods within each of the aforementioned categories can also be found. Moreover, the technical equipment (e.g. culture vessels or physiotherapy equipment etc. which are used frequently in these experiments) may not be specified well or it may present with unstable output over time. Consequently, this non-uniformity in ultrasound exposure experiments leads to poor reproducibility of these experiments and uncertainty in the conditions experienced by sonicated samples, both of which decrease the scientific value of results obtained.
Section snippets
Aim
The main scope of this narrative review is to briefly describe mechanisms of action of ultrasound, make a list of the most commonly used set-ups for therapeutic ultrasound exposure experiments in vitro using cultured cells in culture vessels, describe phenomena that influence the outcomes of these experiments, assess their pros and cons, and to make a few recommendations as to how to perform sonication experiments in vitro in culture vessels to increase the validity and reproducibility of these
Mechanisms of action
Therapeutic ultrasound produces effects in biological samples by different mechanisms of action [30]. In some cases, different mechanisms of action may occur simultaneously. One mechanism of action usually predominates however, ascribing a particular biological response to only one mechanism may be more daring than justified. Mechanisms of action of ultrasound are usually categorized to thermal and non-thermal [31].
Factors influencing outcome and reproducibility of sonication experimetns in vitro
When performing therapeutic ultrasound exposure experiment in vitro using culture vessels attention should be paid to following issues: proper reporting of ultrasound exposure conditions, sufficient description of geometrical set-up of the items involved in the experiment, quality of coupling medium, influence of laboratory glass and plastics in which the sonicated samples are placed, standing wave formation, influence of motion or rotation of the sonicated sample and characteristics of the
Proposals for guidelines
In all studies, the overarching purpose of reporting exposure parameters is to enable other investigators to replicate studies and thereby validate, supplement, or call into question published results [52]. Although some studies include extremely thorough characterization of the ultrasound fields used, in many cases this information is either very limited, poorly explained or missing altogether [53]. Evaluating the acoustic pressure field within an ultrasonic bioreactor is a necessary
Choice of experimental set-up
The most common arrangements for sonication experiments performed in vitro are depicted in Fig. 4.
Many experiments are carried out in a tank filled with degassed water (Fig. 4a–f) to facilitate transmission of ultrasound energy to the region of interest. In this type of experiment, the sonicated cells are usually placed either in conventional glass or plastic culture vessels (Fig. 4a–d) or in culture vessels sealed by Parafilm® or Mylar film (Fig. 4e) or in a home-made sample holder with
Focused transducers
During in vitro sonication experiments the position and size of the sonicated sample relative to the ultrasound field significantly influence the final energy seen by particular cells. For focused shockwave transducers, the maximum acoustic pressure reaches only a very limited region. If this region is smaller than the sonicated sample, different cells are affected by significantly different temporal pressure distributions. In this case, a correlation of the shockwave parameters with the
Influence of coupling medium
Coupling medium is used to facilitate transmission of ultrasound waves to the site of interest. In Fig. 4a–f the coupling medium is degassed water and in Fig. 4g ultrasound gel is used to couple ultrasound from the transducer into the culture vessel containing the biological sample. The ideal coupling medium transmits ultrasound energy efficiently, eliminates air spaces between the transducer head and the tissue, and serves as a lubricant for contact applications [85]. Several studies have been
Influence of sample holder
Laboratory glass and plastics alter the ultrasound exposure conditions in the majority of ultrasound exposure experiments in vitro. Indeed, our team has shown that commonly used culture vessels can either reduce, or even locally increase, the ultrasound intensity experienced by sonicated cells, depending on both material and shape of the holder [91].
Origin of standing waves
Standing waves are set up when a significant proportion of a travelling wave is reflected (e.g. at a water/culture medium-air interface), and the incident and reflected waves are superimposed in such a manner that their peaks and troughs coincide. In such a situation there are fixed positions where the particle displacements induced by the 2 waves always add to reinforce each other and produce a maximum displacement amplitude (termed an antinode), and intervening positions where their summation
Influence of rotation
If attached cells are exposed to travelling waves it may be necessary to rotate the sample holder as cavitating bubbles are pushed by radiation force to the far side of the vessel from the transducer [110]. If the cells are attached on the front wall of the holder, then the cavitating bubbles may become ineffective in causing biological response since they are pushed away. On the other hand, if the cells are attached on the back wall, streaming may theoretically detach the cells. Suitable
Influence of the cells
The biological response of the sonicated sample also depends on a number of intrinsic factors. The same stimuli may result in different biological responses in different cell lines. Moreover, even cells of the same type may show different responses to identical stimuli [116].
If cells of the same cell line are sonicated in the form of attached cells and once in the form of cell suspensions, the biological response may be very different [117], [118]. The influence of trypsin (used to detach the
Rated outcome
We chose a narrative format for this review since there are many factors which needed to be described and writing a systematic review according to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) statement [143] would be very difficult if not impossible. Rather than giving highly detailed information, we have given a brief structured insight into performing in vitro sonication experiments using cells in culture vessels and drawn attention to the main pitfalls that
Limitations
Since narrative reviews deal with a broad range of topics, the methodology of searching for relevant articles inevitably lacks the methodological quality of systematic reviews, which, in contrast, focus on one precisely defined question or topic. Even though we did our best to collect material of good quality, the reader should be aware of the fact that this is the narrative type of review, not a systematic one. As we already stated, this work does not intend to give highly detailed information
Conclusions
The performance of in vitro sonication experiments is an indispensable research method. However, these experiments are accompanied with many potential pitfalls. Attention should always be paid to proper reporting of ultrasound exposure conditions, sufficient description of the experimental set-up, assessment of alteration of ultrasound field by coupling medium, culture vessel and culture medium, consideration of the influence of standing waves and artificial moving with the sonicated sample, as
Declaration of Competing Interest
All authors declare no conflicts of interest.
Acknowledgements
This work was supported by the Grant Project IGA_LF_2020_015 and by the European Regional Development Fund - Project ENOCH (No. CZ.02.1.01/0.0/0.0/16_019/0000868).
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