Table 3.

Factors that affect differential ultracentrifugation (dUC)-based exosome isolation. The efficiency of exosome isolation using differential ultracentrifugation is governed by four main factors: acceleration, the type of rotor in which the samples are placed, the viscosity of the solution in question, and finally the time needed to create the desired pellet [53]. This table summarizes each variable and considerations to keep in mind when isolating exosomes using dUC.

Acceleration (g)	The acceleration of the centrifuge, also known as the g force, refers to the speed and determines the separation efficiency.
Rotor (k)	The k-factor represents the relative pelleting efficiency of a rotor at maximum speed. The lower the k factor, the better the pelleting efficiency of the rotor, and the shorter the centrifugation time. The pelleting time (T) is determined by the equation T = k/s, where T is the time in hours required for centrifugation, s is the sedimentation coefficient in Svedberg units, and k is the k-factor. Sedimentation coefficients depend on the size and shape of the vesicle being isolated, and the viscosity of the sample media. The smaller the s, the longer it takes to pellet the particle. There are two types of rotors that are commonly used for exosome isolation: swinging bucket (SW) and fixed-angle (FA) rotors, principally differing in sedimentation efficiency. A SW rotor stands out horizontally during centrifugation, and thus has a larger sedimentation path than FA rotors. While this lowers the pelleting efficiency of SW rotors (higher k value) resulting in lower yield, SW rotors have better resolution, i.e., they can separate vesicles with small differences in size more effectively than FA rotors.
Viscosity	Reducing viscosity of the sample increases the efficiency of isolation, as the higher the viscosity, the more difficult it would be for the exosomes to travel through the sample and pellet.
Time	The amount of time a biological fluid is centrifuged is determined by the viscosity, rotor g value, and desired purity of the exosomal fraction. The duration can be extended to yield greater quantities of exosome-based contents such as protein and RNA, though this is limited by the possibility of condensing the pellet to such an extreme that they aggregate, making them hard to resuspend and it may thus interfere with the functional integrity of the final product. Longer time of centrifugation also co-precipitates non-exosomal proteins and reduces purity of the end product.