NO875090L - CELL Separation Reagents. - Google Patents

Description

Technical area

The present invention relates to a new material for the separation and purification of biological cells and subcellular components. In particular, the invention relates to a new material for isolating specific cell types or specific subcellular components based on their wear density (buoyant density). Another aspect of the invention relates to a new method and means for use in the isolation of specific cell types and specific subcellular components which provide improved purity of the recovered cells and subcellular components.

Related application

The present application is a continuation-in-part of

US Patent Application 849,819 filed April 9, 1986.

Background for the invention

Separation of components of biological fluids and

tissue is often required for clinical, diagnostic procedures, scientific research and occasionally treatment of patients. In the clinical, diagnostic area, there is, for example, a need for materials and methods that enable rapid isolation of purified lymphocytes for tissue typing procedures, immunological function tests and various other procedures. Basic research also requires purified lymphocytes as well as other cell types from blood. In addition, studies of cultured cells and subcellular components such as plasmids, DNA, chromosomes, mitochondria and other subcellular components also require highly purified preparations.

Separation and purification can be effected in several ways.

However, since the isolated cells are often used in procedures that require viable cells, it is important that the function of the thus isolated cells is unimpaired. In order to ensure viability of the cells and unimpaired biological function of cells and subcellular components, it is important to avoid the introduction of possible interfering substances during the course of the separation procedure. For example, isolated lymphocytes used in histocompatibility tests are stimulated with different mitogens. The medium used must not itself be a mitogen, as this will affect the accuracy of the measurements of DNA synthesis.

Density gradient centrifugation is a technique used for the separation of biological cells and subcellular components. It is highly desirable that the material chosen for forming the gradient has certain characteristics that will confer compatibility with sensitive, biological materials. Gradient materials that have been used in the art include sucrose, dextran, bovine serum albumin (BSA), "Ficoll", iodinated, low molecular weight compounds such as metrizamide and heavy salts such as cesium chloride. Most of these materials, however, have undesirable characteristics that can potentially impair the biochemical functions of the desired, isolated fractions. For example, some currently available blood separation media contain erythrocyte aggregation polymers which can reduce the mitogen responsiveness of isolated lymphocyte preparations (J. Immunol. Meth. 38:43-51, 1980). These materials can also form solutions with an undesirably high osmolality or viscosity. Cell aggregation is often caused by BSA at physiological pH (7.4), and it is undesirable to use a reduced pH (5.1) because this causes other problems such as cell swelling (which affects cell density) and possible impairment of cell function. It is also expensive for use in separations on a large scale. "Ficoll" can similarly cause cellular aggregation which can only be remedied by the use of a dispersant, or undesired by lowering the pH. "Ficoll" is also highly viscous, which makes it difficult to develop a linear, isoosmotic gradient with this. It is also difficult to separate cells with very similar floating densities

some of these materials, even when using discontinuous gradients.

A density gradient material that has been used with some success for cell separation is colloidal silica. Colloidal silica is an aqueous suspension of colloidal particles formed by the polymerization of monosilicic acid from SiC>2 dissolved in water. Individual particles average 130-140 Å in size and generally range from 30 to 220 Å. The colloidal suspension is most stable for storage at pH 8-10 at which the colloidal particles have a net negative charge. However, this is not entirely satisfactory. Colloidal silica solutions precipitate irreversibly upon freezing and form gels in the presence of proteins under certain ionic conditions such as above 0.1 M NaCl at pH 5-7. Unmodified silica gels also exhibit toxicity against numerous cell types including macrophages and red blood cells. Countless attempts have been made to reduce this toxicity and increase the stability of the colloid in salt solutions and protein at physiological pH by coating the colloidal silica with a polymer. Polymers such as dextran (Exp. Cell Res., 50: 355-368 (1968)), polyvinyl alcohol (PVA) (J. Coll. and Interface Sci., 51: 388-393

(1974) , polyethylene glycol (PEG) (Exp. Cell Res., 57: 338-350

(1969), Arch. Biochem. Biophys., 168: 289-301 (1975)), dextran sulfate, methylcellulose, carboxymethylcellulose

(Exp. Cell Res., 50: 355-368, (1968)), polyvinylpyrrolidone (PVP) (Exp. Cell Res., 46: 621-623 (1966); Exp. Cell Res., 50: 355-368 ( 1968); J. Cell Biol., 55: 579-585 (1972);

Exp. Cell Res., 110: 449-457 (1977)) and a mixture of PEG, BSA and "Ficoll" (Arch. Biochem. Biophys., 168: 289-301

(1975) ) has been used as a coating for silica sols like this

as Ludox<®> (registered trademark of DuPont).

Exclusive coating of the silica particles with polymer also presents problems. The coating procedure requires the use of an excess of free polymer in the solution. The excess polymer increases the osmolality and viscosity of the solution. It is also difficult to remove the polymer from the purified biological material. While the morphological characteristics of cells and organelles purified with polymer-coated colloidal silica are generally acceptable, the biochemical characteristics are often impaired. For example, lymphocytes isolated with mixtures of colloidal silica and PVP have a reduced incorporation of radioactive thymidine compared to cells in control medium. Exp. Cell Res., 50: 353 (1968).

Another way to reduce the toxicity of colloidal silica has been to chemically modify the surface of the silica particles. An example of a chemically modified colloidal silica is Ludox<®>AM (registered trademark of DuPont) in which aluminum is incorporated into the colloidal silica. J. Colloid and Interface Science, 55:25 (1975). This preparation is reported to be stable over a wide pH range, however, it is not suitable for cell separation unless it is first thoroughly dialyzed and/or treated with carbon to make it non-toxic to lymphoid cells and useful for separating lymphocytes- subpopulations. J. Immunological Methods, 28:277 (1979). Ludox<®>AM contributes only to a minor extent to the osmolarity, and it is therefore possible to construct isoosmotic gradients using this compound. However, Ludox<®>AM has several disadvantages. The gradient material must be stored under sterile conditions as it will support the growth of various microorganisms. Antibiotics and antifungal agents must be added to the gradient material to inhibit the growth of contaminating microorganisms that may be found in various lymphoid tissues. In order to completely separate subpopulations of lymphoid cells, it is furthermore necessary to use discontinuous gradients. J. Immunological Methods, 28:277-292 (1979). Limitations associated with discontinuous density separation (Ann. Rev. Biophys. Bioeng. 1:93-130, 1972; Int. Rev. Exp. Path 14:91-204, 1975; Automated Cell Identification and Cell Sorting", Academic Press, p. 21 -96, 1970) may contribute to lower lymphocyte recovery which may result in altered lymphocyte ratios (Scand. J. Immunol. 3:61, 1974; Clin. Immunol. Immunopath. 3:584-597, 1975). Furthermore, discontinuous gradients are prepared from individual solutions of varying densities. It is extremely time-consuming to give the different solutions the correct density. Development of density gradients by centrifugation also requires speeds of 20,000 to 30,000 rpm.

Another chemically modified colloidal silica commercially available is Percoll<®> (registered trademark of Pharmacia) which is a silica particle to which a layer of PVP is hydrogen bonded Anal.Biochem., 88: 271-282

(1978). Percoll<®> has been widely used for the separation of blood cell components as well as subcellular organelles from a myriad of sources. As supplied from the manufacturer, Percoll<®> has

a density of 1.130 - 0.005 g/cm<3>, a pH of 9.0 - 0.5 and an osmolality of < 25 mOsM/kg. Percoll<®> is made isoosmotic by adding physiological saline and adjusting the pH

to 7.0-7.4 by adding acid or base. The density must also be carefully adjusted. If cells have a floating density greater than 1.11-1.12 g/cm , the Percoll<®> must be concentrated. The usual technique for using Percoll<®> is either to vaporize the gradient by layering or by using a gradient former, or by centrifugation (which generally requires 20,000 to 30,000 rpm) before adding the sample. Alternatively, cell preparations can be mixed directly with diluted iso-osmotic Percoll<®> before centrifugation.

Several factors limit the ease, performance and utility of Percoll<®>as a blood cell separation medium. Percoll<®>has a strong absorbance in the ultraviolet region ~~~~ due to PVP. This is a significant disadvantage when density gradients are analyzed with respect to nucleic acids and proteins by spectrophotometric methods. PVP also provides

a high background by the Lowry method for protein determination.

Cell Separation: Methods and Selected Applications, vol. I, 115, 134 (1982). Percoll<®> is somewhat stable at physiological pH and ionic strength, but is not stable to autoclaving after it has been made isoosmotic (by addition of salt, acid and base), and in dilute solutions it tends to aggregate. The latter problem is due to dissociation of PVP from the silica surface and can be prevented by adding low concentrations of free PVP. As indicated above, free PVP has a negative effect on at least some cell functions. It is also difficult to separate cells that only have small differences in fluid density

with Percoll<®>.

It has now been found that a new colloidal silica preparation, which is useful for the separation of biological materials, can be prepared using a new chemical modification technique. The material is produced by reacting an organosilane under aqueous conditions with non-porous, colloidal silica at an elevated temperature and an alkaline pH, forming a covalent bond between the silica particle and the organosilane. The material can be used for the isolation of specific cell types or specific subcellular components based on their floating density. The material provides improved purity of the recovered cells and subcellular components and reduced sample processing time. A material combining different sizes of reagent-modified silica particles can also be prepared. This combination material has the further advantage of enabling the separation of cells and cellular components with only tiny differences in fluid density. This type of separation has so far been impossible to achieve. A further material that can be prepared consists of reagent-modified silica particles to which purified antibody has been coupled. This antibody-modified material carries the advantage that cells and cell components can be separated based on their antigenic determinants as well as their complement of any other cellular component, such as peptide-harmonic receptors to which antibodies can be formed.

The material has several desirable characteristics. The reagent modification reduces the toxicity of the colloidal silica and eliminates aggregation of the colloidal silica particles in the presence of physiological salt and protein. The material is compatible with biological material and is suitable for density separation of both cellular and subcellular biological particles. It enables the use of either preformed gradients or in situ density gradient formation and rapid cell separation using centrifugation at relatively low speed. The material is of physiological ionic strength and pH, is isoosmolar, has low viscosity and a density in the range 1.0 to 1.4 g/cm 3. It is stable over a wide range of temperature and pH values. As shown by its stability, the material is completely stable to autoclaving under physiological conditions. Furthermore, it is soluble or dispersible in aqueous solutions, it is easily removed from biological samples and has a low price.

The methods of using this material also bring several advantages. Traditional blood separation procedures require careful blood stratification techniques. The technical skill required to perform blood separation with this material is much less than with other techniques. For example, no layering is necessary with the in situ technique. The material to be separated is simply mixed with the reagent-modified colloidal silica and centrifuged.

To separate mononuclear cells from whole blood, the inherent density difference that exists between mononuclear cells and other cellular elements present in peripheral blood is used. A continuous density gradient suitable for mononuclear cell separation is formed after centrifugation by the sedimentation of the reagent-modified, colloidal silica particles.

Rapid gradient formation is due to the high degree of sedimentation of the particles used (up to 200 Å in diameter) for this type of separation. Cell separation is effected by the movement of the peripheral blood cells during the centrifugation to their respective floating densities within the continuous density gradient. The separation technique is inexpensive and requires little, if any, special equipment other than a clinical centrifuge.

Summary of the invention

According to the invention, a method has been found for the production and use of non-porous, colloidal silica particles coated with a reagent which imparts a non-toxic, non-ionic, hydrophilic surface to the silica particles. The resulting reagent-modified colloidal silica can be used for the separation of cells and subcellular components from all types of body fluids such as blood, bone marrow, spinal and pleural fluids, and semen, as well as dispersed tissue samples, cultured cells and other sources of biological cells and their components. According to one embodiment of the invention, a new technique for the production of a reagent-modified, colloidal silica is provided which includes steps for mixing colloidal silica with an organosilane reagent at elevated temperatures and alkaline pH in aqueous media so that the organosilane is coupled to the colloidal silica. The resulting reagent-modified, colloidal silica is freed of reaction by-products by active carbon adsorption and then deionized. The reagent-modified colloidal silica is then reheated to form additional covalent bonds between the reagent and the reagent-modified colloidal silica. The osmotic pressure and pH are adjusted to be compatible with biological materials. The reagent-modified colloidal silica is then diluted with a buffer to the density suitable for the biological material to be separated. The organosilane, covalently bound to the surface of the silica, reduces the toxicity of the colloidal silica and prevents coagulation of the colloidal silica particles in the presence of protein at physiological pH and salt concentration.

According to another embodiment of the present invention, a new technique is provided for the production of antibody-coupled, reagent-modified, colloidal silica using a thiol reagent which includes the step of mixing the reagent-modified, colloidal silica with a thiol reagent in aqueous media so that thiol groups are incorporated on the surface of the reagent-modified colloidal silica. The thiol groups are further reacted with a succinimide to provide a bond to which protein A is then coupled. An antibody is then coupled to protein A, forming antibody-modified colloidal silica. Use of antibody-modified colloidal silica enables the separation from a cell mixture such as blood of cell components with antigenic determinants to which the antibody can bind.

According to another embodiment of the present invention, a new technique for the production of antibody-modified colloidal silica using carbodiimide is provided which includes the step of reacting reagent-modified colloidal silica with a carbodiimide in aqueous media to form a binding arm to which protein A is then linked via an amide bond. The antibody is then linked to protein A, forming antibody-modified, colloidal silica.

According to another embodiment of the present invention, there is provided a novel technique for separating cellular components from mixtures of biological cells such as whole blood or other biological fluids, which includes the step of contacting a diluted cell mixture with a dilute reagent-modified colloidal silica material having a final density that exceeds the flow density of the cell components of the cell mixture to be separated, by layering the cell mixture on top of the material, centrifuging the cell mixture and material in an oscillating bucket rotor to effect cell separation, and collecting the migrating cell components to a characteristic flow density for a specific cell type where they can form a band at this density. For example, lymphocytes can be separated from other cell types in a blood sample. Typically, lymphocytes have a floating density of 1.060-1.074 g/cm 3 and will form a band of cells at the density interface. Other cell types will penetrate into the gradient material. The cell band can be removed, and the cells washed, pelleted and resuspended in a medium suitable for growth or metabolic studies,

or other experimentation.

According to another embodiment of the present invention, a new technique for separation is provided

of cell components from mixtures of biological cells

such as whole blood or other biological fluids, and which involves mixing a sample of a cell mixture with the

diluted reagent-modified colloidal silica material, and centrifugation in a fixed-angle rotor or a swinging bucket rotor. A continuous density gradient is formed after centrifugation. The component cells migrate to characteristic flow densities within the gradient and form bands of cells at densities characteristic of specific cell types. For example, mononuclear cells can be separated from other cells in a blood sample using this in situ separation technique. The mononuclear cells form a band of cells just below the meniscus. The mononuclear band, as well as other bands of cells, can each be collected, washed, pelleted and resuspended in a medium suitable for growth or metabolic studies, or other experimentation.

According to another embodiment of the present invention, a new technique for separating biological cells or subcellular components with only small differences in fluid density is provided. The technique includes the steps of mixing reagent-modified colloidal silica particles of different sizes with a mixture of cells such as a blood sample, buffy coat or bone marrow, mixing the cell mixture and the reagent-modified colloidal silica material, centrifuging the mixture to develop a density gradient and then collecting the individual bands of cells that form at specific, characteristic flow densities. The cells thus collected are then washed, pelletized and resuspended in a medium suitable for growth or metabolic studies, or other experimentation. For example, B and T lymphocytes can be separated from other cells in a blood sample. B cells have a lower density range than T cells. The T and B cells can be separated by choosing a gradient shape so that the B cell band is at a sharp break in the gradient, and the T cell band separately at a higher density. Inclusion of small reagent-modified colloidal silica particles (those prepared from colloidal silica particles of i 80 Å) in the gradient widens or narrows the lower density region, while inclusion of larger reagent-modified colloidal silica particles (those prepared from colloidal silica particles of f; 200 Å) compresses the higher density region of the gradient. By selecting the appropriate initial density and expanding selected regions of the density gradient with reagent-modified colloidal silica particles prepared from the appropriate particle size, B and T cells can be separated. This solution can also be used to separate NK cells from B and T cells. Separation of B, T and NK cells can be followed by staining the cells with fluorescent monoclonal antibodies for B, T and NK cells.

According to another embodiment of the present invention, a new technique for separating biological cells or cellular components with different antigenic determinants is provided. Colloidal silica with a large particle size (approx. 600 Å) is used for the production of the antibody-modified, colloidal silica particles. The antibody-modified colloidal silica is mixed with a cell mixture. The mixture is incubated for a sufficient time to allow the antibody-modified colloidal silica to bind to the antigenic sites of the component cells. The mixture is then centrifuged through a cell separation material. The antibody-modified colloidal silica with attached cells can be recovered from the bottom of the gradient. Attached cells can be removed from the antibody-modified colloidal silica prepared using a thiol reagent by treatment with a sulfhydryl reducing agent. If the antibody-modified colloidal silica had been prepared by coupling protein A directly to the surface of the reagent-modified colloidal silica via an amide bond, the attached cells can be removed by treatment with ethanolamine. After removal from the antibody-modified colloidal silica, the cells can be washed and resuspended in a medium suitable for growth or metabolic studies, or other experimentation.

Brief description of the drawings

Figures la, b, c, d are graphical representations of the shape of the gradients obtained when distinct populations of dilute reagent-modified colloidal silica are mixed. Figure 2 is a schematic representation of a reagent-modified colloidal silica in which the bound organosilane reagent groups are represented by "R".

Detailed description of the preferred embodiments

Reagent-modified colloidal silica materials have been described which are suitable for the separation of biological cells and cellular components. Use of "distinct populations" as defined hereinafter, of the materials will decrease the time required to obtain isolated cells and cellular components, as well as increase the purity of the resulting isolates. The use of mixtures of distinct populations of the reagent-modified, colloidal silica materials will enable the separation of cell types with very similar floating densities. The use of distinct populations of reagent-modified colloidal silica that have been further modified by coupling to purified monoclonal antibodies will enable the separation of cells based on their complement of antigenic determinants.

The size of the non-porous, colloidal silica particles used in the coupling reaction varies from approx. 30 Å to approx. 600 Å. The starting material for each individual material comprises a majority of colloidal silica particles which all have approx. same diameter. However, this "population" of colloidal silica particles has a size range that can be represented by a distribution curve such as that which can be derived using particle size dilution methods known in the art. Most of the particles in the population will be of the size at the top of the distribution curve, but the entire curve represents the population. The sizes specified for the populations are the sizes specified in the manufacturer's specifications. The actual sizes of the populations as received from the manufacturer may vary from sample to sample. To remedy the size variations, each population is indicated as "approx. x Å" where x is the particle size indication given in the manufacturer's catalogs and product specifications. Colloidal silica particles useful according to the invention include the material sold under the name "Nyacol"

by Nyacol Products, Inc. (Worchester, Mass.), which material is an aqueous suspension of colloidal particles formed by the polymerization of monosilicic acid from SiC3 dissolved in water. Once the particles are covalently bound to the modifier, the particles become a collection of reagent-modified, colloidal silica particles each having a flow density within a defined range. The resulting flow density range and the most preferred flow density of the particle populations after coupling with the modifying reagent are shown in Table 1.

A population of reagent-modified,

colloidal silica particles that can be distinguished from another population by the diameter of the particles used in the coupling reaction and its bulk density after coupling with the modifying reagent are designated as a "distinct population" even though the characteristics of some of the particles in two distinct populations may be similar. One or more distinct populations can be mixed to form a cell separation material mixture with specific separation properties.

The reagent covalently bound to the colloidal silica particles can be any organosilane reagent. Organosilanes are the only compounds that can form stable, covalent siloxane bonds with the silanol (Si-O-Si) groups on the surface of the colloidal silica. The functional group in the organosilane must be non-ionic and hydrophilic. Ionic functional groups will be undesirable as the ionic charge will be sensitive to changes in pH and salt, and thus result in gelation. Hydrophobic functional groups make the colloidal silica sensitive to gelation at neutral pH and physiological salt concentration. Hydrophilic functional groups bind H2O to the colloid surface. The bound H2O serves as a protective shell that is independent of changes in pH, ionic strength and protein, and thus protects the colloid against gelation.

The organosilane imparts a non-toxic, non-ionic, hydrophilic surface to the silica particles. Non-toxic means that the material does not affect the integrity or function of the biological material to be separated. Examples of organosilanes that can be used are listed in Table 2. The preferred organosilane is gamma-glycidoxypropyltrimethoxysilane.

The reagent-modified colloidal silica can be further modified by coupling with an antibody, preferably a monoclonal antibody and preferably a purified monoclonal antibody. First, protein A is linked to the surface of the reagent-modified, colloidal silica or via an amide bond. The antibody is then bound to protein A, forming antibody-modified, colloidal silica. Colloidal silica of any size can be used, but

however, the preferred size is approx. 600 Å colloidal silica. If protein A is coupled directly to the reagent

modified colloidal silica via an amide bond, only the reagent-modified colloidal silica prepared using the organosilane listed in Table 3 can be used.

The material according to the invention can generally be prepared as follows: the colloidal silica is mixed with an organosilane reagent which will stabilize the colloidal state. Preferably, the colloidal silica is heated to approx. 75°C

before and during addition of the reagent. The aqueous colloidal silica solution may be of any suitable concentration so that when the desired amount of reagent is mixed with the colloidal silica, the silica always remains in excess relative to the reagent. This can be carried out by controlled reagent addition to the colloidal silica. The reagent addition rate is preferably from 0.5 to 2.0 ml of reagent per liter of colloidal silica per minute, and preferably 0.7 ml of reagent per liter of colloidal silica per minute.

The amount of reagent used depends on the total number of surface silanols present in the particular colloidal silica to be chemically modified. The total number of surface silanol groups within a particular colloidal silica can be calculated as follows: Total surface silanols = volume (ml) x density (g/ml) x % silica solids (manufacturer's data) x surface area, nm 2 /g (manufacturer's data) x 4 .5 silanols/nm 2 (value from The Chemistry of Silica: Solubility, Polymerization, Colloid and Surface Properties, and Biochemistry, pp. 633-636, 1979). A reagent molecule1 can react and form covalent bonds with one, two or three surface silanol molecules. An estimate of the volume of reagent that must be used to chemically modify a colloidal silica can be calculated as follows: Reagent volume = (total surface silanols/Avogadro's number)/Z) x (reagent molecular weight) x (reagent density) where Z is preferably 1, 2 or 3 and is preferably z = 3. The colloidal silica is "fully modified" when Z = 3. To form "partially modified", colloidal silica for use in the production of antibody-modified colloidal silica prepared with a thiol reagent, approx. 50-75% of the amount of reagent calculated to form fully modified, colloidal silica.

After the reagent addition is complete, the reaction mixture is heated to from 70 to 80°C with stirring for 1 hour to 3 hours. Reaction by-products are preferably removed by passing the reaction mixture through activated carbon and by ionizing the reaction mixture. Ionization of the reagent-modified colloidal silica is preferably carried out by passing the reaction mixture through a cation-exchange resin (hydrogen form) and through an anion-exchange resin (hydroxide form). Any reagent-modified colloidal silica remaining in either the carbon or resin columns is preferably recovered by passing water through the respective columns. Further covalent bonding between the reagent and the silica surface can be facilitated by further heating the reagent-modified colloidal silica under vigorous stirring. This step is preferably carried out at from 90 to 100°C for from 3 hours to 6 hours. It is carried out even more advantageously at approx. 95°C for 3 hours.

The reagent-modified colloidal silica preparation is then adjusted to physiological osmotic pressure and pH.

The osmotic pressure is preferably adjusted during use

of solid sodium chloride and potassium chloride. The pH is preferably adjusted with HEPES buffer in the hydrogen and sodium forms. The density of the resulting solution is then adjusted prior to use with buffer (20 mM, pH 7.4) to form dilute reagent-modified colloidal silica with a final density spanning the density range of the material to be separated.

The buffer in which the reagent-modified colloidal silica is diluted can be any buffer that is "compatible" with the biological material to be separated and purified. To be "compatible", the carrier liquid must be of physiological ionic strength and pH, and not affect the structure or function of the material to be separated. Examples of buffers that can be used include 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (HEPES), N-N-bis-2-hydroxyethyl-2-aminoethanesulfonic acid (BES), bis-2-hydroxyethylimino-TRIS-hydroxymethylmethane-2- bis-2-hydroxyethylamino-2-hydroxymethyl-1,3-propanediol (BIS-TRIS), 1,3-bis-TRIS-hydroxymethylmethylaminopropane (BIS-TRIS-PROPAN), N-2-hyroxyethylpiperazine-N-3-propanesulfonic acid ( EPPS), N-2-hydroxyethylpiperazine-N-2-hydroxypropane-sulfonic acid (HEPPSO), 3-N-morpholinopropanesulfonic acid (MOPS), piperazine-N-N-bis-2-ethanesulfonic acid (PIPES), piperazine-N-N-bis- 2-hydroxypropanesulfonic acid (POPSO), 3-N-TRIS-hydroxy-methylmethylamino-2-hydroxypropanesulfonic acid (TAPSO), N-TRIS-hydroxymethylmethyl-2-aminoethanesulfonic acid (TES).

The reagent-modified, colloidal silica or

the diluted reagent-modified colloidal material can be sterilized using sterilization methods known in the art. The materials are stable against autoclaving, but a preferred method of sterilization is however

time to pass this through a 0.2 micrometer filter. The sterilized product can be used under aseptic conditions for cell separation, for example to separate cells for subsequent growth in culture or for use in tests that require sterility of the starting materials.

The efficiency of in situ separation of component cells from a mixture of cells by continuous density gradient centrifugation is dependent on the density of the resulting mixture of cells and the dilute reagent-modified colloidal silica. When separating mononuclear cells from whole blood, for example, the density of the mixture can be calculated using the following formula:

, ncn ,3 (1.024 q/cm3 (X ml) + (Y1 q/cm3) (Y ml)

in. 067 g/cm - — —

(X ml + Y ml)

where 1.067 g/cm 3 is the resulting density of an optimal mixture of whole blood and dilute reagent-modified colloidal silica, and where 1.024 g/cm 3 is the effective density of whole blood (the density of blood minus the density contribution of red blood cells in the blood) and where X = the volume of blood to be separated and where Y^"= the density of dilute reagent-modified colloidal silica and where Y = the volume of dilute reagent-modified colloidal silica.

Any combination of whole blood and diluted reagent-modified colloidal silica can be used. The only limitation is that the resulting density of the mixture must be approx. 1.067 - 0.001 g/cm<3>. Erythrocyte density is pH dependent. The density of erythrocytes is greatest at pH 7.40 - 0.03 in dilute reagent-modified colloidal silica. Consequently, the most efficient separation of erythrocytes from mononuclear cells is achieved at this pH.

The structure of the reagent-modified colloidal silica is as follows: and R is the reagent, chained to the colloidal silica particle. When the reagent is gamma-glycidoxypropyltrimethoxysilane, the structure of the reaction product is as follows:

in which two X groups form either two -OH groups or preferably one -OH and one further bond, or preferably two further bonds with the silica surface. No cross-linking or polymerization between R groups takes place. The schematic representation of the final, colloidal silica particle is shown in figure 2, in which the bound reagent groups are represented by "R".

In use, the diluted reagent-modified colloidal silica is suitably contacted with a sample containing cells or cellular components to be separated. The choice of which particle size material to use is based on the range of flow densities of the cells or cellular components to be separated, and the centrifugal force and duration that must be used in the separation. Use of a larger particle size is preferred because it reduces the time and g-force required to develop a density gradient compared to smaller particle sizes. Large particle sizes have a greater degree of sedimentation than small particle sizes as the degree of sedimentation is proportional to the square of the particle diameter. Lower centrifugation times and g-forces reduce the damage to the cells or the cellular components arising from the centrifugation.

Cell separation in the reagent-modified, colloidal silica is carried out by movement of the cells or the cellular components during centrifugation against their flow densities within the density gradient. The cell bands that form at the respective flow densities can then be individually transferred to centrifuge tubes with a pipette or preferably with a cell removal tube. The procedures for the manufacture and use of indicated cell removal tubes are described in US patent application 849,698 filed on April 9, 1986, the description of which is hereby incorporated by reference. The cell separation reagent can be removed by washing. The cells can then be pelleted and resuspended in buffer or growth medium.

The invention will be further understood by considering the following examples. However, it should be understood that these examples are given for illustrative reasons and are not limiting, and that many changes can be made in e.g. quantities or choice of material without deviating from the scope of the invention as stated in the requirements.

Example 1

Production of reagent-modified, colloidal silica

Three liters of 70 Å colloidal silica (50 wt% silica)

(supplied from Nyacol Products, Inc., Worchester, Mass.) was heated to 75°C. 210 ml of gamma-glycidoxypropyltrimethoxysilane (supplied from Petrarch Systems, Inc.) was added at 0.2 ml per minute and with continuous stirring. The reaction mixture was heated to 75°C with stirring, and for an additional 60 minutes after the last addition of reagent. The reaction mixture was then passed through an activated carbon column (diameter 8 cm, height 40 cm) to remove

any reaction byproduct. The reagent-modified colloidal silica was then deionized by passing it through first a cation exchange resin column (hydrogen form) (8 cm x 40 cm) and then an anion exchange resin column (hydroxide form) (8 cm x 40 cm) with 3.8 liters per hour. Three liters of water were then passed through the carbon and ion exchange resins to remove any remaining reagent-modified colloidal silica.

To facilitate the formation of additional covalent bonds between the reagent and the silica surface, the reagent-modified colloidal silica was then heated to 95°C with vigorous stirring for 3 hours. 8 to 9 g/l solid sodium chloride and 0.3 to 0.4 g/l potassium chloride and 20 mM HEPES buffer in hydrogen (2.51 g/1) and sodium (2.47 g/1) form were then added to the reagent-modified colloidal silica preparation to adjust the osmotic pressure and pH to physiological levels. The resulting solution was then diluted to a final density of

1.074-1.099 g/cm 3 when mixed with a HEPES-buffered solution of identical osmotic pressure and pH. (See Table 4 in which the properties of two of the dilute reagent-modified colloidal silica materials prepared as indicated above,

have been compared with two commercially available cell separation reagents, Percoll<®>(registered trademark of Pharmacia) and Ludox<®>HS-30 (registered trademark of DuPont)).

Example 2

Separation of mononuclear cells from blood

3 ml of diluted reagent-modified colloidal silica prepared from colloidal silica with a particle diameter of approx. 70 Å and with a density after dilution of approx. 1.074 g/cm 3 , was placed in a 15 ml conical centrifuge tube. 3 ml of blood diluted 1:1 with phosphate-buffered saline (sodium chloride, 8 g/l; potassium chloride, 0.2 g/l; disodium hydrogen phosphate, 1.15 g/l; potassium dihydrogen phosphate, 0.2 g/l, pH 7.4) was layered on the reagent-modified colloidal silica. The tube was centrifuged at 900 x g for 20 minutes at room temperature in a swinging bucket rotor. The mononuclear cell band collected at the meniscus was removed with a cell removal tube or pipette. The mononuclear cells were washed by adding 10 ml of Hank's balanced salt solution (potassium chloride, 0.4 g/l; potassium dihydrogen phosphate, 60 mg/l; sodium chloride, 8.0 g/l; sodium bicarbonate,

0.35 g/l; disodium hydrogen phosphate, 48 mg/l; glucose, 1 g/l; phenol red 10 mg/l, pH 7.4) and centrifugation at 300 x g for 10 minutes at room temperature. The mean percentage recovery of lymphocytes was 6 2.3, the mean viability of the cells was more than 95%, the mean percentage of lymphocytes was 77.6%. (See Table 4 for a comparison with recoveries obtained with the selected commercially available cell separation reagents).

Example 3

Aseptic Separation of Mononuclear Cells from a Large Volume of Anticoagulated Whole Blood 4 to 15 mL of EDTA anticoagulated whole blood was added aseptically to a tube containing 6.7 mL of a filter-sterilized, dilute reagent-modified, colloidal silica with a flow density after dilution of approx. 1.089 to 1.099 g/cm 3. The blood and colloidal silica were mixed gently by inverting the tube several times. The mononuclear cells were separated by centrifugation at 2000 x g for 10 minutes at room temperature in a fixed angle rotor. The mononuclear cell band that formed just below the meniscus was removed aseptically with a sterile cell removal tube.

The recovered cells were washed by adding 10 ml of sterile Hank's balanced salt solution, after which the tube was inverted. The tube was then centrifuged at 300 x g for 10 minutes at room temperature. For maximum lymphocyte recovery, 0.1% BSA was included in the wash buffer. The mean recovery of lymphocytes was 84.8%, the mean viability was 98.0%, and the mean purity of the lymphocyte population was 76.4%. (See Table 5 for a comparison of recoveries obtained with selected commercially available cell separation reagents. See also Table 6 for an analysis of the percentage of blood component cell types recovered with product used according to this example).

Example 4

Separation of mononuclear cells from a small volume of anti-coagulated whole blood

1 to 3 ml of EDTA anti-coagulated whole blood was added to a tube containing 6.7 ml of diluted reagent-modified colloidal silica with a density after dilution of approx. 1.089 to 1.099 g/cm<3>. 1 ml of Hank's buffered saline, pH 7.4, was added. The blood and colloidal silica were carefully mixed by inverting the tube several times. The mono-

nucleated cells were separated by centrifugation at 2000 x g for 10 min at room temperature in a fixed-angle rotor. The mononuclear cell band that formed just below the meniscus was removed with a cell removal tube. The recovered cells were washed by adding 10 ml of Hank's balanced salt solution, and the tube was inverted. The tube then became

centrifuged at 300 x g for 10 min at room temperature. For maximum lymphocyte recovery, 0.1% BSA was included in the wash buffer.

Example 5

Aseptic separation of lymphocytes from a large volume of anticoagulated whole blood 4 to 15 ml of EDTA anticoagulated whole blood was added aseptically to a tube containing 6.7 ml of filter-sterilized, diluted, reagent-modified, colloidal silica with a density after dilution of about. 1.079 to 1.089 g/cm 3 . The blood and the diluted reagent-modified colloidal silica were gently mixed by inverting the tube several times. The mononuclear cells were separated by centrifugation at 2000 x g for 15 min at room temperature in a fixed angle rotor. The monocyte and platelet cell band that formed just below the meniscus was removed with a sterile pipette and a sterile cell removal tube. The lymphocyte band that formed below the previous band was removed with the cell removal tube. The recovered cell bands were each washed by adding 10 ml of sterile Hank's balanced salt solution and inverting the tube. The tube was then centrifuged at 300 x g for 10 minutes at room temperature. For maximum lymphocyte recovery, 0.1% BSA was included in the wash buffer.

Example 6

Separation of lymphocytes from a small volume of anti-coagulated whole blood 1 to 3 ml of EDTA anti-coagulated whole blood was added to a tube containing 6.7 ml of a dilute reagent-modified colloidal silica with a floating density after dilution of approx. 1.079 to 1.089 g/cm 3 . The blood and diluted reagent-modified colloidal silica were gently mixed by inverting the tube several times. The mononuclear cells were separated by centrifugation at 2000 x g for 15 min at room temperature in a fixed angle rotor. The cell band comprising monocytes and platelets that formed just below the meniscus was removed with a pipette and cell removal tube. The lymphocyte band that formed below the previous band was removed with the cell removal tube. The recovered bands of cells were each washed by adding 10 ml of Hank's balanced salt solution, and the tube was inverted. The tube was then centrifuged at 300 x g for 10 minutes at room temperature. For maximum lymphocyte recovery, 0.1% BSA was included in the wash buffer.

Example 7

Separation of B and T lymphocyte cells from whole blood using a clinical trial kit

A total of 6.7 ml of diluted reagent-modified colloidal silica comprising 1.7 ml of reagent-modified colloidal silica prepared from approx. 70 Å colloidal silica,

4.0 ml reagent-modified colloidal silica prepared from 130-200 Å colloidal silica and 1.0 ml reagent-modified colloidal silica prepared from 600 Å colloidal silica,

was mixed with 1 to 5.0 ml of whole blood and centrifuged at 2000 x g for 15 min in a fixed-angle rotor. The B lymphocytes formed a band at a sharp break in the gradient. The B lymphocytes formed a band separately above the T cell band. The B-cell band was removed with a pipette and a cell removal tube. The T cell band was then removed using the cell removal tube. The collected cell bands were each washed with 10 ml of Hank's balanced salt solution, pH 7.4, and were recovered by centrifugation at 300 x g for 10 min at room temperature.

Example 8

Separation of B lymphocytes before fusion with myeloma cells

7

1 to 6 ml of a mouse spleen cell suspension, 1 x 10 to 5 x 10 7 cells/ml in Hank's balanced salt solution, pH 7.4, was mixed with 6.7 ml of diluted reagent-modified colloidal silica and centrifuged at 2000 x g for 10 to 15 minutes in a fixed angle rotor. The B-lymphocytes are less dense than most of the other cells in the spleen and will form a band a few millimeters below the meniscus. The band of cells was collected using a cell removal tube. The cells were washed by addition of 10 ml of Hank's balanced salt solution, pH 7.4 and centrifugation at 300 x g for 10 min at room temperature. The separated cells can then be used directly for fusion with myeloma cells.

Example 9

Separation of cells using silica particles modified with monoclonal antibodies

100 ml of partially reagent-modified colloidal silica particles (prepared from colloidal silica particles of about 600 Å) were reacted under aqueous conditions with 0.5 to 1.0 ml of 3-mercaptopropyltrimethoxysilane to form thiol-modified colloidal silica which now contains thiol groups on the reagent-modified colloidal silica surface. The thiol-modified colloidal silica was reacted under aqueous conditions with 0.25 to 1.50 g of N-succinimidyl, 3-(2-pyridylthio)-proprionate (SPDP) which reacts with the thiols to form SPDP-modified, colloidal silica. 20 to 2000 mg of protein A was then added to the SPDP-modified colloidal silica. SPDP forms an amide bond with protein A and thus links it to the particles to form protein A-modified colloidal silica. 5 to 200 µg of monoclonal antibody (Mab) was then mixed with 0.1-1.0 ml of protein A-modified colloidal silica. Protein A binds Mab to form antibody-modified, colloidal silica. The antibody-modified colloidal silica was mixed with 1 to 5.0 ml of whole blood and incubated for 15 minutes to allow the antibody to bind to the antigen on the cells. The mixture was then separated by density gradient centrifugation on dilute reagent-modified colloidal silica. The cells to which antibody-modified, colloidal silica is bound, had changed in density and can thus be easily separated. The antibody-modified, colloidal silica with

bound cells were collected using a cell removal tube. The cells were removed from the antibody-modified colloidal silica by treatment with dithiothreitol or other sulfhydryl reducing agents such as glutathione or beta-mercaptoethanol. The cells were then washed with 10 ml of Hank's balanced salt solution, pH 7.4, and were collected by centrifugation at 300 x g for 10 min.

Example 10

Separation of cells using silica particles modified with monoclonal antibodies

Fully reagent-modified colloidal silica was prepared using epoxy organosilane or any of the organosilanes listed in Table 3. 100 mL of fully reagent-modified colloidal silica (prepared from colloidal silica particles of about 600 Å) was reacted with 0.5 to 5.0 g of 1,1-carbonyldiimidazole or 0.3 to 3.0 g of s.succinimide under aqueous conditions, forming carbodiimide-modified colloidal silica. The carbodiimide-modified colloidal silica was then reacted directly with 13 to 1300 mg of protein A in water to form protein A-modified colloidal silica. Purified monoclonal antibody (Mab), 5 µg to 200 µg, was then mixed with 0.1 to 1.0 ml of protein A-modified colloidal silica. Protein A binds Mab to form antibody-modified, colloidal silica. The antibody-modified colloidal silica was mixed with 1 to 6.0 ml of whole blood and incubated for 15 minutes to allow the antibody-modified colloidal silica to bind to antigen on the cells.

. The mixture was then separated by density gradient centrifugation through dilute reagent-modified colloidal silica. The cells to which the antibody-modified, colloidal silica was bound, had changed in density and could thus be easily separated. The antibody-modified colloidal silica with attached cells was collected using a cell removal tube. The cells were removed from the antibody-modified colloidal silica by treatment

with ethanolamine. The cells were then washed with 10 ml of Hank's balanced salt solution, pH 7.4, and were collected by centrifugation at 300 x g for 10 min.

Example 11

Separation of X- and Y-bearing sperm cells from semen samples

7

1 to 6 ml of a semen sample containing 1 x 10

to 2 x 10 7 sperm/nl, was mixed with 6.7 ml of diluted reagent-modified, colloidal silica which had a density after dilution of 1.185 g/cm 3 , a density which lies between the densities of X and Y sperm. The gradient material consisted of 1.7 ml of reagent-modified, colloidal silica prepared from colloidal silica of approx. 70 Å and 5.0 ml of reagent-modified, colloidal silica prepared from colloidal silica of approx. 200 Å. The mixture was centrifuged at 2000

x g for 10 to 15 minutes in a fixed angle rotor. The density of Y-sperm is less than X-sperm due to the lower DNA content per sperm. The Y sperm were removed using a pipette and cell removal tube. The X sperm were removed using a cell removal tube.

The separation was monitored by fluorescence staining

of the separated spermatozoa using acridine orange and was quantified using a Fluorescence Activated Cell Sorter (FACS) laser system. The greater DNA content of the X-sperm gives a greater fluorescent image compared to the Y-sperm. Biol. Reprod. 28: 312-321 (1983).

Example 12

Formation of gradient shapes using a combination of particle sizes

A total of 6.0 ml of a mixture of distinct populations of diluted reagent-modified colloidal silica, each with a density of 1.089 g/cm 3 after dilution, was mixed with 3.0 ml of whole blood and centrifuged for 10 minutes in a rotor with fixed angle with 2000 x g at room temperature. Distinct populations of reagent-modified colloidal silica were mixed as indicated in Table 7. Density marker beads (Pharmacia) were used to measure the density at which cells in the blood sample formed bands. The results for the combinations listed in Table 7 are shown in Figure 1.

Claims (22)

1. Cell separation material comprising: (a) a plurality of colloidal silica particles; and (b) an effective amount of a modifying reagent covalently bonded to said colloidal silica particles to form reagent-modified colloidal silica particles having a non-ionic hydrophilic surface. 2. Material according to claim 1, characterized in that the specified modifying reagent comprises an organosilane with a water-soluble functional group. 3. Material according to claim 2, characterized in that the specified organosilane is of the general formula: 4. Material according to claim 3, characterized in that "Y" is selected from the group consisting of OCH2 CHCH2 0, 02 CCH3 , N(CH2 CH2 OH)2 , C02 CH3 , NH(CH2 )2 NH(CH2 )2 C02 CH3 , NHCOCf^NHC(CH3)0, N(CH2 CH2 )2 0 and N(CH=CH2 )2 . 5. Material according to claim 3, characterized in that "X" is selected from the group consisting of H3 CO, Cl, H5 C2 0, H3 CC02 , H3 C and (H3 C)2 . 6. Material according to claim 2, characterized in that the specified organosilane is of the general formula: 7. Material according to claim 6, characterized in that "Y" is selected from the group consisting of OCH2 CHCH2 0,■02 CCH3 , N(CH2 CH2 OH)2 , C02 CH3 , NH(CH2 )2 NH(CH2 )2 C02 CH3 , NHCOCf^ NHC(CH3 > 0, N(CH2 CH2 )2 0 and N(CH=CH2 )2 . 8. Material according to claim 6, characterized in that "X" is selected from the group consisting of H3 C0, Cl, H5 C2 0, H3CC02, H3C and (H3 C)2 - 9. Material according to claim 2, characterized in that the organosilane comprises gamma-glycidoxypropyltrimethoxysilane. 10. Material according to claim 1, characterized in that each of specified colloidal silica particles has approximately the same size and wherein specified reagent-modified colloidal silica particles resulting therefrom comprise a distinct population. 11. Material according to claim 10, characterized in that the indicated size of the colloidal silica particles is between 30 Å and 600 Å in diameter. 12. Material according to claim 10, characterized in that one or more distinct populations of reagent-modified, colloidal silica particles are suspended in a buffer and mixed to form a cell separation material mixture with a desired final density. 13. Method for separating component cell types from a cell mixture, comprising steps for: (a) placing a cell mixture on top of a cell separation material comprising a plurality of colloidal silica particles covalently bound to a modifying reagent, forming reagent-modified colloidal silica particles with a nonionic, hydrophilic surface, which cell separation material is suspended in a buffer; and (b) centrifuging the cell sample through said cell separation material to form at least one component cell band with a characteristic floating density. 14. Method according to claim 13, characterized in that the specified modifying reagent comprises an organosilane with a water-soluble functional group. 15. Method for separating component cell types from a cell mixture comprising steps for: (a) adding a cell mixture to a tube containing a cell separation material comprising a plurality of colloidal silica particles covalently bound to a modifying reagent to form reagent-modified colloidal silica particles having a non-ionic hydrophilic surface, which cell separation material is suspended in a buffer; (b) mixing said cell mixture and said cell separation material by inversion; and (c) centrifuging said cell mixture and said cell separation material to develop a density gradient in which said component cells of said cell mixture form at least one band of cells having a characteristic floating density. 16. Method according to claim 15, characterized in that specified modification reagent is an organosilane. 17. Method according to claim 15, characterized in that at least two cell bands which have a characteristic flow density are formed, one of the indicated two cell bands having a higher density than the other of the indicated two cell bands. 18. Method for separating component cell types from a cell mixture according to claim 15, characterized in that at least two distinct populations of indicated cell separation material are mixed, where each of the indicated populations comprises a majority of colloidal silica particles of approximately uniform size, covalently bound to a modifying reagent during formation of reagent-modified, colloidal silica particles that have a nonionic, hydrophilic surface. 19. Cell separation material produced by a method comprising the steps of: (a) mixing a colloidal silica particle covalently bonded to an organosilane having a water-soluble functional group with a mercaptosilane capable of forming thiol groups on the surface of said particles, forming thiol-modified colloidal silica; (b) reacting said thiol-modified colloidal silica with a succinimide capable of reacting with said thiol groups to form succinimide-modified colloidal silica; (c) reacting said succinimide-modified colloidal silica with protein A to form protein A-modified colloidal silica; and (d) reacting said protein A-modified colloidal silica with a purified antibody to form antibody-modified colloidal silica. 20. Cell separation material produced by the method comprising steps for: (a) mixing a plurality of colloidal silica particles covalently bonded to an organosilane having a water-soluble functional group with a coupling reagent capable of reacting with said colloidal silica particles to form activated particles; (b) reacting said activated particles with protein A to form protein A-modified colloidal silica; and (c) reacting said protein A-modified colloidal silica with an antibody to form antibody-substance f-modified colloidal silica. 21. Cell separation material according to claim 20, characterized in that specified coupling reagent is selected from 1,1-carbonyldiimidazole and succinimide. 22. Method for the separation of specific cell types from a cell mixture comprising the steps of: (a) mixing an antibody-modified colloidal silica and a cell mixture to form a particle-cell mixture; (b) incubating said particle-cell mixture for a sufficient time to allow component cell types to bind to said antibody-modified particles to form an incubated particle-cell mixture and particle-bound cells; (c) mixing said incubated particle-cell mixture and said particle-bound cells with a cell separation material comprising a plurality of colloidal silica particles covalently bound to an organosilane to form reagent-modified colloidal silica particles having a non-ionic, hydrophilic surface, said cell separation material being suspended in a buffer; and (d) centrifuging said particle-bound cells and incubated particle-cell mixture through said cell separation material to form at least one component cell band having a characteristic floating density and at least one band of particle-bound cells. 1. Cell separation material comprising: (a) a plurality of colloidal silica particles; and (b) an effective amount of a modifying reagent covalently bound to said colloidal silica particles to form reagent-modified colloidal silica particles which are non-toxic to biological materials and which have a non-ionic, hydrophilic surface, which is stable in aqueous media and which collectively form a stable colloid in aqueous media. 2. Material according to claim 1, characterized in that the indicated modifying reagent comprises an organosilane which has a water-soluble functional group. 3. Material according to claim 2, characterized in that the organosilane is of the general formula: 4. Material according to claim 3, characterized in that "Y" is selected from the group consisting of OCH2 CHCH2 0, 02 CCH3 , N(CH2 CH2 OH)2 , C02 CH3 , NH(CH2 )2 NH(CH2 )2 C02 CH3 , NHCOCI^NHC(CHg)0, N(CH2 CH2 )2 0 and N(CH=CH2 )2 . 5. Material according to claim 3, characterized in that "X" is selected from the group consisting of H3 C0, Cl, H5 C2 0, H3CC02, H3C and (H3 C)2. 6. Material according to claim 2, characterized in that the specified organosilane is of the general formula: (X) 3 -Si-(CH 2 ) 2 -Y. 7. Material according to claim 6, characterized in that "Y" is selected from the group consisting of OCH2CHCH20, 02CCH3, N(CH2 CH2 OH)2 , C02 CH3 , NH(CH2 )2 NH(CH2 )2 C02 CH3 , NHCOCf^ NHC(CH3 )0, N( CH2 CH2 )2 0 and N(CH=CH2 )2 . 8. Material according to claim 6, characterized in that "X" is selected from the group consisting of H3 CO, Cl, H5 C2 0, H3 CC02 , H3 C and (H3 C)2 . 9. Material according to claim 2, characterized in that the organosilane comprises gamma-glycidoxypropyltrimethoxysilane. 10. Material according to claim 1, characterized in that each of specified colloidal silica particles has approximately the same size and wherein specified reagent-modified colloidal silica particles resulting therefrom comprise a distinct population. 11. Material according to claim 10, characterized in that the stated size of the colloidal silica particles is between 30 Å and 600 Å in diameter. 12. Material according to claim 10, characterized in that one or more distinct populations of reagent-modified, colloidal silica particles are suspended in a buffer and mixed to form a cell separation material mixture with a desired final density. 13. Method for separating component cell types from a cell mixture, comprising steps for: (a) placing a cell mixture on top of a cell separation material comprising a plurality of colloidal silica particles covalently bound to a modifying reagent, forming reagent-modified colloidal silica particles with a nonionic, hydrophilic surface, which cell separation material is suspended in a buffer ; and (b) centrifuging the cell sample through said cell separation material to form at least one component cell band with a characteristic floating density. 14. Method according to claim 13, characterized in that the specified modifying reagent comprises an organosilane with a water-soluble functional group. 15. Method for separating component cell types from a cell mixture comprising steps for: (a) adding a cell mixture to a tube containing a cell separation material comprising a plurality of colloidal silica particles covalently bound to a modifying reagent to form reagent-modified colloidal silica particles having a non-ionic hydrophilic surface, which cell separation material is suspended in a buffer; (b) mixing said cell mixture and said cell separation material by inversion; and (c) centrifuging said cell mixture and said cell separation material to develop a density gradient in which said component cells of said cell mixture form at least one band of cells having a characteristic floating density. 16. Method according to claim 15, characterized in that specified modification reagent is an organosilane. 17. Method according to claim 15, characterized in that at least two cell bands which have a characteristic flow density are formed, one of the indicated two cell bands having a higher density than the other of the indicated two cell bands. 18. Method for separating component cell types from a cell mixture according to claim 15, characterized in that at least two distinct populations of indicated cell separation material are mixed, where each of the indicated populations comprises a majority of colloidal silica particles of approximately uniform size, covalently bound to a modifying reagent during formation of reagent-modified, colloidal silica particles that have a nonionic, hydrophilic surface. 19. Cell separation material produced by a method comprising the steps of: (a) mixing a colloidal silica particle covalently bonded to an organosilane having a water-soluble functional group with a mercaptosilane capable of forming thiol groups on the surface of said particles, forming thiol-modified colloidal silica; (b) reacting said thiol-modified colloidal silica with a succinimide capable of reacting with said thiol groups to form succinimide-modified colloidal silica; (c) reacting said succinimide-modified colloidal silica with protein A to form protein A-modified colloidal silica; and (d) reacting said protein A-modified colloidal silica with a purified antibody to form antibody-modified colloidal silica. 20. Cell separation material produced by the method comprising steps for: (a) mixing a plurality of colloidal silica particles covalently bonded to an organosilane having a water-soluble functional group with a coupling reagent capable of reacting with said colloidal silica particles to form activated particles; (b) reacting said activated particles with protein A to form protein A-modified colloidal silica; and (c) reacting said protein A-modified colloidal silica with an antibody to form antibody-substance f-modified colloidal silica. 21. Cell separation material according to claim 20, characterized in that specified coupling reagent is selected from 1,1-carbonyldiimidazole and succinimide. 22. Method for the separation of specific cell types from a cell mixture comprising the steps of: (a) mixing an antibody-modified colloidal silica and a cell mixture to form a particle-cell mixture; (b) incubating said particle-cell mixture for a sufficient time to allow component cell types to bind to said antibody-modified particles to form an incubated particle-cell mixture and particle-bound cells; (c) mixing said incubated particle-cell mixture and said particle-bound cells with a cell separation material comprising a plurality of colloidal silica particles covalently bound to an organosilane to form reagent-modified colloidal silica particles having a non-ionic, hydrophilic surface, said cell separation material being suspended in a buffer; and (d) centrifuging said particle-bound cells and incubated particle-cell mixture through said cell separation material to form at least one component cell band having a characteristic floating density and at least one band of particle-bound cells.