High gradient magnetic separation of rosette-forming cells

High Gradient Magnetic Separation (HGMS) is a rapid and straightforward technique that has previously been proven effective in extracting erythrocytes from a flowing cell suspension if the red cell hemoglobin is in a paramagnetic state. In this work it was applied to the enrichment of the small population (<2%) of splenocytes from an immune mouse that bound sheep red cells to form rosettes. Samples flowed through the HGMS column in a strong magnetic field where rosettes and free sheep cells were selectively retained. These were subsequently eluted by simply removing the magnetic field. The process required 20–30 min per mouse spleen. Rosettes in the initial sample and in the fractions that passed through, or were retained by, the column were enumerated under the microscope. Under the conditions used here, the retained and eluted cells typically showed a 20–50-fold increase in the frequency of rosetted cells, and the cells that passed through the magnet showed 90–100% depletion of rosettes. The recovery of intact rosettes and the overall cell recovery were generally both in the range of 80–90%.     

Red blood cell magnetophoresis

The existence of unpaired electrons in the four heme groups of deoxy and methemoglobin (metHb) gives these species paramagnetic properties as contrasted to the diamagnetic character of oxyhemoglobin. Based on the measured magnetic moments of hemoglobin and its compounds, and on the relatively high hemoglobin concentration of human erythrocytes, we hypothesized that differential migration of these cells was possible if exposed to a high magnetic field. With the development of a new technology, cell tracking velocimetry, we were able to measure the migration velocity of deoxygenated and metHb-containing erythrocytes, exposed to a mean magnetic field of 1.40 T and a mean gradient of 0.131 T/mm, in a process we call cell magnetophoresis. Our results show a similar magnetophoretic mobility of 3.86 x 10(-6) mm(3) s/kg for erythrocytes with 100% deoxygenated hemoglobin and 3.66 x 10(-6) mm(3) s/kg for erythrocytes containing 100% metHb. Oxygenated erythrocytes had a magnetophoretic mobility of from -0.2 x 10(-6) mm(3) s/kg to +0.30 x 10(-6) mm(3) s/kg, indicating a significant diamagnetic component relative to the suspension medium, in agreement with previous studies on the hemoglobin magnetic susceptibility. Magnetophoresis may open up an approach to characterize and separate cells for biochemical analysis based on intrinsic and extrinsic magnetic properties of biological macromolecules.     

Effects of an inhomogeneous magnetic field on flowing erythrocytes

Effects of an inhomogeneous magnetic field on narrow erythrocyte streams in a wide and transparent laminar buffer flow were studied. The stream line of erythrocytes containing paramagnetic hemoglobin showed distinct displacement toward the stronger magnetic field. The displacement increased in the order, oxygenated erythrocytes (no displacement), erythrocytes containing cyanomethemoglobin, deoxygenated erythrocytes, erythrocytes containing methemoglobin in the high spin state; more precisely the displacement was proportional to the square of the paramagnetic moment of hemoglobin contained in the erythrocytes. In addition, the displacement was proportional to the product of the magnetic flux density and its gradient, and approximately proportional to the hematocrit of the flowing-erythrocyte suspension, and was much larger than that calculated for a single erythrocyte. These phenomena could be successfully interpreted by the interaction of paramagnetic erythrocytes with the inhomogeneous magnetic field, the resistance force (Stokes Law) from the bulk water, and the hydrodynamic interaction between erythrocytes.     

Analysis of the distribution of flowing erythrocytes in a model vessel under an inhomogeneous magnetic field

Changes in the distribution of flowing erythrocytes in a straight cylinder were studied under an inhomogeneous magnetic field. The magnetic field was applied perpendicular to a cylinder, which had a 90° side vessel at the end (oriented towards the magnetic field) to detect changes in the erythrocyte distribution within the cylinder. (1) The attraction of paramagnetic erythrocytes by the magnetic field was demonstrated by an increase in the concentration (or number) of erythrocytes drawn into the side vessel. The flow of diamagnetic, oxygenated erythrocytes was unaffected. (2) The degree of attraction of the paramagnetic erythrocytes was proportional to ``(magnetic susceptibility)' and to ``(magnetic flux density) × (magnetic field gradient)' up to 10 T²/m, but it saturated at high magnetic field. The onset of the saturation depended on the magnetic susceptibility of the erythrocytes. (3) The degree of attraction depended on the hematocrit of the flowing erythrocyte suspension, with a maximum value at a low hematocrit. These phenomena are explained on the basis of the balance between the paramagnetic attractive force of the magnetic field and the collision rate between erythrocytes. Received: 2 May 1996 / Accepted: 1 July 1996     

Distribution of erythrocyte in a model vessel exposed to inhomogeneous magnetic fields

In order to investigate magnetic field effects on blood flow, changes in the flow of erythrocytes in a model branched vessel were observed in an inhomogeneous magnetic field. The magnetic field was applied perpendicular to the straight vessel before branching. When the suspension containing paramagnetic erythrocytes with high spin methemoglobin or deoxygenated hemoglobin flowed in the model vessel, the erythrocytes were attracted towards the stronger magnetic field (i.e. to the side branch) and an excess flow of erythrocytes to the side branch was detected. This excess flow of erythrocytes to the side branch was the highest at a hematocrit of about 5% for the suspension containing erythrocytes with high spin methemoglobin. In the case of mixed suspensions containing erythrocytes with high spin methemoglobin and oxygenated erythrocytes, the excess flow of erythrocytes to the side branch reached its maximum at the "partial hematocrit" for the paramagnetic erythrocyte of around 5% and remained nearly constant with a further increase of the "partial hematocrit." The effect of magnetic field decreased as the flow velocity increased. These results are explained with the paramagnetism of erythrocytes and with the assumption of a hydrodynamic interaction among erythrocytes which are pulled in the direction of the magnetic field. It is suggested that a strong inhomogeneous magnetic field is not totally negligible to the blood circulation.     

Separation of cells labeled with immunospecific iron dextran microspheres using high gradient magnetic chromatography

Immunospecific magnetic microspheres, consisting of ferromagnetic iron dextran conjugated to Protein A, were used to specifically label red blood cells (RBC) for cell separation studies using high gradient magnetic chromatography ( HGMC ). When 10(7)-10(8) RBC labeled with Protein A-iron dextran microspheres were applied to a column containing 30 mg stainless steel wire placed in a 7.5 kilogauss magnetic field, 96 +/- 2% of the cells were retained in the column. These cells could be eluted by removing the magnetic field and mechanically agitating the column. The retention of labeled cells by HGMC was shown to be dependent on the applied magnetic field and the amount of wire packed into the column. HGMC in conjunction with cell labeling with immunospecific iron dextran microspheres have useful applications for the separation of specific cell types.     

Detection of intracellular macromolecule behavior under strong magnetic fields by linearly polarized light

Strong static magnetic fields on the order of 10 T have a diamagnetic force on cell components and generate a clear alignment of a smooth muscle cell assembly, parallel to the direction of the magnetic fields within an exposure period of 3 days. This work shows the effects of diamagnetic torque forces on cell component motion. Linearly polarized light was utilized to detect the displacement of intracellular macromolecules. The polarized light passed through a mass of cells in a magnetic field, and transmission of the light increased and reached a plateau 2 h after the beginning of magnetic field exposure at 14 T. However, no distinct change was observed in transmission of the light under zero magnetic field exposure. The change in polarized light intensity through the lamellar cell assembly under magnetic fields corresponds to behavioral changes in cell components. It was speculated that intracellular macromolecules rotated and showed a displacement due to diamagnetic torque forces during 2-3 h of magnetic field exposure at 14 T.     

Orientation of bull sperms in static magnetic fields

The orientation of bull sperm cells in static magnetic fields was investigated by microscopic observation. The bull sperm, which has a very flat head, was fixed and its motion was stopped by glutaraldehyde. It was oriented with the whole body and the flat head perpendicular to the direction of the magnetic field. The diamagnetic cell components, such as the cell membrane, the DNA in the head, and the microtubule in the tail, were thought to contribute to this orientation, because bull sperm does not have paramagnetic components. For quantitative measurement of the orientation, the intensity of transmitted light through glutaraldehyde-fixed bull sperm suspension in a photometric cell was determined. The intensity changed slightly in proportion to the mean degree of orientation of the sperms. It increased sigmoidally depending on the intensity of the magnetic field and reached 100% at just below 1 T. The magnetic orientation is very strong in comparison to that of erythrocytes or platelets. By studying the response of the bull sperm to the magnetic field, it will be possible to understand its microstructure in more detail.     

Magnetic behavior of human erythrocytes at different hemoglobin states

The effect of a static magnetic field on human erythrocytes at different hemoglobin states (normal, oxidized and reduced hemoglobin) was investigated. Three different blood samples, normal, iron deficiency anemic and beta thalassemia minor, were studied. Measurements of the magnetization curves of the erythrocytes for all blood samples in all states showed diamagnetic behavior; however, oxidation was found to enhance this behavior. These measurements have also shown that the normal and iron deficiency samples in the reduced states exhibit a less diamagnetic response in comparison with the normal state. This result indicates that the reduction process gave rise to a paramagnetic component of the magnetization. Analysis of the measured paramagnetic behavior, using a Brillouin function, gave an effective magnetic moment of 8 muB per reduced hemoglobin molecule for both normal and anemic samples. This result shows that both anemic and normal blood have similar magnetic behavior and the only difference is the number of hemoglobin molecules per erythrocyte. For the beta thalassemia minor blood sample, magnetic measurements showed that both the normal and reduced states have almost the same diamagnetic behavior. However, this diamagnetic response is less than that for the normal state of the iron deficiency anemic sample. This result may indicate a low oxygen intake for the blood in the normal state for the beta thalassemia minor blood. All magnetic measurements were made using a vibrating sample magnetometer using field steps of 0.001 T from 1 T to -1 T.     

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.     

Measurement of the erythrocyte orientation in a flow by spin labeling

When a suspension of erythrocytes labeled in their membrane with a fatty acid paramagnetic molecule is allowed to flow in a flat quartz sample cell, the recorded electron paramagnetic spectra change as a function of the orientation of the cell in the magnetic field. This indicates that the red cells are themselves oriented in the flow. Such spectral variations have been reproduced by a numerical simulation procedure, which allowed us to quantify the proportion of oriented red blood cells by measuring the amplitude of some characteristic lines on the experimental spectra. Orientation rates were then measured as a function of various rheological parameters, such as shear rate, hematocrit and viscosity of the suspending medium. The kinetics of the disorientation process was determined by stopping the flow.     

New method of immobilization of microbial cells by capture on the surface of insoluble pyridinium-type resin

A new method for the immobilization of microbial cells has been developed. Whole cells of Escherichia coli with aspartase activity were immobilized by capture on the surface of cross-linked poly(N-benzyl-4-vinylpyridinium bromide) containing styrene (BVPS resin), an insoluble pyridinium-type resin. When a suspension of the bacterial cells in buffer solution was passed through a glass column containing beads of BVPS resin, the cells were captured on the resin surface and formed an immobilized cell system. A fixed-bed column reactor containing 300 mg of the bacterial cells immobilized by capture on 10 g of BVPS resin beads was used for the preparation of L-aspartic acid from ammonium fumarate. Continuous operation of tne bioreactor produced L-aspartic acid in a quantitative yield when the influent substrate concentration was 0.1M and the flow rate was 0.41-0.83 bed volumes per hour at pH 7.4-7.7 at 30 degrees C.     

Modulation of the Cell Membrane Potential and Intracellular Protein Transport by High Magnetic Fields

To explore cellular responses to high magnetic fields (HMF), we present a model of the interactions of cells with a homogeneous HMF that accounts for the magnetic force exerted on paramagnetic/diamagnetic species. There are various chemical species inside a living cell, many of which may have large concentration gradients. Thus, when an HMF is applied to a cell, the concentration‐gradient magnetic forces act on paramagnetic or diamagnetic species and can either assist or oppose large particle movement through the cytoplasm. We demonstrate possibilities for changing the machinery in living cells with HMFs and predict two new mechanisms for modulating cellular functions with HMFs via (i) changes in the membrane potential and (ii) magnetically assisted intracellular diffusiophoresis of large proteins. By deriving a generalized form for the Nernst equation, we find that an HMF can change the membrane potential of the cell and thus have a significant impact on the properties and biological functionality of cells. The elaborated model provides a universal framework encompassing current studies on controlling cell functions by high static magnetic fields. Bioelectromagnetics. 2021;42:27–36. © 2020 Bioelectromagnetics Society.     

Rapid magnetic purification of rosette-forming lymphocytes.

High gradient magnetic separation, which as previously been shown effective in extracting erythrocytes from a flowing cell suspension, has been used to separate rosetted and unrosetted human peripheral blood lymphocytes. The hemoglobin in the sheep red cells used to form rosettes was first oxidizied to the paramagnetic methemoglobin form. Samples of 50 x 10(6) lymphocytes could be processed in 10 min under sterile conditions with greater than 90% purity of the rosetted cell fraction and maintenance of T cell function in mixed lymphocyte cultures.     

The induction in vitro of immunological tolerance in T lymphocytes: an inhibitory role of macrophages.

Hapten-reactive helper T cells were generated in the spleen of C57BL/6 mice primed with sulfanilated syngeneic IgG (S-MGG). Specific immunological tolerance was induced in vitro in these helper T cells, when spleen cell suspension was passed through Sephadex G-10 column to remove adherent cells and cultured in the presence of soluble S-MGG for 21 to 24 hours. On the other hand, tolerance was not inducible in unfractionated, primed spleen cells. When G-10-passed spleen cells were added to the culture dishes containing phagocytic, adherent cells of the spleen, tolerance was no more inducible in these reconstituted cell population. From these experimental results, it was concluded that macrophages played an interfering role in tolerance induction. The experimental data were also discussed in terms of macrophage function in the recognition of antigen by T lymphocytes.     

NADH production from NAD+ with a formate dehydrogenase system involving immobilized cells of a methylotrophic Arthrobacter strain.

A convenient and economical method of NADH production from NAD+ has been established using a formate dehydrogenase system involving immobilized cells of a methanol-utilizing bacterium. Arthrobacter sp, KM62. Four kinds of cell entrapment were studied. An immobilized cell preparation showing a high NADH production activity was obtained by entrapment in a kappa-carrageenan gel lattice. The NADH-producing activity of the immobilized cells was investigated under various conditions. The NADH-producing activity was evoked on the addition of Triton X-100 to the reaction mixture. The conditions for the continuous production of NADH with an immobilized cell column were also investigated. When a reaction mixture containing 10 mumol (6.63 mg) ml-1 NAD+ was passed through the column (1.2 x 20 cm) containing 1.62 g (as dry weight) of immobilized cells, at a space velocity of 0.125 at 35 degrees C, complete conversion was attained.     

Diamagnetic Susceptibility and Root Growth Responses to Magnetic Fields in Lens culinaris,Glycine soja,and Triticum aestivum

We studied the response of root growth to different magnetic fields and forces. We submitted the seeds of three plant species, Lens culinaris L., Glycine soja Siebold & Zucc., and Triticum aestivum L., which differ in concentrations of paramagnetic (e.g., Fe or Co) and diamagnetic materials (e.g., starchy amyloplasts), to different static magnetic fields and forces. A magnetic field of 176 G reduced root growth of L. culinaris, G. soja, and T. aestivum, 37, 31, and 15%, respectively. A weaker magnetic field of 21 G reduced root growth of L. culinaris and G. soja only 13 and 21%, respectively, whereas it had no significant effect on the cereal T. aestivum. The germinating seeds of L. culinaris and G. soja were less diamagnetic than T. aestivum, and the latter had a smaller paramagnetic component. Since at room temperature, the paramagnetic component was much smaller than the diamagnetic one, the magnetic inhibition of root growth may be linked to the diamagnetic susceptibility, the inhibition being greater for the less diamagnetic materials and for the stronger magnetic forces. These results provide new examples of possible species-specific effects of moderate magnetic fields on plant growth, especially when growth is rapid, such as root growth after germination. We propose a simple hypothesis to relate root growth inhibition with magnetic fields and with the different responses found among species, seasons, and physiological and environmental conditions reported here and in the literature. It is based on a reduced magnetic force acting on the cell biological substances and on the cellular organelles such as amyloplasts, rather than on the cytoplasmic matrix where they are immersed as a consequence of their lower diamagnetic susceptibility. As a result, a nonuniform magnetic field exerts a ponderomotive force on the biological components in the opposite direction to the growing tip. This can result in intracellular magnetophoresis, and can account for inhibition of the root growth rate downwards. This inhibition would be stronger the lower the diamagnetic susceptibility.     

When pigs fly: immunomagnetic separation facilitates rapid determination of Pig-a mutant frequency by flow cytometric analysis

In vivo mutation assays based on the Pig-a null phenotype, that is, the absence of cell surface glycosylphosphatidylinositol (GPI) anchored proteins such as CD59, have been described. This work has been accomplished with hematopoietic cells, most often rat peripheral blood erythrocytes (RBCs) and reticulocytes (RETs). The current report describes new sample processing procedures that dramatically increase the rate at which cells can be evaluated for GPI anchor deficiency. This new method was applied to blood specimens from vehicle, 1,3-propane sultone, melphalan, and N-ethyl-N-nitrosourea treated Sprague Dawley rats. Leukocyte- and platelet-depleted blood samples were incubated with anti-CD59-phycoerythrin (PE) and anti-CD61-PE, and then mixed with anti-PE paramagnetic particles and Counting Beads (i.e., fluorescent microspheres). An aliquot of each specimen was stained with SYTO 13 and flow cytometric analysis was performed to determine RET percentage, RET:Counting Bead ratio, and RBC:Counting Bead ratio. The major portion of these specimens were passed through ferromagnetic columns that were suspended in a magnetic field, thereby depleting each specimen of wild-type RBCs (and platelets) based on their association with anti-PE paramagnetic particles. The eluates were concentrated via centrifugation and the resulting suspensions were stained with SYTO 13 and analyzed on the flow cytometer to determine mutant phenotype RET:Counting Bead and mutant phenotype RBC:Counting Bead ratios. The ratios obtained from pre- and post-column analyses were used to derive mutant phenotype RET and mutant phenotype RBC frequencies. Results from vehicle control and genotoxicant-treated rats are presented that indicate the scoring system is capable of returning reliable mutant phenotype cell frequencies. Using this wild-type cell depletion strategy, it was possible to interrogate ≥ 3 million RETs and ≥ 100 million RBCs per rat in approximately 7 min. Beyond considerably enhancing the throughput capacity of the analytical platform, these blood-processing procedures were also shown to enhance the precision of the measurements.     

Ferritin as a label for high-gradient magnetic separation.

In three model systems, particles the size of cells or smaller have been surface labeled with ferritin to make them slightly paramagnetic, by virtue of the iron in the ferritin. In each case it was possible to show that labeled particles could be magnetically removed from a flowing suspension by the high-gradient magnetic separation (HGMS) technique. The first system of particles consisted of small (1 micron) carboxylate-modified latex spheres to which ferritin was covalently bound to create stable paramagnetic particles analogous to a ferritin-labeled subcellular membrane preparation. In the second system polyacrylamide beads that more closely approximated whole cells in size (5-50 microns) were labeled with immunoferritin. The third system was a biomembrane preparation: erythrocyte ghosts labeled with a ferritin-lectin conjugate. A field of 7 T (tesla) (70 kG) was used in each case, along with buffer flow rates through the HGMS column in the range 0.1-1.0 ml/min.