Linas Mazutis

Single-cell analysis and sorting using droplet-based microfluidics

We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. Compartmentalization of single cells in droplets enables the analysis of proteins released from or secreted by cells, thereby overcoming one of the major limitations of traditional flow cytometry and fluorescence-activated cell sorting. As an example of this approach, we detail a binding assay for detecting antibodies secreted from single mouse hybridoma cells. Secreted antibodies are detected after only 15 min by co-compartmentalizing single mouse hybridoma cells, a fluorescent probe and single beads coated with anti-mouse IgG antibodies in 50-pl droplets. The beads capture the secreted antibodies and, when the captured antibodies bind to the probe, the fluorescence becomes localized on the beads, generating a clearly distinguishable fluorescence signal that enables droplet sorting at ∼200 Hz as well as cell enrichment. The microfluidic system described is easily adapted for screening other intracellular, cell-surface or secreted proteins and for quantifying catalytic or regulatory activities. In order to screen ∼1 million cells, the microfluidic operations require 2–6 h; the entire process, including preparation of microfluidic devices and mammalian cells, requires 5–7 d.

Selective droplet coalescence using microfluidic systems

We report a microfluidic approach, which allows selective and controlled 1 : 1, 2 : 1 or 3 : 1 droplet fusion. A surfactant-stabilized droplet with an interfacial surfactant coverage, Γ, of >98% will fuse spontaneously with a second droplet when Γ of the latter droplet is <16%. However, when Γ of the second droplet is ~66%, the two droplets will not fuse, unless they have previously been brought into contact for critical time τ. Therefore, controlling the number of droplets in contact for time τ allows precise control over the number of fused droplets. We have demonstrated efficient (proportion of droplets coalesced p(c) = 1.0, n > 1000) and selective 1 : 1, 2 : 1 or 3 : 1 droplet fusion (proportion of correctly fused droplets p(s) > 0.99, n > 1000). Coalescence in this regime is induced by hydrodynamic flow causing interface separation and is efficient at different Ca numbers and using different dispersed phases, continuous phases and surfactants. However, when Γ of the second droplet is ~96% coalescence is no longer observed. Droplet-based microfluidic systems, in which each droplet functions as an independent microreactor, are proving a promising tool for a wide range of ultrahigh-throughput applications in biology and chemistry. The addition of new reagents to pre-formed droplets is critical to many of these applications and we believe the system described here is a simple and flexible method to do so, as well as a new tool to study interfacial stability phenomena.

Single-cell barcoding and sequencing using droplet microfluidics

Single-cell RNA sequencing has recently emerged as a powerful tool for mapping cellular heterogeneity in diseased and healthy tissues, yet high-throughput methods are needed for capturing the unbiased diversity of cells. Droplet microfluidics is among the most promising candidates for capturing and processing thousands of individual cells for whole-transcriptome or genomic analysis in a massively parallel manner with minimal reagent use. We recently established a method called inDrops, which has the capability to index >15,000 cells in an hour. A suspension of cells is first encapsulated into nanoliter droplets with hydrogel beads (HBs) bearing barcoding DNA primers. Cells are then lysed and mRNA is barcoded (indexed) by a reverse transcription (RT) reaction. Here we provide details for (i) establishing an inDrops platform (1 d); (ii) performing hydrogel bead synthesis (4 d); (iii) encapsulating and barcoding cells (1 d); and (iv) RNA-seq library preparation (2 d). inDrops is a robust and scalable platform, and it is unique in its ability to capture and profile >75% of cells in even very small samples, on a scale of thousands or tens of thousands of cells.

Dynamics of molecular transport by surfactants in emulsions

We consider the dynamics of equilibration of the chemical potential of a fluorophore in a monodisperse emulsion containing droplets with two initially different concentrations of the fluorophore. Although the exchange mechanism involves a single timescale at the droplet (microscopic) level, the organisation of the droplets determines the exchange dynamics at the population (macroscopic) level. The micelle concentration in the continuous phase and the chemistry of the fluorophore control the microscopic exchange rate while the disorder of the initial condition determines the power-law of the long timescale, recovered in a minimal analytical model. We also show here that an additive in the droplet such as Bovine Serum Albumin (BSA) acts on the microscopic exchange rate and slows down the exchange process by increasing the solubility of the fluorophore in the dispersed phase rather than by creating a viscoelastic layer at the droplet interface.

Simple Microfluidic Approach to Fabricate Monodisperse Hollow Microparticles for Multidrug Delivery

Herein, we report the production of monodisperse hollow microparticles from three different polymers, namely, pH-responsive acetylated dextran and hypromellose acetate succinate and biodegradable poly(lactic-co-glycolic acid), at varying polymer concentrations using a poly(dimethylsiloxane)-based microfluidic device. Hollow microparticles formed during solvent diffusion into the continuous phase when the polymer close to the interface solidified, forming the shell. In the inner part of the particle, phase separation induced solvent droplet formation, which dissolved the shell, forming a hole and a hollow-core particle. Computational simulations showed that, despite the presence of convective recirculation around the droplet, the mass-transfer rate of the solvent dissolution from the droplet to the surrounding phase was dominated by diffusion. To illustrate the potential use of hollow microparticles, we simultaneously encapsulated two anticancer drugs and investigated their loading and release profiles. In addition, by utilizing different polymer shells and polymer concentrations, the release profiles of the model drugs could be tailored according to specific demands and applications. The high encapsulation efficiency, controlled drug release, unique hollow microparticle structure, small particle size (<7 μm), and flexibility of the polymer choice could make these microparticles advanced platforms for pulmonary drug delivery.

Microtubule sliding drives proplatelet elongation and is dependent on cytoplasmic dynein

Bone marrow megakaryocytes produce platelets by extending long cytoplasmic protrusions, designated proplatelets, into sinusoidal blood vessels. Although microtubules are known to regulate platelet production, the underlying mechanism of proplatelet elongation has yet to be resolved. Here we report that proplatelet formation is a process that can be divided into repetitive phases (extension, pause, and retraction), as revealed by differential interference contrast and fluorescence loss after photoconversion time-lapse microscopy. Furthermore, we show that microtubule sliding drives proplatelet elongation and is dependent on cytoplasmic dynein under static and physiological shear stress by using fluorescence recovery after photobleaching in proplatelets with fluorescence-tagged β1-tubulin. A refined understanding of the specific mechanisms regulating platelet production will yield strategies to treat patients with thrombocythemia or thrombocytopenia.

Preparation of monodisperse emulsions by hydrodynamic size fractionation

We describe a microfluidic system allowing fast and efficient size-dependent separation of droplets using hydrodynamics. This system allows efficient fractionation of droplets with a difference in volume as small as 2.33-fold (corresponding to a 1.33-fold difference in diameter of spherical droplets) at >4.5 kHz frequencies. To demonstrate the utility of the system we combined the size-fractionation module with a passive droplet fusion module to create an integrated microfluidic platform allowing the preparation of highly monodisperse emulsions composed of >99% pairwise fused droplets. This type of microfluidic platform should be useful for many droplet-based lab-on-a-chip applications.

MAGIC: A diffusion-based imputation method reveals gene-gene interactions in single-cell RNA-sequencing data

Single-cell RNA-sequencing is fast becoming a major technology that is revolutionizing biological discovery in fields such as development, immunology and cancer. The ability to simultaneously measure thousands of genes at single cell resolution allows, among other prospects, for the possibility of learning gene regulatory networks at large scales. However, scRNA-seq technologies suffer from many sources of significant technical noise, the most prominent of which is ‘dropout’ due to inefficient mRNA capture. This results in data that has a high degree of sparsity, with typically only ~10% non-zero values. To address this, we developed MAGIC (Markov Affinity-based Graph Imputation of Cells), a method for imputing missing values, and restoring the structure of the data. After MAGIC, we find that two- and three-dimensional gene interactions are restored and that MAGIC is able to impute complex and non-linear shapes of interactions. MAGIC also retains cluster structure, enhances cluster-specific gene interactions and restores trajectories, as demonstrated in mouse retinal bipolar cells, hematopoiesis, and our newly generated epithelial-to-mesenchymal transition dataset.

Microfluidic Production of Alginate Hydrogel Particles for Antibody Encapsulation and Release.

Owing to their biocompatibility and reduced side effects, natural polymers represent an attractive choice for producing drug delivery systems. Despite few successful examples, however, the production of monodisperse biopolymer-based particles is often hindered by high viscosity of polymer fluids. In this work, we present a microfluidic approach for production of alginate-based particles carrying encapsulated antibodies. We use a triple-flow micro-device to induce hydrogel formation inside droplets before their collection off-chip. The fast mixing and gelation process produced alginate particles with a unique biconcave shape and dimensions of the mammalian cells. We show slow and fast dissolution of particles in different buffers and evaluate antibody release over time.

DNA Nanoparticles for Improved Protein Synthesis In Vitro.

Abstract The amplification and digital quantification of single DNA molecules are important in biomedicine and diagnostics. Beyond quantifying DNA molecules in a sample, the ability to express proteins from the amplified DNA would open even broader applications in synthetic biology, directed evolution, and proteomics. Herein, a microfluidic approach is reported for the production of condensed DNA nanoparticles that can serve as efficient templates for in vitro protein synthesis. Using phi29 DNA polymerase and a multiple displacement amplification reaction, single DNA molecules were converted into DNA nanoparticles containing up to about 104 clonal gene copies of the starting template. DNA nanoparticle formation was triggered by accumulation of inorganic pyrophosphate (produced during DNA synthesis) and magnesium ions from the buffer. Transcription–translation reactions performed in vitro showed that individual DNA nanoparticles can serve as efficient templates for protein synthesis in vitro.