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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.


Treating biological cells with ultrasound in multiwell culture plates. DuoSon with transducer (identified by *) clamped in position in a laminar flow hood (top) and a close-up of a six-well plate supported by silicone in a water bath with the transducer submerged in culture media (bottom)
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Fig2: Treating biological cells with ultrasound in multiwell culture plates. DuoSon with transducer (identified by *) clamped in position in a laminar flow hood (top) and a close-up of a six-well plate supported by silicone in a water bath with the transducer submerged in culture media (bottom)

Mentions: A six-well culture plate (Costar® tissue-culture treated; Corning®, Tewksbury, MA, USA) was supported in a water bath by silicone rubber (Fig. 2) to minimize reflections [8, 9]. The water bath was placed on a thermostat-controlled hot plate to keep the culture medium in each well of the six-well plate at 37 °C. The entire setup was placed in a laminar flow hood together with the DuoSon to prevent infection (Fig. 2). The transducer was clamped to a scissor stand to allow for straightforward insertion and removal from the culture well. The transducer face was positioned 5 mm from the culture surface in each culture well (Fig. 3). The thickness of the culture plastic at the base of the culture well is 1.27 mm.Fig. 2


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)

Treating biological cells with ultrasound in multiwell culture plates. DuoSon with transducer (identified by *) clamped in position in a laminar flow hood (top) and a close-up of a six-well plate supported by silicone in a water bath with the transducer submerged in culture media (bottom)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Treating biological cells with ultrasound in multiwell culture plates. DuoSon with transducer (identified by *) clamped in position in a laminar flow hood (top) and a close-up of a six-well plate supported by silicone in a water bath with the transducer submerged in culture media (bottom)
Mentions: A six-well culture plate (Costar® tissue-culture treated; Corning®, Tewksbury, MA, USA) was supported in a water bath by silicone rubber (Fig. 2) to minimize reflections [8, 9]. The water bath was placed on a thermostat-controlled hot plate to keep the culture medium in each well of the six-well plate at 37 °C. The entire setup was placed in a laminar flow hood together with the DuoSon to prevent infection (Fig. 2). The transducer was clamped to a scissor stand to allow for straightforward insertion and removal from the culture well. The transducer face was positioned 5 mm from the culture surface in each culture well (Fig. 3). The thickness of the culture plastic at the base of the culture well is 1.27 mm.Fig. 2

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.