Theory and practice of centrifugal elutriation (CE). Factors influencing the separation of human blood cells

Centrifugal elutriation (CE) is currently a widely used preparative cell separation technique. In order to optimize the separation of cells that show only small differences in sedimentation velocity, several conditions that might influence the resolution capacity, such as rotor speed, counterflow, jetstream, cell load, density, and viscosity of the elutriation medium, were analyzed. Experiments carried out with human red blood cells (rbc) indicated that selective losses of rbc from the rotor caused by the jetstream, could be prevented if the separations were carried out at high rotor speeds, as predicted by the theory. In addition, high cell loads (5 X 10(8) rbc) resulted in better separations than low cell loads (5 X 10(7) rbc). Human monocytes were separated into subpopulations that differed only about 0.003 g/mL in density, but have virtually the same size. The separation was carried out either by increasing the density or viscosity of the elutriation medium or by decreasing the rotor speed. In all cases similar results were obtained. These results indicated that under optimal conditions CE can be applied for the separation of cells that differ only slightly in sedimentation velocity.     

Theory and practice of centrifugal elutriation (CE)

Centrifugal elutriation (CE) is currently a widely used preparative cell separation technique. In order to optimize the separation of cells that show only small differences in sedimentation velocity, several conditions that might influence the resolution capacity, such as rotor speed, counterflow, jetstream, cell load, density, and viscosity of the elutriation medium, were analyzed. Experiments carried out with human red blood cells (rbc) indicated that aselective losses of rbc from the rotor caused by the jetstream, could be prevented if the separations were carried out at high rotor speeds, as predicted by the theory. In addition, high cell loads (5×10⁸ rbc) resulted in better separations than low cell loads (5×10⁷ rbc). Human monocytes were separated into subpopulations that differed only about 0.003 g/mL in density, but have virtually the same size. The separation was carried out either by increasing the density or viscosity of the elutriation medium or by decreasing the rotor speed. In all cases similar results were obtained. These results indicated that under optimal conditions CE can be applied for the separation of cells that differ only slightly in sedimentation velocity.     

Correlation between phagocytic and membrane surface properties reflected by partitioning of human peripheral blood monocytes in two-polymer aqueous phases

Human peripheral blood monocyte-enriched fractions (identified by staining for peroxidase and by sizing) were obtained by velocity sedimentation at unit gravity of peripheral blood mononuclear cells. They were then fractionated by countercurrent distribution (a multiple-extraction procedure) in a charged Dextran/poly(ethylene glycol) aqueous phase system. The monocytes remained viable after the separation (order of 90%). Cells obtained from different cavities along the extraction train were tested for their ability to phagocytize latex particles. With increasing partition coefficient (presumably higher charge-associated membrane properties) the ratio of monocytes that phagocytized to monocytes that did not phagocytize increased appreciably. When, however, monocytes were permitted to phagocytize particles prior to countercurrent distribution, an increase in partition coefficient was associated with an appreciable decrease in the above-specified ratio. Control experiments indicate that the observed change in partitioning behavior cannot be ascribed to an alteration in size and/or density of the monocytes as a function of phagocytosis. It may be due to the internalization of charged surface groups during phagocytosis. We conclude that there is a correlation between the surface properties of monocytes (as reflected by partitiartitioning behavior cannot be ascribed to an alteration in size and/or density of the monocytes as a function of phagocytosis. It may be due to the internalization of charged surface groups during phagocytosis. We conclude that there is a correlation between the surface properties of monocytes (as reflected by partitioning) and their ability to ingest particles. Furthermore, an alteration in the surface charge-associated properties of monocytes as a consequence of phagocytosis is indicated by the cells' reduced partition coefficient.     

The evaluation of AKR leukemia virus purity: the requirement for both velocity and isopycnic centrifugation methods.

The purity of AKR murine leukemia virus obtained by isopycnic centrifugation was compared with the purity obtained by combining velocity sedimentation and isopycnic centrifugation methods. Evaluation of AKR and Rauscher viral purity by electron microscopy and by analysis of [³H]uridine-labeled viral RNA demonstrated that the velocity centrifugation step is essential for the removal of contaminants banding in the viral density region (1.19 – 1.15 g/ml). For studies requiring relatively pure oncornavirus preparations, a combination of both velocity sedimentation and isopycnic centrifugation steps are suggested. Viral recovery of about 50% was obtained by the method described.     

A diffusion driven instability in systems that separate particles by velocity sedimentation.

Velocity sedimentation has been used extensively to separate particles according to the magnitude of their sedimentation velocity in suitable media. This technique has been used over a wide range of particle size from protein molecules, viruses, subcellular particles to whole cells. Successful separation demands that collective particle motion should not occur. In practice it is observed that such systems may, under certain circumstances, suffer from a particular type of instability which destroys the normal dependence of sedimentation velocity on particle size and density. The aim of this paper is to identify the critical parameters that determine the development of this instability. Stability criteria are deduced and predictions of the theory compared with published observations. Satisfactory agreement between theory and observation is obtained. It is concluded that the simple stability criterion, namely that stable sedimentation will occur if the total density gradient is in the direction of the sedimenting force, grossly overestimates the particle load that can be separated in practice. Some specific recommendations for optimum particle loading are included. Earlier theoretical and experimental works are briefly reviewed.     

Effector mechanisms in allograft rejection. II. Density, electrophoresis, and size fractionation of allograft-infiltrating cells demonstrating several classes of killer cells.

An analysis of killer cells infiltrating “sponge-matrix” allografts during rejection has been performed by preparative fractionation by density centrifugation, velocity sedimentation, and free flow cell electrophoresis and by the use of heterologous anti-T-cell sera. At the peak of rejection, 7 to 8 days after transplantation, the allograft is infiltrated by several classes of killer cells, most notably by non-T lymphocytes, monocytes/macrophages, and T lymphocytes. The predominant cell types capable of performing in vitro lysis of relevant target cells appeared to be monocytes and non-T lymphocytes. T lymphocytes formed only a minority of the killer cells at this stage of the response. In contrast, as also documented earlier, the predominant killer cells in the regional lymph nodes and the spleen of the graft recipient mice were T lymphocytes (blasts).     

Streptolysin O: Sedimentation Coefficient and Molecular Weight Determinations

The sedimentation coefficient of streptolysin O as determined by sucrose density gradient ultracentrifugation is 3.7S. An approximate molecular weight of 60,500 was calculated from the sedimentation velocity, and a similar value was obtained by Sephadex gel filtration. There was no measurable difference in the sedimentation coefficient of streptolysin O in either the active or reversibly inactive forms, indicating that there were at most only minor conformational differences between the two forms.     

Convection-free boundary sedimentation of dilute protein solutions

Use of the density gradient sedimentation velocity technique appears to be essential for the accurate determination of the mean sedimentation coefficients of dilute protein solutions. When performed on an analytical ultracentrifuge equipped with a photoelectric-scanning-absorption optical system, the density gradient sedimentation velocity technique has been shown to be particularly useful in studying the subunit association-dissociation equilibria of multisubunit enzyme systems. The time factor has been shown to be a major advantage of the density gradient sedimentation velocity technique, as opposed to the sedimentation equilibrium technique, in studying the subunit association-dissociation equilibria of multisubunit enzymes such as rabbit muscle apo-glyceraldehyde-3-phosphate dehydrogenase, which is very unstable in dilute solution.     

The cell-mediated lympholysis-inducing capacity of highly purified human monocytes and T lymphocytes in primary and secondary mixed leukocyte cultures

In this study the capacity of T cells and monocytes to induce cell-mediated lympholysis (CML) in primary and secondary MLC was investigated. The T lymphocytes were enriched by rosetting with sheep red blood cells (E) and further purified by sedimentation at unit gravity, which completely removed the contaminating monocytes. In addition, a highly purified monocyte population was obtained by 1 X G sedimentation of the non-E rosette-forming cells. These purified T cells have a poor CML-inducing capacity in primary and secondary MLC. In contrast, monocytes were very effective in inducing CML in both primary and secondary MLC. Induction of CML by monocytes in primary MLC was inhibited by heterologous anti-Ia-like antisera, indicating that the induction of CML by monocytes was related to the presence of HLA-DR (Ia-like) antigens on these cells.     

A separation chamber to sort cells,nuclei, and chromosomes at moderate g forces. II. Studies on velocity sedimentation and equilibrium density centrifugation of mammalian cells

A separation chamber having a surface of 50 cm² and a height of 2 cm is described for the rapid separation of cells and cell organelles at acceleration forces from 10 to 90g. To eliminate wall sedimentation artifacts, the chamber was positioned 20 cm from the rotor axis in a speed-controlled centrifuge. The chamber has flow deflectors for the undisturbed introduction of the sample layer and the gradient; an antivortex cross prevents swirling upon acceleration and deceleration. To illustrate the use of the separation chamber, examples of velocity sedimentation and of equilibrium density centrifugation are given: (i) human monocytes (70% were 90% pure) are separated from lymphocytes in 10 min at 20g; (ii) nonparenchymal rat liver cells are separated in 10 min at 16g in 97% pure endothelial cells and 99% pure Kupffer cells; (iii) equilibrium density centrifugation of human peripheral blood cells at about 90g permits the separation of erythrocytes, monocytes, lymphocytes, neutrophils, eosinophils, and basophils in one run. B cells are separated from T cells. The movement of swinging buckets is analyzed in mathematical terms and a simple method is offered to determine the position of cells in density gradients with the use of a small programmable calculator.     

Characterization of a CV-1 cell cycle. V. An assessment of velocity sedimentation for cell separation and synchrony.

Velocity sedimentation at unit gravity has been used to enrich populations of logarithmically growing cells in different cell cycle phases. In order to evaluate the degree of synchrony obtained by this method of cell separation, synchronous populations of CV-1 cells, initially obtained by the selective detachment of mitotic cells from roller cultures, were separated by velocity sedimentation. It was found that although the mean cell volume increased linearly, the cells remained heterogeneous with respect to size during all phases of the cell cycle. Since the velocity sedimentation technique depends upon discrimination of cell size, the size heterogeneity of cells throughout the cycle limits the degree of synchrony which can be obtained by this method.     

Monitoring the homogeneity of adenovirus preparations (a gene therapy delivery system) using analytical ultracentrifugation

This study explores the capability of modern analytical ultracentrifugation (AUC) to characterize the homogeneity, under product formulation conditions, of preparations of adenovirus vectors used in gene therapy and to assess the lot-to-lot consistency of this unique drug product. We demonstrate that a single sedimentation velocity run on an adenovirus sample can detect and accurately quantify a number of different forms of virus particles and subvirus particles. These forms include (a) intact virus monomer particles, (b) virus aggregates, (c) empty capsids (ECs), and (d) smaller assembly intermediates or subparticles formed during normal or aberrant virus assembly (or as a result of damage to the intact adenovirus or EC material during all phases of virus production). This information, which is collected on adenovirus samples under the exact formulation conditions that exist in the adenovirus vial, is obtained by direct boundary modeling of the AUC data generated from refractometric and/or UV detection systems using the computer program SEDFIT developed by Peter Schuck. Although both detectors are useful, refractometric detection using the Rayleigh interferometer offers a key advantage for providing accurate concentration information due to the similar response factors for both protein and DNA and its insensitivity to light scattering effects. Additional AUC data obtained from analytical band sedimentation velocity and density gradient sedimentation equilibrium experiments in CsCl with UV detection were also generated. These results further support conclusions concerning the solution properties of adenovirus, the identity of the different virus species, and the overall capability of boundary sedimentation velocity analysis.     

The determination of density and molecular weight distributions of lipoproteins by sedimentation equilibrium.

A method is presented by which an experimental record of total concentration as a function of radial distance, obtained in a sedimentation equilibrium experiment conducted with a noninteracting mixture in the absence of a density gradient, may be analyzed to obtain the unimodal distributions of molecular weight and of partial molar volume when these vary concomitantly and continuously. Particular attention is given to the caracterization of classes of lipoproteins exhibiting Gaussian distributions of these quantities, although the analysis is applicable to other types of unimodal distribution. Equations are also formulated permitting the definition of the corresponding distributions of partial specific volume and of density. The analysis procedure is based on a method (employing Laplace transforms) developed previously, but differs from it in that it avoids the necessity of differentiating experimental results, which introduces error. The method offers certain advantages over other procedures used to characterize and compare lipoprotein samples (exhibiting unimodal distributions) with regard to the duration of the experiment, economy of the sample, and, particularly, the ability to define in principle all of the relevant distributions from one sedimentation equilibrium experiment and an external measurement of the weight average partial specific volume. These points and the steps in the analysis procedure are illustrated with experimental results obtained in the sedimentation equilibrium of a sample of human serum low density lipoprotein. The experimental parameters (such as solution density, column height, and angular velocity) used in the conduction of these experiments were selected on the basis of computer-simulated examples, which are also presented. These provide a guide for other workers interested in characterizing lipoproteins of this class.     

Purification of Giardia muris cysts by velocity sedimentation.

Giardia muris cysts were separated from fecal contaminants in primary isolates by unit gravity velocity sedimentation. Crude isolates obtained by centrifugation over 1.0 M sucrose were overlaid onto a Percoll density gradient, 1.01 to 1.03 g/ml. G. muris cysts were well separated from faster-sedimenting fecal debris and slower-sedimenting Spironucleus muris and bacteria in 1.5 h.     

Characterization by sedimentation analysis of kinetoplast DNA from Trypanosoma cruzi at different stages of culture.

The kinetoplast DNA networks of Trypanosoma cruzi exist under two forms which have been studied by equilibrium density centrifugation in CsCl gradients containing ethidium bromide and by band sedimentation analysis. The relative proportion of the two forms has been measured and varies significantly between the exponential and stationary phase of growth, suggesting that one of these forms is a replicative intermediate. Both forms exhibit very high sedimentation coefficients. The sedimentation velocity ethidium titration was used to measure the superhelix density of the kinetoplast DNA after having established the validity of the method with in vitro closed DNA networks. The superhelix density of the native form of the kinetoplast DNA minicircles is very low and varies according to the physiological state of the trypanosomes. Furthermore, we observed a significant increase of the superhelix density of the kinetoplast DNA of trypanosomes grown in the presence of ethidium.     

Separation of epithelial cells from suspensions of cells from the hamster parotid gland in an isokinetic density gradient of Ficoll in tissue culture medium.

Epithelial cells were separated from suspensions of hamster parotid cells by velocity sedimentation in an isokinetic gradient and by isopycnic sedimentation. Epithelial cells were 48.1 ± 18.0% of the cells in the starting sample suspensions of cells from the disaggregated hamster parotid glands. The purest gradient fractions following velocity sedimentation in a previously described isokinetic gradient contained 98.8 ± 1.8% epithelial cells. The purest fractions obtained from isopycnic sedimentation contained 99.9 ± 0.2% epithelial cells. Purification of parotid epithelial cells by velocity sedimentation in the isokinetic gradient seems preferable to purification using isopycnic centrifugation because a larger proportion of the epithelial cells are obtained in the zone of the gradient which contains highly purified epithelial cells and because velocity sedimentation requires lower centrifugal forces for a shorter period of time.     

Purification of Giardia muris cysts by velocity sedimentation

Giardia muris cysts were separated from fecal contaminants in primary isolates by unit gravity velocity sedimentation. Crude isolates obtained by centrifugation over 1.0 M sucrose were overlaid onto a Percoll density gradient, 1.01 to 1.03 g/ml. G. muris cysts were well separated from faster-sedimenting fecal debris and slower-sedimenting Spironucleus muris and bacteria in 1.5 h.     

Velocity sedimentation of organelles at low centrifugal force in an isokinetic gradient.

Mast-cell granules and polystyrene microspheres (0.600 and 1.011 micrometer in diameter) were sedimented in a previously described [Pretlow (1971) Anal. Biochem. 41, 248--255] isokinetic gradient in a low-speed centrifuge. For the analytical velocity sedimentation of organelles, this gradient offers several advantages over gradients that are commonly used for the sedimentation of organelles: (a) the density gradient (0.0008 g.ml-1.cm-1) is small, and the effective densities of organelles will change relatively little during sedimentation; (b) the densities at all points in the gradient (1.017--1.027 g/ml) are less than those in gradients commonly used for the sedimentation of organelles, the effective densities of sedimenting organelles are consequently relatively large, and the effect of density as a determinant of velocity of sedimentation is less limiting than in conventional gradients; (c) the small slope of the gradient is associated with a relatively slow increase in the viscosity encountered by the sedimenting organelle; (d) the iso-osmotic gradient is not significantly affected by the gradient medium (Ficoll), and the osmolarity can be adjusted to the desired value by the selection of an appropriate salt solution as the solvent for the Ficoll; (e) the gradient will be isokinetic for particles of densities similar to most organelles. An ultracentrifuge is not required for work with this gradient.     

Synchronization of Mouse L-Cells by a Velocity Sedimentation Technique

Differential velocity sedimentation at unit gravity has been used to separate an asynchronous population of mammalian cells into fractions synchronized in all phases of the cell cycle. Better enrichment was obtained for G₁ and S phases than for G₂-M phase. Electronic cell volume measurements of the fractions indicated that the separation was primarily dependent on cell size, and an experimentally determined sedimentation coefficient agreed very well with its predicted value. Sources of dispersion in the separation (including the contribution of cell density heterogeneity) were quantitated and found to be insufficient to explain all of the observed dispersion. Both the limitations and the applications of the technique are discussed.     

Characterization of guinea pig macrophages: I. Mobility and maturation of peritoneal cells following inflammatory stimuli

The wide spectrum of activities of macrophages may, in part, be a reflection of their different subpopulations. Sedimentation velocity was used in separate different guinea pig macrophage populations which were then compared in assays which measure some of the parameters of inflammatory responses. Normal resident, thioglycollate-induced exudates or cells elicited by ip injection of tuberculin into BCG-sensitized animals were studied. In thioglycollate exudates the small, possibly recently derived monocytes were most responsive to chemotactic agents whereas populations with high sedimentation velocity were more phagocytic, responsive to MIF and produced more LAF. By contrast, all macrophage populations or subpopulations from sensitized animals challenged with PPD were unresponsive to lymphocyte-derived chemotactic factor, endotoxin-activated serum, or to N-formyl-l-methionyl-l-phenylalanine in chemotaxis assays. In addition, macrophages from these animals did not migrate from capillary tubes in MIF assays. The lack of migration is presumably due to the action of MIF on these cells in vivo. Cells with low sedimentation velocity (predominantly lymphocytes), however, did migrate from capillary tubes. We conclude that small cells elicited by a nonspecific stimulus are probably attracted to the inflammatory site by chemotaxins and must subsequently enlarge or mature before they are able to respond to MIF, to be activated to produce LAF, or to exhibit extensive phagocytosis.