FACS-Based Functional Protein Screening via Microfluidic Co-encapsulation of Yeast Secretor and Mammalian Reporter Cells

Abstract

in this study, we present a straightforward approach for functional cell-based screening by coencapsulation of secretor yeast cells and reporter mammalian cells in millions of individual agarosecontaining microdroplets. our system is compatible with ultra-high-throughput selection utilizing standard fluorescence-activated cell sorters (FACS) without need of extensive adaptation and optimization. In a model study we co-encapsulated murine interleukin 3 (mIL-3)-secreting S. cerevisiae cells with murine Ba/F3 reporter cells, which express green fluorescent protein (GFP) upon stimulation with mIL-3, and could observe specific and robust induction of fluorescence signal compared to a control with yeast cells secreting a non-functional mIL-3 mutant. We demonstrate the successful enrichment of activating mIL-3 wt-secreting yeast cells from a 1:10,000 dilution in cells expressing the inactive cytokine variant by two consecutive cycles of co-encapsulation and fAcS. this indicates the suitability of the presented strategy for functional screening of high-diversity yeast-based libraries and demonstrates its potential for the efficient isolation of clones secreting bioactive recombinant proteins. The importance of biopharmaceuticals (biologics) in medicine is increasing at a fast pace and the biologics market is predicted to reach nearly 400 billion USD/year by 2025 1. Frequently applied biologics comprise substances such as cytokines, monoclonal antibodies, hormones, soluble receptors, recombinant DNAs, enzymes, and synthetic vaccines. While biologics in targeted therapies often demonstrate remarkable safety and specificity, especially in case of autoimmune diseases 2 and cancer 3 , the discovery of novel molecules and the necessary functional validation still represent a bottleneck in the development of novel biopharmaceuticals. To overcome these shortcomings, powerful display technologies such as phage display 4 and yeast surface display 5 have been developed which allow for the isolation of specific high-affinity molecules and respective genes from complex variant libraries. However, identified binders frequently show poor physiological activity in a biological context. Thus, extensive secondary functional screens are necessary for identification of hit molecules with the desired functional activity. Furthermore, those screens require elaborate subcloning of the surface-displayed hits into soluble expression formats and outcoming clones frequently demand further optimization of physicochemical and pharmacokinetic properties 6. Consequently, implementing functional assays and phenotypic screens in an earlier selection phase appears highly beneficial for the discovery of new potent biologic drugs or even first-in-class medicines with novel molecular mechanisms of action 7. In this context, a major limitation is represented by the relatively low throughput of classical phenotypic screens, falling far behind the performance of high-diversity library-based approaches resting on affinity-driven selection protocols 8. Complex p...

Droplet flow cytometry for single-cell analysis

“…Researchers have also reported the use of DFC for the screening of co-cultures of mammalian cells and yeast cells. Individual yeast secretors (e.g., S. cerevisiae 103 and Pichia pastoris 104 ) and mammalian reporter cells were co-encapsulated and cultivated in agarose microdroplets, which is followed by FACS to select clones secreting bioactive recombinant proteins. These studies demonstrated the feasibility of DFC for screening and isolation of protein variants produced by microbes, which would enable and accelerate the discovery of new functional biologics, such as cytokines, antibodies, and soluble receptors.…”

Application of Microfluidic Technology in Field of Antibody Preparation

“…In this approach, CHO cells, transfection to stably expressing the Trastuzumab IgG, were sorted based on magnetic markers of target proteins (Figure 11A). Another strategy used Gel microdroplet (GMD) -fluorescence activated cell sorting (Yanakieva et al, 2020). P pastoris expressing full-length monoclonal antibodies and mammalian target cells were co-encapsulated in GMDs, then IgG antibodies bound to the epidermal growth factor receptor (EGFR) target cells were detected by fluorescently labeled antibodies for FACS screening (Fang, Chu, Ackerman, & Griswold, 2017) (Figure 11B).…”

Versatile and rapid microfluidics-assisted antibody discovery

“…[12][13][14] Antibodies secreted from single cells are interrogated after compartmentalization in droplets, 15 nanostructures such as nanopens, 16 or microcapillaries. [17][18][19] Most antibody secreting cells are plasma cells obtained from rodent immunization, 20 but also differentiated memory B cells, plasma blasts from human peripheral blood, 21,22 or secretion libraries 23 are applied. Antibodies screened by microfluidics retain their native chain pairing, which can be assessed by next-generation sequencing (NGS), 24 used for direct antibody subcloning or subsequent library generation.…”

“…25 Compatible with the high throughput of microfluidic setups, mostly fluorescence-based methods are applied for real-time adjustable mining of large diversities. Methodologies range from selection for binding on the recombinant target or cells 26 to internalization or functional screening using reporter gene target cells, 23,27,28 as exemplified by the recent work from Gérard et al 20 While broad diversities of neutralizing antibodies against infectious diseases such as HIV, Ebola, or COVID-19 have been identified from convalescent individuals, 22,29,30 microfluidics can also yield tools 16 as well as therapeutic antibodies targeting cancer or immunological diseases. 31,32 Several companies offer custom and customizable microfluidic chips for commercial and academic antibody screening campaigns.…”

“…31,32 Several companies offer custom and customizable microfluidic chips for commercial and academic antibody screening campaigns. 12,20,30 In addition, stand-alone devices have recently been made commercially available, 16,23,33 further driving development and diversification of microfluidic methodologies for therapeutic antibody discovery.…”