Ultrasound-Mediated Transfection of Mammalian Cells

Abstract

Mammalian cells were successfully transfected with plasmid DNA in vitro using ultrasound transmitted through the walls of cell culture flasks or plates. Primary rat fibroblasts or chondrocytes were exposed to ultrasound in the presence of plasmids containing lacZ or neo genes. The transfection efficiency was evaluated by counting the number of β-galactosidase (β-Gal) positive cells or neomycin-resistant colonies. Transfection efficiency was optimized by varying ultrasound conditions, ambient temperatures (room temperature or 37°C), plasmid concentrations, and initial cell populations. Additional experiments were performed to elucidate the mechanism of the ultrasound-mediated transfection. Maximal gene transfection was seen with two ultrasound conditions: 1-MHz carrier frequency 411 ± 189 kPascal continuous wave with 20 or 30 sec of exposure time, and 1 MHz carrier frequency 319 ± 157 kPascal continuous wave with 40 or 60 sec of exposure time. Gene expression was negligible when transfection procedures were performed at room temperature. The average stable transfection rate was 0.34% of surviving cells with a plasmid concentration of 40 μg/ml in primary fibroblasts. The transient transfection rate was 2.4% of surviving cells for primary chondrocytes. Data suggest that increasing plasmid concentration will increase efficiency. Identical treatment with 3.5 MHz produced no transfection, implying that cavitation produced by the ultrasound pressure wave appeared to play a critical role in mediating transfection. Ultrasound-mediated transfection was effective for suspended cells as well as for plated cells. This transfection method is simple, easy to keep sterile, and convenient. Ultrasound-mediated transfection appears to be a promising method for gene transfer into mammalian cells. Transfection of mammalian cells in vitro would be enhanced if the method could be applied directly to culture dishes or flasks without replating and transferring cells or introducing probes or other devices into the culture medium. Kim et al. have developed a method that uses ultrasonic waves to transfect plasmids into fibroblasts, chondrocytes, and presumably other mammalian cells. They report that ultrasonic power and duration, ambient temperature, and plasmid concentration are critical to achieving maximal expression, whereas initial cell density is less important. They show that cavitation is a key component of ultrasound-mediated transfection. Stable transfection of fibroblasts resulted in marker gene expression in 0.34% of the surviving fibroblasts.