Kara E. McCloskey

The use of magnetite-doped polymeric microspheres in calibrating cell tracking velocimetry

Continuous magnetic separation, in which there is no accumulation of mass in the system, is an inherently dynamic process, requiring advanced knowledge of the separable species for optimal instrument operation. By determining cell magnetization in a well-defined field, we may predict the cell trajectory behavior in the well-characterized field environments of our continuous separators. Magnetization is determined by tracking the migration of particles with a technique known as cell tracking velocimetry (CTV). The validation of CTV requires calibration against an external standard. Furthermore, such a standard, devoid of the variations and instabilities of biological systems, is needed to reference the method against day-to-day shifts or trends. To this end, a method of synthesizing monodisperse, magnetite-doped polymeric microspheres has been developed. Five sets of microspheres differing in their content of magnetite, and each of approximately 2.7 microm diameter, are investigated. An average gradient of 0.18 T/mm induces magnetic microsphere velocities ranging from 0.45 to 420 microns/s in the CTV device. The velocities enable calculation of the microsphere magnetization. Magnetometer measurements permit the determination of magnetization at a flux density comparable to that of the CTV magnets analysis region, 1.57 T. A comparison of the results of the CTV and magnetometer measurements shows good agreement.

Magnetophoretic Cell Sorting Is a Function of Antibody Binding Capacity

Antibody binding capacity (ABC) is a term representing a cellapos;s ability to bind antibodies, correlating with the number of specific cellular antigens expressed on that cell. ABC allows magnetically conjugated antibodies to bind to the targeted cells, imparting a magnetophoretic mobility on each targeted cell. This enables sorting based on differences in the cell magnetophoretic mobility and, potentially, a magnetic separation based on the differences in the cell ABC values. A cellapos;s ABC value is a particularly important factor in continuous magnetic cell separation. This work investigates the relationship between ABC and magnetic cell separation efficiency by injection of a suspension of immunomagnetically labeled quantum simply cellular calibration microbeads of known ABC values into fluid flowing through a quadrupole magnetic sorter. The elution profiles of the outlet streams were evaluated using UV detectors. Optimal separation flow rate was shown to correlate with the ABC of these microbeads. Comparing experimental and theoretical results, the theory correctly predicted maximum separation flow rates but overestimated the separation fractional recoveries.

Magnetophoretic mobilities correlate to antibody binding capacities

METHODS A methodology and a mathematical theory have been developed, which allow quantitation of the expression levels of cellular surface antigens using immunomagnetic labels and cell tracking velocimetry (CTV) technology. RESULTS Quantum Simply Cellular (QSC) microbeads were immunomagnetically labeled with anti-CD2 fluorescein isothiocyanate (FITC) antibodies and anti-FITC MACS paramagnetic nanoparticles. Magnetophoretic mobility has been defined as the magnetically induced velocity of the labeled cell or microbead divided by the magnetophoretic driving force, proportional to the magnetic energy density gradient. DISCUSSION Using computer imaging and processing technology, the mobility measurements were accomplished by microscopically recording and calculating the velocity of immunomagnetically labeled QSC microbeads in a nearly constant magnetic energy gradient. A calibration curve correlating the measured magnetophoretic mobility of the immunomagnetically labeled microbeads to their antibody binding capacities (ABC) has been obtained. CONCLUSION The results, in agreement with theory, indicate a linear relationship between magnetophoretic mobility and ABC for microbeads with less than 30,000 ABC. The mathematical relationships and QSC standardization curve obtained allow determination of the number of surface antigens on similarly immunomagnetically labeled cells.

Study of magnetic particles pulse-injected into an annular SPLITT-like channel inside a quadrupole magnetic field

Advantages of the continuous magnetic flow sorting for biomedical applications over current, batch-wise magnetic separations include high throughput and a potential for scale-up operations. A continuous magnetic sorting process has been developed based on the quadrupole magnetic field centered on an annular flow channel. The performance of the sorter has been described using the conceptual framework of split-flow thin (SPLITT) fractionation, a derivative of field-flow fractionation (FFF). To eliminate the variability inherent in working with a heterogenous cell population, we developed a set of monodisperse magnetic microspheres of a characteristic magnetization, and a magnetophoretic mobility, similar to those of the cells labeled with a magnetic colloid. The theory of the magnetic sorting process has been tested by injecting a suspension of the magnetic beads into the carrier fluid flowing through the sorter and by comparing the theoretical and experimental recovery versus total flow-rate profiles. The position of the recovery maxima along the total flow-rate axis was a function of the average bead magnetophoretic mobility and the magnetic field intensity. The theory has correctly predicted the position of the peak maxima on the total flow-rate axis and the dependence on the bead mobility and the field intensity, but has not correctly predicted the peak heights. The differences between the calculated and the measured peak heights were a function of the total flow-rate through the system, indicating a fluid-mechanical origin of the deviations from the theory (such as expected of the lift force effects in the system). The well-controlled elution studies using the monodisperse magnetic beads, and the SPLITT theory, provided us with a firm basis for the future sorter evaluation using cell mixtures.

Quantification of Cellular Properties from External Fields and Resulting Induced Velocity: Cellular Hydrodynamic Diameter

An experimental technique is discussed in which the size distribution of a population of cells is determined by calculating each cells settling velocity. The settling velocity is determined from microscopically obtained images which were recorded on SVHS tape. These images are then computer imaged and processed, and the cells location and velocity are determined using a computer algorithm referred to as cell tracking velocimetry (CTV). Experimental data is presented comparing the distribution of human lymphocytes and a human breast cancer cell line, MCF-7, determined using a Coulter counter and the CTV approach.

Quantification of cellular properties from external fields and resulting induced velocity: magnetic susceptibility.

An experimental technique is discussed in which the magnetic susceptibility of immunomagnetically labeled cells can be determined on a cell-by-cell basis. This technique is based on determining the magnetically induced velocity that an immunomagnetically labeled cell has in a well-defined magnetic energy gradient. This velocity is determined through the use of video recordings of microscopic images of cells moving in the magnetic energy gradient. These video images are then computer digitized and processed using a computer algorithm, cell tracking velocimetry, which allows larger numbers (>10(3)) of cells to be analyzed.

In vitro derivation and expansion of endothelial cells from embryonic stem cells.

Vascular endothelial cells or endothelial progenitor cells derived from stem cells could potentially lead to a variety of clinically relevant applications, including cell-based therapies and tissue engineering. Embryonic stem (ES) cells serve as an excellent in vitro system for studying differentiation events and for developing methods of generating various specialized cells for future regenerative therapeutic applications. Two obstacles associated with using embryonic stem cells include (1) isolating homogeneous populations of differentiated cells and (2) obtaining terminally differentiated cell populations that are capable of proliferating further. Here, we describe methods for isolating purified proliferating populations of endothelial cells from mouse ES cells using Flk-1-positive cells, vascular endothelial growth factor supplementation, and a highly selective manual selection technique. This methodology, although rigorous, overcomes two current obstacles in ES derivation and culture by generating highly purified (>96%) populations of actively proliferating endothelial cells from mouse ES cells. Using this in vitro derivation procedure, millions of cells at various stages of differentiation may be obtained and expanded up to 25 population doublings.

Measurement of CD2 expression levels of IFN-α–treated fibrosarcomas using cell tracking velocimetry

METHODS A methodology and a mathematical relationship have been developed that allow quantitation of the expression levels of cellular surface antigens, in terms of antibody binding capacities (ABC). This methodology uses immunomagnetically labeled cells and calibration microbeads combined with cell tracking velocimetry (CTV) technology to measure magnetophoretic mobilities corresponding to cellular ABC. The mobility measurements were accomplished by microscopically recording and calculating the velocity of immunomagnetically labeled QSC microbeads and cells in a nearly constant magnetic energy gradient. RESULTS Transformed fibrosarcoma cells were given controlled treatments of interferon-alpha in order to manipulate CD2 antigen expression levels. These cells were then immunomagnetically labeled with anti-CD2 FITC antibodies and anti-FITC MACS paramagnetic nanoparticles. Measured magnetophoretic mobilities were used to calculate ABC for these cells, corresponding to CD2 expression levels. CONCLUSION The results from CTV and flow cytometry (FCM) qualitatively verify that these fibrosarcoma cells express elevated levels of CD2 molecules with increasing interferon-alpha treatment from 0 to 24 h. The mean basal CD2 expression level, in terms of ABC, was calculated to be 27,000 from CTV analysis, whereas FCM indicates a comparable ABC value of 33,000.

Endothelial progenitor cells from embryonic stem cells

Stem cell research has gained significant interest due to their potential to repopulate damaged or diseased cell tissues. Pluripotent embryonic stem (ES) cells, able to rapidly and stably proliferate in vitro, provide an excellent system for studying cellular differentiation events. Our lab is particularly interested in stem cell differentiation of the endothelial lineage. Recent evidence has demonstrated that induction of Flk-1 positive expressing cells can be achieved by culturing ES cells on type IV collagen-coated dishes. Vascular endothelial growth factor (VEGF), a cytokine for the Flk-1 receptor, can stimulate endothelial cells to mimic vasculogenesis through cell migration and the formation of line, or tube-like structures in vitro. ES cells were; induced to differentiate on collagen type IV coated-dishes. Flk-l+ cells were then sorted and replated on collagen type IV and treated with VEGF. Spontaneous tube-like formations were observed in the unsorted endothelial progenitor cells without VEGF exposure. Two distinct cell morphologies arose from the Flkl+ sorted cells: a cobblestone morphology and smooth muscle-like cells that stained positive for /spl alpha/-smooth muscle actin. Current efforts are being made to further characterize the maturing cell types arising from Flk-1+ cell populations and develop methodology to isolate a pure population of endothelial cells.