A novel scheme for the separation and live recovery of one cell type from a mixture of cells using a cell affinity chromatography (CAC) system is demonstrated. An anti-murine IgG was chemically immobilized to a cellophane support via a carbonyldiimidazole (CDI) link. Murine splenocytes flowed over the support, and B-cells were allowed to attach at a shear rate of 15 s-1. Once loading was terminated, the support was washed at a shear rate of 315 s-1 to remove nonspecifically bound cells. Elution of the B-cells was initiated by the transmembrane diffusion of hydrochloric acid (pH 1), supplied to the side of the membrane opposite the cells. At the same time, a shear flow of normal saline was established on the cell side of the membrane, and cells, freed by acid, were retrieved. Results showed that, on average, 250 cells/mm2 attached to antibody immobilized on cellophane surfaces, at a shear rate of 15 s-1, and that attached cells were successfully displaced by acid supplied to the side of the membrane opposite that holding the cells. On average, at least 60% of the B-cells removed by this elution appeared viable, based on a Trypan Blue dye exclusion assay.

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PubMedDate not supplied

Effects of secondary flow caused by a curved channel on plasma protein adsorption to artificial surfaces.

The effects of secondary flow induced by a curved channel on fibrinogen deposition and replacement on a glass surface were studied. Platelet adhesion to surface-bound fibrinogen was also studied to indicate how secondary flow may affect thrombogenesis on artificial surfaces. A saline pre-wetted channel with straight and curved sections was exposed to flowing plasma at a Reynolds number of 28.6. Results show that fibrinogen deposited on the surface at a shear rate of 175 s-1 was replaced faster in regions of secondary flow (Dean numbers from 11 to 19) than in adjacent regions of shear flow. Platelets adhered only to those surfaces where fibrinogen had been detected.

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PubMedDate not supplied

Capture of rare cells in suspension with antibody-coated polystyrene beads.

A method for the separation of one cell type present in small number from a predominant mixture of cell types using macroscopic polystyrene beads is demonstrated. An antibody specific to murine leukocytes (CD45) was adsorbed to the surface of the beads. Beads and murine hybridoma B cells were placed in test tubes and periodically inverted at fixed time intervals, causing the beads to settle through the suspension under creeping flow conditions. Capture was dependent upon interception: the captured cells must have traveled along streamlines that brought them to within a cell radius of the bead surface. B cells attached to 99-micrometer beads (maximum shear rate 8.1 s-1) were captured with greater efficiency but in lesser quantity than those attached to 170-micrometer beads (maximum shear rate 13.9 s-1). Cell capture unexpectedly reached a plateau in less than 2 h, a phenomenon that appears to involve changes in both the cells and the beads. Capture of cells was effective out to dilutions of 1:10 000 with purity in the captured population of better than 74%. This method allows for the study of physical parameters important for cell attachment and capture as well as for practical separation of rare cells.

Animals↗

PubMedDate not supplied

Separated flows in artificial organs. A cause of early thrombogenesis?

Separated flow is unavoidable in artificial blood-wetted devices. Surfaces bound by separated flows cause abnormal protein adsorption, then platelet adhesion and activation, and eventually thrombogenesis and embolization. A prolonged abnormal adsorption pattern is expected, especially in separated flows, as blood first displaces a wetting liquid during start-up of a device. The authors obtained patterns of immunoglobulin G (IgG), fibrinogen, and high molecular weight kininogen (HMK) adsorption in and near a separated flow. The flow was induced in flowing saline, replaced at time zero by plasma. The separated flow was induced behind a 4 mm bar introduced into a steady shear flow (Re = 26.4) in an apparatus designed so that the surface behind the bar was a standard glass microscope slide. The staining technique revealed the distribution of each protein of interest over the surface of the slide, and was applied to slides residing in the flow for 1, 5, 10, 30, and 60 min after the introduction of plasma (final dilution, 3.5% and 8.5%). Results show the expected, rapid disappearance of fibrinogen from surfaces near (but not in) the separated region, and prolonged appearance and even more prolonged disappearance of fibrinogen from the surface bounding the separated region. Slides removed from the apparatus, when exposed to a platelet suspension, showed that platelets adhered where fibrinogen was present on the surface.

Adsorption↗

PubMedDate not supplied

Extracorporeal cellular immunotherapy.

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Adsorption↗