A Microfluidic Platform for Rapid Isolation of Cells Based on Secretion of Antigen Specific Antibodies
CitationDing, Ruihua. 2017. A Microfluidic Platform for Rapid Isolation of Cells Based on Secretion of Antigen Specific Antibodies. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
AbstractDroplet microfluidics provides compartmentalization of cells into picoliter-sized droplets, enabling economical, sensitive and massively parallel biological assays. Droplets can be actively manipulated for different applications. In particular, droplet sorting allows rapid isolation of individual desired droplets from a large excess of undesired droplets. Droplet sorting is particularly powerful when combined with in-droplet assays to identify cells that secrete molecules of interest. However, the accuracy of droplet sorting is limited due to droplet coalescence.
In this dissertation I present a droplet filter design that effectively separates droplets by size. The droplet filter has a sharp size cut-off and can distinguish droplets differing in volume by as little as 20%. A simple model explains the behaviour of the droplets as they pass through the filter. We apply the filter upstream of a droplet sorter and show it improves sorting efficiency.
We also develop a droplet-based system to accurately identify and isolate individual cells that secrete antigen-specific antibodies. This system eliminates the inefficient and time-consuming steps of cell fusion and clonal expansion required in standard monoclonal antibody generation methods. I present use of this system, coupled with filter-improved droplet sorting, to isolate cells that secrete antigen-specific antibodies. The platform is capable of screening a million droplets within one day and is able to isolate rare antigen-specific cells that consist of 0.1% of the total population. We apply this system to frozen primary rat cells. After sorting, we perform single-cell RT-PCR to identify the IgG-encoding sequences from each isolated cell. A novel ELISA assay is used to verify the target-binding ability of the encoded IgG molecules.
Citable link to this pagehttp://nrs.harvard.edu/urn-3:HUL.InstRepos:42061470
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