Isolation and purification of canine adipose microvascular endothelial cells.
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Adipose Tissue↗
Biomedical subjects
M Zborowski
Publications and source records attributed to M Zborowski.
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.
Antigens, Surface↗
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 magnet's analysis region, 1.57 T. A comparison of the results of the CTV and magnetometer measurements shows good agreement.
Cells, Cultured↗
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.
Magnetics↗
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 cell's 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 cell's 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.
Algorithms↗
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.
Cells, Cultured↗
Flow rate optimization for the quadrupole magnetic cell sorter.
The quadrupole magnetic cell sorter is a form of split-flow thin-channel (SPLITT) separation device. It employs a quadrupole magnetic field and annular channel geometry. Immunomagnetic labels are used to bind to specific receptors on the surface of the cells of interest. It is the interaction of these labels with the magnetic field that brings about the selective isolation of these cells. The SPLITT separation devices have generally been based on parallel-plate geometry, usually with effectively constant field strength applied across the channel thickness. The nonconstant field strength and annular channel geometry of the magnetic cell sorter require that a new strategy be developed for optimization of inlet and outlet flow rates. We present such a strategy here based on a consideration of certain specific cell trajectories within the system.
Cell Separation↗
Detection of rare MCF-7 breast carcinoma cells from mixtures of human peripheral leukocytes by magnetic deposition analysis.
BACKGROUND: The presence of malignant breast cancer cells in bone marrow or peripheral blood is a prognostic factor. We tested the capacity of a novel magnetic cell analyzer to detect rare cancer cells in mixtures with human peripheral leukocytes. METHODS: Human peripheral leukocytes were spiked with cells of the MCF-7 line, and the cell mixture was labeled with anti-epithelial membrane antigen antibody and a magnetic colloid. The MCF-7 cells were selectively captured on a magnetic deposition substrate from the flowing leukocyte and MCF-7 cell mixture. RESULTS: The recovery of the MCF-7 cells from the original mixture ranged from 20% to 60%. The limit of detection of the MCF-7 cells was 10(-6) (n = 9). The morphology of the captured cancer cells was well preserved and comparable to that observed in cytospin smears. All deposited cells were located in a small area of 1.4 mm x 6 mm and could be quickly identified with an optical microscope following Wright's staining. CONCLUSIONS: This is a proof-of-principle study using a simplified model of rare cancer cells in a leukocyte mixture. The clinical relevance of the method will be tested in the future by extension to patient bone marrow samples and using antibody cocktails to increase specificity against the breast carcinoma cells.
Antibodies, Monoclonal↗
Continuous, flow-through immunomagnetic cell sorting in a quadrupole field.
A flow-through quadrupole magnetic cell separator has been designed, built, and evaluated by using a cell model system of human peripheral T lymphocytes (CD4+, CD8+, and CD45+ cells). The immunomagnetic labeling was accomplished by using a sandwich of mouse anti-human monoclonal antibody conjugated to fluorescein isothiocyanate and rat anti-mouse polyclonal antibody conjugated to a colloidal magnetic nanoparticle. The feed and sorted fractions were analyzed by FACScan flow cytometry. The magnetically labeled cells were separated from nonlabeled ones in a flow-through cylindrical column within a quadrupole field, which exerted a radial, outward force on the magnetic cells. The flow rate of the cell samples was 0.1-0.75 ml/min, and the flow rate of sheath fluid was 1.5-33.3 times that of the sample flow rate. The maximum shear stress exerted on the cell was less than 1 dyne/cm2, which was well below the level that would threaten cell integrity and membrane disruption. The maximum magnetic field was 0.765 T at the channel wall, and the gradient was 0.174 T/mm. The highest purity of selected cells was 99.6% (CD8 cells, initial purity of 26%), and the highest recovery of selected cells was 79% (CD4 cells, initial purity of 20%). The maximum throughput of the quadrupole magnetic cell separator was 7,040 cells/s (CD45 cells, initial purity of 5%). Theoretical calculations showed that the throughput can be increased to 10(6) cells/s by a scale-up of the current prototype.
Animals↗
Lymphocyte fractionation using immunomagnetic colloid and a dipole magnet flow cell sorter.
The relationship between cell function and surface marker expression is a subject of active investigation in biology and medicine. These investigations require separating cells of a homogeneous subset into multiple fractions of varying marker expression. We have developed a novel cell sorter, the dipole magnet flow sorter (DMFS), which separates selected T lymphocyte subpopulations, targeted by immunomagnetic colloid, into multiple fractions according to cell surface marker expression, as determined by flow cytometry. A narrow stream of cells is introduced into a sheath of carrier fluid in a rectangular channel while subjected to a perpendicular magnetic force. The special design of the pole pieces ensures a constant magnetic force acting on the magnetically labeled cells in the separation area. Cells are spread across the flow in relation to their magnetophoretic mobility. Separation is achieved by control of the positions of the effluent stream boundaries, which separate fluid volumes with cells of different magnetophoretic mobility. CD4 and CD8 T lymphocytes labeled with primary antibody-fluorescein isothiocyanate (FITC) conjugate and anti-FITC-magnetic colloid are the chosen cell systems. Flow cytometry analysis shows that, for CD4 cells, a three-fold increase in total marker number per cell is observed when comparing the highest to the lowest fluorescence fractions. Similarly, a four-fold increase in total marker number is observed for CD8 cells. We also observed the separation of two dissimilar cell types that differed in expression of the CD4 marker, monocytes and T helper lymphocytes. We believe that this type of separation is applicable to any cells in suspension for which a suitable antibody exists and, due to the comparatively gentle nature of the process, is particularly suitable for the sorting of fragile cells.
Biomedical Engineering↗
Theoretical analysis of cell separation based on cell surface marker density.
A theoretical analysis was performed to determine the number of fractions a multidisperse, immunomagnetically labeled cell population can be separated into based on the surface marker (antigen) density. A number of assumptions were made in this analysis: that there is a proportionality between the number of surface markers on the cell surface and the number of immunomagnetic labels bound; that this surface marker density is independent of the cell diameter; and that there is only the presence of magnetic and drag forces acting on the cell. Due to the normal distribution of cell diameters, a "randomizing" effect enters into the analysis, and an analogy between the "theoretical plate" analysis of distillation, adsorption, and chromatography can be made. Using the experimentally determined, normal distribution of cell diameters for human lymphocytes and a breast cancer cell line, and fluorescent activated cell screening data of specific surface marker distributions, examples of theoretical plate calculations were made and discussed.
Antigens, Surface↗
A mechanistic model of plasma filtration.
A model describing the sieving and transmembrane pressure behavior of plasma filtration is developed and numerically simulated. The model assumes a mechanistic criteria for particle passage through a membrane with cylindrical pores. The initial pore diameter distribution and porosity are assumed to be known. Model inputs include the particle diameter distribution, concentration and total flow rate of the permeate plasma solution. Outputs of the model include transmembrane pressure, the time-averaged sieving coefficients, and size distributions of the deposited particles and accumulated filtrate particles. Optimal filtration is characterized by high, stable sieving coefficients for desired particles, high retention of larger particles and relatively small increases in transmembrane pressure. These characteristics are realized for membranes with mean pore diameters equal to or slightly larger than mean permeate particle diameters. Simulations demonstrate that the incorporation of membrane properties into models of plasma filtration is both significant and readily possible.
Biophysical Phenomena↗
Applications of magnetic targeting in diagnosis and therapy--possibilities and limitations: a mini-review.
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Animals↗
Immunomagnetic isolation of magnetoferritin-labeled cells in a modified ferrograph.
Pan T, helper, and cytotoxic lymphocytes were isolated from the human peripheral blood mononuclear cell fraction by antibody staining, ferritin labeling, and deposition on glass slides. Two distinct forms of ferritin were used: one was native horse spleen ferritin, and the other was magnetoferritin. Magnetoferritin was obtained by reconstituting the horse spleen ferritin iron core with superparamagnetic magnetite instead of the usual paramagnetic ferrihydrite crystal. The cell deposition on microscopic glass slides in the magnetic field was obtained by an instrument that was adapted from an industrial magnetic deposition analyzer, the ferrograph. The identity of cells in the magnetic deposits was confirmed by comparing the cell fractions in the feed and in the eluate with the use of flow cytometry. The immunostaining protocol amplified the number of ferritin molecules per cell surface antigen 20-70 times. Magnetoferritin, but not native ferritin, imparted a sufficient magnetic moment to cells to deplete the labeled cell population between 67 and 88% of its initial concentration in a magnetic field of 1.67 Tesla (T), a field gradient of 2.57 T/mm, and a flow rate of 0.01 ml/min. This study showed that the magnetic moment of magnetoferritin was sufficient for immunomagnetic isolation of lymphocytes from mononuclear cell preparations in the modified ferrograph.
Animals↗
Analytical magnetapheresis of ferritin-labeled lymphocytes.
Analytical magnetapheresis is a technique for analyzing magnetic particles in suspension. The magnetically susceptible particles form a deposition pattern from the suspending medium under carefully controlled flow and magnetic field conditions. This technique was used to determine the effective magnetic volumetric susceptibility, delta chi, of human lymphocytes labeled with an iron-rich protein, ferritin. Dynabeads M450, monodisperse polymeric beads doped with magnetite, of a diameter 4.5 microns, close to that of human lymphocytes, were used as a reference. The experiment showed an almost complete deposition of ferritin-labeled lymphocytes at an average flow velocity of 0.28 mm/s, a representative magnetic field of 1.67 T, and a magnetic field gradient of 2.57 T/mm. The calculated delta chi was (2.92 +/- 0.24) x 10(-6)[SI] (ferritin-labeled lymphocytes), and the corresponding number of ferritin molecules per lymphocyte was (1.75 +/- 0.44) x 10(7). In comparison, an almost complete deposition of the Dynabeads was observed at a much higher average flow velocity, 15 mm/s, a much lower field, 0.164 T, and a much lower field gradient, 0.025 T/mm. These results corresponded to a much higher delta chi = 0.245[SI] (Dynabeads M450). These results offer important guidelines in evaluating the use of ferritin as a soluble magnetic cell label.
Calibration↗
Extracorporeal techniques of endotoxin removal: a review of the art and science.
The incidence of sepsis in the United States has risen in the past few years, and mortality from sepsis has risen over the past several decades. These increases have occurred despite the progress made in antibiotic therapy. The high mortality rate may not be related to the bacteria themselves but rather to the host's response. Of particular concern is the bacterial release of endotoxins, the development of endotoxic shock, and the resulting multiorgan failure, which has a high fatality rate and is the leading cause of death in patients admitted to intensive care units. Removing or neutralizing endotoxins are the two major therapeutic approaches to treating sepsis. To date, pharmacological methods have not been successful. Extracorporeal techniques are based on filtration, sorption, or both. Results with hemofiltration have not been definitive, in part because endotoxin levels have not been measured. Sorption devices, based on polymyxin B and anion exchange matrices, do remove endotoxins. Clinical studies in Japan on septic shock or multiorgan failure with polymyxin-B-bound fibers in direct hemoperfusion continue to suggest the utility of extracorporeal techniques for endotoxin removal. Continued effort is required and warranted to assess the clinical efficacy of these techniques.
Endotoxins↗
Defective leukocyte function-related cryoprecipitable protein in cryoglobulinemia.
Cryoprecipitable proteins (CPP), purified from plasmas removed during plasmapheresis treatment of patients with type II and type III cryoglobulinemia (CG), were evaluated in vitro for their effect on normal leukocyte function. CPP reduced normal mononuclear cell blastogenesis and polymorphonuclear cell phagocytosis responses in a dose-dependent fashion. At the maximum concentration studied, 5,000 micrograms/ml, CPP were shown to suppress normal blastogenesis and phagocytosis 30-60% and 50-80% below the albumin control, respectively. A 50% suppression of normal blastogenesis and phagocytosis was observed at 500 micrograms/ml for type III CPP. Leukocyte functions in CG patients were evaluated and compared to those of healthy controls. Blastogenesis and phagocytosis were significantly inhibited in CG patients. These studies suggest that CG patients have defective leukocyte function and that CPP have suppressive effects on normal leukocyte function. Our studies suggest that removal of CPP in CG patients may prove beneficial not only in reducing typical symptoms of CG but also in reducing the potential inhibitory effects of CPP on leukocyte function.
Adult↗
A comparison of efficacy of Tołpa Torf Preparation (TTP) in the treatment of cervicitis with or without surgery.
Tołpa Torf Preparation (TTP) is an immunomodulating drug produced by Torf Corporation, Wrocław and registered for human use in Poland. TTP enhances the process of tissue regeneration. Authors evaluate TTP effectiveness in the treatment of inflammatory states of the cervix, especially cervical erosions and the influence of this preparation of the macroscopic, cytological and bacteriological state of the cervix. TTP was used in 31 patients with the diagnosis of cervical erosion. All patients treated as yet were classified into 3 groups, depending on the treatment of cervical erosion used previously. TTP was administered orally in the dose of 5 mg (in 10 ml of water) daily during 10 days and locally in the form of tampons soaked with 1% TTP solution in the volume of 5 ml also during 10 days. TTP administered this way has beneficial therapeutic effects on the healing of cervical erosion accelerating the process of epithelialization and bringing normalization of the cytological picture. Especially beneficial in the treatment of cervical erosion is combined use of TTP and electrocoagulation or curettage--the healing time can be shortened by half.
Adjuvants, Immunologic↗