IADR Abstract Archives
Cell Culture Vessels Affect the Evaluation of Ultrasound on Cells
Objectives: The lack of standardisation to study ultrasound exposure conditions has led to inter-study variation and prevents meaningful comparisons. The given study aimed to investigate the effect of low-frequency ultrasound exposure on osteogenic differentiation in vitro.
Methods: A Duoson device at 45 kHz frequency (continuous wave at 10, 25 or 75 mW/cm2 intensity) was used to treat Saos-2 cells, cultured in 6-well plates or 35 mm Petri dishes. The distance between the transducer and cells through the culture medium was 5 mm. The vibration and amplitude at the above acoustic intensities were measured, and the effect on osteogenic differentiation, metabolic activity and cell attachment was studied.
Results: Higher vibration amplitude was identified at the inner edge of the culture vessels than the outer edge. Increasing the intensity from 10 to 75 mW/cm2 increased the average vibrational amplitude by 127 %. Vibrometer and modelling data showed that standing waves existed in both culture platforms. Within 6-well plates, higher vibration amplitudes and acoustic pressures were recorded compared with the 35 mm dishes. Following ten days of exposure to ultrasound, cells seeded in 6-well plates showed reduced total well cell metabolic activity and reduced matrix mineralisation compared with 35 mm dishes. Furthermore, a dose-dependent response was observed within 35 mm dishes. Transmission electron microscopy demonstrated higher ultrasound intensities resulted in reduced cell attachment, damaged mitochondria and endoplasmic reticulum.
Conclusions: The ultrasound dose generated by the same transducer is propagated differently according to the culture system used, which may lead to different biological responses. This work highlighted the need to develop a standardised culture system to generate reliable data to allow direct inter-study comparisons.
Methods: A Duoson device at 45 kHz frequency (continuous wave at 10, 25 or 75 mW/cm2 intensity) was used to treat Saos-2 cells, cultured in 6-well plates or 35 mm Petri dishes. The distance between the transducer and cells through the culture medium was 5 mm. The vibration and amplitude at the above acoustic intensities were measured, and the effect on osteogenic differentiation, metabolic activity and cell attachment was studied.
Results: Higher vibration amplitude was identified at the inner edge of the culture vessels than the outer edge. Increasing the intensity from 10 to 75 mW/cm2 increased the average vibrational amplitude by 127 %. Vibrometer and modelling data showed that standing waves existed in both culture platforms. Within 6-well plates, higher vibration amplitudes and acoustic pressures were recorded compared with the 35 mm dishes. Following ten days of exposure to ultrasound, cells seeded in 6-well plates showed reduced total well cell metabolic activity and reduced matrix mineralisation compared with 35 mm dishes. Furthermore, a dose-dependent response was observed within 35 mm dishes. Transmission electron microscopy demonstrated higher ultrasound intensities resulted in reduced cell attachment, damaged mitochondria and endoplasmic reticulum.
Conclusions: The ultrasound dose generated by the same transducer is propagated differently according to the culture system used, which may lead to different biological responses. This work highlighted the need to develop a standardised culture system to generate reliable data to allow direct inter-study comparisons.