8 Methods Involved in Separation of Whole Cells (With Diagram)

We have been concerned with methods used to separate and isolate particles re­leased from disrupted cells and tissues.

Until quite re­cently, little attention was directed to a related problem—namely, how to separate whole viable cells from one another when the tissue or culture being studied is heterogeneously composed.

For example, an organ such as the liver is composed of many differ­ent types of cells, including hepatic cells, Kupffer cells, connective tissue cells, smooth muscle cells, blood cells, and so on. Therefore, a homogenate of liver tissue contains subcellular particles from diverse kinds of cells.

Even a culture of the same cell type may be heterogeneous with regard to cell ages and therefore is representative of a broad spec­trum of morphological characteristics and/or physio­logical activity.

The development of methods for separating different types of cells present in a tissue has lagged behind technological advances in the area of subcellular fractionation. In the following section, we will examine some of the problems associated with whole-cell separations and some of the more impor­tant methods that have evolved to effect such separa­tions.

1. Tissue Disaggregation:

If the tissue to be fractionated consists of suspensions of individual cells (e.g., a culture of microorganisms, certain tissue cultures, blood cells, and certain tu­mors), the problem of whole-cell separation is far less difficult than when the cells comprise a solid tissue (such as liver, kidney, brain, etc.). It is therefore not surprising that, to date, most efforts directed toward whole-cell separations have involved natural suspen­sions of cells as the starting material. Some success has been obtained with solid tissues by employing chemical agents that induce tissue disaggregation— primarily digestive enzymes and chelating agents.

These materials weaken the connections between neighboring cells, making it possible to mechanically disperse the tissue into individual cells without appre­ciable cell breakage. The tissue may be treated after its removal from the animal, although perfusion of the organ with a solution of the disaggregating agent prior to the organ’s excision is more often preferred.

Once the tissue has been reduced to a suspension of individual cells, fractionation into subpopulations fol­lows. If the goal of the experiment is to isolate a par­ticular cell subpopulation for further study, then the remaining cells in the suspension may be selectively destroyed or removed by chemical means. For exam­ple, the leukocytes of blood may be separated from the erythrocytes by selective destruction of the erythro­cytes using osmotic or chemical lysis. Purification of a particular subpopulation of cells may also be achieved by taking advantage of differential cell agglutinability in the mixed population.

Simply freezing and thawing a suspension of cells may differentially lyse specific subpopulations. In general, chemical procedures cause some changes in all the cells in the mixed popu­lation, so that the method of choice is more generally one that achieves a separation by mild physical means.

Among the most popular of the latter methods are adherence and filtration, conventional and zonal centrifugation, centrifugal elutriation, unit gravity separation, countercurrent distribution, electrophoresis, and fluorescence-activated cell sorting.

2. Adherence and Filtration:

Separations of cells using differences in adherence phenomena or filtration properties are among the old­est physical procedures used. Some cells readily ad­here to glass beads, nylon wool, glass wool, and so on and may be separated from nonadhering cells by pass­ing the cell suspension through a hollow glass column packed with these materials.

Success has also been ob­tained by coating glass or plastic beads with antibod­ies, antigens, or haptens so that cells will be differen­tially adsorbed to the beads on the basis of chemical interactions between the plasma membrane and the coating material. Sieves of varying pore diameter can also be used to separate populations of cells on the ba­sis of differences in cell diameter.

3. Conventional and Zonal Centrifugation:

Because of their relatively large size (i.e., in compari­son with organelles and macromolecules) whole cells sediment quite rapidly. Consequently, attempts to fractionate suspensions of cells using centrifugation involve rotation at low rpm (i.e., small RCF) for short periods of time (typically less than 500 g for a few min­utes).

As with subcellular centrifugal fractionations, greatest resolution is obtained using density gradi­ents in which the mixture of cells in the starting zone is separated into subpopulations on the basis of differ­ences in average cell size and/or density.

Most cells be­have like miniature osmometers, so that strict atten­tion must be paid to the selection of gradient solute. Salts are rarely used to prepare density gradients for cell separations because of their deleterious osmotic effects.

Large, impermeable, and biologically inert polymers such as Percoll and Ficoll are the more fre­quent choices. Because they offer the advantage of greatly increased sample size, reograd zonal rotors have been used with great success for separating dif­ferent cell populations.

4. Centrifugal Elutriation (Counter-Streaming Centrifugation):

Centrifugal elutriation is an ingenious technique pio­neered in the 1940s by P. E. Lindahl and brought to its present state of the art principally through the work of C. R. McEwen. In centrifugal elutriation, a suspen­sion of cells is pumped into a specially designed rotor chamber through a marginally located entry port (Fig. 12-15), and this is followed by a continuous flow of suspending medium.

Centrifugal sedimentation of the cells is opposed by the centripetal flow of the sus­pending medium. Both effects vary in magnitude across the radial dimension of the rotor chamber, be­cause (1) the centrifugal force increases with distance from the rotor axis and (2) the rate of centripetal liq­uid flow varies according to the cross-sectional area of the chamber (the area increases exponentially as the liquid travels toward the rotor axis).

Depending on its initial position in the chamber and its sedimentation coefficient, each cell will either sediment radially un­der the centrifugal force or be carried centripetally by the liquid flow. As a result, the cells migrate through the chamber to positions where these two forces can­cel one another.

Some ceils (e.g., the smallest ones) may be swept from the rotor chamber entirely and others form a zone within the rotor chamber and can be collected for further study. Centrifugal elutriation has been successfully applied to separations of blood cells, algae, yeasts, and other cells in culture.

5. Unit Gravity Separation:

The separation of particles on the basis of sedimenta­tion rate differences may not require centrifugation if the particles are sufficiently large. For example, whole cells sediment fairly quickly even at 1 g (i.e., at unit gravity). Unit gravity procedures have been used effectively to separate different types of blood cells, tissue culture cells, and populations of microorga­nisms into subpopulations.

The separation is achieved by layering the mixture of cells onto the top of a sta­tionary density gradient and allowing the cells to set­tle through the gradient for some period of time. The gradient and separate cell populations are then col­lected as a series of fractions. Devices used to sepa­rate cells in density gradients at unit gravity are called “sta-put” devices and vary from simple cylin­drical chambers to more elaborate apparatuses having moving, conical end caps. The principle is illustrated in Figure 12-16.

Not only is it possible to separate heterogeneous mixtures of cells using this simple approach, but a population of a single cell type (e.g., a cell culture) can be fractionated according to cell age when age and size are related. In this way, events that occur during successive phases of the cell cycle can be studied by examining the cells present in different collected fractions.

A number of methods for separating and isolating the molecular constituents of cells. Some of these methods have been appro­priately modified and applied with varying degrees of success to separations of the different kinds of cells that make up a tissue; countercurrent distribution and electrophoresis.

6. Countercurrent Distribution:

Cells (and other particles) may be separated from one another on the basis of differences in their partition between two immiscible liquids; this technique is called countercurrent distribution (CCD). Naturally, if the cells are to be separated without undue damage, the milieu selected must be compatible with the cells with respect to ionic composition, concentration, os­motic pressure, and so on.

This demand appreciably restricts the selection of immiscible liquids usable for CCD separations of cells, especially in comparison with the range of choices available when countercur­rent distribution is employed for molecular separa­tions.

Greatest success has been ob­tained with phases consisting of polyethylene glycol and aqueous solutions of dextran (polyglucose in which most glycosidic linkages are 1→6). The tech­nique has been especially fruitful in separations of dif­ferent microorganisms and different blood cells.

7. Electrophoresis:

Electrophoresis is one of the most popular methods used for separating different molecular species, espe­cially proteins. However, electropho­resis can be used to separate whole cells. Cell separa­tions using this technique are based on the fact that the plasma membranes of cells contain ionized groups (e.g., proteins, sialic acid, and short carbohydrate chains) that impart a net electrostatic charge to the cell surface.

Different types of cells possess different net charges so that when they are placed in the appro­priate conductive medium and subjected to an electri­cal current, they will migrate through the medium at different rates. Hence, they become separated into subpopulations that can be collected for further study.

Various chemical substances may be applied to the cells to selectively alter their normal surface charge distribution and assist in their electrophoretic separa­tion. Electrophoresis has been applied successfully to separations of microorganisms, blood cells, ascites tu­mor cells, HeLa cells, and other cells in culture.

8. Fluorescence-Activated Cell Sorting:

Fluorescent dyes such as fluorescein can react with and bind to the surfaces of cells; the type and quantity of dye bound vary for different kinds of cells. This dif­ferential property has been used for years to visually distinguish different types of cells in a mixed popula­tion and very recently has been employed in an ele­gant instrument that physically separates the cells.

The instrument, known as a fluorescence-activated (or “multi parameter”) cell sorter and depicted dia- grammatically in Figure 12-17, has a complex history, but its development may be credited to the combined contributions of M. J. Fulwyler, L. A. Herzenberg, R. G. Sweet, W. A. Bonner, and H. R. Hulett. The fluorescein-treated suspension of cells is mixed with electrolyte solution (“sheath fluid”) and forced down­ward through a tiny nozzle vibrating at 40,000 cycles per second.

The vibrations of the nozzle break the emerging stream into uniform droplets approximately equal in number to the frequency of vibration (i.e., 40,000 droplets per second). The population density of the original cell suspension and the flow rate are adjusted so that each droplet contains no more than one cell (indeed, most droplets contain no cells).

Just prior to droplet formation, the stream is illumi­nated with an argon-ion laser beam that excites the fluorescent material in the cell surfaces. Two detec­tors respectively measure the amount of fluorescent light arid the volume of the cell and trigger an electri­cal pulse that charges each cell-containing droplet as it is formed.

The amount and sign of the electrostatic charge borne by the droplet depend on the size of the entrained cell and the number of fluorescein mole­cules bound to its surface. These charge parameters can be selected by the operator and effectively divide the droplets into three classes: positively charged, negatively charged, and uncharged.

The droplets then pass between two electrostatic plates; the charged droplets are appropriately deflected as they pass through the field between these plates and the un­charged droplets continue on their original course. Fi­nally, the three droplet streams are collected in reser­voirs.

The left and right streams contain different population of cells and the undeflected center stream consists primarily of empty droplets, unwanted cells, and debris. The fluorescence-activated cell sorter can separate about 5000 cells per second.