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

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 andgt 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 pL droplets can secrete molecules to measurable concentrations in andlt one hour These droplets can be formed analyzed and selected at rates droplets per second allowing for high speed analysis We will develop a platform to measure single cell secretion of Tumor Necrosis Factor Alpha TNF Interferon Gamma IFN 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 PUBLIC HEALTH RELEVANCE The immune system is regulated by and performs its protective functions through the actions of many individual cells Our research aims to develop new methods to rapidly study individual cells of the immune system so that medical researchers will more quickly discover chemicals and methods to regulate the immune response These chemicals and methods will eventually lead to drugs and therapies to treat a variety of diseases such as rheumatoid arthritis and certain types of cancer