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.     

Temperature regulation during centrifugal elutriation and its effect on cell separation

Centrifugal elutriation appears to be a promising method for cell separation. The quality of the separation may be limited by the control of temperature within the separation chamber, which affects the fluid viscosity and rotor speed. The factors affecting the temperature regulations have been re-examined. At flow rates between 10 and 40 mL/min the temperature within the chamber was primarily dependent on the temperature of the fluid flowing into the rotor. Increases in the temperature of the fluid while it flowed through the rotor were observed and were greater at higher rotor speeds and lower flow rates. This heating, caused by friction at the rotating seal, could raise the fluid temperature within the chamber by as much as 6 degrees C. Fluctuations in the temperature of the centrifuge produced temperature variations of only 0.3 degrees C in the fluid in the elutriation chamber. Small increases in the rate of elutriation of cells, concomitant with centrifuge cooling and speed fluctuations, were detected by optical density measurements. However, neither the modal volume nor coefficient of variation of the collected cells were affected.     

Temperature regulation during centrifugal elutriation and its effect on cell separation

Centrifugal elutriation appears to be a promising method for cell separation. The quality of the separation may be limited by the control of temperature within the separation chamber, which affects the fluid viscosity and rotor speed. The factors affecting the temperature regulation have been re-examined. At flow rates between 10 and 40 mL/min the temperature within the chamber was primarily dependent on the temperature of the fluid flowing into the rotor. Increases in the temperature of the fluid while it flowed through the rotor were observed and were greater at higher rotor speeds and lower flow rates. This heating, caused by friction at the rotating seal, could raise the fluid temperature within the chamber by as much as 6°C. Fluctuations in the temperature of the centrifuge produced temperature variations of only 0.3°C in the fluid in the elutriation chamber. Small increases in the rate of elutriation of cells, concomitant with centrifuge cooling and speed fluctuations, were detected by optical density measurements. However, neither the modal volume nor coefficient of variation of the collected cells were affected.     

THE RESOLUTION OF MIXTURES OF VIABLE MAMMALIAN CELLS INTO HOMOGENEOUS FRACTIONS BY ZONAL CENTRIFUGATION

Large-scale separation of mixtures of mammalian cells was obtained with the A-1X zonal centrifuge rotor and density gradients consisting of Ficoll dissolved in modified Eagle's MEM suspension-culture medium. The cells remained viable as tested by plating efficiency or by motility observed with time-lapse photography. Rabbit thymocyte and HeLa cell mixtures were separated with 99 and 89 per cent purity, respectively. Mixtures of thymocytes and suspension-cultured, human acute leukemia cells (Roswell Park strain LKID) were separated with 93 and 91% purity, respectively. HeLa cells were isolated 92% pure from a mixture with horse leukocytes. A book of charts giving the sedimentation position and velocity versus time of cells in the A rotor under standard conditions of gradient composition, angular velocity, and temperature was prepared with the use of a computer program based on the differential sedimentation equation. The charts are used to estimate the centrifugation time necessary for maximum separation of cells. The success achieved in separating mixtures of cells points to the future possibility of large-scale fractionation of solid tissues, especially tumor tissues, into preparations cf viable cells of a single type.     

Centrifugal elutriation (counterstreaming centrifugation) of cells

Lindahl first described the separation of cells by velocity sedimentation utilizing a special technique (counterstreaming centrifugation) that was later modified slightly and renamed centrifugal elutriation. Centrifugal elutriation has been applied, with variable degrees of success, to the separation of hemopoietic cells, mouse tumor cells, testicular cells, and a variety of other specialized cells as well as cells in particular phases of the cell cycle. The capacity of the elutriator to separate large numbers of cells is its chief advantage. The purities of the separated cells have not been compared with the purities of cells separated by other methods in most cases; such comparisons would permit more sophisticated comparison of elutriation with other techniques for velocity sedimentation.     

Centrifugal elutriation (counterstreaming centrifugation) of cells.

Lindahl first described the separation of cells by velocity sedimentation utilizing a special technique (counterstreaming centrifugation) that was later modified slightly and renamed centrifugal elutriation. Centrifugal elutriation has been applied, with variable degrees of success, to the separation of hemopoietic cells, mouse tumor cells, testicular cells, and a variety of other specialized cells as well as cells in particular phases of the cell cycle. The capacity of the elutriator to separate large numbers of cells is its chief advantage. The purities of the separated cells have not been compared with the purities of cells separated by other methods in most cases; such comparisons would permit more sophisticated comparison of elutriation with other techniques for velocity sedimentation.     

Zonal unit-gravity elutriation. A new technique for separating large cells and multicellular complexes from cell suspensions

A new and simple technique, zonal unit-gravity elutriation, has been devised for separating very large cells, multicellular complexes, or small organisms from suspensions consisting mainly of small cells. The separation vessel is a conical chamber with an entrance at the lower, narrower part of the cone and an exit at the upper, wider part of the cone via a dome-shaped lid. A baffle at the entrance prevents turbulence from incoming fluid. Chambers of differing widths and wall slopes are chosen depending on the sedimentation rate of the particles to be separated. A small volume of the cell suspension is placed in the chamber on the bench in a cold-room. Medium stabilized by a shallow density gradient is pumped into the base of the chamber and ascends, creating a decreasing velocity gradient. Cells sediment at unit-gravity against this ascending counterstream, and are separated into bands according to sedimentation velocity. By adjusting the flow rate of the medium, different sizes of cells can be separated. Tumor cells can be enriched, and larger blast cells can be separated from small cells in lymphoid cell suspensions. The procedure produces complete separation of thymic nurse cells (epithelial-lymphoid complexes) from free thymocytes in digested thymus suspensions and produces substantial enrichment of thymic rosettes (macrophage-lymphoid complexes). A very favorable situation for applying this technique is the isolation of Taenia taeniaformis larvae, which can be completely purified from infected liver suspensions, representing a 4 X 10(5)-fold enrichment of the parasites, with high recovery, in a single 30 min operation.     

Cell cycle synchronization of animal cells and nuclei by centrifugal elutriation

Synchronization of cells and nuclei is a powerful technique for the exact study of regulatory mechanisms and for understanding cell cycle events. Counterflow centrifugal elutriation is a biophysical cell separation technique in which cell size and sedimentation density differences of living cells are exploited to isolate subpopulations in various stages of cell cycle. Here, a protocol is described for the separation of phase-enriched subpopulations from exponentially growing Chinese hamster ovary cells at high-resolution power of elutriation. The efficiency of elutriation is confirmed by measuring the DNA content fluorimetrically and by flow cytometry. The resolution power of elutriation is demonstrated by the ability to fractionate nuclei of murine pre-B cells. The installation and elutriation by collecting 16-30 synchronized fractions, including particle size analysis, can be achieved in 4-5 h.     

Isolation of cell cycle fractions by counterflow centrifugal elutriation

Counterflow centrifugal elutriation (CCE) has been used to fractionate cell populations on the basis of sedimentation properties, with minimal perturbation of metabolic function. Therefore, it is an ideal method for the isolation of cell cycle phase specific populations. We present modifications of the standard Beckman centrifugal elutriation system which permit standardization of the elutriation procedure and eliminate inter-run variability. We provide elutriation parameters for the cell cycle fractionation of a variety of cultured cell lines and suggest ways to improve the quality of the cell separations. In addition, we describe protocols for the fractionation of up to 3.50 X 10(8) cells in the small (JE-6B) Beckman elutriation system. This represents a four- to eight-fold increase in cell numbers over current cell fractionation procedures. Cell cycle populations containing greater than 95% G1, greater than 80% S, and greater than 70% G2/M were consistently obtained using these protocols. Finally, we analyzed phase-enriched fractions from several cultured cell lines for the cell cycle regulation of the enzyme thymidine kinase. The data confirm previous findings that CCE is an excellent means of obtaining physiologically unperturbed cell cycle phase specific fractions.     

Characterization of the separation properties of the Beckman elutriator system

The role of fluid flow in the elutriation process was visualized by pumping dye solution through the Beckman JE-6 elutriator rotor. Three major fluid flow disturbances were observed in the separation chambers, namely; jet-streaming, ripple flow, and whirl flow. In order to evaluate the effects of these non-ideal fluid flow patterns on the separation of homogeneous populations of particles or cells, 12--35 micron diameter latex spheres and 9L rat brain tumor cells were fractionated with the Beckman elutriator system. The elutriator system was evaluated on the basis of: (1) recovery, (2) elution loss during loading, (3) homogeneity of the size distributions, and (4) the relationship of the median volume of eluted particles or cells to the rotor speed and the collection fluid velocity. Both a conventional collection method (two 40-mL fractions at ech collection rotor speed) and a long collection method (10--15 40-mL fractions at several collection rotor speeds) were compared to determine if collection procedures could compensate for some of the difficulties caused by the non-ideal fluid flow patterns. Although more than 90% of the particles or cells were always recovered, about 5% eluted during the loading procedure. Neither collection method altered this phenomenon. The long collection method significantly improved the homogeneity of the collected populations, but this was accompanied by a reduction in cell yield. The median particle or cell volume of each fraction agreed with that expected under ideal fluid flow conditions except at high and low rotor speeds when the conventional collection method was used.     

Characterization of the separation properties of the beckman elutriator system

The role of fluid flow in the elutriation process was visualized by pumping dye solution through the Beckman JE-6 elutriator rotor. Three major fluid flow disturbances were observed in the separation chambers, namely; jet-streaming, ripple flow, and whirl flow. In order to evaluate the effects of these non-ideal fluid flow patterns on the separation of homogeneous populations of particles or cells, 12–35 μm diameter latex spheres and 9L rat brain tumor cells were fractionated with the Beckman elutriator system. The elutriator system was evaluated on the basis of: (1) recovery, (2) elution loss during loading, (3) homogeneity of the size distributions, and (4) the relationship of the median volume of eluted particles or cells to the rotor speed and the collection fluid velocity. Both a conventional collection method (two 40-mL fractions at each collection rotor speed) and a long collection method (10–15 40-mL fractions at several collection rotor speeds) were compared to determine if collection procedures could compensate for some of the difficulties caused by the non-ideal fluid flow patterns. Although more than 90% of the particles or cells were always recovered, about 5% eluted during the loading procedure. Neither collection method altered this phenomenon. The collected populations, but this was accompanied by a reduction in cell yield. The median particle or cell volume of each fraction agreed with that expected under ideal fluid flow conditions except at high and low rotor speeds when the conventional collection method was used.     

Application of gravitational sedimentation to efficient cellular recycling in continuous alcoholic fermentation

A mathematical model for the sedimentation velocity in an inclined parallel plate sedimenter is proposed. The parameters of the alcoholic fermentation broth (cell density of Saccharomyces cerevisiae, density of the fermentation medium, viscosity of the broth at various alcohol and biomass contents) were determined experimentally. The sedimentation velocities were predicted under the various operational conditions and parameters, both of the broth (the alcohol concentration and cell content) and the sedimenter prototype (length, distance between the plates, and slope). The proposed model for the sedimentation velocity presented a good correlation with the experimental results of continuous sedimentation. These sedimenter prototypes were assembled and tested for efficiency of separation of yeast cell under conditions considered for interest for continuous alcoholic fermentation. A selective filter for the overflow composed of calcium alginate gel improved operation. A high operational stability, high separation efficiency (over 98%), and adequate settler residence times (about 20 min) were attained. The operational results permitted the operation of continuous alcoholic fermentation with cellular recycling effected exclusively by gravitational sedimentation, this characterizing a process of enormous industrial interest because of the operational simplicity and low operational and maintenance costs. (c) 1993 John Wiley & Sons, Inc.     

Separation and concentration of phytoplankton populations using centrifugal elutriation

Centrifugal elutriation is a technique for separating particles on the basis of their sedimentation velocity, an expression of size, shape, and specific gravity. Unialgal cultures, mixtures of two phytoplankton cultures, and natural seawater samples were elutriated to determine the feasibility of this technique for collecting fractions of different cell cycle phases, separating two phytoplankton species, and concentrating cells from dilute samples. Elutriation resulted in the separation of a culture of Dunaliella tertiolecta and Phaeodactylum tricornutum into homogeneous fractions of each species. Cells in the natural seawater sample were concentrated by nearly 2 orders of magnitude. Centrifugal elutriation provides an alternative cell separation and concentration technique when large numbers of cells are required.     

Separation of plant cell organelles by zonal centrifugation in reorienting density gradients

Preparative separations of plant cell organelles (glyoxysomes, proplastids, mitochondria, and endoplasmic reticulum) in an ordinary refrigerated centrifuge were obtained by sucrose density gradient centrifugation in a zonal rotor loaded and unloaded in the static manner. The quality of the separation which was monitored by marker enzymes and electron microscopy compares to analytical separations in swinging-bucket rotors. Membrane alterations observed in glyoxysomes and mitochondria are traced back to sucrose as a major component of the homogenization and density gradient medium.     

Zonal unit-gravity elutriation

A new and simple technique, zonal unit-gravity elutriation, has been devised for separating very large cells, multicellular complexes, or small organisms from suspensions consisting mainly of small cells. The separation vessel is a conical chamber with an entrance at the lower, narrower part of the cone and an exit at the upper, wider part of the cone via a dome-shaped lid. A baffle at the entrance prevents turbulence from incoming fluid. Chambers of differing widths and wall slopes are chosen depending on the sedimentation rate of the particles to be separated. A small volume of the cell suspension is placed in the chamber on the bench in a cold-room. Medium stabilized by a shallow density gradient is pumped into the base of the chamber and ascends, creating a decreasing velocity gradient. Cells sediment at unit-gravity against this ascending counterstream, and are separated into bands according to sedimentation velocity. By adjusting the flow rate of the medium, different sizes of cells can be separated. Tumor cells can be enriched, and larger blast cells can be separated from small cells in lymphoid cell suspensions. The procedure produces complete separation of thymic nurse cells (epithelial-lymphoid complexes) from free thymocytes in digested thymus suspensions and produces substantial enrichment of thymic rosettes (macrophage-lymphoid complexes). A very favorable situation for applying this technique is the isolation ofTaenia taeniaformis larvae, which can be completely purified from infected liver suspensions, representing a 4×10⁵-fold enrichment of the parasites, with high recovery, in a single 30 min operation.     

Separation of mouse testis cells by equilibrium density centrifugation in Renografin gradients

Mouse testis cells have been separated by equilibrium density centrifugation in gradients of Renografin. Intact testis cells were not damaged by the separation procedure provided that, following separation, the osmolarity was reduced gradually. The various cell types were identified microscopically and by ³H-thymidine labelling with similar results. The present technique has demonstrated that significant variations in cell density occur during spermatogenesis. Approximately ten-fold enrichments of nearly all testis cell types were achieved by equilibrium density separation of testis cell suspensions. More homogeneous cell populations were prepared by density gradient centrifugation of cell fractions obtained from velocity sedimentation separations. Overall enrichments of spermatogonia, by 29-fold; pachytene spermatocytes, 45-fold; dividing meiotic cells, 170-fold; round spermatids, 30-fold; step 11–13 elongating spermatids, 12-fold; Leydig cells, 70-fold; and cytoplasmic fragments, 55-fold, were obtained. In this study, a method for preparation of cell suspensions was also developed to produce higher yields of spermatogonia and young primary spermatocytes; however, the density distribution of these cells was altered.     

Metrizamide in D2O — A novel solute for the separation of labeled and unlabeled proteins by equilibrium density gradient centrifugation

Metrizamide(2-(3-acetamido-5-N-methylacetamido-2,4,6-triiodobenzamido)-2-deoxy-d-glucose) dissolved in D₂O was found to be a very suitable medium for the separation of labeled and unlabeled proteins by equilibrium gradient sedimentation. It is nontoxic, and has little influence on the activity of enzymes. Solutions in the density range of 1.3–1.45 g cm^?3 have low viscosities. Since the spontaneous equilibrium gradient, which is dependent on the angular velocity, occurs only after a long time of centrifugation in metrizamide solutions, the equilibrium density gradient sedimentation of proteins can be performed at the highest available speed with any preformed shallow gradient. Examples for the separation of proteins of different densities are given.     

Influence of flow velocity and cell concentration on dynamic adhesion of erythrocytes to glass surface

Comparative studies were carried out on dynamic adhesion of 51Cr-labelled erythrocytes to the surface of glass beads in the presence of serum in the medium (50 microng of protein/ml) and in protein-free medium. The influence of cell concentration (within the range 4 X 10(5) to 8 X 10(6)/ml) and of cellular flow velocity (within the range 1.5-0.4 cm/min) on the value of adhesion was investigated. It was found that when serum was present in the medium, the decisive influence on erythrocyte adhesion was exerted by the velocity with which the cells pass though the glass bead layer. Cell concentration under these conditions has only a very slight effect. When the medium does not contain serum, erythrocyte adhesion to the bead layer seems to depend on both cell concentration and flow velocity. Preliminary data were obtained concerning the release of 51Cr from the bead layer after erythrocyte adhesion.     

Separation of cells by velocity sedimentation

A system for fractionating populations of living cells by velocity sedimentation in the earth's gravitational field is described. The cells start in a thin band near the top of a shallow gradient of 3% to 30% fetal calf serum in phosphate buffered saline at 4°C. Cell separation takes place primarily on the basis of size and is approximately independent of cell shape. A sharply-defined upper limit, called the streaming limit, exists for the cell concentration in the starting band beyond which useful cell separations cannot be achieved. This limit, which varies with the type of cell being sedimented, can be significantly increased by proper choice of gradient shape. For sheep erythrocytes (sedimentation velocity of 1.6 mm/hour) it is 1.5 × 10⁷ cells/ml. Measured and calculated sedimentation velocities for sheep erythrocytes are shown to be in agreement. The technique is applied to a suspension of mouse spleen cells and it is shown, using an electronic cell counter and pulse height analyzer, that cells are fractionated according to size across the gradient such that the sedimentation velocity (in mm/hour) approximately equals r²/4 where r is the cell radius in microns. Since cells of differing function also often differ in size, the system appears to have useful biological applications.     

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.