Low-intensity ultrasound (LIUS) enhances the proliferation rate of various mammalian stem cells through mechanical stimulation. This study quantitively finds suitable LIUS stimulation parameters for increasing the proliferation rate of human adipose-derived mesenchymal stem cells (hAdMSCs) for mass production. Various stimulation conditions of LIUS were assessed based on the beam pattern of the ultrasonic transducer and the attenuation of the sound waves. Using optimal LIUS stimulation parameters for enhancing proliferation of hAdMSCs taken from bromodeoxyuridine (BrdU) incorporation assay, long-term culture of hAdMSCs was performed for 16 days. The resultant hAdMSCs were characterized for various biomarkers such as CD34−, CD45−, CD73+, CD95+, CD105+ and cytological staining and a cytokine array assay. LIUS stimulation parameters found for enhancing the hAdMSCs proliferation were the frequency of 5 MHz, an intensity of 300 mWcm−2, a duration of 10 min per day, and continuous waves with a 100% duty cycle. The LIUS stimulated hAdMSCs group showed a 3.25-fold increase in the cell number compared to the control group after 16 days of culture. By confirming the effects of quantitatively measured LIUS stimulation on the enhancement of hAdMSCs proliferation, this study may be a foundation for the applications of LIUS stimulation in the industrial-scale production of hAdMSCs.

Background: Low-intensity ultrasound (LIUS) has been used to increase the proliferation rate of various stem cells including human adipose-derived mesenchymal stem cells (hAdMSCs). hAdMSCs is now commercially produced for various therapeutic applications. The purpose of this study was to show feasibility of enhancing the productivity of cell culture during 16-day cell culturing and increasing proliferation rate of hAdMSCs by LIUS stimulation with appropriate ultrasound parameters. Methods: Beam patterns of 5 and 10 MHz ultrasound transducers were measured to confirm the area of stimulation. The intensity of sound waves transmitted through a Petri-dish was measured in situ for quantitative evaluation. Bromodeoxyuridine (BrdU) incorporation assay was performed to search for appropriate parameters for LIUS stimulation of hAdMSCs. Cell culture medium supplemented with 8% fetal bovine serum (FBS) in a 35 mm Petri-dish was used for 16 days with subculture from 2 to 6 passage. Results: A frequency of 5 MHz, an intensity of 300 , a duration of 10 minutes per day, and continuous waves with 100% duty cycle were the best parameters according to the BrdU assay of proliferation rate of hAdMSCs. LIUS stimulation group had about 3.25-fold greater number of cells from passage 2 to 6 compared with the control group. Doubling time was decreased to 4.44 hours in average. Cell viability was the same between the control and LIUS stimulation groups.Conclusions: This study of enchanced proliferation rate and cell culture productivity of hAdMSCs by LIUS stimulation may lay the foundation for the application of LIUS stimulation in cell therapeutic industry by reducing the production cost and time required for cell therapy.