Single-cell sorting based on secreted products for functionally defined cell therapies

Abstract

Cell therapies have emerged as a promising new class of “living” therapeutics over the last decade and have been particularly successful for treating hematological malignancies. Increasingly, cellular therapeutics are being developed with the aim of treating almost any disease, from solid tumors and autoimmune disorders to fibrosis, neurodegenerative disorders and even aging itself. However, their therapeutic potential has remained limited due to the fundamental differences in how molecular and cellular therapies function. While the structure of a molecular therapeutic is directly linked to biological function, cells with the same genetic blueprint can have vastly different functional properties (e.g., secretion, proliferation, cell killing, migration). Although there exists a vast array of analytical and preparative separation approaches for molecules, the functional differences among cells are exacerbated by a lack of functional potency-based sorting approaches. In this context, we describe the need for next-generation single-cell profiling microtechnologies that allow the direct evaluation and sorting of single cells based on functional properties, with a focus on secreted molecules, which are critical for the in vivo efficacy of current cell therapies. We first define three critical processes for single-cell secretion-based profiling technology: (1) partitioning individual cells into uniform compartments; (2) accumulating secretions and labeling via reporter molecules; and (3) measuring the signal associated with the reporter and, if sorting, triggering a sorting event based on these reporter signals. We summarize recent academic and commercial technologies for functional single-cell analysis in addition to sorting and industrial applications of these technologies. These approaches fall into three categories: microchamber, microfluidic droplet, and lab-on-a-particle technologies. Finally, we outline a number of unmet needs in terms of the discovery, design and manufacturing of cellular therapeutics and how the next generation of single-cell functional screening technologies could allow the realization of robust cellular therapeutics for all patients.

SEC-seq: Association of molecular signatures with antibody secretion in thousands of single human plasma cells

“…Linking secreted protein function with specific nucleic acid sequences of heavy and light chains of IgG (Fig. 3d) or alpha and beta chains of T cell receptors (TCRs) can enable functional discovery workflows for new monoclonal antibodies or engineered TCR-based therapies 6 . A parallel study is also exploring the transcriptome that underlies secretion of high levels of vascular endothelial growth factor by mesenchymal stromal cells (unpublished work), which can elucidate features associated with therapeutic sub-populations of cells.…”

“…Two recent instruments that employ these approaches are the Beacon system from Berkeley Lights, and Isoplexis' Isolight system. Both systems use microscopic imaging to analyze the secreted products from cells, with dynamic range and the number of color channels constrained by the cameras and filter sets used [4][5][6] . In contrast, the gold standard in single-cell analysis, flow cytometers, leverage laser-based excitation and PMT-based detection to achieve sensitive multiplexed measurements with high dynamic range.…”

Secretion encoded single-cell sequencing (SEC-seq) uncovers gene expression signatures associated with high VEGF-A secretion in mesenchymal stromal cells

“…However, there is a lack of methods to probe the heterogeneity in secretory functions and link these to specific gene expression networks. Methods to sort therapeutic cell populations based on functional potency and uncover the single-cell level gene expression driving this potency can be transformative for the next generation of cell therapies 2,5 .…”

“…Cell function is defined by a myriad of biomolecules that they secrete. Over 3000 proteins are predicted to be secreted from human cells 1 , and secreted proteins such as immunoglobulins, cytokines, chemokines, extracellular matrix proteins, proteases, morphogens and growth factors span a diversity of critical functions 2 . For example, mesenchymal stromal cells (MSCs) have been widely evaluated as therapeutics because they secrete bioactive factors including growth and neurotrophic factors (VEGF, HGF, GDNF), cytokines (IDO, PGE2, TGF-β, IL-10), and extracellular vesicles, which promote immunomodulation and regeneration 3,4 .…”

Single-cell Electrochemical Aptasensor Array

“…[1][2][3] In the search for early cancer detection, high-precision medicine and therapy, the technologies most used today for sensitive detection of target analytes and monitoring the variation of these species are mainly including two types. One is based on the identification of molecular differences at the singlecell level, such as flow cytometry, fluorescence-activated cell sorting, next generation proteomics, lipidomic studies, [4][5][6][7] another is based on capturing or detecting single tumor cells from fresh or fixed primary tumors and metastatic tissues, and rare circulating tumors cells (CTCs) from blood or bone marrow, for example, dielectrophoresis technique, 8 microfluidic based microposts chip, 9 electrochemical (EC) approach. Compared to other methods, EC sensors have the merits of easy operation, high sensitivity, and portability.…”