Thesis

Separation and characterization of mammalian cells by velocity sedimentation

Unit gravity (Sta-Put) sedimentation and reorienting gradient zonal centrifugation were used to separate and characterize mammalian cells by velocity sedimentation. The development of both techniques as well as density grad-theory have been reviewed. Zone resolution for both techniques was compared by utilizing a model system of polystyrene beads (of known diameters and densities) in minimum slope gradients of sucrose at unit gravity or predesigned isokinetic gradients of Ficoll in the zonal rotor. Both minimum Slope and isokinetic gradients resulted in constant velocities during sedimentation. Slightly better zone resolution was obtained by unit gravity sedimentation, presumably as a consequence of the mildness of the technique. Nevertheless, both techniques yielded a high level of resolution. Biological application of both techniques was demonstrated by separating and characterizing three sizedensity subpopulations of cells present in the 129/J mouse ascites teratocarcinoma tumor. Buoyant density centrifugation revealed that the tumor is comprised of two distinct density classes of cells with average densities of 1.17 gm/ml and 1.04 gm/ml and average diameters of 15.7 um and 19.9. Unit gravity sedimentation also revealed the presence of independently varying cell densities as well as cell sizes in the tumor. Isolinetic gradients of Ficoll in the reorienting gradient zonal rotor were utilized to probe size variations within each density class. Cells comprising the less dense (1.04 gm/ml) subpopulation of the tumor were shown to have two discrete internal size classes with average diameters of 24 and 9 pm. The denser subpopulation of cells (1.17 gm/ml) appears to be formed by a continuous spectrum of sizes. This work demsonstrates the versatility of both techniques in distinguishing size-density classes in a heterogenous cell population.

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