Compositions and methods for cell-like calibration particles

Abstract

A method includes calibrating a cytometry device for analyzing target cells by inserting a hydrogel particle into the cytometry device. The hydrogel particle has at least one of a background fluorescence characteristic or a spectral characteristic that is substantially similar to at least one of a background fluorescence characteristic or a spectral characteristic of the target cells. The method further includes measuring at least one characteristic of the hydrogel particle using the cytometry device.

Description

[0001] Cross-references to related applications

[0002] This application claims priority and benefits to U.S. Provisional Application No. 62 / 965,494, filed January 24, 2020, entitled “Compositions and Methods for Cell-Like Calibration Particles,” the entire disclosure of which is incorporated herein by reference.

[0003] This application relates to U.S. Patent No. 9,915,598, issued March 13, 2018, entitled “Hydrogel Particles with Tunable Optical Properties”, and to U.S. Patent No. 9,714,897, issued July 25, 2017, entitled “Hydrogel Particles with Tunable Optical Properties and Method of Using the Same Thereof,” the entire disclosure of each of which is incorporated herein by reference for all purposes. Technical Field

[0004] This disclosure relates to flow cytometry, and more specifically to hydrogel bead substrates that exhibit cell-like autofluorescence, thereby enabling more precise fluorescence and spectral calibration and compensation. Background Technology

[0005] Flow cytometry and hematologic analysis are techniques that allow for the rapid separation, counting, and characterization of individual cells and are commonly used in a variety of applications in clinical and laboratory settings. The techniques rely on directing a light beam onto a focused stream of fluid. In some implementations, multiple detectors are then aimed at the point where the stream passes through the beam: one detector is aligned with the beam (forward scattering, or "FSC") and several detectors are perpendicular to the beam (side scattering, or "SSC"). FSC is typically correlated with cell volume, while SSC depends on the internal complexity or granularity of the particles (i.e., the shape of the nucleus, the amount and type of cytoplasmic granules, or membrane roughness). Due to these correlations, different specific cell types exhibit different FSCs and SSCs, allowing for cell type differentiation in flow cytometry. These measurements form the basis of cell counting analysis. In other forms of cell counting, cells are imaged, and descriptive characteristics such as size / shape / volume and, in some cases, biochemical characteristics are recorded. In addition to these measurements, cells are often analyzed in multiple fluorescence channels or using a spectrometer. These detection modalities are used to differentiate biomarker curves and other biological characteristics between different cell populations.

[0006] Most synthetic or polymeric products used for cell analysis are made from plastic materials, such as polystyrene (latex), which is an opaque polymer that typically has fixed forward and side scatter profiles based on particle diameter. Additionally, polystyrene has high auto-fluorescence in important detection channels, which leads to background fluorescence signals even in the absence of fluorophores or associated biomarkers. In other cases, the auto-fluorescence of polystyrene is much lower than the cell material, leading to inaccurate compensation and spectral separation. Overall, the inherent auto-fluorescence of polystyrene makes it unsuitable for use in fluorescence calibration and compensation in many cases. Specifically, rare or low-expressed biomarkers cannot be properly distinguished from polystyrene particles, and thus cannot be used as controls / standards. Additionally, auto-fluorescence from polystyrene particles can lead to false fluorescence resonance energy transfer (FRET), which leads to poor signal-to-noise ratios for dyes that rely on FRET functionality (e.g., tandem dyes). Auto-fluorescence interference caused by polystyrene is exacerbated in spectral analysis, which can resolve the complete spectral profile of a given target versus a separate fluorescence channel. In summary, these inherent limitations of polystyrene make it less effective as a substrate for calibration and compensation using a range of fluorescent species, particularly those that exhibit excitation or emission profiles in the violet and ultraviolet ranges.

[0007] Several key cell counting instrument setup procedures rely on the ability of calibration particles to mimic cells as closely as possible. In cytometry, compensation is a numerical correction for signal overlap between channels of different fluorophore emission spectra. Compensation is critical when analyzing different biochemical targets using multiple unique fluorophores, as it is important to distinguish true signal responses from “spill over” signals or interference from different fluorescence channels. In some known implementations, fluorescence compensation uses polystyrene-based controls to demonstrate fluorescence resolution for a given set of antibodies / fluorophores. However, due to the auto-fluorescence of polystyrene, there are entire classes of fluorophores (e.g., tandem dyes, UV / violet responsive dyes) that cannot be effectively compensated for using existing bead-based polystyrene products. The auto-fluorescence and poor performance of polystyrene fundamentally limits the complexity and diversity of fluorophores used in the cell analysis process.

[0008] Accordingly, there is a need for a substrate that more closely mimics the auto-fluorescence of actual cells. SUMMARY

[0009] In some embodiments, a method includes calibrating a cell counting device for analyzing target cells by inserting a hydrogel particle into the cell counting device. The hydrogel particle has at least one of an auto fluorescence property or a spectral property that is substantially similar to at least one of an auto fluorescence property or a spectral property of the target cells. The method further includes measuring at least one property of the hydrogel particle using the cell counting device.

[0010] In some embodiments of the disclosure, a composition includes a hydrogel particle having an auto fluorescence profile or a spectral profile that is more similar to that of a cell than that of polystyrene (e.g., latex), as measured by a cell counting device.

[0011] In other embodiments, the disclosure provides methods of producing a hydrogel particle having an auto fluorescence property or a spectral property that is substantially similar to a corresponding auto fluorescence property or spectral property of a target cell. The disclosure also sets forth methods of preparing a hydrogel particle having a predetermined auto fluorescence property and / or spectral property. The disclosure also sets forth methods of calibrating a cell counting device for analyzing target cells, the method comprising: a) inserting a hydrogel particle having an auto fluorescence property and / or spectral property that is substantially similar to a corresponding auto fluorescence property and / or spectral property of the target cells into the cell counting device; and b) measuring a fluorescence property and / or spectral property of the hydrogel particle using the cell counting device, thereby calibrating a cell counting device for analyzing the target cells.

[0012] In some embodiments, a method includes calculating a compensation value for a cell count measurement of a target cell, and modifying the cell count measurement of the target cell based on the compensation value. Calculating a compensation value for a cell count measurement of a target cell includes inserting a first hydrogel particle into the cell counting device at a first time. The first hydrogel particle has at least one of a background fluorescence property or a spectral property that is substantially similar to at least one of a background fluorescence property or a spectral property of the target cells. At least one property of the first hydrogel particle is measured using the cell counting device. The calculation further includes inserting a second hydrogel particle into the cell counting device at a second time different from the first time, and measuring at least one property of the second hydrogel particle using the cell counting device. The calculation further includes comparing the measured at least one property of the first hydrogel particle to the measured at least one property of the second hydrogel particle to determine the compensation value. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1A(A) hydrogel particles of the disclosure, (B) exemplary optical properties of polystyrene beads, are shown.

[0014] Figure 1B Optical properties of polystyrene beads are shown in contrast to the optical properties of the hydrogel particles of (A). Figure 1A

[0015] Figure 2 Differences between polystyrene and scattering curves of actual / target cells or hydrogel particles of the disclosure are depicted according to some embodiments.

[0016] Figure 3 The ability to engineer the autofluorescence or spectral properties of the hydrogel to match the autofluorescence or spectral properties of the target cell population is shown according to some embodiments.

[0017] Figure 4 The ability to independently adjust each property of the engineered hydrogel to match the passive optical scattering, autofluorescence, biomarker, and fluorescent properties of any target cell is shown according to some embodiments.

[0018] Figure 5 An exemplary method of producing hydrogel particles is shown according to some embodiments.

[0019] Figure 6 is a plot of intensity versus wavelength / channel that demonstrates the principle of fluorescence compensation.

[0020] Figures 7A-7C includes a graph of data showing a comparison of human cells to polystyrene and hydrogel particles on a range of fluorescence and spectral detectors according to some embodiments.

[0021] Figure 8A is a spectral curve of lymphocytes stained with an antibody (“Ab”) modified with Alexa 700, and Figure 8B is a spectral curve of hydrogel particles of the disclosure according to some embodiments, showing that they are in agreement with the spectral curve of the lymphocytes of Figure 8A

[0022] Figure 9 is a flowchart showing a method for calibrating a cytometry device for analyzing target cells according to some embodiments.

[0023] Figure 10 is a flowchart showing the process of using the hydrogels of the disclosure to calibrate and calculate fluorescence compensation and / or spectral separation according to some embodiments.

[0024] Figures 11A-11D ​​is a bar graph showing a comparison between cell stains, hydrogel compensation beads, and known (polystyrene-based) products, according to some embodiments.

[0025] Figure 12 is a comparison of auto-fluorescence of polystyrene and cell controls to exemplary hydrogels of the present disclosure having various compositions and properties, according to some embodiments. DETAILED DESCRIPTION

[0026] Several known calibration measurements for flow cytometers, such as inter-laser delay, fluorescence response, sort time, and fluorescence compensation, use polystyrene beads. These calibration measurements are critical for the accurate performance of the cytometer and any downstream analysis or sorting of cell populations. While polystyrene is robust and low cost compared to using cell controls, it exhibits intrinsically different optical and fluorescent behavior compared to cells. As a result, polystyrene beads represent a poor substitute for cell controls in all but the most basic calibration processes.

[0027] To overcome the limitations of polystyrene, cells are sometimes used during instrument setup and calibration, however such methods suffer from batch-to-batch variability, high cost, poor shelf life, and biohazardous shipping / handling restrictions. Variations in cell size and differences between user-prepared cells make them unsuitable for certain instrument calibration controls. Additionally, cell control materials are often challenging to source when examining rare diseases.

[0028] Compared to polystyrene, the particles of the present invention exhibit cell-like auto-fluorescence and spectral profiles, allowing for more sensitive instrument calibration, better fluorescence compensation, and better overall experimental data resolution. The particles are also synthetically manufactured, allowing for high batch-to-batch precision without any of the drawbacks of using cell controls.

[0029] As shown in Figures 1A-1B and Figure 2 , polystyrene particles are fundamentally limited in the optical properties they can have, such as forward and side scatter. This is largely due to the fact that they are opaque, as opposed to cells, so side scatter is a direct function of particle size, rather than internal cellular complexity. For example, Figure 1A (A) shows exemplary optical properties of the engineered hydrogel particles of the present disclosure, where light from an excitation light source can interact with the internal structure of the engineered hydrogel particles to produce side scatter information about that internal structure. In contrast, Figure 1A (B) and Figure 1B show exemplary optical properties of polystyrene beads, where light from an excitation light source does not interact with the internal structure of the polystyrene beads, so the resulting side scatter information is limited. Additionally, as Figure 2As shown, polystyrene beads have a 3-4 order of magnitude difference in side scatter curves compared to actual cells (e.g., target biological cells). Additionally, polystyrene has high auto-fluorescence in many channels, even without fluorophores, which results in poor detector resolution (see, e.g., Figures 7A-7C In other cases, polystyrene has low auto-fluorescence when compared to cellular material, resulting in inaccurate calculation of stain indices, compensation, or spectral separation. This phenomenon makes it very difficult or impossible to accurately measure rare or poorly expressed biomarkers in a sample. This also results in poor compensation performance in channels where polystyrene emits auto-fluorescence. Due to these limitations of polystyrene, users must often rely on purified cell lines to calibrate fluorescence intensity, fluorescence compensation, inter-laser delay, sort delay, size, and cell complexity for immunophenotyping experiments. This is a lengthy and labor-intensive process that greatly increases the cost of flow cytometry validation and research pipelines. More importantly, these calibration cell lines introduce biological variability, leading to differences in interpretation of immunophenotyping data.

[0030] In order to use multiple fluorophores for a given biomarker phenotyping experiment, the fluorophores should be distinguishable on a cytometric instrument. The fluorescence profile of a given antibody, when bound to cells containing cognate biomarkers / antigens, can be used for comparison to other antibody-fluorophore combinations used in the same “panel” of reagents. Due to the challenges of using cells for fluorescence compensation, polystyrene beads are often used as a proxy during fluorescence compensation setup. However, the background auto-fluorescence of polystyrene results in poor detector resolution, inaccurate compensation matrix calculations, background auto-fluorescence, and poor lower limits of detection.

[0031] Embodiments of the present disclosure provide compositions comprising hydrogel particles having background fluorescence properties (e.g., auto-fluorescence) that are substantially similar to those of target cells (e.g., human cells) and overcome various shortcomings of polystyrene discussed above. The hydrogel particles described herein can have background spectral profiles that are substantially similar to those of target cells. The present inventors have unexpectedly discovered that the fluorescence properties of the hydrogel particles can be independently tuned by varying the composition of the hydrogel particles. Furthermore, the authors have discovered that the background fluorescence properties of the hydrogel particles can be tuned without affecting the baseline optical properties of the particles (i.e., auto-fluorescence can be tuned independently of forward scatter (FSC) and side scatter (SSC)). This property enables the hydrogels to accurately mimic both the optical and auto-fluorescence properties of target cells as measured by cytometric devices.

[0032] The present disclosure also provides methods of making hydrogel particles, wherein the hydrogel particles have fluorescence properties that are substantially similar to fluorescence properties of a target cell. The present disclosure also provides methods of making hydrogel particles, wherein the hydrogel particles have predetermined optical or fluorescence properties. Also provided is a method of calibrating a cell counting device for analyzing a target cell, the method comprising: a) inserting a hydrogel particle having fluorescence properties that are substantially similar to fluorescence properties of the target cell into the device; b) measuring the fluorescence properties of the hydrogel particle using the cell counting device, thereby calibrating a cell counting device for analyzing the target cell. Known cell counting devices include commercially available devices for performing flow cytometry, fluorescence activated cell sorting (FACS), hematology, and high content imaging.

[0033] Hydrogel

[0034] The hydrogel particles of the present disclosure comprise a hydrogel. A hydrogel is a material comprising a three-dimensional network of macromolecules that allows it to swell in the presence of water and to shrink in the absence of water (or by reducing the amount of water), but which is not soluble in water. Swelling (i.e., water uptake) is a result of the presence of hydrophilic functional groups attached to or dispersed in the macromolecular network. Crosslinking between adjacent macromolecules results in the water insolubility of these hydrogels. Crosslinking can be due to chemical (e.g., covalent) or physical (e.g., van der Waals forces, hydrogen bonding, ionic forces, etc.) bonds. While some in the polymer industry can refer to one or more macromolecular materials described herein as a “xerogel” in a dry state and as a “hydrogel” in a hydrated state, for the purposes of the present disclosure, the term “hydrogel” refers to the macromolecular material whether dehydrated or hydrated. A hydrogel of particular value is characterized in that, whether dehydrated or hydrated, the material retains its general shape. Thus, if a hydrogel has an approximately spherical shape in a dehydrated condition, it will be spherical in a hydrated condition.

[0035] According to some embodiments, the disclosed hydrogels of the present disclosure can comprise, for example, greater than about 30% water, greater than about 40% water, greater than about 50% water, greater than about 55% water, greater than about 60% water, greater than about 65% water, greater than about 70% water, greater than about 75% water, greater than about 80% water, or greater than about 85% water.

[0036] Hydrogels prepared synthetically can be prepared by polymerizing monomeric materials to form a backbone and crosslinking the backbone with a crosslinking agent. Common hydrogel monomers include lactic acid, glycolic acid, acrylic acid, 1-hydroxyethyl methacrylate, ethyl methacrylate, propylene glycol methacrylate, acrylamide, N-vinyl pyrrolidone, methyl methacrylate, glycidyl methacrylate, methacrylic acid diols, ethylene glycol, fumaric acid, and the like. Common crosslinking agents include tetramethylene glycol dimethacrylate and N,N'- 15 methylenebisacrylamide. In some embodiments, the hydrogel particles of the present disclosure are prepared by polymerization of acrylamide.

[0037] In some embodiments, the hydrogel comprises a mixture of at least one monofunctional monomer and at least one bifunctional monomer.

[0038] The monofunctional monomer can be a monofunctional acrylic monomer. Non-limiting examples of monofunctional acrylic monomers are acrylamide; methacrylamide; N-alkyl acrylamides (such as N-ethyl acrylamide, N-isopropyl acrylamide, or N-tert-butyl acrylamide); N-alkyl methacrylamides (such as N-ethyl methacrylamide or N-isopropyl methacrylamide); N,N-dialkyl acrylamides (such as N,N-dimethyl acrylamide and N,N-diethyl acrylamide); N-[(dialkylamino)alkyl] acrylamides (such as N-[3-dimethylamino)propyl] acrylamide or N-[3-(diethylamino)propyl] acrylamide); N-[(dialkylamino)alkyl] methacrylamides (such as N-[3-dimethylamino)propyl] methacrylamide or N-[3-(diethylamino)propyl] methacrylamide); (dialkylamino)alkyl acrylates (such as 2-(dimethylamino)ethyl acrylate, 2-(dimethylamino)propyl acrylate, or 2-(diethylamino)ethyl acrylate); and (dialkylamino)alkyl methacrylates (such as 2-(dimethylamino)ethyl methacrylate).

[0039] The bifunctional monomer is any monomer that can be polymerized with the monofunctional monomer of the present disclosure to form a hydrogel as described herein that further contains a second functional group that can participate in a second reaction (e.g., conjugation of a fluorophore).

[0040] In some embodiments, the bifunctional monomer is selected from the group consisting of allyl alcohol, allyl isothiocyanate, allyl chloride, and allyl maleimide.

[0041] The bifunctional monomer can be a bifunctional acrylic monomer. Non-limiting examples of bifunctional acrylic monomers are N,N'-methylenebisacrylamide, N,N'-methylenebisacrylamide, N,N'-ethylenebisacrylamide, N,N'-ethylenebismethacrylamide, N,N' propylenebisacrylamide, and N,N'-(l,2-dihydroxyethylidene)bisacrylamide.

[0042] Higher order branched and linear co-monomers can be replaced in the polymer mixture to adjust the refractive index while maintaining the polymer density, as described in U.S. Patent No. 6,657,030, the contents of which are incorporated by reference herein in their entirety.

[0043] In some embodiments, the hydrogel comprises molecules that modulate the optical properties of the hydrogel. Molecules that are capable of altering the optical properties of a hydrogel are discussed further below.

[0044] Naturally occurring hydrogels that can be used in the present disclosure include various polysaccharides that can be obtained from natural sources, such as plants, algae, fungi, yeasts, marine invertebrates, and arthropods. Non-limiting examples include agarose, dextran, chitin, cellulose-based compounds, starch, derivatized starch, and the like. These typically have repeating glucose units as a major part of the polysaccharide backbone.

[0045] The polymerization of the hydrogel can be initiated by a persulfate salt. The persulfate salt can be any water-soluble persulfate salt. Non-limiting examples of water-soluble persulfate salts are ammonium persulfate and alkali metal persulfate salts. Alkali metals include lithium, sodium, and potassium. In some preferred embodiments, the persulfate salt is ammonium persulfate or potassium persulfate, more preferably, it is ammonium persulfate.

[0046] The polymerization of the hydrogel can be accelerated by an accelerator. The accelerator can be a tertiary amine. The tertiary amine can be any water-soluble tertiary amine. Preferably, the tertiary amine is N,N,N',N' tetramethylethylenediamine or 3-dimethylamino)propionitrile, more preferably, it is N,N,N',N' tetramethylethylenediamine (TEMED).

[0047] Hydrogel particles

[0048] In one aspect, the hydrogel particles of the present disclosure comprise a hydrogel and are produced by polymerizing droplets (see Figure 5 ). Microfluidic methods of producing a plurality of droplets, including fluidic and rigidified droplets, are known and described in U.S. Patent Application Publication No. 2011 / 0218123 and U.S. Patent No. 7,294,503, the entire contents of each of which are incorporated by reference herein in their entirety. Such methods provide a plurality of droplets containing a first fluid and substantially surrounded by a second fluid, wherein the first fluid and the second fluid are substantially immiscible (e.g., droplets containing a water-based liquid are substantially surrounded by an oil-based liquid). In another form, the hydrogel particles are produced via precipitation or chemical polymerization. In another form, the hydrogel particles are produced via membrane emulsification. In another form, the hydrogel particles are formed via piezoelectric dispersion.

[0049] The plurality of fluid droplets (e.g., prepared using a microfluidic device) can be polydisperse (e.g., have a range of different sizes), or in some cases, the fluid droplets can be monodisperse or substantially monodisperse, e.g., have a uniform diameter distribution, e.g., such that no more than about 10%, about 5%, about 3%, about 1%, about 0.03%, or about 0.01% of the droplets have an average diameter that is greater than about 10%, about 5%, about 3%, about 1%, about 0.03%, or about 0.01% of the average diameter. As used herein, the average diameter of a population of droplets refers to the arithmetic average of the diameters of the droplets.

[0050] Accordingly, the present disclosure provides a population of hydrogel particles comprising a plurality of hydrogel particles, wherein the population of hydrogel particles is substantially monodisperse.

[0051] The term microfluidic refers to a device, apparatus, or system that includes at least one fluidic channel having a cross-sectional dimension less than 1 mm and a length to largest cross-sectional dimension perpendicular to the channel of at least about 3: 1. Microfluidic devices that include microfluidic channels are particularly suitable for preparing a plurality of monodisperse droplets.

[0052] Non-limiting examples of microfluidic systems that can be used with the present application include those disclosed in U.S. Patent Application Publication No. 2006 / 0163385 ("Forming and Control of Fluidic Species"), U.S. Patent Application Publication No. 2005 / 0172476 ("Method and Apparatus for Fluid Dispersion"), U.S. Patent Application Publication No. 2007 / 000342 ("Electronic Control of Fluidic Species"), International Patent Application Publication No. WO 2006 / 096571 ("Method and Apparatus for Forming Multiple Emulsions"), U.S. Patent Application Publication No. 2007 / 0054119 ("Systems and Methods of Forming Particles"), International Patent Application Publication No. WO 2008 / 121342 ("Emulsions and Techniques for Formation"), and International Patent Application Publication No. WO 2006 / 078841 ("Systems and Methods for Forming Fluidic Droplets Encapsulated in Particles Such as Colloidal Particles"), the entire contents of each of which are incorporated herein by reference in their entireties.

[0053] Droplet size is related to microfluidic channel size. Microfluidic channels can have any size, for example, have a maximum dimension perpendicular to fluid flow that is less than about 5 mm or 2 mm, or less than about 1 mm, or less than about 500 μm, less than about 200 μm, less than about 100 μm, less than about 60 μm, less than about 50 μm, less than about 40 μm, less than about 30 μm, less than about 25 μm, less than about 10 μm, less than about 3 μm, less than about 1 μm, less than about 300 nm, less than about 100 nm, less than about 30 nm, or less than about 10 nm.

[0054] Droplet size can be adjusted by adjusting the relative flow rates. In some embodiments, the droplet diameter is equal to the width of the channel, or within about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the width of the channel.

[0055] The hydrogel particles of the present disclosure are substantially similar in size to the droplets from which they are composed. Thus, in some embodiments, the hydrogel particles have a diameter of less than about 1 pm, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 600, 800, or less than 1000 pm. In some embodiments, the hydrogel particles have a diameter of greater than about 1 pm, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 600, 800, or greater than 1000 pm. In typical embodiments, the hydrogel particles have a diameter in the range of 5 pm to 100 pm.

[0056] In some embodiments, the hydrogel particles of the present disclosure are spherical in shape.

[0057] In some embodiments, the hydrogel particles of the present disclosure have material modulus properties (e.g., elasticity) that more closely resemble target cells than polystyrene beads of the same diameter.

[0058] In some embodiments, the hydrogel particles of the present disclosure do not comprise agarose.

[0059] Optical properties

[0060] Passive and non-passive optical properties (e.g., fluorescent properties)

[0061] The three main modes of deconvolution for flow cytometry are two passive optical properties of the particles (forward scatter, FSC, corresponding to refractive index or RI; and side scatter, SSC) and fluorescence, a non-passive optical property (i.e., a property imparted by molecules that are not the base polymer component, such as fluorophores, fluorescers, or quantum dots, and represent biomarkers present on the surface of a given cell type, which are typically measured using antibodies with conjugated fluorophores. Thus, allowing the hydrogel particles of the present disclosure to mimic the composition of a particular cell type with respect to these three modes can be used to provide a synthetic, robust calibrator for flow cytometry.

[0062] In some embodiments, the disclosed hydrogel particles have a refractive index (RI) greater than about 1.10, greater than about 1.15, greater than about 1.20, greater than about 1.25, greater than about 1.30, greater than about 1.35, greater than about 1.40, greater than about 1.45, greater than about 1.50, greater than about 1.55, greater than about 1.60, greater than about 1.65, greater than about 1.70, greater than about 1.75, greater than about 1.80, greater than about 1.85, greater than about 1.90, greater than about 1.95, greater than about 2.00, greater than about 2.10, greater than about 2.20, greater than about 2.30, greater than about 2.40, greater than about 2.50, greater than about 2.60, greater than about 2.70, greater than about 2.80, or greater than about 2.90.

[0063] In some embodiments, the disclosed hydrogel particles have a refractive index (RI) less than about 1.10, less than about 1.15, less than about 1.20, less than about 1.25, less than about 1.30, less than about 1.35, less than about 1.40, less than about 1.45, less than about 1.50, less than about 1.55, less than about 1.60, less than about 1.65, less than about 1.70, less than about 1.75, less than about 1.80, less than about 1.85, less than about 1.90, less than about 1.95, less than about 2.00, less than about 2.10, less than about 2.20, less than about 2.30, less than about 2.40, less than about 2.50, less than about 2.60, less than about 2.70, less than about 2.80, or less than about 2.90.

[0064] SSC of the disclosed hydrogel particles is most meaningfully measured in comparison to target cells. In some embodiments, the SSC of the disclosed hydrogel particles is within 30%, within 25%, within 20%, within 15%, within 10%, within 5%, or within 1% of the SSC of the target cells, as measured by a cytometry device.

[0065] FSC of the disclosed hydrogel particles is most meaningfully measured in comparison to target cells. In some embodiments, the FSC of the disclosed hydrogel particles is within 30%, within 25%, within 20%, within 15%, within 10%, within 5%, or within 1% of the FSC of the target cells, as measured by a cytometry device.

[0066] FSC of the hydrogel can be adjusted by incorporating high refractive index molecules in the hydrogel. Preferred high refractive index molecules include colloidal silica, alkyl acrylates, and alkyl methacrylates. Thus, in some embodiments, the hydrogel particles of the present disclosure comprise alkyl acrylates and / or alkyl methacrylates.

[0067] Alkyl acrylates or alkyl methacrylates may contain 1 to 18, 1 to 8, or 2 to 8 carbon atoms in an alkyl group (such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, 2-ethylhexyl, heptyl or octyl). The alkyl group may be branched or straight-chain.

[0068] High refractive index molecules may also include ethylene compounds, such as vinyl aromatics like styrene and methylstyrene, optionally substituted on the aromatic ring by alkyl groups (such as methyl, ethyl or tert-butyl) or by halogens (such as chlorostyrene).

[0069] In some implementations, the FSC is adjusted by changing the water content present during the hydrogel formation process.

[0070] FSC is related to particle volume, and therefore can be adjusted by changing the particle diameter as described in this article.

[0071] SSCs can be engineered by encapsulating nanoparticles within a hydrogel to mimic organelles in target cells. In some embodiments, the hydrogel particles of this disclosure comprise one or more types of nanoparticles selected from: polymethyl methacrylate (PMMA) nanoparticles, polystyrene (PS) nanoparticles, and silica nanoparticles.

[0072] Functionalization of hydrogel particles

[0073] Hydrogel particles can be functionalized to mimic the optical and fluorescent properties of labeled cells. In some embodiments, the hydrogel particles comprise a bifunctional monomer, and the functionalization of the hydrogel particles occurs via said bifunctional monomer. In a typical embodiment, the functionalized hydrogel particles comprise free amine groups.

[0074] Hydrogel particles can be functionalized with fluorescent dyes of fluorescent substances known in the art, including The Fluorescent dyes described in the Handbook—A Guide to Fluorescent Probes and Labelling Techniques—are incorporated herein by reference in their entirety. Functionalization can be mediated by compounds containing free amine groups (e.g., allylamine), which can be incorporated into hydrogel particles during formation.

[0075] Non-limiting examples of known fluorescent dyes include: 6-carboxy-4',5'- dichloro-2',7'-dimethoxyfluorescein succinimidyl ester; 5-(and-6)-carboxyfluorescein; 5-carboxyfluorescein; 6-carboxyfluorescein; 5-(and-6)-carboxyfluorescein; 5-carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl) ether, -alanine- carboxamide or succinimidyl ester; 5-carboxyfluorescein succinimidyl ester; 6- carboxyfluorescein succinimidyl ester; 5-(and-6)-carboxyfluorescein succinimidyl ester; 5-(4,6-dichlorotriazinyl)aminofluorescein; 2',7'-difluorofluorescein; erythrosin-5- isothiocyanate; erythrosin-5-isothiocyanate; 6-(fluorescein-5-carboxamido)hexanoic acid or succinimidyl ester; 6-(fluorescein-5-(and-6)-carboxamido)hexanoic acid or succinimidyl ester; fluorescein-5-EX succinimidyl ester; fluorescein-5- isothiocyanate; fluorescein-6-isothiocyanate; 488 carboxylic acid or succinimidyl ester; Oregon 488 isothiocyanate; Oregon 488-X succinimidyl ester; Oregon 500 carboxylic acid; Oregon 500 carboxylic acid, succinimidyl ester or triethylammonium salt; Oregon 514 carboxylic acid; Oregon 514 carboxylic acid or succinimidyl ester; Rhodamine Green™ carboxylic acid, succinimidyl ester or hydrochloride; Rhodamine Green™ carboxylic acid, trifluoroacetamide or succinimidyl ester; Rhodamine Green™-X succinimidyl ester or hydrochloride; Rhodol Green™ carboxylic acid, N,0-bis-(trifluoroacetamide) or succinimidyl ester; bis-(4-carboxy-piperidinyl)sulfonerhodamine or bis(succinimidyl ester); 5-(and-6) carboxynaphthofluorescein, 5-(and-6) carboxynaphthofluorescein succinimidyl ester; 5-carboxyrhodamine 6G hydrochloride; 6-carboxyrhodamine 6G hydrochloride, 5-carboxyrhodamine 6G succinimidyl ester; 6-carboxyrhodamine 6G succinimidyl ester; 5-(and-6)-carboxyrhodamine 6G succinimidyl ester; 5-carboxy-2',4',5',7'-tetrabromosulfonerythyl fluorescein succinimidyl ester or bis-(diisopropylethylammonium) salt; 5-carboxytetramethyl rhodamine; 6-carboxytetramethyl rhodamine; 5-(and-6)-carboxytetramethyl rhodamine; 5-carboxytetramethyl rhodamine succinimidyl ester; 6-carboxytetramethyl rhodamine succinimidyl ester; 5-(and-6)-carboxytetramethyl rhodamine succinimidyl ester; 6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimidyl ester; 6-carboxy-X rhodamine succinimidyl ester; 5-(and-6)-carboxy-X rhodamine succinimidyl ester; 5-carboxy-X-rhodamine triethylammonium salt; Lissamine™ rhodamine B sulfonyl chloride; Malachite Green; Isothiocyanate salt; mono(thiosuccinimidyl ester); 21 carboxylic acid or succinimidyl ester; 7 carboxylic acid or succinimidyl ester; Rhodamine Red™-X succinimidyl ester; 6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoic acid; succinimidyl ester; tetramethylrhodamine-5-isothiocyanate; tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas sulfonyl; Texas sulfonyl chloride; Texas STP ester or sodium salt; Texas succinimidyl ester; Texas succinimidyl ester; and X-rhodamine-5-(and-6)-isothiocyanate.

[0076] Other examples of fluorescent dyes include those commercially available from Invitrogen dyes, including but not limited to FL; TMR STP ester; TR-X STP ester; 630 / 650-X STP ester; 650 / 665-X STP ester; 6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3- propionic acid succinimidyl ester; 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3,5- dipropionic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid succinimidyl ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid; thiosuccinimidyl or sodium salt; 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3- propionyl)amino)hexanoic acid; 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3- propionyl)amino)hexanoic acid or succinimidyl ester; N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)sulfanylalanine, succinimidyl ester or triethylammonium salt; 6-4,4-difluoro-l,3-dimethyl-5-(4- methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid; 4,4-difluoro-5,7-diphenyl-4- bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester; 4,4-difluoro-5-phenyl-4-bora- 3a,4a-diaza-s-indacene-3-propionic acid; succinimidyl ester; 6-((4,4-difluoro-5-phenyl-4-bora- 3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoic acid or succinimidyl ester; 4,4-difluoro- 5-(4-phenyl-l,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester; 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester; 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryloxy)acetyl)aminohexanoic acid or succinimidyl ester; 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3- propionic acid; 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid; succinimidyl ester;4,4-difluoro-l,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8- propionic acid; 4,4-difluoro-l,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8- propionic acid succinimidyl ester; 4,4-difluoro-5-(2-diethyl)-4-bora-3a,4a-diaza-s- indacene-3-propionic acid succinimidyl ester; 6-(((4-(4,4-difluoro-5-(2-diethyl)-4- bora-3a,4a-diaza-s-indacene-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimidyl ester; and 6-(((4,4-difluoro-5-(2-diethyl)-4-bora-3a,4a-diaza-s-indacene-3- yl)styryloxy)acetyl)aminohexanoic acid or succinimidyl ester.

[0077] Fluorescent dyes can also include, for example, Alexa Fluor® fluorescent dyes, commercially available from Invitrogen, including but not limited to Alexa Fluor® 350 carboxylate; Alexa Fluor® 430 carboxylate; Alexa Fluor® 488 carboxylate; Alexa Fluor® 532 carboxylate; Alexa Fluor® 546 carboxylate; Alexa Fluor® 555 carboxylate; Alexa Fluor® 568 carboxylate; Alexa Fluor® 594 carboxylate; Alexa Fluor® 633 carboxylate; Alexa Fluor® 647 carboxylate; Alexa Fluor® 660 carboxylate; and Alexa Fluor® 680 carboxylate. 350 carboxylate; Alexa 430 carboxylate; Alexa 488 carboxylate; Alexa 532 carboxylate; Alexa 546 carboxylate; Alexa 555 carboxylate; Alexa 568 carboxylate; Alexa 594 carboxylate; Alexa 633 carboxylate; Alexa 647 carboxylate; Alexa 660 carboxylate; and Alexa 680 carboxylate. Fluorescent dyes of the present disclosure can also be, for example, cyanine dyes, commercially available from Amersham-Pharmacia Biotech, including but not limited to Cy3 NHS ester; Cy 5 NHS ester; Cy5.5 NHS ester; and Cy7 NHS ester.

[0078] Tandem dyes (such as those containing PE-Cy5 or other combinations) can also be effectively utilized in the present disclosure due to low auto-fluorescence. Typically, polystyrene auto-fluorescence interferes with the fluorescence resonance energy transfer (FRET) signal required for utilizing tandem or aggregate dyes.

[0079] Target cells

[0080] Hydrogel particles of the present disclosure behave similarly to target cells in procedures for staining and analysis, such as by flow cytometry or FACS.

[0081] In some embodiments, the target cell is an immune cell. Non-limiting examples of immune cells include B lymphocytes (also known as B cells), T lymphocytes (also known as T cells), natural killer (NK) cells, lymphokine-activated killer (LAK) cells, monocytes, macrophages, neutrophils, granulocytes, mast cells, platelets, Langerhans cells, stem cells, dendritic cells, peripheral blood mononuclear cells, tumor infiltrating (TIL) cells, genetically modified immune cells (including hybridomas), pharmacologically modified immune cells, and derivatives, precursor cells, or progenitor cells of any of the cell types listed herein.

[0082] In some embodiments, the target cell includes all cells of a particular cell class that share a characteristic. For example, the target cell can be a lymphocyte, including NK cells, T cells, and B cells. The target cell can be an activated lymphocyte.

[0083] In some embodiments, the target cell is a primary cell, a cultured cell, an established cell, a normal cell, a transformed cell, an infected cell, a stably transfected cell, a transiently transfected cell, a proliferating cell, or a terminally differentiated cell.

[0084] In one embodiment, the target cell is a primary neuronal cell. A variety of neurons can be target cells. As non-limiting examples, the target cell can be a primary neuron; an established neuron; a transformed neuron; a stably transfected neuron; or a motor or sensory neuron.

[0085] In other embodiments, the target cell is selected from the group consisting of a primary lymphocyte, a monocyte, and a granulocyte.

[0086] The target cell can be virtually any type of cell, including prokaryotic and eukaryotic cells.

[0087] Suitable prokaryotic target cells include, but are not limited to, bacteria such as E. coli, various Bacillus species, and extremophilic bacteria such as thermophiles.

[0088] Suitable eukaryotic target cells include, but are not limited to, fungi, such as yeast and filamentous fungi, including species of Saccharomyces, Aspergillus, Trichoderma, and Neurospora; plant cells, including cells of corn, sorghum, tobacco, rapeseed, soybean, cotton, tomato, potato, alfalfa, sunflower, and the like; and animal cells, including fish, bird, and mammal. Suitable fish cells include, but are not limited to, cells from species of salmon, trout, tilapia, tuna, carp, flounder, halibut, swordfish, cod, and zebrafish. Suitable bird cells include, but are not limited to, cells of chicken, duck, quail, pheasant, and turkey, as well as other jungle fowl or wild birds. Suitable mammal cells include, but are not limited to, cells from species of horse, cow, water buffalo, deer, sheep, rabbit, rodent (e.g., mouse, rat, hamster, and guinea pig), goat, pig, primate, marine mammal (including dolphin and whale); as well as cell lines (e.g., human cell lines of any tissue or stem cell type) and stem cells (including pluripotent and non-pluripotent) and non-human fertilized eggs.

[0089] Suitable cells also include those cell types associated with a variety of diseases, even those in a non-diseased state. Thus, suitable eukaryotic cell types include, but are not limited to, all types of tumor cells (e.g., melanoma, myeloid leukemia, lung cancer, breast cancer, ovarian cancer, colon cancer, kidney cancer, prostate cancer, pancreatic cancer, and testicular cancer), cardiomyocytes, dendritic cells, endothelial cells, epithelial cells, lymphocytes (T and B cells), mast cells, eosinophils, vascular intimal cells, macrophages, natural killer cells, red blood cells, hepatocytes, white blood cells (including mononuclear leukocytes), stem cells (e.g., hematopoietic, neural, skin, lung, kidney, liver, and cardiac muscle stem cells (e.g., for screening against differentiation and dedifferentiation factors)), osteoclasts, chondrocytes and other connective tissue cells, keratinocytes, melanocytes, hepatocytes, renal cells, and adipocytes. In certain embodiments, the cells are primary disease state cells, such as primary tumor cells. Suitable cells also include known research cells, including, but not limited to, Jurkat T cells, NIH 3T3 cells, CHO, COS, and the like. See the ATCC Cell Line Catalogue, which is specifically incorporated herein by reference.

[0090] In some embodiments, the target cell is a tumor microvesicle or a tumor macrovesicle. Tumor microvesicles, also known as tumor secretory microvesicles or tumor secretory exosomes, can be found in circulating blood and can have immunosuppressive activity. Tumor microvesicles typically have a diameter in the range of 30-200 nm in size. Larger tumor microvesicles can be referred to as tumor macrovesicles and can have a diameter in the range of 3-10 pm in size.

[0091] Examples

[0092] Example 1: Production of Hydrogel Particles

[0093] Photomasks for UV lithography were sourced from CADart Services Inc. and designed using AutoCad (AutoDesk, inc.). SU-8 photoresist (Microchem, inc.) was photocrosslinked using a collimated UV light source (OAI, inc.) on 4" silicon wafers to create masters for microfluidic device fabrication. PDMS (polydimethylsiloxane, Sigma Aldrich, inc.) was prepared and formed using standard published methods for soft lithography and microfluidic device fabrication (see McDonald JC et al., 2000, Electrophoresis 21 :27-40).

[0094] Droplets were formed using flow-focusing geometry, where two oil channels concentrate the central flow of aqueous monomer solution to break droplets in a water-in-oil emulsion. A fluorohydrocarbon oil (Novec 7500 3M, inc.) was used as the external continuous phase liquid for droplet formation. To stabilize the droplets prior to polymerization, 0.5% w / w of surfactant was added to the oil phase (Krytox 157FSH, ammonium carboxylate salt of Dupont). To prepare the basic polyacrylamide gel particles, a central phase containing an aqueous monomer solution of N-acrylamide (1-20% w / v), crosslinker (N,N'-bisacrylamide, 0.05-1% w / v), accelerator, and ammonium persulfate (1% w / v) was used. An accelerator (N,N,N',N'-tetramethylethylenediamine 2% volume %) was added to the oil phase to initiate hydrogel particle polymerization after droplet formation.

[0095] Several co-monomers were added to the basic gel formulation to increase functionality. In one example, an aryl-acrylate was added to modulate the auto-fluorescent properties of the particles. In other examples, polystyrene nanoparticles were added to the hydrogel matrix at low concentrations to modulate the auto-fluorescent properties of the particles. The fluorescent properties were also modulated by adjusting the crosslinking density of the particles, by engineering the kinetics of the crosslinking and curing process (e.g., changing one of temperature, time, and / or concentration of one or more accelerators). The co-monomers, nanoparticle additives, and crosslinking density of the basic gel formulation were adjusted to affect the fluorescent and spectral properties of the particles, creating a formulation model that mimics the cellular-like background optical response. Specifically, the type of chemical side groups present on the various co-monomers incorporated into the gel matrix affect the fluorescent and spectral properties of the particles, as do the concentration of co-monomers, additives, and the crosslinking density of the core polymer.

[0096] Chemometric multiplexing of hydrogel particles is achieved by secondary labeling using co-monomers containing chemical orthogonal pendants (amines, carboxyls, maleimides, epoxides, alkynes, etc.).

[0097] We formed droplets at an average rate of 5 kHz and collected them in a fluorocarbon oil phase. After 30 minutes of polymerization at 50 °C, we washed the resulting hydrogel particles from the oil into an aqueous solution.

[0098] Example 2: Multidimensional tuning of hydrogel particle optical properties

[0099] As Figure 3 shown, the autofluorescence properties of the hydrogel particles can be tuned in multiple dimensions to match the cellular-like background autofluorescence / spectroscopic profile of a particular cell type (unlike polystyrene beads). Independent tuning of autofluorescence and forward scatter can be achieved by tuning / selectively modifying the co-monomer composition, the composition of nanoparticle additives, and / or the crosslinking density of the hydrogel particles. Cells are deconvolved using a combination of optical parameters (such as FSC and SSC or secondary labels). The hydrogel particles are tuned to precisely match the SSC and FSC of a particular cell type, unlike polystyrene beads (such as Figure 1A (B) and Figure 1B shown). Figure 1A (A) shows that cells and the engineered hydrogels described herein are translucent, allowing internal features to be resolved by side scatter (SSC). In contrast, polystyrene beads are opaque and have a defined side scatter determined by diameter, as shown in Figure 1A (B). The hydrogel particles can be further tuned with the ratio of specific chemical pendants and secondary labels, allowing precise matching of any cell type without suffering from biological noise as with fixed cell lines (see Figure 4 ). Specifically, as shown in Figure 4 , the multiplexing capability of the functionalized hydrogels allows for the addition of antigens and other biomarkers to the hydrogel base polymer, adding further “cell-like” dimensions to the product.

[0100] Example 3: Comparison of engineered hydrogel particles to polystyrene particles and cells.

[0101] Hydrogel particles were formed using the methods described above and measured in all fluorescence channels on a Beckman Coulter Cytoflex instrument. 5um polystyrene beads (BD Biosciences) were measured in parallel. Cells obtained from commercial suppliers were run in phosphate buffered saline and measured on a Beckman Coulter Cytoflex instrument. Figure 6is a plot of intensity versus wavelength / channel that illustrates the principle of fluorescence compensation. Specifically, Figure 6 The concept of fluorescence spillover and compensation is illustrated. As shown, Figure 6 Channel A (A) shows the highest intensity for the model fluorophore, while Channels B and C show spillover or residual emission signals from the single fluorophore. When combined with other fluorophores emitting in these channels, such values can be subtracted from the measured fluorescence signal to calculate a more accurate or “true” fluorescence signal intensity.

[0102] Figures 7A-7C Helpful in comparing fluorescence characteristics between lymphocytes, polystyrene beads, and the engineered hydrogel autofluorescent hydrogel of the present disclosure (“FlowCytes”). Each plot represents a standard fluorescence detection channel in common experimental procedures and an exemplary antigenic or biological target named in that detection channel. Channels are as follows:

[0103] • Channel FL1 - A - Thiazole Orange - A (DNA binding photosensitizer)

[0104] • Channel FL2 - A - PerCP - A (PerCP-conjugated antibody, where PerCP is peridinin-chlorophyll-protein, a fluorescent complex)

[0105] • Channel FL3 - A - CD4 apc-a (cluster of differentiation (CD) 4 Allophycocyanin (APC) antibody)

[0106] • Channel FL4 - A - APC - A700 - A (conjugated antibody)

[0107] • Channel FL5 - A - APC - A750 - A (conjugated antibody)

[0108] • Channel FL6 - A - BV421 - 1 (Bright Violet 421 antibody conjugate)

[0109] • Channel FL7 - A - BV510 - A (Bright Violet 421 antibody conjugate)

[0110] • Channel FL8 - A - Purple 610 - A fluorescent nanoparticle dye

[0111] • Channel FL9 - A - Purple 660 - A fluorescent nanoparticle dye

[0112] • Channel FL10 - A - PE - A (phycoerythrin antibody)

[0113] • Channel FL11 - A - ECO - A

[0114] • Channel FL12 - A - 7AAO - A (7-aminoactinomycin D)

[0115] like Figures 7A-7C As shown, compared to polystyrene beads, flow cytometry cells exhibit more cell-like autofluorescence (i.e., their associated autofluorescence characteristics are closer to those of lymphocytes). This allows for measurements of a larger dynamic range and more accurate fluorescence compensation on the same instrument. For example, Figure 7C The flow cytometry results showed lower autofluorescence in the UV and violet spectra and were more cell-like (i.e., more similar to lymphocytes than polystyrene beads). Furthermore, the flow cytometry exhibited a relatively high signal-to-noise ratio, which facilitated better detection of poorly expressed or “fuzzy” biomarkers by reducing background noise and increasing the dynamic range of a given detector. The hydrogel described herein also allows for the use of synthetic bead products with fluorescent substances that are excited or emitted in the violet and UV ranges, a property that current polystyrene-based products cannot match.

[0116] Figure 8A The spectral curves of lymphocytes stained with Ab modified with Alexa 700 are shown. Figure 8B This is a spectral profile of the hydrogel particles (flow cytometry) of this disclosure stained with Ab modified with Alexa 700 according to the implementation scheme. Figures 8A-8B As shown, the stained flow cytometry cells exhibit cell-like spectral characteristics, and the peak matching is r. 2 =1.

[0117] Figure 9 This is a flowchart illustrating a method for calibrating a cell counting device used to analyze target cells, according to some implementation schemes. For example... Figure 9 As shown, method 900 optionally includes obtaining or generating hydrogel particles at 902 having at least one background fluorescence characteristic and / or at least one spectral characteristic, said at least one background fluorescence characteristic and / or at least one spectral characteristic being substantially similar (e.g., within 10%) to a corresponding at least one fluorescence characteristic and / or at least one spectral characteristic of a target cell (e.g., human cell). At 904, method 900 includes inserting at least one hydrogel particle (e.g., a plurality of hydrogel particles, optionally in an aqueous medium or solution) into a cell counting device. The method further includes measuring the fluorescence characteristics of the hydrogel particles at 906 using the cell counting device.

[0118] Figure 10 This is a flowchart illustrating the process of calibrating and calculating fluorescence compensation and spectral separation using the hydrogel described in this disclosure. Figure 10As shown, the method 1000 includes modifying the hydrogel particles to bind antibody-fluorophore conjugates or DNA-binding dyes (e.g., anti-kappa light chain antibody) to the hydrogel particles at 1008. At 1010, separate reagents are bound and the hydrogel particles are inserted into a cytometer device to measure their fluorescence and / or spectral properties. A fluorescence compensation matrix and / or spectral separation table for the plurality of separate fluorophores is then calculated at 1012.

[0119] Figures 11A-11D is a bar graph showing a comparison between cell staining, hydrogel compensation beads, and known (polystyrene-based) bead products, according to some embodiments. In all cases described, the hydrogel beads of the present disclosure show more cell-like characteristics, resulting in superior compensation and spectral separation performance. Figure 11A The staining index and resolution performance of hydrogel compensation beads compared to known products is described. As shown in Figure 11A The staining index of the hydrogel compensation beads of the present disclosure is more cell-like than known compensation bead products, as shown in Figure 11B The mean fluorescence intensity (MFI) of stained hydrogels compared to known compensation products is described. As shown in Figure 11B The stained hydrogel compensation beads have a more cell-like MFI than known compensation bead products, as shown in Figure 11C The background autofluorescence of hydrogels compared to known compensation products is described. As shown in Figure 11C The unstained hydrogel compensation beads have a more cell-like background autofluorescence than known compensation bead products over a wide range of channels, as shown in Figure 11D The bleed-through performance of hydrogels compared to known compensation products is described. Figure 11D The fluorescence channel bleed-through of the hydrogel compensation beads of the present disclosure is superior to known compensation bead products, as shown in

[0120] Figure 12 is a chart showing two exemplary methods of modulating the autofluorescence of hydrogel particles according to some embodiments: (1) modulating the percentage of resonant comonomer additive or (2) changing the crosslinking density of the hydrogel. Figure 12 The autofluorescence of polystyrene and cell controls is compared to that of these exemplary hydrogel particles. As seen in Figure 12 The hydrogel particles made with 5% resonant comonomer additive have cell-like autofluorescence (1050) while the polystyrene control has undesirably high autofluorescence (9781), as seen in the left table of Figure 12 The hydrogel particles made with 10% crosslinking density have cell-like autofluorescence (1104) while the polystyrene control has undesirably high autofluorescence (9781), as seen in the right table of

[0121] In some embodiments, the composition comprises an aqueous solution and hydrogel particles suspended in the aqueous solution. The hydrogel particles have at least one of a background autofluorescence that is substantially similar to the background autofluorescence of the target cells or a spectral profile that is substantially similar to the spectral profile of the target cells. These particular properties have been engineered using a combination of co-monomer additives, adjusted kinetics of solidification (influenced by time, temperature, and chemical accelerants, which can thus be adjusted by changing them), and low concentrations of nanoparticle additives. These properties (autofluorescence and spectral profile) are characterized using a non-passive optical excitation channel, thus distinguishing it from passive optical features like SSC and FSC.

[0122] The hydrogel particles can also have an SSC that is within 10% of the SSC of the target cells, as measured by a cytometry device. The hydrogel particles can also have an FSC that is within 10% of the FSC of the target cells, as measured by a cytometry device.

[0123] The hydrogel particles can also have a refractive index that is greater than about 1.15, or greater than about 1.3, or greater than about 1.7.

[0124] The hydrogel particles can also have a diameter that is less than about 100 pm, or less than about 10 pm, or less than about 1 pm.

[0125] In some embodiments, the hydrogel particles contain polymeric nanoparticle additives.

[0126] In some embodiments, the hydrogel particles are chemically functionalized. For example, the hydrogel particles can comprise free amine groups.

[0127] In some embodiments, the hydrogel particles comprise allyl amines.

[0128] In some embodiments, the target cells are immune cells.

[0129] In some embodiments, the hydrogel particles are produced by polymerizing droplets.

[0130] In some embodiments, the hydrogel particles are produced by polymerizing droplets, and the hydrogel particles are subsequently modified by conjugating or attaching a fluorophore / fluorochrome. The modified hydrogel particles can have a fluorescence profile that matches (e.g., is substantially similar to or within 10% of) the fluorescence profile of the target cells.

[0131] In some embodiments, a population of hydrogel particles comprises a plurality of hydrogel particles, each hydrogel particle from the plurality of hydrogel particles having at least one of a background autofluorescence or spectral profile that is substantially similar to that of a target cell. The population of hydrogel particles can be substantially monodisperse. In some such embodiments, no more than 10% of the hydrogel particles have an average diameter that is greater than about 10% of the average diameter of the population of hydrogel particles.

[0132] In some embodiments, a method comprises calibrating a cytometry device for analyzing target cells by inserting at least one hydrogel particle (e.g., a plurality of hydrogel particles, optionally in an aqueous medium or solution) into the cytometry device. The at least one hydrogel particle has at least one of a background fluorescence property (e.g., autofluorescence) or spectral property that is substantially similar to at least one of a background fluorescence property or spectral property of a target cell. The method further comprises measuring at least one property (e.g., a calibration-related property) of the hydrogel particle using the cytometry device. The at least one property can comprise one or more of: interlaser delay, fluorescence response, sort time, or fluorescence compensation. The method optionally further comprises adjusting one of fluorescence compensation or spectral unmixing based on the measured property. Spectral unmixing is the process of decomposing the spectral features of a mixed pixel into a set of final members and their respective abundances. The calculation of compensation and spectral unmixing using the described cell-like reagents allows for the multiplexing of an expanded range of fluorophores by reducing noise and increasing cell-like accuracy of a given fluorophore. In some embodiments, the method further comprises, prior to inserting the hydrogel particle into the cytometry device: binding a fluorophore-containing reagent to the hydrogel particle to form a complex, measuring at least one property of the complex, and calculating fluorescence compensation or spectral unmixing based on the at least one measured property. Optionally, the method further comprises using the modified hydrogel particle to assess viability of a target cell.

[0133] In some embodiments, the hydrogel particle has been modified to bind to an antibody conjugated to a fluorophore (e.g., a fluorescent).

[0134] In some embodiments, the hydrogel particle is a modified hydrogel particle that has been modified to bind to at least one of an intercalating nucleic acid labeling reagent or an amine-reactive nucleic acid labeling reagent.

[0135] The hydrogel particle can have an SSC that is within 10% of the SSC of a target cell, as measured by a cytometry device. Alternatively or additionally, the hydrogel particle can have an FSC that is within 10% of the FSC of a target cell, as measured by a cytometry device.

[0136] In some embodiments, the hydrogel particles can have a refractive index greater than about 1.15, or greater than about 1.3, or greater than about 1.7.

[0137] In some embodiments, the hydrogel particles can have a diameter less than about 100 pm, or a diameter less than about 10 pm, or a diameter less than about 1 pm.

[0138] In some embodiments, the hydrogel particles comprise a polymeric nanoparticle additive.

[0139] In some embodiments, the hydrogel particles are chemically functionalized hydrogel particles.

[0140] In some embodiments, the hydrogel particles comprise free amine groups.

[0141] In some embodiments, the hydrogel particles comprise allyl amines.

[0142] In some embodiments, the target cells are immune cells.

[0143] In some embodiments, the method further comprises polymerizing the droplets to produce the hydrogel particles.

[0144] In some embodiments, the hydrogel particles are hydrogel particles that have been modified by conjugating or attaching one of a fluorophore or a fluorescent substance, and the modified hydrogel particles match a fluorescence or spectral profile of the cells.

[0145] In some embodiments, a method comprises calculating a compensation value for a cell count measurement of a target cell, and modifying the cell count measurement of the target cell based on the compensation value. Calculating the compensation value for the cell count measurement of the target cell comprises inserting a first hydrogel particle into a cell counting device at a first time. The first hydrogel particle has at least one of a background fluorescence characteristic or a spectral characteristic that is substantially similar to at least one of a background fluorescence characteristic or a spectral characteristic of the target cell. Measuring at least one characteristic of the first hydrogel particle using the cell counting device. The calculating further comprises inserting a second hydrogel particle into the cell counting device at a second time different from the first time, and measuring at least one characteristic of the second hydrogel particle using the cell counting device. The calculating further comprises comparing the measured at least one characteristic of the first hydrogel particle to the measured at least one characteristic of the second hydrogel particle to determine the compensation value.

[0146] In some embodiments, a method includes calculating a plurality of adjustment values for a cell count measurement of a target cell, and modifying the cell count measurement of the target cell based on the plurality of adjustment values. Calculating a plurality of adjustment values for a cell count measurement of a target cell includes inserting two hydrogel particles into the cell counting device: a first hydrogel particle from a hydrogel particle having at least one of a background fluorescence characteristic or a spectral characteristic that is substantially similar to at least one of a background fluorescence characteristic or a spectral characteristic of the target cell; and a second hydrogel particle from a hydrogel particle configured to bind with or pre-bound with a reagent that is a reagent that produces at least one of a fluorescent signal that is different from the background fluorescence characteristic or a spectral signal that is different from the spectral characteristic. Calculating a plurality of adjustment values for a cell count measurement of a target cell also includes measuring at least one characteristic of the first hydrogel particle and at least one characteristic of the second hydrogel particle using the cell counting device, and comparing the measured at least one characteristic of the first hydrogel particle and the measured at least one characteristic of the second hydrogel particle to determine a fluorescence overlap with at least one additional reagent and a spectral overlap with the at least one additional reagent. The cell count measurement of the target cell is then modified based on the plurality of adjustment values (e.g., including or based on the fluorescence overlap with at least one additional reagent and / or the spectral overlap with the at least one additional reagent).

[0147] Although shown and described herein in the context of cell counting device calibration and cell count measurement compensation, the cell-like hydrogel particles described herein can also be used in other applications to improve their performance and / or accuracy. For example, additional applications that are compatible with the cell-like hydrogel particles of the present disclosure include, but are not limited to: (1) setting the lower limit of detection (“LLOD”) of an instrument (examples of instruments include, but are not limited to: a flow cytometer, a hematology analyzer, a cytometry analyzer, or an image-based cell counter) to determine the true signal-to-noise ratio of ambiguous or poorly expressed biomarkers; (2) photomultiplier tube (“PMT”) gain adjustment to capture cell-like fluorescence linearity; (3) mean fluorescence intensity (“MFI”) calculation, and (4) instrument settings and quality control (“QC”) for fluorescence detection (active optical properties, rather than passive optical properties).

[0148] While various specific embodiments have been shown and described, it will be understood that various changes can be made without departing from the spirit and scope of the application.

[0149] As used throughout the specification and the appended claims, the following terms and expressions take the following meanings:

[0150] The indefinite articles “a” and “an,” as used herein in a context relating to a patent application, are not intended to convey a limitation on a number of embodiments. The indefinite articles “a” and “an” are intended to mean that there is at least one of the items being described, unless otherwise is clearly indicated by the context of the description.

[0151] “at least one” and “one or more of’ are used interchangeably to mean that an item can include one or more than one of the listed elements.

[0152] Unless otherwise indicated, it will be understood that all numbers in the description and claims, expressing quantities of ingredients, ratios, and values of properties such as reaction conditions, are to be, unless otherwise indicated, construed either to be approximations of functional or experimental results, and are intended to be within reasonable experimental error.

[0153] As used herein, the terms “about” and “approximately” generally mean plus or minus 10% of the stated value, e.g., about 250 pm would include 225 pm to 275 pm, and about 1,000 pm would include 900 pm to 1,100 pm.

[0154] Unless specifically stated otherwise, or as is apparent from the context of usage, discussions herein utilizing terms such as “about,” “approximately,” “generally,” “substantially,” or the like are meant to enable a skill in the art to recreate like or similar results. Thus, unless otherwise indicated, terms such as “about,” “approximately,” “generally,” and “substantially” are intended to convey that exact numerical values need not be achieved perfectly, but that a reasonable or desired range can be considered to be within a range that is acceptable to one of skill in the art. Thus, as used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Further, the use of the term “and / or” to describe a relationship between items in the context of a list of items (e.g., A, B, and / or C) indicates that any of the items can be present or none of the items are present.

Claims

1. A method of calibrating a cytometry device for analyzing target cells, the method comprising: (a) inserting a hydrogel particle into the cytometry device, the hydrogel particle having optical properties that are substantially similar to corresponding optical properties of the target cells, the optical properties being forward scatter (FSC), side scatter (SSC), or both, the hydrogel particle further having a background fluorescence property that is substantially similar to a background fluorescence property of the target cells, wherein the background fluorescence property of the hydrogel particle is provided by a resonant comonomer, encapsulated autofluorescent polymer nanoparticles, or a modulated hydrogel cross-linking density, such that the background fluorescence property is modulated independently of the optical properties; and (b) measuring fluorescence of the hydrogel particle using the cytometry device; whereby the cytometry device is calibrated.

2. The method of claim 1, further comprising: Prior to step (a): conjugating an antibody to a fluorophore is bound to the hydrogel particle; and After step (b), calculating fluorescence compensation based on the measured fluorescence of the hydrogel particle bound to the fluorophore.

3. The method of claim 1, wherein the hydrogel particle is bound to an antibody conjugated to a fluorophore.

4. The method of claim 1, wherein the hydrogel particle has a refractive index greater than about 1.

15.

5. The method of claim 1, wherein the hydrogel particle has a refractive index greater than about 1.

3.

6. The method of claim 1, wherein the hydrogel particle has a refractive index greater than about 1.

7.

7. The method of claim 1, wherein the hydrogel particle has a diameter less than about 100 pm.

8. The method of claim 1, wherein the hydrogel particle has a diameter less than about 10 pm.

9. The method of claim 1, wherein the hydrogel particle has a diameter less than about 1 pm.

10. The method of claim 1, wherein the hydrogel particle comprises a free amine group.

11. The method of claim 1, wherein the hydrogel particle comprises an allyl amine.

12. The method of claim 1, wherein the target cells are immune cells.

13. The method of claim 1, further comprising polymerizing droplets to produce the hydrogel particle.

14. The method of claim 1, wherein step (b) comprises measuring a property selected from the group consisting of interlaser delay, fluorescence response, sort time, and fluorescence compensation. ​

Images