Functional single-cell hybridoma screening using droplet-based microfluidics

Abstract

Monoclonal antibodies can specifically bind or even inhibit drug targets and have hence become the fastest growing class of human therapeutics. Although they can be screened for binding affinities at very high throughput using systems such as phage display, screening for functional properties (e.g., the inhibition of a drug target) is much more challenging. Typically these screens require the generation of immortalized hybridoma cells, as well as clonal expansion in microtiter plates over several weeks, and the number of clones that can be assayed is typically no more than a few thousand. We present here a microfluidic platform allowing the functional screening of up to 300,000 individual hybridoma cell clones within less than a day. This approach should also be applicable to nonimmortalized primary B-cells, as no cell proliferation is required: Individual cells are encapsulated into aqueous microdroplets and assayed directly for the release of antibodies inhibiting a drug target based on fluorescence. We used this system to perform a model screen for antibodies that inhibit angiotensin converting enzyme 1, a target for hypertension and congestive heart failure drugs. When cells expressing these antibodies were spiked into an unrelated hybridoma cell population in a ratio of 1∶10,000 we observed a 9,400-fold enrichment after fluorescence activated droplet sorting. A wide variance in antibody expression levels at the single-cell level within a single hybridoma line was observed and high expressors could be successfully sorted and recultivated.

Stationary nanoliter droplet array with a substrate of choice for single adherent/nonadherent cell incubation and analysis

“…Much of the work thus far has been directed toward improving droplet manipulation capabilities (12)(13)(14)(15)(16). With these methods, droplets are mobile, and thus cytometry is performed under flow conditions (17), making continuous monitoring of single cells difficult. Continuous monitoring may be achieved by using stationary indexed droplets, but many current droplet immobilization techniques are limited by pressure coupling between droplet generation and capture events, as well as the requirement to adjust droplet volume to nanowell size (6,18,19).…”

Droplet-Based Microfluidics: Enabling Impact on Drug Discovery

“…This resulted in a 15-fold reduction in the experimental time needed for the analysis of four targets compared with traditional phage-display formats. 76 Other recent efforts in this area include the functional screening of hybridoma cells for the release of antibodies inhibiting a drug target at a screening rate of 300,000 cell clones per day 77 and chromatin immunoprecipitation on a chip to select for antibodies against specific proteins. 78 In 2007, Maerkl and Quake 79 introduced the MITOMI (Mechanically Induced Trapping of Molecular Interactions) platform (see Fig.…”

A microfluidic device for on-chip agarose microbead generation with ultralow reagent consumption

“…Water-inoil microdroplets allow the generation of microreactors (pl to fl volume), which permits the compartmentalization of reactions for multiple chemical and biochemical applications such as protein crystallization, 1 organic molecule 2 or nanoparticle synthesis, 3 genome sequencing, 4 and single cell and single molecule analysis. [5][6][7] With respect to molecular biology operations, previous work showed the efficiency of reagents compartmentalization for polymerase chain reaction (PCR), 8,9 single cell analysis, [10][11][12] or transcriptome analysis. 13 Generation of monodisperse microdroplets 12 is one way to carry out high throughput analysis 14 and execute independent reactions in parallel.…”