Traditional multiwell plates and petri dishes limit the evaluation of the effects of ultrasound on cells in vitro

Dhanak Gupta, Jill Savva, James H Chandler, Richard Shelton, Ben Scheven, Helen Mulvana, Pietro Valdastri, Margaret Lucas, Damien Walmsley

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Ultrasound accelerates healing in fractured bone; however, the mechanisms responsible are poorly understood. Experimental setups and ultrasound exposures vary or are not adequately characterized across studies, resulting in inter-study variation and difficulty in concluding biological effects. This study investigated experimental variability introduced through the cell culture platform used. Continuous wave ultrasound (45 kHz; 10, 25 or 75 mW/cm2, 5 min/d) was applied, using a Duoson device, to Saos-2 cells seeded in multiwell plates or Petri dishes. Pressure field and vibration quantification and finite-element modelling suggested formation of complex interference patterns, resulting in localized displacement and velocity gradients, more pronounced in multiwell plates. Cell experiments revealed lower metabolic activities in both culture platforms at higher ultrasound intensities and absence of mineralization in certain regions of multiwell plates but not in Petri dishes. Thus, the same transducer produced variable results in different cell culture platforms. Analysis on Petri dishes further revealed that higher intensities reduced vinculin expression and distorted cell morphology, while causing mitochondrial and endoplasmic reticulum damage and accumulation of cells in sub-G1 phase, leading to cell death. More defined experimental setups and reproducible ultrasound exposure systems are required to study the real effect of ultrasound on cells for development of effective ultrasound-based therapies not just limited to bone repair and regeneration.
Original languageEnglish
Pages (from-to)1745-1761
Number of pages17
JournalUltrasound in Medicine & Biology
Issue number9
Early online date24 Jun 2022
Publication statusPublished - 1 Aug 2022

Bibliographical note

Funding Information:
This work is funded by the Engineering and Physical Sciences Research Council and part of the Ultrasurge Project under Grant EP/R045291/1. We thank Dr. Yao Jiang, School of Biomedical Sciences, University of Birmingham for assisting with flow cytometry.

Publisher Copyright:
© 2022 The Authors


  • Ultrasound
  • Saos-2 cells
  • Osteogenic differentiation
  • Bone
  • Standing waves
  • Experimental reproducibility

ASJC Scopus subject areas

  • Radiological and Ultrasound Technology
  • Biophysics
  • Acoustics and Ultrasonics


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