Cellular Response to Heterogeneously Charged Polystyrene
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bacteriological grade polystyrene or silicon dioxide. The domain sizes (3.3 gim) were chosen to accommodate the cell type used such that any cell of the given type would be forced to sit across 4-5 domains of alternating charge polarities of similar magnitudes. A minimal interdomain spacing was used to minimize the active sites (receptors) on the cells experiencing charge gradients. The response of human monocyte-derived-macrophages cultured on these two surfaces, to stimulation by opsonized Zymosan particles, was analyzed by light microscopy, scanning electron microscopy, confocal microscopy and enzyme-linked immunosorbent assays. EXPERIMENT
Model surfaces A substrate was designed and fabricated with the following characteristics: Base materials: Quartz (Hoya Corp., Tokyo, Japan ), 0.4 mm thick. Electrodes: Parallel electrodes, 3.3 um wide, with a 0.7 um separation, 150 A thick, formed of commercially pure titanium. Electrodes formed in arrays of 8.9 mm by 8.9 mm (approximately 2300 parallel electrodes) with connecting pads permitting alternate electrodes to be independently charged. Cover coating: 900 A silicon dioxide. For experiments on polystyrene, an additional 6000 A (dry film thickness) spin cast coating of NUNC® ( Naperville, IL ) polystyrene (8 wt % in toluene) was applied. Culture assemblies: Each substrate was incorporated into a culture assembly (well) by being adhered to the open bottom of a 8.9 by 8.9 mm square polystyrene well (NUNC®), 9 mm deep, using the same polystyrene/toluene solution. Sixteen such wells were assembled together in a suitable support and transfer structure which permitted easy transport between incubator and microscope while supplying a choice of four regulated constant potentials between ± 30 and ± 75 volts (Kepco Inc.) to each -.--. * set of alternating electrodes on each substrate
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Figure 1: Schematic arrangement of individual substrate and sixteen well assembly. Surface Characterization The surface texture of the polystyrene exposed to the cells was characterized using a noncontact optical interferometry profilometer (TOPO 3-D®; Wyko, Tucson, AZ). Water contact angle of polystyrene at each selected level of induced surface charge density was measured using an optical goniometer. The electrostatic field pattern at the material-tissue culture medium interface was numerically computed with Boundary Element Analysis (BEASY®, Computational Mechanics, Southampton, England). 134
Cell type and culture conditions Human blood monocytes were obtained from the Red Cross and elutriated. The cells were cultured in polypropylene tubes in Dulbeco's Modified Minimum Essential Medium (ABI Inc., Columbia, MD) with 20% human AB serum (ABI Inc, Columbia, MD), 10% fetal bovine serum (GIBCO; Lot#29N7361; Cat. No.200-6140), macrophage colony stimulating factor (100 ng/ml; Lot# FAP-007), and antibiotics (Penn.Strep. and Fungizone). The cells were fed every 48 hrs by replacing half of the old medium with fresh medium and used on the 4th day of culture. Human Immunoglobul
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