Acoustic streaming induced by MHz-frequency ultrasound extends the volume limit of cell suspension culture.

Large-scale cell suspension culture technology opens up opportunities for numerous medical and bioengineering applications. For these purposes, scale-up of the culture system is paramount. For initial small-scale culture, a simple static suspension culture (SSC) is generally employed. However, cell sedimentation due to the lack of agitation limits the culture volume feasible for SSC. Thus, when scaling up, cell suspensions must be manually transferred from the culture flask to another vessel suitable for agitation, which increases the risk of contamination and human error. Ideally, the number of culture transfer steps should be kept to a minimum. The present study describes the fabrication of an ultrasonic suspension culture system that stirs cell suspensions with the use of acoustic streaming generated by ultrasound irradiation at a MHz frequency. This system was applied to 100-mL suspension cultures of Chinese hamster ovary cells-a volume ten-fold larger than that generally used. The cell proliferation rate in this system was 1.88/day when applying an input voltage of 40 V to the ultrasonic transducer, while that of the SSC was 1.14/day. Hence, the proposed method can extend the volume limit of static cell suspension cultures, thereby reducing the number of cell culture transfer steps.

Suspension culture in a T-flask with acoustic flow induced by ultrasonic irradiation.

Suspension culture is an essential large-scale cell culture technique for biopharmaceutical development and regenerative medicine. To transition from monolayer culture on the culture surface of a flask to suspension culture in a bioreactor, a pre-specified cell number must first be reached. During this period of preparation for suspension culture, static suspension culture in a flask is generally performed because the medium volume is not large enough to use a paddle to circulate the medium. However, drawbacks to this static method include cell sedimentation, leading to high cell density near the bottom and resulting in oxygen and nutrient deficiencies. Here, we propose a suspension culture method with acoustic streaming induced by ultrasonic waves in a T-flask to create a more homogeneous distribution of oxygen, nutrients, and waste products during the preparation period preceding large-scale suspension culture in a bioreactor. To demonstrate the performance of the ultrasonic method, Chinese hamster ovary cells were cultured for 72 h. Results showed that, on average, the cell proliferation was improved by 40% compared with the static method. Thus, the culture time required to achieve a 1000-fold increase could be reduced by 32 h (a 14% reduction) compared with the static method. Furthermore, the ultrasonic irradiation did not compromise the metabolic activity of the cells cultured using the ultrasonic method. These results demonstrate the effectiveness of the ultrasonic method for accelerating the transition to large-scale suspension culture.