Functional single-cell hybridoma screening using droplet-based microfluidics.
El Debs, B., Utharala, R., Balyasnikova, I.V., Griffiths, A.D. & Merten, C.A.
Proc Natl Acad Sci U S A. 2012 Jul 2. Research Highlight in Nature Methods
9(9): 866. 2012. Epub 2012 Jul 2.
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.
Miniaturization and Parallelization of Biological and Chemical Assays in Microfluidic Devices.
Vyawahare, S., Griffiths, A.D. & Merten, C.A.
Chem Biol. 2010 Oct 29;17(10):1052-65.
Microfluidic systems are an attractive solution for the miniaturization of biological and chemical assays. The typical sample volume can be reduced up to 1 million-fold, and a superb level of spatiotemporal control is possible, facilitating highly parallelized assays with drastically increased throughput and reduced cost. In this review, we focus on systems in which multiple reactions are spatially separated by immobilization of reagents on two-dimensional arrays, or by compartmentalization in microfabricated reaction chambers or droplets. These systems have manifold applications, and some, such as next-generation sequencing are already starting to transform biology. This is likely the first step in a biotechnological transformation comparable to that already brought about by the microprocessor in electronics. We discuss both current applications and likely future impacts in areas such as the study of single cells/single organisms and high-throughput screening.
High-throughput screening of enzymes by retroviral display using droplet-based microfluidics.
Granieri, L., Baret, J.C., Griffiths, A.D. & Merten, C.A.
Chem Biol. 2010 Mar 26;17(3):229-35.
During the last 25 years, display techniques such as phage display have become very powerful tools for protein engineering, especially for the selection of monoclonal antibodies. However, while this method is extremely efficient for affinity-based selections, its use for the selection and directed evolution of enzymes is still very restricted. Furthermore, phage display is not suited for the engineering of mammalian proteins that require posttranslational modifications such as glycosylation or membrane anchoring. To circumvent these limitations, we have developed a system in which structurally complex mammalian enzymes are displayed on the surface of retroviruses and encapsulated into droplets of a water-in-oil emulsion. These droplets are made and manipulated using microfluidic devices and each droplet serves as an independent reaction vessel. Compartmentalization of single retroviral particles in droplets allows efficient coupling of genotype and phenotype. Using tissue plasminogen activator (tPA) as a model enzyme, we show that, by monitoring the enzymatic reaction in each droplet (by fluorescence), quantitative measurement of tPA activity in the presence of different concentrations of the endogenous inhibitor PAI-1 can be made on-chip. On-chip fluorescence-activated droplet sorting allowed the processing of 500 samples per second and the specific collection of retroviruses displaying active wild-type tPA from a model library with a 1000-fold excess of retroviruses displaying a non-active control enzyme. During a single selection cycle, a more than 1300-fold enrichment of the active wild-type enzyme was demonstrated.
Droplet-based microfluidic platforms for the encapsulation and screening of Mammalian cells and multicellular organisms.
Clausell-Tormos, J., Lieber, D., Baret, J.C., El-Harrak, A., Miller, O.J., Frenz, L., Blouwolff, J., Humphry, K.J., Koster, S., Duan, H., Holtze, C., Weitz, D.A., Griffiths, A.D. & Merten, C.A.
Chem Biol. 2008 May;15(5):427-37. Research Highlight in Nature Methods 5(7): 580-581. 2008.
High-throughput, cell-based assays require small sample volumes to reduce assay costs and to allow for rapid sample manipulation. However, further miniaturization of conventional microtiter plate technology is problematic due to evaporation and capillary action. To overcome these limitations, we describe droplet-based microfluidic platforms in which cells are grown in aqueous microcompartments separated by an inert perfluorocarbon carrier oil. Synthesis of biocompatible surfactants and identification of gas-permeable storage systems allowed human cells, and even a multicellular organism (C. elegans), to survive and proliferate within the microcompartments for several days. Microcompartments containing single cells could be reinjected into a microfluidic device after incubation to measure expression of a reporter gene. This should open the way for high-throughput, cell-based screening that can use >1000-fold smaller assay volumes and has approximately 500x higher throughput than conventional microtiter plate assays.
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