Detachment of RAW264.7 macrophages from a culture dish using ultrasound excited by a Langevin transducer

Abstract

To study the relationship between macrophages and antigens, an efficient culture method for macrophages is important. During culture, macrophages adhering to the culture surface are difficult to harvest by general trypsinization. Thus, prolonged trypsinization or cell scraping has been used to detach macrophages. However, prolonged trypsinization has a negative effect on cell viability, and the detachment efficiency with cell scrapers depends highly on the skill of a technician. Therefore, we developed a macrophage-detaching method by combining trypsin-EDTA and ultrasonic vibration to detach cells from a ubiquitous culture vessel. We fabricated a device that propagated ultrasound to a φ-35-mm culture dish from underneath. To demonstrate our concept, RAW264.7cells were used as model cells and exposed to several detaching conditions to evaluate the effects of our developed method. In addition to the proposed method, as traditional detaching methods, simple trypsinization with trypsin-EDTA and manual cell scraping were performed. Furthermore, to determine the optimal intensity of the ultrasound, input voltages into the ultrasound transducer of 200, 225, and 250V were used. As a result, the number of live cells detached by the developed method with an input amplitude of 225V was approximately 4.8 times more than that by simple trypsinization and approximately 4.3 times more than that by scraping. Furthermore, the proliferation and phagocytosis level of the cells were increased by the developed method at 225V, while no significant difference was found in metabolism. Thus, the developed method improves culture efficiency and cell functions without causing metabolic disorders.