ABSTRACTINVESTIGATING BLOOD CELL COMMUNICATION WITH NOVEL SAMPLE HANDLING AND MICROFLUIDIC TECHNOLOGYByKari B. AndersonPuringergic receptor signaling events in platelets is a major determinant in platelet function. ADP binding to the P2Y-type receptors on platelet membranes, and the subsequent pathways evoked from this type of binding, is well-established. However, an understanding activity of the ATP-sensitive P2X1 platelet receptor is incomplete due to a number of obstacles involved with studying this receptor, in particular, the high concentrations of ATP already present in the washed platelet sample matrix. To circumvent this problem, most studies aimed at investigating P2X1 platelet activity require that the platelet samples contain apyrase in order to reduce already-existing levels of ATP and ready the P2X1 receptor for exogenously added ATP. Of course, one drawback to this method is that the apyrase will also rapidly degrade any added ATP. Here, we describe a method which, ironically, employs the reported P2X1 inhibitor NF449 to sensitize platelets in the absence of any added apyrase. Sensitization is verified by spectrofluorimetric determination of Ca2+ entry into the platelets after stimulation with concentrations of ATP ranging from 67 nM to 10 μM. Results suggest that sensitization of the P2X1 receptor by NF449 is not necessarily dependent upon the inhibitor concentration, but rather the ratio of the inhibitor and exogenously-added ATP concentrations. The sensitization by the NF449 was also found to be highly time-dependent. In order to study the platelet in the presence of red blood cells (RBCs) and endothelial cells a microfluidic approach was taken, but before vascular mimic could be created, some new microfluidic technologies were explored. A simple and inexpensive approach to fabricate polystyrene devices that is based upon molding polystyrene (PS) from Petri dishes against PDMS molds was developed. The ability to incorporate microchannels in polystyrene and integrate the resulting device with standard laboratory equipment such as an optical plate reader for analyte readout and pipets for fluid propulsion is described. A simple approach for sample and reagent delivery to the device channels using a standard, multi-channel micropipette and a PDMS-based injection block is detailed. Integration of the microfluidic device with these off-chip functions (sample delivery and readout) enables high-throughput screens and analyses. The device was sealed against a PDMS-base and compared against PDMS-based microchips in terms of their absorption of an anti-platelet drug, clopidogrel. Furthermore, these polystyrene devices were used to monitor two endothelial cell processes. One experiment involved the fluorescence measurement of nitric oxide (NO) produced within an endothelial cell line following stimulation with ATP. The result was a four-fold increase in NO production (as compared to a control), with this receptor-based mechanism of NO production verifying the maintenance of cell receptors following immobilization onto the PS substrate. The ability to monitor cellular uptake was also demonstrated by optical determination of Ca2+ into endothelial cells following stimulation with the Ca2+ ionophore A20317. The result was a significant increase (42%) in the calcium uptake in the presence of the ionophore, as compared to a control (17%) (p < 0.05). The ability to successfully culture cells on chip and measure analytes associated with that PS may be a useful material for microfluidic device fabrication. The PS device was further integrated with a polyester membrane in order to study drug transport across the membrane. However, significant absorption of clopidogrel still occurred so a device made only of PS was fabricated using an epoxy mold and hydraulic press to embed channels and seal devices. The result was a device with millimeter sized channels, but the ability to rapidly fabricate these devices was lost. Fluidic devices fabricated using conventional soft lithography are well suited as prototyping methods. Unfortunately, the prototypes tend to lack the ruggedness and reusability of methods associated with mass production techniques. 3 Dimensional (3D) printing, commonly used for producing design prototypes in industry, allows for one step production of devices. 3D printers build a device layer by layer based on 3D computer models. Here, a reusable, high throughput, 3D printed fluidic device was created that enables flow and incorporates a membrane above a channel in order to study drug transport and affect cells. The device contains 8 parallel channels, 3 mm wide 1.5 mm deep, connected to a syringe pump through standard, threaded fittings. The device was also printed to allow integration with commercially available cell inserts whose bottoms are constructed of a porous polycarbonate membrane; this insert enables molecular transport to occur from the channel to above the well. When concentrations of antibiotics, levofloxacin and linezolid, are pumped through the channels, approximately 18-21% of the drug migrates through the membrane. Finally, we show that mammalian cells cultured on this membrane can be affected by reagents flowing through the channels. Specifically, saponin was used to compromise cell membranes and a fluorescent label used to monitor the extent, resulting in a 4-fold increase in fluorescence for saponin treated cells.
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