Harvesting Cells and Subcellular Components: Continuous-Flow Centrifugation

This discussion of methods for the differential isola­tion and separation of cells, and subcellular compo­nents would not be complete without a description of the use of centrifugation for harvesting large quanti­ties of particulate material from large-volume suspen­sions.

The technique is generally known as continuous-flow centrifugation.

The most common application of continuous-flow centrifugation is the harvesting of bacteria, algae, protozoa, and other cells grown in multi-liter cultures as a preliminary to chemical, physiological, or mor­phological analysis.

However, the technique is also fre­quently employed:

(1) To collect cell-free culture media prior to the isolation and assay of cellular excretion products such as enzymes, vitamins, and hormones;

(2) To separate blood;

(3) To remove the larger subcellu­lar components such as nuclei, chloroplasts, and mito­chondria from large volumes of tissue homogenates; and

(4) To collect precipitates from large volumes of aqueous suspensions.

During continuous-flow centrifugation, the suspen­sion of particles is introduced into the spinning centri­fuge rotor as a continuous, uninterrupted stream. As the suspension passes through the rotor, particles are sedimented out of the stream and are trapped and concentrated within specific rotor chambers, while the clarified supernatant leaves the rotor and is col­lected separately.

Continuous-flow centrifuge rotors thereby eliminate the need for a series of batch sepa­rations when very large volumes of particle suspen­sions must be processed. When processing is com­pleted, the rotor is simply decelerated and opened, and the trapped cells or particles removed.

Although cells or other particles present in multi- liter volumes can be harvested using conventional rotors, that ap­proach is far less efficient. Even the largest conven­tional rotors generally accommodate only a few liters of suspension, so that a succession of spins is neces­sary when larger volumes of material must be han­dled.

Equally important, the increased size and weight of these rotors restricts their maximum oper­ating speeds and may necessitate extended centrifu­gation time to ensure total particle “cleanout.” Be­cause at any instant continuous-flow rotors contain only a small fraction of the total volume of material to be centrifuged, they may be quite small. Thus, in addi­tion to eliminating the need for successive runs, continuous-flow rotors can be operated at much higher speeds (hence greater RCF), providing more rapid and efficient particle cleanout.

The RCF experienced by the particles as they enter the collection chambers of the rotor depends on rotor speed and causes the particles to sediment at specific rates. If the rate of particle sedimentation is greater than the rate at which the surrounding liquid moves toward a centripetal exit port in the chamber, then the particles become trapped in the rotor.

However, if the sedimentation rate is less than the rate of centripetal flow, the particles are carried toward the exit ports and out of the rotor. Usually, flow rate and rotor speed are selected to provide maximum cleanout of the par­ticle suspension.

However, for heterogeneous popula­tions of particles, the flow rate and rotor speed can of­ten be adjusted so that a differential fractionation of the particles is achieved, that is, depending on their sizes, shapes, and densities, some particles will be trapped in the rotor, while others are conducted out of the rotor with the supernatant.