This proposal will develop a method of magnetic cell sorting for positive and negative cell selection in a continuous flow. Results in the previous funding period support this approach. A high yield, high purity magnetic separation method would have a significant positive impact on research in cell biology and other applications. The applicants have selected two promising designs: dipole and quadrupole magnetic separators, and two promising soluble magnetic labels: colloidal iron dextran and molecular magnetoferritin. They have developed tools for motion analysis in the magnetic and flow fields, using particle tracking velocimetry (PTV).
The specific aims are (1) To optimize magnetic sorter performance. The two sorters will be optimized for maximum magnetic force over the largest volume of the sample. Hydrodynamic focusing will be evaluated for use in the dipole separator. Field-flow fractionation will be used to optimize performance of the quadrupole separator. The effects of magnetic susceptibility distribution, related to cell surface antigen density, on the recovery and purity of sorted fractions will be studied with human peripheral lymphocytes. Novel reference magnetic susceptibility beads based on Dynabeads will be developed to standardize the sorter. The best performing sorter will be scaled up for use with clinical samples.
The second aim i s to determine the distribution of cell magnetic susceptibilities by PTV. Results of previous studies suggest the deviation from linearity between the cell magnetic moment and field at high field gradients leads to large error in cell susceptibility measurement. Higher order contributions of B to cell magnetization will be analyzed to increase the precision of the measurement. Cell susceptibility frequency distributions within and between cell subtypes will be measured for iron dextran and magnetoferritin immunomagnetic labels, and compared to the antigen density distribution by flow cytometry.
Aim 3 will test the magnetic flow sorter performance on large cell volumes. The prototype will be tested for enrichment of stem cells from peripheral blood mononuclear cells.
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