Characterizing cell interactions at scale with made-to-order droplet ensembles (MODEs).

Cell-cell interactions are important to numerous biological systems, including tissue microenvironments, the immune system, and cancer. However, current methods for studying cell combinations and interactions are limited in scalability, allowing just hundreds to thousands of multicell assays per experiment; this limited throughput makes it difficult to characterize interactions at biologically relevant scales. Here, we describe a paradigm in cell interaction profiling that allows accurate grouping of cells and characterization of their interactions for tens to hundreds of thousands of combinations. Our approach leverages high-throughput droplet microfluidics to construct multicellular combinations in a deterministic process that allows inclusion of programmed reagent mixtures and beads. The combination droplets are compatible with common manipulation and measurement techniques, including imaging, barcode-based genomics, and sorting. We demonstrate the approach by using it to enrich for chimeric antigen receptor (CAR)-T cells that activate upon incubation with target cells, a bottleneck in the therapeutic T cell engineering pipeline. The speed and control of our approach should enable valuable cell interaction studies.

High diversity droplet microfluidic libraries generated with a commercial liquid spotter.

Droplet libraries consisting of many reagents encapsulated in separate droplets are necessary for applications of microfluidics, including combinatorial chemical synthesis, DNA-encoded libraries, and massively multiplexed PCR. However, existing approaches for generating them are laborious and impractical. Here, we describe an automated approach using a commercial array spotter. The approach can controllably emulsify hundreds of different reagents in a fraction of the time of manual operation of a microfluidic device, and without any user intervention. We demonstrate that the droplets produced by the spotter are similarly uniform to those produced by microfluidics and automate the generation of a ~ 2 mL emulsion containing 192 different reagents in ~ 4 h. The ease with which it can generate high diversity droplet libraries should make combinatorial applications more feasible in droplet microfluidics. Moreover, the instrument serves as an automated droplet generator, allowing execution of droplet reactions without microfluidic expertise.

An Oil-Free Picodrop Bioassay Platform for Synthetic Biology.

Droplet microfluidics enables massively-parallel analysis of single cells, biomolecules, and chemicals, making it valuable for high-throughput screens. However, many hydrophobic analytes are soluble in carrier oils, preventing their quantitative analysis with the method. We apply Printed Droplet Microfluidics to construct defined reactions with chemicals and cells incubated under air on an open array. The method interfaces with most bioanalytical tools and retains hydrophobic compounds in compartmentalized reactors, allowing their quantitation.

Printed droplet microfluidics for on demand dispensing of picoliter droplets and cells.

Although the elementary unit of biology is the cell, high-throughput methods for the microscale manipulation of cells and reagents are limited. The existing options either are slow, lack single-cell specificity, or use fluid volumes out of scale with those of cells. Here we present printed droplet microfluidics, a technology to dispense picoliter droplets and cells with deterministic control. The core technology is a fluorescence-activated droplet sorter coupled to a specialized substrate that together act as a picoliter droplet and single-cell printer, enabling high-throughput generation of intricate arrays of droplets, cells, and microparticles. Printed droplet microfluidics provides a programmable and robust technology to construct arrays of defined cell and reagent combinations and to integrate multiple measurement modalities together in a single assay.

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers.

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Compact and modular multicolour fluorescence detector for droplet microfluidics.

Multicolour fluorescence detection is often necessary in droplet microfluidics, but typical detection systems are complex, bulky, and expensive. We present a compact and modular detection system capable of sub-nanomolar sensitivity utilizing an optical fibre array to encode spectral information recorded by a single photodetector.

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device.

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 µm to 50 µm in diameter while using large channels that are robust against fouling and clogging.

MP4, a vasodilatory PEGylated hemoglobin.

A vasodilatory hemoglobin (Hb)-based O(2) carrier (HBOC) has been developed by surface conjugation polyethylene glycol to tetrameric human Hb (MP4, Sangart, San Diego). Because the NO-binding kinetics of MP4 are similar to vasoconstrictive HBOCs, we propose that the decoupling of NO scavenging from vascular response is a consequence of MP4's high O(2) affinity (p50 = 5 mmHg) and unique surface chemistry. The release of ATP from erythrocytes is vasodilatory and the application of a high O(2) affinity HBOC minimizes ATP interference with intravascular ATP signaling. A second potential mechanism of action for MP4 involves the surface conjugation of polyethylene glycol (PEG) to tetrameric human Hb. It has been shown that the addition of unconjugated high molecular weight (Mw) PEG to cultured lung endothelial cells causes an immediate and significant reduction in endothelial permeability; an effect opposite to that of endothelial agonists such as cell-free Hb. It appears that some of the benefits of the PEG-endothelium interaction are carried onto molecules such as PEGylated Hb and PEGylated albumin, as demonstrated by favorable hemodynamic responses in vivo. PEGylation of ß93 cysteine residues, as in MP4, has also been reported to increase the nitrite reductase activity of Hb and enhance conversion of endogenous nitrite to bioactive NO.

Erythrocytic ATP release in the presence of modified cell-free hemoglobin.

The red blood cell (RBC) has been proposed as an O(2) sensor through a direct link between the desaturation of intracellular hemoglobin (Hb) and ATP release, leading to vasodilation. We hypothesized that the addition of cell-free Hb to the extracellular space provides a supplementary O(2) source that reduces RBC desaturation and, consequently, ATP release. In this study, the saturation of RBC suspensions was lowered by additions of deoxygenated hemoglobin-based oxygen carrier (HBOC) and then assayed for extracellular ATP. When an acellular human Hb intramolecularly cross-linked between alpha subunits (alphaalphaHb, p50 = 33 mmHg) was added to the red cell suspension, ATP production was significantly less than that in the presence of a lower p50 HBOC (Hb cross-linked between beta subunits, betabetaHb, p50 = 8 mmHg). These results provide a potential mechanism for the O(2) affinity of HBOCs to interfere with a vasodilatory signal.

A quantitative framework for the design of acellular hemoglobins as blood substitutes: implications of dynamic flow conditions.

The delivery of oxygen to tissue by cell-free carriers eliminates intraluminal barriers associated with red blood cells. This is important in arterioles, since arteriolar tone controls capillary perfusion. We describe a mathematical model for O(2) transport by hemoglobin solutions and red blood cells flowing through arteriolar-sized tubes to optimize values of p50, Hill number, hemoglobin molecular diffusivity and concentration. Oxygen release is evaluated by including an extra-luminal resistance term to reflect tissue oxygen consumption. For low consumption (i.e., high resistance to O(2) release) a hemoglobin solution with p50=15 mmHg, n=1, D(HBO2)=3 x 10(-7) cm(2)/s delivers O(2) at a rate similar to that of red blood cells. For high consumption, the p50 must be decreased to 5 mmHg. The model predicts that regardless of size, hemoglobin solutions with higher p50 will present excess O(2) to arteriolar walls. Oversupply of O(2) to arteriolar walls may cause constriction and paradoxically reduced capillary perfusion.