UCLA

Cell therapy is a rapidly advancing field in which living cells are used to treat a variety of diseases and disorders. One of the key challenges in developing effective cell therapeutics is understanding how cells secrete factors to communicate with neighboring or distant cells to coordinate responses in immunomodulation, tissue regeneration, and paracrine effects. Single-cell secretion analysis is an important tool in this context, as it allows for the direct measurements of the secretion of specific molecules from individual cells, which can provide insights into their functional behavior. Although recent technological advances in microfluidics and molecular biology have enabled precise measurement of secretion at the single-cell level, there are no widely available approaches to isolate viable cells based on this phenotype for further downstream transcriptomic, molecular, or functional characterization of secreting cells. The ability to selectively bind to antigenic peptides and secrete cytokines can define populations of cells with therapeutic potential in emerging engineered T cell receptor (TCR) immunotherapies. In Chapter 2, we leverage cavity-containing microscale hydrogel microparticles, called nanovials, each coated with millions of peptide-major histocompatibility complex (pMHC) monomers to isolate low and high affinity antigen-reactive T cells. A subset of T cells, activated by nanovials upon TCR binding, secrete cytokines, which are captured and stained on nanovials, allowing flow sorting based on a combination of affinity and function. The TCRs of sorted cells on nanovials are sequenced, recovering paired TCR αβ-chains using microfluidic emulsion-based single-cell sequencing. By labeling nanovials having different pMHCs with unique oligonucleotide-barcodes we could link TCR sequence to cognate pMHC with 100% accuracy. We identified an expanded repertoire of functional TCRs specific to viral antigens that were not recovered or did not have matching target information using standard techniques. Next, we explore whether the selection of single-cells based on extracellular vesicle (EV) secretion can yield superior cell therapeutic potential using nanovial technology. In this approach, we characterized and sorted single mesenchymal stem cells (MSCs) based on their EV secretion level followed by transcriptomic analysis to investigate molecular underpinnings of EV biogenesis. MSCs with high EV secretion phenotype were transcriptionally distinct, expressing markers associated with EV biogenesis, positive regulation of cell proliferation and vascular regeneration. We further applied this technique to evaluate differences in therapeutic efficacy between high and low EV secretors in in vivo myocardial infarction model. High secretor treatment group showed enhanced functional and tissue remodeling outcomes compared to low secretor treatment group by decreasing the infarct size by 2-fold. Nanovial-based multiparametric analysis of cells based on their ability to secrete molecules opens a new frontier in functional screening for improved discovery and development of cell therapies. This technology is not just limited to cytokine or extracellular vesicle secretion, but also can be further expanded in profiling other secreted markers or even different cell types. With such easily translatable features, nanovials can accelerate the discovery therapeutically important base cells for genetic modification and elucidate the link between molecular mechanism, cellular structure and function for the development of next generation of cell therapies.