Intellectual merit: The cells of every organ or organism release (secrete) a wide variety of molecules into their surroundings. Secretion changes over time as cells grow, change structure and function, interact with other cells, and when they become damaged or diseased. Molecules produced by cells play key roles in the growth of embryos, infection, immune response and healing, and in regulating internal body functions despite fluctuating environmental conditions, aging, injury and illness. A tool to measure the types and numbers of molecules secreted by individual cells and their changes in space and time would help researchers understand how cells and organisms function and malfunction, in biomedical, biological and bio-industrial contexts. The tool must be able to sort individual cells based on their secretion behaviors and recover them undamaged for further use, e.g. for tissue engineering or medical treatment. Currently, no technology is simultaneously: 1) Sensitive enough to quantitatively measure many important molecules secreted by single cells at the very low concentrations typical in living organisms, 2) Fast enough to measure changes in extracellular molecular concentrations over periods of minutes, 3) Able to sort and recover live cells for further use, 4) Able to use cells as they occur naturally, without needing genetic modification or an external supply of chemical labels, 5) Capable of quick extension to conduct many different measurements simultaneously on each cell or the same measurement on many cells (high-throughput), 6) Inexpensive enough for wide use in research laboratories, clinics, and industry. The proposed Real Time Secretion-Single Cell Analyzer (RTS-SCA) will be a significant breakthrough that will enable researchers to directly measure secretion dynamics, investigate the degree of variation in secretion behavior within populations of cells and the selection of cells with desired properties. RTS-SCA will find applications in biological and chemical engineering, developmental, immune and cell biology, computational biology, pharmacology and medicine. The technology to be developed is a critical first step towards developing micro- or nano-probes that could later be used as diagnostic tools in living tissues.
Broader impact: Bioengineering, developmental and cell biology, biomedical research, and applied bioagriculture and bioindustry all require a currently unavailable capability to dynamically quantify secretion at the single cell or tissue level. This ability will advance basic understanding of cell functions and interactions, and aid tissue engineering, therapy development and drug discovery. The technology will allow improved versions of common research techniques, such as flow cytometry, polymerase chain reaction, gel electrophoresis, gene sequencing or microarray analysis. Immediate applications of the RTS-SCA include the analysis of the key signaling molecules in embryonic development, immunology, wound healing, tissue function regulation, and diseases like cancer, in which both cancer and host-tissue cells send complex chemical messages to each other. Bioengineering and pharmacology need to identify and select particular cells that produce molecules of interest at high levels, and to determine optimal stimulation and culture conditions for molecule production. The RTS-SCA could help in optimizing cell-secretion behaviors, essential for the development of tissue regeneration therapies. The RTS-SCA?s ability to quantify the complex kinetics of insulin release, which involves disrupted pulsatile secretion from pancreatic tissues, could accelerate research in treatments for type-2 diabetes, a disease which accounts for 10% of the U.S. health-care expenditure. Since periodic insulin secretion is an indicator of healthy pancreas function, RTS-SCA would help in selecting tissue fragments for transplant therapy for type-1 diabetes. Dynamic analysis of molecules is especially important in immune system responses and diseases, many of which are mediated by currently unmeasurable time-varying secreted molecules, examples include atherosclerosis, allergies, rheumatoid arthritis and multiple sclerosis. The RTS-SCA will help understand how immune system cells sense and respond to external challenges facilitating design of more effective treatments and vaccines.
Dissemination Plan: The scientists from Indiana University-Bloomington (IUB) and University of California San Diego (UCSD) have established collaborations with two major research centers, The IUB Center for Genomics and Bioinformatics and The Moores UCSD Cancer Center Microscopy Shared Resource, covering research needs in the Midwest, West Coast, and across the USA. To encourage the RTS-SCA's wide use, each center will receive an operating RTS-SCA. These centers will train undergraduates, graduate students, postdoctoral researchers, faculty and external partners to use the RTS-SCA for their research, maintain the instrument, advertise the instrument to outside collaborators, and provide appropriate outreach support through their networks.
