Russell Biotech, Inc.
trussell@russellbiotech.com
(215) 589-6412

Technology

• Display high specificity for targeted cells and very low non-specific binding/trapping of non-targeted cells.
• Work directly in undiluted whole blood and bone marrow as well as cell suspensions.
• Have greater magnetic susceptibility than superparamagnetic particles.
• Exhibit reaction kinetics (cell capture + magnetic incubation) on the order of seconds to minutes.
• Remove platelets directly from undiluted whole blood in minutes prior to Ficoll gradient centrifugation to yield platelet free mononuclear cells for research.
• Significantly improve the purity of sorted cells and the speed of Flow Cell Sorting by rapidly removing unwanted cells in a pre-sort depletion step.
• Are extremely user friendly compared to existing technologies.

CURRENT TECHNOLOGY
Existing magnetic cell separation technology is based on superparamagnetic particles. Such particles can range in diameter from around 50nm to 5 micron or larger provided the individual magnetic crystal sub-unit within the particle is less than 25nm in diameter. Superparamagnetic particles are magnetic only when placed in a magnetic field. It is this property of superparamagnetic particles that permits the isolation of specific cell populations and the resuspension of the particle bound cells following the removal of the magnetic field.

However, the primary attribute of these particles, superparamagnetism, is also responsible for some of the technology’s drawbacks. Small particles with diameters of 50 to 200 nm require high gradient magnetic fields which can lead to cumbersome apparatus and relatively long magnetic incubation times. Larger superparamagnetic particles with diameters of ~5 micron, in contrast overcome this problem, however, mixing times are significantly increased. In addition, superparamagnetic particles often lead to non-specific binding/trapping of non-targeted cells, thus reducing the specificity of the technology. These attributes limit the utility of superparamagnetic particles.

QUICK-SEP TECHNOLOGY
Quick-Sep ferromagnetic particles* (FMP) retain the positive attributes of superparamagnetic particles, however they eliminate the properties that impede their utility.

Quick-Sep FMP are metallic particles that have a density of 9g/cm3 and are composed of a nickel core with a nickel oxide outer shell. The particles can be produced in a range of diameters from 50 nm to 1.5 microns.

Studies using a Coulter N4 particle analyzer have shown, remarkably, that FMP of diameters from 0.05 to 1.5 micron can be dispersed by vortexing after exposure to a magnetic field. This property was formerly attributed only to superparamagnetic particles.

The high magnetic susceptibility of ferromagnetic particles, as compared to superparamagnetic particles, provides rapid magnetic collection of bound cells on the order of seconds to minutes. Also, because of the magnetic properties of FMP, the procedure does not require elaborate equipment.

Finally, a desirable attribute of metallic particles is the complete lack of non-specific binding of non-targeted cells. Quantitative recovery of non-targeted cells is possible using FMP. Particles coated with CD15-antibody were used to deplete greater than 99% of the granulocytes, and a subset of monocytes known to be CD15 positive, from whole blood with quantitative recovery of the non-targeted lymphocytes (see table). A separate experiment demonstrated that the CD15 targeted population can also be isolated quantitatively (data not shown). In addition, FMP coupled to CD15, CD45 and CD4 depleted >99.5% of the leukocyte population (see table).

Lack of Binding to Non-Targeted Cells

+Analyzed by forward vs 90 degree light scatter (Flow Cytometry).
Absolute cell count was determined using TruCOUNT beads from BD.
* Percent Depleted

Method
The cell separation procedure using FMP is revolutionary as it is extremely user-friendly, rapid, and well suited to the research environment found in universities and biotech and pharmaceutical companies. Basically, FMP conjugated with the appropriate specific agent, e.g. bound monoclonal antibody, are added to the sample that might be whole blood, bone marrow, or a tissue culture cell suspension. The particles and sample are mixed (range: a few seconds to 4 minutes) and then immediately placed in a magnetic field for a few seconds up to 2 minutes. The magnetic supernatant is removed and is a source of non-targeted cells while the pellet can be re-suspended and used as a source of targeted cells.

*patent pending