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Ultrasound field characterization and bioeffects in multiwell culture plates.

Patel US, Ghorayeb SR, Yamashita Y, Atanda F, Walmsley AD, Scheven BA - J Ther Ultrasound (2015)

Bottom Line: Calculations were performed using Fourier transform and average intensity plotted against distance from the transducer.The ultrasonic output demonstrated considerable lateral spread of the ultrasound field from the exposed well toward the adjacent culture wells in the multiwell culture plate; this correlated well with the dose-dependent increase in the number of cultured cells where significant biological effects were also seen in adjacent untreated wells.Significant thermal variations were not detected in adjacent untreated wells.

View Article: PubMed Central - PubMed

Affiliation: School of Dentistry, College of Medical and Dental Sciences, University of Birmingham, St Chad's Queensway, Birmingham, B4 6NN UK.

ABSTRACT

Background: Ultrasound with frequencies in the kilohertz range has been demonstrated to promote biological effects and has been suggested as a non-invasive tool for tissue healing and repair. However, many challenges exist to characterize and develop kilohertz ultrasound for therapy. In particular there is a limited evidence-based guidance and standard procedure in the literature concerning the methodology of exposing biological cells to ultrasound in vitro.

Methods: This study characterized a 45-kHz low-frequency ultrasound at three different preset intensity levels (10, 25, and 75 mW/cm(2)) and compared this with the thermal and biological effects seen in a 6-well culture setup using murine odontoblast-like cells (MDPC-23). Ultrasound was produced from a commercially available ultrasound-therapy system, and measurements were recorded using a needle hydrophone in a water tank. The transducer was displaced horizontally and vertically from the hydrophone to plot the lateral spread of ultrasound energy. Calculations were performed using Fourier transform and average intensity plotted against distance from the transducer. During ultrasound treatment, cell cultures were directly exposed to ultrasound by submerging the ultrasound transducer into the culture media. Four groups of cell culture samples were treated with ultrasound. Three with ultrasound at an intensity level of 10, 25, and 75 mW/cm(2), respectively, and the final group underwent a sham treatment with no ultrasound. Cell proliferation and viability were analyzed from each group 8 days after three ultrasound treatments, each separated by 48 h.

Results: The ultrasonic output demonstrated considerable lateral spread of the ultrasound field from the exposed well toward the adjacent culture wells in the multiwell culture plate; this correlated well with the dose-dependent increase in the number of cultured cells where significant biological effects were also seen in adjacent untreated wells. Significant thermal variations were not detected in adjacent untreated wells.

Conclusions: This study highlights the pitfalls of using multiwell plates when investigating the biological effect of kilohertz low-frequency ultrasound on adherent cell cultures.

No MeSH data available.


The calculated spatial-average intensity from ultrasound produced from the DuoSon. Spatial-average intensity calculated when a 10 a, 25 b, and 75 mW/cm2c ultrasound beam is produced from the DuoSon transducer. Dimensions of the transducer and culture wells are to a 1:1 scale with the horizontal axis. A diagrammatic representation of the culture wells in a six-well plate have been superimposed to demonstrate proximity of the culture wells to each other and their spatial relationship to the ultrasound beam and average intensities. Intensity without Fourier analysis is shown as mean ± SD
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Fig4: The calculated spatial-average intensity from ultrasound produced from the DuoSon. Spatial-average intensity calculated when a 10 a, 25 b, and 75 mW/cm2c ultrasound beam is produced from the DuoSon transducer. Dimensions of the transducer and culture wells are to a 1:1 scale with the horizontal axis. A diagrammatic representation of the culture wells in a six-well plate have been superimposed to demonstrate proximity of the culture wells to each other and their spatial relationship to the ultrasound beam and average intensities. Intensity without Fourier analysis is shown as mean ± SD

Mentions: Maximum voltage and frequency measurements of ultrasound produced from the DuoSon are shown in Table 1. These values were used to calculate spatial-average intensities as described in the methods section. Beam plots of calculated intensities are shown in Fig. 4a–c. The data indicate that the measurements recorded where the transducer and hydrophone were centrally aligned showed some resemblance to the intensities quoted by the manufacturers. Measurements made horizontally away from the long axis of the transducer showed a gradual reduction of the average intensity. Figure 4 also displays the size of the transducer and positioning of culture wells in a six-well plate which are a 1:1 scale with the horizontal axis. Horizontal measurements show that at 20 and 25 mm from the central axis of the transducer, the calculated intensities without Fourier analysis were 7.75 and 5.2 mW/cm2, respectively, when an ultrasound beam using the preset 10 mW/cm2 mode is selected. An ultrasound beam produced using the preset 25 mW/cm2 mode recorded an average intensity of 19 and 12.5 mW/cm2, and when using the 75 mW/cm2 mode, 61.5 and 58.5 mW/cm2 was recorded at 20 and 25 mm, respectively, from the central axis of the transducer. The beam plots of the 10 and 25 mW/cm2 modes (Fig. 4a, b) are similar in form, as opposed to that of the 75 mW/cm2 mode (Fig. 4c). The 75 mW/cm2 mode produces an ultrasound beam which has a flatter peak. These data imply that when biological cells cultured in dishes of a six-well plate are treated with ultrasound, adjacent culture wells will also be exposed to an ultrasound field.Table 1


Ultrasound field characterization and bioeffects in multiwell culture plates.

Patel US, Ghorayeb SR, Yamashita Y, Atanda F, Walmsley AD, Scheven BA - J Ther Ultrasound (2015)

The calculated spatial-average intensity from ultrasound produced from the DuoSon. Spatial-average intensity calculated when a 10 a, 25 b, and 75 mW/cm2c ultrasound beam is produced from the DuoSon transducer. Dimensions of the transducer and culture wells are to a 1:1 scale with the horizontal axis. A diagrammatic representation of the culture wells in a six-well plate have been superimposed to demonstrate proximity of the culture wells to each other and their spatial relationship to the ultrasound beam and average intensities. Intensity without Fourier analysis is shown as mean ± SD
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4490766&req=5

Fig4: The calculated spatial-average intensity from ultrasound produced from the DuoSon. Spatial-average intensity calculated when a 10 a, 25 b, and 75 mW/cm2c ultrasound beam is produced from the DuoSon transducer. Dimensions of the transducer and culture wells are to a 1:1 scale with the horizontal axis. A diagrammatic representation of the culture wells in a six-well plate have been superimposed to demonstrate proximity of the culture wells to each other and their spatial relationship to the ultrasound beam and average intensities. Intensity without Fourier analysis is shown as mean ± SD
Mentions: Maximum voltage and frequency measurements of ultrasound produced from the DuoSon are shown in Table 1. These values were used to calculate spatial-average intensities as described in the methods section. Beam plots of calculated intensities are shown in Fig. 4a–c. The data indicate that the measurements recorded where the transducer and hydrophone were centrally aligned showed some resemblance to the intensities quoted by the manufacturers. Measurements made horizontally away from the long axis of the transducer showed a gradual reduction of the average intensity. Figure 4 also displays the size of the transducer and positioning of culture wells in a six-well plate which are a 1:1 scale with the horizontal axis. Horizontal measurements show that at 20 and 25 mm from the central axis of the transducer, the calculated intensities without Fourier analysis were 7.75 and 5.2 mW/cm2, respectively, when an ultrasound beam using the preset 10 mW/cm2 mode is selected. An ultrasound beam produced using the preset 25 mW/cm2 mode recorded an average intensity of 19 and 12.5 mW/cm2, and when using the 75 mW/cm2 mode, 61.5 and 58.5 mW/cm2 was recorded at 20 and 25 mm, respectively, from the central axis of the transducer. The beam plots of the 10 and 25 mW/cm2 modes (Fig. 4a, b) are similar in form, as opposed to that of the 75 mW/cm2 mode (Fig. 4c). The 75 mW/cm2 mode produces an ultrasound beam which has a flatter peak. These data imply that when biological cells cultured in dishes of a six-well plate are treated with ultrasound, adjacent culture wells will also be exposed to an ultrasound field.Table 1

Bottom Line: Calculations were performed using Fourier transform and average intensity plotted against distance from the transducer.The ultrasonic output demonstrated considerable lateral spread of the ultrasound field from the exposed well toward the adjacent culture wells in the multiwell culture plate; this correlated well with the dose-dependent increase in the number of cultured cells where significant biological effects were also seen in adjacent untreated wells.Significant thermal variations were not detected in adjacent untreated wells.

View Article: PubMed Central - PubMed

Affiliation: School of Dentistry, College of Medical and Dental Sciences, University of Birmingham, St Chad's Queensway, Birmingham, B4 6NN UK.

ABSTRACT

Background: Ultrasound with frequencies in the kilohertz range has been demonstrated to promote biological effects and has been suggested as a non-invasive tool for tissue healing and repair. However, many challenges exist to characterize and develop kilohertz ultrasound for therapy. In particular there is a limited evidence-based guidance and standard procedure in the literature concerning the methodology of exposing biological cells to ultrasound in vitro.

Methods: This study characterized a 45-kHz low-frequency ultrasound at three different preset intensity levels (10, 25, and 75 mW/cm(2)) and compared this with the thermal and biological effects seen in a 6-well culture setup using murine odontoblast-like cells (MDPC-23). Ultrasound was produced from a commercially available ultrasound-therapy system, and measurements were recorded using a needle hydrophone in a water tank. The transducer was displaced horizontally and vertically from the hydrophone to plot the lateral spread of ultrasound energy. Calculations were performed using Fourier transform and average intensity plotted against distance from the transducer. During ultrasound treatment, cell cultures were directly exposed to ultrasound by submerging the ultrasound transducer into the culture media. Four groups of cell culture samples were treated with ultrasound. Three with ultrasound at an intensity level of 10, 25, and 75 mW/cm(2), respectively, and the final group underwent a sham treatment with no ultrasound. Cell proliferation and viability were analyzed from each group 8 days after three ultrasound treatments, each separated by 48 h.

Results: The ultrasonic output demonstrated considerable lateral spread of the ultrasound field from the exposed well toward the adjacent culture wells in the multiwell culture plate; this correlated well with the dose-dependent increase in the number of cultured cells where significant biological effects were also seen in adjacent untreated wells. Significant thermal variations were not detected in adjacent untreated wells.

Conclusions: This study highlights the pitfalls of using multiwell plates when investigating the biological effect of kilohertz low-frequency ultrasound on adherent cell cultures.

No MeSH data available.