UCLA

Variations in protein secretions between individual cells directly influences their functional activity in many applications. However, the inherent complexity associated with the accurate detection and quantification of secreted factors leaves these proteins vastly underutilized as phenotypic markers today. Here we report on the development, optimization, and application of novel, easy-to-use, droplet-based technologies for the detection and isolation of single viable immune cells based on their secreted factors. We first demonstrate that rapid compartmentalization significantly improves the resolution and accuracy of single-cell secretion studies. Using an ultra-high throughput microfluidic step emulsifier, we encapsulated 1,000 cells/second into uniformly sized 70 �m droplets, providing an environment to rapidly concentrate molecules and prevent intercellular diffusion. When combined with a commercially available cytokine capture reagent, droplet encapsulation reduced >99% of false positive signals generated during a screen of IL-2 production from primary T cells. Cells remained viable throughout analysis and could be sorted using standard fluorescence activated cell sorting, providing an easy method of isolating functionally unique clones for downstream analysis. Next, we detail the development of a novel lab-on-a-particle platform which enables the assessment of single cell secretions using only standard laboratory equipment and without the need for specialized knowledge of microfluidic device fabrication or operation. In this approach precisely engineered hydrogel nanovials are utilized as a surface to bind cells, partition them into uniform compartments, and capture their secreted products. The utility of this platform is initially highlighted by screening differences in the total antibody production levels of individual cells within a producer cell line (CHO-DP12), revealing that antibody titers can be increased by 50% through a single selection and expansion of high-producers. Subsequently, we highlight the platform versatility through the successful detection of antigen specific antibodies from hybridoma and primary plasma cells, as well as cytokine secretions from primary T cells. Finally, we detail methods to optimize parameters for the sorting of our hydrogel nanovials across commonly utilized commercial flow sorters. As a whole, this work explores methods to democratize droplet based single cell analysis to develop open-source screening tools which will advance basic immunological science and inform novel cell based biotechnology applications.