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

Desislava Yanakieva,Adrian Elter,Jens Bratsch,Harald Kolmar,Karlheinz Friedrich,Stefan Becker

doi:10.1038/s41598-020-66927-5

Desislava Yanakieva, Adrian Elter + Show 4 more

Open Access

https://doi.org/10.1038/s41598-020-66927-5

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Abstract

In this study, we present a straightforward approach for functional cell-based screening by co-encapsulation of secretor yeast cells and reporter mammalian cells in millions of individual agarose-containing 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.

Highlights

The importance of biopharmaceuticals in medicine is increasing at a fast pace and the biologics market is predicted to reach nearly 400 billion USD/year by 20251
We demonstrate the fluorescence-activated cell sorters (FACS)-based selection of yeast cells secreting functional murine interleukin-3 by pairwise co-encapsulation with murine reporter cells which respond to mIL-3 stimulation by expression of green fluorescent protein (GFP)
Flow cytometry analysis confirmed cell population separation of the non-activated (0 ng/mL mIL3) and the weakly-activated (0.625 ng/mL mIL-3) reporter cells (Fig. 2B), which is an essential prerequisite for efficient positive selection utilizing FACS

Summary

Introduction

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 20251. 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]. In droplet-based microfluidics, cells and reactants are compartmentalized in small volume aqueous droplets, utilizing a microfluidic chip with distinct channels for the aqueous phase(s) and for the continuous phase - inert carrier fluid (oil)[16] This technology opens the door to drug screening on a single-cell level, allowing for a drastic increase in throughput and, most importantly, for genotype-phenotype coupling. Compartmentalization of individual cells in distinct droplets entraps all secreted molecules and analytes, preventing interference with other cells and their products[17]

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