Droplet-based Analysis of Secretion and Sorting (DASS) for characterization and selection of individual immune cells

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DESCRIPTION (provided by applicant): Methods to analyze the products secreted from single cells will be of particular value for studies of the immune system. A successful antibody response requires activation of the individual cells that encode useful antibodies. Similarly, a successful Cytotoxic-T-Lymphocyte (CTL) response requires activation of the individual cells that encode appropriate T-cell receptors. It has been difficult to study secretion from single cell because the cell and its secreted products must be maintained in the same compartment. Multi-well plates are not suitable because the output of a single cell is too dilute for measurement in the relatively large well volumes (>2 uL). We have developed water-in-oil droplets as a powerful tool to study molecules secreted from single cells: A single cell in one of these small volume (40 pL) droplets can secrete molecules to measurable concentrations in < one hour. These droplets can be formed, analyzed and selected at rates ~1000 droplets per second, allowing for high-speed analysis. We will develop a platform to measure single-cell secretion of Tumor Necrosis Factor Alpha (TNF-α Interferon Gamma (IFN-γ), and Granzyme B, key functional and regulatory molecules of the immune system. A major advantage of our water-in-oil droplet platform, relative to well-based systems, is that living cells of interest can be rapidly identifie and collected. These cells can then be used for further study, or they can be expanded to high cell numbers for use in adoptive cell therapy research. These assays will be generally useful to study immune system activation and regulation. In addition, the assays will enable the isolation of individual T-cells with the ability to kill infected or cancerous cells. This will provide a powrful means to determine the protein sequence of useful T- cell receptors. These sequences can be used for Chimeric T-Cell therapy, or used to create antibody-like molecules to enable identification of their cancer-cell targets, some of which might prove to be useful cancer-cell markers. In a second application, droplet-selected CTLs will be released and expanded, and the resulting expanded population will be greatly enriched for cells with autologous anti-cancer-cell activity. This process, or features of it, might be important in developing more effective active adoptive cell therapy protocols. In Phase II research, we will make these tools available through commercializing prototype microfluidic detection and instruments, and by selling quality-controlled assay reagents.