Thin-shelled double emulsion droplets and uses thereof

Abstract

Certain embodiments of the invention provide improved double emulsion droplets. Also provided are methods, systems and devices for making improved double emulsion droplets and methods of using the improved double emulsion droplets, e.g., to screen for desired antibodies.

Description

[0001] 07810.002W01

[0002] THIN-SHELLED DOUBLE EMULSION DROPLETS AND USES THEREOF

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] This application claims priority to United States Provisional Application Number 63 / 708,563 that was filed on October 17, 2024. The entire content of the application referenced above is hereby incorporated by reference herein.

[0005] BACKGROUND

[0006] Previous methods to create double emulsion droplets of suitable size for fluorescence- activated cell sorting (FACS) with high cell loading have not been demonstrated. A primary issue with loading cells into double emulsion droplets is the thickness of the oil shell, as the oil shell thickness takes away from volume for cells. Accordingly, improved methods to create double emulsion droplets, and improved double emulsion droplets, are needed.

[0007] SUMMARY

[0008] Accordingly, provided herein are improved double emulsion droplets. Also provided are methods, systems and devices for making improved double emulsion droplets and methods of using the improved double emulsion droplets, e.g., to screen for desired antibodies.

[0009] BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1. Figure 1A-1C depicts images of droplets according to certain embodiments of the invention. Figure 1 A provides images of certain droplets compared to traditional double emulsions. Figure IB provides results of quantitative comparisons of OD and ID of certain droplets of the invention in microns. Figure 1C provides one version of a microfluidic channel used to make droplets of the invention.

[0011] Figure 2. Figure 2 depicts a schematic example of functional workflows for certain embodiments of the invention (e.g., a 2 cell example).

[0012] Figure 3. Figure 3 depicts results demonstrating the lack of antibody leakage from droplets (see, e.g., Example 1).

[0013] Figure 4. Figure 4 depicts results demonstrating NF AT function in droplet with anti CD3 antibodies (see, e.g, Example 2).

[0014] Figure 5. Figure 5A-5B depicts results of demonstration of NF AT function in droplets with anti CD3 antibodies and optimization of CD28 concentration. In Figure 5A, GFP+% is the 07810.002W01 percentage of cells which are above the GFP threshold. In Figure 5B, MFI is mean fluorescence intensity (arbitrary units, AU), (see, e.g., Example 3)

[0015] Figure 6. Figure 6A-6B depicts results of optimization in plates (see, e.g., Example 4). In Figure 6A, GFP+% is the percentage of cells that are above the GFP threshold. In Figure 6B, MFI is mean fluorescence intensity (arbitrary units, AU). The X axis is concentration of optiprep, %.

[0016] Figure 7. Figure 7A-7E depicts results related to determining the optimal concentration of antibodies to be used in droplets (see, e.g, Example 5).

[0017] Figure 8. Figure 8 depicts results related to the optimization of conditions in drops and plates for functional screens (see, e.g, Example 6).

[0018] Figure 9. Figure 9A depicts NF AT activation in drops compared to NF AT activation in plates from one week earlier. Figure 9B depicts NF AT activation in drops compared to NF AT activation in plates from the same day.

[0019] Figure 10. Figure 10 (and see, e.g., Example 8) depicts sorting of droplets via FACS, including an image of a FACS Aria II flow cytometer, images of sorted drops, and an example of sorted populations enriched for hits.

[0020] DETAILED DESCRIPTION

[0021] Provided herein are improved double emulsion droplets. In certain embodiments, the invention provides a population of double emulsion droplets described herein that comprises a first population of desired cells.

[0022] In certain embodiments, the droplets have an oil shell that is less than 40% of the total droplet volume.

[0023] In certain embodiments, the droplets have a protein-impermeable shell.

[0024] In certain embodiments, the droplets have an antibody-impermeable shell.

[0025] In certain embodiments, the droplets have a DNA and RNA-impermeable shell.

[0026] In certain embodiments, the population comprises a second population of desired cells.

[0027] In certain embodiments, the population comprises a third population of desired cells.

[0028] In certain embodiments, the first, second and / or third population of desired cells is a population of antibody producing cells.

[0029] In certain embodiments, the first, second and / or third population of desired cells is a population of reporter cells.

[0030] In certain embodiments, the first, second and / or third population of desired cells is a 07810.002W01 population of cancer cells.

[0031] In certain embodiments, the droplets have an oil shell that is about 20-30% of the total droplet volume.

[0032] In certain embodiments, the droplets have an oil shell that is about 25-30% of the total droplet volume.

[0033] In certain embodiments, the droplets have an oil shell that is about 30-40% of the total droplet volume.

[0034] Also provided herein are devices and systems for making the double emulsion droplets described herein.

[0035] Also provided herein is the use of the double emulsion droplets described herein for screening antibodies.

[0036] Also provided herein are double emulsion droplets made by the processes described herein.

[0037] In certain embodiments, the range for the oil shell thickness is about 10 %-70 %, preferably 20 %-30 %, 25 %-30 %, or 30 %-40 % of the total droplet volume.

[0038] For a 55 um OD droplet (e.g., of a suitable size for sorting with a 130 um nozzle on a FACS Aria), the oil shell thickness may be approximately 4.5 um, 3 um, 2 um, or lum.

[0039] For a 40 um OD droplet (e.g. of a suitable size for sorting with a 100 um nozzle on a FACS Aria), the oil shell thickness may be approximately 3 um, 2.25 um, 1.5 um, or 0.75 um.

[0040] The size of the droplets can be, e.g., from 10-15 um up to about 150-200 um.

[0041] As described herein, the oil shell thickness and the droplet size contribute to the stability, cell loading efficiency, and overall performance (e.g., by affecting clogging).

[0042] The droplets described herein, and the methods of making the droplets (which may be referred to as Xcell Drops), are an improvement over conventional double emulsion droplets because they have a thinner oil shell that provides additional room for cells. Because screening throughput is proportional to the percentage of cells loaded with all the cells required for the screen, increasing cell loading in drops is very important. The thinner shells may also help Xcell Drops to take longer to settle in solution, improving sortability on FACS. Xcell Drops are designed to be compatible with antibody discovery assays.

[0043] As described herein, Xcell Drops are a novel type of thin-shelled double emulsion made with microfluidics, tailored specifically for drug discovery, and formulated to be compatible with single cells. Improving on traditional double emulsions, Xcell Drops have 2-3 times more internal volume for cells and will enable an increase in cell co-loading that will facilitate the 07810.002W01 screening of millions of antibodies for function, e.g., in a single day. Xcell Drops are made using techniques that increase cell loading while ensuring thin oil shells and sortability on FACS. Unlike alternative technologies prone to antibody leakage, such as hydrogels, core-shell particles, and nanovials, Xcell Drops have an antibody -impermeable shell that provides assay fidelity and minimizes false-positives. As described herein, Xcell Drops are designed to be compatible with commercial FACS instruments, thereby eliminating the need for expensive specialized droplet sorters and making high-throughput screening technology accessible across industry and academia.

[0044] An important goal of screening with Xcell Drops is to enable multiple drug candidates per program, thereby increasing the likelihood of success in clinical trials and bringing better therapies to patients.

[0045] Certain aspects of the current invention are described below, which can involve the following steps: prepare cells and reagents; make droplets; incubate droplets; screen and sort droplets; sequence antibodies from droplets.

[0046] Xcell Drops

[0047] Xcell Drops are double emulsions characterized as having thin oil shells that lead to higher cell loading and better sortability. They also prevent antibody and other large molecule leakage from droplets and can be used to perform high throughput screens of drug function. They are designed to be compatible with FACS and offer superior sortability.

[0048] Media composition

[0049] Similar to other double emulsions, Xcell Drops are composed of two fluids: an inner aqueous phase and an outer oil phase. The inner aqueous phase encapsulates the cells and screening reagents, while the oil phase separates the inner aqueous phase from the outer aqueous phase. Because the droplets are contained in an outer bulk aqueous phase, they are compatible with commercial FACS instruments, a significant advantage over alternative droplet types.

[0050] Surfactants can also be used in the inner aqueous phase, oil phase, and outer aqueous phase in order to stabilize the droplets and prevent breakup during droplet formation, incubation, and sorting. Surfactants should be chosen to provide maximum droplet stability as well as maximum cell viability and assay compatibility.

[0051] Shell thickness

[0052] Xcell Drops are characterized by a shell thickness that is decreased compared to typical double emulsions. A typical double emulsion would have an oil shell that is between 50% and 80% of the total droplet volume. In contrast, Xcell Drops have an oil shell that is less than 40% 07810.002W01 of the total droplet volume. The shell thickness is defined as ~~ where OD is the outer diameter of the droplet and ID is the inner diameter of the droplet inner aqueous core. The decreased shell thickness offers many beneficial properties for Xcell Drops. These include increased cell loading for a given OD, thanks to an increased ID that provides a larger volume for cells, as well as increased settling time that leads to better sortability.

[0053] Another key advantage of Xcell Drops is that they enhance cell loading compared to other double emulsions. For cell loading, having a larger droplet inner aqueous volume allows for more cells to be loaded into the drop, leading to a higher screening throughput. Due to the cubic scaling of droplet volume from droplet diameter, small differences in shell thickness can lead to a large difference in droplet inner aqueous volume. For a 55 pm OD droplet, a droplet with a 7.5 pm shell would have about half the internal aqueous volume of a droplet with a 2.5 pm shell. For two cell types loaded into these droplets at 1E7 cells / ml, the 7.5 pm shell droplet would have cell co-loading of only 7.7%, whereas the 2.5 pm shell droplet would have cell coloading of 19.1%, an increase of 2.5x.

[0054] Multiple components are involved in creating Xcell Drops with decreased shell thickness compared to traditional double emulsions that also have high cell loading. These include surfactant optimization, decreased cell clumping, device geometry, and choice of flow control, which are expanded on herein.

[0055] Molecule leakage properties

[0056] The oil shell of Xcell Drops prevents leakage of proteins, including antibodies, as well as RNA and DNA. The oils used prevent leakage of large molecules like proteins, RNA, and DNA. However, some small molecules may still pass through the oil, helping to equilibrate droplet pH, O2, CO2, and other important molecules inside of the droplet to the outer bulk media condition.

[0057] Xcell Drops should be incubated in an outer aqueous media that is as similar as possible to the inner aqueous media. For example, if the inner aqueous media contains media A and additive B, the outer aqueous media should also contain media A and additive B. This will help to provide additional small molecule nutrients to cells inside of the drop and avoid droplet breakup due to osmotic stress. However, it may be acceptable in certain embodiments and preferable in some to have additional surfactants in the outer aqueous media in order to prevent droplet breakup. Additionally, it may be acceptable in certain embodiments and preferable to exclude some large molecule reagents such that they do not permeate the oil shell, in order to increase droplet stability. Any additives outside of the drop should be compatible with the cells or assay occurring inside of the drop, as the additives may leak inside. 07810.002W01

[0058] Improvements in droplet settling time

[0059] The time for droplet settling is very important for performing droplet sorts, as when droplets settle they can clog the sorter, be sorted at inconsistent rates, or require constant replenishing that takes away from time sorting. Droplet settling time is proportional to the difference between the droplet density and media density.

[0060] The oil is the densest part of a double emulsion. HFE-7500 is the most common oil used for double emulsions and has a density of 1614 kg / m3, about 1.6x that of water. Because droplet volume scales with the cube of the droplet diameter, seemingly small changes to shell thickness lead to a disproportionately large change in oil volume, and thus a disproportionately large change in droplet density. For a droplet with a 55 pm outer diameter (OD), a change in shell thickness from 7.5 pm to 3.5 pm will lead to a density decrease from 1374 kg / m3to 1199 kg / m3, about a 15% difference in density. Decreasing the shell thickness further to 1.5 pm will lead to a further density decrease to 1087 kg / m3, for a total of 26% difference in density. ^particle Pmedia) <

[0061] The settling velocity ot a particle can be estimated as v = - - - where g

[0062] ^^media is the gravitational constant on Earth, D is the particle diameter, Pparticleand Pmediaare the density of the particle and media respectively, and the density of the media. It should be noted that this is an approximation of settling time but is a commonly used approximation in the literature. Using this equation, the settling time can be estimated of droplets of different shell thicknesses in a FACS tube used for sorting droplets, assuming a settling distance of 2 cm (corresponding to a typical amount of media in a FACS tube). For a 55 pm OD droplet with a

[0063] 7.5 pm shell, the settling time is calculated at 32 seconds, whereas for the same droplet with a

[0064] 3.5 pm shell would be 58 seconds, or a 1.5 pm shell at 126 seconds. These differences in settling time can be the difference in keeping the droplets suspended in between mixing, yielding improved sorting performance.

[0065] Instrument design to make Xcell Drops with thin shells and high cell loading Media overview and flowrates

[0066] A minimum of four media inlets should be utilized for the instrument to make Xcell Drops: outer continuous (or sheath) phase, oil phase, and two inner aqueous phases. Two inner aqueous phases are used to keep separate different components, like antibody-producing cells and function-reporting cells, prior to encapsulation in droplets.

[0067] The container volume of the outer continuous phase should be large, approximately 50 ml, to allow for droplet production over several hours at the higher flowrates required of the 07810.002W01 continuous phase. As the flowrates will be lower, the container volume of the oil phase can be lower, between 1 and 15 ml, and inner aqueous phases can also be lower, between 1 and 15 ml.

[0068] The inner aqueous phases can be mixed to keep cells in suspension, minimizing cell clumping and keeping the concentration of cells uniform throughput droplet creation. The phases can be mixed by any appropriate method (magnetic stirring, tube rotation, shaking, etc). A cell density matching reagent can be used to further keep cells in suspension during droplet creation. The density matching reagent can be used at as high of a concentration such that cell performance is not decreased and droplet stability remains high. One or more cell declumping reagents can also be added to minimize cell clumping.

[0069] Flowrates should be appropriately chosen to obtain highly stable and uniform droplets at the desired droplet size and shell thickness. For the inner aqueous phases and oil phase, the ratio of flowrates is approximately equal to the ratio of volumes of the inner aqueous phase and oil phase, which will control the oil shell thickness compared to the overall droplet size. For a given droplet ID and inner aqueous volume, the oil phase volume can be estimated with a mass balance by multiplying the inner aqueous phase volume by the ratio of the oil phase flowrate to the total of the two inner aqueous phase flowrates. By adding the volume of the oil shell to the volume of the inner aqueous phase, the droplet OD and shell thickness can then be estimated.

[0070] For a 55 pm OD Xcell Drop created with a 6.6 pl / min total inner aqueous phase flowrate, a 3.3 pl / min oil phase flowrate (50% of inner aqueous phase flowrate) would lead to an oil shell thickness of approximately 3.5 pm (6.4% of drop OD), whereas a 1.2 pl / min oil phase flowrate (18% of inner aqueous phase flowrate) would lead to an oil shell thickness of approximately 1.5 pm (2.7% of drop OD). For different droplet sizes, the ratio of flowrates to create a shell thickness of a given % of drop OD will remain the same.

[0071] The continuous phase flowrate should be between 5x and 50x, preferably between 5x and lOx, the flowrate of the total inner aqueous phase. It should be chosen so as to produce uniform droplets. Too low of a flowrate will often create droplets with two inner aqueous cores per droplet, while too high of a flowrate can lead to non-uniformity of droplets and excess oil droplets.

[0072] Media components of Xcell Drops

[0073] For the inner aqueous phase / dispersed phase: cell media base, cell density matching reagent (e.g., 0 - 30%), protein solution, like FBS or BSA (e.g., 0.1%, 1%, 10%), cells, surfactants, e.g., water-soluble, cell-compatible (e.g., 0%, 0.1%, 1%, 2%, 10%), other assay reagents, e.g., activating antibodies (e.g., anti-CD28 for CD3 NF AT activation screen), cell 07810.002W01 declumping reagents.

[0074] For the oil phase / shell phase: oil, e.g., O2, CO2, HsO+permeable, high solubility of O2, not antibody, protein, RNA, DNA permeable but with some small molecule permeability, e.g., a fluorinated oil like HFE-7500 or FC-40, or as another option mineral oil; surfactant, e.g., oilsoluble, cell-compatible. Does not affect cell or assay performance (e.g., 0.1%, 1%, 2%, 10%), such as RAN-008, Fluoro-surf, dSurf, Krytox, EA surfactant.

[0075] For the outer aqueous phase / continuous phase: cell media, which can be the same composition as inner aqueous phase, protein solution, which can be the same composition as inner aqueous phase, surfactant, which can be water-soluble, cell-compatible, e.g., typically higher than or equal to the surfactant composition inside the drop, e.g., 0.1%, 1%, 2%, 10%. In certain embodiments, the protein solution is not included, which may increase droplet stability.

[0076] Pressure driven flow

[0077] High control over flowrates is important when producing Xcell Drops. This is particularly true for the oil phase when operated at low flowrates, which helps to maintain uniform thin shells on the Xcell Drops. Pressure controllers from Fluigent have been used to maintain uniform flowrates, but those from Elveflow or an OEM could also work. The pressure controllers should operate with a maximum pressure of 2 bar at minimum, ideally at 7 bar or higher. The pressure source, such as an air compressor or house air-line, should be higher than the maximum pressure of the pressure controllers. The pressure controllers should have a feedback loop with flowrate controllers (Fluigent, Elveflow, OEM, etc) which control the pressure based on the measured flowrate to a desired setpoint flowrate.

[0078] Oil can first be allowed to wet the channel, prior to making Xcell Drops, by starting the oil flowrate at 2-1 Ox the final oil flow rate. If this is not done then transient wetting may occur and Xcell Drops may be non-uniform, inner aqueous phases be unmixed, and be of the incorrect size. Continuous and aqueous flow rates should start at the final flowrate for making Xcell Drops as the oil flow rate is slowly decreased. Once Xcell Drop production is steady at the desired flow rates, a few minutes should pass before collecting drops so that the outlet tubing can clear of droplets made while the flowrates are being varied.

[0079] Regarding the temperature incubation of medias, all or some of the medias used to create drops may be kept at 37°C improve cell viability and performance during Xcell Drop creation. Alternatively, all or some of the medias used to create drops may be kept at 0 - 10°C to improve cell viability while minimizing the creation of cellular products like antibodies, etc, prior to entering the Xcell Drops. 07810.002W01

[0080] Regarding chip design to make Xcell Drops with thin shells and high cell loading, with respect to the inlets and outlets: at least 3 inlets, comprised of connections for 1. Aqueous continuous phase, 2. Oil shell phase, 3. Aqueous inner phase. At least 1 outlet to collect created Xcell Drops.

[0081] Regarding the mixing of two or more aqueous inner phases prior to droplet creation, this could be done on-chip or via a T-junction with tubing immediately upstream of the chip. Separate incompatible components (e.g. antibody-producing cells and function-reporting cells; enzyme-producing cells and substrate; etc) will be mixed just prior to droplet generation to maximize assay fidelity.

[0082] Break-off of Xcell Drops: 1. The aqueous inner phase, containing assay reagents and cells, meets the oil shell phase. 2. A single emulsion drop forms with the aqueous inner phase encapsulated in the oil shell phase. 3. The single emulsion drop then meets the aqueous continuous phase. 4. An Xcell Drop forms with oil shell phase encapsulated by the aqueous continuous phase, which simultaneously encapsulates the aqueous inner phase. Alternatively, steps 1-4 can occur simultaneously, with single emulsion drop forming concurrently with the final Xcell Drop.

[0083] Minimizing wetting: in certain embodiments, during steps 1-2, the channel walls must be uniformly hydrophobic so that the oil shell phase will coat the channel and encapsulate the inner aqueous phase. During steps 3-4, in certain embodiments, the channel walls must be uniformly hydrophilic so that the aqueous continuous phase will coat the channel and encapsulate the outer shell phase (which encapsulates the inner aqueous phase). The channel base material can be chosen to be hydrophobic and selectively surface-coated at the location of steps 3-4 to be hydrophilic. Alternatively, the channel base material can be chosen to be hydrophilic and selectively surface-coated at the location of steps 1-2 to be hydrophobic. Alternatively the height and width dimensions of the channel can be chosen to be large enough such that wetting is unlikely to occur, preventing the need for surface coating.

[0084] Cell filters / cell declumping: cell filters should be placed upstream of droplet creation. Cell filters can be of any type commonly used in the literature, such as pillar arrays. The filters should be designed to capture at least any clumps larger than the nozzle size, to avoid clogging the droplet generator. Filters should have enough area to capture all the large cell clumps that would occur during the experiment. Cells can also be filtered manually before they are put into the device using a 30 pm filter, 70 pm filter, etc. Protocols which decrease cell clumping, including the use of cell declumping reagents, can be used to minimize the amount of clumps the 07810.002W01 device is exposed to.

[0085] Inertial microfluidics: inertial microfluidics techniques could be used to increase cell ordering prior to entering into drops, increasing cell loading frequency, or to help break up clumps into single cells.

[0086] Device dimensionless parameters: nozzle width (Or.); the nozzle width is the dominant parameter that controls droplet size. All other parameters can be defined as dimensionless parameters based off the nozzle width, e.g., for 50 pm Xcell Drops, Or. ~ 50 pm. Nozzle length (Or. L, norm Or.L / Or.) (e.g., 1.5, 3, 10, >10. Aspect Ratio (AR = H / Or.), defined by channel height (H), e.g., Inlet Aspect Ratio (ARin) or 0.5 - 1 and Outlet Aspect Ratio (ARout) of >2. Width of outlet after nozzle (Wout,norm = Wout / Or.), e.g., >2. Width of dispersed phase at nozzle (Wd,norm = Wd / Or.) (Dispersed phase is another word for inner aqueous phase) Ideally large to minimize clogging of cells upstream of the nozzle, e.g., greater than 1, e.g., greater than 3. Width of oil phase at nozzle (Wo,norm = Wo / Or.), e.g., 1. Width of continuous phase at nozzle (W c,norm Wc / Or.) (Continuous phase is another word for outer aqueous phase), e.g., 0.5, 1, 2.

[0087] Droplet screening

[0088] The following is a list of assays (non-exclusive) that Xcell Drops are or may be compatible with:

[0089] Fluorescence reporter of function. Ex. NF AT, NFkB, STAT3. Fluorescence reporter activated downstream of the biological pathway such that the cell creates a fluorescent reporter product. The fluorescent reporter product may either be stored inside the cell, changing the fluorescence of the cell, and / or be released into the Xcell Drop, changing the fluorescence of the entire drop. This may be done with engineered cell lines or engineered primary cells.

[0090] Binding. Drug binds to a target on a call, increasing fluorescence localized to the cell. Cytokine release. Drug increases or decreases cytokine production in the target cell. Cytokine is released from the cell and captured, increasing fluorescence.

[0091] Cell death. Drug kills the cell, allowing a reagent to enter through the dead cell membrane and make the dead cell fluorescent.

[0092] Internalization. Drug is internalized into the cell, making the cell fluorescent.

[0093] Enzyme function. Enzyme converts substrate into a product, thereby modifying the fluorescence properties of the substrate, typically by making it more fluorescence.

[0094] RNA expression. RNA expression levels are used to determine drug function. Technologies include Dropseq, Indrop, DARTseq, or any other technology for RNA expression.

[0095] Drug concentration. Screen and / or optimize drug production rate from cells. Find drugs 07810.002W01 with naturally high production rates for cell-based drug manufacturing. Know the drug concentration reporter cells are exposed to during other screens.

[0096] Specificity. Vary target antigen level. Vary cell and tissue types. Screen directly on heterogeneous patient primary cells. Measure toxicity and tissue localization. Perform functional screen on cells where function is not desired.

[0097] Multiplexed versions of any combination of these assays is also contemplated.

[0098] Optimization of cells and reagents for droplet based assays. Media conditions should be chosen to be suitable for all cell types and / or reagents present in the droplet, e.g., media should be suitable for both antibody-producing cells and function-reporting cells. Antibody-producing cells and function-reporting cells should be compatible and minimally interfere with each other.

[0099] Droplet sorting. Xcell Drops may be sorted on commercial FACS instruments. In general, for maximum sorting rates and sorting efficiency, the maximum OD should not be more than i the FACS nozzle diameter. For example, for a 130 pm nozzle the maximum OD of the drop should not be more than 65 pm. Xcell Drops may also be sorted on other commercial droplet sorters, such as those from Atrandi or Sphere Fluidics, or with a custom droplet sorter. Xcell Drops have a thin oil shell that may make them take longer to settle and clump during sorting. Xcell Drops can be sorted into individual wells, where they can be sequenced individually, or can be sorted into a single well for pooled sequencing or single cell sequencing, e.g. with lOx Genomics’ Chromium.

[0100] Antibody sequencing. Antibodies may be sequenced from Xcell Drops.

[0101] Droplet breaking for sequencing. Xcell Drops may be broken by any method which breaks a large percentage of drops while keeping RNA or DNA (depending on sequencing method) high. This includes placing Xcell Drops into a -80 Celsius freezer, allowing drops to freeze, and then thawing the drops such that the ice crystals will break the drops. This also includes using perfluoro-octanol to destabilize the droplet oil shell. After breakup the drops will release their contents and can be further processed for sequencing.

[0102] Cell recovery. Droplet breaking. Xcell Drops may be broken by any method which breaks a large percentage of drops while keeping cell viability high. This includes using perfluoro-octanol to destabilize the droplet oil shell.

[0103] Compound(s) that could be included (if applicable). Antibodies, antibody-drug conjugates, bispecifics, multispecifics, peptides, small molecules, large molecule (nucleotide, protein) to prevent leakage from drop, labeling with nucleotide would allow for sequencing of hits. 07810.002W01

[0104] Cell type(s) that may be used (if applicable):

[0105] • 1 antibody producing cell and 1 function reporting cell;

[0106] • 1 cell that both produces antibody and reports function;

[0107] • 1 cell that produces antibody and a separate reagent that reads out antibody function;

[0108] • 1 antibody producing cell and 1 function reporting cell with a separate reagent that reads out antibody function;

[0109] • 1 antibody producing cell, 1 function reporting cell (e.g. T cell), and 1 other participating cell (e.g. cancer cell) which participates in the assay but does not report function or make antibody;

[0110] • Function reporting cell can be cell line or primary cell and can be engineered or not engineered;

[0111] • Antibody producing cell can be cell line or primary cell (e.g. B cell) and can be engineered or not engineered;

[0112] • Other participating cell can be cell line or primary cell (e.g. patient cancer cell) and can be engineered or not engineered;

[0113] • Function reporting cell and drug produced via cell-free synthesis.

[0114] Cells can be engineered to produce more or less antibody to maximize ability to differentiate compounds. Cells can be engineered to have differing levels of target receptors to find compounds that are effective with more or less levels of receptors. Importantly, this can be used for a toxicity screen, where compounds should be more effective against cells with higher levels of the key target receptors and ineffective against cells with lower levels of the key target receptors. Primary cells can be used as reporter cells to find compounds that are effective against realistic target cells. If there are several antibody producing cells in the same drop, positive drops will bring some negative hits into the sorted fraction.

[0115] The term “Fc region” refers to a C-terminal region of an immunoglobulin heavy chain polypeptide. The Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. The term includes native sequence of Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at about position Cys226, or from about position Pro230, to the carboxyl-terminus of the Fc region (using herein the numbering system according 07810.002W01 to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991). The C-terminal lysine (Lys447) of the Fc region may or may not be present. One or more C-terminal residue(s) of Fc region may be absent or replaced by other amino acid substitution(s).

[0116] A “variant Fc” or “engineered Fc” refers to an Fc region that has been modified relative to a parent, native Fc region. A variant Fc may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising a modification or substitution at one or more amino acid positions in the Fc region. An engineered Fc also include modified glycosylation in the Fc region.

[0117] The terms “Fc receptor” or “FcR” refer to a receptor that binds to the Fc region. There are three main classes of Fc receptors: (1) FcyR which binds to IgG, (2) FcaR which binds to IgA, and (3) FcaR which binds to IgE. The FcyR family includes several members, such as Fcyl (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD16A), and FcyRIIIB (CD16B). The Fey receptors differ in their affinity for IgG and also have different affinities for the IgG subclasses (e.g., IgGl, IgG2, IgG3, and IgG4).

[0118] The term “antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to, chimeric antibody, humanized antibody, human antibody, monoclonal antibody, single-domain antibody, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigenbinding activity. An antibody may include a full-length immunoglobulin molecule or a portion of a full-length immunoglobulin molecule that contains an antigen binding site that specifically binds an antigen of a target of interest. The immunoglobulin can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule. The immunoglobulins can be derived from any species (e.g., human, or mouse). An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fab, Fab', Fab'-SH, F(ab')2 and scFv.

[0119] The term “antigen binding domain” or “binding domain” as used herein refers to one or more fragments of an antibody that retains the ability to specifically bind a target antigen. Examples of antigen binding domain includes, but not limited to, Fab (a monovalent fragment consisting of the VL, VH, CL and CHI domains), single-chain variable fragment (scFv), single- 07810.002W01 domain antibody (nanobody or VHH), a VL (light chain variable region), and a VH (heavy chain variable region).

[0120] A scFv is a fusion protein of the variable region of the heavy (VH) and light chains (VL) of an immunoglobulin that is connected by means of a linker peptide. The linker is usually short, about 10-25 amino acids in length. If flexibility is important, the linker will contain a significant number of glycines. If solubility is important, serines or threonines may be utilized in the linker. The linker may link the amino-terminus of the VH to the carboxy-terminus of the VL, or the linker may link the carboxy -terminus of the VH to the amino-terminus of the VL.

[0121] A Fv fragment is an antibody fragment, and contains a complete antigen recognition and binding site. The six CDRs of both the variable regions (VH and VL) interact with each other to form an antigen -binding site. However, a variable region (or a half Fv, which contains only three antigen-specific CDRs) alone is also known to be able to recognize and bind to an antigen, although its affinity is lower than the affinity of the entire binding site.

[0122] A Fab fragment (also referred to as F(ab)) also contains a light chain constant region and heavy chain constant region CHI. For example, papain digestion of an antibody produces two kinds of fragments: an antigen-binding fragment, called a Fab fragment, containing the variable regions of a heavy chain and light chain, which serve as an antigen-binding domain; and the remaining portion, which is called an “Fc” because it is readily crystallized. A Fab' fragment is different from a Fab fragment in that a Fab' fragment also has several residues derived from the carboxyl terminus of a heavy chain CHI region, which contains one or more cysteine residues from the hinge region of an antibody. A Fab' fragment is, however, structurally equivalent to Fab in that both are antigen-binding fragments which comprise the variable regions of a heavy chain and light chain. Herein, an antigen-binding fragment comprising the variable regions of a heavy chain and light chain which may serve as an antigen-binding domain, and which is equivalent to that obtained by papain digestion, is referred to as a “Fab-like antibody,” even when it is not identical to an antibody fragment produced by protease digestion. Fab'-SH is Fab' with one or more cysteine residues having free thiol groups in its constant region. A F(ab') fragment is produced by cleaving the disulfide bond between the cysteine residues in the hinge region of F(ab')2. Other chemically crosslinked antibody fragments are also known to those skilled in the art. Pepsin digestion of an antibody yields two fragments; one is a F(ab')2 fragment which comprises two antigen-binding domains, and the other is the remaining fragment (referred to as pFc'). Herein, an antibody fragment equivalent to that obtained by pepsin digestion is referred to as a “F(ab')2-like antibody” when it comprises two antigen- 07810.002W01 binding domains. Such antibody fragments can also be produced, for example, by genetic engineering.

[0123] A small, functional antibody known as a heavy-chain antibody (HCAb) was first reported in cam elid serum in 1993. Unlike conventional antibodies with a heterotetrametric structure, camelid-derived HCAb is devoid of light-chain polypeptides and lacks the first constant domain (CHI) in heavy-chains. The antigen-binding fragment in HCAbs contains only one single-variable domain. This domain is termed VHH and is also known as a single-domain antibody (sdAb) or nanobody (Nb). Uniquely, the monomeric state of these single domain antibodies provides the ability to recognize and bind antigens independently. With a smaller biomolecule in the size range of 12-15 kDa and a higher affinity and stability, single domain antibodies are contemplated for use herein.

[0124] The term “chimeric” antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species. In some embodiments, a chimeric antibody is a monoclonal antibody comprising a variable region from one source or species (e.g., mouse) and a constant region derived from a second source or species (e.g., human).

[0125] A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human framework regions (FRs). In certain embodiments, a humanized antibody will comprise at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non- human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.

[0126] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a group of substantially homogeneous antibodies, that is, an antibody group wherein the antibodies constituting the group are homogeneous except for naturally occurring mutants that may exist in a small amount. Monoclonal antibodies are highly specific and interact with a single antigenic site. Furthermore, each monoclonal antibody targets a single antigenic determinant (epitope) on an antigen, as compared to common polyclonal antibody preparations that typically contain various antibodies against diverse antigenic determinants. In addition to their specificity, monoclonal antibodies are advantageous in that they are typically produced from hybridoma cultures not contaminated with other immunoglobulins. 07810.002W01

[0127] The terms “identical” or percent “identity,” in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues, e.g., at least 60% identity, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater, that are identical over a specified region when compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using a sequence comparison algorithm or by manual alignment and visual inspection. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, Clustal X, Clustal W, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. The parameters employed for an alignment to achieve maximal alignment can be determined by one of skill in the art. For sequence comparison of polypeptide sequences for purposes of this application, the BLASTP algorithm standard protein BLAST for aligning two proteins sequence with the default parameters is used.

[0128] The terms “protein,” “peptide” and “polypeptide” are used interchangeably herein.

[0129] The term “variant” polypeptide refers to a polypeptide derived from the native protein but has substitution of one or more amino acids at one or more sites in the native protein, or deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and / or C -terminal end of the native protein; or deletion or addition of one or more amino acids at one or more sites in the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art.

[0130] Thus, the polypeptides of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the polypeptides can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art.

[0131] Individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in an encoded sequence are “conservatively modified variations,” where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following five groups each contain amino acids that are conservative substitutions for one 07810.002W01 another: Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine (R), Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q).

[0132] The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base which is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucl. Acids Res., 19:508 (1991); Ohtsuka et al., JBC, 260:2605 (1985); Rossolini et al., Mol. Cell. Probes, 8:91 (1994). A "nucleic acid fragment" is a fraction of a given nucleic acid molecule. Deoxyribonucleic acid (DNA) in the majority of organisms is the genetic material while ribonucleic acid (RNA) is involved in the transfer of information contained within DNA into proteins. The term "nucleotide sequence" refers to a polymer of DNA or RNA that can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers. The terms "nucleic acid," "nucleic acid molecule," "nucleic acid fragment," "nucleic acid sequence or segment," or "polynucleotide" may also be used interchangeably with gene, cDNA, DNA and RNA encoded by a gene.

[0133] A "variant" of a molecule is a sequence that is substantially similar to the sequence of the native molecule. For nucleotide sequences, variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis that encode the native protein, as well as those that encode a polypeptide having amino acid 07810.002W01 substitutions. Generally, nucleotide sequence variants of the invention will have at least 40, 50, 60, to 70%, e.g, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g, 81%-84%, at least 85%, e.g, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%, sequence identity to the native (endogenous) nucleotide sequence.

[0134] “Conservatively modified variations” of a particular nucleic acid sequence refers to those nucleic acid sequences that encode identical or essentially identical amino acid sequences, or where the nucleic acid sequence does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance the codons CGT, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded protein. Such nucleic acid variations are "silent variations" which are one species of "conservatively modified variations." Every nucleic acid sequence described herein which encodes a polypeptide also describes every possible silent variation, except where otherwise noted. One of skill will recognize that each codon in a nucleic acid (except ATG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule by standard techniques. Accordingly, each "silent variation" of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0135] The invention encompasses isolated or substantially purified nucleic acid or protein compositions. In the context of the present invention, an "isolated" or "purified" DNA molecule or an "isolated" or "purified" polypeptide is a DNA molecule or polypeptide that exists apart from its native environment and is therefore not a product of nature. An isolated DNA molecule or polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell. For example, an "isolated" or "purified" nucleic acid molecule or protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In one embodiment, an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. A protein that is substantially free of cellular material 07810.002W01 includes preparations of protein or polypeptide having less than about 30%, 20%, 10%, 5%, (by dry weight) of contaminating protein. When the protein of the invention, or biologically active portion thereof, is recombinantly produced, culture medium may represent less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein-of-interest chemicals. Fragments and variants of the disclosed nucleotide sequences and proteins or partial-length proteins encoded thereby are also encompassed by the present invention.

[0136] A “vector" is defined to include, inter alia, any plasmid, cosmid, phage or binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication).

[0137] "Expression cassette" as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a nontranslated RNA, in the sense or antisense direction. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development.

[0138] Such expression cassettes will comprise the transcriptional initiation region of the invention linked to a nucleotide sequence of interest. Such an expression cassette is provided with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.

[0139] "Promoter" refers to a nucleotide sequence, usually upstream (5') to its coding sequence, which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. "Promoter" includes a minimal 07810.002W01 promoter that is a short DNA sequence comprised of a TATA- box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression. "Promoter" also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements that is capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a DNA sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments. A promoter may also contain DNA sequences that are involved in the binding of protein factors that control the effectiveness of transcription initiation in response to physiological or developmental conditions.

[0140] "Expression" refers to the transcription and / or translation in a cell of an endogenous gene, transgene, as well as the transcription and stable accumulation of sense (mRNA) or functional RNA. In the case of antisense constructs, expression may refer to the transcription of the antisense DNA only. Expression may also refer to the production of protein.

[0141] The term "substantial identity" in the context of a peptide indicates that a peptide comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, at least 90%, 91%, 92%, 93%, or 94%, or 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window.

[0142] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that has the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0143] The invention will now be illustrated by the following non-limiting Examples.

[0144] Example 1.

[0145] To demonstrate the lack of antibody leakage between the Xcell Drops, a fluorescently- 07810.002W01 labeled antibody is introduced inside the Xcell Drops and fluorescence intensity is measured to determine if any fluorescence signal is transferred to Xcell Drops that never received antibody.

[0146] N-hydroxy-succinimidyl-ester (NHS) fluorescein (FITC) is prepared by weighing approximately 10 milligrams (mg) of lyophilized NHS-FITC and resuspending it in dimethylsulfoxide (DMSO). The final concentration of NHS-FITC resuspended in DMSO is 10 mg per milliliter (mL).

[0147] Anti-CD28 IgG antibody protein with a molecular weight of 150 kiloDaltons (kDa) is then labeled with the 10 mg per mL NHS-FITC DMSO solution. 100 microliters (pL) of unlabeled anti-CD28 IgG protein is added to Phosphate Buffered Saline (PBS), containing 50 millimolar (mM) NaHCO3, to a concentration of 1 mg per mL. A 20-fold molar excess of NHS- FITC is added to the anti-CD28 IgG antibody protein PBS-NaHCO3 solution. This mixture is then incubated for 1 hour at 20 °C in the absence of light. Following this incubation, the NHS- FITC anti-CD28 IgG protein solution is passed through a 50 kDa filter assembly that is centrifuged at 12,000 x gravity (g) for 10 minutes at 20 °C. PBS is added to the concentrator holding the 150kDa anti-CD28 IgG antibody protein and spun again at 12,000 x g for 10 minutes 20 °C. Media is then added to the concentrator to achieve an antibody concentration of approximately 1 mg per mL. Labeled anti-CD28 IgG protein can be stored short-term at 4 degrees Celsius or less than or equal to negative 20 degrees Celsius.

[0148] Xcell Drops with a diameter of approximately 55 micrometers (pm) are produced using a continuous flow rate of lOOpL per minute of 2 percent Poloxamer 188 suspended in cell media. The flow rate of the oil (2 percent RAN-008 in HFE-7500) is set to 2.5pL per minute and the flow rate of the inner aqueous is set to 3.3pL per minute, containing either cell media or cell media containing NHS-FITC-labeled anti-CD28 IgG protein. Drops containing only cell media and drops containing cell media containing NHS-FITC-labeled anti-CD28 IgG protein are mixed at a ~1:1 ratio and incubated overnight at 37 degrees Celsius.

[0149] Xcell Drop fluorescence intensity is measured at time 0 hour (immediately after mixing) and 24 hours post-mixing using fluorescence microscopy on a EVOS M7000 microscope and quantified in ImageJ. The intensity of the NHS-FITC-negative drops remained unchanged 24 hours after mixing. This indicates there is no detectable transfer of NHS-FITC-labeled anti- CD28 IgG protein to the Xcell Drops only containing cell media. There was a trend toward increased fluorescence in the NHS-FITC-labeled anti-CD28 IgG protein Xcell Drops 24 hours after mixing which can be attributed to changes in pH within the inner aqueous upon exposure to a lower pH of the sheath fluid. 07810.002W01

[0150] Example 2.

[0151] To demonstrate the ability to detect T cell activation in trans by an antibody delivered inside of a Xcell Drop, fluorescence of GFP produced under the control of an NF AT promoter in Jurkat cells in response to activating monoclonal antibodies is measured in both plate and Xcell Drop formats.

[0152] The Jurkat NFAT-GFP cell line is a human T cell line that contains green fluorescent protein (GFP) under the control of a Nuclear factor of activated T cells (NF AT) promoter. When a Jurkat NFAT-GFP cell receives an activation signal through T cell activation pathways initiating with surface receptors such as CD28 and CD3, the NF AT promoter induces the expression of GFP. GFP expression can then be measured by fluorescence microscopy to quantify the expression of the protein as a proxy for T cell activation.

[0153] To label Jurkat NFAT-GFP cells, cells are counted and resuspended in a solution of Phosphate Buffered Saline (PBS) containing 0.1 percent beta mercaptoethanol and a dilution (1 to 1000) of CellTrace Yellow (CTY). Jurkat NFAT-GFP cells are placed at 4 degrees Celsius in the absence of light for 20 minutes. Following CTY incubation, the dye to quenched with a solution of cell media (RPMI-ATCC Blend) containing 10 percent Fetal bovine serum (FBS) for 5 minutes at 20 degrees Celsius in the absence of light. The cell suspension is then centrifuged and resuspended in RPMI-ATCC Blend with 10 percent FBS with puromycin and counted.

[0154] For Xcell Drops:

[0155] Labeled Jurkat NFAT-GFP cells will be delivered in an “Aql” solution containing 15% Optiprep, 0.1 percent beta mercaptoethanol in cell media (RPMI-ATCC Blend with 10 percent FBS without puromycin). A separate “Aq2” solution containing antibody is mixed on chip at a 1 : 1 ratio with the Aql solution to prevent antibody from being exposed to cells prior to encapsulation in Xcell Drops.

[0156] After mixing labeled Jurkat NFAT-GFP cells with antibody and creating Xcell Drops there was a final concentration of (1) anti-CD3 antibody (OKT3) of 1 nanomolar (nM) and anti- CD28 antibody of 10 nM, or (2) IgG2a negative control antibody of InM and anti-CD28 antibody of lOnM.

[0157] A microfluidic chip is used for all Aql and Aq2 mixing and Xcell Drop formation. The flow rate of Aql is set to 3.3 microliters (pL) per minute. The flow rate of Aq2 is set to 3.3pL per minute. The flow rate of the sheath fluid is set to lOOpL per minute. The flow rate of the oil is set to 2.5pL per minute. 07810.002W01

[0158] For Plate:

[0159] The same Aql and Aq2 cell suspensions and solutions are combined in the same 1 to 1 ratio as used for the production of Xcell Drops. Aql is diluted such that the final cell concentration per well is 400,000 cells per milliliter (mL).

[0160] Following overnight incubation of both Xcell drops and plate containing labeled Jurkat NFAT-GFP cells and antibody combinations, cells are imaged on an EVOS M7000 microscope following transfer to microscope slides to minimize background fluorescence. At thresholds where the negative control (condition 2) showed 0 percent GFP positivity, corresponding to 100% screen specificity, the plate format demonstrated higher sensitivity to OKT3 antibody resulting in a GFP positivity of 25.8% compared to the Xcell drop format at 9.3%. These conditions would allow detection of T cell activation with sufficient sensitivity and specificity.

[0161] Example 3.

[0162] To determine the optimal level of T cell stimulation antibodies for the detection of T cell activation Xcell Drops, fluorescence of GFP produced under the control of an NF AT promoter in Jurkat cells in response to activating monoclonal antibodies is measured in a cell culture plate format.

[0163] To label Jurkat NFAT-GFP cells, cells are counted and resuspended in a solution of Phosphate Buffered Saline (PBS) containing 0.1 percent beta mercaptoethanol and a dilution (1 to 1000) of CellTrace Yellow (CTY). Jurkat NFAT-GFP cells are placed at 4 degrees Celsius in the absence of light for 20 minutes. Following CTY incubation, the dye is quenched with a solution of cell media (RPMI-ATCC Blend) containing 10 percent Fetal bovine serum (FBS) for 5 minutes at 20 degrees Celsius in the absence of light. The cell suspension is then centrifuged and resuspended in RPMI-ATCC Blend with 10 percent FBS with puromycin and counted.

[0164] Variables in this study are the concentration of anti-CD3 (OKT3) antibody (1 or 10 nanomolar (nM)) and anti-CD28 antibody (0,1,10,100, or 1000 nM). A corresponding negative control for OKT3 is used in this study (IgG2a) at the same concentrations. A positive control for NF AT activation condition is treated with 5 ng per mL of Phorbol 12-myristate 13-acetate (PMA) and 1 pM lonomycin. The combinations of the aforementioned variables creates a total of 21 conditions to be tested. Labeled Jurkat NFAT-GFP cells were mixed with the 16 conditions and plated at a final concentration of 428,000 cells per milliliter (mL).

[0165] Following overnight incubation of labeled Jurkat NFAT-GFP cells and antibody combinations, cells are imaged on an EVOS M7000 microscope following transfer to 07810.002W01 microscope slides to minimize background fluorescence. A threshold of 400 was used across all conditions to obtain percent GFP positivity. The positive control for NF AT activity resulted in a 59.5 percent GFP positivity. At InM of OKT3 in the absence of anti-CD28, Jurkat NFAT-GFP cells achieved 31.3 percent GFP. This increased at 1 and 10 nM anti-CD28 to 40.0 and 50.8 percent GFP, respectively. 100 and 1000 nM anti-CD28 yielded 39.7 and 47.3 percent GFP, respectively. At lOnM of OKT3 in the absence of anti-CD28, Jurkat NFAT-GFP cells achieved 32.9 percent GFP. This increased at 1 and 10 nM anti-CD28 to 42.9 and 45.7 percent GFP, respectively. 100 and 1000 nM anti-CD28 yielded 38.4 and 54.89 percent GFP, respectively. 10 and 1000 nM of anti-CD28 yielded similar GFP positivity for both 1 and 10 nM OKT3 antibody stimulation, thus lOnM anti-CD28 and 1 or lOnM OKT3 will be used in future experiments.

[0166] Example 4.

[0167] To determine the maximum level of density gradient reagent that allows for the detection of T cell activation Xcell Drops, fluorescence of GFP produced under the control of an NF AT promoter in Jurkat cells in response to activating monoclonal antibodies is measured in a cell culture plate format.

[0168] To label Jurkat NFAT-GFP cells, cells are counted and resuspended in a solution of Phosphate Buffered Saline (PBS) containing 0.1 percent beta mercaptoethanol and a dilution (1 to 1000) of CellTrace Yellow (CTY). Jurkat NFAT-GFP cells are placed at 4 degrees Celsius in the absence of light for 20 minutes. Following CTY incubation, the dye is quenched with a solution of cell media (RPMI-ATCC Blend) containing 10 percent Fetal bovine serum (FBS) for 5 minutes at 20 degrees Celsius in the absence of light. The cell suspension is then centrifuged and resuspended in RPMI-ATCC Blend with 10 percent FBS with puromycin and counted.

[0169] Variables in this study are the concentration of Optiprep (0,10,15, and 20 percent) and the concentration of anti-CD3 (OKT3) antibody (1 or 10 nanomolar (nM)). A corresponding negative control for OKT3 is used in this study (IgG2a) at the same concentrations. Anti-CD28 antibody is included in all conditions at a concentration of 10 nM. The combinations of the aforementioned variables creates a total of 16 conditions to be tested. Labeled Jurkat NFAT- GFP cells were mixed with the 16 conditions and plated at a final concentration of 433,000 cells per milliliter (mL).

[0170] Following overnight incubation of labeled Jurkat NFAT-GFP cells and antibody combinations, cells are imaged on an EVOS M7000 microscope following transfer to microscope slides to minimize background fluorescence. A threshold of 400 was used across all 07810.002W01 conditions to obtain percent GFP positivity. With respect to the concentration of Optiprep used, stimulation of Jurkat NFAT-GFP cells with lOnM of OKT3 in conjunction with lOnM anti- CD28 antibody resulted in a maximal GFP positivity of approximately 65% in the absence of Optiprep. With InM of OKT3 in conjunction with lOnM anti-CD28 antibody stimulation resulted in a maximal GFP positivity of approximately 54% in the absence of Optiprep. For both 1 and 10 nM of OKT3 stimulation with 10 nM anti-CD28, the GFP positivity decreased with the addition of Optiprep between 10 - 20% to a range of 29 - 39% GFP positivity with 1 nM OKT3 and a range of 37 - 40% with 10 nM OKT3. The GFP positivity appeared to plateau at the range of 10 - 20% Optiprep, without a distinct trend with increasing Optiprep. As 15 percent Optiprep gives better cell suspension than lower Optiprep concentrations, this concentration is selected for future experiments.

[0171] Example 5.

[0172] A purpose of this Example was to determine the NF AT activation of antibodies in a plate experiment, to compare with Xcell Drop conditions. This includes anti-CD3 antibodies such as OKT3, EHT3a, UCHT1, and SK7 as well as an anti-CD28 antibody.

[0173] Unstained NFAT-luc-GFP cell line cultured in RPMI-ATCC blend media containing 0.1% beta mercaptoethanol (BME) were plated at a concentration of 500,000 cells per milliliter (mL) in a relative volume of 6 microliters (uL) per well in a 96-well U-bottom plate. Optiprep is added to a final concentration of 15 percent for all wells.

[0174] In “Plate 1,” wells A1-A5 contain cells treated with OKT3 antibody at a concentration range of .01 to 100 nanomolar (nM), in ten-fold increments, without anti-CD28 antibody present. Wells B1-B5 contain cells treated with HIT3a antibody at a concentration range of .01 to 100 nM, in ten-fold increments, without anti-CD28 antibody present. Wells C1-C5 contain cells treated with UCHT1 antibody at a concentration range of .01 to 100 nM, in ten-fold increments, without anti-CD28 antibody present. Wells D1-D5 contain cells treated with SK7 antibody at a concentration range of .01 to 100 nM, in ten-fold increments, without anti-CD28 antibody present. Wells El -5 contain cells treated with a IgG2a isotype antibody (negative control antibody) at a concentration range of .01 to 100 nM, in ten-fold increments, without anti- CD28 antibody present.

[0175] In “Plate 1,” Wells A7-11 contain cells treated with OKT3 antibody at a concentration range of .01 to 100 nM, in ten-fold increments, with 1 nanogram (ng) per mL anti-CD28 antibody present. Wells B7-B11 contain cells treated with HIT3a antibody at a concentration 07810.002W01 range of .01 to 100 nM, in ten-fold increments, with 1 ng per mL anti-CD28 antibody present. Wells C7-11 contain cells treated with UCHT1 antibody at a concentration range of .01 to 100 nM, in ten-fold increments, with 1 ng per mL anti-CD28 antibody present. Wells D7-D11 contain cells treated with SK7 antibody at a concentration range of .01 to 100 nM, in ten-fold increments, with 1 ng per mL anti-CD28 antibody present. Wells E7-11 contain cells treated with a negative control antibody at a concentration range of .01 to 100 nM, in ten-fold increments, with 1 ng per mL anti-CD28 antibody present.

[0176] In “Plate 2,” wells A1-A5 contain cells treated with OKT3 antibody at a concentration range of .01 to 100 nM, in ten-fold increments, with 10 ng per mL anti-CD28 antibody present. Wells B1-B5 contain cells treated with HIT3a antibody at a concentration range of .01 to 100 nM, in ten-fold increments, with 10 ng per mL anti-CD28 antibody present. Wells C1-C5 contain cells treated with UCHT1 antibody at a concentration range of .01 to 100 nM, in ten-fold increments, with 10 ng per mL anti-CD28 antibody present. Wells D1-D5 contain cells treated with SK7 antibody at a concentration range of .01 to 100 nM, in ten-fold increments, with 10 ng per mL anti-CD28 antibody present. Wells El-5 contain cells treated with a negative control antibody at a concentration range of .01 to 100 nM, in ten-fold increments, with 10 ng per mL anti-CD28 antibody present.

[0177] In “Plate 2,” wells A7-9 contain triplicate replicates of cells not treated without positive or negative antibodies. Wells B7-B9 contain triplicate replicates of cells treated with only 1 ng per mL of anti-CD28 antibody. Wells C7-C9 contain triplicate replicates of cells treated with only 10 ng per mL of anti-CD28 antibody. Wells D7-D9 contain triplicate replicates of cells treated with 5 ng per mL phorbol myristate acetate (PMA) and 1 uM ionomycin.

[0178] Plates are incubated overnight at 37 degrees Celsius before being analyzed on a Fortessa cytometer for GFP expression. Jurkat NFAT-luc-GFP cells were not stained prior with a CellTrace dye and solely gated on forward and side scatter to identify the population.

[0179] A dose-response curve of the percentage of NF AT activation, as measured by GFP expression, by flow cytometry gating was determined. Positive antibodies OKT3, HIT3a, UCHT1, and SK7 and negative antibody IgG2a were added to Jurkat NFAT-luc-GFP cells at concentrations of .01, 0.1, 1, 10 and 100 nM. The negative antibody had no appreciable activation of NF AT while OKT3, HIT3a, UCHT1, and SK7 exhibited varying degrees of activation over the range of antibody concentrations.

[0180] When comparing GFP expression between samples of varying anti-CD28 antibody concentration, a slight reduction in the half maximal effective concentration (EC50) (measured 07810.002W01 between 0.01 and 1 nM positive antibody) was seen in OKT3, HIT3a, UTCH1, and SK7 antibodies when combined with either 1 and 10 ng per mL anti-CD28 antibody.

[0181] When comparing the degree of NF AT activation (through GFP expression) by anti-CD3 antibodies, OKT3 produced the highest percentage of GFP positive cells, followed by HIT3a, UTCH1, and lastly SK7. This held true for .01, .1, and 1 nM antibody concentrations.

[0182] The results of this Example allow for the use of anti-CD28 antibody at a concentration of 10 ng per mL for future experiments with anti-CD3 antibodies.

[0183] Example 6.

[0184] To optimize conditions for antibody screening in Xcell drops, several variables will be evaluated. Sheath fluid with RPMI versus thaw media (consisting of RPMI-ATCC blend without puromycin) will be compared to evaluate the level of GFP positivity resulting from positive antibody hits. The anti-CD3 antibody OKT3 will be compared at two concentrations, 0.1 nanomolar (nM) and 1 nM, previously used in plate format to evaluate the level of GFP positivity resulting from positive antibody hits. Tubing used in the PDMS device will be evaluated to prevent displacement of tubing during operations of the machine during droplet formation. Lastly, varying temperatures during the experiment will be compared to evaluate the effect on GFP positivity resulting from positive antibody hits.

[0185] To label Jurkat NFAT-GFP cells, cells are gently pipetted several times to reduce cell clumping. Jurkat NFAT-GFP cells are then counted, centrifuged at 300 x gravity (g) at 20 degrees Celsius for 5 minutes. The cells are resuspended in a solution of Phosphate Buffered Saline (PBS) containing 0.1 percent beta mercaptoethanol (BME) and either a dilution (1 to 1000) of CellTrace Yellow (CTY). Jurkat NFAT-GFP cells are placed at 4 degrees Celsius in the absence of light for 20 minutes.

[0186] Following CTY incubation, labeled Jurkat NFAT-GFP cells are washed with the same staining volume with thaw media (RPMI - ATCC blend containing 10 percent Fetal Bovine Serum (FBS)). to quench the dye. Cells are then spun down at 300 x g for 5 minutes at 20 degrees Celsius. Cells are resuspended in .5 to 1 milliliter (mL) of the thaw media and are counted.

[0187] To generate droplets, 4 conditions are tested labeled “Gl”, “G2”, “G3”, and “G4 ” Condition Gl contains an aqueous solution 1 (Aql) composed of 4 million Jurkat NF AT-luc- GFP cells labeled with CellTrace Yellow, 15 percent Optiprep, 0.1 percent beta mercaptoethanol (BME), and thaw media. Gl aqueous solution 2 (Aq2) is composed of 2 nM OKT3 positive 07810.002W01 antibody, 20 nM anti-CD28 positive antibody, 15 percent Optiprep, and thaw media. Sheath fluid for condition G1 is composed of 2 percent Pol oxamer 188 in RPMI media. Condition G2 contains an aqueous solution 1 (Aql) composed of 4 million Jurkat NFAT-luc-GFP cells labeled with CellTrace Yellow, 15 percent Optiprep, 0.1 percent beta mercaptoethanol (BME), and thaw media. G1 aqueous solution 2 (Aq2) is composed of 2 nM OKT3 positive antibody, 20 nM anti- CD28 positive antibody, 15 percent Optiprep, and thaw media. Sheath fluid for condition G1 is composed of 2 percent Pol oxamer 188 in thaw media. Condition G3 contains an aqueous solution 1 (Aql) composed of 4 million Jurkat NFAT-luc-GFP cells labeled with CellTrace Yellow, 15 percent Optiprep, 0.1 percent beta mercaptoethanol (BME), and thaw media. G1 aqueous solution 2 (Aq2) is composed of 0.2 nM OKT3 positive antibody, 20 nM anti-CD28 positive antibody, 15 percent Optiprep, and thaw media. Sheath fluid for condition G1 is composed of 2 percent Pol oxamer 188 in RPMI media. Condition G4 contains an aqueous solution 1 (Aql) composed of 4 million Jurkat NFAT-luc-GFP cells labeled with CellTrace Yellow, 15 percent Optiprep, 0.1 percent beta mercaptoethanol (BME), and thaw media. G1 aqueous solution 2 (Aq2) is composed of 0.2 nM OKT3 positive antibody, 20 nM anti-CD28 positive antibody, 15 percent Optiprep, and thaw media. Sheath fluid for condition G1 is composed of 2 percent Pol oxamer 188 in thaw media.

[0188] Droplet flow rate for conditions Gl, G2, G3, and G4 are 3.3 microliter (uL) per minute for Aql, 3.3 uL per minute for Aq2, 100 uL per minute for the sheath fluid, and 4 uL per minute for oil.

[0189] For plate conditions (specifically testing the effect of temperature on GFP positivity), a cell suspension of Jurkat NFAT-luc-GFP cells at a concentration of 800,000 cells per mL is mixed at a 1 : 1 ratio with the above Aq2 solutions, individually. IOOUL of the combined solutions are dispensed per well. After overnight incubation, cells were imaged on a EVOS M7000 microscope for GFP expression and quantified with ImageJ.

[0190] When comparing sheath fluid with RPMI versus thaw media, RPMI sheath fluid gave similar GFP positivity compared to thaw media. Droplet conditions Gl and G3 with RPMI sheath had GFP positivity of 42.59 and 25.0 percent, respectively. Conditions G2 and G4 with thaw media sheath had GFP positivity of 35.56 and 24.00 percent, respectively. When comparing the effect of varying experimental temperatures on GFP positivity from the plate format, cells kept on ice had the highest overall GFP positivity for 0.1 and 1 nM OK3 antibody concentrations (42.31 and 60.42 percent, respectively). Cells kept at 20 degrees Celsius had the second highest overall GFP positivity for 0.1 and 1 nM OK3 antibody concentrations (26.83 and 07810.002W01

[0191] 48.57 percent, respectively). Cells kept at 37 degrees Celsius had the lowest overall GFP positivity for 0.1 and 1 nM OK3 antibody concentrations (13.04 and 29.82 percent, respectively). Overnight viabilities for all conditions were above 82 percent.

[0192] Example 7.

[0193] To compare the results for multiple antibody conditions between Xcell Drops and plates, using purified antibodies as a proof-of-concept, three anti-CD3 antibodies (positive antibodies) and one IgG2a isotype antibody (negative antibody) are used in this Example. Clone and concentrations of antibodies were determined from previous studies.

[0194] To label Jurkat NFAT-GFP cells, cells are gently pipetted several times to reduce cell clumping. Jurkat NFAT-GFP cells are then counted, centrifuged at 300 x gravity (g) at 20 degrees Celsius for 5 minutes. The cells are resuspended in a solution of Phosphate Buffered Saline (PBS) containing 0.1 percent beta mercaptoethanol (BME) and either a dilution (1 to 1000) of CellTrace Yellow (CTY) or a dilution (1 to 1000) of CellTrace Deep Red (CTDR). Jurkat NFAT-GFP cells are placed at 4 degrees Celsius in the absence of light for 20 minutes.

[0195] Following CTY and CTDR incubation, labeled Jurkat NFAT-GFP cells are washed with the same staining volume with RPMI - ATCC blend containing 10 percent Fetal Bovine Serum (FBS) to quench the dye. Cells are then spun down at 300 x g for 5 minutes at 20 degrees Celsius. Cells are resuspended in .5 to 1 milliliter (mL) of RPMI- ATCC blend and are counted.

[0196] To generate droplets, 8 conditions are tested labeled “Gl”, “G2”, “G3”, “G4”, “G5”, “G6”, “G7”, and “G8”. Condition Gl contains an aqueous solution 1 (Aql) composed of 2 million Jurkat NFAT-luc-GFP cells labeled with CellTrace Yellow, 15 percent Optiprep, 0.1 percent BME, and thaw media. Gl aqueous solution 2 (Aq2) is composed of 2 nM OKT3 positive antibody, 20 nM anti-CD28 positive antibody, 15 percent Optiprep, and thaw media. Sheath fluid for condition Gl is composed of 2 percent Pol oxamer 188 in RPMI media. Condition G2 contains Aql composed of 2 million Jurkat NFAT-luc-GFP cells labeled with CellTrace Yellow, 15 percent Optiprep, 0.1 percent BME, and thaw media. G2 Aq2 is composed of 2 nM UCHT1 positive antibody, 20 nM anti-CD28 positive antibody, 15 percent Optiprep, and thaw media. Sheath fluid for condition G2 is composed of 2 percent Pol oxamer 188 in RPMI media. Condition G3 contains Aql composed of 2 million Jurkat NFAT-luc-GFP cells labeled with CellTrace Yellow, 15 percent Optiprep, 0.1 percent BME, and thaw media. G3 Aq2 is composed of 2 nM SK7 positive antibody, 20 nM anti-CD28 positive antibody, 15 percent Optiprep, and thaw media. Sheath fluid for condition G3 is composed of 2 percent Poloxamer 07810.002W01

[0197] 188 in RPMI media. Condition G4 contains Aql composed of 1 million Jurkat NFAT-luc-GFP cells labeled with CellTrace Deep Red, 15 percent Optiprep, 0.1 percent BME, and thaw media. G4 Aq2 is composed of 2 nM IgG2a negative antibody, 20 nM anti-CD28 positive antibody, 15 percent Optiprep, and thaw media. Sheath fluid for condition G4 is composed of 2 percent Poloxamer 188 in RPMI media. Condition G5 contains an aqueous solution 1 (Aql) composed of 2 million Jurkat NFAT-luc-GFP cells labeled with CellTrace Yellow, 15 percent Optiprep, 0.1 percent beta mercaptoethanol (BME), and thaw media. G5 Aq2 is composed of 0.2 nM OKT3 positive antibody, 20 nM anti-CD28 positive antibody, 15 percent Optiprep, and thaw media. Sheath fluid for condition G5 is composed of 2 percent Poloxamer 188 in RPMI media. Condition G6 contains Aql composed of 2 million Jurkat NFAT-luc-GFP cells labeled with CellTrace Yellow, 15 percent Optiprep, 0.1 percent BME, and thaw media. G6 Aq2 is composed of 0.2 nM UCHT1 positive antibody, 20 nM anti-CD28 positive antibody, 15 percent Optiprep, and thaw media. Sheath fluid for condition G6 is composed of 2 percent Poloxamer 188 in RPMI media. Condition G7 contains Aql composed of 2 million Jurkat NFAT-luc-GFP cells labeled with CellTrace Yellow, 15 percent Optiprep, 0.1 percent BME, and thaw media. G7 Aq2 is composed of 0.2 nM SK7 positive antibody, 20 nM anti-CD28 positive antibody, 15 percent Optiprep, and thaw media. Sheath fluid for condition G7 is composed of 2 percent Poloxamer 188 in RPMI media. Condition G8 contains Aql composed of 1 million Jurkat NFAT-luc-GFP cells labeled with CellTrace Deep Red, 15 percent Optiprep, 0.1 percent BME, and thaw media. G8 Aq2 is composed of 0.2 nM IgG2a negative antibody, 20 nM anti-CD28 positive antibody, 15 percent Optiprep, and thaw media. Sheath fluid for condition G8 is composed of 2 percent Poloxamer 188 in RPMI media.

[0198] Droplet flow rate for conditions Gl, G2, G3, G4, G5, G6, G7, G8 are 3.3 microliter (uL) per minute for Aql, 3.3 uL per minute for Aq2, 100 uL per minute for the sheath fluid, and 4 uL per minute for oil.

[0199] FACS sorting is performed on a FACS Aria Fusion with a 130 micron (pm) nozzle, and a neutral density filter of 1.5. Sorting is performed into 96-well plates containing lOOuL of PBS. Once gates have been set to capture GFP, CTY, and CTDR, drop delay is optimized to maximize sort efficiency. A drop delay of 16.8 was selected after testing a range of drop delays from 16.3 to 17.3 where 16.8 gave the highest sort efficiency of 68 percent. Comparing all the drop delays tested, sort efficiency ranged from 26 to 68 percent. Drop size was approximately 55pm outside diameter / 45pm inside diameter / 5 pm ST.

[0200] For plate conditions, a cell suspension of Aql Jurkat NFAT-luc-GFP cells stained with 07810.002W01

[0201] CellTrace Yellow at a concentration of 800,000 cells per mL is mixed with Aq2 in the plate for a final concentration of 400,000 cells per mL. Additionally, a cell suspension of Aql Jurkat NFAT-luc-GFP cells stained with CellTrace Deep Red at a concentration of 800,000 cells per mL is mixed with Aq2 in the plate for a final concentration of 400,000 cells per mL.

[0202] Plate samples were read after overnight incubation on the EVOS M700 microscope. Plate samples were then run on the FACS Aria II for cell sorting and analysis.

[0203] The percentage of cells from Xcell drops containing a positive antibody (e.g. OKT3, UCHT1, or SK7) and labeled with CellTrace Yellow were determined, before and after flow cytometry sorting. The percentages are derived from scanning fluorescence microscopy and ImageJ analysis of CellTrace Yellow and CellTrace Deep Red cells. Post sorting, all conditions with positive antibodies at 0.1 or 1 nM concentrations had an increase in the proportion of cells with positive antibodies.

[0204] The comparison of dose-response curves from positive antibodies OKT3, UCHT1, and SK7 at InM and 0.1 nM concentrations from this Example and the previous Example were determined. We observed good correlation between NF AT signaling in Xcell Drops and plates, both when droplet data was compared to plate data from the 9 / 16 and 9 / 9 data sets.

[0205] Example 8.

[0206] This Example was designed to optimize enrichment of droplets containing cells via FACS sorting based on previous droplet stability study results.

[0207] To label Jurkat NF AT-GFP cells, cells are gently pipetted several times to reduce cell clumping. Jurkat NF AT-GFP cells are then counted, centrifuged at 300 x gravity (g) at 20 degrees Celsius for 5 minutes. The cells are resuspended in a solution of Phosphate Buffered Saline (PBS) containing 0.1 percent beta mercaptoethanol (BME) and either a dilution (1 to 1000) of CellTrace Yellow (CTY) or a dilution (1 to 1000) of CellTrace Deep Red (CTDR). Jurkat NF AT-GFP cells are placed at 4 degrees Celsius in the absence of light for 20 minutes.

[0208] Following CTY and CTDR incubation, labeled Jurkat NF AT-GFP cells are washed with the same staining volume with thaw media to quench the dye. Mix the cells and incubate for 5 minutes at 20 degrees Celsius in the absence of light. Cells are then spun down at 300 x g for 5 minutes at 20 degrees Celsius. Cells are resuspended in .5 to 1 milliliter (mL) of thaw media and are counted.

[0209] To generate droplets, 2 conditions are tested with “Gl” pertaining to the InM OKT3- containing condition and “G2” pertaining to the IgG2a negative control. Condition “Gl” 07810.002W01 contains Aql, composed of 2 million NFAT-luc-GFP cell stained with either CTY or CTDR, 15 percent Optiprep, .1 percent BME in RPMI-ATCC Blend and Aq2, composed of 2nM anti-CD3 OKT3 antibody, 20nM anti-CD28 antibody, 15 percent Optiprep in RPMI-ATCC blend media. The sheath fluid composition for condition “Gl” is 2 percent Poloxamer 188 in RPMI-ATCC blend media. Condition “G2” contains Aql, composed of 1 million NFAT-luc-GFP cell stained with CTY, 15 percent Optiprep, .1 percent BME in RPMI-ATCC Blend and Aq2, composed of 2nM IgG2a antibody, 20nM anti-CD28 antibody, 15 percent Optiprep in RPMI-ATCC blend media. The sheath fluid composition for condition “G2” is 2 percent Poloxamer 188 in RPMI- ATCC blend media. Droplet flow rates for both Gl and G2 conditions are 3.3 uL per mL for Aql and Aq2, lOOuL per minute for the sheath flow rate, and 4ul per minute for the oil flow rate.

[0210] FACS sorting is performed on a FACS Aria Fusion with a 130 micron (pm) nozzle, and a neutral density filter of 1.5. Sorting is performed into 96-well plates containing 100 uL of PBS. Once gates have been set to capture GFP, CTY, and CTDR, drop delay is optimized to maximize sort efficiency. A drop delay of 16.5 was selected after testing a range of drop delays from 16.3 to 17.3 where 16.5 gave the highest sort efficiency of 72 percent. Sort efficiency for the experiment ranged from 50 to 75 percent. Drop size was approximately 55pm outside diameter / 45pm inside diameter / 5 pm ST.

[0211] To enrich droplets with stained cells, condition “Gl” cells were used. 2000 drops were sorted which yielded 984 drops post-sort that contain cells (49.2 percent recovery). To sort for GFP+ drops, drops from conditions Gl and G2 were mixed at either a 1 to 10 or 1 to 100 ratio (G1 / G2). For drops mixed at a 1 to 10 ratio, 9.85 percent of drops in the mix were from Gl (containing anti-CD3 OKT3 antibody) via detection of CTDR. 1000 drops were sorted for GFP+ and a subset of images were quantified for enrichment of Gl drops, resulting in 150 out of 201 quantified post-sort drops with CTDR (75 percent Gl drops post-sort). This is a 7.6-fold enrichment of Gl drops over the pre-sort condition, demonstrating an enrichment of anti-CD3 OKT3 antibody. A more stringent GFP+ gating scheme for drops mixed at a 1 to 10 ratio resulted in 181 drops sorted, with 64 out of 72 post-sort drops with CTDR (88.9 percent Gl post-sort). This is a 9-fold enrichment of drops with anti-CD3 OKT3 antibody over the pre-sort condition. For drops mixed at a 1 to 100 ratio, 2.1 percent of drops in the mix contained anti- CD3 OKT3 antibody via detection of CTDR. 256 drops were sorted resulting in 61 out of 99 post-sort drops with CTDR (61.6% percent Gl post-sort). This is a 29.3-fold enrichment of drops with anti-CD3 OKT3 antibody over the pre-sort condition. For the standard GFP+ gate, 07810.002W01 the true positive rate (TPR) for anti-CD3 OKT3 antibody in this experiment was approximately

[0212] 23 percent with a false positive rate (FPR) for negative control IgG2a antibody of approximately .5 to 1 percent for the GFP+ gate.

[0213] Example 9.

[0214] The DNA product from Xcell Drop screening and sequencing can be used as an input to create new cell lines for additional follow up screens. This minimizes the cost and time of additional cell engineering steps and can enable low throughput screens in other formats.

[0215] One embodiment to enable further low throughput screens is as follows:

[0216] 1. Isolate individual Xcell Drops from positive functional screens by sorting individual drops into wells in a 96 well or 384 well plate.

[0217] 2. Lyse individual functionally positive Xcell Drops by using freeze-thaw, drying, perfluoro-octanol, or another method.

[0218] 3. Capture mRNA from the cell and reverse transcribe to create DNA.

[0219] 4. PCR amplify Variable Heavy (VH) and Variable Light (VL) genes using degenerate or family-specific primers that flank the framework regions.

[0220] 5. Clone VH and VL PCR products into dual mammalian cell expression plasmids or lentiviral plasmids.

[0221] 6. Engineer antibody-producing cell lines using created plasmids or lentivirus.

[0222] 7. Perform further screens using new antibody-producing cell lines.

[0223] All publications, patents, and non-patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

CLAIMSWhat is claimed is:

1. A population of double emulsion droplets described herein that comprises a first population of desired cells.

2. The population of double emulsion droplets of claim 1, wherein the droplets have an oil shell that is less than 40% of the total droplet volume.

3. The population of double emulsion droplets of claim 1 or 2, wherein the droplets have a protein-impermeable shell.

4. The population of double emulsion droplets of any one of claims 1-3, wherein the droplets have an antibody-impermeable shell.

5. The population of double emulsion droplets of any one of claims 1-4, wherein the droplets have a DNA and RNA-impermeable shell.

6. The population of double emulsion droplets of any one of claims 1-5, wherein the population comprises a second population of desired cells.

7. The population of double emulsion droplets of claim 6, wherein the population comprises a third population of desired cells.

8. The population of double emulsion droplets of claim 6 or 7, wherein the first, second and / or third population of desired cells is a population of antibody producing cells.

9. The population of double emulsion droplets of claim 6 or 7, wherein the first, second and / or third population of desired cells is a population of reporter cells.

10. The population of double emulsion droplets of claim 6 or 7, wherein the first, second and / or third population of desired cells is a population of cancer cells.07810.002W0111. The population of double emulsion droplets of any one of claims 1-10, wherein the droplets have an oil shell that is about 20-30% of the total droplet volume.

12. The population of double emulsion droplets of any one of claims 1-10, wherein the droplets have an oil shell that is about 25-30% of the total droplet volume.

13. The population of double emulsion droplets of any one of claims 1-10, wherein the droplets have an oil shell that is about 30-40% of the total droplet volume.

14. A device for making the double emulsion droplet of any one of claims 1-13.

15. The use of the double emulsion droplet of any one of claims 1-13 for screening antibodies.

Images