Single cell magnetometry by magnetophoresis vs. bulk cell suspension magnetometry by SQUID-MPMS - a comparison.

Paramagnetic constituents of a cell have strong effect on cell's volume magnetic susceptibility even at low volume fraction because of their high susceptibility relative to that of the diamagnetic cell constituents. The effect can be measured at a single cell level by measuring cell terminal velocity in viscous media using a microscope equipped with a well-defined field and gradient magnet configuration (referred to as magnetophoretic analysis by cell tracking velocimetry, CTV). The sensitivity of such a microscopic-scale magnetometry was compared to that of a reference method of superconducting quantum interference-magnetic properties measurement system (SQUID-MPMS) using a red blood cell (RBC) suspension model. The RBC hemoglobin oxygen saturation determines the hemoglobin molecular magnetic susceptibility (diamagnetic when fully oxygenated, paramagnetic when fully deoxygenated or converted to methemoglobin). The SQUID-MPMS measurements were performed on an average of 5,000 RBCs in 20 µL physiological phosphate buffer at room temperature, those by CTV on a single cell track in a mean magnetic field of 1.6 T and mean gradient of 240 T/m, repeated for an average of 1,000 tracks per sample. This suggests 5,000× higher sensitivity of cell susceptometry by magnetophoretic analysis than by SQUID-MPMS. The magnetophoretic mean RBC magnetic susceptibilities were in the range determined by SQUID-MPMS (lower limit) and theory (upper limit). The ability of magnetophoretic analysis to resolve susceptibility peaks in a mixed cell populations was confirmed for an oxy RBC and met RBC mixture. Magnetophoretic analysis by CTV provides new tool for studies of emergence of paramagnetic reaction products in the cell.

Continuous, intrinsic magnetic depletion of erythrocytes from whole blood with a quadrupole magnet and annular flow channel; pilot scale study.

The ability to separate RBCs from the other components of whole blood has a number of useful clinical and research applications ranging from removing RBCs from typical clinical blood draw, bone marrow transplants to transfusions of these RBCs to patients after significant blood loss. Viewed from a mechanistic/process perspective, there are three routine methodologies to remove RBCs: 1) RBCs lysis, 2) separation of the RBCs from the nucleated cells (i.e., stem cells) based on density differences typically facilitated through centrifugation or sedimentation agents, and 3) antibody based separation in which a targeted RBC is bound with an affinity ligand that facilitates its removal. More recently, several microfluidic based techniques have also been reported. In this report, we describe the performance of continuous RBC separation achieved by the deflection of intrinsically magnetic, deoxygenated RBCs as they flow through a magnetic energy gradient created by quadrupole magnet. This quadrupole magnetic, with aperture of 9.65 mm, has a maximum field of B0 = 1.36 T at the pole tips and a constant field gradient of B0 /r0 = 286 T/m. The annular flow channel, contained within this quadrupole magnet, is 203 mm long, has an inner radius of 3.98 mm, and an inner, outer radius of 4.36 mm, which corresponds to an annulus radius of 380 micrometer. At the entrance and exit to this annular channel, a manifold was designed which allows a cell suspension and sheath fluid to be injected, and a RBC enriched exit flow (containing the magnetically deflected RBCs) and a RBC depleted exit flow to be collected. Guided by theoretical models previously published, a limited number of operating parameters; total flow rate, flow rate ratios of flows in and flow out, and ratios of RBC to polystyrene control beads was tested. The overall performance of this system is consistent with our previously presented, theoretical models and our intuition. As expected, the normalized recovery of RBCs in the RBC exit fraction ranged from approximately 95% down to 60%, as the total flow rate through the system increased from 0.1 to 0.6 ml/min. At the cell concentrations studied, this corresponds to a flow rate of 1.5 × 106 -9 × 106 cells/min. While the throughput of these pilot scale studies are slow for practical applications, the general agreement with theory, and the small cross-sectional area in which the actual separation is achieved, 77 mm2 (annulus radius times the length), and corresponding volume of approximately 2 mls, suggests the potential to scale-up a system for practical applications exists and is actively being pursued.

Tessellated permanent magnet circuits for flow-through, open gradient separations of weakly magnetic materials.

Emerging microfluidic-based cell assays favor label-free red blood cell (RBC) depletion. Magnetic separation of RBC is possible because of the paramagnetism of deoxygenated hemoglobin but the process is slow for open-gradient field configurations. In order to increase the throughput, periodic arrangements of the unit magnets were considered, consisting of commercially available Nd-Fe-B permanent magnets and soft steel flux return pieces. The magnet design is uniquely suitable for multiplexing by magnet tessellation, here meaning the tiling of the magnet assembly cross-sectional plane by periodic repetition of the magnet and the flow channel shapes. The periodic pattern of magnet magnetizations allows a reduction of the magnetic material per channel with minimal distortion of the field cylindrical symmetry inside the magnet apertures. A number of such magnet patterns are investigated for separator performance, size and economy with the goal of designing an open-gradient magnetic separator capable of reducing the RBC number concentration a hundred-fold in 1 mL whole blood per hour.

Circular Halbach array for fast magnetic separation of hyaluronan-expressing tissue progenitors.

Connective tissue progenitors (CTPs) are a promising therapeutic agent for bone repair. Hyaluronan, a high molecular mass glycosaminoglycan, has been shown by us to be a suitable biomarker for magnetic separation of CTPs from bone marrow aspirates in a canine model. For the therapy to be applicable in humans, the magnetic separation process requires scale-up without compromising the viability of the cells. The scaled-up device presented here utilizes a circular Halbach array of diametrically magnetized, cylindrical permanent magnets. This allows precise control of the magnetic field gradient driving the separation, with theoretical analysis favoring a hexapole field. The separation vessel has the external diameter of a 50 mL conical centrifuge tube and has an internal rod that excludes cells from around the central axis. The magnet and separation vessel (collectively dubbed the hexapole magnet separator or HMS) was tested on four human and four canine bone marrow aspirates. Each CTP-enriched cell product was tested using cell culture bioassays as surrogates for in vivo engraftment quality. The magnetically enriched cell fractions showed statistically significant, superior performance compared to the unenriched and depleted cell fractions for all parameters tested, including CTP prevalence (CTPs per 10(6) nucleated cells), proliferation by colony forming unit (CFU) counts, and differentiation by staining for the presence of osteogenic and chondrogenic cells. The simplicity and speed of the HMS operation could allow both CTP isolation and engraftment during a single surgical procedure, minimizing trauma to patients and lowering cost to health care providers.

Magnetic separation of algae genetically modified for increased intracellular iron uptake.

Algae were investigated in the past as a potential source of biofuel and other useful chemical derivatives. Magnetic separation of algae by iron oxide nanoparticle binding to cells has been proposed by others for dewatering of cellular mass prior to lipid extraction. We have investigated feasibility of magnetic separation based on the presence of natural iron stores in the cell, such as the ferritin in Auxenochlorella protothecoides (A. p.) strains. The A. p. cell constructs were tested for inserted genes and for increased intracellular iron concentration by inductively coupled plasma atomic absorption (ICP-AA). They were grown in Sueoka's modified high salt media with added vitamin B1 and increasing concentration of soluble iron compound (FeCl3 EDTA, from 1× to 8× compared to baseline). The cell magnetic separation conditions were tested using a thin rectangular flow channel pressed against interpolar gaps of a permanent magnet forming a separation system of a well-defined fluid flow and magnetic fringing field geometry (up to 2.2 T and 1,000 T/m) dubbed "magnetic deposition microscopy", or MDM. The presence of magnetic cells in suspension was detected by formation of characteristic deposition bands at the edges of the magnet interpolar gaps, amenable to optical scanning and microscopic examination. The results demonstrated increasing cellular Fe uptake with increasing Fe concentration in the culture media in wild type strain and in selected genetically-modified constructs, leading to magnetic separation without magnetic particle binding. The throughput in this study is not sufficient for an economical scale harvest.

Feasibility study of red blood cell debulking by magnetic field-flow fractionation with step-programmed flow.

Emerging applications of rare cell separation and analysis, such as separation of mature red blood cells from hematopoietic cell cultures, require efficient methods of red blood cell (RBC) debulking. We have tested the feasibility of magnetic RBC separation as an alternative to centrifugal separation using an approach based on the mechanism of magnetic field-flow fractionation (MgFFF). A specially designed permanent magnet assembly generated a quadrupole field having a maximum field of 1.68 T at the magnet pole tips, zero field at the aperture axis, and a nearly constant radial field gradient of 1.75 T/mm (with a negligible angular component) inside a cylindrical aperture of 1.9 mm (diameter) and 76 mm (length). The cell samples included high-spin hemoglobin RBCs obtained by chemical conversion of hemoglobin to methemoglobin (met RBC) or by exposure to anoxic conditions (deoxy RBC), low-spin hemoglobin obtained by exposure of RBC suspension to ambient air (oxy RBC), and mixtures of deoxy RBC and cells from a KG-1a white blood cell (WBC) line. The observation that met RBCs did not elute from the channel at the lower flow rate of 0.05 mL/min applied for 15 min but quickly eluted at the subsequent higher flow rate of 2.0 mL/min was in agreement with FFF theory. The well-defined experimental conditions (precise field and flow characteristics) and a well-established FFF theory verified by studies with model cell systems provided us with a strong basis for making predictions about potential practical applications of the magnetic RBC separation.

Open Gradient Magnetic Red Blood Cell Sorter Evaluation on Model Cell Mixtures.

The emerging applications of biological cell separation to rare circulating tumor cell (CTC) detection and separation from blood rely on efficient methods of red blood cell (RBC) debulking. The two most widely used methods of centrifugation and RBC lysis have been associated with the concomitant significant losses of the cells of interest (such as progenitor cells or circulating tumor cells). Moreover, RBC centrifugation and lysis are not well adapted to the emerging diagnostic applications, relying on microfluidics and micro-scale total analytical systems. Therefore, magnetic RBC separation appears a logical alternative considering the high iron content of the RBC (normal mean 105 fg) as compared to the white blood cell iron content (normal mean 1.6 fg). The typical magnetic forces acting on a RBC are small, however, as compared to typical forces associated with centrifugation or the forces acting on synthetic magnetic nanoparticles used in current magnetic cell separations. This requires a significant effort in designing and fabricating a practical magnetic RBC separator. Applying advanced designs to the low cost, high power permanent magnets currently available, and building on the accumulated knowledge of the immunomagnetic cell separation methods and devices, an open gradient magnetic red blood cell (RBC) sorter was designed, fabricated and tested on label-free cell mixtures, with potential applications to RBC debulking from whole blood samples intended for diagnostic tests.

Microfluidic chip for graduated magnetic separation of circulating tumor cells by their epithelial cell adhesion molecule expression and magnetic nanoparticle binding.

The enumeration of circulating tumor cells (CTCs) in the peripheral bloodstream of metastatic cancer patients has contributed to improvements in prognosis and therapeutics. There have been numerous approaches to capture and counting of CTCs. However, CTCs have potential information beyond simple enumeration and hold promise as a liquid biopsy for cancer and a pathway for personalized cancer therapy by detecting the subset of CTCs having the highest metastatic potential. There is evidence that epithelial cell adhesion molecule (EpCAM) expression level distinguishes these highly metastatic CTCs. The few previous approaches to selective CTC capture according to EpCAM expression level are reviewed. A new two-stage microfluidic device for separation, enrichment and release of CTCs into subpopulations sorted by EpCAM expression level is presented here. It relies upon immunospecific magnetic nanoparticle labeling of CTCs followed by their field- and flow-based separation in the first stage and capture as discrete subpopulations in the second stage. To fine tune the separation, the magnetic field profile across the first stage microfluidic channel may be modified by bonding small Vanadium Permendur strips to its outer walls. Mathematical modeling of magnetic fields and fluid flows supports the soundness of the design.

Sequential CD34 cell fractionation by magnetophoresis in a magnetic dipole flow sorter.

Cell separation and fractionation based on fluorescent and magnetic labeling procedures are common tools in contemporary research. These techniques rely on binding of fluorophores or magnetic particles conjugated to antibodies to target cells. Cell surface marker expression levels within cell populations vary with progression through the cell cycle. In an earlier work we showed the reproducible magnetic fractionation (single pass) of the Jurkat cell line based on the population distribution of CD45 surface marker expression. Here we present a study on magnetic fractionation of a stem and progenitor cell (SPC) population using the established acute myelogenous leukemia cell line KG-1a as a cell model. The cells express a CD34 cell surface marker associated with the hematopoietic progenitor cell activity and the progenitor cell lineage commitment. The CD34 expression level is approximately an order of magnitude lower than that of the CD45 marker, which required further improvements of the magnetic fractionation apparatus. The cells were immunomagnetically labeled using a sandwich of anti-CD34 antibody-phycoerythrin (PE) conjugate and anti-PE magnetic nanobead and fractionated into eight components using a continuous flow dipole magnetophoresis apparatus. The CD34 marker expression distribution between sorted fractions was measured by quantitative PE flow cytometry (using QuantiBRITE PE calibration beads), and it was shown to be correlated with the cell magnetophoretic mobility distribution. A flow outlet addressing scheme based on the concept of the transport lamina thickness was used to control cell distribution between the eight outlet ports. The fractional cell distributions showed good agreement with numerical simulations of the fractionation based on the cell magnetophoretic mobility distribution in the unsorted sample.

Development of multistage magnetic deposition microscopy.

Magnetic deposition microscropy (MDM) combines magnetic deposition and optical analysis of magnetically tagged cells into a single platform. Our multistage MDM uses enclosed microfabricated channels and a magnet assembly comprising four zones in series. The enclosed channels alleviate the problem plaguing previous versions of MDM: scouring of the cell deposition layer by the air-liquid interface as the channel is drained. The four-zone magnet assembly was designed to maximize capture efficiency, and experiments yielded total capture efficiencies of >99% of fluorescent- and magnetically-labeled Jurkat cells at reasonable throughputs (10(3) cells/min). A digital image processing protocol was developed to measure the average pixel intensities of the deposited cells in different zones, indicative of the marker expression. Preliminary findings indicate that the multistage MDM may be suitable for depositing cells and particles in successive zones according to their magnetic properties (e.g., magnetic susceptibilities or magnetophoretic mobilities). The overall goal is to allow the screening of multiple disease conditions in a single platform.

Characterization of nonspecific crossover in split-flow thin channel fractionation.

Split-flow thin channel (SPLITT) fractionation is a technique for continuous separation of particles or macromolecules in a fluid stream into fractions according to the lateral migration induced by application of a field perpendicular to the direction of flow. Typical applications have involved isolation of different fractions from a polydisperse sample. Some specialized applications involve the separation of the fraction influenced by the transverse field from the fraction that is not. For example, immunomagnetically labeled biological cells may be separated from nonlabeled cells with the application of a transverse magnetic field gradient. In such cases, it may be critically important to minimize contamination of the labeled cells with nonlabeled cells while at the same time maximizing the throughput. Such contamination is known as nonspecific crossover (NSC) and refers to the real or apparent migration of nonmobile particles or cells across stream lines with the mobile material. The possible mechanisms for NSC are discussed, and experimental results interpreted in terms of shear-induced diffusion (SID) caused by viscous interactions between particles in a sheared flow. It is concluded that SID may contribute to NSC, but that further experiments and mathematical modeling are necessary to more fully explore the phenomenon.

Negative selection of hematopoietic progenitor cells by continuous magnetophoresis.

OBJECTIVE: To evaluate a negative selection technique for the hematopoietic progenitor cell enrichment from clinical leukapheresis product using continuous magnetophoresis. METHODS: The leukapheresis product was labeled with a tetrameric antibody cocktail (TAC) and magnetic colloid against nonprogenitor leukocytes (StemSep enrichment cocktail kit, Stem Cell Technologies, Vancouver, Canada). The separation of hematopoietic progenitor cells was performed by flow-through magnetophoresis in an annular channel placed coaxially inside a quadrupole magnetic field, in a split-flow thin-cell fractionation configuration (referred to as quadrupole magnetic flow sorter, QMS). The TAC antibody cocktail and the magnetic colloid were titrated to determine minimum effective antibody and magnetic reagent concentrations by measuring cell magnetophoretic mobility (m) distribution using cell tracking velocimetry. RESULTS: Leukapheresis products from eight donors having initial CD34+ cell purity between 0.37 and 9.7% were enriched to the final purity of 30 to 85% and yield of 49 to 84% with a maximum throughput of 6.7 x 10(4) cells/s. The progenitor cell enrichment was accompanied by a more than 3.5 log(10) T-lymphocyte depletion, a significant factor considering the intended application to allogeneic transplantation. Cell colony-forming unit assays showed that there was no deterioration of progenitor cell proliferation and differentiation following the QMS enrichment process. CONCLUSIONS: The negative selection method of hematopoietic progenitor cells by continuous magnetophoresis is a promising approach to a process scale-up, important for clinical applications.

Hemoglobin degradation in malaria-infected erythrocytes determined from live cell magnetophoresis.

During intra-erythrocytic development, malaria trophozoites digest hemoglobin, which leads to parasite growth and asexual replication while accumulating toxic heme. To avoid death, the parasite synthesizes insoluble hemozoin crystals in the digestive vacuole through polymerization of beta-hematin dimers. In the process, the heme is converted to a high-spin ferriheme whose magnetic properties were studied as early as 1936 by Pauling et al. Here, by magnetophoretic cell motion analysis, we provide evidence for a graduated increase of live cell magnetic susceptibility with developing blood-stage parasites, compatible with the increase in hemozoin content and the mechanism used by P. falciparum to avoid heme toxicity. The measured magnetophoretic mobility of the erythrocyte infected with a late-stage schizont form was m = 2.94 x 10(-6) mm3 s/kg, corresponding to the net volume magnetic susceptibility (relative to water) of Deltachi = 1.80 x 10(-6), significantly higher than that of the oxygenated erythrocyte (-0.18x10(-6)) but lower than that of the fully deoxygenated erythrocyte (3.33x10(-6)). The corresponding fraction of hemoglobin converted to hemozoin, calculated based on the known magnetic susceptibilities of hemoglobin heme and hemozoin ferriheme, was 0.50, in agreement with the published biochemical and crystallography data. Magnetophoretic analysis of live erythrocytes could become significant for antimalarial drug susceptibility and resistance determination.

Continuous flow magnetic cell fractionation based on antigen expression level.

Cell separation is important in medical and biological research and plays an increasingly important role in clinical therapy and diagnostics, such as rare cancer cell detection in blood. The immunomagnetic labeling of cells with antibodies conjugated to magnetic nanospheres gives rise to a proportional relationship between the number of magnetic nanospheres attached to the cell and the cell surface marker number. This enables the potential fractionation of cell populations by magnetophoretic mobility (MM). We exploit this feature with our apparatus, the Dipole Magnet Flow Fractionator (DMFF), which consists of an isodynamic magnetic field, an orthogonally-oriented thin ribbon of cell suspension in continuous sheath flow, and ten outlet flows. From a sample containing a 1:1 mixture of immunomagnetically labeled (label+) and unlabeled (label-) cells, we achieved an increase in enrichment of the label+ cell fraction with increasing outlet numbers in the direction of the magnetic field gradient (up to 10-fold). The total recovery of the ten outlet fractions was 90.0+/-7.7%. The mean MM of label+ cells increased with increasing outlet number by up to a factor of 2.3. The postulated proportionality between the number of attached magnetic beads and the number of cell surface markers was validated by comparison of MM measured by cell tracking velocimetry (CTV) with cell florescence intensity measured by flow cytometry.

Tailoring the surface charge of dextran-based polymer coated SPIONs for modulated stem cell uptake and MRI contrast.

Tracking stem cells in vivo using non-invasive techniques is critical to evaluate the efficacy and safety of stem cell therapies. Superparamagnetic iron oxide nanoparticles (SPIONs) enable cells to be tracked using magnetic resonance imaging (MRI), but to obtain detectable signal cells need to be labelled with a sufficient amount of iron oxide. For the majority of SPIONs, this can only be obtained with the use of transfection agents, which can adversely affect cell health. Here, we have synthesised a library of dextran-based polymer coated SPIONs with varying surface charge from -1.5 mV to +18.2 mV via a co-precipitation approach and investigated their ability to be directly internalised by stem cells without the need for transfection agents. The SPIONs were colloidally stable in physiological solutions. The crystalline phase of the particles was confirmed with powder X-ray diffraction and their magnetic properties were characterised using SQUID magnetometry and magnetic resonance. Increased surface charge led to six-fold increase in uptake of particles into stem cells and higher MRI contrast, with negligible change in cell viability. Cell tracking velocimetry was shown to be a more accurate method for predicting MRI contrast of stem cells compared to measuring iron oxide uptake through conventional bulk iron quantification.

Magnetophoretic cell sorting is a function of antibody binding capacity.

Antibody binding capacity (ABC) is a term representing a cell'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 cell'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.

Enrichment of circulating melanoma cells (CMCs) using negative selection from patients with metastatic melanoma.

Circulating tumor cells have emerged as prognostic biomarkers in the treatment of metastatic cancers of epithelial origins viz., breast, colorectal and prostate. These tumors express Epithelial Cell Adhesion Molecule (EpCAM) on their cell surface which is used as an antigen for immunoaffinity capture. However, EpCAM capture technologies are of limited utility for non-epithelial cancers such as melanoma. We report a method to enrich Circulating Melanoma Cells (CMCs) that does not presuppose malignant cell characteristics. CMCs were enriched by centrifugation of blood samples from healthy (N = 10) and patient (N = 11) donors, followed by RBC lysis and immunomagnetic depletion of CD45-positive leukocytes in a specialized magnetic separator. CMCs were identified by immunocytochemistry using Melan-A or S100B as melanoma markers and enumerated using automated microscopy image analyses. Separation was optimized for maximum sensitivity and recovery of CMCs. Our results indicate large number of CMCs in Stage IV melanoma patients. Analysis of survival suggested a trend toward decreased survival with increased number of CMCs. Moreover, melanoma-associated miRs were found to be higher in CMC-enriched fractions in two patients when compared with the unseparated samples, validating this method as applicable for molecular analyses. Negative selection is a promising approach for isolation of CMCs and other EpCAM -negative CTCs, and is amenable to molecular analysis of CMCs. Further studies are required to validate its efficacy at capturing specific circulating cells for genomic analysis, and xenograft studies.

A quantitative determination of magnetic nanoparticle separation using on-off field operation of quadrupole magnetic field-flow fractionation (QMgFFF).

Quadrupole Magnetic Field-Flow Fractionation (QMgFFF) is a technique for characterization of sub-micrometer magnetic particles based on their retention in the magnetic field from flowing suspensions. Different magnetic field strengths and volumetric flow rates were tested using on-off field application and two commercial nanoparticle preparations that significantly differed in their retention parameter, λ (by nearly 8-fold). The fractograms showed a regular pattern of higher retention (98.6% v. 53.3%) for the larger particle (200 nm v. 90 nm) at the higher flow rate (0.05 mL/min v. 0.01 mL/min) at the highest magnetic field (0.52 T), as expected because of its lower retention parameter. The significance of this approach is a demonstration of a system that is simpler in operation than a programmed field QMgFFF in applications to particle mixtures consisting of two distinct particle fractions. This approach could be useful for detection of unwanted particulate contaminants, especially important in industrial and biomedical applications.

Erythrocyte enrichment in hematopoietic progenitor cell cultures based on magnetic susceptibility of the hemoglobin.

Using novel media formulations, it has been demonstrated that human placenta and umbilical cord blood-derived CD34+ cells can be expanded and differentiated into erythroid cells with high efficiency. However, obtaining mature and functional erythrocytes from the immature cell cultures with high purity and in an efficient manner remains a significant challenge. A distinguishing feature of a reticulocyte and maturing erythrocyte is the increasing concentration of hemoglobin and decreasing cell volume that results in increased cell magnetophoretic mobility (MM) when exposed to high magnetic fields and gradients, under anoxic conditions. Taking advantage of these initial observations, we studied a noninvasive (label-free) magnetic separation and analysis process to enrich and identify cultured functional erythrocytes. In addition to the magnetic cell separation and cell motion analysis in the magnetic field, the cell cultures were characterized for cell sedimentation rate, cell volume distributions using differential interference microscopy, immunophenotyping (glycophorin A), hemoglobin concentration and shear-induced deformability (elongation index, EI, by ektacytometry) to test for mature erythrocyte attributes. A commercial, packed column high-gradient magnetic separator (HGMS) was used for magnetic separation. The magnetically enriched fraction comprised 80% of the maturing cells (predominantly reticulocytes) that showed near 70% overlap of EI with the reference cord blood-derived RBC and over 50% overlap with the adult donor RBCs. The results demonstrate feasibility of label-free magnetic enrichment of erythrocyte fraction of CD34+ progenitor-derived cultures based on the presence of paramagnetic hemoglobin in the maturing erythrocytes.

Magnetic Pressure as a Scalar Representation of Field Effects in Magnetic Suspensions.

Magnetic microsphere suspensions undergo complex motion when exposed to finite sources of the magnetic field, such as small permanent magnets. The computational complexity is compounded by a difficulty in choosing a suitable choice of visualization tools because this often requires using the magnetic force vector field in three dimensions. Here we present a potentially simpler approach by using the magnetic pressure. It is a scalar quantity, pm = B2/2µ0, and its usefulness has been already demonstrated in applications to magnetohydrodynamics and ferrohydrodynamics (where B is the applied field and µ0 = 4π×10-7 T.m/A). The equilibrium distribution of the magnetic bead plug in aqueous suspension is calculated as an isosurface of the magnitude of the magnetic pressure pm = const, in the field of two permanent magnet blocks calculated from closed formulas. The geometry was adapted from a publication on the magnetic bead suspensions in microsystems and the predicted bead plug distribution is shown to agree remarkably well with the experiment.