The main goal of this project is to develop a novel in vitro technique for measurement of total cell volume in samples of islets of Langerhans that will be useful for assessing the quality of islet preparations for pancreatic transplantation and research. DNA content is currently used as a measure of total cell volume. It has been proposed that the quality of islet preparations can be represented by the ratio of viable to total cell volume, which is related to the ratio of oxygen consumption rate (OCR) to DNA content, currently determined separately. The new method will replace DNA measurement and will be useful in several applications: (1) in a small, stand alone device useful with small aliquots, {2) in a small device capable of simultaneous measurement of both OCR and total cell volume in the same container, and (3) in a configuration useful for assay of a large islet preparation. The method will provide an estimate of total cell volume with greater accuracy, rapidity and convenience than is now possible. The technology, known as Ultrasonic Streaming Pulsed Doppler (USPD), measures the reflectivity of suspended cells from which total volume is inferred by use of a calibration curve. The method also induces flow and the reflections occur at frequencies below that of the incident energy making return signals much easier to detect than would be possible in still fluids. This flow may also provide necessary mixing and eliminate the need for a stirrer. The primary aim in Phase I will be to developed the fundamental calibrated relationship between islet total volume and reflectivity that will be the basis of the methodology. This will be developed with a series of reflectivity experiments with polystyrene micro spheres and rat and porcine islets.
Other aims are to determine regimes of operational parameters in which the method can operate without damage to islets, to design transducers that can operate through container walls and to determine the practicality of using the streaming flows to provide mixing. The technique will be demonstrated with samples of islets in very small test vessels and with larger islet preparation samples prior to transplantation. The major goals for Phase II will be demonstration of the technology with human islets and development of diagnostic devices for commercial application. The technique will have other applications in medicine and bioprocessing wherever information on cell concentration is required rapidly.
