Understanding the formation and behaviors of droplets toward consideration of changeover during cell manufacturing

Abstract

Preventing the contamination of processed cells is required for achieving reproducible manufacturing. A droplet is one of the potential causes contamination in cell manufacturing. The present study elucidates the formation mechanism and characteristics of droplets based on the observation and detection of droplets on the base surface of the biological safety cabinet (BSC) where cell processing is conducted under unidirectional airflow. Pouring fluorescent solution into the vessel using a measuring pipette was conducted to visualize the formation of droplets by videos as well as visual detection by blacklight irradiation on the base surface of the BSC. The experiments revealed that airborne and non-airborne droplets emerged from bursting bubbles, which formed when the entire solution was pushed out of the measuring pipette. Therefore, the improving procedure of pouring technique when entire solution was not pushed out of the pipette realized no formation of the droplets due to the prevention of emergence of bubble. In addition, an alternative procedure in which the entire solution was poured into the deep point of the test tube prevented the flying of non-airborne droplets outside the tube, while airborne droplets that escaped the tube rode the airflow of BSC. These results suggested a method for the prevention of the droplet formation, as well as the deposit control of droplets onto the surface in BSC, leading to cleanup area in the BSC for changeover with environment continuity.

Keywords: Airborne and non-airborne droplets; BSC, biological safety cabinet; Bursting bubble; CPA, cell processing area; Changeover; Pouring the solution.

Fig. 1

Schematic illustration of experimental design. Schematic illustration of the experimental design to confirm the attached droplets on the base surface of a biological safety cabinet.

Fig. 2

Observations of droplet formation. Still images of the observations of droplets during pouring of the solution with the airflow turned off (A) and airflow turned on (B) in the biological safety cabinet. Scale bars: 50 μm. Closed and dot squares indicated the tips of pipet and culture dish, respectively. Solid and dotted arrows indicated the typical airborne and non-airborne droplets, respectively.

Fig. 3

Effect of pushing out on droplet formation. Still images of the observations of droplets without (A) and with (B) pushing out the entire solution. Scale bars: 50 μm. Detection of the droplets on the surface of the workbench in the biological safety cabinet without (C) and with (D) pushing out the entire solution. Red circles indicate the points where the solution was poured.

Fig. 4

Effect of wall on droplet behaviors. Still images of the observations of the droplets during pouring of the solution without (A) and with (B) pushing out the entire solution at the shallow point, and completely pouring the solution at deep point in the tube (C). White and yellow arrows indicate the typical airborne and non-airborne droplets, respectively. Scale bars: 50 μm. Detection of the droplets on the base surface of the biological safety cabinet without (D) and with (E) pushing out the entire solution at the shallow point, and pushing out the entire solution at the deep point in the tube (F). Red circles indicate the points where the solution was poured.

Fig. 5

Schematic illustration of changeover with environment continuity. Schematic illustration of changeover based on the treatment of droplets with pouring method and classification. Changeover with environment continuity by inhibition of droplet formation (A). Changeover with environment continuity after treatment using the base surface according to the classification of the droplets (B).