Hydrogel particles with tunable optical properties and methods of using the same

Abstract

This application describes hydrogel particles and their use in cell counting applications. The hydrogel particles described herein are selectively tunable to possess at least one optical property that is substantially similar to the at least one optical property of target cells. In this regard, in one aspect, the hydrogel particles provided herein are used as calibration reagents for detecting target cells in a sample.

Description

[0001] This invention application is a divisional application based on a patent application with application date of February 8, 2016, application number 2016800199084, and invention name “Hydrogel particles with tunable optical properties and methods of using the same”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 62 / 114,004, filed February 9, 2015, and U.S. Provisional Application No. 62 / 184,192, filed June 24, 2015, the disclosures of each of which are incorporated herein by reference in their entirety. Background Art

[0004] Flow cytometry is a technique that can quickly separate, count, and characterize individual cells, and is commonly used in clinical and laboratory environments for a variety of applications. The technique relies on directing a beam of light onto a focused stream of fluid in a liquid. Multiple detectors are then aimed at the following points, where the liquid stream passes through the beam at the point: one point is in line with the beam (forward scatter or FSC), and several points are perpendicular to the beam (side scatter or SSC). FSC is related to cell volume, and SSC depends on the intrinsic complexity of the particle (e.g., the shape of the nucleus, the number and type of cytoplasmic particles, or the roughness of the cell membrane surface). Due to these correlations, different specific cell types exhibit different FSC and SSC, making it possible to distinguish cell types in flow cytometry.

[0005] However, the ability to identify specific cell types relies on proper instrument calibration, which relies on using purified cells of the target cell type. Obtaining these purified cells can require expensive and laborious processes that are prone to batch variability. Therefore, there is a need in the art for synthetic compositions with tunable optical properties that can mimic specific cell types in instruments such as flow cytometers. Summary of the Invention

[0006] In one aspect of the present invention, hydrogel particles comprising polymerized monomers and having at least one surface are provided. The hydrogel particles have at least one optical property substantially similar to at least one optical property of a target cell. In one embodiment, the optical property is a side scatter waveform (SSC), a forward scatter waveform (FSC), a fluorescence emission waveform, or a combination thereof. The target cell can be any target cell specified by the user. For example, in one embodiment, the target cell is an immune cell, a stem cell, or a cancer cell.

[0007] In another aspect, a method for calibrating a cytometer for analyzing target cells is provided. In one embodiment, the method comprises inserting into the device a hydrogel particle having at least one optical property substantially similar to that of a target cell, wherein the hydrogel particle comprises polymerized monomers and has at least one surface. The method further comprises measuring the at least one optical property of the hydrogel particle using the cytometer. In one embodiment, the at least one optical property serves as a reference for detecting target cells in a sample.

[0008] In yet another aspect, a method for detecting target cells in a sample is provided. The method comprises inserting hydrogel particles having at least one optical property substantially similar to that of target cells into a device, wherein the hydrogel particles comprise polymerized monomers. The method further comprises measuring the at least one optical property of the hydrogel particles using the cell counting device. A sample comprising a cell population is inserted into the cell counting device, and at least one optical property of individual cells in the population is measured. Finally, based on the optical property measurement results, a determination is made as to whether the target cell or population is present in the sample.

[0009] In one embodiment of the methods provided herein, the hydrogel particles comprise a biodegradable monomer. In further embodiments, the biodegradable monomer is a monosaccharide, a disaccharide, a polysaccharide, a peptide, a protein, or a protein domain. In even further embodiments, the biodegradable monomer is functionalized with acrylamide or acrylate.

[0010] The present invention comprises:

[0011] 1. A method for calibrating a cell counting device for analyzing target cells, comprising:

[0012] inserting into the device a hydrogel particle having at least one optical property substantially similar to that of a target cell, wherein the hydrogel particle comprises polymerized monomers and has at least one surface;

[0013] The at least one optical property of the hydrogel particles is measured using the cell counting device.

[0014] 2. A method for detecting target cells in a sample, comprising:

[0015] inserting into the device a hydrogel particle having at least one optical property substantially similar to that of a target cell, wherein the hydrogel particle comprises a polymerized monomer;

[0016] measuring the at least one optical property of the hydrogel particles using the cell counting device;

[0017] inserting a sample comprising a cell population into the cell counting device;

[0018] measuring the at least one optical property of individual cells in the cell population;

[0019] Whether the target cell or a population thereof is present in the sample is determined based on the optical property measurement results.

[0020] 3. The method of claim 2, further comprising: if the target cell or target cell population is present in the sample, sorting it into a separate container.

[0021] 4. The method of any one of items 1-3, further comprising: inserting a population of hydrogel particles into the cell counting device, wherein each of the hydrogel particles has at least one optical property substantially similar to that of the target cell.

[0022] 5. The method of claim 4, wherein the at least one optical property is the same for each of the population of hydrogel particles.

[0023] 6. The method of any one of items 1 to 5, wherein the at least one optical property is side scatter (SSC).

[0024] 7. The method of any one of items 1 to 4, wherein the at least one optical property is forward scattering (FSC).

[0025] 8. The method of any one of items 1-4, wherein the at least one optical property is fluorescence emission.

[0026] 9. The method of any one of items 1 to 5, wherein the at least one optical property is FSC and SSC.

[0027] 10.The method of any one of items 1 to 6, wherein the monomer is selected from the group consisting of hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinyl pyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glyceryl methacrylate (GMA), ethylene glycol methacrylate, ethylene glycol, fumaric acid, 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate, poly(ethylene glycol) methacrylate, methoxy-poly(ethylene glycol) ) methacrylate, methacrylic acid, sodium methacrylate, glyceryl methacrylate, hydroxypropyl methacrylate, hydroxybutyl methacrylate, phenyl acrylate, phenyl methacrylate, benzyl acrylate, benzyl methacrylate, 2-phenylethyl acrylate, 2-phenylethyl methacrylate, 2-phenoxyethyl acrylate, 2-phenoxyethyl methacrylate, phenylthioethyl acrylate, phenylthioethyl methacrylate, 2,4,6-tribromophenyl acrylate, 2,4,6-tribromophenyl methacrylate, pentabromophenyl acrylate, pentabromophenyl methacrylate, pentachlorophenyl acrylate, pentachlorophenyl methacrylate, 2,3-dibromopropyl acrylate, 2,4,6-tribromophenyl methacrylate, 3-dibromopropyl acrylate, 2-naphthyl acrylate, 2-naphthyl methacrylate, 4-methoxybenzyl acrylate, 4-methoxybenzyl methacrylate, 2-benzyloxyethyl acrylate, 2-benzyloxyethyl methacrylate, 4-chlorophenoxyethyl acrylate, 4-chlorophenoxyethyl methacrylate, 2-phenoxyethoxyethyl acrylate, 2-phenoxyethoxyethyl methacrylate, N-phenylacrylamide, N-phenylmethacrylamide, N-benzylacrylamide, N-benzylmethacrylamide, N,N-dibenzylacrylamide, N,N-dibenzylmethacrylamide, N-diphenylmethacrylamide, N-(4-methylphenyl)methacrylamide, N-1-naphthylacrylamide, N-4- Nitrophenylacrylamide, N-(2-phenylethyl)acrylamide, N-triphenylmethylacrylamide, N-(4-hydroxyphenyl)acrylamide, N,N-methylphenylacrylamide, N,N-phenylphenylethylacrylamide, N-diphenylmethylacrylamide, N-(4-methylphenyl)methylacrylamide, N-1-naphthylmethacrylamide, N-4-nitrophenylmethacrylamide, N-(2-phenylethyl)methacrylamide, N-triphenylmethylmethacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenylmethacrylamide, N,N′-phenylphenylethylmethacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridine, or a combination thereof.

[0028] 11. The method of any one of items 1 to 9, wherein the monomer is a biodegradable monomer.

[0029] 12. The method of claim 11, wherein the biodegradable monomer is a monosaccharide, a disaccharide, a polysaccharide, a peptide, a protein, or a protein domain.

[0030] 13. The method of claim 12, wherein the biodegradable monomer is a protein or protein domain comprising at least one unnatural amino acid.

[0031] 14. The method of claim 11 , wherein the biodegradable monomer is a structural polysaccharide.

[0032] 15. The method of claim 11, wherein the biodegradable monomer is agar, agarose, alginic acid, alguronic acid, α-glucan, amylopectin, amylose, arabinoxylan, β-glucan, callose, capsullan, carrageenan polysaccharide, cellodextrin, animal cellulose, cellulose, chitin, chitosan, laminarin, curdlan, cyclodextrin, α-cyclodextrin, dextrin, dextran, polysucrose, fructan, fucoidan, galactoglucomannan, galactomannan, galactosaminegalactose, gellan gum, dextran, glucomannan, glucuron Acid xylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hydroxypropyl methylcellulose, icodextrin, inulin, kefir polysaccharide, laminarin, mushroom polysaccharide, fructan polysaccharide, lichen polysaccharide, mannan, mixed glucan, paramylon, pectic acid, pectin, pentastarch, phytoglycogen, oyster mushroom glucan, polydextrose, polysaccharide peptide, metalloporphyrin, pullulan, schizophyllan, levulose, sizosan, brunei gum, xanthan gum, xylan, xyloglucan, zymosan, or a combination thereof.

[0033] 16. The method of claim 11, wherein the biodegradable monomer is chitosan or hyaluronic acid.

[0034] 17. The method of claim 12, wherein the protein is a structural protein, a domain thereof, or a combination thereof.

[0035] 18. The method of claim 12, wherein the protein is a proteoglycan, a domain thereof, or a combination thereof.

[0036] 19. The method of claim 12, wherein the protein is an extracellular matrix component.

[0037] 20. The method of claim 18, wherein the proteoglycan is decorin, biglycan, testoglycan, bikunitz inhibitor, fibromodulin, lumican, a domain thereof, or a combination thereof.

[0038] 21. The method of claim 19, wherein the protein is collagen, elastin or proteoglycan.

[0039] 22. The method of claim 21, wherein the protein is collagen.

[0040] 23. The method of claim 22, wherein the collagen is collagen type I, collagen type II, collagen type III, domains thereof, or a combination thereof.

[0041] 24. The process of any one of items 1 to 23, wherein the monomer is functionalized.

[0042] 25. The method of claim 24, wherein the monomer is functionalized with an acrylate or acrylamide.

[0043] 26. The process of any one of items 1 to 25, wherein the monomer is difunctional.

[0044] 27. The method of any one of items 1 to 26, wherein the hydrogel particles are functionalized on the at least one surface.

[0045] 28. The method of claim 27, wherein said hydrogel particles are functionalized with an antibody or fragment thereof.

[0046] 29. The method of claim 27, wherein the hydrogel is functionalized with at least one cell surface marker.

[0047] 30. The method of claim 29, wherein the cell surface marker is one of the cell surface markers listed in Table 2, or a combination thereof.

[0048] 30. The method of claim 27, wherein the hydrogel particles are functionalized with a fluorophore on at least one of the surfaces.

[0049] 31. The method of any one of items 1 to 30, wherein a substance is encapsulated by the hydrogel particles.

[0050] 32. The method of claim 31 , wherein the substance is a bead.

[0051] 33. The method of claim 32, wherein the beads are fluorescent beads.

[0052] 34. The method of claim 33, wherein the fluorescent beads emit fluorescence at one wavelength, two wavelengths, three wavelengths, four wavelengths, five wavelengths, six wavelengths, seven wavelengths, eight wavelengths, or nine wavelengths.

[0053] 35. The method of any one of items 32-34, wherein the beads have a diameter of about 500 nm to about 10 μm.

[0054] 36. The method of claim 31 , wherein the substance is a biomolecule.

[0055] 37. The method of claim 36, wherein the biomolecule is a nucleic acid, a protein, a peptide, a carbohydrate, or a combination thereof.

[0056] 38. The method of any one of items 32-35, further comprising: calibrating one or more properties of the cell counting device using the hydrogel particles.

[0057] 39. The method of claim 38, wherein one or more properties of the cell counting device are a cell sorting system, a laser source, optical properties, flow rate, or a combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] FIG1 illustrates the optical properties of the disclosed hydrogel particles compared to polystyrene beads.

[0059] Figure 2 Methods for preparing the hydrogel particles labeled herein are described.

[0060] FIG3 provides bright field and fluorescence images of the hydrogel particles labeled in the present application.

[0061] FIG4 illustrates the use of the hydrogel particles of the present application as markers for cell types exhibiting various optical scattering properties.

[0062] Figure 5 Data showing the relationship between flow cytometric inter-droplet delay and hydrogel particle diameter are provided.

[0063] FIG6 provides bright field images (6A and 6C) and fluorescence images (6B and 6D) of Chinese hamster ovary cells (6A and 6B) and hydrogel particles (6C and 6D) of the present application.

[0064] Figure 7 Data are presented showing a comparison of human oral mucosal cells with hydrogel particles encapsulating varying amounts of DNA, as measured by fluorescence activated cell sorting (FACS).

[0065] Figure 8 Data are presented for hydrogel particles encapsulating different concentrations of nanoparticles, demonstrating that side scatter is modulated independently of forward scatter.

[0066] Figure 9 Data are presented for hydrogel particles prepared with varying percentages of polymer, demonstrating modulation of the refractive index as measured by forward scattering.

[0067] Figure 10 One embodiment of hydrogel parameters adjusted to match and / or mimic desired cell population metrics is shown.

[0068] 11 and 12 are schematic diagrams showing embodiments of how to adjust the forward scattering properties, side scattering properties, and surface properties of hydrogel particles.

[0069] FIG13 is a scatter plot of various hydrogel particles (A) and (B) and a commercial blood sample (C). DETAILED DESCRIPTION

[0070] The indefinite articles “a” and “an” and the definite article “the” are intended to include both the singular and the plural, unless the context clearly indicates otherwise.

[0071] "At least one" and "one or more" are used interchangeably to indicate that the article may include one or more than one of the listed elements.

[0072] Unless otherwise indicated, all numbers expressing amounts, ratios, and numerical properties of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about."

[0073] Several critical calibration measurements of flow cytometers require precise time resolution, such as setting the offset time between lasers and calculating the delay time between detection and sorting of objects. Due to the fluid conditions within the instrument, accurately setting these timing parameters requires the use of calibration particles that are the same size as the cells to be analyzed. Timing calibration is typically performed using polystyrene beads with variable fluorescence intensity to calibrate the response of the excitation source and set the timing delay between lasers and the sorting delay. Flow cytometers can also be calibrated using forward scatter and side scatter signals, which are universal measures of the size and granularity or complexity of the target sample. These calibrations are critical for the accurate performance of the cytometer and for any downstream analysis or sorting of cell populations. The disclosed hydrogel particles exhibit tunable scattering properties and are suitable for use as calibration reagents for a variety of mammalian or bacterial cell types. For clinical, food safety, and research purposes, scattering is a standard metric for distinguishing cell types in heterogeneous mixtures.

[0074] While polystyrene particles can be used to set inter-laser delay and sorting delay for some applications, many eukaryotic cell types fall outside the size range of commercially available polystyrene particles (1-20 μm), making it nearly impossible to accurately calibrate a flow cytometer for these target cells. Furthermore, as shown in Figure 1, polystyrene particles are fundamentally limited in their optical properties, such as side scatter, a general measure of cellular complexity. Polystyrene particles are therefore limited in two of the most important passive optical measurements used in flow cytometry: FSC (forward scatter) and SSC (side scatter), which measure target cell size and complexity, respectively. Due to these limitations of polystyrene, users must rely on purified cell lines to calibrate their experiments for fluorescence intensity, inter-laser delay, sorting delay, size, and cellular complexity. This is a time-consuming and labor-intensive process that significantly increases the cost of flow cytometry validation and research pipelines. Most importantly, these calibration cell lines introduce biological variation, leading to discrepancies in data interpretation.

[0075] Furthermore, when these instruments are used in clinical applications, such as for isolating human regulatory T cells or stem cells for downstream cell therapy, quality control (QC) of the calibrated flow cytometer is also a key consideration. The FDA stipulates that the sterility, identity, purity, and potency of cell therapy products must be confirmed before administration to patients (Riley et al. (2009). Immunity 30, pp. 656-665). Therefore, contamination of cell populations with polystyrene QC particles is also a problem, as polystyrene has been implicated in certain cancers. In addition, cell populations contaminated by QC standards that are enzymatically degraded or digested in vivo after administration to patients may need to overcome the contamination issues caused by them.

[0076] The present invention addresses these and other needs, as discussed below.

[0077] In one aspect, a composition comprising a hydrogel particle plurality is provided, wherein the individual hydrogel particles of the hydrogel particle plurality each have one or more optical properties that are substantially similar to one or more optical properties of a target cell. The individual hydrogel particles of the hydrogel particle plurality each comprise a hydrogel synthesized by polymerizing one or more monomers, i.e., thereby forming a homopolymer or copolymer. As discussed further below, the use of bifunctional monomers allows the hydrogel to be further derivatized, for example, with a fluorescent dye, a cell surface marker or an antigenic epitope binding fragment thereof, or a combination thereof. An example of a hydrogel parameter adjusted to meet / match a desired cell subpopulation indicator is described in Figure 10Methods for modulating hydrogel properties are described herein. The ability to adjust a range of parameters, including hydrogel components and their concentrations, provides the ability to tune the particles to mimic a wide range of cells, such as one of the cell types described herein.

[0078] As provided above, in one aspect, the present invention provides individual hydrogel particles each having one or more optical properties that are substantially similar to one or more optical properties of a target cell. In one embodiment, the one or more optical properties are a side scatter waveform, a forward scatter waveform, or a secondary marker waveform, such as a fluorescent marker waveform, such as a fluorescent marker waveform of a fluorescently labeled antibody bound to the surface of the hydrogel particle. As used herein, "substantially similar" means at least 40% similar, at least 50% similar, at least 60% similar, at least 70% similar, at least 80% similar, at least 90% similar, at least 95% similar, at least 96% similar, at least 97% similar, at least 98% similar, or at least 99% similar.

[0079] The present invention is based in part on the unexpected discovery that one or more optical properties of hydrogel particles can be independently adjusted by varying the composition of the hydrogel particles, e.g., varying the amount of initial monomer (or comonomer) in the composition; varying the surface functionalization; varying the amount of polymerization initiator; or varying the amount of crosslinker. For example, side scatter (SSC) can be adjusted without significantly affecting forward scatter (FSC), and vice versa. Furthermore, the optical properties of hydrogel particles (e.g., refractive index) can be adjusted without significantly affecting particle density. This is a surprising and useful property because hydrogel particles used as cell surrogates in cell counting methods such as flow cytometry or (fluorescence activated cell sorting system) FACS require a minimum density in order to function in these assays.

[0080] In another aspect, a method for preparing hydrogel particles is provided, wherein the hydrogel particles have one or more optical properties that are substantially similar to the optical properties of one or more target cells. In one embodiment, the hydrogel particles have a predetermined optical property. In one embodiment, the optical property is SSC, FSC, fluorescence emission, or a combination thereof.

[0081] In yet another aspect, a method for calibrating a cell counting device for analyzing target cells is provided. In one embodiment, the method comprises (a) inserting hydrogel particles having at least one optical property substantially similar to that of target cells into the device; and b) measuring the at least one optical property of the hydrogel particles using the cell counting device, thereby calibrating the cell counting device for analyzing target cells. Cell counting devices are known in the art and include commercially available devices for performing flow cytometry and FACS.

[0082] As provided above, in one aspect of the present invention, a composition comprising a population of hydrogel particles is provided. A hydrogel is a material comprising a three-dimensional network of macromolecules that swells in the presence of water and shrinks in the absence (or reduced amount) of water, but is insoluble in water. Swelling, i.e., water absorption, is due to the presence of hydrophilic functional groups attached to or dispersed within the macromolecular network. Crosslinking between adjacent macromolecules renders these hydrogels insoluble in aqueous media. Crosslinking can be due to chemical bonding (i.e., covalent bonds) or physical bonding (i.e., van der Waals forces, hydrogen bonding, ionic forces, etc.). Synthetically prepared hydrogels can be prepared by polymerizing monomeric substances to form a backbone, and crosslinking the backbone with a crosslinking agent. The term "hydrogel" as used herein refers to a macromolecular substance, regardless of whether it is dehydrated or hydrated. A particularly valuable feature of hydrogels is that the substance maintains its general shape regardless of whether it is dehydrated or hydrated. Thus, if a hydrogel has a nearly spherical shape in the dehydrated state, it will also be spherical in the hydrated state.

[0083] In one embodiment, the hydrogel particles disclosed herein comprise 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, greater than about 85% water, greater than about 90% water, or greater than about 95% water. In another embodiment, the water content of the hydrogel particles is from about 10% to about 95% by weight, or from about 20% to about 95% by weight, or from about 30% to about 95% by weight, or from about 40% to about 95% by weight, or from about 50% to about 95% by weight, or from about 60% to about 95% by weight, or from about 70% to about 95% by weight, or from about 80% to about 95% by weight.

[0084] The hydrogels provided herein are in the form of particles and are synthesized as follows: one or more monomers provided herein are polymerized. The synthesis is performed to form individual hydrogel particles. In one embodiment, the monomeric material (monomer) is polymerized to form a homopolymer. However, in another embodiment, copolymers of different monomeric units (i.e., comonomers) are synthesized and used in the methods provided herein. In one embodiment, the monomers and comonomers used in the methods and compositions described herein are difunctional monomers or include difunctional monomers (where comonomers are used). In one embodiment, the hydrogel is synthesized in the presence of a crosslinking agent. In a further embodiment, the hydrogel is synthesized in the presence of a polymerization initiator.

[0085] The amount of monomer can be changed by the user of the present invention, for example, to obtain a specific optical property that is substantially similar to a target cell. In one embodiment, the presence of monomeric component (that is, monomer, comonomer, bifunctional monomer, or a combination thereof, for example, the bis / acrylamide of various cross-linking ratios, allylamine or other comonomers or alginate providing chemical functionality for secondary labeling / conjugation) is 10 weight % to about 95 weight % of a hydrogel. In a further embodiment, the presence of monomeric component is about 15 weight % to about 90 weight % of a hydrogel, or about 20 weight % to about 90 weight % of a hydrogel.

[0086] Examples of various monomers and crosslinking chemistries that can be used in the present invention are provided in the Thermo Scientific crosslinking technology handbook entitled "Easy molecular bonding crosslinking technology" (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163- CrosslinkingReagents-Handbook.pdf , the disclosure of which is incorporated herein by reference in its entirety for all purposes. For example, a hydrazine coupling reaction (e.g., with an NHS ester compound) or an EDC coupling reaction (e.g., with a maleimide compound) can be used to construct the hydrogel of the present invention.

[0087] In one embodiment, the monomers used in the hydrogels provided herein are lactic acid, glycolic acid, acrylic acid, 1-hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinyl pyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glyceryl methacrylate (GMA), glyceryl methacrylate, ethylene glycol, fumaric acid, derivatized versions thereof, or combinations thereof.

[0088] In one embodiment, one or more of the following monomers are used herein to form the hydrogels of the present invention: 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate, poly(ethylene glycol) methacrylate, methoxy-poly(ethylene glycol) methacrylate, methacrylic acid, sodium methacrylate, glyceryl methacrylate, hydroxypropyl methacrylate, hydroxypropyl methacrylate, or a combination thereof.

[0089] In another embodiment, one or more of the following monomers are used in the present application to form a tunable hydrogel: phenyl acrylate, phenyl methacrylate, benzyl acrylate, benzyl methacrylate, 2-phenylethyl acrylate, 2-phenylethyl methacrylate, 2-phenoxyethyl acrylate, 2-phenoxyethyl methacrylate, phenylthioethyl acrylate, phenylthioethyl methacrylate, 2,4,6-tribromophenyl acrylate, 2,4,6-tribromophenyl methacrylate, pentabromophenyl acrylate, pentabromophenyl methacrylate , pentachlorophenyl acrylate, pentachlorophenyl methacrylate, 2,3-dibromopropyl acrylate, 2,3-dibromopropyl methacrylate, 2-naphthyl acrylate, 2-naphthyl methacrylate, 4-methoxybenzyl acrylate, 4-methoxybenzyl methacrylate, 2-benzyloxyethyl acrylate, 2-benzyloxyethyl methacrylate, 4-chlorophenoxyethyl acrylate, 4-chlorophenoxyethyl methacrylate, 2-phenoxyethoxyethyl acrylate, 2-phenoxyethoxyethyl methacrylate, N-phenylacrylamide, N- Phenylmethacrylamide, N-benzylacrylamide, N-benzylmethacrylamide, N,N-dibenzylacrylamide, N,N-dibenzylmethacrylamide, N-diphenylmethacrylamide, N-(4-methylphenyl)methacrylamide, N-1-naphthylacrylamide, N-4-nitrophenylacrylamide, N-(2-phenylethyl)acrylamide, N-triphenylmethacrylamide, N-(4-hydroxyphenyl)acrylamide, N,N-methylphenylacrylamide, N,N-phenylphenylethylacrylamide, N-diphenylmethylmethacrylamide , N-(4-methylphenyl)methyl methacrylamide, N-1-naphthyl methacrylamide, N-4-nitrophenyl methacrylamide, N-(2-phenylethyl) methacrylamide, N-triphenylmethyl methacrylamide, N-(4-hydroxyphenyl) methacrylamide, N,N-methylphenyl methacrylamide, N,N′-phenylphenylethyl methacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridine, as described in U.S. Patent No. 6,657,030, which is incorporated herein by reference in its entirety for all purposes.

[0090] Both synthetic monomers and biological monomers can be used in the hydrogels provided herein to form synthetic hydrogels, biological hydrogels, or hybrid hydrogels comprising synthetic and biological components (e.g., peptides, proteins, monosaccharides, disaccharides, polysaccharides, primary amine thiol groups, carbonyl groups, carbohydrates, carboxylic acids present on biomolecules). For example, proteins, peptides, or carbohydrates can be used as independent monomers to form hydrogels that include or do not include synthetic monomers (or polymers) and are combined with chemically compatible comonomers and crosslinking agents (see, for example, the Thermo Scientific crosslinking technology manual entitled "Easy molecular bonding crosslinking technology," available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf , the disclosure of which is incorporated herein by reference in its entirety for all purposes. Compatible crosslinkers include, but are not limited to, amines, carboxyls, and other reactive chemical pendant groups. Representative reactive groups useful in the hydrogels and monomers described herein are listed in Table 1 below.

[0091]

[0092]

[0093] In general, any form of polymerization chemistry / method conventionally known to those skilled in the art can be used to form the polymer. In some embodiments, polymerization can be catalyzed by ultraviolet-induced free radical formation and reaction order. In other embodiments, the disclosed hydrogel particles are prepared by polymerization of acrylamide or polymerization of acrylates. For example, in one embodiment, the acrylamide is an acrylamide derivatized with a polymerizable carbohydrate, as described in U.S. Patent No. 6,107,365, the disclosure of which is incorporated herein by reference in its entirety for all purposes. As described herein and as known to those skilled in the art, the specific attachment of acrylamide groups to sugars facilitates application to a variety of monosaccharides or higher order polysaccharides, for example, synthetic polysaccharides found in serum or tissues or polysaccharides derived from natural sources such as glycoproteins.

[0094] In one embodiment, acrylate functionalized polyethylene glycol monomers are used as hydrogel monomers. For example, in one embodiment, the PEG is an acrylate or acrylamide functionalized PEG.

[0095] In some embodiments, the hydrogel particles comprise a monofunctional monomer polymerized with at least one difunctional monomer. An example includes, but is not limited to, the use of acrylamide and bisacrylamide (difunctional monomers) to form a polyacrylamide polymer. In another embodiment, the hydrogel particles provided herein comprise a difunctional monomer polymerized with a second difunctional monomer. An example includes, but is not limited to, forming a polymer with a mixed composition comprising compatible chemicals such as acrylamide, bisacrylamide, and structural homologues of bisacrylamide comprising numerous additional chemicals. The reactions of chemically compatible monomers, difunctional monomers, and mixed combinations are obvious to the art and follow chemical reaction principles known to those skilled in the art. (Refer to Thermo Manual and Acrylamide Polymerization Manual). See, for example, the Thermo Scientific Crosslinking Technology Manual entitled "Easy molecular bonding crosslinking technology" (available at tools.lifetechnologies.com / content / sfs / brochurs / 1602163-Crosslinking- Reagents-Handbook.pdf ), and the Polyacrylamide Emulsion Handbook (SNF Floerger, available at snf.com.au / downloads / Emulsion_Handbook_E.pdf), the disclosures of each of which are incorporated herein by reference in their entirety for all purposes.

[0096] In one embodiment, the hydrogel particles provided herein comprise a polymerizable monofunctional monomer and are monofunctional acrylic monomers. Non-limiting examples of monofunctional acrylic monomers for use herein 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]acrylamide, such as N-[3-(dimethylamino)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.

[0097] A bifunctional monomer is any monomer that can be polymerized with a monofunctional monomer to form the hydrogel described herein, wherein the bifunctional monomer further comprises a second functional group that can participate in a second reaction, for example, conjugation of a fluorophore or a cell surface receptor (or a domain thereof).

[0098] In some embodiments, the difunctional monomer is selected from the group consisting of: allylamine, allyl alcohol, allyl isothiocyanate, allyl chloride, and allylmaleimide.

[0099] The difunctional monomer may be a difunctional acrylic monomer. Non-limiting examples of difunctional acrylic monomers are N,N'-methylenebisacrylamide, N,N'-methylenebismethacrylamide, N,N'-ethylenebisacrylamide, N,N'-ethylenebismethacrylamide, N,N'-propylenebisacrylamide and N,N'-(1,2-dihydroxyethylene)bisacrylamide.

[0100] Higher order branched chain and linear comonomers can be substituted in the polymer blend to adjust the refractive index while maintaining polymer density as described in US Pat. No. 6,657,030, which is incorporated herein by reference in its entirety for all purposes.

[0101] In some embodiments, the hydrogel comprises molecules that adjust the optical properties of the hydrogel. Molecules capable of changing the optical properties of a hydrogel are discussed further below.

[0102] In one embodiment, individual hydrogel particles or a population thereof comprise a biodegradable polymer as a hydrogel monomer. In one embodiment, the biodegradable polymer is a poly(ester) based on polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL), and copolymers thereof. In one embodiment, the biodegradable polymer is a carbohydrate or protein, or a combination thereof. For example, in one embodiment, a monosaccharide, disaccharide, or polysaccharide (e.g., glucose, sucrose, or maltodextrin), a peptide, or a protein (or a domain thereof) is used as a hydrogel monomer. Other biodegradable polymers include poly(hydroxyalkyl esters) of the PHB-PHV class, other poly(esters), and natural polymers, such as modified poly(saccharides), such as starch, cellulose, and chitosan. In another embodiment, the biocompatible polymer is an adhesive protein, cellulose, a carbohydrate, starch (e.g., maltodextrin, 2-hydroxyethyl starch, alginic acid), dextran, lignin, poly(hydroxyethyl) acid, amino acids, or chitin. Such biodegradable polymers are commercially available, for example, from Sigma Aldrich (St. Louis, MO).

[0103] In one embodiment, the protein comprises only natural amino acids. However, the present invention is not limited thereto. For example, self-assembling artificial proteins and proteins containing non-natural amino acids (e.g., those incorporated into non-ribosomal peptides or synthetically introduced via synthetic methods, see, for example, Zhang et al. (2013). Current Views is in Structural Biology 23, pp. 581-587, the disclosure of which is incorporated herein by reference in its entirety for all purposes) or their protein domains can also be used as hydrogel monomers. The range of non-natural (unnatural) amino acids that can be incorporated into these compositions is well known to those skilled in the art (Zhang et al. (2013). Current Views is in Structural Biology 23, pp. 581-587; the disclosure of which is incorporated herein by reference in its entirety for all purposes). In one embodiment, a biodegradable polymer is used as a comonomer, i.e., in the monomer mixture. In one embodiment, the biodegradable polymer is a difunctional monomer.

[0104] In one embodiment, the biomonomer is functionalized with acrylamide or acrylate. For example, in one embodiment, the polymerizable acrylamide-functionalized biomolecule is an acrylamide- or acrylate-functionalized protein (e.g., an acrylamide-functionalized collagen or a functionalized collagen domain), an acrylamide- or acrylate-functionalized peptide, or an acrylamide- or acrylate-functionalized monosaccharide, disaccharide, or polysaccharide.

[0105] Any monosaccharide, disaccharide or polysaccharide (functionalized or otherwise) can be used as a hydrogel monomer. In one embodiment, a monosaccharide, disaccharide or polysaccharide functionalized with acrylamide or acrylate is used as a polymerizable hydrogel monomer. In one embodiment, a structural polysaccharide is used as a polymerizable hydrogel monomer. In a further embodiment, the structural polysaccharide is arabinoxylan, cellulose, chitin or pectin. In another embodiment, alginic acid (alginate) is used as a polymerizable hydrogel monomer. In another embodiment, glycosaminoglycans (GAGs) are used as polymerizable monomers in the hydrogels provided herein. In a further embodiment, the GAGs are chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparan sulfate or hyaluronic acid (also referred to in the art as hyaluronidase or hyaluronate / ester), which are used as polymerizable hydrogel monomers. The additional range of compatible biomonomers and their reactive chemistries is known to those skilled in the art and follows general chemical reactivity principles.

[0106] Additional ranges of biocompatible monomers that can be incorporated are known in the art, see, for example, the disclosure of non-degradable biocompatible monomers in Shastri (2003). Current Pharmaceutical Biotechnology 4, pp. 331-337, which is incorporated herein by reference in its entirety for all purposes. Other monomers are provided in de Moraes Porto (2012). Polymer Biocompatibility, Polymerization, Dr. Ailton De Souza Gomes (Ed.), ISBN: 978-953-51-0745-3; InTech, DOI: 10.5772 / 47786; Heller et al. (2010). Journal of Polymer Science Part A: Polymer Chemistry 49, pp. 650-661; Final Report for Biocompatible Materials (2004), The Board of the Biocompatible Materials and the Molecular Engineering in Polymer Science programmes, ISBN 91-631-4985-0, the disclosures of each of which are fully incorporated herein by reference.

[0107] In one embodiment, the biocompatible monomers used in the hydrogels described herein include ethylene glycol methacrylate (EGDMA), 2-hydroxyethyl methacrylate (HEMA), methyl methacrylate (MMA), methacryloxymethyltrimethylsilane (TMS-MA), N-vinyl-2-pyrrolidone (N-VP), styrene, or a combination thereof.

[0108] Naturally occurring hydrogels useful in the present invention include various polysaccharides obtainable from natural sources, such as plants, algae, fungi, yeast, marine invertebrates, and arthropods. Non-limiting examples include agarose, dextran, chitin, cellulose-based compounds, starch, derivatized starch, and the like. These will typically have repeating glucose units as the major portion of the polysaccharide backbone. The crosslinking chemistry of these polysaccharides is known in the art, see, for example, the Thermo Scientific crosslinking technology manual entitled "Easy molecular bonding crosslinking technology," available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking- Reagents-Handbook.pdf ).

[0109] In one embodiment, hyaluronic acid is used as a hydrogel monomer (either as a single monomer or as a comonomer). In one embodiment, hyaluronic acid is functionalized, for example, with acrylate or acrylamide. Hyaluronic acid is a high molecular weight GAG composed of repeating disaccharide units of N-acetylglucosamine and glucuronic acid linked together by alternating β-1,4 glycosidic bonds and β-1,3 glycosidic bonds. In the human body, hyaluronate / ester is present in several soft connective tissues, including skin, umbilical cord, synovial fluid, and vitreous. Therefore, in one embodiment, when it is desired to simulate skin cells, umbilical cord cells, or vitreous cells, in one embodiment, hyaluronic acid is used as a hydrogel monomer. Methods for making hydrogel particles are described in Xue et al. (2012). Soft Matter. 8, pp. 3280-3294, the disclosure of which is incorporated herein by reference in its entirety for all purposes. As described herein, hyaluronic acid can be derivatized with various reactive arms (reactive handles) depending on the desired crosslinker and other monomers used to form the hydrogel particles.

[0110] In yet other embodiments, chitosan, a linear polysaccharide composed of randomly distributed β-(1-4)-linked D-glucosamine (deacetylated units) and N-acetyl-D-glucosamine (acetylated units), is used as a hydrogel monomer (either as the sole monomer or as a comonomer).

[0111] Other polysaccharides useful as monomers or comonomers in hydrogels include, but are not limited to, agar, agarose, alginic acid, alguronic acid, α-glucan, pullulan, amylose, arabinoxylan, β-glucan, callose, capsullan, carrageenan polysaccharides (e.g., kappa, iota, or lambda types), cellodextrin, animal cellulose, cellulose, chitin, chitosan, laminarin, curdlan, cyclodextrin, α-cyclodextrin, dextrin, polysucrose, fructan, fucoidan, galactoglucomannan, galactomannans, galactosaminegalactose, gellan gum, dextran, glucomannan, Glucuronoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hydroxypropyl methylcellulose, icodextrin, inulin, kefir polysaccharide, laminarin, mushroom polysaccharide, fructan polysaccharide, lichen polysaccharide, mannan, mixed glucan, paramylon, pectic acid, pectin, pentastarch, phytoglycogen, oyster mushroom glucan, polydextrose, polysaccharide peptide, metalloporphyrin, pullulan, schizophyllan, levulose, sizosan, brunei gum, xanthan gum, xylan, xyloglucan, zymosan, or its combination. As described in full, according to required cross-linking agent and / or other comonomer for hydrogel, polysaccharide can be further functionalized. For example, in one embodiment, one or more polysaccharides described herein are functionalized with acrylate or acrylamide.

[0112] In one embodiment, individual hydrogel particles or their colonies comprise peptides, proteins, protein domains or their combinations as hydrogel monomers or their colonies. In further embodiments, the protein is a structural protein, or its domain, for example, such as silk protein, elastin, titin or collagen, or its domain. In one embodiment, the protein is an extracellular matrix (ECM) component (e.g., collagen, elastin, proteoglycan). In even further embodiments, the structural protein is collagen. In further embodiments, the collagen is collagen type I, collagen type II, or collagen type III or its combination. In another embodiment, the hydrogel monomer comprises a proteoglycan. In further embodiments, the proteoglycan is decorin, biglycan, testoglycan, bikunitz inhibitor, fibromodulin, lumican, or its domain.

[0113] In another embodiment, acrylate-functionalized structural protein hydrogel monomers are used as components of the hydrogels provided herein (e.g., acrylate-functionalized proteins or protein domains, e.g., silk protein, elastin, titin, collagen, proteoglycan, or functionalized domains thereof). In a further embodiment, acrylate-functionalized structural protein hydrogel monomers include proteoglycans, e.g., decorin, biglycan, testoglycan, bikunitz inhibitor, fibromodulin, lumican, or domains thereof.

[0114] In one embodiment, PEG monomers and oligopeptides can mimic extracellular matrix proteins for use in the hydrogels provided herein, for example, with multi-arm PEG functionalized with vinyl sulfone, integrin binding peptides, and bis-cysteine ​​matrix metalloproteinase peptides, as described by Lutolf et al. (2003). Proc. Natl. Acad. Sci. USA 100, 5413-5418, which is fully incorporated herein by reference for all purposes. In this particular embodiment, the hydrogel is formed by a Michael-type addition reaction between the dithiolated oligopeptide and the vinyl sulfone groups on the PEG. The range of other compatible chemicals that can be incorporated into the present invention is obvious to those skilled in the art and follows general chemical reactivity principles, see, for example, the Thermo Scientific crosslinking technology manual entitled "Easy molecular bonding crosslinking technology" (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf ).

[0115] Other biologically active domains in natural proteins can also be used as hydrogel monomers or portions thereof. For example, cell adhesion integrin binding domains, controlled release affinity binding domains, or transglutaminase crosslinking domains can be used in the hydrogels provided herein. Details of the preparation of these hydrogels can be found in Martino et al. (2009). Biomaterials 30, 1089; Martino et al. (2011). Sci. Trans. Med. 3, 100ra89; Hu and Messersmith (2003). J. Am. Chem. Soc. 125, 14298, each of which is fully incorporated herein by reference for all purposes.

[0116] In one embodiment, recombinant DNA methods are used to produce proteins designed to gel in response to changes in pH and temperature, for example, by the methods described in Petka et al. (1998). Science 281, pp. 389-392, which is incorporated herein by reference in its entirety for all purposes. Briefly, the protein consists of terminal leucine zipper domains flanking water-soluble polyelectrolyte segments. In near-neutral aqueous solutions, coiled-coil aggregates of the terminal domains form a three-dimensional hydrogel polymer network.

[0117] Common crosslinking agents that can be used to crosslink the hydrogels provided herein include, but are not limited to, ethylene glycol dimethacrylate (EGDMA), tetraethylene glycol dimethacrylate, and N,N'-15-methylenebisacrylamide. The range of additional crosslinking agents that can be used will be apparent to those skilled in the art and will follow general chemical reactivity principles, see, for example, the Thermo Scientific crosslinking technology manual entitled "Easy molecular bonding crosslinking technology" (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf ).

[0118] In one embodiment, the polymerization of the hydrogel is initiated by persulfate or an equivalent initiator that catalyzes the formation of free radicals. The range of compatible initiators is known to those skilled in the art and follows general chemical reactivity principles, see, for example, the Thermo Scientific crosslinking technology manual entitled "Easy molecular bonding crosslinking technology" (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdfThe persulfate can be any water-soluble persulfate. Non-limiting examples of water-soluble persulfates include ammonium persulfate and alkali metal persulfates. Alkali metals include lithium, sodium, and potassium. In some embodiments, the persulfate is ammonium persulfate or potassium persulfate. In further embodiments, the polymerization of the hydrogel provided herein is initiated by ammonium persulfate.

[0119] The polymerization of the hydrogel can be accelerated by accelerators that catalyze the formation of polymerization-labile chemical side groups. The range of accelerators that can be used is known to those skilled in the art and follows general chemical reactivity principles, see, for example, the ThermoScientific crosslinking technology manual entitled "Easy molecular bonding crosslinking technology" (available at tools.lifetechnologies.com / content / sts / brochures / 1602163- Crosslinking-Reagents-Handbook.pdf In one embodiment, the accelerator is a tertiary amine. The tertiary amine can be any water-soluble tertiary amine. In one embodiment, the accelerator is used for the polymerization reaction and is N,N,N',N'-tetramethylethylenediamine, 3-dimethylamino)propionitrile, or N,N,N',N'-tetramethylethylenediamine (TEMED). In another embodiment, the accelerator is used for the polymerization reaction and is azobis(isobutyronitrile) (AIBN).

[0120] As discussed above, the hydrogels used in the compositions and methods of the present application may include any of the monomeric units and crosslinking agents described herein, and in one aspect, are prepared as hydrogel particles by making droplets (see, e.g., Figure 2 Microfluidic methods for preparing a plurality of droplets (including fluidic droplets and fossilized droplets) are known to those skilled in the art and are described in U.S. Patent Publication No. 2011 / 0218123 and U.S. Patent No. 7,294,503, each of which is incorporated herein by reference in its entirety for all purposes. Such methods provide a plurality of droplets comprising a first fluid and substantially surrounded by a second fluid, wherein the first fluid and the second fluid are substantially immiscible (e.g., comprising droplets of a water-based liquid substantially surrounded by an oil-based liquid).

[0121] A plurality of fluidic droplets (e.g., prepared using a microfluidic device) can be polydisperse (e.g., having a range of different sizes), or in some cases, the fluidic droplets can be monodisperse or substantially monodisperse, e.g., having a uniform distribution of diameters, 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 mean of the diameters of the droplets. The average diameter of the particles can be measured, for example, by light scattering techniques. In one embodiment, the average diameter of the hydrogel particles is adjusted by, for example, varying the flow rates of the first and second fluids within a channel of the microfluidic device, or varying the volume of a channel of the microfluidic device.

[0122] Additionally, the present application provides a population of hydrogel particles comprising a plurality of hydrogel particles, wherein the population of hydrogel particles is substantially monodisperse.

[0123] The term microfluidics refers to a device, apparatus, or system comprising at least one fluidic channel having a cross-sectional dimension of less than 1 mm and a ratio of length to maximum cross-sectional dimension perpendicular to the channel of at least about 3:1. Microfluidic devices comprising microfluidic channels are particularly well-suited for producing a plurality of monodisperse droplets.

[0124] Non-limiting examples of microfluidic systems that can be used in the present invention are disclosed in U.S. Patent Application Publication No. 2006 / 0163385; U.S. Patent Application Publication No. 2005 / 0172476; U.S. Patent Application Publication No. 2007 / 000342; International Patent Application Publication No. WO2006 / 096571; U.S. Patent Application Publication No. 2007 / 0054119; U.S. Patent No. 7,776,927; and International Patent Application Publication No. WO2006 / 078841, each of which is incorporated herein by reference in its entirety for all purposes.

[0125] Droplet size is related to the general size of microfluidics. The microfluidic channel can have any size, for example, a maximum dimension perpendicular to the liquid flow of 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.

[0126] The droplet size can be adjusted by adjusting the relative flow rates. In some embodiments, the droplet diameter is equal to the channel width, or within about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the channel width.

[0127] The disclosed hydrogel particles are substantially similar in size to the droplets formed therefrom. Thus, in some embodiments, the hydrogel has a diameter of less than about 1 μm, less than about 2 μm, less than about 5 μm, less than about 10 μm, less than about 15 μm, less than about 20 μm, less than about 25 μm, less than about 30 μm, less than about 35 μm, less than about 40 μm, less than about 45 μm, less than about 50 μm, less than about 60 μm, less than about 70 μm, less than about 80 μm, less than about 90 μm, less than about 100 μm, less than about 120 μm, less than about 150 μm, less than about 200 μm, less than about 250 μm, less than about 300 μm, less than about 350 μm, less than about 400 μm, less than about 450 μm, less than about 500 μm, less than about 600 μm, less than about 800 μm, or less than 1000 μm. In some embodiments, the diameter of the hydrogel is greater than about 1 μm, greater than about 2 μm, greater than about 5 μm, greater than about 10 μm, greater than about 15 μm, greater than about 20 μm, greater than about 25 μm, greater than about 30 μm, greater than about 35 μm, greater than about 40 μm, greater than about 45 μm, greater than about 50 μm, greater than about 60 μm, greater than about 70 μm, greater than about 80 μm, greater than about 90 μm, greater than about 100 μm, greater than about 120 μm, greater than about 150 μm, greater than about 200 μm, greater than about 250 μm, greater than about 300 μm, greater than about 350 μm, greater than about 400 μm, greater than about 450 μm, greater than about 500 μm, greater than about 600 μm, greater than about 800 μm, or greater than 1000 μm. In one embodiment, the diameter of the hydrogel particles is between 5 μm and 100 μm.

[0128] In some embodiments, the disclosed hydrogel particles are spherical in shape.

[0129] In some embodiments, the disclosed hydrogel particles do not comprise agarose.

[0130] In one embodiment, the hydrogel particles are prepared by suspension polymerization, which is also known in the art as bead polymerization, bead polymerization, or granulation polymerization (see Elbert (2011). Acta Biomater. 7, pp. 31-56, which is incorporated herein by reference in its entirety for all purposes). In suspension polymerization, the monomer is insoluble in the continuous phase, for example, an aqueous monomer solution in an oil continuous phase. In suspension polymerization, polymerization initiation occurs within the monomer-rich droplets and requires the presence of more than one free radical per droplet at any time. In one embodiment, the monomer phase includes a monomer that can be a difunctional monomer or multiple monomer species (comonomers, which can be multiple difunctional monomers). In one embodiment, the monomer phase includes an initiator and / or a crosslinker.

[0131] Emulsion polymerization can also be used to form the hydrogel particles described herein. However, similar to suspension polymerization, in emulsion polymerization, the monomer has poor solubility in the continuous phase, and polymerization initiation occurs outside the monomer liquid (see Elbert (2011). Acta Biomater. 7, pp. 31-56, which is incorporated herein by reference in its entirety for all purposes). In emulsion polymerization embodiments, the initiator causes chain growth of monomer (or comonomer) dissolved in the continuous phase or, in the presence of a surfactant, monomer contained in micelles.

[0132] In another embodiment, the hydrogel particles are formed by precipitation polymerization, as described, for example, in Elbert (2011). Acta Biomater. 7, pp. 31-56, which is incorporated herein by reference in its entirety for all purposes. Precipitation polymerization is a technique that exploits the difference in solubility between monomers and polymers to produce microparticles. Specifically, it is known that larger polymer chains are generally less soluble than smaller polymer chains. Therefore, for a given molecular weight, phase separation may be advantageous. Precipitation polymerization initially begins as a solution polymerization in a single-phase, homogeneous system. In one embodiment, immediately after the start of polymerization, a relatively high concentration of polymer chains is present, favoring phase separation through nucleation. As polymerization proceeds, the concentration of polymer chains decreases, and existing particles capture chains before nucleation of new particles can occur. Consequently, particle nucleation occurs only briefly after the reaction begins, which, in one embodiment, results in a narrower particle size distribution. Additional methods include, but are not limited to, photolithographic particle formation (Helgeson et al. (2011). Curr. Opin. Colloid. Interface Sci. 16, pp. 106-117, which is incorporated herein by reference in its entirety for all purposes), membrane emulsification (e.g., by the micosieve emulsification process described by Nanomi BV (Netherlands), and microchannel emulsification (Sugiura et al. (2002). Languimir 18, pp. 5708-5712, which is incorporated herein by reference in its entirety), and bulk emulsification (SNF Floerger, available at snf.com.au / downloads / Emulsion_Handbook_E.pdf, which is incorporated herein by reference in its entirety).

[0133] In one embodiment, hydrogel particles are formed within a microfluidic device having two oil channels that meet at a central stream of an aqueous monomer solution. In this embodiment, droplets form at the interface of the two channels and the central stream, breaking up the droplets in the water-in-oil emulsion. In one embodiment, once the droplets are formed, they are stabilized prior to polymerization, for example, by adding a surfactant to the oil phase. However, in another embodiment, the droplets are destabilized prior to polymerization. In one embodiment, polymerization of the monomer is triggered by adding a promoter (e.g., N,N,N',N'-tetramethylethylenediamine) to one or both of the oil channels after the initial droplet formation.

[0134] The aqueous monomer solution provided above can include a single monomer species or multiple monomer species. The aqueous monomer solution can include a comonomer, a difunctional monomer, or a combination thereof. In one embodiment, the monomer or population of monomers can include a difunctional monomer, for example, one of the monomers described above. As described below, the comonomer can be used to adjust forward scattering or side scattering, for example, by adjusting the refractive index of the hydrogel particles.

[0135] In one embodiment, the center stream of the aqueous monomer solution comprises a crosslinker, such as N,N'-bisacrylamide. In a further embodiment, the center stream of the aqueous monomer solution comprises a crosslinker and an accelerator in addition to the monomer. In yet further embodiments, the aqueous monomer solution comprises an initiator, such as an oxidizing agent, such as ammonium persulfate.

[0136] Forward scattering is adjusted by adjusting the refractive index of the gel by adding comonomers allyl acrylate and allyl methacrylate (see also Figures 11 and 12). Forward scattering can also be adjusted by containing side scattering nanoparticles of sufficient optical resolution / size / density, including but not limited to, higher density colloidal suspensions of silica and / or PMMA particles. The side scattering of the droplets is adjusted as follows: a colloidal suspension of silica nanoparticles and / or PMMA (poly(methyl methacrylate)) particles (~100 nm) is added to the central aqueous phase prior to polymerization (Figures 11 and 12).

[0137] In one embodiment, a bead, a plurality of beads, a biomolecule, or a plurality of biomolecules are embedded (encapsulated) within a hydrogel particle. In one embodiment, the encapsulated beads or biomolecules are used to mimic one or more intracellular organelles of a target cell, or the cell itself after the cell has engulfed the particle. In one embodiment, encapsulation or embedding of the beads or biomolecules is accomplished during hydrogel particle formation. For example, the beads can be suspended at an appropriate concentration such that, on average, one bead is embedded / encapsulated within a single hydrogel particle. The bead suspension can be contained, for example, within an aqueous solution of monomer. Similarly, a biomolecule or mixture of biomolecules can be incorporated into an aqueous solution of monomer to encapsulate one or more biomolecules.

[0138] Alternatively, once the hydrogel particles are formed, for example, by the methods described above, in one embodiment, they can be further processed, for example, by embedding a bead, a plurality of beads, a biomolecule, or a plurality of biomolecules within the hydrogel particles.

[0139] Thus, in one aspect of the present invention, there is provided a hydrogel comprising an embedded substance.

[0140] In one embodiment, the embedded material is an embedded molecule, such as a biomolecule. The biomolecule can be a single substance or a plurality of different substances. For example, proteins, peptides, carbohydrates, nucleic acids, or combinations thereof can be encapsulated within the hydrogel particles of the present invention. In addition, different nucleic acid molecules (e.g., having different sequences or nucleic acid types, such as genomic DNA, messenger RNA, or DNA-RNA hybrids) can be encapsulated by the hydrogel particles of the present invention. These can include any protein or nucleic acid as two forms of biomaterials that contain unstable chemical side groups (or can be modified by the supplier (e.g., Integrated DNA Technology chemical side group modification). Such side groups are compatible with common reactive chemistries in comonomer compositions (e.g., acrylate chemistries, NHS esters, primary amines, copper-catalyzed click chemistry (Sharpless)). The range of available embedded molecules containing compatible chemistries will be understood by those skilled in the art.

[0141] In one embodiment, different subpopulations of hydrogel particles are created, each having a different concentration of a biomolecule. In a further embodiment, the biomolecule is a nucleic acid, a protein, an intracellular ion such as calcium (or other biomolecules selected by the user, e.g., calcium). In another embodiment, different subpopulations of hydrogel particles are created, each having a different concentration of a drug substance. In one embodiment, the drug substance is a biomolecule (i.e., a biopharmaceutical, an antibody drug, an antibody drug conjugate, a protein / enzyme, a peptide, a non-ribosomal peptide, or related molecules) or a small molecule synthetic drug (e.g., a type I / II / III polyketide, a non-ribosomal peptide with biologically active properties, or other small molecule entities generally categorized by those skilled in the art).

[0142] In this regard, the present invention is particularly suitable for determining the resolution of an assay in which individual nucleic acid or protein contents in a cell are stained. In one embodiment, different populations of hydrogel particles provided herein are encapsulated with known varying amounts of intracellular material, such as nucleic acids or proteins. The intracellular material in individual hydrogel particles is stained, and the fluorescence of individual hydrogels from the various populations is measured using a cell counting device. This enables the generation of a standard curve to establish the sensitivity and dynamic range of an intracellular assay. Once established, samples can be run through a cytometer to detect the presence of target cells and to quantify the amount of intracellular material in each target cell. In one embodiment, the embedded material is an infectious disease biomarker, such as one of the infectious disease biomarkers listed in the Infectious Disease Biomarker Database (IDBD, see Yang et al. (2008) IDBD: Infectious Disease Biomarker Database. Nucleic Acid Res. 36, pp. D455-D460, which is incorporated herein by reference in its entirety for all purposes). In further embodiments, the infectious disease biomarker is a biomarker of: gastrointestinal infection, respiratory tract infection, nervous system infection, genitourinary infection, viral infection, hemorrhagic fever, zoonotic disease, arbovirus, antibiotic resistance, or bioterrorism. In further embodiments, the viral infection is Ebola virus infection.

[0143] In one embodiment, the method provided herein is used to determine the sensitivity and / or dynamic range of a cellular nucleic acid quantification assay. In this embodiment, for a sample, the cell type (presence or absence) within the sample and the amount of cellular nucleic acid within the cell are queried.

[0144] In another embodiment, the present invention provides a means for determining the resolution and / or sensitivity of an intracellular protein quantification assay. In one embodiment, hydrogel particles are encapsulated with known amounts of a protein at various concentrations and then stained with an appropriate antibody against the protein. The fluorescence of each particle is measured to determine the sensitivity and / or dynamic range of the assay. This fluorescence value is then compared to the fluorescence value obtained from cells in the sample to determine the presence of the target cell, whether it contains the intracellular protein, and the amount of the protein.

[0145] In one embodiment, individual hydrogel particles are tuned to possess at least one optical property substantially similar to that of circulating tumor cells or fetal cells present in maternal blood. The individual particles are imbued with known amounts of the biomolecule to be analyzed. The particles are used to generate a standard curve for a biomolecule detection assay for a specific cell type.

[0146] As provided above, in one aspect of the present invention, a hydrogel comprising an embedded substance is provided. In one embodiment, the embedded substance is a bead or a plurality of beads. In one embodiment, the hydrogel particle is embedded with a single bead. In another embodiment, for an individual hydrogel, the average number of embedded beads in the plurality of hydrogel particles is one.

[0147] In the case of one or more beads embedded in a hydrogel particle, in one embodiment, the optical properties of the one or more beads are combined with the FSC and SSC properties of the hydrogel particle for quality control of flow cytometry experiments. For example, in one embodiment, the embedded beads are used as controls to calibrate a flow cytometer system, including laser source, optical properties, and flow rate. In another embodiment, the embedded beads are used as a means to quantify the amount of fluorescence in a sample, such as a specific cell. In this regard, various concentrations of embedded beads can be used to generate a fluorescence standard curve to determine whether cells express certain markers and at what expression levels.

[0148] In one embodiment, beads having a diameter of about 1 μm to about 3 μm, about 2 μm to about 4 μm, or about 3 μm to about 7 μm are embedded in the hydrogel provided herein. For example, in one embodiment, the beads have a diameter of about 3 μm to about 3.5 μm. In a further embodiment, the beads are fluorescent beads. In another embodiment, the beads have a diameter of about 1 μm to about 2.5 μm or about 1.5 μm to about 3 μm. In a further embodiment, the beads are fluorescent beads and can be internally dyed or dyed on their surface. In even further embodiments, the fluorescent beads are internally dyed. Without wishing to be bound by any theory, it is believed that internal dyeing isolates the fluorophore from environmental interactions that may cause changes in the fluorescent output.

[0149] As provided above, in one embodiment, the embedded beads are fluorescent beads, and in a further embodiment, the fluorescent beads are internally dyed. One skilled in the art will be able to select an appropriate fluorophore for use with the embedded beads. In one embodiment, the beads are derivatized with one or more of the following fluorescent dyes: 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein succinimidyl ester; 5-(and-6)-carboxyeosin; 5-carboxyfluorescein; 6-carboxyfluorescein; 5-(and-6)-carboxyfluorescein; S-carboxyfluorescein-di-(5-carboxymethoxy-2-nitrobenzyl) ether, -alanine-carboxamide, or succinimidyl ester; 5-carboxyfluorescein succinimidyl ester; 6-carboxyfluorescein succinimidyl ester; Fluorescein imide ester; 5-(and -6)-carboxyfluorescein succinimidyl ester; 5-(4,6-dichlorotriazinyl)aminofluorescein; 2',7'-difluorofluorescein; eosin-5-isothiocyanate; erythrosine-5-isothiocyanate; 6-(fluorescein-5-formylamino)hexanoic acid or succinimidyl ester; 6-(fluorescein-5-(and -6)-formylamino)hexanoic acid or succinimidyl ester; fluorescein-S-EX succinimidyl ester; fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate; 488 carboxylic acid, or succinimide ester; 488 isothiocyanates; 488-X succinimidyl ester; 500 carboxylic acid; 500 carboxylic acid, succinimide ester or triethylammonium salt; 514 carboxylic acid; 514 carboxylic acid or succinimidyl ester; rhodamine green TM Carboxylic acid, succinimidyl ester, or hydrochloride; Rhodamine green TM Carboxylic acid, trifluoroacetamide, or succinimidyl ester; Rhodamine green TM -X succinimidyl ester or hydrochloride; Rodel green TMCarboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester; bis-(4-carboxypiperidinyl)sulfone rhodamine 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′-tetrabromosulfonefluorescein succinimidyl ester or di-(diisopropylethylammonium) salt; 5-carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine; 5-(and-6)-carboxytetramethylrhodamine; 5-carboxytetramethylrhodamine succinimide ester; 6-carboxytetramethylrhodamine succinimide ester; 5-(and-6)-carboxytetramethylrhodamine succinimide ester; 6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimide ester; 6-carboxy-X-rhodamine succinimide ester; 5-(and-6)-carboxy-X-rhodamine succinimide ester; 5-carboxy-X-rhodamine triethylammonium salt; Lissamine TM Rhodamine B sulfonyl chloride; Malachite green; Isothiocyanate; Mono(sulfosuccinimide ester); 21 carboxylic acid or succinimide ester; 7-carboxylic acid or succinimide ester; Rhodamine Red TM-X succinimide ester; 6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoic acid; succinimide ester; tetramethylrhodamine-5-isothiocyanate; tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; sulfonyl group; Sulfonyl chloride; -X STP ester or sodium salt; -X succinimide ester; -X succinimidyl ester; and X-rhodamine-5-(and -6) isothiocyanate, 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-sym-indacen-3-propionic acid succinimide ester; 4,4-difluoro-4-bora-3a,4a-diaza-sym-indacen-3,5-dipropionic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sym-indacen-3-pentanoic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sym-indacen-3-pentanoic acid succinimide ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sym-indacen-3-propionic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3 a,4a-diaza-sym-indacen-3-propionic acid succinimide ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sym-indacen-3-propionic acid; sulfosuccinimide ester or sodium salt; 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sym-indacen-3-propionyl)amino)hexanoic acid; 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sym-indacen-3-propionyl)amino)hexanoic acid or succinimide ester; N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sym-indacen-3-propionyl)sulfoalanine, succinimide Ester or triethylammonium salt; 6-4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-sym-indacene-3-propionic acid; 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-sym-indacene-3-propionic acid succinimide ester; 4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-sym-indacene-3-propionic acid; succinimide ester; 6-((4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-sym-indacene-3-propionyl)aminohexanoic acid or succinimide ester; 4,4-difluoro-5-(4- phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-sym-indacen-3-propionic acid succinimide ester; 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-sym-indacen-3-propionic acid succinimide ester; 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-sym-indacen-3-yl)phenyloxy)acetyl)aminohexanoic acid or succinimide ester; 4,4-difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-sym-indacen-3-propionic acid; 4,4-difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-sym-indacen-3-propionic acid;Succinimide ester; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-sym-indacen-8-propionic acid; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-sym-indacen-8-propionic acid succinimide ester; 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-sym-indacen-3-propionic acid succinimide ester; 6-(( (4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacen-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimide ester; and 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacen-3-yl)phenyloxy)acetyl)amino)hexanoic acid or succinimide ester, Alexa fluor dyes commercially available from Invitrogen, including but not limited to; 350 carboxylic acid; 430 carboxylic acid; 488 carboxylic acid; 532 carboxylic acid; 546 carboxylic acid; 555 carboxylic acid; 568 carboxylic acid; 594 carboxylic acid; 633 carboxylic acid; 647 carboxylic acid; 660 carboxylic acid; and 680 carboxylic acid, a cyanine dye commercially available from Amersham-Pharmacia Biotech, which includes, but is not limited to, Cy3 NHS ester; Cy5 NHS ester; Cy5.5 NHS ester; and Cy7 NHS ester.

[0150] Other fluorophores useful in the present invention are given in Table 2 below.

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162] Commercially available beads include, but are not limited to, those sold by Bangs Laboratories, Inc., Sperhotech Inc., Thermo Scientific, Inc., and equivalent suppliers, which can be used in combination with the hydrogel particles described herein. One skilled in the art can select beads of appropriate bead diameter, fluorescence emission and / or excitation spectrum, and / or fluorescence intensity based on experiments. For example, quality control beads used in conjunction with a blue, red, or ultraviolet laser can be embedded in one or more hydrogel particles provided herein. For example, for a blue laser (Catalog No. A-16500 (2.5 μm), A-16503 (6.0 μm)), a red laser (Catalog No. A-16501 (2.5 μm), A-16504 (6.0 μm)), or an ultraviolet laser (Catalog No. A-16502 (2.5 μm), A-16505 (6.0 μm)), Alignflow TM Flow cytometry localization beads can be embedded in one or more hydrogel particles provided herein.

[0163] In one embodiment, fluorescent beads that can be excited at any wavelength in the range of 365 nm to 650 nm are embedded in the hydrogel particles. In one embodiment, the beads are "rainbow particles" that contain a mixture of fluorophores, such as 4 fluorophores, 5 fluorophores, 6 fluorophores, 7 fluorophores, or 8 fluorophores. In view of this, the user selects which wavelength to use to excite the particles based on the fluorophores to be interrogated. Rainbow particles are commercially available, for example, from BD Biosciences (Cat. Nos. 556298 (mid-range FL1 fluorescence), 556286 (6 colors, 3.0-3.4 μm), 556288 (6 colors, 6.0-6.4 μm), 559123 (8 colors)) and Spherotech of various diameters (e.g., Cat. Nos. RCP20-5 (4 colors), RCP-30-5 (6 peaks), RCP-30-5A (8 peaks)

[0164] Cell sorting beads can be embedded in one or more hydrogel particles provided in the present application. In one embodiment, the cell sorting beads are similar to biological samples in size, emission wavelength, and intensity, and can be used to calibrate the cell sorting system of a flow cytometer, including laser source, optical properties, and flow rate. In one embodiment, the cell sorting beads are embedded in one or more hydrogel particles and can be used for ultraviolet laser, blue laser, green / yellow laser or red laser. In one embodiment, when using a green laser, the embedded beads are excited at 570nm and emitted at 575nm, but can also be excited at 488nm. Commercial cell sorting beads are purchased from, for example, Life Technologies (catalog numbers C-16506 (ultraviolet laser), C-16508 (blue laser), C-16509 (green-yellow laser), C-16507 (red laser)).

[0165] Compensation control beads can also be embedded in one or more of the hydrogel particles provided herein. Accurate compensation is an important parameter for efficient multicolor analysis in flow cytometry. However, cell-based compensation controls are not completely effective because many antigens are not highly expressed, and sparsely stained cells can lead to inaccurate compensation settings.

[0166] In one embodiment, the compensation control beads include fluorescent antibody conjugate capture capability (positive compensation beads) or are inert (negative compensation beads). The compensation beads are mixed with human, mouse, rat, hamster, or rabbit antibodies conjugated to a fluorophore; the two components provide a clear high-signal positive control with a moderate negative colony, which can then be used to appropriately set the compensation, regardless of the concentration of the cells in the actual experiment. Once the antibody is mixed with the beads, the antibody is embedded in one or more hydrogel particles provided herein. Commercially available compensation beads are purchased from, for example, Life Technologies (Cat. Nos. A-10344, A-10389, A10497, A10513) and Spherotech (Cat. Nos. CMIg-P-08-2K, CMIg-P-30-2K, CMIg-P-50-3K, CMIg-P-70-3K).

[0167] In one embodiment, the hydrogel particles containing embedded / encapsulated beads are used as a reference for cell-based assays, such as phagocytosis assays, cytotoxicity assays, motility assays, cell viability assays, and the like. Phagocytosis is the process by which cells engulf solid particles, forming an internal capsule called a phagosome. In view of this, the hydrogel particles can be adjusted to have one or more optical properties that are substantially similar to those of a phagocytic cell before and after engulfment of the particle by the phagocytic cell. Thus, in one embodiment, the hydrogel particles provided herein are used as control particles for a phagocytosis assay. In a further embodiment, (i) one or more optical properties of the hydrogel particle are substantially similar to those of a phagocytic cell before uptake of the particle, and (ii) one or more optical properties of the second hydrogel particle are substantially similar to those of a phagocytic cell after uptake of the particle. In view of this, a control is generated for measuring particles taken up by phagocytic cells.

[0168] In one embodiment, the phagocytes are professional phagocytes. In another embodiment, the phagocytes are non-professional phagocytes (i.e., cells that consume dying cells and foreign organisms). In further embodiments, the non-professional phagocytes are epithelial cells, endothelial cells, fibroblasts, or mesenchymal cells. In one embodiment, the hydrogel particles are configured to have one or more optical properties substantially similar to those of professional phagocytes (before and / or after particle absorption) listed in Table 3 below.

[0169]

[0170]

[0171] In one embodiment, a population of hydrogel particles of the present invention embedded with a substance, such as nucleic acids or beads, is used as a control reagent in a genomic cytometry assay. Thus, a specific number of copies of a particular chromosome, RNA sequence, and / or DNA sequence can be simulated by the embedded substance. The hydrogel particles can then be used as a control for a sample of genetic information to be probed, such as the number of copies of a chromosome, RNA sequence, and / or DNA sequence.

[0172] The three main modes of deconvolution in flow cytometry are two passive optical properties of the particles (forward scatter, FSC, corresponding to the refractive index, or RI; and side scatter, SSC) and the biomarkers present on the surface of a given cell type. Therefore, the composition of the hydrogel particles of the present application that allows them to mimic specific cell types for these three modes is used to provide a synthetic, powerful calibration agent for flow cytometry.

[0173] In one embodiment, 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.

[0174] In another embodiment, the disclosed hydrogel particles have a refractive index (RI) of about 1.10 to about 3.0, or about 1.15 to about 3.0, or about 1.20 to about 3.0, or about 1.25 to about 3.0, or about 1.30 to about 3.0, or about 1.35 to about 3.0, or about 1.4 to about 3.0, or about 1.45 to about 3.0, or about 1.50 to about 3.0, or about 1.6 to about 3.0, or about 1.7 to about 3.0, or about 1.8 to about 3.0, or about 1.9 to about 3.0, or about 2.0 to about 3.0.

[0175] In some embodiments, the disclosed hydrogel particles have a refractive index (RI) of 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.

[0176] It is most meaningful to measure the SSC of the disclosed hydrogel particles compared to the 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 cell counting device.

[0177] In one embodiment, the SSC of a hydrogel particle is modulated by incorporating a high refractive index molecule (or a population thereof) into the hydrogel. In one embodiment, the high refractive index molecule is disposed within the hydrogel particle, and in further embodiments, the high refractive index molecule is a silica gel, an alkyl acrylate, an alkyl methacrylate, or a combination thereof. Thus, in some embodiments, the disclosed hydrogel particles are alkyl acrylate and / or alkyl methacrylate. In one embodiment, the concentration of the monomer is adjusted to further modulate the refractive index of the hydrogel particle.

[0178] The alkyl acrylate or alkyl methacrylate may contain 1 to 18 carbon atoms, 1 to 8 carbon atoms, or 2 to 8 carbon atoms in the 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.

[0179] High refractive index molecules may also include vinyl aromatics such as styrene and methylstyrene, which are optionally substituted on the aromatic ring with alkyl groups such as methyl, ethyl or tert-butyl, or with halogens, such as chlorostyrene.

[0180] In some embodiments, FSC is adjusted by adjusting the percentage of monomer present in the composition, thereby varying the water content present during hydrogel formation. In one embodiment, when using monomers and comonomers, the ratio of monomer to comonomer is adjusted to alter the forward scattering properties of the hydrogel particles. This is illustrated in both Figures 11 and 12.

[0181] It is most meaningful to measure the FSC of the disclosed hydrogel particles compared to the 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 cell counting device.

[0182] FSC is related to particle volume and can therefore be adjusted by varying particle diameter, as described herein. Generally, it has been observed that larger objects refract more light than smaller objects, resulting in a higher forward scatter signal (and vice versa). Therefore, in one embodiment, particle diameter is varied to adjust the FSC properties of the hydrogel particles. For example, in one embodiment, an increase in hydrogel particle diameter is achieved by utilizing larger microfluidic channels during fluid formation.

[0183] SSCs can be designed by encapsulating nanoparticles within a hydrogel to mimic organelles in target cells. In some embodiments, the disclosed hydrogel particles comprise one or more types of nanoparticles selected from the group consisting of polymethyl methacrylate (PMMA) nanoparticles, polystyrene (PS) nanoparticles, and silica nanoparticles. See also Figures 11 and 12, which illustrate that the addition of various concentrations of nanoparticles allows for tuning the side scatter of the particles. Without wishing to be bound by theory, the ability to selectively modulate both forward and side scatter of the hydrogels described herein may provide a powerful platform for mimicking a wide range of cell types.

[0184] Although the present invention primarily describes the modification of optical properties, the invention is not limited thereto. For example, hydrogel particles can be made and adjusted to adjust the capacity of the particles, for example, to calibrate a Coulter counter. In one embodiment, the capacity of the hydrogel particles is adjusted by varying the amount of hydrogel monomer in the composition. For example, polyaniline, polyacetylene; polyphenylene vinylene; polypyrrole (X=NH) and polythiophene (X=S) comonomers; and polyaniline (X=NH / N) and polyphenylene sulfide (X=S) comonomer concentrations can all be adjusted to vary the capacity. In one embodiment, the concentration of one or more of these monomers is increased to increase the capacity of the hydrogel particles.

[0185] In some embodiments, the disclosed hydrogel particles have material modulus properties (eg, elasticity) that more closely resemble those of target cells than do polystyrene beads of the same diameter.

[0186] After the hydrogel particles are formed, one or more particle surfaces can be functionalized, for example, to simulate one or more optical properties of a target cell or to label a target cell. Functionalized hydrogel particles can also include embedded beads or substances such as biomolecules, as described above. In one embodiment, one or more hydrogel particles are functionalized with one or more fluorescent dyes, one or more cell surface markers (or their epitope binding regions), or a combination thereof. In one embodiment, the hydrogel particles are formed by polymerizing at least one bifunctional monomer, and after formation, the hydrogel particles include one or more functional groups that can be used to further connect a cell surface marker, an epitope binding region of a cell surface marker, a fluorescent dye, or a combination thereof. In one embodiment, the free functional group is an amine, a carboxyl, a hydroxyl, or a combination thereof. Depending on the desired functionalization, it will be understood that a variety of bifunctional monomers can be used, for example, to functionalize the particles using different chemical methods and different molecules.

[0187] The hydrogel particles can be functionalized with any fluorescent dye known in the art, including The Fluorescent dyes are listed in Handbook - A Guide to Fluorescent Probes and Labeling Technologies, which is incorporated herein by reference in its entirety for all purposes. Functionalization can be mediated by compounds containing free amine groups, such as allylamine, which can be introduced into the difunctional monomers used to form the hydrogel, as discussed above.

[0188] Non-limiting examples of known fluorescent dyes that can be used to functionalize the surface of the hydrogel particles described herein include: 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein succinimidyl ester; 5-(and-6)-carboxyeosin; 5-carboxyfluorescein; 6-carboxyfluorescein; 5-(and-6)-carboxyfluorescein; S-carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl) ether, -alanine-carboxamide, or succinimidyl ester; 5-carboxyfluorescein succinimidyl ester; 6-carboxyfluorescein Fluorescein succinimidyl ester; 5-(and -6)-carboxyfluorescein succinimidyl ester; 5-(4,6-dichlorotriazinyl)aminofluorescein; 2',7'-difluorofluorescein; eosin-5-isothiocyanate; erythrosine-5-isothiocyanate; 6-(fluorescein-5-formylamino)hexanoic acid or succinimidyl ester; 6-(fluorescein-5-(and -6)-formylamino)hexanoic acid or succinimidyl ester; fluorescein-S-EX succinimidyl ester; fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate; 488 carboxylic acid, or succinimide ester; 488 isothiocyanates; 488-X succinimidyl ester; 500 carboxylic acid; 500 carboxylic acid, succinimide ester or triethylammonium salt; 514 carboxylic acid; 514 carboxylic acid or succinimidyl ester; rhodamine green TM Carboxylic acid, succinimidyl ester or hydrochloride; Rhodamine green TM Carboxylic acid, trifluoroacetamide, or succinimidyl ester; Rhodamine green TM -X succinimidyl ester or hydrochloride; Rodel green TMCarboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester; bis-(4-carboxypiperidinyl)sulfone rhodamine 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′-tetrabromosulfonefluorescein succinimidyl ester or bis-(diisopropylethyl)-1,2-dimethoxy ... ammonium) salt; 5-carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine; 5-(and-6)-carboxytetramethylrhodamine; 5-carboxytetramethylrhodamine succinimidyl ester; 6-carboxytetramethylrhodamine succinimidyl ester; 5-(and-6)-carboxytetramethylrhodamine 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; isothiocyanates; Mono(sulfosuccinimide ester); 21 carboxylic acid or succinimide ester; 7-carboxylic acid or succinimidyl ester; Rhodamine Red TM -X succinimidyl ester; 6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoic acid; succinimidyl ester; tetramethylrhodamine-5-isothiocyanate; tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; sulfonyl group; Sulfonyl chloride; -X STP ester or sodium salt; -X succinimide ester; -X succinimidyl ester; and X-rhodamine-5-(and -6) isothiocyanate.

[0189] Other examples of fluorescent dyes for use in the hydrogel particles described herein include, but are not limited to, 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-sym-indacen-3-propionic acid succinimide ester; 4,4-difluoro-4-bora-3a,4a-diaza-sym-indacen-3,5-dipropionic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sym-indacen-3-pentanoic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sym-indacen-3-pentanoic acid succinimide ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sym-indacen-3-propionic acid; 4,4-difluoro-5,7-dimethyl Methyl-4-bora-3a,4a-diaza-sym-indacene-3-propionic acid succinimide ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sym-indacene-3-propionic acid; sulfosuccinimide ester or sodium salt; 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sym-indacene-3 propionyl)amino)hexanoic acid; 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sym-indacene-3-propionyl)amino)hexanoic acid or succinimide ester; N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sym-indacene Indacene-3-propionyl) cysteic acid, succinimide ester or triethylammonium salt; 6-4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-sym-indacene-3-propionic acid; 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-sym-indacene-3-propionic acid succinimide ester; 4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-sym-indacene-3-propionic acid; succinimide ester; 6-((4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-sym-indacene-3 -propionyl)amino)hexanoic acid or succinimide ester; 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-sym-indacen-3-propionic acid succinimide ester; 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-sym-indacen-3-propionic acid succinimide ester; 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-sym-indacen-3-yl)phenylvinyloxy)acetyl)aminohexanoic acid or succinimide ester; 4,4-difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-sym-indacen-3-propionic acid;4,4-Difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-sym-indacen-3-propionic acid; succinimide ester; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-sym-indacen-8-propionic acid; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-sym-indacen-8-propionic acid succinimide ester; 4,4-difluoro-5-(2-thienyl)-4-bora-3 a,4a-diaza-sym-indacen-3-propionic acid succinimide ester; 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-sym-indacen-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimide ester; and 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-sym-indacen-3-yl)phenyloxy)acetyl)amino)hexanoic acid or succinimide ester. ;

[0190] In one embodiment, fluorescent dyes used to derivatize the surface of one or more hydrogel particles include, but are not limited to, Alexa fluor dyes commercially available from Invitrogen, which include, but are not limited to 350 carboxylic acid; 430 carboxylic acid; 488 carboxylic acid; 532 carboxylic acid; 546 carboxylic acid; 555 carboxylic acid; 568 carboxylic acid; 594 carboxylic acid; 633 carboxylic acid; 647 carboxylic acid; 660 carboxylic acid; and In another embodiment, fluorescent dyes used in the hydrogel particles and methods described herein include cyanine dyes commercially available from Amersham-Pharmacia Biotech, including but not limited to Cy3 NHS ester; Cy5 NHS ester; Cy5.5 NHS ester; and Cy7 NHS ester.

[0191] One skilled in the art can select one or more suitable dyes based on the desired spectral excitation and emission properties of the hydrogel particles.

[0192] The hydrogel particles, in one embodiment, are derivatized with one or more cell surface markers (see, e.g., Tables 4 and 7-8) or fragments thereof (e.g., the extracellular portion thereof in the case of transmembrane proteins), for example, by attaching one or more cell surface markers, their extracellular portions or complementary binding regions to the particles via free amines, free amines present on the surface of the hydrogel particles, free carboxyl groups and / or free hydroxyl groups. Functionalization of the hydrogel particles with dyes or cell surface molecules can also be performed via linkers such as streptavidin / biotin conjugates.

[0193] According to the target cell, individual hydrogel particles are derivatized with one or more cell surface markers or fragments thereof (e.g., in the case of transmembrane proteins, their extracellular portions) to further simulate the structural properties of the target cell. Tables 4 and 7-8 provided below list non-limiting lists of cell surface markers that can be used for derivatizing hydrogel particles according to the target cell. Although cell surface markers are provided, it should be understood that portions of cell surface markers, such as receptor binding moieties, ligand binding moieties, or the extracellular portions of markers can also be used for derivatizing hydrogel particles (at free functional groups, as described above). See also Figures 11 and 12, which show that hydrogel surface modification together with selective regulation of FSC and / or SSC with, for example, cell surface receptors enables the manufacture of hydrogel particles with desired properties.

[0194]

[0195] Cell types include, but are not limited to, various cell lines such as CHO, HEK-293, BHK-21, NSO, MDCK, VERO, MRC-S, W1-38 and Sp2 / 0 mouse myeloma (hybridoma). Tables 5 and 6 each provide other cell types for use in the hydrogel particles described herein.

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0218]

[0219] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0220]

[0221] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0222]

[0223] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0224]

[0225] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0226]

[0227] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0228]

[0229] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0230]

[0231] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0232]

[0233] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0234]

[0235] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0236]

[0237] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0238]

[0239] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0240]

[0241] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0242]

[0243] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0244]

[0245] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0246]

[0247] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0248]

[0249] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0250]

[0251] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0252]

[0253] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0254]

[0255] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0256]

[0257] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0258]

[0259] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0260]

[0261] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0262]

[0263] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0264]

[0265] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0266]

[0267] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0268]

[0269] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0270]

[0271] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0272]

[0273] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0274]

[0275] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0276]

[0277] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0278]

[0279] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0280]

[0281] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0282]

[0283] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0284]

[0285] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0286]

[0287] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0288]

[0289] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0290]

[0291] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0292]

[0293] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0294]

[0295] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0296]

[0297] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0298]

[0299] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0300]

[0301] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0302]

[0303] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0304]

[0305] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0306]

[0307] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0308]

[0309] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0310]

[0311] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0312]

[0313] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0314]

[0315] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0316]

[0317] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0318]

[0319] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0320]

[0321] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0322]

[0323] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0324]

[0325] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0326]

[0327] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0328]

[0329] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0330]

[0331] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0332]

[0333] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0334]

[0335] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0336]

[0337] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0338]

[0339] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0340]

[0341] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0342]

[0343] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0344]

[0345] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0346]

[0347] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0348]

[0349] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0350]

[0351] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0352]

[0353] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0354]

[0355] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0356]

[0357] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0358]

[0359] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0360]

[0361] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0362]

[0363] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0364]

[0365] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0366]

[0367] Table 8. Cell surface markers used with the hydrogel particles described in this application.

[0368]

[0369] In one embodiment, a population of hydrogel particles is used to determine the dynamic range and / or sensitivity of detecting a specific cell surface marker or combination thereof on a target cell population. For example, a population of hydrogel particles can be adjusted to have the SSC and / or FSC waveform of a target cell, and a subpopulation of hydrogel particles can be derivatized with a specific number of copies of a cell surface marker, such as a cell surface receptor or a domain thereof, such as a surface antigen binding region thereof. For example, individual subpopulations of hydrogel particles can each be derivatized to have a unique number of copies, e.g., one subpopulation will contain 100 copies of a cell surface marker, a second subpopulation will contain 1,000 copies of the same cell surface marker, a third subpopulation will contain 10,000 copies of the same cell surface marker, and so on. The population of hydrogel particles is fluorescently stained for each cell surface marker, and the hydrogel particles in each subpopulation are detected. Given this, the subpopulations of hydrogel particles can be used to generate a standard curve for fluorescence emission for target cells having each cell marker. The cell surface marker can be any cell surface marker provided herein, or a binding region thereof, or a cell surface marker known to those skilled in the art.

[0370] The hydrogel particles of the present application behave similarly to target cells during processes such as staining and analysis by flow cytometry or FACS. For example, in one embodiment, the hydrogel particles have one or more optical properties substantially similar to those of one of the cell types listed in Table 1, Table 2, or Table 3.

[0371] In some embodiments, the target cell is an immune cell. Non-limiting examples of immune cells include B lymphocytes, also referred to as B cells, T lymphocytes, also referred to 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, drug-modified immune cells, and derivatives, precursors or progenitor cells of any cell type listed herein.

[0372] In some embodiments, target cells include all cells of a particular class of cells having shared properties. For example, target cells can be lymphocytes, including NK cells, T cells, and B cells. Target cells can be activated lymphocytes.

[0373] 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.

[0374] 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.

[0375] In other embodiments, the target cell is selected from the group consisting of: primary lymphocytes, monocytes, and granulocytes.

[0376] The target cell can be essentially any type of cell, including prokaryotic and eukaryotic cells.

[0377] Suitable prokaryotic target cells include, but are not limited to, bacteria such as Escherichia coli (E. coli), various Bacillus species, and extremophilic bacteria such as thermophiles.

[0378] Suitable eukaryotic target cells include, but are not limited to, fungi such as yeast and filamentous fungi, including the genera Saccharomyces, Aspergillus, Trichoderma, and Neurospora; plant cells, including those from corn, sorghum, tobacco, canola, soybean, cotton, tomato, potato, truncatula, sunflower, etc.; and animal cells, including fish, birds, and mammals. Suitable fish cells include, but are not limited to, those from the following species: salmon, trout, tilapia, tuna, carp, flounder, halibut, swordfish, cod, and zebrafish. Suitable avian cells include, but are not limited to, cells from chickens, ducks, quail, pheasants, and turkeys, and other jungle or game birds. Suitable mammalian cells include, but are not limited to, cells from horses, cows, buffalo, deer, sheep, rabbits, rodents such as mice, rats, hamsters and guinea pigs, goats, pigs, primates, marine mammals including dolphins and whales, and cell lines, such as human cell lines of any tissue or stem cell type, and stem cells, including totipotent and non-totipotent cells, and non-human fertilized eggs.

[0379] Suitable cells also include those cell types involved in numerous disease states (even if also in non-disease states). Therefore, suitable eukaryotic cell types include, but are not limited to, all types of tumor cells (for example, melanoma, myeloid leukemia, lung cancer, breast cancer, ovarian cancer, colon cancer, kidney cancer, prostate cancer, pancreatic cancer and testicular cancer), myocardial cells, dendritic cells, endothelial cells, epithelial cells, lymphocytes (T cells and B cells), mast cells, eosinophils, vascular endothelial cells, macrophages, natural killer cells, red blood cells, hepatocytes, leukocytes (including mononuclear leukocytes), stem cells such as hematopoietic stem cells, neural hepatocytes, skin hepatocytes, lung hepatocytes, kidney hepatocytes, liver hepatocytes and myocardial stem cells (for screening differentiation factors and dedifferentiation factors), osteoclasts, chondrocytes and other connective tissue cells, keratinocytes, melanocytes, hepatocytes, kidney cells, and adipocytes. In some embodiments, cell is a primary disease state cell, for example, a primary tumor cell. Suitable cells also include known research cells, including, but not limited to, Jurkat T cells, NIH3T3 cells, CHO, COS, etc. See the ATCC cell line catalog, which is expressly incorporated herein by reference.

[0380] In some embodiments, the target cells are tumor microvesicles or tumor multiporous liposomes. Tumor cell microvesicles, also known as tumor-secreted microvesicles or tumor-secreted exosomes, are found in circulating blood and have immunosuppressive activity. Tumor cell microvesicles typically range in size from 30 to 200 nm in diameter. Larger tumor microvesicles, referred to as tumor macrovesicles, can range in size from 3 to 10 μm in diameter.

[0381] The hydrogel particles described herein can be used in any flow cytometer known to those skilled in the art. For example, one or more of the flow cytometers listed in Table 9 below can be used with the hydrogels and assays described herein.

[0382]

[0383]

[0384]

[0385]

[0386]

[0387] Example

[0388] The present invention is further illustrated by referring to the following examples. However, it should be noted that these examples, like the above embodiments, are illustrative and should not be interpreted as limiting the scope of the present invention in any way.

[0389] Example 1: Production of hydrogel particles

[0390] UV photolithography photomasks were obtained from CADart Services Inc. and designed using AutoCad (AutoDesk, Inc.). SU-8 photoresist (Microchem, Inc.) was photocrosslinked on a 4″ silicon wafer using a calibrated UV light source (OAI, Inc.) to create masks for microfluidic device fabrication. PDMS (polydimethylsiloxane, Sigma Aldrich, Inc.) was prepared and patterned using soft lithography and standard published methods for microfluidic device fabrication (see, McDonald JC, et al., 2000, Electrophoresis 21: 27-40).

[0391] Droplets were formed using a flow focusing geometry in which two oil channels converged to form a central stream of an aqueous monomer solution, thereby breaking the droplets in the water-in-oil emulsion. For droplet formation, a fluorocarbon oil (Novec 7500 3M, Inc.) was used as the external continuous phase liquid. To stabilize the droplets before polymerization, a surfactant was added to the oil phase (ammonium carboxylate salt of Krytox 157FSH, Dupont) at 0.5% w / w. To prepare basic polyacrylamide gel particles, a central phase of an aqueous monomer solution containing N-acrylamide (1-20% w / v), a crosslinker (N,N′-bisacrylamide, 0.05-1% w / v), an accelerator, and ammonium persulfate (1% w / v) was used. An accelerator (N,N,N′,N′-tetramethylethylenediamine (2% vol%)) was added to the oil phase to trigger the polymerization of the hydrogel particles after droplet formation.

[0392] Several comonomers were added to the alkaline gel formulation to increase functionality. After gel formation, allylamine provided primary amine groups for secondary labeling. We tuned the refractive index of the gel, and thus the forward scattering, by adding the comonomers allyl acrylate and allyl methacrylate. The side scattering of the droplets was modulated by adding a colloidal suspension of silica nanoparticles and / or PMMA (poly(methyl methacrylate)) particles (~100 nm) to the central aqueous phase before polymerization.

[0393] Stoichiometric multiplication of hydrogel particles is achieved by utilizing comonomers containing chemically orthogonal pendant groups (amine, carboxyl, maleimide, epoxide, alkyne, etc.) for secondary labeling.

[0394] Droplets were formed at an average rate of 5 kHz and collected in a fluorocarbon oil phase. Polymerization was performed at 50°C for 30 minutes and the resulting hydrogel particles were washed from the oil phase into an aqueous solution.

[0395] Example 2: Production and imaging of 12 11m hydrogel particles

[0396] Water containing 5% acrylamide, 0.25% bisacrylamide, 0.05% allylamine, and 0.1% ammonium persulfate was flowed through the central channel and converged by oil containing 0.1% TEMED through a 10 micron nozzle to produce 10 μm hydrogel particles, as shown in FIG. Figure 3A After polymerization, the particles were washed in water, as shown. Figure 3B As shown, they were conjugated to relevant dyes. Fluorescent hydrogel particles were visualized using fluorescence microscopy, as shown Figure 3C shown.

[0397] Example 3: Multidimensional modulation of optical properties of hydrogel particles

[0398] As shown in Figure 4, hydrogel particles can be tuned in multiple dimensions to match specific cell types, unlike polystyrene beads. Optical parameters such as FSC and SSC ( Figure 4A ) or a combination of secondary markers to deconvolute the cells. The hydrogel particles are adjusted to match the SSC and FSC of a specific cell type unlike polystyrene beads (brown), which are limited in size (FSC) and side scatter ( Figure 4B The hydrogel particles are further functionalized with specific chemical side groups and secondary labels in stoichiometrically adjusted ratios, allowing precise matching of cell types without compromising biological noise, while still behaving like fixed cell lines ( Figure 4C ).

[0399] Example 4: Relationship between flow cytometer delay time and hydrogel particle diameter

[0400] like Figure 5 As shown, the inter-droplet delay of the flow cytometer can be accurately correlated with the diameter of the hydrogel particles. The data shown are for hydrogel particles with diameters of 3 μm, 6 μm, 10 μm, 32 μm, and 50 μm when using a flow cytometer nozzle size of 70 to 100 μm.

[0401] Example 5: Comparison of hydrogel particles containing encapsulated DNA and cells

[0402] To form hydrogel particles containing encapsulated DNA, 40 μg / mL to 1000 μg / mL of recombinant calf thymus DNA was added to a polymer mixture containing 20% ​​19:1 (acrylamide:bisacrylamide) in water and 0.1% allylamine. 0.4% ammonium persulfate was added to the mixture prior to droplet formation. The hydrogel particles were formed as described in Example 1. When imaged using a commercial imaging cytometer and propidium iodide, the hydrogel particles containing 200 μg / mL of encapsulated calf thymus DNA showed cell-like staining, which was compared to Chinese hamster ovary cells stained using the same procedure. Images were taken using a Nexcelom Cellometer TM Obtained (Figure 6).

[0403] Cells obtained from buccal mucosal swabs were washed in PBS and stained with propidium iodide. In parallel, colonies of hydrogel particles containing a range of DNA concentrations were stained in the same manner. Both cell and particle suspensions were analyzed on a flow cytometer (488 / 590 nm excitation / emission). Flow cytometric analysis of cheek cells and the same range of encapsulated DNA particles showed that the particles exhibited a range of cell-like fluorescent properties ( Figure 7 , left panel). The intensity of staining showed a linear correlation with the median intensity, as measured by flow cytometry ( Figure 7 , right).

[0404] Example 6: Modulating Side Scattering of Hydrogel Particles

[0405] Silica gel was added to the aqueous portion of the polymer mixture at 12.5%, 6.25%, 3.125% and 0%, and hydrogel particles were formed as described in Example 1. Forward scatter and side scatter data were obtained using flow cytometry. The results showed that the side scatter signal ( Figure 8 , left) increases with the percentage of encapsulated nanoparticles, while forward scattering ( Figure 8 , right panel) remained generally unchanged, indicating no correlation with modulation of side and forward scatter.

[0406] Example 7: Adjusting forward scattering of hydrogel particles

[0407] In this experiment, the percentage of acrylamide:bisacrylamide in the hydrogel composition was varied from 10 to 40% to adjust the refractive index of the hydrogel particles, as measured by forward scattering in a flow cytometer. Figure 9 As shown, forward scatter increases with increasing percentage of acrylamide:bisacrylamide relative to .

[0408] Example 8: Adjusting the Optical Properties of Hydrogel Particles

[0409] Regulate hydrogel particles to match the example of the optical properties of required cell subtypes. Comonomers can be combined with nanoparticles to use passive optical measurement to regulate the forward scattering and side scattering properties of the hydrogel in a flow cytometer. By combining these properties with chemically unstable comonomers (e.g., allylamine, acrylic acid), it is possible to increase and label (if necessary) other fluorophores / proteins / biological side groups so as to match cell sub-population dyeing in addition to scattering properties. These are three main types of flow cytometry that can be utilized to determine the measurements of cells. Other side groups, for example, comprising heavy metals, can be used for, for example, Cy-TOF (cytometry, time-of-flight mass spectrometry) calibration. Finally, biocompatible materials can be encapsulated to simulate subcellular organelle dyeing.

[0410] Example 9: Adjusting the Optical Properties of Hydrogel Particles

[0411] A 50 nm nanoparticle colloidal suspension was incorporated into a hydrogel matrix to mimic the optical properties of lymphocytes and monocytes ( Figure 13A and 13B The percentage composition of the suspension was changed to match the blood cell subpopulation from the blood sample control (Streck) ( Figure 13C ).

[0412] Specifically, the acrylamide monomer concentration of the hydrogel particles (0.7-0.8 M) was adjusted to increase the particles' forward scatter, thereby matching the blood cell subpopulation. The percentage of bisacrylamide crosslinker was also varied to affect forward scatter (1-5%). Silica nanoparticles were used in the composition at 5% or 10% to adjust side scatter. The results of this experiment are shown in Figure 13.

[0413] ********

[0414] All documents, patents, patent applications, publications, product specifications, and procedures cited throughout this application are hereby incorporated by reference in their entirety for all purposes.

[0415] The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art to make and use the invention in the best manner known to the inventors. Modifications and variations of the embodiments of the invention described above are possible without departing from the invention, as will be apparent to those skilled in the art based on the above teachings. It should be understood, therefore, that the invention may be practiced in any manner not specifically described herein, within the scope of the claims and their equivalents.

Claims

1. A population of hydrogel particles, each hydrogel particle comprising: (i) polymers comprising polymerized monomers and comonomers; (ii) one or more biomolecules; and (iii) nanoparticles encapsulated in the hydrogel, wherein the optical properties of the population of hydrogel particles are at least 40% similar to corresponding properties of target cells, the optical properties being provided by the comonomer, the ratio of monomer to comonomer, or the encapsulated nanoparticles.

2. The population of hydrogel particles of claim 1 , wherein the polymerized monomer is lactic acid, glycolic acid, acrylic acid, 1-hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate, propylene glycol methacrylate, acrylamide, N-vinyl pyrrolidone, methyl methacrylate, glycidyl methacrylate, glyceryl methacrylate, ethylene glycol methacrylate, ethylene glycol, fumaric acid, 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate, poly(ethylene glycol) methacrylate, Methoxy-poly(ethylene glycol) methacrylate, methacrylic acid, sodium methacrylate, glyceryl methacrylate, hydroxypropyl methacrylate, hydroxybutyl methacrylate, phenyl acrylate, phenyl methacrylate, benzyl acrylate, benzyl methacrylate, 2-phenylethyl acrylate, 2-phenylethyl methacrylate, 2-phenoxyethyl acrylate, 2-phenoxyethyl methacrylate, phenylthioethyl acrylate, phenylthioethyl methacrylate, 2,4,6-tribromophenyl acrylate, 2,4,6-tribromophenyl methacrylate, pentabromophenyl acrylate, pentabromophenyl methacrylate, pentachlorophenyl acrylate, pentachlorophenyl methacrylate , 2,3-dibromopropyl acrylate, 2,3-dibromopropyl methacrylate, 2-naphthyl acrylate, 2-naphthyl methacrylate, 4-methoxybenzyl acrylate, 4-methoxybenzyl methacrylate, 2-benzyloxyethyl acrylate, 2-benzyloxyethyl methacrylate, 4-chlorophenoxyethyl acrylate, 4-chlorophenoxyethyl methacrylate, 2-phenoxyethoxyethyl acrylate, 2-phenoxyethoxyethyl methacrylate, N-phenylacrylamide, N-phenylmethacrylamide, N-benzylacrylamide, N-benzylmethacrylamide, N,N-dibenzylacrylamide, N,N-dibenzylmethacrylamide, N-diphenylmethacrylamide , N-(4-methylphenyl)methacrylamide, N-1-naphthylmethacrylamide, N-4-nitrophenylmethacrylamide, N-(2-phenylethyl)methacrylamide, N-triphenylmethylmethacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenylacrylamide, N,N-phenylphenylethylacrylamide, N-diphenylmethylmethacrylamide, N-(4-methylphenyl)methylmethacrylamide, N-1-naphthylmethacrylamide, N-4-nitrophenylmethacrylamide, N-(2-phenylethyl)methacrylamide, N-triphenylmethylmethacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenylmethacrylamide, N,N'-phenylphenylethyl methacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridine, methacrylamide, N-alkylacrylamide, N-alkylmethacrylamide, N,N-dialkylacrylamide, N-[(dialkylamino)alkyl]acrylamide, N-[(dialkylamino)alkyl]methacrylamide, (dialkylamino)alkyl acrylate or (dialkylamino)alkyl methacrylate.

3. The population of hydrogel particles according to claim 1, wherein the polymerized monomer is acrylamide.

4. The population of hydrogel particles of claim 1 , wherein the comonomer comprises one or more pendant chemical groups capable of reacting with a dye molecule.

5. The population of hydrogel particles of claim 1 , wherein the comonomer is allylamine, allyl acrylate, allyl methacrylate, allyl alcohol, allyl isothiocyanate, allyl chloride, allylmaleimide, or bisacrylamide.

6. The population of hydrogel particles according to claim 5, wherein the bisacrylamide is N,N'-methylenebisacrylamide, N,N'-methylenebismethacrylamide, N,N'-ethylenebisacrylamide, N,N'-ethylenebismethacrylamide, N,N'-propylenebisacrylamide and N,N'-(1,2-dihydroxyethylene)bisacrylamide.

7. The population of hydrogel particles of claim 1 , wherein the polymer comprises polymerized acrylamide and bisacrylamide.

8. The population of hydrogel particles of claim 7, wherein the optical properties are provided by the ratio of acrylamide to bisacrylamide.

9. The population of hydrogel particles of claim 1 , wherein the encapsulated nanoparticles are polymethyl methacrylate, polystyrene, or silica.

10. The population of hydrogel particles of claim 1 , wherein the encapsulated nanoparticles are polymethyl methacrylate.

11. The population of hydrogel particles of claim 1 , wherein the encapsulated nanoparticles are polystyrene.

12. The population of hydrogel particles of claim 1, wherein the encapsulated nanoparticles are silica.

13. The population of hydrogel particles of claim 7, wherein the encapsulated nanoparticles are silica.

14. The population of hydrogel particles of claim 1, comprising biomolecules of a single species.

15. The population of hydrogel particles of claim 1, comprising biomolecules of a plurality of different substances.

16. The population of hydrogel particles of claim 1, wherein each hydrogel particle comprises a surface, and the surface is functionalized with one or more biomolecules.

17. The population of hydrogel particles of claim 16, wherein the surface is functionalized with biomolecules of a single species.

18. The population of hydrogel particles of claim 16, wherein the surface is functionalized with biomolecules of a plurality of different species.

19. The population of hydrogel particles of claim 1, wherein each hydrogel particle in the population of hydrogel particles comprises a known concentration of one or more biomolecules.

20. The population of hydrogel particles of claim 16, wherein the surface is functionalized with one or more of: (a) a cell surface marker, (b) a nucleic acid sequence, (c) a polypeptide, (d) an antibody or epitope binding region thereof, or (e) a combination thereof.

21. The population of hydrogel particles of claim 20, wherein the surface is functionalized with one or more cell surface markers.

22. The population of hydrogel particles of claim 20, wherein the surface is functionalized with one or more antibodies or epitope binding regions thereof.

23. The population of hydrogel particles of claim 1, wherein the one or more biomolecules are encapsulated within the hydrogel particles.

24. The population of hydrogel particles of claim 23, wherein the one or more encapsulated biomolecules are nucleic acids or peptides.

25. The population of hydrogel particles of claim 24, wherein the one or more encapsulated biomolecules are nucleic acids.

26. The population of hydrogel particles of claim 1, wherein one or more biomolecules are encapsulated in the hydrogel particles and the surface of the hydrogel particles is functionalized with the one or more biomolecules.

27. The population of hydrogel particles of claim 26, wherein the one or more biomolecules encapsulated in the hydrogel particles and the one or more biomolecules on the surface of the hydrogel particles are biomolecules of different substances.

28. The population of hydrogel particles of claim 21, wherein the cell surface marker is IL15, IL21, CD137, or a combination thereof.

29. The population of hydrogel particles of claim 21, wherein the one or more cell surface markers are CD4, CD8, CD16, CD56, CD19, or a combination thereof.

30. The population of hydrogel particles of claim 29, wherein the target cell is a T cell, a natural killer cell, or a B cell.

31. The population of hydrogel particles of claim 21, wherein the one or more cell surface markers is hemoglobin.

32. The population of hydrogel particles of claim 31 , wherein the hemoglobin is human hemoglobin.

33. The population of hydrogel particles of claim 31, wherein the target cells are red blood cells or monocytes.

34. The population of hydrogel particles of claim 1, wherein the elasticity of the hydrogel particles is at least 40% similar to the elasticity of target cells.

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