Functional single-cell hybridoma screening using droplet-based microfluidics

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

Combi-Seq: Multiplexed transcriptome-based profiling of drug combinations using deterministic barcoding in single-cell droplets

AbstractAnti-cancer therapies often exhibit only short-term effects. Tumors typically develop drug resistance causing relapses that might be tackled with drug combinations. Identification of the right combination is challenging and would benefit from high-content, high-throughput combinatorial screens directly on patient biopsies. However, such screens require a large amount of material, normally not available from patients. To address these challenges, we developed a scalable microfluidic workflow to screen hundreds of drug combinations in picoliter-size droplets using transcriptome changes as a readout for drug effects. We devised a deterministic combinatorial DNA barcoding approach to encode treatment conditions, enabling the gene expression-based readout of drug effects in a highly multiplexed fashion. We applied our method to screen the effect of 420 drug combinations on the transcriptome of K562 cells using only ∼250 single cell droplets per condition, to successfully predict synergistic and antagonistic drug pairs, as well as their pathway activities.</jats:p

Combi-seq for multiplexed transcriptome-based profiling of drug combinations using deterministic barcoding in single-cell droplets

Anti-cancer therapies often exhibit only short-term effects. Tumors typically develop drug resistance causing relapses that might be tackled with drug combinations. Identification of the right combination is challenging and would benefit from high-content, high-throughput combinatorial screens directly on patient biopsies. However, such screens require a large amount of material, normally not available from patients. To address these challenges, we present a scalable microfluidic workflow, called Combi-Seq, to screen hundreds of drug combinations in picoliter-size droplets using transcriptome changes as a readout for drug effects. We devise a deterministic combinatorial DNA barcoding approach to encode treatment conditions, enabling the gene expression-based readout of drug effects in a highly multiplexed fashion. We apply Combi-Seq to screen the effect of 420 drug combinations on the transcriptome of K562 cells using only similar to 250 single cell droplets per condition, to successfully predict synergistic and antagonistic drug pairs, as well as their pathway activities.LBM