CELL PARTITIONING WITH TWO PHASE AQUEOUS POLYMERS

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Partitioning of cells and membranes in tow-polymer aqueous phase systems by means of counter current distribution (CCD) has been shown empirically to be a sensitive, versatile technique for the characterization and fractionation of cell subpopulations on the basis of surface or membrane properties. It is propsed to continue evaluation and development of a thermodynamic model of cell partitioning in such systems, since the partition theory developed to date predicts much higher resolution separations than are observed. Different phase systems will be examined containing variety of anions, especially phosphate and sulfate. The systems will be characterized in terms of polymer content, salt composition, density and viscosity of the phases, interfacial tensions, and also electrostatic and surface potentials of the phases. The effects of poymer molecular weight and polymer chemical composition will be examined by using PEG-dextran and ficoll-dextran systems with polymers of various molecular weights. Other determinants of partitioning will be examined including measurement of three phase contact angles on single cells adsorbed at the phase boundary, cell-phase droplet interactions and influence of fluid shear and convection in determining partition. Well characterized model particles of defined sizes and surface properties such as monodisperse polystyrene latices will be used to evaluate some of the predicted primary determinants of particle phase partitioning. The experimental and system variables of the CCD method will be evaluated and new techniques explored for the separation of cell populations which differ only slightly in surface properties. The knowledge gained from the basic studies will be used to examine the partitioning behavior of erythrocytes from patients with multiple sclerosis (MS), hereditary spherocytosis and paroxysmal nocturnal hemoglobinuria (PNH). In the case of MS is is hoped to develop a test for the diagnosis of the disease which in addition could yield new mechanistic information on MS, and with PNH preliminary CCD studies have enabled us to nondestructively isolate abnormal RBC subpopulations.