Synthetic cell mimics with modified surface charges

Abstract

The disclosure provides charged cell mimics and their uses. In some embodiments, the charged cell mimics are composed of polymer particles, such as hydrogel particles. In one aspect of the disclosure, a population of charged cell mimics described herein can be used to validate an instrument for charge analysis, for example, an electrophoretic light scattering (ELS) instrument. In another aspect of the disclosure, charged cell mimics can be used in a method to measure charge properties of a biological particle, for example, a biological cell or an extracellular vesicle.

Description

SYNTHETIC CELL MIMICS WITH MODIFIED SURFACE CHARGES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 549,031, filed February 2, 2024, the content of which is herein incorporated by reference in its entirety. FIELD OF THE INVENTION

[0002] The present disclosure relates to compositions, such as compositions of cell-like particles engineered to have a surface charge, and methods of using the same. In some embodiments, disclosed cell-like particles, also referred to herein as charged cell mimics, comprise polymeric structures, such as hydrogel particles. BACKGROUND OF THE INVENTION

[0003] Many biological functions of cells are regulated by their surface charge. The surface charge of a cell is determined by the composition and dynamic status of the cell membrane, and can have either a positive, neutral, or negative electrical state. The surface charge may differ depending upon the particular cell type or differentiation state, or between malignant and benign cells. For example, the surface charge profile of cancer cells is distinct from that of non-malignant cells and, in some cancers, may be used as a biomarker for cancer detection and treatment (see, e.g., Nishino M., et al PLoS ONE.2020; 15: e0236373). Additionally, the cell surface charge affects membrane- regulated cell functions such as endocytosis, muscle cell contraction, nutrient transport, cell signaling, and insulin release (see, e.g., Ma Y., et al. Front. Immunol. (2017), 8:1513; Shi X., et al. Nature. (2013), 493:111–115; Chanut F. PLoS Biol. (2006), 4: e53; Fu Z., Curr. Diabetes Rev. (2012), 9:25–53; Rorsman P., et al, Physiol. Rev. (2018), 98:117–214; Ouyang L, et al. Cells. (2021) 10:1519).

[0004] As the surface charge of cells plays an important role in cellular function, its measurement can be used as a diagnostic tool to characterize, e.g., cellular state. The most common parameter to characterize surface charge of cells is by determining the zeta potential. Zeta potential measurements are routinely performed using electrophoretic light scatting (ELS) methods (also referred to as laser doppler electrophoresis and phase analysis light scattering). ELS measures electrophoretic mobility (EPM) which is converted by formula to a zeta potential. As EPMmeasurements are highly dependent on experimental conditions (e.g., dispersion conditions, temperature), it is difficult to compare measurements between experiments performed on different instruments or even on the same instrument.

[0005] Accordingly, there exists a need for methods to validate and standardize ELS instruments to ensure robustness of zeta potential measurements between instruments and experiments. SUMMARY OF THE INVENTION

[0006] In some aspects, the disclosure provides a method for validating an instrument for charge analysis of biological cells, said method comprising the steps of: inserting into the instrument a population of hydrogel particle cell mimics comprising a predetermined zeta potential that is substantially similar to the zeta potential of corresponding biological cells, measuring electrophoretic mobility (EPM) of the population of hydrogel particle cell mimics using the instrument, and determining any difference between synthetic zeta potential calculated from the EPM of the population of hydrogel particle mimics and the predetermined zeta potential, thereby validating the instrument for analysis of the biological cells.

[0007] In some embodiments of the foregoing or related aspects, the instrument is an electrophoretic light scattering (ELS) instrument. In some embodiments, the method further comprises inserting a biological sample comprising a population of the corresponding biological cells into the instrument and measuring an EPM of the biological sample. In some embodiments, the method further comprising determining a biological zeta potential for the biological sample based on the measured EPM of the biological sample, said biological zeta potential being adjusted based on the difference between the synthetic zeta potential and the predetermined zeta potential determined for the hydrogel particle cell mimics. In some embodiments, the population of hydrogel particle cell mimics that is inserted into the instrument is suspended in a first buffer, wherein the biological sample is suspended in a second buffer, and wherein the first buffer and the second buffer are substantially the same. In some embodiments, the population of hydrogel particle cell mimics that is inserted into the instrument is at substantially the same concentration as the population of corresponding biological cells. In some embodiments, the hydrogel particle cell mimics in the population are of substantially the same size as the corresponding biological cells. In some embodiments, the hydrogel particle cell mimics in the population have a substantially similar optical property to the corresponding biological cells. In some embodiments, thesubstantially similar optical property is selected from forward side scatter, side scatter, autofluorescence, fluorescence, and a combination thereof.

[0008] In some aspects, the disclosure provides a method for measuring a zeta potential of a population of biological particles, comprising (i) inserting into an ELS instrument a population of hydrogel particle cell mimics comprising a predetermined zeta potential, (ii) measuring electrophoretic mobility (EPM) of the population of hydrogel particle cell mimics using the ELS instrument, (iii) repeating (i)-(ii) for a panel of populations of hydrogel particle cell mimics, each population comprising a distinct zeta potential, (iv) generating a calibration curve from the EPM and predetermined zeta potential for the panel, (v) measuring EPM of the population of biological particles, and (vi) determining the zeta potential of the population of biological particles based on a comparison of the EPM measurement to the calibration curve. In some embodiments, the hydrogel particle cell mimics in the populations of steps (i)-(iii) are substantially the same size as the biological particles. In some embodiments, the populations of hydrogel particle cell mimics in the populations of steps (i)-(iii) are at substantially the same concentration as the population of biological particles.

[0009] In some embodiments of the foregoing or related aspects, the population of biological particles comprises cells. In some embodiments, the population of biological particles comprises pre-apoptotic cells. In some embodiments, the pre-apoptotic cells comprise surface-exposed phosphatidylserine. In some embodiments, the population of biological particles comprises extracellular vesicles. In some embodiments, the biological particles or the biological cells comprise a surface label. In some embodiments, the hydrogel particle cell mimics comprise a surface label. In some embodiments, the surface label comprises a biomarker. In some embodiments, the biomarker is a nucleic acid, protein, peptide, carbohydrate, or combination thereof.

[0010] In some embodiments of the foregoing or related aspects, the hydrogel particle cell mimics comprise a neutrally charged monomer and a charged monomer.

[0011] In some aspects, the disclosure provides a charged cell mimic comprising a hydrogel particle comprising a neutral monomer and a charged monomer, wherein the hydrogel particle comprises a predetermined zeta potential and wherein the predetermined zeta potential is substantially similar to a zeta potential of a target biological cell. In some embodiments, the target biological cell is an apoptotic cell or a pre-apoptotic cell. In some embodiments, the targetbiological cell comprises a biomarker. In some embodiments, the hydrogel particle comprises the biomarker. In some embodiments, the charged cell mimic comprises an antibody or antigen binding fragment thereof linked to the hydrogel particle, the antibody or antigen binding fragment thereof selected from the group consisting of: an anti-CD3 antibody or antigen binding fragment thereof, an anti-CD8 antibody or antigen binding fragment thereof, and an anti-CD28 antibody or antigen binding fragment thereof.

[0012] In some embodiments of any of the foregoing or related aspects, the hydrogel particle has a diameter of about 10 μm to about 100 μm. In some embodiments, the hydrogel particle has a diameter of about 1 μm to about 10 μm. In some embodiments, the hydrogel particle has a diameter of about 0.1 μm to about 1 μm.

[0013] In some embodiments of any of the foregoing or related aspects, the hydrogel particle comprises a charged monomer, wherein the charged monomer comprises a positive charge, such as a positively charged monomer. In some embodiments, the positively charged monomer comprises (i) a tertiary amine salt thereof, or (ii) an aminium or salt thereof.

[0014] In some embodiments, the positively charged monomer comprises 3- (methacryloylamino)propyl]trimethylammonium. In some embodiments, the hydrogel particle comprises a net positive surface charge at a pH of about 6 to about 8. In some embodiments, the hydrogel particle comprises a predetermined zeta potential of about +10 mV to about +100mV.

[0015] In some embodiments of any of the foregoing or related aspects, the hydrogel particle comprises a charged monomer, wherein the charged monomer comprises a negative charge. In some embodiments, the charged monomer comprises (i) a carboxylate or salt thereof, or (ii) a sulfonate or a salt thereof. In some embodiments, the charged monomer is selected from mono-2- (methacryloyloxy)ethyl succinate and acrylate. In some embodiments, the hydrogel particle comprises a net negative surface charge at a pH of about 6 to about 8. In some embodiments, the hydrogel particle comprises a predetermined zeta potential of about -10 mV to about -100 mV.

[0016] In some embodiments of any of the foregoing or related aspects, the hydrogel particle comprises a neutral surface charge at a pH of about 6 to about 8.

[0017] In some embodiments of any of the foregoing or related aspects, the hydrogel particle comprises a predetermined zeta potential of less than about -10 mV or greater than about +10 mV.

[0018] In some embodiments of any of the foregoing or related aspects, the neutrally charged monomer comprises hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate(HEMA), propylene glycol methacrylate, acrylamide, N-vinylpyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glycerol methacrylate (GMA), 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, glycerol 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-phenyl acrylamide, N-phenyl methacrylamide, N-benzyl acrylamide, N-benzyl methacrylamide, N,N-dibenzyl acrylamide, N,N-dibenzyl methacrylamide, N-diphenylmethyl acrylamide, N-(4-methylphenyl)methyl acrylamide, N-1-naphthyl acrylamide, N-4-nitrophenyl acrylamide, N-(2-phenylethyl)acrylamide, N-triphenylmethyl acrylamide, N-(4- hydroxyphenyl)acrylamide, N,N-methylphenyl acrylamide, N,N-phenyl phenylethyl acrylamide, N-diphenylmethyl methacrylamide, N-(4-methyl phenyl)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'-phenyl phenylethyl methacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2- vinylpyridine, or a combination thereof.

[0019] In some embodiments of any of the foregoing or related aspects, the hydrogel particle comprises a ratio of the neutrally charged monomer and the charged monomer, wherein the ratio is about 100:1 to about 10:1.

[0020] In some embodiments of any of the foregoing or related aspects, the hydrogel particle comprises another property that is substantially similar to the biological cells or biologicalparticles, said other property selected from the group consisting of: volume, FSC, SSC, background fluorescence; fluorescence profile; shared surface marker, and refractive index.

[0021] In some aspects, the disclosure comprises a charged cell mimic comprising a hydrogel particle comprising a neutral monomer, a charged monomer, and an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen binding fragment thereof is selected from the group consisting of an anti-CD3 antibody or fragment thereof, an anti-CD8 antibody or fragment thereof, and an anti-CD28 antibody or fragment thereof, wherein the hydrogel particle comprises a predetermined zeta potential, and wherein the predetermined zeta potential is substantially similar to a zeta potential of a target biological cell.

[0022] In some embodiments of any of the foregoing or related aspects, the hydrogel particle comprises a main body, the main body comprising a plurality of macropores.

[0023] In some aspects, the disclosure provides a method of inducing an immune cell response, comprising contacting an immune cell with the charged cell mimic described herein, wherein the immune cell response comprises activation and / or expansion of the immune cell.

[0024] In some aspects, the disclosure provides a method of inducing an immune cell response, comprising contacting an immune cell with a charged cell mimic, wherein the charged cell mimic comprises a neutral monomer, a charged monomer, and an antibody or an antigen-binding fragment thereof, wherein the hydrogel particle comprises a predetermined zeta potential, wherein the predetermined zeta potential is substantially similar to a zeta potential of the immune cell, and wherein the immune cell response comprises activation and / or expansion of the immune cell. In some embodiments, the antibody or antigen binding fragment thereof is selected from the group consisting of: an anti-CD3 antibody or fragment thereof, an anti-CD8 antibody or fragment thereof, and an anti-CD28 antibody or fragment thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying figures, which are incorporated herein and form a part of the specification, illustrate some, but not the only or exclusive, example embodiments and / or features. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than limiting.

[0026] FIG.1A is a graphshowing the zeta-potential distribution for exemplary hydrogel particle cell mimic particles described herein having a positive charge.

[0027] FIG.1B is a graph showing the zeta-potential distribution for exemplary hydrogel particle cell mimic particles described herein having a negative charge.

[0028] FIG. 2 is a graph showing the zeta potential of charged hydrogel particles in buffers with different ionic strength. The methods of the present disclosure are capable of producing particles with zeta-potential across the charge spectrum.

[0029] FIG.3A shows phase contrast microscopy of positively charged hydrogel particles (P4) in 1X PBS.

[0030] FIG.3B shows phase contrast microscopy of positively charged hydrogel particles (P4) in DI water.

[0031] FIG.4 is a graph showing the zeta potential of an uncharged control hydrogel particle (C) and negatively charged hydrogel particles (N1, N2, and N3) at pH 4.0, pH 7.0, and pH 10.0.

[0032] FIG.5 is a graph showing the zeta potential of an uncharged control hydrogel particle (C) and positively charged hydrogel particles (P1, P2, P3, and P4) at pH 4.0, pH 7.0, and pH 10.0.

[0033] FIG. 6 shows a flow cytometry scatter plot of uncharged hydrogel particles (control), negatively charged hydrogel particles, and positively charged hydrogel particles prior to lyophilization.

[0034] FIG. 7 shows a flow cytometry scatter plot of uncharged hydrogel particles (control), negatively charged hydrogel particles, and positively charged hydrogel particles post- lyophilization. DETAILED DESCRIPTION OF THE INVENTION Definitions

[0035] The indefinite articles “a” and “an” and the definite article “the” are intended to include both the singular and the plural, unless the context in which they are used clearly indicates otherwise.

[0036] As used herein, the term "or" is to be interpreted in its broadest sense and is not limited to mutually exclusive alternatives. The term "or" encompasses both the conjunctive and disjunctive cases, unless explicitly stated otherwise or clear from the context.

[0037] “At least one” and “one or more” are used interchangeably to mean that the article may include one or more than one of the listed elements.

[0038] Unless otherwise indicated, it is to be understood that all numbers expressing quantities, ratios, and numerical properties of ingredients, reaction conditions, and so forth, used in the specification and claims are contemplated to be able to be modified in all instances by the term “about”.

[0039] "About" will be understood by persons of ordinary skill and will vary to some extent depending on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill given the context in which it is used, "about" will mean up to ±10% of the particular value.

[0040] The term “including all ranges and subranges there between” or equivalents, are used herein to denote the intention that disclosure of any range or series of possible values, inherently also discloses all ranges and subranges encompassed by the highest and lowest values disclosed. This term includes the entire range from highest to lowest disclosed values, as well as subranges from any two or more disclosed points. This term is also intended to disclose any subranges encompassed anywhere within the highest and lowest disclosed values, including between two points that are explicitly recited in the document, up to one decimal point. Thus, disclosure of values 0, 5, 10, 15, 20, including all ranges and subranges therebetween, should be interpreted as also encompassing a range from 0-20, a range from 0-5 or 5-15, as well as a range from 2-16, or 3.1 to 19.8, etc.

[0041] “Substantially similar,” as used herein, denotes 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.

[0042] As provided above, in one aspect of the invention, compositions comprising a plurality of hydrogel particles are provided. As used herein, the term “hydrogel” refers to a material comprising a macromolecular three-dimensional network that allows it to swell when in the presence of water, to shrink in the absence of (or by reduction of the amount of) water, but not dissolve in water. The swelling, i.e., the absorption of water, is a consequence of the presence of hydrophilic functional groups attached to or dispersed within the macromolecular network. Crosslinks between adjacent macromolecules result in the aqueous insolubility of these hydrogels. The cross-links may be due to chemical (i.e., covalent) or physical (i.e., Van Der Waal forces, hydrogen-bonding, ionic forces, etc.) bonds. Synthetically prepared hydrogels can be prepared by polymerizing a monomeric material to form a backbone and cross-linking the backbone with acrosslinking agent. The term “hydrogel” encompasses the macromolecular material whether dehydrated or in a hydrated state. A characteristic of a hydrogel that is of particular value is that the material retains the general shape, whether dehydrated or hydrated. Thus, if the hydrogel has an approximately spherical shape in the dehydrated condition, it will be spherical in the hydrated condition. In some embodiments, a hydrogel particle disclosed herein comprises greater than about 30%, greater than about 40%, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, or greater than about 95% water. In another embodiment, a hydrogel particle has a water content of about 10 percent by weight to about 95 percent by weight, or about 20 percent by weight to about 95 percent by weight, or about 30 percent by weight to about 95 percent by weight, or about 40 percent by weight to about 95 percent by weight, or about 50 percent by weight to about 95 percent by weight, or about 60 percent by weight to about 95 percent by weight, or about 70 percent by weight to about 95 percent by weight, or about 80 percent by weight to about 95 percent by weight.

[0043] As used herein, the term “cell mimic” refers to a synthetic particle that substantially replicates one or more optical properties and / or surface charge characteristics of a target biological cell. In some examples, the cell mimic exhibits an optical or surface charge characteristic that closely resembles the corresponding property of a specified cell type when analyzed by flow cytometry, microscopy, electrophoretic mobility measurements, or other optical and electrokinetic detection methods. Such properties include, but are not limited to, optical properties, for example, forward scatter (FSC), side scatter (SSC), and fluorescence emission profiles that fall within a predetermined range of similarity to the target cell type; and surface charge characteristics, as measured by zeta potential or other electrokinetic methods, that approximate the charge distribution and magnitude typically observed on the surface of the target biological cell type under physiological or specified experimental conditions.

[0044] “Surface charge,” as used herein, refers to the measure of electric charge accumulated over a surface and is measured in coulombs per square meter (C / m2). Methods to measure surface charge are known in the art (see, e.g., Delgado, et al (2005) Pure Appl. Chem.77:1753-1805).

[0045] “Zeta potential,” as used herein, refers to the electrical potential difference between the bulk of a liquid and the stationary layer of fluid attached to a dispersed particle. More specifically, it is measured at the slipping plane, which is the boundary between the mobile and immobile partsof the electric double layer surrounding a particle in solution. Zeta potential is typically expressed in units of volts (V) or millivolts (mV). Methods to measure zeta potential include, but are not limited to electrophoretic light scattering (ELS), also known as laser doppler electrophoresis and phase analysis light scattering, electroacoustic techniques, streaming potential measurements, and electroosmotic flow analysis.

[0046] .

[0047] “Double layer,” “electrical double layer,” “DL,” and “EDL,” are used interchangeably herein to refer to the structure of charges at the surface of an object in a fluid. The EDL comprises a first layer of ions adsorbed to the surface and a second layer of ions attracted to the surface such that the ions diffuse with the object (the “diffuse layer”) (see, e.g., Delgado, et al (2005) Pure Appl. Chem.77:1753-1805).

[0048] A “slipping plane,” as used herein, also known as the shear plane, refers to the theoretical boundary within the electrical double layer surrounding a dispersed particle in a liquid medium. This plane separates the stationary layer of fluid attached to the dispersed particle (Stern layer) from the mobile diffuse layer of ions in the bulk solution. It is the interface between the immobile fluid adhering to the dispersed particle and the mobile fluid in solution.

[0049] An “iso-electric point,” as used herein, refers to the pH at which a particle, molecule, or surface carries no net electrical charge in an aqueous medium. At the iso-electric point, the zeta potential of the particle, molecule, or surface is zero, indicating minimal electrostatic repulsion between particles.

[0050] The “electrophoretic mobility” or “EPM” as used herein refers to the mobility of a particle in response to an applied electric field. EPM is typically defined as the rate of migration (e.g., centimeters per second) per unit of electric field strength (e.g., volts per centimeter). EPM is determined by measuring the particle’s velocity when subjected to a time dependent electric field. The velocity is termed the electrophoretic velocity and the proportionality constant between the electrophoretic velocity and the electric field is the EPM. The EPM of a particle is directly proportional to the net charge of the particle and inversely proportional to the molecular size of the particle and the viscosity of the electrophoresis medium. Using appropriate models, for example, the Helmholtz-Smoluchowski (HS) equation and Hückel equation, EPM can be used to calculate zeta potential. See, e.g., Egorova, Electrophoresis. 1994 Aug-Sep;15(8-9):1125-31 andPolaczyk et al., Colloids and Surfaces A: Physicochemical and Engineering Aspects. 2020 Feb 586:124097.

[0051] An “electrophoretic light scattering instrument” or “ELS instrument” refers to an instrument to analyze the temporal variation of light scattered by a dispersion of particles in a liquid subject to an externally applied electric field. In some embodiments, an ELS instrument is validated according to a method described herein, the method comprising inserting into the instrument a sample comprising a population of charged cell mimics having a predetermined zeta potential, measuring an EPM of the population, and determining a difference between an observed zeta potential calculated from the EPM and the predetermined zeta potential. In some embodiments, the ELS instrument is “validated” if the observed zeta potential is substantially similar to the predetermined zeta potential (e.g., the difference between the observed zeta potential and the predetermined zeta potential is less than a standard deviation calculated for the observed zeta potential, e.g., the difference is less than about ±5% of the predetermined zeta potential, e.g., the observed zeta potential is less than about ±0.5 mV, less than about ±1 mV, less than about ±5 mV, or less than about ±10 mV of the predetermined zeta potential). Overview

[0052] As appreciated by the skilled artisan, an ELS experiment is typically performed by illuminating a sample cell containing optical windows and electrodes with a light source and detecting the intensity of light scattered from particles in the sample at a well-defined detection angle. When voltage is applied to the electrodes, the particles migrate to the electrode of opposite charge and attain a certain velocity. The magnitude and direction of the velocity depends upon the size and surface charge of the particles, as well as solution viscosity and conductivity. The velocity of dispersed particles in the sample causes a frequency shift of incident light such that the scattered light is red- or blue-shifted (i.e., the optical frequency increases or decreases respectively) depending on the direction of the particle migration relative to the scattering angle. The EPM is calculated from the frequency shift of incident light.

[0053] The EPM in turn is used to calculate zeta potential of particles in the sample. The zeta potential is the electric potential at the slipping plane of particles in the sample. The presence of a charge on the surface of a particle will influence the distribution of ions in the surrounding interfacial region. The liquid layer surrounding the particles exists as two parts: an inner region inwhich counterions are strongly attracted to the particle and an outer diffuse region in which they are less associated with the particle. Within the diffuse region there is a slipping plane between counterions that move with the particle and those that move independently of the particle. The magnitude of the zeta potential depends on the summation of charges at the slipping plane, which includes the particle valence charge, the charge from surface-absorbed counterions, and charge from non-absorbed associated ions moving with the particle. As these charges are dependent on the dispersant conditions (e.g., pH, ionic strength, additive concentrations, etc.), the absolute value of the zeta potential likewise depends on the experimental conditions.

[0054] The present disclosure is based, at least in part, on methods and compositions for validating an ELS instrument in order to achieve zeta potential measurements that are standardized between different experiments and instruments. As described herein, a method for validating an ELS instrument comprises the steps of inserting into the instrument a population of charged cell mimics (e.g., polymeric particle comprising a neutral monomer and a charged monomer) described herein comprising a predetermined zeta potential, measuring the EPM of the population, and determining the difference between a zeta potential calculated from the EPM and the predetermined zeta potential. In some embodiments, a difference that is less than about ± 5% of the predetermined zeta potential indicates the ELS is providing an accurate measure of EPM. In some embodiments, a zeta potential calculated from the EPM that is less than about ±0.5 mV to about ±1 mV relative to the predetermined zeta potential indicates the ELS is providing an accurate measure of EPM. In some embodiments, a zeta potential calculated from the EPM that is less than about ±0.5 mV to about ±5 mV relative to the predetermined zeta potential indicates the ELS is providing an accurate measure of EPM. In some embodiments, a zeta potential calculated from the EPM that is less than about ±0.5 mV to about ±10 mV relative to the predetermined zeta potential indicates the ELS is providing an accurate measure of EPM. In some embodiments, a zeta potential calculated from the EPM that is less than about ±0.5 mV, less than about ±1 mV, less than about ±5 mV, or less than about ±10 mV relative to the predetermined zeta potential indicates the ELS is providing an accurate measure of EPM. In some embodiments, the difference is between an average zeta potential calculated from the EPM and the predetermined zeta potential, wherein a difference that is less than one standard deviation from the average zeta potential indicates the instrument is validated.

[0055] It is to be appreciated that an advantage of validating the ELS instrument with the charged cell mimics described herein as compared to charged beads (e.g., charged polystyrene beads) is that the charged cell mimics are prepared to have substantially similar characteristics to biological cells (e.g., a substantially similar size, shape, refractive index, elastic modulus, etc.). Without being bound by theory, the movement of biological cells in an alternate electric field will vary based upon size and hydrodynamic drag, and the variation will result in different zeta potential measurements depending upon the particular ELS instruments and / or set of experimental conditions. By validating an ELS instrument using a charged cell mimic described herein having substantially similar characteristics to the biological cells in the population, the skilled artisan is able to confirm the ELS instrument is providing accurate zeta potential measurements for the biological cells in the population. Moreover, in some embodiments, the difference between zeta potential calculated from the EPM and the predetermined zeta potential of the population charged cell mimics is used as a reference point to compare EPM measurements made on different ELS instruments. Charged Cell Mimics of the Disclosure

[0056] In some embodiments, the disclosure provides a charged cell mimic comprising a charged monomer and / or comonomer. In some embodiments, the charged cell mimic comprises polymer comprising a charged monomer and / or comonomer. In some embodiments, the polymer is cross- linked to form a particle. In some embodiments, the polymer is cross-linked to form a hydrogel particle. In some embodiments, the comonomer is a neutral monomer described herein. In some embodiments, the polymer comprises more than one comonomer. In some embodiments, the polymer comprises a charged monomer, a neutral comonomer, and a bifunctional comonomer. In some embodiments, the bifunctional comonomer comprises a second functional group that can participate in a second reaction, e.g., conjugation to a fluorophore or a biomolecule described herein.

[0057] In some embodiments, the charged cell mimic comprises a w / w ratio of the charged monomer to comonomer (e.g., neutral monomer and / or bifunctional comonomer) of about 1:100 to about 100:1. In embodiments, the w / w ratio of charged monomer to comonomer (e.g., neutral monomer and / or bifunctional comonomer) is about 1:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1, including all rangesand subranges therebetween. In embodiments, the w / w ratio of charged monomer to comonomer (e.g., neutral monomer and / or bifunctional comonomer) is about 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, or 1:100, including all ranges and subranges therebetween. Positively Charged Cell Mimics

[0058] In some embodiments, the charged cell mimic comprises a net positive charge at neutral pH. In some embodiments, the charged cell mimic comprises a cationic monomer, wherein the charged cell mimic is characterized by a net positive surface charge at neutral pH. In some embodiments, the charged cell mimic comprises a zeta potential of greater than about +5 mV. In some embodiments, the charged cell mimic comprises a zeta potential of greater than about +10 mV. In some embodiments, the charged cell mimic comprises a zeta potential of greater than about +20 mV. In some embodiments, the charged cell mimic comprises a zeta potential of greater than about +30 mV.

[0059] In some embodiments, the charged cell mimic comprises a zeta potential of about +5 mV to about +100 mV. In some embodiments, the charged cell mimic comprises a zeta potential of about +10 mV to about +100 mV. In some embodiments, the charged cell mimic comprises a zeta potential of about +20 mV to about +100 mV.

[0060] In some embodiments, a positively charged cell mimic of the disclosure has a zeta potential of about +1 mV, +2 mV, +3 mV, +4 mV, +5 mV, +6 mV, +7 mV, +8 mV, +9 mV, +10 mV, +11 mV, +12 mV, +13 mV, +14 mV, +15 mV, +16 mV, +17 mV, +18 mV, +19 mV, +20 mV, +21 mV, +22 mV, +23 mV, +24 mV, +25 mV, +26 mV, +27 mV, +28 mV, +29 mV, +30 mV, +31 mV, +32 mV, +33 mV, +34 mV, +35 mV, +36 mV, +37 mV, +38 mV, +39 mV, +40 mV, +41 mV, +42 mV, +43 mV, +44 mV, +45 mV, +46 mV, +47 mV, +48 mV, +49 mV, +50 mV, +51 mV, +52 mV, +53 mV, +54 mV, +55 mV, +56 mV, +57 mV, +58 mV, +59 mV, +60 mV, +61 mV, +62 mV, +63 mV, +64 mV, +65 mV, +66 mV, +67 mV, +68 mV, +69 mV, +70 mV, +71 mV, +72 mV, +73 mV, +74 mV, +75 mV, +76 mV, +77 mV, +78 mV, +79 mV, +80 mV, +81 mV, +82 mV, +83 mV, +84 mV, +85 mV, +86 mV, +87 mV, +88 mV, +89 mV, +90 mV, +91 mV, +92 mV, +93 mV, +94 mV, +95 mV, +96 mV, +97 mV, +98 mV, +99 mV, or +100 mV, including all ranges and subranges therebetween.

[0061] In embodiments, a positively charged cell mimic of the disclosure has a zeta potential of about -2 mV, -3 mV, -4 mV, -5 mV, -6 mV, -7 mV, -8 mV, -9 mV, -10 mV, -11 mV, -12 mV, -13 mV, -14 mV, -15 mV, -16 mV, -17 mV, -18 mV, -19 mV, -20 mV, -21 mV, -22 mV, -23 mV, -24 mV, -25 mV, including all ranges and subranges therebetween, in 1X PBS at ambient temperature.

[0062] In embodiments, a positively charged cell mimic of the disclosure has a zeta potential of about +15 mV, +16 mV, +17 mV, +18 mV, +19 mV, +20 mV, +21 mV, +22 mV, +23 mV, +24 mV, +25 mV, +26 mV, +27 mV, +28 mV, +29 mV, +30 mV, +31 mV, +32 mV, +33 mV, +34 mV, +35 mV, +36 mV, +37 mV, +38 mV, +39 mV, +40 mV, +41 mV, +42 mV, +43 mV, +44 mV, +45 mV, +46 mV, +47 mV, +48 mV, +49 mV, +50 mV, +51 mV, +52 mV, +53 mV, +54 mV, +55 mV, +56 mV, +57 mV, +58 mV, +59 mV, +60 mV, +61 mV, +62 mV, +63 mV, +64 mV, or +65 mV, including all ranges and subranges therebetween, in deionized water at ambient temperature.

[0063] In embodiments, a positively charged cell mimic of the disclosure has a zeta potential of about +5 mV, +6 mV, +7 mV, +8 mV, +9 mV, +10 mV, +11 mV, +12 mV, +13 mV, +14 mV, +15 mV, +16 mV, +17 mV, +18 mV, +19 mV, +20 mV, +21 mV, +22 mV, +23 mV, +24 mV, +25 mV, +26 mV, +27 mV, +28 mV, +29 mV, +30 mV, +31 mV, +32 mV, +33 mV, +34 mV, +35 mV, including all ranges and subranges therebetween, at acidic pH at ambient temperature, such as a pH less than 7. In embodiments acidic pH is pH 1, pH 1.5, pH 2, pH 2.5, pH 3, pH 3.5, pH 4, pH 4.5, pH 5, pH 5.5, pH 6, pH 6.5, or pH 6.9, including all ranges and subranges therebetween.

[0064] In embodiments, a positively charged cell mimic of the disclosure has a zeta potential of about +4 mV, +5 mV, +6 mV, +7 mV, +8 mV, +9 mV, +10 mV, +11 mV, +12 mV, +13 mV, +14 mV, +15 mV, +16 mV, +17 mV, +18 mV, +19 mV, +20 mV, +21 mV, +22 mV, +23 mV, +24 mV, or +25 mV, including all ranges and subranges therebetween at neutral pH at ambient temperature, such as pH 7 ± 0.5.

[0065] In embodiments, a positively charged cell mimic of the disclosure has a zeta potential of about +2 mV, +3 mV, +4 mV, +5 mV, +6 mV, +7 mV, +8 mV, +9 mV, +10 mV, +11 mV, +12 mV, +13 mV, +14 mV, +15 mV, +16 mV, +17 mV, +18 mV, +19 mV, +20 mV, +21 mV, +22 mV, +23 mV, +24 mV, +25 mV, including all ranges and subranges therebetween, at basic pH at ambient temperature, such as greater than pH 7. In embodiments basic pH is pH 7.5, pH 8, pH 8.5,pH 9, pH 9.5, pH 10, pH 10.5, pH 11, pH 11.5, pH 12, pH 12.5, pH 13, pH 13.5, or pH 14, including all ranges and subranges therebetween.

[0066] In embodiments, a positively charged cell mimic of the disclosure comprises a cationic monomer. In embodiments, the cationic monomer comprises an acrylate moiety. In embodiments, the cationic monomer comprises a methacrylate moiety. In embodiments, the cationic monomer comprises an acrylamide moiety. In embodiments, the cationic monomer comprises a methacrylamide moiety.

[0067] In embodiments, the cationic monomer comprises a (meth)acrylamide moiety or a (meth)acrylate moiety; and a quaternary ammonium group. In embodiments, the cationic monomer comprises an acrylamide moiety and a quaternary ammonium group. In embodiments, the cationic monomer comprises a methacrylamide moiety and a quaternary ammonium group. In embodiments, the cationic monomer comprises an acrylate moiety and a quaternary ammonium group. In embodiments, the cationic monomer comprises a methacrylate moiety and a quaternary ammonium group.

[0068] In embodiments, a spacer group comprising 1, 2, 3, 4, 5, or 6 carbon atoms separates the acrylamide moiety from the quaternary ammonium group. In embodiments, a spacer group comprising 1, 2, 3, 4, 5, or 6 carbon atoms separates the methacrylamide moiety from the quaternary ammonium group. In embodiments, a spacer group comprising 1, 2, 3, 4, 5, or 6 carbon atoms separates the acrylate moiety from the quaternary ammonium group. In embodiments, a spacer group comprising 1, 2, 3, 4, 5, or 6 carbon atoms separates the methacrylate moiety from the quaternary ammonium group.

[0069] In embodiments, the quaternary ammonium group has the formula: [R1R2R3R4N]+, where R1, R2, and R3are each independently selected from the group consisting of methyl, ethyl, propyl, and butyl; and R4is selected from the group consisting of H and C1-C6alkyl; In embodiments, the quaternary ammonium group is positively charged.

[0070] In embodiments, the quaternary ammonium group comprises trimethylammonium. In embodiments, the quaternary ammonium group comprises tetraethylammonium. In embodiments, the quaternary ammonium group comprises tetrapropylammonium. In embodiments, the quaternary ammonium group comprises tetrabutylammonium.

[0071] In embodiments, the cationic monomer comprises 3- (methacryloylamino)propyl]trimethylammonium, 3-Acrylamidopropyl)trimethylammonium chloride (APTAC), Allylamine hydrochloride, (3-Acrylamidopropyl)trimethylammonium chloride, [2-(Methacryloyloxy)ethyl]trimethylammonium chloride, Lysine (meth)acrylate, Poly(L-lysine) (meth)acrylate, Poly(ethylenimine) (meth)acrylate, Poly(amidoamine) (meth)acrylate, or a combination thereof. In embodiments, a positively charged cell mimic of the disclosure comprises the cationic monomer 3-(methacryloylamino)propyl]trimethylammonium.

[0072] In embodiments, the cationic monomer comprises Compound 1 as represented in Table 1, which has the structure:Compound 1.

[0073] In embodiments, the cationic monomer comprises 3- Acrylamidopropyl)trimethylammonium chloride (APTAC), which has the structure:Compound 4. Negatively Charged Cell Mimics

[0074] In some embodiments, the charged cell mimic comprises a net negative charge at neutral pH. In some embodiments, the charged cell mimic comprises an anionic monomer, wherein the charged cell mimic is characterized by a net negative surface charge at neutral pH. In some embodiments, the charged cell mimic comprises a zeta potential of less than about -5 mV. In some embodiments, the charged cell mimic comprises a zeta potential of less than about -10 mV. In some embodiments, the charged cell mimic comprises a zeta potential of less than about -20 mV. In some embodiments, the charged cell mimic comprises a zeta potential of less than about -30 mV.

[0075] In some embodiments, a negatively charged cell mimic of the disclosure has zeta potential of about -5 mV to about -100 mV. In some embodiments, the charged cell mimic comprises a zetapotential of about -10 mV to about -100 mV. In some embodiments, the charged cell mimic comprises a zeta potential of about -20 mV to about -100 mV.

[0076] In some embodiments, the a negatively charged cell mimic of the disclosure has a zeta potential of about -1 mV, -2 mV, -3 mV, -4 mV, -5 mV, -6 mV, -7 mV, -8 mV, -9 mV, -10 mV, - 11 mV, -12 mV, -13 mV, -14 mV, -15 mV, -16 mV, -17 mV, -18 mV, -19 mV, -20 mV, -21 mV, -22 mV, -23 mV, -24 mV, -25 mV, -26 mV, -27 mV, -28 mV, -29 mV, -30 mV, -31 mV, -32 mV, -33 mV, -34 mV, -35 mV, -36 mV, -37 mV, -38 mV, -39 mV, -40 mV, -41 mV, -42 mV, -43 mV, -44 mV, -45 mV, -46 mV, -47 mV, -48 mV, -49 mV, -50 mV, -51 mV, -52 mV, -53 mV, -54 mV, -55 mV, -56 mV, -57 mV, -58 mV, -59 mV, -60 mV, -61 mV, -62 mV, -63 mV, -64 mV, -65 mV, -66 mV, -67 mV, -68 mV, -69 mV, -70 mV, -71 mV, -72 mV, -73 mV, -74 mV, -75 mV, -76 mV, -77 mV, -78 mV, -79 mV, -80 mV, -81 mV, -82 mV, -83 mV, -84 mV, -85 mV, -86 mV, -87 mV, -88 mV, -89 mV, -90 mV, -91 mV, -92 mV, -93 mV, -94 mV, -95 mV, -96 mV, -97 mV, -98 mV, -99 mV, or -100 mV,, including all ranges and subranges therebetween.

[0077] In embodiments, a negatively charged cell mimic of the disclosure has a zeta potential of about -5 mV, -6 mV, -7 mV, -8 mV, -9 mV, -10 mV, -11 mV, -12 mV, -13 mV, -14 mV, -15 mV, -16 mV, -17 mV, -18 mV, -19 mV, -20 mV, -21 mV, -22 mV, -23 mV, -24 mV, -25 mV, -26 mV, -27 mV, including all ranges and subranges therebetween, in 1X PBS at ambient temperature.

[0078] In embodiments, a negatively charged cell mimic of the disclosure has a zeta potential of about -4 mV, -5 mV, -6 mV, -7 mV, -8 mV, -9 mV, -10 mV, -11 mV, -12 mV, -13 mV, -14 mV, -15 mV, -16 mV, -17 mV, -18 mV, including all ranges and subranges therebetween, in deionized water at ambient temperature.

[0079] In embodiments, a negatively charged cell mimic of the disclosure has a zeta potential of about -1 mV, -1.1 mV, -1.2 mV, -1.3 mV, -1.4 mV, -1.5 mV, -1.6 mV, -1.7 mV, -1.8 mV, -1.9 mV, -2 mV, -2.1 mV, -2.2 mV, -2.3 mV, -2.4 mV, -2.5 mV, -2.6 mV, -2.7 mV, -2.8 mV, -2.9 mV, -3 mV, -3.1 mV, -3.2 mV, -3.3 mV, -3.4 mV, -3.5 mV, -3.6 mV, -3.7 mV, -3.8 mV, -3.9 mV, -4 mV, -4.1 mV, -4.2 mV, -4.3 mV, -4.4 mV, -4.5 mV, -4.6 mV, -4.7 mV, -4.8 mV, -4.9 mV, -5 mV, including all ranges and subranges therebetween, at acidic pH at ambient temperature, such as a pH less than 7. In embodiments acidic pH is pH 1, pH 1.5, pH 2, pH 2.5, pH 3, pH 3.5, pH 4, pH 4.5, pH 5, pH 5.5, pH 6, pH 6.5, or pH 6.9, including all ranges and subranges therebetween.

[0080] In embodiments, a negatively charged cell mimic of the disclosure has a zeta potential of about -2 mV, -3 mV, -4 mV, -5 mV, -6 mV, -7 mV, -8 mV, -9 mV, -10 mV, -11 mV, -12 mV, -13mV, -14 mV, -15 mV, -16 mV, -17 mV, -18 mV, -19 mV, -20 mV, -21 mV, -22 mV, -23 mV, -24 mV, -25 mV, -26 mV, -27 mV, -28 mV, -29 mV, -30 mV, including all ranges and subranges therebetween, at neutral pH at ambient temperature, such as pH 7 ± 0.5.

[0081] In embodiments, a negatively charged cell mimic of the disclosure has a zeta potential of about -2 mV, -3 mV, -4 mV, -5 mV, -6 mV, -7 mV, -8 mV, -9 mV, -10 mV, -11 mV, -12 mV, -13 mV, -14 mV, -15 mV, -16 mV, -17 mV, -18 mV, -19 mV, -20 mV, -21 mV, -22 mV, -23 mV, -24 mV, -25 mV, -26 mV, -27 mV, -28 mV, -29 mV, -30 mV, including all ranges and subranges therebetween, at basic pH at ambient temperature, such as greater than pH 7. In embodiments basic pH is pH 7.5, pH 8, pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5, pH 11, pH 11.5, pH 12, pH 12.5, pH 13, pH 13.5, or pH 14, including all ranges and subranges therebetween.

[0082] In embodiments, a negatively charged cell mimic of the disclosure comprises an anionic monomer. In embodiments, the negatively charged monomer comprises an anionic ionizable functional group. In embodiments, the anionic ionizable functional group is selected from carboxylate, phosphate, sulfate, sulfonate, phenolate, thiolate, enolate, alkoxide, or phosphonate. In embodiments, the anionic ionizable functional group comprises carboxylate.

[0083] In embodiments, the anionic monomer comprises a methacrylate moiety or a vinyl moiety; and an anionic ionizable functional group selected from carboxylic acid, sulfonic acid, and phosphonic acid. In embodiments, the anionic monomer comprises a methacrylate moiety and a succinate group. In embodiments, the anionic monomer comprises a methacrylate moiety and a carboxylate group.

[0084] In embodiments, a spacer group comprising 1, 2, 3, 4, 5, or 6 carbon atoms separates the methacrylate or vinyl moiety from the anionic ionizable functional group. In embodiments, a spacer group comprising 1, 2, 3, 4, 5, or 6 carbon atoms separates the methacrylate or vinyl moiety from the anionic ionizable functional group. In embodiments, a spacer group comprising 1, 2, 3, 4, 5, or 6 carbon atoms separates the methacrylate or vinyl moiety from the anionic ionizable functional group. In embodiments, a spacer group comprising 1, 2, 3, 4, 5, or 6 carbon atoms separates the methacrylate or vinyl moiety from the anionic ionizable functional group.

[0085] In embodiments, the anionic monomer has the formula: R1−O−CH2−CH2 −O−C(=O)−CH(R2)=CH2, where R1 is selected from the group consisting of -COOH, -SO3H, - PO3H2, and their corresponding anions;R2 is selected from the group consisting of hydrogen, C1-C6 alkyl, and C1-C6 substituted alkyl. Inembodiments, the anionic monomer is negatively charged.

[0086] In embodiments, the anionic monomer comprises mono-2-(Methacryloyloxy)ethylsuccinate (MMES), sodium acrylate, acrylic acid, mono-2-(Methacryloyloxy)ethyl succinatesodium styrene sulfonate, 2-Acrylamido-2-methyl-1-propanesulfonic acid sodium salt, 2-sulfoethyl methacrylate, vinylphosphonic acid, or a combination thereof.

[0087] In embodiments, the anionic monomer comprises mono-2-(Methacryloyloxy)ethylsuccinate (MMES), which has the structure:Compound 2.

[0088] In embodiments, a charged cell mimic of the disclosure comprises a zwitterionic monomer.In embodiments, the zwitterionic monomer comprises a cationic moiety and an anionic moiety. Inembodiments, the cationic moiety comprises a quaternary ammonium group. In embodiments, theanionic moiety comprises a sulfonate group.

[0089] In embodiments, the zwitterionic monomer comprises 3-[(3-Acrylamidopropyl)dimethylammonio]propane-1-sulfonate, 3-[[2-(Methacryloyloxy)ethyl]dimethylammonio]propane-1-sulfonate, or a combination thereof.Physical Features of Charged Cell Mimics

[0090] In some embodiments, the charged cell mimic comprises an isoelectric point greater thanabout 9. In some embodiments, the charged cell mimic comprises an isoelectric point of about pH8 to about pH 9. In some embodiments, the charged cell mimic comprises an isoelectric point ofabout pH 7 to about pH 8. In some embodiments, the charged cell mimic comprises an isoelectric point less than about pH 7. In some embodiments, the charged cell mimic comprises an isoelectric point of about pH 6 to about pH 7. In some embodiments, the charged cell mimic comprises an isoelectric point of about pH 5 to about pH 6. In some embodiments, the charged cell mimic comprises an isoelectric point of about pH 4 to about pH 5. In some embodiments, the charged cellmimic comprises an isoelectric point of less than about pH 4. In some embodiments, the chargedcell mimic has an isoelectric point of about pH 1, pH 2, pH 3, pH 4, pH 5, pH 6, pH 7, pH 8, pH 9, including all ranges and subranges therebetween.

[0091] In some embodiments, the charged cell mimic comprises a longest diameter of less than about 1 μm. In some embodiments, the charged cell mimic comprises a longest diameter of greater than about 1 μm. In some embodiments, the charged cell mimic comprises a longest diameter of about 1μm to about 100 μm. In some embodiments, the charged cell mimic comprises a longest diameter of about 1 μm to about 50 μm. In some embodiments, the charged cell mimic comprises a longest diameter of about 1 μm to about 10 μm.

[0092] In some embodiments, the charged cell mimic is spherical. In some embodiments, the charged cell mimic is non-spherical.

[0093] In some embodiments, the charged cell mimic is substantially spherical and comprises a diameter having a length. In some embodiments, the length is less than 1 μm. In some embodiments, the length is greater than 1 μm. In some embodiments, the length is about 1 μm to about 100 μm. In some embodiments, the length is about 1 μm to about 50 μm. In some embodiments, the length is about 1 μm to about 10 μm.

[0094] In some embodiments, the charged cell mimic comprises an embedded substance. In one embodiment, the embedded substance is an embedded molecule, for example a biomolecule. The biomolecule can be a single species or a plurality of different species. For example, a protein, peptide, carbohydrate, nucleic acid or combination thereof can be encapsulated within a hydrogel particle of the invention. Moreover, different nucleic acid molecules (e.g., of varying sequences or nucleic acid type such as genomic DNA, messenger RNA or DNA-RNA hybrids) can be encapsulated by the hydrogel particle of the invention. These can be comprised of any protein or nucleic acid as both forms of biological material contain labile chemical side-groups (or can be modified by commercial vendors (e.g., Integrated DNA Technology chemical side group modifications). Such side-groups are compatible with reaction chemistries commonly found in co- monomer compositions (e.g. acrylate chemistry, NHS-ester, primary amines, copper catalyzed click chemistry (Sharpless)). The range of possible embedded molecules which contain compatible chemistries is understood by those skilled in the art.

[0095] In one embodiment, different subpopulations of hydrogel particles are fabricated, each with a different concentration of biomolecule. In a further embodiment, the biomolecule is a nucleic acid, a protein, an intracellular ion such as calcium acid (or other biomolecule of the user’schoosing, for example, calcium). In another embodiment, different subpopulations of hydrogel particles are fabricated, each with a different concentration of a drug substance. The drug substance in one embodiment is a biomolecule (i.e., a biologic, antibody, antibody drug conjugate, protein / enzyme, peptide, non-ribosomal peptide, or related molecule) or a small molecule synthetic drug (e.g., Type I / II / III polyketide, non-ribosomal peptide with bioactive properties, or other small molecule entity as generally classified by those skilled in the art).

[0096] In some embodiments, the charged cell mimic comprises a characteristic substantially similar to a biological particle described herein (e.g., a biological cell or an extracellular vesicle described herein). In some embodiments, the characteristic is selected from a size, a shape, a surface charge, surface labeling, elastic modulus, refractive index, and a combination thereof.

[0097] In some embodiments, the charged cell mimic comprises a surface charge substantially similar to the biological particle. In some embodiments, the surface charge is positive. In some embodiments, the surface charge is negative.

[0098] In some embodiments, the charged cell mimic comprises another property that is substantially similar to the biological particle (e.g., a biological cell or an extracellular vesicle described herein), said other property selected from the group consisting of: volume, forward scattering (FSC), side scattering (SSC), background fluorescence, fluorescence profile, surface labeling, refractive index (RI), elastic modulus, and a combination thereof.

[0099] In one embodiment, the RI of the charged cell mimic is 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, including all ranges and subranges therebetween. In another embodiment, the RI is 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, including all ranges and subranges therebetween. In someembodiments, the RI is 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, including all ranges and subranges therebetween.

[0100] In some embodiments, the SSC of the charged cell mimic is within 30%, within 25%, within 20%, within 15%, within 10%, within 5%, or within 1% (including all ranges and subranges therebetween) that of the biological particle (e.g., a biological cell or an extracellular vesicle described herein), as measured by a cytometric device.

[0101] High-refractive index molecules can also include vinylarenes such as styrene and methylstyrene, optionally substituted on the aromatic ring with an alkyl group, such as methyl, ethyl or tert-butyl, or with a halogen, such as chlorostyrene.

[0102] In some embodiments, FSC is modulated by adjusting the percentage of monomer present in the composition, thereby altering the water content present during hydrogel formation. In one embodiment, where a monomer and co-monomer are employed, the ratio of monomer and co- monomer is adjusted to change the hydrogel particle’s forward scatter properties.

[0103] The FSC of a disclosed hydrogel particle is most meaningfully measured in comparison to that of the biological particle (e.g., a biological cell or an extracellular vesicle described herein). In some embodiments, a disclosed hydrogel particle has an FSC within 30%, within 25%, within 20%, within 15%, within 10%, within 5%, or within 1% that of a target cell, as measured by a cytometric device.

[0104] FSC is related to particle volume, and thus can be modulated by altering particle diameter, as described herein. Generally, it has been observed that large objects refract more light than smaller objects leading to high forward scatter signals (and vice versa). Accordingly, particle diameter in one embodiment is altered to modulate FSC properties of a hydrogel particle. For example, hydrogel particle diameter is increased in one embodiment is altered by harnessing larger microfluidic channels during particle formation.

[0105] SSC can be engineered by encapsulating nanoparticles within hydrogels to mimic organelles in a target cell. In some embodiments, a hydrogel particle of the disclosure comprisesone or more types of nanoparticles selected from the group consisting of: polymethyl methacrylate (PMMA) nanoparticles, polystyrene (PS) nanoparticles, and silica nanoparticles. Without wishing to be bound by theory, the ability to selectively tune both forward and side scatter of a hydrogel, as described herein, allows for a robust platform to mimic a vast array of cell types. Techniques for tuning the FSC, SSC, RI of a polymer particle are described in US Patent No.9,714,897, which is hereby incorporated by reference in its entirety for all purposes.

[0106] In some embodiments, the charged cell mimic is fabricated and adjusted to tune the capacitances. In one embodiment, a charged cell mimic’s capacitance is adjusted by altering the amount of hydrogel monomer in the composition. For example, polyaniline, polyacetylene; polyphenylene vinylene; polypyrrole (X = NH) and polythiophene (X = S) co-monomers; and polyaniline (X = NH / N) and polyphenylene sulfide (X = S) co-monomer concentrations can all be adjusted to alter capacitance. In one embodiment, the concentration of one or more of these monomers is increased to increase the capacitance of the charged cell mimic.

[0107] In some embodiments, a charged cell mimic of the disclosure has material modulus properties (e.g., elasticity) substantially similar to that of the biological particle (e.g., a biological cell or an extracellular vesicle described herein), e.g., as compared to a polystyrene bead of the same diameter.

[0108] After the charged cell mimic is formed, one or more of the particle’s surfaces can be functionalized, for example, to mimic one or more charge properties of a biological particle (e.g., a biological cell or an extracellular vesicle described herein) comprising a surface label. The charged cell mimic can also include an embedded bead or substance such as a biomolecule as described above. In one embodiment, the charged cell mimic is functionalized with one or more fluorescent dyes, one or more cell surface markers / immunostimulatory biomolecules (or epitope binding regions thereof), or a combination thereof. In one embodiment, the charged cell mimic is formed by polymerizing at least one bifunctional monomer and after formation, the particle includes one or more functional groups that can be used for further attachment of a cell surface marker, an epitope binding region of a cell surface marker, a fluorescent dye, or combination thereof. The free functional group, in one embodiment, is an amine group, a carboxyl group, a hydroxyl group or a combination thereof. Depending on the functionalization desired, it is to be understood that multiple bifunctional monomers can be used, for example, to functionalize the particle using different chemistries and with different molecules.

[0109] In some embodiments, the charged cell mimic is functionalized with any fluorescent dye known in the art, including fluorescent dyes listed in The Molecular Probes® Handbook-A Guide to Fluorescent Probes and Labeling Technologies, incorporated herein by reference in its entirety for all purposes. Functionalization can be mediated by a compound comprising a free amine group, e.g. allylamine, which can be incorporated into a bifunctional monomer used to form the hydrogel, as discussed below.

[0110] Depending on the biological particle (e.g., biological cell or extracellular vesicle), individual charged cell mimics can be derivatized with one or more cell surface markers, or fragments thereof, for example, extracellular portions thereof in the case of transmembrane proteins to further mimic the structural properties of the biological particle. Biological Particles

[0111] In some embodiments, the biological particle comprises a cell. The present disclosure encompasses all known cell types. In some embodiments the biological particle is a cell form a unicellular organism. In some embodiments the biological particle is a cell from a multi-cellular organism. In some embodiments, the biological particle is a cell from a vertebrate animal. In some embodiments, the biological sample is a cell from a mammal. In some embodiments, the biological particle is a cell from a primate. In some embodiments, the biological particle is a cell from a human. A non-limiting disclosure of cells that can be mimicked by the charged cell particles of the present disclosure can be found in United States Patent 9,714,897, which is hereby incorporated by reference for all purposes.

[0112] In some embodiments, the biological sample can further distinguish between different stages / responses amongst same / similar cell types. For example, in some embodiments, the cell is apoptotic. In some embodiments, the cell is pre-apoptotic. Exposure of phosphatidylserine on the outer leaflet of the plasma membrane is a surface change present in many apoptotic cells. Increased phosphatidylserine at the cell surface renders it more negative. In some embodiments, the cell (e.g., the apoptotic cell or the pre-apoptotic cell) comprises surface-exposed phosphatidylserine. Thus, in some embodiments, the cell (e.g., the apoptotic cell or the pre-apoptotic cell) comprises a negative surface charge. In some embodiments, the cell is a malignant cell. In some embodiments, the malignant cell comprises a negative surface charge.

[0113] In some embodiments, the biological particle comprises an extracellular vesicle. In some embodiments, the biological particle is a tumor microvesicle or tumor macrovesicle. Tumor microvesicles, also known as tumor-secreted microvesicles or tumor-secreted exosomes, can be found in circulating blood and may have immune-suppressive activities. Tumor microvesicles typically range in size from 30-200 nm in diameter. Larger tumor micro vesicles may be referred to as tumor macro vesicles and can range in size from 3-10 μm in diameter.

[0114] In some embodiments, the biological particle comprises a surface label. In some embodiments, the surface label comprises a fluorophore. In some embodiments, the surface label comprises a biomarker. In some embodiments, the biomarker is selected from a nucleic acid, protein, peptide, carbohydrate, or combination thereof.

[0115] In some embodiments, the surface label alters the surface charge of the biological particle. In some embodiments, the surface label increases positive charge at the cell surface. In some embodiments, the surface label increased negative charge at the cell surface. In some embodiments, the method comprises measuring the surface charge of the biological particle prior to and after conjugation of the surface label. In some embodiments, the surface charge is altered prior to and after the conjugation of the surface label. In some embodiments, the difference in the surface charge prior to the conjugation and the surface charge after the conjugation is used to determine the degree of surface labeling. Methods of Making Charged Cell Mimics

[0116] The polymers provided herein, in the form of particles, are synthesized by polymerizing a charged monomer described herein. In some embodiments, the synthesis is carried out to form individual polymer particles. In some embodiments, the charged monomer is polymerized with a neutral monomer to form a copolymer. In some embodiments, the charged monomer is polymerized with a charged monomer, a first comonomer, and a second comonomer to form a copolymer. In some embodiments, the first comonomer is a neutral monomer. In some embodiments, the second copolymer is bifunctional (i.e., comprises a chemical functionality for secondary labeling / conjugation). In some embodiments, the polymer is synthesized by cross- linking the copolymer.

[0117] The amount of monomer can be varied by the user of the invention, for example to obtain a particular optical property that is substantially similar to that of a target cell. In one embodiment,the monomeric component (e.g., the charged monomer, the neutral co-monomer, and / or the bifunctional comonomer) is present at about 10 percent by weight to about 95 percent weight of a hydrogel. In a further embodiment, the monomeric component is present at about 15 percent by weight to about 90 percent weight of the hydrogel, or about 20 percent by weight to about 90 percent weight of the hydrogel, including all ranges and subranges there between.

[0118] Examples of various monomers and cross-linking chemistries available for use with the present invention are provided in the Thermo Scientific Crosslinking Technical Handbook 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 by reference in its entirety for all purposes. For example, hydrazine (e.g., with an NHS ester compound) or EDC coupling reactions (e.g., with a maleimide compound) can be used to construct the hydrogels of the invention.

[0119] In some embodiments, a charged cell mimic of the disclosure comprises a particle formed from a polymer matrix. In some embodiments, the polymer matrix comprises a plurality of polymers. In some embodiments, the polymers of the plurality are covalently cross-linked. In some embodiments, the polymers of the plurality are non-covalently (physically) cross-linked. In some embodiments, the polymers of the plurality are cross-linked (e.g., covalently or non-covalently) to form a hydrogel.

[0120] The hydrogels provided herein, in the form of particles, are synthesized by polymerizing one or more of the monomers provided herein. The synthesis is carried out to form individual hydrogel particles. The monomeric material (monomer) in one embodiment is polymerized to form a homopolymer. However, in another embodiment copolymers of different monomeric units (i.e., co-monomers) are synthesized and used in the methods provided herein. The monomer or co- monomers used in the methods and compositions described herein, in one embodiment, is a bifunctional monomer or includes a bifunctional monomer (where co-monomers are employed). In one embodiment, the hydrogel is synthesized in the presence of a crosslinker. In a further embodiment, embodiment, the hydrogel is synthesized in the presence of a polymerization initiator.

[0121] In some embodiments, the polymers of the plurality comprise a charged monomer described herein and a comonomer described herein. In some embodiments, the polymers of the plurality comprise a charged monomer, a neutral monomer, and a bifunctional monomercomprising a reactive group for conjugation, such as for conjugation to a biomolecule or a fluorophore.

[0122] In some embodiments, the charged monomer comprises an ionizable functional group. In some embodiments the comonomer is nonionic at neutral pH.

[0123] In some embodiments, the ionizable functional group is anionic. In some embodiments, the ionizable functional group comprises a net negative charge at neutral pH. In some embodiments, the ionizable functional group is selected from a carboxylate, a phosphate, a sulfate, a sulfonate, a phenolate, a thiolate, an enolate, an alkoxide, and a phosphonate.

[0124] In some embodiments, the ionizable functional group is cationic. In some embodiments, the ionizable functional group comprises a net positive charge at neutral pH. In some embodiments, the ionizable functional group is a tertiary amine.

[0125] In some embodiment, the polymer comprises a monomer selected from lactic acid, glycolic acid, acrylic acid, 1-hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinylpyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glycerol methacrylate (GMA), glycol methacrylate, ethylene glycol, fumaric acid, a derivatized version thereof, and a combination thereof.

[0126] In some embodiments, the polymer comprises a poly(acrylamide). In some embodiments, the poly(acrylamide) is polymerized from a neutral acrylamide monomer and a charged acrylamide monomer. In some embodiments, the poly(acrylamide) is polymerized from a charged acrylamide monomer and a monomer selected from acrylamide, N-phenyl acrylamide, N-phenyl methacrylamide, N-benzyl acrylamide, N-benzyl methacrylamide, N,N-dibenzyl acrylamide, N,N-dibenzyl methacrylamide, N-diphenylmethyl acrylamide, N-(4-methylphenyl)methyl acrylamide, N-1-naphthyl acrylamide, N-4-nitrophenyl acrylamide, N-(2-phenylethyl)acrylamide, N-triphenylmethyl acrylamide, N-(4-hydroxyphenyl)acrylamide, N,N-methylphenyl acrylamide, N,N-phenyl phenylethyl acrylamide, N-diphenylmethyl methacrylamide, N-(4-methyl phenyl)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'-phenyl phenylethyl methacrylamide, and a combination thereof. In some embodiments, the monomer comprises acrylamide, which has the structure:Compound 3.

[0127] In some embodiments, the monomer comprises an alkyl methacrylate, e.g., an alkyl methacrylate comprising 1 to 18, 1 to 8, or 2 to 8, carbon atoms in the alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tertbutyl, 2-ethylhexyl, heptyl or octyl groups. The alkyl group may be branched or linear. In some embodiments, the charged acrylamide monomer comprises an ionizable functional group described herein. In some embodiments, the charged acrylamide monomer comprises a cationic functional group.

[0128] In embodiments, a positively charged cell mimic of the disclosure comprises a cationic monomer. In embodiments, the cationic monomer comprises an acrylate moiety. In embodiments, the cationic monomer comprises a methacrylate moiety. In embodiments, the cationic monomer comprises an acrylamide moiety. In embodiments, the cationic monomer comprises a methacrylamide moiety.

[0129] In embodiments, the cationic monomer comprises a (meth)acrylamide moiety or a (meth)acrylate moiety; and a quaternary ammonium group. In embodiments, the cationic monomer comprises an acrylamide moiety and a quaternary ammonium group. In embodiments, the cationic monomer comprises a methacrylamide moiety and a quaternary ammonium group. In embodiments, the cationic monomer comprises an acrylate moiety and a quaternary ammonium group. In embodiments, the cationic monomer comprises a methacrylate moiety and a quaternary ammonium group.

[0130] In embodiments, a spacer group comprising 1, 2, 3, 4, 5, or 6 carbon atoms separates the acrylamide moiety from the quaternary ammonium group. In embodiments, a spacer group comprising 1, 2, 3, 4, 5, or 6 carbon atoms separates the methacrylamide moiety from the quaternary ammonium group. In embodiments, a spacer group comprising 1, 2, 3, 4, 5, or 6 carbon atoms separates the acrylate moiety from the quaternary ammonium group. In embodiments, a spacer group comprising 1, 2, 3, 4, 5, or 6 carbon atoms separates the methacrylate moiety from the quaternary ammonium group.

[0131] In embodiments, the quaternary ammonium group has the formula: [R1R2R3R4N]+, where R1, R2, and R3are each independently selected from the group consisting of methyl, ethyl, propyl, and butyl; andR4is selected from the group consisting of H and C1-C6alkyl; In embodiments, the quaternary ammonium group is positively charged.

[0132] In embodiments, the quaternary ammonium group comprises trimethylammonium. In embodiments, the quaternary ammonium group comprises tetraethylammonium. In embodiments, the quaternary ammonium group comprises tetrapropylammonium. In embodiments, the quaternary ammonium group comprises tetrabutylammonium.

[0133] In embodiments, the cationic monomer comprises 3- (methacryloylamino)propyl]trimethylammonium, 3-Acrylamidopropyl)trimethylammonium chloride (APTAC), Allylamine hydrochloride, (3-Acrylamidopropyl)trimethylammonium chloride, [2-(Methacryloyloxy)ethyl]trimethylammonium chloride, Lysine (meth)acrylate, Poly(L-lysine) (meth)acrylate, Poly(ethylenimine) (meth)acrylate, Poly(amidoamine) (meth)acrylate, or a combination thereof. In embodiments, the cationic monomer comprises 3- (methacryloylamino)propyl]trimethylammonium.

[0134] In embodiments, a positively charged cell mimic of the disclosure comprises the cationic monomer Compound 1, which has the structure:Compound 1.

[0135] In embodiments, a positively charged cell mimic of the disclosure comprises APTAC, which has the structure:Compound 4.

[0136] In embodiments, a precursor solution of a positively charged cell mimic of the disclosure comprises a cationic monomer in a total concentration of about 0.01M, 0.02M, 0.03M, 0.04M, 0.05M, 0.06M, 0.07M, 0.08M, 0.09M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2M, 2.1M, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M, 2.7M, 2.8M, 2.9M, 3M, 3.1M, 3.2M, 3.3M, 3.4M, 3.5M, 3.6M, 3.7M, 3.8M, 3.9M,4M, 4.1M, 4.2M, 4.3M, 4.4M, 4.5M, 4.6M, 4.7M, 4.8M, 4.9M, or 5M, including all ranges and subranges therebetween.

[0137] In embodiments, a precursor solution of a positively charged cell mimic of the disclosure comprises APTAC in a total concentration of about 0.01M, 0.02M, 0.03M, 0.04M, or 0.05M, including all ranges and subranges therebetween.

[0138] In embodiments, a negatively charged cell mimic of the disclosure comprises an anionic monomer. In embodiments, the negatively charged monomer comprises an anionic ionizable functional group. In embodiments, the anionic ionizable functional group is selected from carboxylate, phosphate, sulfate, sulfonate, phenolate, thiolate, enolate, alkoxide, or phosphonate. In embodiments, the anionic ionizable functional group comprises carboxylate.

[0139] In embodiments, the anionic monomer comprises a methacrylate moiety or a vinyl moiety; and an anionic ionizable functional group selected from carboxylic acid, sulfonic acid, and phosphonic acid. In embodiments, the anionic monomer comprises a methacrylate moiety and a succinate group. In embodiments, the anionic monomer comprises a methacrylate moiety and a carboxylate group.

[0140] In embodiments, a spacer group comprising 1, 2, 3, 4, 5, or 6 carbon atoms separates the methacrylate or vinyl moiety from the anionic ionizable functional group. In embodiments, a spacer group comprising 1, 2, 3, 4, 5, or 6 carbon atoms separates the methacrylate or vinyl moiety from the anionic ionizable functional group. In embodiments, a spacer group comprising 1, 2, 3, 4, 5, or 6 carbon atoms separates the methacrylate or vinyl moiety from the anionic ionizable functional group. In embodiments, a spacer group comprising 1, 2, 3, 4, 5, or 6 carbon atoms separates the methacrylate or vinyl moiety from the anionic ionizable functional group.

[0141] In embodiments, the anionic monomer has the formula: R1−O−CH2−CH2−O−C(=O)−CH(R2)=CH2, where R1is selected from the group consisting of -COOH, -SO3H, - PO3H2, and their corresponding anions; R2 is selected from the group consisting of hydrogen, C1-C6 alkyl, and C1-C6 substituted alkyl. In embodiments, the anionic monomer is negatively charged.

[0142] In embodiments, the anionic monomer comprises mono-2-(Methacryloyloxy)ethyl succinate (MMES), sodium acrylate, acrylic acid, mono-2-(Methacryloyloxy)ethyl succinate sodium styrene sulfonate, 2-Acrylamido-2-methyl-1-propanesulfonic acid sodium salt, 2- sulfoethyl methacrylate, vinylphosphonic acid, or a combination thereof.

[0143] In embodiments, a negatively charged cell mimic of the disclosure comprises mono-2-(Methacryloyloxy)ethyl succinate (MMES), which has the structure:Compound 2.

[0144] In embodiments, a precursor solution of a negatively charged cell mimic of the disclosurecomprises an anionic monomer in a total concentration of about 0.01M, 0.02M, 0.03M, 0.04M, 0.05M, 0.06M, 0.07M, 0.08M, 0.09M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2M, 2.1M, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M, 2.7M, 2.8M, 2.9M, 3M, 3.1M, 3.2M, 3.3M, 3.4M, 3.5M, 3.6M, 3.7M, 3.8M, 3.9M,4M, 4.1M, 4.2M, 4.3M, 4.4M, 4.5M, 4.6M, 4.7M, 4.8M, 4.9M, or 5M, including all ranges andsubranges therebetween.

[0145] In embodiments, a precursor solution of a negatively charged cell mimic of the disclosurecomprises MMES in a total concentration of about 0.01M, 0.02M, 0.03M, 0.04M, or 0.05M, including all ranges and subranges therebetween.

[0146] In embodiments, the zwitterionic monomer comprises 3-[(3-Acrylamidopropyl)dimethylammonio]propane-1-sulfonate, 3-[[2- (Methacryloyloxy)ethyl]dimethylammonio]propane-1-sulfonate, or a combination thereof.

[0147] In embodiments, a precursor solution of a charged cell mimic of the disclosure comprisesa zwitterionic monomer in a total concentration of about 0.01M, 0.02M, 0.03M, 0.04M, 0.05M, 0.06M, 0.07M, 0.08M, 0.09M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2M, 2.1M, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M, 2.7M, 2.8M, 2.9M, 3M, 3.1M, 3.2M, 3.3M, 3.4M, 3.5M, 3.6M, 3.7M, 3.8M, 3.9M,4M, 4.1M, 4.2M, 4.3M, 4.4M, 4.5M, 4.6M, 4.7M, 4.8M, 4.9M, or 5M, including all ranges andsubranges therebetween.

[0148] In embodiments, a precursor solution of a charged cell mimic of the disclosure comprisesa cationic monomer and an anionic monomer. In embodiments, a precursor solution of a charged cell mimic of the disclosure comprises a cationic monomer and an anionic monomer in a w / w ratioof about 1:1, 2:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, including all ranges and subranges therebetween.

[0149] In embodiments, a precursor solution of a charged cell mimic of the disclosure comprises a cationic monomer at a concentration of 0.1-5M and an anionic monomer at a concentration of 0.1-5M. In embodiments, a precursor solution of a charged cell mimic of the disclosure comprises a cationic monomer at a total concentration of about 0.01M, 0.02M, 0.03M, 0.04M, 0.05M, 0.06M, 0.07M, 0.08M, 0.09M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, including all ranges and subranges therebetween; and an anionic monomer at a total concentration about 0.01M, 0.02M, 0.03M, 0.04M, 0.05M, 0.06M, 0.07M, 0.08M, 0.09M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, including all ranges and subranges therebetween. Table 1: Exemplary Cationic, Anionic, and Zwitterionic Monomers

[0150] In some embodiments, the polymer comprises an acrylic polymer. In some embodiments, the acrylic polymer is polymerized from a neutral acrylate monomer and a charged acrylatemonomer. In some embodiments, the acrylic polymer is polymerized from a charged acrylate monomer and an acrylate monomer selected from PEG-acrylate, 2-methyl acrylate, 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, 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, glycerol methacrylate, hydroxypropyl methacrylate, hydroxybutyl methacrylate, and a combination thereof. In some embodiments, the acrylate monomer is PEG-acrylate. In some embodiments, the acrylate monomer is 2-hydroxyethyl methacrylate. In some embodiments, the acrylate monomer is 2-methyl methacrylate. In some embodiments, the charged acrylate monomer comprises an ionizable functional group described herein. In some embodiments, the charged acrylate monomer comprises a cationic functional group. In some embodiments, the charged acrylate monomer comprises an anionic functional group. In some embodiments, the charged acrylate monomer is unsubstituted (i.e., unsubstituted acrylate).

[0151] In another embodiment, the polymer comprises a monomer selected from 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-phenyl acrylamide, N-phenyl methacrylamide, N-benzyl acrylamide, N-benzyl methacrylamide, N,N-dibenzyl acrylamide, N,N-dibenzyl methacrylamide, N-diphenylmethyl acrylamide, N-(4-methylphenyl)methyl acrylamide, N-1-naphthyl acrylamide, N-4-nitrophenyl acrylamide, N-(2-phenylethyl)acrylamide, N-triphenylmethyl acrylamide, N-(4- hydroxyphenyl)acrylamide, N,N-methylphenyl acrylamide, N,N-phenyl phenylethyl acrylamide, N-diphenylmethyl methacrylamide, N-(4-methyl phenyl)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'-phenyl phenylethyl methacrylamide, N-vinylcarbazole, 4-vinylpyridine, and 2-vinylpyridine, as described in U.S. Patent No.6,657,030, which is incorporated by reference in its entirety herein for all purposes.

[0152] In embodiments, a precursor solution for a charged cell mimic of the disclosure comprises the acrylic monomer in a total concentration of about 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2M, 2.1M, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M, 2.7M, 2.8M, 2.9M, 3M, 3.1M, 3.2M, 3.3M, 3.4M, 3.5M, 3.6M, 3.7M, 3.8M, 3.9M, 4M, 4.1M, 4.2M, 4.3M, 4.4M, 4.5M, 4.6M, 4.7M, 4.8M, 4.9M, or 5M, including all ranges and subranges therebetween.

[0153] In embodiments, a precursor solution for a charged cell mimic of the disclosure comprises acrylamide in a total concentration of about 0.7M, 0.8M, 0.9M, 1M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2M, 2.1M, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M, 2.7M, 2.8M, 2.9M, 3M, including all ranges and subranges therebetween.

[0154] Both synthetic monomers and bio-monomers can be used in the hydrogels provided herein, to form synthetic hydrogels, bio-hydrogels, or hybrid hydrogels that comprise a synthetic component and a bio-component (e.g., peptide, protein, monosaccharide, disaccharide, polysaccharide, primary amines, sulfhydryls, carbonyls, carbohydrates, carboxylic acids present on a biomolecule). For example, proteins, peptides or carbohydrates can be used as individual monomers to form a hydrogel that includes or does not include a synthetic monomer (or polymer) and in combination with chemically compatible co-monomers and crosslinking chemistries (see for example, the Thermo Scientific Crosslinking Technical Handbook 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 by reference in its entirety for all purposes.). Compatible crosslinking chemistries include, but are not limited to, amines, carboxyls, and other reactive chemical side groups. Representative reactive groups amenable for use in the hydrogels and monomers described herein are provided in Table 2, below.

[0155] In one embodiment, an acrylate-functionalized poly(ethylene) glycol monomer is used as a hydrogel monomer. For example, the PEG in one embodiment is an acrylate or acrylamide functionalized PEG.

[0156] In some embodiments, a hydrogel particle comprises a monofunctional monomer polymerized with at least one bifunctional monomer. One example includes, but is not limited to, the formation of poly-acrylamide polymers using acrylamide and bis-acrylamide (a bifunctional monomer). In another embodiment, a hydrogel particle provided herein comprises a bifunctionalmonomer polymerized with a second bifunctional monomer. One example includes, but is not limited to, the formation of polymers with mixed composition containing compatible chemistries such as acrylamide, bis-acrylamide, and bis-acrylamide structural congeners containing a wide range of additional chemistries. The range of chemically compatible monomers, bifunctional monomers, and mixed compositions will be apparent to those skilled in the art and follows chemical reactivity principles know to those skilled in the art. (reference Thermo handbook and acrylamide polymerization handbook). See, for example, the Thermo Scientific Crosslinking Technical Handbook entitled “Easy molecular bonding crosslinking technology,” (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents- Handbook.pdf) and the Polyacrylamide Emulsions Handbook (SNF Floerger, available at snf.com.au / downloads / Emulsion_Handbook_E.pdf), the disclosure of each of which is incorporated by reference in its entirety for all purposes.

[0157] In one embodiment, a hydrogel particle provided herein comprises a polymerizable monofunctional monomer and is a monofunctional acrylic monomer. Non-limiting examples of monofunctional acrylic monomers for use herein are acrylamide; methacrylamide; N- alkylacrylamides such as N-ethylacrylamide, N-isopropylacrylamide, or N-tert-butylacrylamide; N-alkylmethacrylamides such as N-ethylmethacrylamide or N-isopropylmethacrylamide; N,N- dialkylacrylamides such as N,N-dimethylacrylamide and N,N-diethyl-acrylamide; N- [(dialkylamino)alkyl]acrylamides such as N-[3dimethylamino)propyl]acrylamide or N-[3- (diethylamino)propyl]acrylamide; N-[(dialkylamino)alkyl]methacrylamides such as N-[3- dimethylamino)propyl]methacrylamide or N-[3-(diethylamino)propyl]methacrylamide; (dialkylamino)alkyl acrylates such as 2-(dimethylamino)ethyl acrylate, 2-(dimethylamino)propyl acrylate, or 2-(diethylamino)ethyl acrylates; and (dialkylamino)alkyl methacrylates such as 2- (dimethylamino)ethyl methacrylate.

[0158] A bifunctional monomer is any monomer that can polymerize with a monofunctional monomer of the disclosure to form a hydrogel as described herein that further contains a second functional group that can participate in a second reaction, e.g., conjugation of a fluorophore or cell surface receptor (or domain thereof).

[0159] In some embodiments, a bifunctional monomer is selected from the group consisting of allyl amine, allyl alcohol, allyl isothiocyanate, allyl chloride, and allyl maleimide.

[0160] A bifunctional monomer can be a bifunctional acrylic monomer. Non-limiting examples of bifunctional acrylic monomers are N,N'-methylenebisacrylamide, N,N'-methylene bismethacrylamide, N,N'-ethylene bisacrylamide, N,N'-ethylene bismethacrylamide, N,N'- propylenebisacrylamide, and N,N'-(1,2-dihydroxyethylene) bisacrylamide.

[0161] Higher order branched chain and linear co-monomers can be substituted in the polymer mix to adjust the refractive index while maintaining polymer density, as described in U.S. Patent No.6,657,030, incorporated herein by reference in its entirety for all purposes. Additional Hydrogel Constituents

[0162] In some embodiments, a hydrogel comprises a molecule that modulates the optical properties of the hydrogel. Molecules capable of altering optical properties of a hydrogel are discussed further below.

[0163] In one embodiment, an individual hydrogel particle or a plurality thereof comprises a biodegradable polymer as a hydrogel monomer. In one embodiment, the biodegradable polymer is a poly(esters) based on polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL), and their copolymers. In one embodiment, the biodegradable polymer is a carbohydrate or a protein, or a combination thereof. For example, in one embodiment, a monosaccharide, disaccharide or polysaccharide, (e.g., glucose, sucrose, or maltodextrin) peptide, protein (or domain thereof) is used as a hydrogel monomer. Other biodegradable polymers include poly(hydroxyalkanoate)s of the PHB-PHV class, additional poly(ester)s, and natural polymers, for example, modified poly(saccharide)s, e.g., starch, cellulose, and chitosan. In another embodiment, the biocompatible polymer is an adhesion protein, cellulose, a carbohydrate, a starch (e.g., maltodextrin, 2- hydroxyethyl starch, alginic acid), a dextran, a lignin, a polyaminoacid, an amino acid, or chitin. Such biodegradable polymers are available commercially, for example, from Sigma Aldrich (St. Louis, MO).

[0164] The protein in one embodiment comprises only natural amino acids. However, the invention is not limited thereto. For example, self-assembling artificial proteins and proteins with non-natural amino acids (e.g., those incorporated into non-ribosomal peptides or synthetically introduced via synthetic approaches, see for example, Zhang et al. (2013). Current Opinion in Structural Biology 23, pp. 581-587, the disclosure of which is incorporated by reference in its entirety for all purposes), or protein domains thereof, can also be used as hydrogel monomers. Therange of non-natural (unnatural) amino acids that can be incorporated into such compositions is known to those skilled in the art (Zhang et al. (2013). Current Opinion in Structural Biology 23, pp.581-587; incorporated by reference in its entirety for all purposes). The biodegradable polymer in one embodiment, is used as a co-monomer, i.e., in a mixture of monomers. The biodegradable polymer in one embodiment is a bifunctional monomer.

[0165] The biomonomer, in one embodiment, is functionalized with acrylamide or acrylate. For example, in one embodiment, the polymerizable acrylamide functionalized biomolecule is an acrylamide or acrylate functionalized protein (for example, an acrylamide functionalized collagen or functionalized collagen domain), an acrylamide or acrylate functionalized peptide, or an acrylamide or acrylate functionalized monosaccharide, disaccharide or polysaccharide.

[0166] Any monosaccharide, disaccharide or polysaccharide (functionalized or otherwise) can be used as a polymer monomer. In one embodiment, an acrylamide or acrylate functionalized monosaccharide, disaccharide or polysaccharide 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 an arabinoxylan, cellulose, chitin or a pectin. In another embodiment, alginic acid (alginate) is used as a polymerizable hydrogel monomer. In yet another embodiment, a glycosaminoglycan (GAG) is used as a polymerizable monomer in the hydrogels provided herein. In a further embodiment, the GAG is chondroitin sulfate, dermatan sulfate, keratin sulfate, heparin, heparin sulfate or hyaluronic acid (also referred to in the art as hyaluron or hyaluronate) is used as a polymerizable hydrogel monomer. The additional range of compatible biomonomers and their reactive chemistries are known by individuals skilled in the art and follow general chemical reactivity principles.

[0167] An additional range of biocompatible monomers that can be incorporated are known in the art, see, for example the non-degradable biocompatible monomers disclosed in Shastri (2003). Current Pharmaceutical Biotechnology 4, pp. 331-337, incorporated by reference herein 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, ISBN91-631-4985-0, the disclosure of each of which are hereby incorporated by reference in their entirety.

[0168] Biocompatible monomers for use with the hydrogels described herein include in one embodiment, ethylene glycol dimethacrylate (EGDMA), 2-hydroxyethyl methacrylate (HEMA), methylmethacrylate (MMA), methacryloxymethyltrimethylsilane (TMS-MA), N-vinyl-2- pyrrolidon (N-VP), styrene, or a combination thereof.

[0169] Naturally occurring hydrogels useful in this invention include various polysaccharides available from natural sources such as plants, algae, fungi, yeasts, marine invertebrates and arthropods. Non-limiting examples include agarose, dextrans, chitin, cellulose-based compounds, starch, derivatized starch, and the like. These generally will have repeating glucose units as a major portion of the polysaccharide backbone. Cross-linking chemistries for such polysaccharides are known in the art, see for example Thermo Scientific Crosslinking Technical Handbook entitled “Easy molecular bonding crosslinking technology,” (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents- Handbook.pdf).

[0170] Hyaluronan in one embodiment is used as a hydrogel monomer (either as a single monomer or as a co-monomer). Hyaluronan in one embodiment, is functionalized, for example with acrylate or acrylamide. Hyaluronan is a high molecular weight GAG composed of disaccharide repeating units of N-acetylglucosamine and glucuronic acid linked together through alternating β-1,4 and β- 1,3 glycosidic bonds. In the human body, hyaluronate is found in several soft connective tissues, including skin, umbilical cord, synovial fluid, and vitreous humor. Accordingly, in one embodiment, where one or more optical properties of a skin cell, umbilical cord cell or vitreous humor cell is desired to be mimicked, in one embodiment, hyaluronan is used as a hydrogel monomer. Methods for fabricating hydrogel particles are described in Xu et al. (2012). Soft Matter. 8, pp.3280-3294, the disclosure of which is incorporated herein in its entirety for all purposes. As described therein, hyaluronan can be derivatized with various reactive handles depending on the desired cross-linking chemistry and other monomers used to form a hydrogel particle.

[0171] In yet other embodiments, chitosan, a linear polysaccharide composed of randomly distributed β-(1-4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit), is used as a hydrogel monomer (either as a single monomer or as a co-monomer).

[0172] Other polysaccharides for use as a hydrogel monomer or co-monomer include but are not limited to, agar, agarose, alginic acid, alguronic acid, alpha glucan, amylopectin, amylose, arabinoxylan, beta-glucan, callose, capsulan, carrageenan polysaccharides (e.g., kappa, iota or lambda class), cellodextrin, cellulin, cellulose, chitin, chitosan, chrysolaminarin, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, ficoll, fructan, fucoidan, galactoglucomannan, galactomannan, galactosaminogalactan, gellan gum, glucan, glucomannan, glucuronoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hypromellose, icodextrin, inulin, kefiran, laminarin, lentinan, levan polysaccharide, lichenin, mannan, mixed-linkage glucan, paramylon, pectic acid, pectin, pentastarch, phytoglycogen, pleuran, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, sinistrin, sizofiran, welan gum, xanthan gum, xylan, xyloglucan, zymosan, or a combination thereof. As described throughout, depending on the desired cross-linking chemistry and / or additional co-monomers employed in the hydrogel, the polysaccharide can be further functionalized. For example, one or more of the polysaccharides described herein in one embodiment is functionalized with acrylate or acrylamide.

[0173] In one embodiment, an individual hydrogel particle or a plurality thereof comprises a peptide, protein, a protein domain, or a combination thereof as a hydrogel monomer or plurality thereof. In a further embodiment, the protein is a structural protein, or a domain thereof, for example, such as silk, elastin, titin or collagen, or a domain thereof. In one embodiment, the protein is an extracellular matrix (ECM) component (e.g., collagen, elastin, proteoglycan). In even a further embodiment, the structural protein is collagen. In yet a further embodiment, the collagen is collagen type I, collagen type II or collagen type III or a combination thereof. In another embodiment, the hydrogel monomer comprises a proteoglycan. In a further embodiment, the proteoglycan is decorin, biglycan, testican, bikunin, fibromodulin, lumican, or a domain thereof.

[0174] In another embodiment, an acrylate-functionalized structural protein hydrogel monomer is used as a component of the hydrogel provided herein (e.g., an acrylate functionalized protein or protein domain, for example, silk, elastin, titin, collagen, proteoglycan, or a functionalized domain thereof). In a further embodiment, the acrylate functionalized structural protein hydrogel monomer comprises a proteoglycan, e.g., decorin, biglycan, testican, bikunin, fibromodulin, lumican, or a domain thereof.

[0175] In one embodiment PEG monomers and oligopeptides can be that mimic extracellular matrix proteins are used in the hydrogels provided herein, for example, with vinyl sulfone-functionalized multi-arm PEG, integrin binding peptides and bis-cysteine matrix metalloproteinase peptides as described by Lutolf et al. (2003). Proc. Natl. Acad. Sci. U.S.A. 100, 5413-5418, incorporated by reference in its entirety for all purposes. In this particular embodiment, hydrogels are formed by a Michael-type addition reaction between the di-thiolated oligopeptides and vinyl sulfone groups on the PEG. The range of additional compatible chemistries that can be incorporated here will be apparent to those skilled in the art and follow general chemical reactivity principles, see for example Thermo Scientific Crosslinking Technical Handbook entitled “Easy molecular bonding crosslinking technology,” (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents- Handbook.pdf).

[0176] Other bioactive domains in natural proteins can also be used as a polymer monomer or portion thereof. For example, a cell-adhesive integrin binding domain, a controlled release affinity binding domain or a transglutaminase cross-linking domain can be used in the hydrogels provided herein. Details for producing such 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 incorporated by reference in its entirety for all purposes.

[0177] In one embodiment, recombinant DNA methods are used to create proteins, designed to gel in response to changes in pH or temperature, for example, by the methods described by Petka et al. (1998). Science 281, pp. 389-392, incorporated by reference in its entirety for all purposes. Briefly, the proteins consist of terminal leucine zipper domains flanking a water-soluble polyelectrolyte segment. In near-neutral aqueous solutions, coiled-coil aggregates of the terminal domains form a three-dimensional hydrogel polymer network.

[0178] In some embodiments, the forward scatter of a hydrogel particle described herein is modulated by adjusting the refractive index of the gel by adding co-monomers allyl acrylate and allyl methacrylate. Forward scatter can also be modulated with side scattering nanoparticles containing sufficient optical resolution / size / density including, but not limited to, higher density colloidal suspensions of silica and / or PMMA particles. Side scattering of the droplets can be tuned by adding a colloidal suspension of silica nanoparticles and / or PMMA (poly(methyl methacrylate)) particles (~ 100 nm) to the central aqueous phase prior to polymerization.

[0179] In some embodiments, the polymeric particle (e.g., hydrogel particle) is functionalized with a fluorophore. In some embodiments, the polymeric particle (e.g., hydrogel particle) isfunctionalized with a fluorescent dye selected from 6-carboxy-4',5'-dichloro-2',7'- dimethoxyfluorescein succinimidyl ester; 5-(6)-carboxyeosin; 5-carboxyfluorescein; 6- carboxyfluorescein; 5-(6)-carboxyfluorescein; S-carboxyfluorescein-bis-(5-carboxymethoxy-2- nitrobenzyl)ether, β-alanine-carboxamide, or succinimidyl ester; 5-carboxy fluorescein succinimidyl ester; 6-carboxyfluorescein succinimidyl ester; 5-(6)-carboxyfluorescein succinimidyl ester; 5-(4,6-dichlorotriazinyl)amino fluorescein; 2',7'-difluoro fluorescein; eosin-5- isothiocyanate; erythrosin5-isothiocyanate; 6-(fluorescein-5-carboxamido) hexanoic acid or succinimidyl ester; 6-(fluorescein-5-(and-6)-carboxamido) hexanoic acid or succinimidyl ester; fluorescein-S-EX succinimidyl ester; fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate; Oregon Green® 488 carboxylic acid or succinimidyl ester; Oregon Green® 488 isothiocyanate; Oregon Green® 488-X succinimidyl ester; Oregon Green® 500 carboxylic acid; Oregon Green® 500 carboxylic acid, succinimidyl ester, or triethylammonium salt; Oregon Green® 514 carboxylic acid; Oregon Green® 514 carboxylic acid or succinimidyl ester; Rhodamine Green™ carboxylic acid, succinimidyl ester, or hydrochloride; Rhodamine Green™ carboxylic acid, trifluoroacetamide, or succinimidyl ester; Rhodamine Green™-X succinimidyl ester or hydrochloride; Rhodol Green™ carboxylic acid, N,O-bis-(trifluoroacetyl), or succinimidyl ester; bis-(4-carboxypiperidinyl) sulfone rhodamine or di(succinimidyl ester); 5-(6)carboxynaphtho fluorescein; 5-(6)carboxynaphthofluorescein succinimidyl ester; 5-carboxyrhodamine 6G hydrochloride; 6-carboxyrhodamine 6G hydrochloride, 5-carboxyrhodamine 6G succinimidyl ester; 6-carboxyrhodamine 6G succinimidyl ester; 5-(6)-carboxyrhodamine 6G succinimidyl ester; 5-carboxy-2',4',5',7'-tetrabromosulfonefluorescein succinimidyl ester or bis- (diisopropylethylammonium) salt; 5-carboxytetramethylrhodamine; 6- carboxytetramethylrhodamine; 5-(6)-carboxytetramethylrhodamine; 5- carboxytetramethylrhodamine succinimidyl ester; 6-carboxytetramethylrhodamine succinimidyl ester; 5-(6)-carboxytetramethylrhodamine succinimidyl ester; 6-carboxy-X-rhodamine; 5- carboxy-X-rhodamine succinimidyl ester; 6-carboxy-X-rhodamine succinimidyl ester; 5-(6)- carboxy-X-rhodamine succinimidyl ester; 5-carboxy-X-rhodamine triethylammonium salt; LissamineTMrhodamine B sulfonyl chloride; malachite green; isothiocyanate; NANOGOLD® mono(sulfosuccinimidyl ester); QSY® 21carboxylic acid or succinimidyl ester; QSY® 7 carboxylic acid or succinimidyl ester; Rhodamine RedTM-X succinimidyl ester; 6- (tetramethylrhodamine-5-(and-6)-carboxamido) hexanoic acid or succinimidyl ester;tetramethylrhodamine-5-isothiocyanate; tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(6)-isothiocyanate; Texas Red® sulfonyl; Texas Red® sulfonyl chloride; Texas Red®-X STP ester or sodium salt; Texas Red®-X succinimidyl ester; Texas Red®-X succinimidyl ester; and X-rhodamine-5-(6) isothiocyanate, BODIPY® dyes commercially available from Invitrogen, including, but not limited to BODIPY® FL; BODIPY® TMR STP ester; BODIPY® TR-X STP ester; BODIPY® 630 / 650-X STP ester; BODIPY® 650 / 665-X STP ester; 6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid or succinimidyl ester; 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3,5-dipropionic acid; 4,4-difluoro- 5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid; 4,4-difluoro-5,7-dimethyl-4-bora- 3a,4a-diaza-s-indacene-3-pentanoic acid or succinimidyl ester; 4,4-difluoro-5,7-dimethyl-4-bora- 3a,4a-diaza-s-indacene-3propionic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s- indacene-3-propionic acid or succinimidyl ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s- indacene-3propionic acid, sulfosuccinimidyl ester, or sodium salt; 6-((4,4-difluoro-5,7-dimethyl- 4-bora-3a,4a-diaza-s-indacene-3propionyl)amino)hexanoicacid; 6-((4,4-difluoro-5,7-dimethyl-4- bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoic acid or succinimidyl ester; N-(4,4- difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl) cysteic acid, succinimidyl ester, or triethylammonium salt; 6-4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a- 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid; 4,4-difluoro-5,7- diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid or succinimidyl ester; 4,4-difluoro-5- phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid or succinimidyl ester; 6-((4,4-difluoro-5- phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino) hexanoic acid or succinimidyl ester; 4,4-difluoro-5-(4-phenyl-1,3butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid or succinimidyl ester; 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid or succinimidyl ester; 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3- yl)styryloxy)acetyl)aminohexanoic acid or succinimidyl ester; 4,4-difluoro-5-styryl-4-bora-3a,4a- diaza-s-indacene-3-propionic acid; 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3- propionic acid or succinimidyl ester; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s- indacene-8-propionic acid; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8- propionic acid or succinimidyl ester; 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3- propionic acid or succinimidyl ester; 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s- indacene-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimidyl ester; and 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryloxy)acetyl) aminohexanoic acid or succinimidyl ester, Alexa fluor dyes commercially available from Invitrogen, including but not limited to Alexa Fluor® 350 carboxylic acid; Alexa Fluor® 430 carboxylic acid; Alexa Fluor® 488 carboxylic acid; Alexa Fluor® 532 carboxylic acid; Alexa Fluor® 546 carboxylic acid; Alexa Fluor® 555 carboxylic acid; Alexa Fluor® 568 carboxylic acid; Alexa Fluor® 594 carboxylic acid; Alexa Fluor® 633 carboxylic acid; Alexa Fluor® 647 carboxylic acid; Alexa Fluor® 660 carboxylic acid; and Alexa Fluor® 680 carboxylic acid, cyanine dyes commercially available from Amersham-Pharmacia Biotech, including, but not limited to Cy3 NHS ester; Cy 5 NHS ester; Cy5.5 NHS ester; and Cy7 NHS ester. Polymerization

[0180] In general, any form of polymerization chemistry / methods known by those skilled in the art, can be employed to form polymers. In some embodiments, polymerization can be catalyzed by ultraviolet light-induced radical formation and reaction progression. In other embodiments, a hydrogel particle of the disclosure is produced by the polymerization of acrylamide or the polymerization of acrylate. For example, the acrylamide in one embodiment is a polymerizable carbohydrate derivatized acrylamide as described in U.S. Patent No. 6,107,365, the disclosure of which is incorporated by reference in its entirety for all purposes. As described therein and known to those of ordinary skill in the art, specific attachment of acrylamide groups to sugars is readily adapted to a range of monosaccharides and higher order polysaccharides, e.g., synthetic polysaccharides or polysaccharides derived from natural sources, such as glycoproteins found in serum or tissues.

[0181] Common cross linking 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 chemistries which can be used will be apparent to those skilled in the art and follow general chemical reactivity principles, see for example Thermo Scientific Crosslinking Technical Handbook entitled “Easy molecular bonding crosslinking technology,” (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents- Handbook.pdf).

[0182] In one embodiment, polymerization of a hydrogel is initiated by a persulfate or an equivalent initiator that catalyzes radical formation. The range of compatible initiators are known to those skilled in the art and follow general chemical reactivity principles, see for example Thermo Scientific Crosslinking Technical Handbook entitled “Easy molecular bonding crosslinking technology,” (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163- Crosslinking-Reagents-Handbook.pdf). The persulfate can be any water-soluble persulfate. Non- limiting examples of water-soluble persulfates are 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 a further embodiment, polymerization of the hydrogel provided herein is initiated by ammonium persulfate.

[0183] Polymerization of a hydrogel can be accelerated by an accelerant which can catalyze the formation of polymerization-labile chemical side groups. The range of possible accelerants is known to those skilled in the art and follow general chemical reactivity principles see for example Thermo Scientific Crosslinking Technical Handbook entitled “Easy molecular bonding crosslinking technology,” (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf). The accelerant in one embodiment, is a tertiary amine. The tertiary amine can be any water-soluble tertiary amine. In one embodiment, an accelerant is used in the polymerization reaction and is 3- (dimethylamino)propionitrile, or N,N,N',N'-tetramethylethylenediamine (TEMED). In another embodiment, an accelerant is used in the polymerization reaction and isazobis (isobutyronitrile) (AIBN).

[0184] As discussed above, the hydrogel for use in the compositions and methods described herein can include any of the monomeric units and crosslinkers as described herein, and in one aspect, are produced as hydrogel particles by polymerizing droplets. Microfluidic methods of producing a plurality of droplets, including fluidic and rigidified droplets, are known to those of ordinary skill in the art, and described in US Patent Publication No. 2011 / 0218123 and U.S. Patent No. 7,294,503, each incorporated herein by reference in their entireties for all purposes. Such methods provide for a plurality of droplets containing a first fluid and being substantially surrounded by a second fluid, where the first fluid and the second fluid are substantially immiscible (e.g., droplets containing an aqueous-based liquid being substantially surrounded by an oil based liquid).

[0185] A plurality of fluidic droplets (e.g., prepared using a microfluidic device) may be polydisperse (e.g., having a range of different sizes), or in some cases, the fluidic droplets may be monodisperse or substantially monodisperse, e.g., having a homogenous distribution of diameters, for instance, 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 greater than about 10%, about 5%, about 3%, about 1%, about 0.03%, or about 0.01% of the average diameter. The average diameter of a population of droplets, as used herein, refers to the arithmetic average of the diameters of the droplets. Average diameters of the particles can be measured, for example, by light scattering techniques. Average diameters of hydrogel particles in one embodiment, are tailored, for example by varying flow rates of the fluid streams of the first and second fluids within the channel(s) of a microfluidic device, or by varying the volume of the channel(s) of the microfluidic device.

[0186] Accordingly, the disclosure provides population of hydrogel particles comprising a plurality of hydrogel particles, wherein the population of hydrogel particles is substantially monodisperse.

[0187] The term microfluidic refers to a device, apparatus or system including at least one fluid channel having a cross-sectional dimension of less than 1 mm, and a ratio of length to largest cross- sectional dimension perpendicular to the channel of at least about 3:1. A micro fluidic device comprising a micro fluidic channel is especially well suited to preparing a plurality of mono disperse droplets.

[0188] Non-limiting examples of microfluidic systems that may be used with 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. WO 2006 / 096571; U.S. Patent Application Publication No. 2007 / 0054119; U.S. Patent No. 7,776,927; and International Patent Application Publication No. WO 2006 / 078841, each incorporated herein by reference in their entireties for all purposes.

[0189] Droplet size is related to microfluidic channel size. The micro fluidic channel may be of any size, for example, having a largest dimension perpendicular to fluid flow of less than about 5 mm or about 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 thanabout 1 μm, less than about 300 nm, less than about 100 nm, less than about 30 nm, or less than about 10 nm, including all ranges and subranges therebetween.

[0190] Droplet size can be tuned by adjusting the relative flow rates. In some embodiments, drop diameters are equivalent to the width of the channel, or within about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% the width of the channel, including all ranges and subranges therebetween.

[0191] The dimensions of a hydrogel particle of the disclosure are substantially similar to the droplet from which it was formed. Therefore, in some embodiments, a hydrogel particle (e.g., either outer body or inner bead) has a diameter of less than about 1 μm, 2 μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 120 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 600 μm, 800 μm, 900 μm, or less than 1000 μm in diameter, including all ranges and subranges therebetween. In some embodiments, a hydrogel particle (e.g., either outer body or inner bead) has a diameter of greater than about 1 μm, 2 μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 120 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 600 μm, 800 μm, 900 μm, or less than 1000 μm in diameter, including all ranges and subranges therebetween. In one embodiment, a hydrogel particle has a diameter in the range of 5 μm to 100 μm.

[0192] The manufacture of hydrogel particles in one embodiment, is carried by suspension polymerization, which is also referred to in the art as pearl, bead or granular polymerization (see Elbert (2011). Acta Biomater. 7, pp. 31-56, incorporated by reference herein in its entirety for all purposes). In suspension polymerization, the monomer is insoluble in the continuous phase, for example an aqueous monomer solution in a continuous oil phase. In suspension polymerization, polymerization initiation occurs within the monomer-rich droplets and with greater than one radical per droplet at any time. The monomer phase in one embodiment includes a monomer which can be a bifunctional monomer or a plurality of monomer species (co-monomers, which can be a plurality of bifunctional monomers. The monomer phase in one embodiment, includes an initiator and / or a crosslinking agent.

[0193] Emulsion polymerization can also be used to form the hydrogel particles described herein. In emulsion polymerization, the monomer has poor solubility in the continuous phase, similar to suspension polymerization, however, polymerization initiation occurs outside the monomerdroplets (see Elbert (2011). Acta Biomater. 7, pp. 31-56, incorporated by reference herein in its entirety for all purposes). In emulsion polymerization embodiments, the initiator causes chain growth of the monomer (or co-monomers) dissolved in the continuous phase or monomer contained in micelles if surfactants are present.

[0194] In another embodiment, hydrogel particles are formed by precipitation polymerization, for example as described in Elbert (2011). Acta Biomater. 7, pp. 31-56, incorporated by reference herein in its entirety for all purposes. Precipitation polymerization is a technique that takes advantage of the differences in the solubility of monomer and polymer to produce microparticles. Specifically, it is known that larger polymer chains generally have lower solubility than smaller ones. Accordingly, above a specific molecular weight, phase separation may be favored. Precipitation polymerization initially begins as solution polymerizations in a single phase, homogenous system. Shortly after the start of the polymerization, in one embodiment, a relatively high concentration of polymer chains is present, favoring phase separation by nucleation. As polymerization proceeds, the concentration of polymer chains is low and existing particles capture the chains before nucleation of new particles can occur. Thus, nucleation of particles occurs only for a brief period of time shortly after the start of the reaction, which in one embodiment, results in a narrow size distribution of particles. Additional methods include but are not limited to lithographic particle formation (Helgeson et al. (2011). Curr. Opin. Colloid. Interface Sci. 16, pp. 106-117, incorporated by reference herein in its entirety for all purposes) membrane emulsification (e.g., by the microsieve emulsification technology techniques described by Nanomi B.V. (Netherlands)) and microchannel emulsification (Sugiura et al. (2002). Languimir 18, pp. 5708- 5712, incorporated by reference herein in its entirety) and bulk emulsification (SNF Floerger, available at snf.com.au / downloads / Emulsion_Handbook_E.pdf, incorporated by reference herein in its entirety).

[0195] In one embodiment, hydrogel particles are formed within a microfluidic device having two oil channels that focus on a central stream of aqueous monomer solution. In this embodiment, droplets form at the interface of the two channels and central stream to break off droplets in water- in-oil emulsion. Once droplets are formed, in one embodiment, they are stabilized prior to polymerization, for example, by adding a surfactant to the oil phase. However, in another embodiment, droplets are not stabilized prior to polymerization. Polymerization of the monomerin one embodiment is triggered by adding an accelerator (e.g., N,N,N’,N’- tetramethylethylenediamine) to one or both of the oil channels after initial droplets are formed.

[0196] The aqueous monomer solution as provided above can include a single monomer species or a plurality of monomer species. The aqueous monomer solution can include co-monomers, a bifunctional monomer or a combination thereof. In one embodiment, the monomer or plurality of monomers can include a bifunctional monomer, for example, one of the monomers described above. As described below, co-monomers can be used to modulate forward scatter or side scatter, for example, by adjusting the refractive index of the hydrogel particle.

[0197] In one embodiment, the central stream of aqueous monomer solution comprises a cross- linker, for example, N,N'-bisacrylamide. In a further embodiment, the central stream of aqueous monomer solution comprises a cross-linker and an accelerator, in addition to the monomer. In yet a further embodiment, the aqueous monomer solution comprises an initiator, for example an oxidizing agent such as ammonium persulfate. Methods of Use Charged Cell Mimics for Instrument Validation

[0198] In some embodiments, a population of charged cell mimics described herein is used as a standard in a method to validate an instrument for measuring a property of a population of biological particles. In some embodiments, the property is surface charge. In some embodiments, the property is zeta potential. In some embodiments, the method comprises determining zeta potential using an ELS instrument. In some embodiments, the method comprises determining zeta potential using a dynamic light scattering (DLS) instrument. In some embodiments, the method comprises determining zeta potential using a particle analyzer.

[0199] In some embodiments, the population of charged cell mimics is used in a method to validate an ELS instrument. In some embodiments, the method comprises inserting into the ELS instrument a sample comprising the population of charged cell mimics suspended in a test solution, wherein the population comprises a predetermined zeta potential, measuring an EPM of the sample, and determining a difference between a zeta potential calculated from the EPM and the predetermined zeta potential.

[0200] In some embodiments, the predetermined zeta potential is measured for the population of charged cell mimics suspended in a reference solution characterized by a property selected frompH, ionic strength, temperature, buffer formulation, and a combination thereof. In some embodiments, the test solution comprises at least one property that is substantially similar to the property of the reference solution. In some embodiments, the test solution comprises a temperature that is substantially similar to the temperature of the reference solution. In some embodiments, the test solution comprises a pH that is substantially similar to the pH of the reference solution. In some embodiments, the test solution comprises an ionic strength that is substantially similar to the ionic strength of the reference solution. In some embodiments, the test solution comprises a buffer formulation that is substantially similar to the buffer formulation of the reference solution. In some embodiments, a difference between the zeta potential calculated from the EPM and the predetermined zeta potential of less than about 20%, about 15%, about 10%, about 5%, or about 1% indicates the ELS instrument is validated.

[0201] In some embodiments, the method further comprises inserting into the validated ELS instrument a test sample comprising a test population of biological particles and measuring an EPM. Persons having skill in the art will recognize the types of biological particles that can be analyzed via an ELS instrument. In some embodiments, the biological particles are cells. In some embodiments, the biological particles are extracellular vesicles.

[0202] In some embodiments, the charged cell mimics of the population comprise a cationic monomer, wherein the population comprises a predetermined zeta potential of about +5 mV to about +100 mV. In some embodiments, the charged cell mimics of the population comprise an anionic monomer, wherein the population comprises a predetermined zeta potential of about -10 mV to about -100 mV. Charged Cell Mimics to Measure Zeta Potential of Biological Particles

[0203] In some embodiments, a population of charged cell mimics described herein is used in a method to measure a property of a test sample comprising a population of biological particles.

[0204] In some embodiments, the method comprises (i) inserting into an ELS device a population of charged cell mimics comprising a predetermined zeta potential, (ii) measuring electrophoretic mobility (EPM) of the population of charged cell mimics using the ELS device, repeating (i)-(ii) for a panel of populations of charged cell mimics, each population comprising a distinct surface charge, (iv) generating a calibration curve from the EPM measurement and the surface charge for each population in the panel, (v) measuring EPM of the population of biological particles, and (vi)determining the surface charge of the population of biological particles based on a comparison of the EPM measurement to the calibration curve.

[0205] In some embodiments, the population of charged cell mimics in the panel are each independently suspended in a solution at a concentration, wherein the solution and / or the concentration is substantially similar for each population in the panel. For example, in some embodiments, the panel comprises a first population of charged cell mimics comprising a negative surface charge and a second population of charged cell mimics comprising a negative surface charge, wherein the first population of charged cell mimics is suspended in a first solution at a first concentration and the second population of biological particles is suspended in a second solution at a second concentration. In some embodiments, the first concentration and the second concentration are substantially the same. In some embodiments, the first solution and the second solution are substantially the same. In some embodiments, the first solution and the second solution are of substantially similar pH. In some embodiments, the first solution and the second solution are of substantially similar temperature. In some embodiments, the first solution and the second solution are of substantially similar ionic strength. In some embodiments, the first solution and the second solution are of substantially similar composition (e.g., substantially similar buffer components). In some embodiments, the population of biological particles are suspended in a solution substantially similar to the solution for each population in the panel. In some embodiments, the population of biological particles are suspended in a solution at a concentration that is substantially similar to the concentration of each population in the panel. In some embodiments, the biological particles and the charged cell mimics in the panel are substantially similar in shape. In some embodiments, the biological particles and the charged cell mimics in the panel are substantially similar in size (e.g., hydrodynamic radius). Charged Cell Mimics as Inducers of Immune Activation

[0206] In some embodiments, a population of charged cell mimics described herein is used for activation and / or expansion of a population of immune cells. Existing methods used to activate and subsequently expand immune cells (e.g., T-cells) in vitro include treatment of the immune cells with immunostimulatory molecules presented on beads (e.g., polystyrene beads). For example, a standard method to activate and expand T cells in vitro involves culturing the T cells with beads presenting an antibody that engages the T cell receptor (e.g., an anti-CD3 antibody)and providing an antibody that engages a co-stimulatory receptor (e.g., an anti-CD28 antibody). However, such methods are prone to inducing cell exhaustion and / or requiring multi-step processes to remove activation agents from culture due to incompatibility with long-term cell survival.

[0207] Accordingly, the present disclosure provides methods for improving the in vitro activation and expansion of immune cells using a charged cell mimic described herein comprising an immunostimulatory molecule. Methods of using polymer particles for immune cell activation have been previously described in Applicant’s related patent applications, including U.S. 18 / 386,107, and PCT / US2023 / 077961, each of which is incorporated by reference in its entirety for all purposes. In some embodiments, this present disclosure describes further enhancements to immune cell activation through the modulation of particle surface charge.

[0208] In an embodiment, the charged cell mimic is functionalized with an immunostimulatory molecule to form a synthetic biomolecule presenting particles. Without being bound by theory, it is believed the charged surface of a charged cell mimic described herein provides enhanced an activation event of an immune cell, e.g., as compared to a nonionic particle. In some embodiments, the charged cell mimics are functionalized with one or more cell surface markers, or fragments thereof, for example, extracellular portions thereof in the case of transmembrane proteins. In some embodiments, the charged cell mimics described herein are functionalized with a an immunostimulatory biomolecules that stimulates an activation event of a T cell. In some embodiments, these synthetic biomolecule presenting particles comprise one or more of an activation biomolecule, a stimulatory biomolecule, a costimulatory biomolecule and / or a T cell homeostasis factor. In some embodiments, the activation events is selected from cell expansion, cell proliferation, cell differentiation, activation maintenance, cell maturation, cell receptor clustering, synapse formation (e.g., between a lymphocyte and a tumor cell), cytokine production, gene expression, protein expression, and any other appropriate occurrence by which the target cell is activated upon recognition of or stimulation by the proper antigen, antibody, immunoglobulin (e.g., CD3, CD19, CD20, CD28, CD80, CD86, CD69, CD154, CD137, IgM, IgG, IgE, IgA, IgD, or antibodies targeting said biomolecules), toll-like receptors (TLR, such as, for example, TLR1- 13), or the like.

[0209] Though the present disclosure is described with reference to immune cells, and in particular, to a T cell, the disclosure is not intended to be so limited in its scope of application.Non-limiting examples of immune cells include B lymphocytes, also called B cells, T lymphocytes, also called 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, gene modified immune cells including hybridomas, drug modified immune cells, and derivatives, precursors or progenitors of any of the cell types listed herein. Additionally, the method may be used for any number of cells or analytes, such as one, at least one, a plurality, etc.

[0210] In some embodiments, the charged cell mimic comprises a biomolecule that support the growth and / or activation of the immune cells. These biomolecules may be selected based on particular cell surface markers of interest. These markers of interest may be one or more cell surface markers, or fragments thereof, for example, extracellular portions thereof in the case of transmembrane proteins, for example. For instance, the biomolecules may be antibodies or antigen-binding fragments thereof related to the particular cell surface marker of interest. In another instance, the biomolecules may be one or more cell surface markers, extracellular portions or ligand binding regions thereof.

[0211] In some embodiments, the biomolecule is selected from cytokines, growth factors, cytokine receptors, extracellular matrix, transcription factors, secreted polypeptides and other molecules, and growth factor receptors, or fragments thereof. In some embodiments, the charged cell mimic comprises a fibroblast growth factor (bFGF), an acidic fibroblast growth factor (aFGF), an epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-I), insulin-like growth factor-II (IGF-II), a platelet-derived growth factor-AB (PDGF), a vascular endothelial cell growth factor (VEGF), activin-A, a bone morphogenic protein (BMP), a chemokine, a morphogen, a neutralizing antibody, a heregulin, an interferon, a macrophage-derived cytokine, an interleukin, an interleukin receptor, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL- 15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, Il-23, IL-24, IL-25, IL-26, IL-28, IL-29, IL- 30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, tumor necrosis factor, TNFα, TNFβ, TNFR1, TNFR2, IFAR1, IFAR2, TGFR1, TGFR2, FGF, granulocyte macrophage colony- stimulating factor, chemokines (e.g. CCL1, CCL2, CCL3, CCL, CCL5, and CXCL8), CD27 ligand (CD27L), CD40L, CD137L, TNF-related apoptosis-inducing ligand (TRAIL), TNF-related activation-induced cytokine (TRANCE), TNF-related weak inducer of apoptosis (TWEAK), B cell activating factor (BAFF), LIGHT (homologous to lymphotoxin, exhibits inducible expression andcompetes with herpes simplex virus glycoprotein D for binding to herpesvirus entry mediator, a receptor expressed on T lymphocytes), TNF-like cytokine 1A (TL1A), glucocorticoid-induced TNF receptor-related protein ligand (GITRL), transforming growth factor α (TGF-α), TGF-β, vascular endothelial growth factor (VEGF), nerve growth factor (NGF), macrophage colony- stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN- α, IFN-β, IFN-γ, or any combination thereof.

[0212] In some embodiments, the charged cell mimic contains growth factor, cytokines or hormone precursors that must be processed by a protease to release the active growth factor. In some embodiments the corresponding proteases capable of producing the active growth factor may be added to the growth media, naturally secreted by the target cells or included in the composition of the particles.

[0213] In some embodiments, the biomolecules are attached to the surface of the charged cell mimic. In some embodiments, the biomolecules are in the matrix of the particle itself. Functionalization of a charged cell mimic with a cell surface molecule can also occur through a linker, such as by a streptavidin / biotin conjugate, a biotin / streptavidin conjugate, a streptavidin / biotin / streptavidin conjugate, and / or a biotin / streptavidin / biotin conjugate. For instance, when the charged cell mimic comprises acrylamide, a streptavidin-biotin linkage can be exploited to attach particular biomolecules to the surface of the charged cell mimic. Of course, other known binding / linkage methods can be used without departing from the spirit of the present disclosure.

[0214] In some embodiments, the charged cell mimic is engineered to degrade to provide such biomolecule to the immune cell. The rate of degradation can be modulated to provide slow degradation of the charged cell mimic and thus slow release of the biomolecule to the immune cell. In some embodiments, the biomolecules are attached to both the surface of the charged cell mimic and in the matrix of the charged cell mimic. In some embodiments, the biomolecules on the surface and in the matrix of the charged cell mimic are the same. In some embodiments, the biomolecules on the surface and in the matrix of the charged cell mimic are different.

[0215] In some embodiments, the charged cell mimic comprises enhanced porosity. Compared to non-porous particles, the alteration of pore size distribution allows more surface area per unit hydrogel particle or more surface area per unit volume for advanced cell therapy. The porosity of the porous particle may be controlled by adjusting manufacturing parameters. For instance, theporosity may be controlled through the use of a porogen. The generation of pores offers a number of advantages over nonporous structures. This includes enhanced nutrient transport and higher surface to area to volume ratio. This 3-dimensional scaffold mimics a bioreactor. This bioreactor is achieved by allowing the porous hydrogels to absorb water, maintain an optimal ion nutrient gradient, and maintain an optimal osmotic pressure which favors cellular growth and cell activation. The creation and use of polymer particles with enhanced porosity is described in PCT / US2023 / 077961, which is hereby incorporate by reference in its entirety for all purposes.

[0216] In some embodiments, the cells can support culturing or proliferation based on proximity of a charged cell mimic to a cell of interest. In one example, the charged cell mimic can be conjugated to the cell of interest, whether via direct or indirect conjugation. In another example, the charged cell mimic can be proximal to but not in contact with the cell of interest. The charged cell mimic and the cell of interest can be separated by less than 1 nm, less than 1 micron, less than 1 millimeter, or any appropriate separation distance by which the activation event can still occur.

[0217] Culturing or proliferation may be distant from an area in which the cell of interest is located (i.e., culturing or proliferation can occur remotely). The distance can be at least 1 millimeter, at least 1 centimeter, at least 1 meter, etc. For example, the charged cell mimic may be introduced intramuscularly or intravenously, and the action is in a lymph node or distant immune organ or other target organ. Alternatively, the charged cell mimic may be introduced on one side of a membrane and the action maybe on another side of a membrane (for e.g., via a semi-permeable membrane).

[0218] In some embodiments, the immune cell is contacted with the charged cell mimic in a culturing media. In some embodiments, the media is substantially isotonic as compared to the cells being cultured. In some embodiments, the base medium comprises salts, essential amino acids, a carbon source that can be metabolized by the target cells, and human serum. In some embodiments, for instance when the target cell is a T cell, the base medium comprises cytokines such as IL-2, IL-7, and IL-15. All these ingredients are supplied in an amount that will support respective target cells. In some embodiments, the immune cell is contacted with a defined culture media comprising human serum (hS), and a charged cell mimic described herein, and wherein the composition is essentially free of cells.

[0219] In some embodiments, the disclosure provides a cell culture composition comprising a charged cell mimic, as described herein, and at least one immune cell. In embodiments, the cellculture composition may comprise a hydrogel charged cell mimic comprising a matrix comprising a polymerized monomer, said matrix comprising a plurality of micropores and a plurality of macropores and one or more immunostimulatory biomolecules, and at least one immune cell. The one or more immunostimulatory biomolecules may be selected from the group consisting of an anti-CD3 antibody or antigen-binding fragment thereof, an anti-CD28 antibody or antigen-binding fragment thereof, an anti-CD19 antibody or antigen-binding fragment, an anti-41BBL antibody or antigen-binding fragment, an anti-OX40L antibody or antigen-binding fragment, an anti-CD2 antibody or antigen-binding fragment, an anti-CD335 antibody or antigen-binding fragment, an anti-CD16 antibody or antigen-binding fragment, an anti-CD56 antibody or antigen-binding fragment, an anti-CD20 antibody or antigen-binding fragment, an anti-CD80 antibody or antigen- binding fragment, an anti-CD86 antibody or antigen-binding fragment, an anti-CD69 antibody or antigen-binding fragment, an anti-CD154 antibody or antigen-binding fragment, an anti-CD137 antibody or antigen-binding fragment, an IgM antibody or antigen-binding fragment, an IgG antibody or antigen-binding fragment, an IgE antibody or antigen-binding fragment, an IgA antibody or antigen-binding fragment, an IgD antibody or antigen-binding fragment, and / or toll- like receptors. In some embodiments, the cells and the charged cell mimics are cultured in media comprising synthetic media supplements and are serum-free.

[0220] In some embodiments, the disclosure provides a cell culture composition comprising an immune cell, a defined culture media comprising human serum (hS), and a charged cell mimic as described herein. In some embodiments, the disclosure provides a cell culture composition comprising a natural killer cell, a defined culture media comprising human serum (hS), and a charged cell mimic as described herein comprising one or more of an interleukin and / or a member of the tumor necrosis factor superfamily. In some embodiments, the disclosure provides a cell culture composition comprising a natural killer cell, a defined culture media comprising human serum (hS), and a charged cell mimic as described herein comprising one or more of IL-15, IL-21, CD137L, and / or CD137. In some embodiments, the disclosure provides a cell culture composition comprising a natural killer cell, a defined culture media comprising human serum (hS), and different charged cell mimics as described herein comprising one or more of IL-15, IL-21, CD137L, and / or CD137. In some embodiments, the disclosure provides a cell culture composition comprising a B cell, a defined culture media comprising human serum (hS), and n charged cell mimic as described herein comprising an antibody or antigen-binding fragment thereof thatspecifically binds to CD19. In some embodiments, the disclosure provides a cell culture composition comprising a T cell, a defined culture media comprising human serum (hS), and a synthetic charged cell mimic as described herein. In some embodiments, the disclosure provides a cell culture composition comprising a T cell, a defined culture media comprising human serum (hS), and a charged cell mimic as described herein comprising one or more antibodies or antigen- binding fragments thereof that specifically bind CD3 and one or more antibodies or antigen- binding fragments thereof that specifically bind CD28.

[0221] In some embodiments, the charged cell mimics form a single monolayer in the cell culture. In some embodiments, the charged cell mimics form a multi-layer support in the cell culture.

[0222] In some embodiments, the cell culture comprises a single type of charged cell mimic. In some embodiments, the cell culture comprises a combination of different types of charged cell mimics.

[0223] In some embodiments, the cell culture comprises between about 1 x 105and about 1 x 108charged cell mimics per mL of cell culture. In some embodiments, the cell culture comprises about 1 x 105, about 1 x 106, about 1 x 107, or about 1 x 108charged cell mimics per mL of cell culture. In some embodiments, the cell culture comprises at least about 1 x 101charged cell mimics per mL of cell culture, e.g., at least about 1 x 101, at least about 1 x 102, at least about 1 x 103, at least about 1 x 104, at least about 1 x 105, at least about 1 x 106, at least about 1 x 107, at least about 1 x 108, at least about 1 x 109, at least about 1 x 1010, at least about 1 x 1011, at least about 1 x 1012, at least about 1 x 1013, at least about 1 x 1014, at least about 1 x 1015, at least about 1 x 1016, at least about 1 x 1017, at least about 1 x 1018, at least about 1 x 1019, at least about 1 x 1020, or more. In some embodiments, the cell culture comprises from about 1 x 105to about 1 x 108charged cell mimics per mL of cell culture (e.g., 1 x 105, 2 x 105, 3 x 105, 4 x 105, 5 x 105, 6 x 105, 7 x 105, 8 x 105, 9 x 105, 1 x 106, 2 x 106, 3 x 106, 4 x 106, 5 x 106, 6 x 106, 7 x 106, 8 x 106, 9 x 106, 1 x 107, 2 x 107, 3 x 107, 4 x 107, 5 x 107, 6 x 107, 7 x 107, 8 x 107, 9 x 107, including all values and subranges therein). In some embodiments, the cell culture comprises about 1 x 105, about 1 x 106, about 1 x 107, or about 1 x 108charged cell mimics per mL of cell culture. In some embodiments, the cell culture comprises a similar concentration of charged cell mimics as APC cells used in traditional cell culturing methods.

[0224] In some embodiments, the charged cell mimics of the present disclosure are applied to the cell culture at a dilution of about 1:1 to about 1:1000. In some embodiments, the charged cellmimics are applied to the cell culture at a dilution of about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, about 1:100, about 1:200, about 1:300, about 1:400, about 1:500, about 1:600, about 1:700, about 1:800, about 1:900, or about 1:1000.

[0225] In some embodiments, the charged cell mimics of the present disclosure and the immune cells are cultured for at least about 30 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, about 6 hours, about 6.5 hours, about 7 hours, about 7.5 hours, about 8 hours, about 8.5 hours, about 9 hours, about 9.5 hours, about 10 hours, about 10.5 hours, about 11 hours, about 11.5 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, 2, days, 36 hours, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 13 days, 14 days, or more, including all ranges and subranges therebetween.

[0226] In some embodiments, culturing the target cell with a charged cell mimic of the present disclosure increases target cell proliferation by about 1% to about 10000% compared to culturing of the target cell without the charged cell mimic. In some embodiments, target cell proliferation is increased by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 200%, about 300%, about400%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1000%, about 2000%, about 3000%, about 4000%, about 5000%, about 6000%, about 7000%, about 8000%, about 9000%, or about 10000%, including all ranges and subranges therebetween, as compared to culturing of the target cell without the charged cell mimic. In some embodiments, cell proliferation can be at least 100,000x the initial cell population.

[0227] In some embodiments, culturing the immune cell with a charged cell mimic of the present disclosure increases immune cell activation by about 1% to about 10000% compared to culturing of the immune cell without the charged cell mimic. In some embodiments, immune cell proliferation is increased by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1000%, about 2000%, about 3000%, about 4000%, about 5000%, about 6000%, about 7000%, about 8000%, about 9000%, or about 10000% compared to culturing of the immune cell without the charged cell mimic. In some embodiments, cell activation can be at least 100,000x the initial cell population. Charged Cell Mimics as Inducers of T cell Activation

[0228] In some embodiments, a population of charged cell mimics described herein are functionalized with a biomolecule for activation and / or expansion of T cells. Generally, T cell activation is triggered by a peptide antigen bound to a major histocompatibility complex (MHC) molecule on the surface of an antigen presenting cell (APC), a T cell receptor / CD3 complex (TCR / CD3). While this is the primary signal in T cell activation, other receptor-ligand interactions between APC and T cells are also required for full activation. For example, TCR stimulation in the absence of other molecular interactions can induce an anergic state such that these cells cannot respond to a complete activation signal upon restimulation. Thus, optimal functionality may be conferred through the use of a second signaling molecule, such as a membrane bound protein or APC secretion product. For these membrane-bound proteins, such second interactions are usually adhesive in nature and enhance the contact between the two cells. Other signaling molecules (e.g., further activation signaling from APC to T cells) may also be relevant. For example, CD28 is a surface glycoprotein that is present in 80% of peripheral T cells in humans and is present in both quiescent and activated T cells. CD28 binds to B7-1 (CD80) or B7-2 (CD86) and is one of the most potent of the known costimulatory molecules. Combined with TCR engagement, CD28 ligation on T cells induces the production of interleukin-2 (IL-2). Secreted IL-2 is an important factor for ex vivo T cell expansion.

[0229] In some embodiments, the biomolecule that can stimulate T cell expansion and / or activation is a polypeptide or fragment thereof. In some embodiments, the polypeptide or fragment thereof that can stimulate T cell expansion and / or activation is a peptide antigen. In some embodiments, the biomolecule that can stimulate T cell expansion and / or activation is a component of an MHC molecule. In some embodiments, the biomolecule that can stimulate T cell expansion and / or activation is a component of a T cell receptor / CD3 complex. In some embodiments, thebiomolecule that can stimulate T cell expansion and / or activation is an antibody that specifically binds a component of a T cell receptor / CD3 complex. In some embodiments, the charged cell mimic of the present disclosure comprises an antibody or antigen-binding fragment therefore that specifically binds to CD3.

[0230] In some embodiments, the charged cell mimic of the present disclosure comprises one or more T cell activation molecules and one or more T cell costimulatory molecules. In some embodiments, the charged cell mimic of the present disclosure comprises one or more antibodies or antigen-binding fragments thereof that specifically bind T cell activation molecules and one or more T cell costimulatory molecules. In some embodiments, the charged cell mimic of the present disclosure comprises a T cell activation molecule of CD3 and a T cell costimulatory molecule selected from CD28, ICOS, CD27, CD40, CD40L, CD137L, and CD137 (or antibodies targeting said activation / costimulatory molecules. In some embodiments, the charged cell mimic of the present disclosure comprises one or more antibodies or antigen-binding fragments thereof that specifically bind to CD3 and one or more antibodies or antigen-binding fragments thereof that specifically bind to CD28, ICOS, CD27, CD40, CD40L, CD137L, CD137, the like, or combinations thereof.

[0231] In some embodiments, the receptor molecule on the charged cell mimic would be a MHC- tetramer (MHC class I or class II) and the CD3 and CD28 molecules would be encapsulated within and / or attached to the surface of the charged cell mimic such that the primary recognition would be dictated by antigen-specificity by the MHC tetramer with the CD3, CD28 stimulation of such targeted cells occurring later with the consequence that only Ag-specific cells are co-stimulated allowing for lower magnitude of Cytokine Release Syndrome.

[0232] In some embodiments, the T cell activation molecule may be an anti-CD3 antibody or an antigen-binding fragment thereof, an anti-macrophage scavenger receptor (MSR1) antibody or an antigen-binding fragment thereof, an anti-T cell receptor (TCR) antibody or an antigen-binding fragment thereof, an anti-CD2 antibody or an antibody thereof, antigen-binding fragments, anti- CD47 antibodies or antigen-binding fragments thereof, major histocompatibility complex (MHC) molecules loaded with MHC peptides or multimers thereof, and MHC-immunoglobulin (Ig) conjugates or multimers thereof, or combinations thereof.

[0233] In some embodiments, the charged cell mimic comprises one or more T cell costimulatory molecules including, but not limited to, CD28, 4.1BB (CD137), OX40 (CD134), CD27(TNFRSF7), GITR (CD357), CD30 (TNFRSF8), HVEM (CD270), LTβR (TNFRSF3), DR3 (TNFRSF25), ICOS (CD278), PD1 (CD279), CD226 (DNAM1), CRTAM (CD355), TIM1 (HAVCR1, KIM1), CD2 (LFA2, OX34), SLAM (CD150, SLAMF1), 2B4 (CD244, SLAMF4), Ly108 (NTBA, CD352, SLAMF6), CD84 (SLAMF5), Ly9 (CD229, SLAMF3) and / or CRACC (CD319, BLAME). In some embodiments, the charged cell mimic comprises one or more antibodies or antigen-binding fragments thereof that specifically bind to CD28, 4.1BB (CD137), OX40 (CD134), CD27 (TNFRSF7), GITR (CD357), CD30 (TNFRSF8), HVEM (CD270), LTβR (TNFRSF3), DR3 (TNFRSF25), ICOS (CD278), PD1 (CD279), CD226 (DNAM1), CRTAM (CD355), TIM1 (HAVCR1, KIM1), CD2 (LFA2, OX34), SLAM (CD150, SLAMF1), 2B4 (CD244, SLAMF4), Ly108 (NTBA, CD352, SLAMF6), CD84 (SLAMF5), Ly9 (CD229, SLAMF3) and / or CRACC (CD319, BLAME). In some embodiments, the charged cell mimic of the present disclosure comprises an anti-CD28 antibody or antigen-binding fragment thereof.

[0234] In some embodiments, the charged cell mimic of the present disclosure comprises one or more polypeptides that promote expansion of a particular T cell subtype while simultaneously inhibiting the development of the other subset. In some embodiments, the polypeptide that promotes expansion of a particular T cell subtype is a cytokine. In some embodiments, the cytokine is an interleukin, interferon, lymphotoxin, a member of the TNF superfamily, or an antibody or antigen-binding fragment thereof that binds to one of the foregoing. In some embodiments, the cytokine is selected from a list including, but not limited to, IL-1, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21, interferon γ, IFN alpha, IFN beta, lymphotoxin α, TNFα, TNFβ or a combination thereof.

[0235] In some embodiments, the charged cell mimic of the present disclosure comprises one or more T cell homeostasis factors. In some embodiments, the T cell homeostasis factor is selected from a list including, but not limited to, transforming growth factor β (TGF-β), or agonists thereof, mimetics thereof, variants thereof, functional fragments thereof, or a combination thereof. In some embodiments, the T cell homeostasis factor is IL-2, an agonist, mimetic, variant, or functional fragment or a combination thereof.

[0236] In some embodiments, the charged cell mimic comprises a CD3 and a CD28 biomolecule or fragment thereof. In some embodiments, the charged cell mimic comprises an anti-CD3 and an anti-CD28 antibody or antigen-binding fragment thereof.

[0237] In some embodiments, the T cell stimulated and / or expanded and or depleted / removed by the charged cell mimic of the present disclosure is selected from the nonlimiting group consisting of natural killer (NK) cells, CD3 + T cells, CD4 + T cells, CD8 + T cells, and regulatory T cells (Treg), or a combination thereof. In some embodiments, the T cell is a helper T cell. In some embodiments, the T cell is a cytotoxic T cell. In some embodiments, the T cell is a Th1 or a Th2 cell. In some embodiments, the T cell is a recombinant T cell. In some embodiments, the recombinant T cell is a CAR T cell. In embodiments, T cells depleted / removed by the charged cell mimics of the present disclosure are CD25+ regulatory T cells and / or CD4+ T cells.

[0238] In some embodiments, the T cell is freshly collected from a subject. In some embodiments, the T cells are derived from any appropriate source within an animal. The animals from which the T cells are harvested may be vertebrate or invertebrate, mammalian or non-mammalian, human or non-human. Examples of animal sources include, but are not limited to, primates, rodents, canines, felines, equines, bovines, and porcines. In some embodiments, the T cell is a cultured cell line. In some embodiments, the T cell is an established cell line. In some embodiments, the T cell is cultured from a preserved or frozen sample. In some embodiments, the charged cell mimic of the present disclosure induces the expansion, proliferation, and / or activation of any appropriate T cell. In some embodiments, the T cell does not expand, proliferate, and / or activate in culture without the charged cell mimic. In some embodiments, the T cells, or subsets thereof are eliminated as a consequence of incubating with the charged cell mimic.

[0239] In some embodiments, the disclosure provides a cell culture composition comprising a T cell, a defined culture media comprising human serum (hS), and a charged cell mimic comprising one or more antibodies or antigen-binding fragments thereof that specifically bind CD3 and one or more antibodies or antigen-binding fragments thereof that specifically bind CD28. EXAMPLES

[0240] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby. Example 1: Synthesis and Zeta Potential Characterization of Charged Hydrogel Particles

[0241] Charged hydrogel particles were formulated using microfluidic droplet generation. Photomasks for UV lithography were sourced from CADart Services Inc. and were designed using AutoCad (AutoDesk, Inc.). SU-8 photo resist (Microchem, Inc.) was photo crosslinked on 4" silicon wafers using a collimated UV light source (OAI, Inc.) to create masters for microfluidic device fabrication. PDMS (polydimethylsiloxane, Sigma Aldrich, Inc.) was prepared and formed using standard published methods for soft lithography and microfluidic device fabrication (See, McDonald JC, et al., 2000, Electrophoresis 21 :27-40).

[0242] Droplets were formed using flow-focusing geometry where two oil channels focus a central stream of aqueous monomer solution to break off droplets in a water-in-oil emulsion. A fluorocarbon-oil (Novec 75003M, Inc.) was used as the outer, continuous phase liquid for droplet formation. To stabilize droplets before polymerization, a surfactant was added at 0.5% w / w to the oil phase (ammonium carboxylate salt of Krytox 157 FSH, Dupont). To make the polyacrylamide gel particle, a central phase of an aqueous monomer solution containing acrylamide monomer (Compound 3 at 3-18% w / v), a charged monomer (Compound 1 or Compound 2 at 0.25-2.5% w / v), a cross-linker (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 in order to trigger hydrogel particle polymerization after droplet formation.

[0243] Droplets were formed at an average rate of 5 kHz and were collected in the fluorocarbon oil phase. Polymerization was completed at 50 °C for 30 minutes, and the resulting hydrogel particles were washed from the oil into an aqueous solution.

[0244] The particles were suspended in 1X PBS. Zeta potential measurements were performed on an Anton-Paar Litesizer 500 at 25 °C.

[0245] As shown in FIG. 1A, hydrogel particles containing Compound 1 (positively charged monomer) had an average zeta potential (from duplicate measurements) of about +10.9 mV. The hydrogel particles containing Compound 1 were prepared from an aqueous monomer solution containing acrylamide monomer at 9% w / v and Compound 1 at 0.264% w / v. As shown in FIG. 1B, hydrogel particles containing Compound 2 (negatively charged monomer) had an average zetapotential (from duplicate measurements) of about -31.9 mM. The hydrogel particles containing Compound 2 were prepared from an aqueous monomer solution containing acrylamide monomer at 9% w / v and Compound 2 at 0.276% w / v. Example 2: Synthesis and Zeta Potential Characterization of Charged Hydrogel Cell Mimics

[0246] Synthesis: Charged hydrogel cell mimics were synthesized by adding charged co- monomers into acrylamide / bis-acrylamide hydrogel particles synthesized with microfluidic emulsion polymerization. First, an aqueous solution was prepared as a precursor to the particles. Monomer (acrylamide), crosslinker (bis-acrylamide, 0.03M), radical initiator (ammonium persulfate, 0.2 w / v%) and pH buffer(Tris-HCl, 100mM) were mixed in DI water. The charged co- monomers, mono-2-(Methacryloyloxy)ethyl succinate (MMES, Sigma Aldrich, Inc.) or (3- Acrylamidopropyl)trimethylammonium chloride (APTAC, Sigma Aldrich, Inc.) were added afterwards. The final molar concentration for acrylamide, MMES and APTAC is summarized in Table 3 below. Table 3. Molar concentration of charged cell mimic components

[0247] Total molar concentration of monomers was kept constant and hydrogel particles with different levels of surface charge were created by the molar fraction of charged co-monomers. Specifically, negatively charged cell mimics were synthesized with approximately 3.2% (N1), 8.1% (N2), 20.2% (N3) molar of MMES. Positively charged cell mimics were similarly synthesized with approximately 3.2% (P1), 8.1% (P2), 20.2% (P3), 40.3% (P4) molar of APTAC.

[0248] Then, the microfluidic device used for emulsion polymerization was prepared as follows: Photomasks for UV lithography were sourced from CADart Services Inc. and were designed using AutoCad (AutoDesk, Inc.). SU-8 photo resist (Microchem, Inc.) was photo crosslinked on 4" silicon wafers using a collimated UV light source (OAI, Inc.) to create masters for microfluidic device fabrication. PDMS (polydimethylsiloxane, Sylgard 184, Dow Corning) was prepared and formed using standard published methods for soft lithography and microfluidic device fabrication (McDonald JC, et al., 2000, Electrophoresis 21:27-40).

[0249] Finally, to form monodisperse hydrogel particles, the aforementioned aqueous solution was dispersed into a water-in-oil emulsion with microfluidic flow-focusing. A continuous oil phase, consisting of a fluorocarbon-oil (Novec 75003M, Inc.) and 2% w / w of fluorosurfactant (Krytox 157 FSH, Dupont) was pressurized into two oil channels, focusing a central stream of the aqueous phase, breaking off droplets. The droplet size (diameter) was kept at a constant 20 μm. After collecting the emulsion for 3 hours in a centrifuge tube, the aqueous phase was then polymerized by sparging with argon for 20 minutes, followed by incubation at 75°C for 20 minutes. The resulting hydrogel particles were then washed into a suspension in 1X PBS with a demulsifier (PFO, 1H,1H,2H,2H-Perfluoro-1-octanol).

[0250] Zeta potential of charged hydrogel cell mimics: The particle suspensions were diluted to between 1e6particles / mL and 3e6particles / mL, buffer exchanged to desired buffer condition, then injected into zeta potential cuvettes (Omega cuvette, Anton-Paar). Then, zeta potential was measured in triplicates with electrophoretic light scattering (Litesizer 500, Anton-Paar).

[0251] First, the zeta potential of charged hydrogel cell mimics was measured in 1X PBS and DI water to demonstrate the effect of ionic strength on the surface charge. Particle suspensions in 1X PBS were washed twice with DI water to yield the buffer exchanged sample. This lowered ionic strength and reduced the conductivity of the suspension from around 15mS / cm to 0.2-0.6mS / cm. Lowered ionic strength is known to increase the thickness of surface electric double layers and zeta potential, which was observed for both positive and negatively charged hydrogel particles. FIG.2 shows increased zeta potential of both positively and negatively charged hydrogel particles in response to decreasing ionic strength. In addition, FIG. 3 shows the swelling ratio of the particles increased due to increased electrostatic potential in the polymer network, with FIG. 3A showing exemplary positively charged hydrogel particle P4 in 1X PBS, and FIG.3B showing the exemplary positively charged hydrogel particle P4 in DI water. These effects showcased stimuli- responsiveness towards ionic strength change with multiple readouts, which can be utilized to monitor ionic strength in various cell studies, such as cell culture or flow cytometry.

[0252] Next, the particles were exchanged into a series of pH buffers. NIST traceable pH standards with pH = 4, 7 and 10 (pH=4 KHP, 0.05M, VWR, BDH5018; pH=7 Phosphate, 0.1M, VWR, BDH5046; pH=10 Carb-bicarb, 0.1M, VWR, BDH 5072) were diluted to the same molar concentration at 0.05M, with similar conductivity at ~6 mS / cm. Particle suspensions in 1X PBS were washed once with the diluted buffer. Zeta potential is known to be inversely responsive to pH changes. For example, a particle that is negatively charged at neutral has an isoelectric point below 7 and vice versa.

[0253] FIG. 4 shows comparative zeta potential measurements of an uncharged control hydrogel particle (C) and negatively charged hydrogel particles (N1, N2, and N3) at pH 4.0, pH 7.0, and pH 10.0. The negatively charged cell mimics in this study showed a clear pH-responsiveness in the range tested, with zeta potential close to neutral at pH = 4, suggesting a common isoelectric point close to 4.

[0254] FIG. 5 shows comparative zeta potential measurements of an uncharged control hydrogel particle (C) and positively charged hydrogel particles (P1, P2, P3, and P4) at pH 4.0, pH 7.0, andpH 10.0. Contrarily, the positively charged cell mimics were less responsive in the range tested and remained significantly charged at pH = 10, indicating an even higher isoelectric point. Similar to the ionic strength study, the pH-responsiveness of these particles can be used to monitor pH changes in a culture media or flow cytometry buffer. The results for all tested samples and buffer conditions are summarized in Table 4 below. Table 4. Zeta potential (mV) of charged cell mimics in different pH environments

[0255] Lyophilization and reconstitution of charged hydrogel cell mimics: Charged hydrogel cell mimics were exchanged into a lyophilization buffer containing 1wt% BSA, 10wt% trehalose dihydrate, 0.05% sodium azide, 1X PBS, and lyophilized overnight. The particles were then reconstituted with 1X PBS and buffer exchanged into staining buffer (BD Pharmingen). A Cytek Aurora flow cytometer was then used to evaluate the scatter profile and aggregation of these particles before and after lyophilization and reconstitution.

[0256] FIG. 6 shows flow cytometry plots of uncharged hydrogel particles (control), negatively charged hydrogel particles, and positively charged hydrogel particles before lyophilization. FIG. 7 shows flow cytometry plots of uncharged hydrogel particles (control), negatively charged hydrogel particles, and positively charged hydrogel particles after lyophilization. Table 5 belowshows doublet percentage flow cytometry measurements of control, negatively charged, and positively charged hydrogel particles. Table 5. Flow cytometric measurements of charged hydrogel particles

[0257] Both negatively charged and control cell mimics showed very low doublets, but positively charged cell mimics were significantly more aggregated after lyophilization and reconstitution. In addition, P3 and P4 had significant changes in scatter profile after reconstitution.

[0258] Together, these data show pH-responsiveness of the exemplary charged cell mimics. Additionally, the capacity of the cell mimics to undergo lyophilization and reconstitution is shown, especially the negatively charged particles. Without dedication to any particular theory, these changes indicate that a cell mimic containing cationic monomer can be modified to include ananionic monomer to reduce aggregation, thereby improving the counting and staining performance when the cell mimic is used as counting or biomarker reference. Example 3: Validation of an ELS instrument

[0259] The charged hydrogel particles as described in Example 1 or Example 2 are used to validate an ELS instrument prior to measuring zeta potential of a sample of cells. The charged hydrogel particles are suspended in the same buffer conditions as described in Example 1, such that the zeta potential measurement on the ELS instrument is expected to be substantially similar to the zeta potential measurements reported in Example 1. The sample of charged hydrogel particles containing Compound 1 is inserted into the ELS instrument and the zeta potential calculated by the instrument is determined. If the average zeta potential (e.g., based on duplicate measurements) is about ±5% of the zeta potential reported in Example 1 (i.e., +10.9 mV), the ELS instrument is validated. For further validation, the sample of charged hydrogel particles containing Compound 2 is inserted into the ELS instrument and the zeta potential calculated by the instrument is determined. If the average zeta potential (e.g., based on duplicate measurements) is about ±5% of the zeta potential reported in Example 1 (i.e., -31.9 mV), further confirmation is provided that the ELS instrument is providing robust zeta potential measurements.

[0260] All, documents, patents, patent applications, publications, product descriptions, and protocols which are cited throughout this application are incorporated herein by reference in their entireties for all purposes.

[0261] The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art the best way known to the inventors to make and use the invention. Modifications and variation of the above-described embodiments of the invention are possible without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. It is therefore understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described. NUMBERED EMBODIMENTS OF THE INVENTION

[0126] Notwithstanding the appended claims, the disclosure sets forth the following numbered embodiments:

[0262] Embodiment I-1. A method for validating an instrument for charge analysis of biological cells, said method comprising the steps of: inserting into the instrument a population of polymer particle cell mimics comprising a predetermined zeta potential that is substantially similar to the zeta potential of corresponding biological cells, measuring electrophoretic mobility (EPM) of the population of polymer particle cell mimics using the instrument, and determining any difference between synthetic zeta potential calculated from the EPM of the population of polymer particle mimics and the predetermined zeta potential, thereby validating the instrument for analysis of the biological cells.

[0263] Embodiment I-2. The method of embodiment I-1, wherein the instrument is an electrophoretic light scattering (ELS) instrument.

[0264] Embodiment I-3. The method of embodiment I-1 or I-2, further comprising inserting a biological sample comprising a population of the corresponding biological cells into the instrument and measuring an EPM of the biological sample.

[0265] Embodiment I-4. The method of embodiment I-3, further comprising determining a biological zeta potential for the biological sample based on the measured EPM of the biological sample, said biological zeta potential being adjusted based on the difference between the synthetic zeta potential and the predetermined zeta potential determined for the polymer particle cell mimics.

[0266] Embodiment I-5. The method of embodiment I-3 or I-4, wherein the population of polymer particle cell mimics that is inserted into the instrument is suspended in a first buffer, wherein the biological sample is suspended in a second buffer, and wherein the first buffer and the second buffer are substantially the same.

[0267] Embodiment I-6. The method of any one of embodiments I-3 to I-5, wherein the population of polymer particle cell mimics that is inserted into the instrument is at substantially the same concentration as the population of corresponding biological cells.

[0268] Embodiment I-7. The method of any one of embodiments I-1 to I-6, wherein the polymer particle cell mimics in the population are of substantially the same size as the corresponding biological cells.

[0269] Embodiment I-8. The method of any one of embodiments I-1 to I-7, wherein the polymer particle cell mimics in the population have a substantially similar optical property to the corresponding biological cells.

[0270] Embodiment I-9. The method of embodiment I-8, wherein the substantially similar optical property is selected from forward side scatter, side scatter, autofluorescence, and a combination thereof.

[0271] Embodiment I-10. A method for measuring a zeta potential of a population of biological particles, comprising (i) inserting into an ELS instrument a population of polymer particle cell mimics comprising a predetermined zeta potential, (ii) measuring electrophoretic mobility (EPM) of the population of polymer particle cell mimics using the ELS instrument, (iii) repeating (i)-(ii) for a panel of populations of polymer particle cell mimics, each population comprising a distinct zeta potential, (iv) generating a calibration curve from the EPM and predetermined zeta potential for the panel, (v) measuring EPM of the population of biological particles, and (vi) determining the zeta potential of the population of biological particles based on a comparison of the EPM measurement to the calibration curve.

[0272] Embodiment I-11. The method of embodiment I-10, wherein the polymer particle cell mimics in the populations of steps (i)-(iii) are substantially the same size as the biological particles.

[0273] Embodiment I-12. The method of any one of embodiments I-1 to I-11, wherein the polymer particle cell mimics have a diameter of about 10 μm to about 100 μm.

[0274] Embodiment I-13. The method of any one of embodiments I-1 to I-11, wherein the polymer particle cell mimics have a diameter of about 1 μm to about 10 μm.

[0275] Embodiment I-14. The method of any one of embodiments I-1 to I-11, wherein the polymer particle cell mimics have a diameter of about 0.1 μm to about 1 μm.

[0276] Embodiment I-15. The method of any one of embodiments I-1 to I-14, wherein the polymer particle cell mimics comprise a net positive surface charge at a pH of about 6 to about 8.

[0277] Embodiment I-16. The method of any one of embodiments I-1 to I-15, wherein the polymer particle cell mimics comprise a predetermined zeta potential of about +10 mV to about +100mV.

[0278] Embodiment I-17. The method of any one of embodiments I-1 to I-14, wherein the polymer particle cell mimics comprise a net negative surface charge at a pH of about 6 to about 8.

[0279] Embodiment I-18. The method of any one of embodiments I-1 to I-14 and I-17, wherein the polymer particle cell mimics comprise a predetermined zeta potential of about -10 mV to about -100 mV.

[0280] Embodiment I-19. The method of any one of embodiments I-1 to I-14, wherein the polymer particle cell mimics comprise a neutral surface charge at a pH of about 6 to about 8.

[0281] Embodiment I-20. The method of any one of embodiments I-1 to I-14, wherein the polymer particle cell mimics comprise a predetermined zeta potential of less than about -10 mV or greater than about +10 mV.

[0282] Embodiment I-21. The method of any one of embodiments I-10 to I-20, wherein the population of biological particles comprises cells.

[0283] Embodiment I-22. The method of embodiment I-21, wherein the population of biological particles comprises pre-apoptotic cells.

[0284] Embodiment I-23. The method of embodiment I-22, wherein the pre-apoptotic cells comprise surface-exposed phosphatidylserine.

[0285] Embodiment I-24. The method of any one of embodiments I-10 to I-20, wherein the population of biological particles comprises extracellular vesicles.

[0286] Embodiment I-25. The method of any one of embodiments I-1 to I-24, wherein the biological particles or the biological cells comprise a surface label.

[0287] Embodiment I-26. The method of any one of embodiments I-1 to I-25, wherein the polymer particle cell mimics comprise a surface label.

[0288] Embodiment I-27. The method of embodiment I-25 or I-26, wherein the surface label comprises a biomarker.

[0289] Embodiment I-28. The method of embodiment I-27, wherein the biomarker is a nucleic acid, protein, peptide, carbohydrate, or combination thereof.

[0290] Embodiment I-29. The method of any one of embodiments I-10 to I-28, wherein the populations of polymer particle cell mimics in the populations of steps (i)-(iii) are at substantially the same concentration as the population of biological particles.

[0291] Embodiment I-30. The method of any one of embodiments I-1 to I-29, wherein the polymer particle cell mimics comprise a neutrally charged monomer and a charged monomer.

[0292] Embodiment I-31. The method of any one of embodiments I-1 to I-29, wherein the polymer particle cell mimics comprise a charged monomer.

[0293] Embodiment I-32. The method of embodiment I-30 or I-31, wherein the charged monomer comprises a positive charge.

[0294] Embodiment I-33. The method of embodiment I-32, wherein the charged monomer comprises (i) a tertiary amine group or salt thereof, or (ii) an aminium or salt thereof.

[0295] Embodiment I-34. The method of embodiment I-32 or I-33, wherein the charged monomer is 3-(methacryloylamino)propyl]trimethylammonium.

[0296] Embodiment I-35. The method of embodiment I-33, wherein the charged monomer comprises a negative charge.

[0297] Embodiment I-36. The method of embodiment I-35, wherein the charged monomer comprises (i) a carboxylate or salt thereof, or (ii) a sulfonate or a salt thereof.

[0298] Embodiment I-37. The method of embodiment I-36, wherein the charged monomer is selected from mono-2-(methacryloyloxy)ethyl succinate andacrylate.

[0299] Embodiment I-38. The method of any one of embodiments I-30 to I-37, wherein the neutrally charged monomer comprises hydroxyethyl methacrylate, ethyl methacrylate, 2- hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N- vinylpyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glycerol methacrylate (GMA), 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, glycerol 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-phenyl acrylamide, N-phenyl methacrylamide, N-benzyl acrylamide, N-benzyl methacrylamide, N,N- dibenzyl acrylamide, N,N-dibenzyl methacrylamide, N-diphenylmethyl acrylamide, N-(4- methylphenyl)methyl acrylamide, N-1-naphthyl acrylamide, N-4-nitrophenyl acrylamide, N-(2-phenylethyl)acrylamide, N-triphenylmethyl acrylamide, N-(4-hydroxyphenyl)acrylamide, N,N- methylphenyl acrylamide, N,N-phenyl phenylethyl acrylamide, N-diphenylmethyl methacrylamide, N-(4-methyl phenyl)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'-phenyl phenylethyl methacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridine, or a combination thereof.

[0300] Embodiment I-39. The method of any one of embodiments I-30 to I-38, comprising a ratio of the neutrally charged monomer and the charged monomer, wherein the ratio is about 100:1 to about 10:1.

[0301] Embodiment I-40. The method of any one of embodiments I-1 to I-39, wherein the ELS instrument is a zeta potential analyzer.

[0302] Embodiment I-41. The method of any one of embodiments I-1 to I-40, wherein the polymer particle cell mimics comprise another property that is substantially similar to the biological cells or biological particles, said other property selected from the group consisting of: volume, FSC, SSC, background fluorescence; fluorescence profile; shared surface marker, and refractive index.

[0303] Embodiment I-42. A charged cell mimic comprising a polymer particle comprising a neutral monomer and a charged monomer, wherein the polymer particle comprises a predetermined zeta potential and wherein the predetermined zeta potential is substantially similar to a zeta potential of a target biological cell.

[0304] Embodiment I-43. The charged cell mimic of embodiment I-42, wherein the target biological cell is an apoptotic cell or a pre-apoptotic cell.

[0305] Embodiment I-44. The charged cell mimic of embodiment I-42, wherein the target biological cell comprises a biomarker.

[0306] Embodiment I-45. The charged cell mimic of embodiment I-44, wherein the polymer particle comprises the biomarker.

[0307] Embodiment I-46. The charged cell mimic of embodiment I-42, comprising an antibody or antigen binding fragment thereof linked to the polymer particle, the antibody or antigen binding fragment thereof selected from the group consisting of: an anti-CD3 antibody or antigen bindingfragment thereof, an anti-CD8 antibody or antigen binding fragment thereof, and an anti-CD28 antibody or antigen binding fragment thereof.

[0308] Embodiment I-47. The charged cell mimic of any one of embodiments I-42 to I-46, wherein the polymer particle is substantially the same size as the target biological cell.

[0309] Embodiment I-48. The charged cell mimic of any one of embodiments I-42 to I-47, wherein the polymer particle has a substantially similar optical property to the target biological cell.

[0310] Embodiment I-49. The charged cell mimic of embodiment I-48, wherein the substantially similar optical property is selected from forward side scatter, side scatter, autofluorescence, and a combination thereof.

[0311] Embodiment I-50. The charged cell mimic of any one of embodiments I-42 to I-49, wherein the polymer particle has a diameter of about 10 μm to about 100 μm.

[0312] Embodiment I-51. The charged cell mimic of any one of embodiment I-50, wherein the polymer particle has a diameter of about 1 μm to about 10 μm.

[0313] Embodiment I-52. The charged cell mimic of any one of embodiments I-42 to I-49, wherein the polymer particle has a diameter of about 0.1 μm to about 1 μm.

[0314] Embodiment I-53. The charged cell mimic of any one of embodiments I-42 to I-52, wherein the polymer particle comprises a net positive surface charge at a pH of about 6 to about 8.

[0315] Embodiment I-54. The charged cell mimic of any one of embodiments I-42 to I-53, wherein the predetermined zeta potential is about +10 mV to about +100mV.

[0316] Embodiment I-55. The charged cell mimic of any one of embodiments I-42 to I-53, wherein the polymer particle comprises a net negative surface charge at a pH of about 6 to about 8.

[0317] Embodiment I-56. The charged cell mimic of any one of embodiments I-42 to I-52 and I- 55, wherein the predetermined zeta potential is about -10 mV to about -100 mV.

[0318] Embodiment I-57. The charged cell mimic of any one of embodiments I-42 to I-52, wherein the polymer particle cell mimics comprise a neutral surface charge at a pH of about 6 to about 8.

[0319] Embodiment I-59. The charged cell mimic of any one of embodiments I-42 to I-57, wherein the target biological cell is pre-apoptotic.

[0320] Embodiment I-60. The charged cell mimic of embodiment I-59, wherein the target biological cell comprises surface-exposed phosphatidylserine.

[0321] Embodiment I-61. The charged cell mimic of embodiment I-44 or I-45, wherein the biomarker is a nucleic acid, protein, peptide, carbohydrate, or combination thereof.

[0322] Embodiment I-62. The charged cell mimic of any one of embodiments I-42 to I-61, wherein the charged monomer comprises a positive charge.

[0323] Embodiment I-63. The charged cell mimic of embodiment I-62, wherein the charged monomer comprises (i) a tertiary amine group or salt thereof, or (ii) an aminium or salt thereof.

[0324] Embodiment I-64. The charged cell mimic of embodiment I-62 or I-63, wherein the charged monomer is selected from 3-(methacryloylamino)propyl]trimethylammonium.

[0325] Embodiment I-65. The charged cell mimic of any one of embodiments I-42 to I-61, wherein the charged monomer comprises a negative charge.

[0326] Embodiment I-66. The charged cell mimic of embodiment I-65, wherein the charged monomer comprises (i) a carboxylate or salt thereof, or (ii) a sulfonate or a salt thereof.

[0327] Embodiment I-67. The charged cell mimic of embodiment I-65 or I-66, wherein the charged monomer is selected from mono-2-(methacryloyloxy)ethyl succinate andacrylate.

[0328] Embodiment I-68. The charged cell mimic of any one of embodiments I-42 to I-67, wherein the neutrally charged monomer comprises hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N- vinylpyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glycerol methacrylate (GMA), 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, glycerol 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-phenyl acrylamide, N-phenyl methacrylamide, N-benzyl acrylamide, N-benzyl methacrylamide, N,N- dibenzyl acrylamide, N,N-dibenzyl methacrylamide, N-diphenylmethyl acrylamide, N-(4- methylphenyl)methyl acrylamide, N-1-naphthyl acrylamide, N-4-nitrophenyl acrylamide, N-(2- phenylethyl)acrylamide, N-triphenylmethyl acrylamide, N-(4-hydroxyphenyl)acrylamide, N,N- methylphenyl acrylamide, N,N-phenyl phenylethyl acrylamide, N-diphenylmethyl methacrylamide, N-(4-methyl phenyl)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'-phenyl phenylethyl methacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridine, or a combination thereof.

[0329] Embodiment I-69. The charged cell mimic of any one of embodiments I-42 to I-68, comprising a ratio of the neutrally charged monomer and the charged monomer, wherein the ratio is about 100:1 to about 10:1.

[0330] Embodiment I-70. The charged cell mimic of any one of embodiments I-42 to I-69, wherein the polymer particle comprises another property that is substantially similar to the target biological cell, said other property selected from the group consisting of: volume, FSC, SSC, background fluorescence; fluorescence profile; shared surface marker, and refractive index.

[0331] Embodiment I-71. A charged cell mimic comprising a polymer particle comprising a neutral monomer, a charged monomer, and an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen binding fragment thereof is selected from the group consisting of an anti-CD3 antibody or fragment thereof, an anti-CD8 antibody or fragment thereof, and an anti- CD28 antibody or fragment thereof, wherein the polymer particle comprises a predetermined zeta potential, and wherein the predetermined zeta potential is substantially similar to a zeta potential of a target biological cell.

[0332] Embodiment I-72. The charged cell mimic of embodiment I-71, wherein the polymer particle comprises a main body, the main body comprising a plurality of macropores.

[0333] Embodiment I-73. A method of inducing an immune cell response, comprising

[0334] contacting an immune cell with the charged cell mimic of any one of embodiments I-42 to I-72, wherein the immune cell response comprises activation and / or expansion of the immune cell.

[0335] Embodiment I-74. A method of inducing an immune cell response, comprising contacting an immune cell with a charged cell mimic, wherein the charged cell mimic comprises a neutral monomer, a charged monomer, and an antibody or an antigen-binding fragment thereof, wherein the polymer particle comprises a predetermined zeta potential, wherein the predetermined zeta potential is substantially similar to a zeta potential of the immune cell, and wherein the immune cell response comprises activation and / or expansion of the immune cell.

[0336] Embodiment I-75. The method of embodiment I-74, wherein the antibody or antigen binding fragment thereof is selected from the group consisting of: an anti-CD3 antibody or fragment thereof, an anti-CD8 antibody or fragment thereof, and an anti-CD28 antibody or fragment thereof. Embodiments 1-91:

[0337] 1. A method for validating an instrument for charge analysis of biological cells, said method comprising the steps of:

[0338] inserting into the instrument a population of hydrogel particle cell mimics comprising a predetermined zeta potential that is substantially similar to the zeta potential of corresponding biological cells, measuring electrophoretic mobility (EPM) of the population of hydrogel particle cell mimics using the instrument, and determining any difference between a synthetic zeta potential calculated from the EPM of the population of hydrogel particle cell mimics and the predetermined zeta potential, thereby validating the instrument for analysis of biological cells.

[0339] 2. The method of embodiment 1, wherein the instrument is an electrophoretic light scattering (ELS) instrument.

[0340] 3. The method of embodiments 1 or 2, comprising inserting a biological sample into the instrument, the biological sample comprising a population of the corresponding biological cells and measuring an EPM of the biological sample, thereby generating a measured EPM of the biological sample.

[0341] 4. The method of any one of embodiments 1-3, further comprising determining a biological zeta potential for the biological sample based on the measured EPM of the biological sample, said biological zeta potential being adjusted based on the difference between the syntheticzeta potential and the predetermined zeta potential determined for the hydrogel particle cell mimics.

[0342] 5. The method of any one of embodiments 1-4, wherein the population of hydrogel particle cell mimics that is inserted into the instrument is suspended in a first buffer, wherein the biological sample is suspended in a second buffer, and wherein the first buffer and the second buffer are substantially the same.

[0343] 6. The method of any one of embodiments 1-5, wherein the population of hydrogel particle cell mimics that is inserted into the instrument is at substantially the same concentration as the population of corresponding biological cells.

[0344] 7. The method of any one of embodiments 1-6, wherein the hydrogel particle cell mimics in the population are of substantially the same size as the corresponding biological cells.

[0345] 8. The method of any one of embodiments 1-7, wherein the hydrogel particle cell mimics in the population comprise a substantially similar optical property to the corresponding biological cells.

[0346] 9. The method of embodiment 8, wherein the substantially similar optical property is selected from forward side scatter, side scatter, autofluorescence, and a combination thereof.

[0347] 10. The method of embodiment 1, wherein the hydrogel particle cell mimics have a diameter of about 0.1 μm to about 1 μm, about 1 μm to about 10 μm, or about 10 μm to about 100 μm.

[0348] 11. The method of any one of embodiments 1-10, wherein the hydrogel particle cell mimics have a net positive surface charge at acidic pH, neutral pH, or basic pH.

[0349] 12. The method of embodiment 11, wherein the predetermined zeta potential of the hydrogel particle cell mimics is at least about +2 mV, +3 mV, +4 mV, +5 mV, +6 mV, +7 mV, +8 mV, +9 mV, +10 mV, +11 mV, +12 mV, +13 mV, +14 mV, +15 mV, +16 mV, +17 mV, +18 mV, +19 mV, +20 mV, +21 mV, +22 mV, +23 mV, +24 mV, +25 mV, +26 mV, +27 mV, +28 mV, +29 mV, +30 mV, +31 mV, +32 mV, +33 mV, +34 mV, +35 mV, +36 mV, +37 mV, +38 mV, +39 mV, +40 mV, +41 mV, +42 mV, +43 mV, +44 mV, +45 mV, +46 mV, +47 mV, +48 mV, +49 mV, +50 mV.

[0350] 13. The method of embodiment 11, wherein the predetermined zeta potential of the hydrogel particle cell mimics is about +2 mV, +3 mV, +4 mV, +5 mV, +6 mV, +7 mV, +8 mV, +9 mV, +10 mV, +11 mV, +12 mV, +13 mV, +14 mV, +15 mV, +16 mV, +17 mV, +18 mV, +19mV, +20 mV, +21 mV, +22 mV, +23 mV, +24 mV, +25 mV, +26 mV, +27 mV, +28 mV, +29 mV, +30 mV, +31 mV, +32 mV, +33 mV, +34 mV, +35 mV, +36 mV, +37 mV, +38 mV, +39 mV, +40 mV, +41 mV, +42 mV, +43 mV, +44 mV, +45 mV, +46 mV, +47 mV, +48 mV, +49 mV, +50 mV in deionized water.

[0351] 14. The method of embodiment 11, wherein the predetermined zeta potential of the hydrogel particle cell mimics is about +7 mV, +8 mV, +9 mV, +10 mV, +11 mV, +12 mV, +13 mV, +14 mV, +15 mV, +16 mV, +17 mV, +18 mV, +19 mV, +20 mV, +21 mV, +22 mV, +23 mV, +24 mV, or +25 mV at pH 4, pH 7, or pH 10.

[0352] 15. The method of any of embodiments 1-10, wherein the hydrogel particle cell mimics have a net negative surface charge at acidic pH, neutral pH, or basic pH.

[0353] 16. The method of embodiment 15, wherein the predetermined zeta potential of the hydrogel particle cell mimics is at least about -1 mV, -2 mV, -3 mV, -4 mV, -5 mV, -6 mV, -7 mV, -8 mV, -9 mV, -10 mV, -11 mV, -12 mV, -13 mV, -14 mV, -15 mV, -16 mV, -17 mV, -18 mV, -19 mV, -20 mV, -21 mV, -22 mV, -23 mV, -24 mV, -25 mV, -26 mV, -27 mV, -28 mV, -29 mV, -30 mV, -31 mV, -32 mV, -33 mV, -34 mV, -35 mV, -36 mV, -37 mV, -38 mV, -39 mV, -40 mV, -41 mV, -42 mV, -43 mV, -44 mV, -45 mV, -46 mV, -47 mV, -48 mV, -49 mV, -50 mV.

[0354] 17. The method of embodiment 15, wherein the predetermined zeta potential of the hydrogel particle cell mimics is about -15 mV, -16 mV, -17 mV, -18 mV, -19 mV, -20 mV, -21 mV, -22 mV, -23 mV, -24 mV, -25 mV, -26 mV, -27 mV, -28 mV, -29 mV, -30 mV, -31 mV, -32 mV, -33 mV, -34 mV, -35 mV, -36 mV, -37 mV, -38 mV, -39 mV, or -40 mV in deionized water.

[0355] 18. The method of embodiment 15, wherein the predetermined zeta potential of the hydrogel particle cell mimics is about -1 mV, -2 mV, -3 mV, -4 mV, -5 mV, -6 mV, or -7 mV at pH 4.

[0356] 19. The method of embodiment 15, wherein the predetermined zeta potential of the hydrogel particle cell mimics is about -2 mV, -3 mV, -4 mV, -5 mV, -6 mV, -7 mV, -8 mV, -9 mV, -10 mV, -11 mV, -12 mV, -13 mV, -14 mV, -15 mV, -16 mV, -17 mV, -18 mV, -19 mV, -20 mV, -21 mV, -22 mV, -23 mV, -24 mV, or -25 mV at pH 7 or pH 10.

[0357] 20. A method for measuring a zeta potential of a population of biological particles, comprising: (i) inserting into an ELS instrument a population of hydrogel particle cell mimics comprising a predetermined zeta potential, (ii) measuring electrophoretic mobility (EPM) of the population of hydrogel particle cell mimics using the ELS instrument, (iii) repeating (i)-(ii) for apanel of populations of hydrogel particle cell mimics, each population comprising a distinct zeta potential, (iv) generating a calibration curve from the EPM and predetermined zeta potential for the panel, (v) measuring EPM of the population of biological particles, and (vi) determining the zeta potential of the population of biological particles based on a comparison of the EPM measurement to the calibration curve.

[0358] 21. The method of embodiment 20, wherein the hydrogel particle cell mimics in the populations of steps (i)-(iii) are substantially the same size as the biological particles.

[0359] 22. The method of embodiment 20 or 21, wherein the population of hydrogel particle cell mimics have an average diameter of about 0.1 μm to about 1 μm, about 1 μm to about 10 μm, or about 10 μm to about 100 μm.

[0360] 23. The method of any one of embodiments 20-22, wherein the population of hydrogel particle cell mimics has a net positive surface charge at acidic pH, neutral pH, or basic pH.

[0361] 24. The method of embodiment 23, wherein the predetermined zeta potential of the population of hydrogel particle cell mimics is at least about +2 mV, +3 mV, +4 mV, +5 mV, +6 mV, +7 mV, +8 mV, +9 mV, +10 mV, +11 mV, +12 mV, +13 mV, +14 mV, +15 mV, +16 mV, +17 mV, +18 mV, +19 mV, +20 mV, +21 mV, +22 mV, +23 mV, +24 mV, +25 mV, +26 mV, +27 mV, +28 mV, +29 mV, +30 mV, +31 mV, +32 mV, +33 mV, +34 mV, +35 mV, +36 mV, +37 mV, +38 mV, +39 mV, +40 mV, +41 mV, +42 mV, +43 mV, +44 mV, +45 mV, +46 mV, +47 mV, +48 mV, +49 mV, +50 mV.

[0362] 25. The method of embodiment 23, wherein the predetermined zeta potential of the predetermined zeta potential of the hydrogel particle cell mimics is about +2 mV, +3 mV, +4 mV, +5 mV, +6 mV, +7 mV, +8 mV, +9 mV, +10 mV, +11 mV, +12 mV, +13 mV, +14 mV, +15 mV, +16 mV, +17 mV, +18 mV, +19 mV, +20 mV, +21 mV, +22 mV, +23 mV, +24 mV, +25 mV, +26 mV, +27 mV, +28 mV, +29 mV, +30 mV, +31 mV, +32 mV, +33 mV, +34 mV, +35 mV, +36 mV, +37 mV, +38 mV, +39 mV, +40 mV, +41 mV, +42 mV, +43 mV, +44 mV, +45 mV, +46 mV, +47 mV, +48 mV, +49 mV, +50 mV in deionized water.

[0363] 26. The method of embodiment 23, wherein the predetermined zeta potential of the population of hydrogel particle cell mimics is about +7 mV, +8 mV, +9 mV, +10 mV, +11 mV, +12 mV, +13 mV, +14 mV, +15 mV, +16 mV, +17 mV, +18 mV, +19 mV, +20 mV, +21 mV, +22 mV, +23 mV, +24 mV, or +25 mV at pH 4, pH 7, or pH 10.

[0364] 27. The method of any one of embodiments 20-22, wherein the population of hydrogel particle cell mimics has a net negative surface charge at acidic pH, neutral pH, or basic pH.

[0365] 28. The method of embodiment 27, wherein the predetermined zeta potential of the population of hydrogel particle cell mimics is at least about -1 mV, -2 mV, -3 mV, -4 mV, -5 mV, -6 mV, -7 mV, -8 mV, -9 mV, -10 mV, -11 mV, -12 mV, -13 mV, -14 mV, -15 mV, -16 mV, -17 mV, -18 mV, -19 mV, -20 mV, -21 mV, -22 mV, -23 mV, -24 mV, -25 mV, -26 mV, -27 mV, -28 mV, -29 mV, -30 mV, -31 mV, -32 mV, -33 mV, -34 mV, -35 mV, -36 mV, -37 mV, -38 mV, -39 mV, -40 mV, -41 mV, -42 mV, -43 mV, -44 mV, -45 mV, -46 mV, -47 mV, -48 mV, -49 mV, or -50 mV.

[0366] 29. The method of embodiment 27, wherein the predetermined zeta potential of the population of hydrogel particle cell mimics is about -15 mV, -16 mV, -17 mV, -18 mV, -19 mV, - 20 mV, -21 mV, -22 mV, -23 mV, -24 mV, -25 mV, -26 mV, -27 mV, -28 mV, -29 mV, -30 mV, -31 mV, -32 mV, -33 mV, -34 mV, -35 mV, -36 mV, -37 mV, -38 mV, -39 mV, or -40 mV in deionized water.

[0367] 30. The method of embodiment 27, wherein the predetermined zeta potential of the population of hydrogel particle cell mimics is about -1 mV, -2 mV, -3 mV, -4 mV, -5 mV, -6 mV, or -7 mV at pH 4.

[0368] 31. The method of embodiment 27, wherein the predetermined zeta potential of the population of hydrogel particle cell mimics is about -2 mV, -3 mV, -4 mV, -5 mV, -6 mV, -7 mV, -8 mV, -9 mV, -10 mV, -11 mV, -12 mV, -13 mV, -14 mV, -15 mV, -16 mV, -17 mV, -18 mV, - 19 mV, -20 mV, -21 mV, -22 mV, -23 mV, -24 mV, or -25 mV at pH 7 or pH 10.

[0369] 32. The method of any one of embodiments 20-31, wherein the population of biological particles comprises biological cells.

[0370] 33. The method of embodiment 20, wherein the population of biological particles comprises pre-apoptotic cells.

[0371] 34. The method of embodiment 33, wherein the pre-apoptotic cells comprise surface- exposed phosphatidylserine.

[0372] 35. The method of any one of embodiments 20-34, wherein the population of biological particles comprises extracellular vesicles.

[0373] 36. The method of any one of embodiments 20-35, wherein the biological particles or the biological cells comprise a surface label.

[0374] 37. The method of any one of embodiments 20-36, wherein the hydrogel particle cell mimics comprise a surface label.

[0375] 38. The method of embodiment 37, wherein the surface label comprises a biomarker.

[0376] 39. The method of embodiment 38, wherein the biomarker is a nucleic acid, protein, peptide, carbohydrate, or combination thereof.

[0377] 40. The method of any one of embodiments 20-39, wherein the populations of hydrogel particle cell mimics in the populations of steps (i)-(iii) are at substantially the same concentration as the population of biological particles.

[0378] 41. The method of any one of embodiments 20-40, wherein the hydrogel particle cell mimics comprise a neutral monomer and a charged monomer.

[0379] 42. The method of embodiment 41, wherein the charged monomer comprises a cationic monomer that has a positive charge.

[0380] 43. The method of embodiment 42, wherein the cationic monomer comprises (i) a tertiary amine salt thereof, or (ii) an aminium or salt thereof.

[0381] 44. The method of embodiment 42, wherein the cationic monomer comprises a methacrylate moiety and a quaternary ammonium group.

[0382] 45. The method of embodiment 44, wherein the quaternary ammonium group comprises trimethylammonium, tetraethylammonium, tetrapropylammonium, or tetrabutylammonium.

[0383] 46. The method of embodiment 42, wherein the cationic monomer comprises 3- (methacryloylamino)propyl]trimethylammonium, 3-Acrylamidopropyl)trimethylammonium chloride (APTAC), Allylamine hydrochloride, (3-Acrylamidopropyl)trimethylammonium chloride, [2-(Methacryloyloxy)ethyl]trimethylammonium chloride, Lysine (meth)acrylate, Poly(L-lysine) (meth)acrylate, Poly(ethylenimine) (meth)acrylate, Poly(amidoamine) (meth)acrylate, or a combination thereof.

[0384] 47. The method of any one of embodiments 20-40, wherein the charged monomer comprises an anionic monomer that has a negative charge.

[0385] 48. The method of embodiment 47, wherein the anionic monomer comprises an anionic ionizable functional group selected from the group consisting of carboxylate, phosphate, sulfate, sulfonate, phenolate, thiolate, enolate, alkoxide, and phosphonate.

[0386] 49. The method of embodiment 47, wherein the anionic monomer comprises a methacrylate moiety and a succinate group; or a methacrylate moiety and a carboxylate group.

[0387] 50. The method of embodiment 47, wherein the anionic monomer is selected from mono-2-(Methacryloyloxy)ethyl succinate (MMES), sodium acrylate, acrylic acid, mono-2- (Methacryloyloxy)ethyl succinate sodium styrene sulfonate, 2-Acrylamido-2-methyl-1- propanesulfonic acid sodium salt, 2-sulfoethyl methacrylate, vinylphosphonic acid, or a combination thereof.

[0388] 51. The method of any one of embodiment 42-50, wherein the neutral monomer comprises hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinylpyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glycerol methacrylate (GMA), 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, glycerol 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-phenyl acrylamide, N-phenyl methacrylamide, N-benzyl acrylamide, N-benzyl methacrylamide, N,N-dibenzyl acrylamide, N,N-dibenzyl methacrylamide, N-diphenylmethyl acrylamide, N-(4-methylphenyl)methyl acrylamide, N-1-naphthyl acrylamide, N-4-nitrophenyl acrylamide, N-(2-phenylethyl)acrylamide, N-triphenylmethyl acrylamide, N-(4- hydroxyphenyl)acrylamide, N,N-methylphenyl acrylamide, N,N-phenyl phenylethyl acrylamide, N-diphenylmethyl methacrylamide, N-(4-methyl phenyl)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'-phenyl phenylethyl methacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2- vinylpyridine, or a combination thereof.

[0389] 52. The method of any one of embodiments 41-51, comprising a w / w ratio of the neutral monomer and the charged monomer, wherein the w / w ratio is about 1:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, or 50:1.

[0390] 53. The method of any one of embodiments 20-52, wherein the ELS instrument is a zeta potential analyzer.

[0391] 54. The method of any one of embodiments 20-53, wherein the population of hydrogel particle cell mimics comprise another property that is substantially similar to the biological cells or biological particles, said other property selected from the group consisting of: volume, FSC, SSC, background fluorescence; fluorescence profile; shared surface marker, and refractive index.

[0392] 55. A charged cell mimic comprising a hydrogel particle comprising a neutral monomer and a charged monomer, wherein the hydrogel particle comprises a predetermined zeta potential and wherein the predetermined zeta potential is substantially similar to a zeta potential of a target biological cell.

[0393] 56. The charged cell mimic of embodiment 56, comprising a w / w ratio of the neutral monomer and the charged monomer, wherein the w / w ratio is about 1:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, or 50:1.

[0394] 57. The charged cell mimic of embodiment 55 or 56, wherein the hydrogel particle has a diameter of about 0.1 μm to about 1 μm, about 1 μm to about 10 μm, or about 10 μm to about 100 μm.

[0395] 58. The charged cell mimic of any one of embodiments 55-57, wherein the hydrogel particle comprises a cationic monomer and has a net positive surface charge at acidic pH, neutral pH, or basic pH.

[0396] 59. The charged cell mimic of embodiment 58, wherein the predetermined zeta potential of the hydrogel particle is at least about +2 mV, +3 mV, +4 mV, +5 mV, +6 mV, +7 mV, +8 mV, +9 mV, +10 mV, +11 mV, +12 mV, +13 mV, +14 mV, +15 mV, +16 mV, +17 mV, +18 mV, +19 mV, +20 mV, +21 mV, +22 mV, +23 mV, +24 mV, +25 mV, +26 mV, +27 mV, +28 mV, +29 mV, +30 mV, +31 mV, +32 mV, +33 mV, +34 mV, +35 mV, +36 mV, +37 mV, +38mV, +39 mV, +40 mV, +41 mV, +42 mV, +43 mV, +44 mV, +45 mV, +46 mV, +47 mV, +48 mV, +49 mV, or +50 mV.

[0397] 60. The charged cell mimic of embodiment 58, wherein the predetermined zeta potential of the hydrogel particle is about +2 mV, +3 mV, +4 mV, +5 mV, +6 mV, +7 mV, +8 mV, +9 mV, +10 mV, +11 mV, +12 mV, +13 mV, +14 mV, +15 mV, +16 mV, +17 mV, +18 mV, +19 mV, +20 mV, +21 mV, +22 mV, +23 mV, +24 mV, +25 mV, +26 mV, +27 mV, +28 mV, +29 mV, +30 mV, +31 mV, +32 mV, +33 mV, +34 mV, +35 mV, +36 mV, +37 mV, +38 mV, +39 mV, +40 mV, +41 mV, +42 mV, +43 mV, +44 mV, +45 mV, +46 mV, +47 mV, +48 mV, +49 mV, or +50 mV in deionized water.

[0398] 61. The charged cell mimic of embodiment 58, wherein the predetermined zeta potential of the hydrogel particle is about +7 mV, +8 mV, +9 mV, +10 mV, +11 mV, +12 mV, +13 mV, +14 mV, +15 mV, +16 mV, +17 mV, +18 mV, +19 mV, +20 mV, +21 mV, +22 mV, +23 mV, +24 mV, or +25 mV at pH 4, pH 7, or pH 10.

[0399] 62. The charged cell mimic of embodiment 58, wherein the cationic monomer comprises (i) a tertiary amine salt, or (ii) an aminium or salt thereof.

[0400] 63. The charged cell mimic of embodiment 58, wherein the cationic monomer comprises a methacrylate moiety and a quaternary ammonium group.

[0401] 64. The charged cell mimic of embodiment 63, wherein the quaternary ammonium group comprises trimethylammonium, tetraethylammonium, tetrapropylammonium, or tetrabutylammonium.

[0402] 65. The charged cell mimic of embodiment 58, wherein the cationic monomer is selected from the group consisting of 3-(methacryloylamino)propyl]trimethylammonium, 3- Acrylamidopropyl)trimethylammonium chloride, and 3-Acrylamidopropyl)trimethylammonium chloride (APTAC).

[0403] 66. The charged cell mimic of any one of embodiments 55-57, wherein the hydrogel particle comprises an anionic monomer and have a net negative surface charge at acidic pH, neutral pH, or basic pH.

[0404] 67. The charged cell mimic of embodiment 66, wherein the predetermined zeta potential of the hydrogel particle is at least about -1 mV, -2 mV, -3 mV, -4 mV, -5 mV, -6 mV, -7 mV, -8 mV, -9 mV, -10 mV, -11 mV, -12 mV, -13 mV, -14 mV, -15 mV, -16 mV, -17 mV, -18 mV, -19 mV, -20 mV, -21 mV, -22 mV, -23 mV, -24 mV, -25 mV, -26 mV, -27 mV, -28 mV, -29mV, -30 mV, -31 mV, -32 mV, -33 mV, -34 mV, -35 mV, -36 mV, -37 mV, -38 mV, -39 mV, -40 mV, -41 mV, -42 mV, -43 mV, -44 mV, -45 mV, -46 mV, -47 mV, -48 mV, -49 mV, or -50 mV.

[0405] 68. The charged cell mimic of embodiment 66, wherein the predetermined zeta potential of the hydrogel particle is about -15 mV, -16 mV, -17 mV, -18 mV, -19 mV, -20 mV, - 21 mV, -22 mV, -23 mV, -24 mV, -25 mV, -26 mV, -27 mV, -28 mV, -29 mV, -30 mV, -31 mV, -32 mV, -33 mV, -34 mV, -35 mV, -36 mV, -37 mV, -38 mV, -39 mV, or -40 mV in deionized water.

[0406] 69. The charged cell mimic of embodiment 66, wherein the predetermined zeta potential of the hydrogel particle is about -1 mV, -2 mV, -3 mV, -4 mV, -5 mV, -6 mV, or -7 mV at pH 4.

[0407] 70. The charged cell mimic of embodiment 66, wherein the predetermined zeta potential of the hydrogel particle is about -2 mV, -3 mV, -4 mV, -5 mV, -6 mV, -7 mV, -8 mV, -9 mV, -10 mV, -11 mV, -12 mV, -13 mV, -14 mV, -15 mV, -16 mV, -17 mV, -18 mV, -19 mV, -20 mV, -21 mV, -22 mV, -23 mV, -24 mV, or -25 mV at pH 7 or pH 10.

[0408] 71. The charged cell mimic of embodiment 66, wherein the anionic monomer comprises an anionic ionizable functional group selected from the group consisting of carboxylate, phosphate, sulfate, sulfonate, phenolate, thiolate, enolate, alkoxide, and phosphonate.

[0409] 72. The charged cell mimic of 66, wherein the anionic monomer comprises a methacrylate moiety and a succinate group; or a methacrylate moiety and a carboxylate group.

[0410] 73. The method of embodiment 66, wherein the anionic monomer is selected from the group consisting of mono-2-(methacryloyloxy)ethyl succinate (MMES) and acrylate.

[0411] 74. The charged cell mimic of embodiment 66, wherein the anionic monomer comprises mono-2-(methacryloyloxy)ethyl succinate (MMES).

[0412] 75. The charged cell mimic of any one of embodiments 56-74, wherein the neutral monomer comprises hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinylpyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glycerol methacrylate (GMA), 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, glycerol 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-phenyl acrylamide, N-phenyl methacrylamide, N-benzyl acrylamide, N-benzyl methacrylamide, N,N-dibenzyl acrylamide, N,N-dibenzyl methacrylamide, N-diphenylmethyl acrylamide, N-(4-methylphenyl)methyl acrylamide, N-1-naphthyl acrylamide, N-4-nitrophenyl acrylamide, N-(2-phenylethyl)acrylamide, N-triphenylmethyl acrylamide, N-(4- hydroxyphenyl)acrylamide, N,N-methylphenyl acrylamide, N,N-phenyl phenylethyl acrylamide, N-diphenylmethyl methacrylamide, N-(4-methyl phenyl)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'-phenyl phenylethyl methacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2- vinylpyridine, or a combination thereof.

[0413] 76. The charged cell mimic of any one of embodiments 56-75, wherein the target biological cell is an apoptotic cell or a pre-apoptotic cell.

[0414] 77. The charged cell mimic of any one of embodiments 56-76, wherein the target biological cell comprises a biomarker.

[0415] 78. The charged cell mimic of embodiment 77, wherein the hydrogel particle comprises the biomarker.

[0416] 79. The charged cell mimic of any one of embodiments 56-78, comprising an antibody or antigen binding fragment thereof linked to the hydrogel particle, the antibody or antigen binding fragment thereof selected from the group consisting of: an anti-CD3 antibody or antigen binding fragment thereof, an anti-CD8 antibody or antigen binding fragment thereof, and an anti-CD28 antibody or antigen binding fragment thereof.

[0417] 80. The charged cell mimic of any one of embodiments 56-79, wherein the hydrogel particle is substantially the same size as the target biological cell.

[0418] 81. The charged cell mimic of any one of embodiments 56-80, wherein the hydrogel particle comprises a substantially similar optical property to the target biological cell.

[0419] 82. The charged cell mimic of embodiment 81, wherein the substantially similar optical property is selected from forward side scatter, side scatter, autofluorescence, and a combination thereof.

[0420] 83. The charged cell mimic of any one of embodiments 56-82, wherein the target biological cell is pre-apoptotic.

[0421] 84. The charged cell mimic of embodiment 83, wherein the target biological cell comprises surface-exposed phosphatidylserine.

[0422] 85. The charged cell mimic of any one of embodiments 78-84, wherein the biomarker is a nucleic acid, protein, peptide, carbohydrate, or combination thereof.

[0423] 86. The charged cell mimic of any one of embodiments 56-85, wherein the hydrogel particle comprises another property that is substantially similar to the target biological cell, said other property selected from the group consisting of: volume, FSC, SSC, background fluorescence; fluorescence profile; shared surface marker, and refractive index.

[0424] 87. A charged cell mimic comprising a hydrogel particle comprising a neutral monomer, a charged monomer, and an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen binding fragment thereof is selected from the group consisting of an anti- CD3 antibody or fragment thereof, an anti-CD8 antibody or fragment thereof, and an anti-CD28 antibody or fragment thereof, wherein the hydrogel particle comprises a predetermined zeta potential, and wherein the predetermined zeta potential is substantially similar to a zeta potential of a target biological cell.

[0425] 88. The charged cell mimic of embodiment 87, wherein the hydrogel particle comprises a main body, the main body comprising a plurality of macropores.

[0426] 89. A method of inducing an immune cell response, comprising

[0427] contacting an immune cell with the charged cell mimic of any one of the preceding embodiments, wherein the immune cell response comprises activation and / or expansion of the immune cell.

[0428] 90. A method of inducing an immune cell response, comprising contacting an immune cell with a charged cell mimic, wherein the charged cell mimic comprises a neutral monomer, a charged monomer, and an antibody or an antigen-binding fragment thereof, wherein the hydrogel particle comprises a predetermined zeta potential, wherein the predetermined zeta potential is substantially similar to a zeta potential of the immune cell, and wherein the immune cell response comprises activation and / or expansion of the immune cell.

[0429] 91. The method of embodiment 90, wherein the antibody or antigen binding fragment thereof is selected from the group consisting of: an anti-CD3 antibody or fragment thereof, an anti- CD8 antibody or fragment thereof, and an anti-CD28 antibody or fragment thereof.

Claims

CLAIMS 1. A method for validating an instrument for charge analysis of biological cells, said method comprising the steps of: inserting into the instrument a population of hydrogel particle cell mimics comprising a predetermined zeta potential that is substantially similar to the zeta potential of corresponding biological cells, measuring electrophoretic mobility (EPM) of the population of hydrogel particle cell mimics using the instrument, and determining any difference between a synthetic zeta potential calculated from the EPM of the population of hydrogel particle cell mimics and the predetermined zeta potential, thereby validating the instrument for analysis of biological cells.

2. The method of claim 1, wherein the instrument is an electrophoretic light scattering (ELS) instrument.

3. The method of claim 1, comprising inserting a biological sample into the instrument, the biological sample comprising a population of the corresponding biological cells and measuring an EPM of the biological sample, thereby generating a measured EPM of the biological sample.

4. The method of claim 3, further comprising determining a biological zeta potential for the biological sample based on the measured EPM of the biological sample, said biological zeta potential being adjusted based on the difference between the synthetic zeta potential and the predetermined zeta potential determined for the hydrogel particle cell mimics.

5. The method of claim 3, wherein the population of hydrogel particle cell mimics that is inserted into the instrument is suspended in a first buffer, wherein the biological sample is suspended in a second buffer, and wherein the first buffer and the second buffer are substantially the same.

6. The method of claim 3, wherein the population of hydrogel particle cell mimics that is inserted into the instrument is at substantially the same concentration as the population of corresponding biological cells.

7. The method of claim 1, wherein the hydrogel particle cell mimics in the population are of substantially the same size as the corresponding biological cells.

8. The method of claim 1, wherein the hydrogel particle cell mimics in the population comprise a substantially similar optical property to the corresponding biological cells.

9. The method of claim 8, wherein the substantially similar optical property is selected from forward side scatter, side scatter, autofluorescence, and a combination thereof.

10. The method of claim 1, wherein the hydrogel particle cell mimics have a diameter of about 0.1 μm to about 1 μm, about 1 μm to about 10 μm, or about 10 μm to about 100 μm.

11. The method of claim 1, wherein the hydrogel particle cell mimics have a net positive surface charge at acidic pH, neutral pH, or basic pH.

12. The method of claim 11, wherein the predetermined zeta potential of the hydrogel particle cell mimics is at least about +2 mV, +3 mV, +4 mV, +5 mV, +6 mV, +7 mV, +8 mV, +9 mV, +10 mV, +11 mV, +12 mV, +13 mV, +14 mV, +15 mV, +16 mV, +17 mV, +18 mV, +19 mV, +20 mV, +21 mV, +22 mV, +23 mV, +24 mV, +25 mV, +26 mV, +27 mV, +28 mV, +29 mV, +30 mV, +31 mV, +32 mV, +33 mV, +34 mV, +35 mV, +36 mV, +37 mV, +38 mV, +39 mV, +40 mV, +41 mV, +42 mV, +43 mV, +44 mV, +45 mV, +46 mV, +47 mV, +48 mV, +49 mV, +50 mV.

13. The method of claim 11, wherein the predetermined zeta potential of the hydrogel particle cell mimics is about +2 mV, +3 mV, +4 mV, +5 mV, +6 mV, +7 mV, +8 mV, +9 mV, +10 mV, +11 mV, +12 mV, +13 mV, +14 mV, +15 mV, +16 mV, +17 mV, +18 mV, +19 mV, +20 mV, +21 mV, +22 mV, +23 mV, +24 mV, +25 mV, +26 mV, +27 mV, +28 mV, +29 mV,+30 mV, +31 mV, +32 mV, +33 mV, +34 mV, +35 mV, +36 mV, +37 mV, +38 mV, +39 mV, +40 mV, +41 mV, +42 mV, +43 mV, +44 mV, +45 mV, +46 mV, +47 mV, +48 mV, +49 mV, +50 mV in deionized water.

14. The method of claim 11, wherein the predetermined zeta potential of the hydrogel particle cell mimics is about +7 mV, +8 mV, +9 mV, +10 mV, +11 mV, +12 mV, +13 mV, +14 mV, +15 mV, +16 mV, +17 mV, +18 mV, +19 mV, +20 mV, +21 mV, +22 mV, +23 mV, +24 mV, or +25 mV at pH 4, pH 7, or pH 10.

15. The method of claim 1, wherein the hydrogel particle cell mimics have a net negative surface charge at acidic pH, neutral pH, or basic pH.

16. The method of claim 15, wherein the predetermined zeta potential of the hydrogel particle cell mimics is at least about -1 mV, -2 mV, -3 mV, -4 mV, -5 mV, -6 mV, -7 mV, -8 mV, -9 mV, -10 mV, -11 mV, -12 mV, -13 mV, -14 mV, -15 mV, -16 mV, -17 mV, -18 mV, -19 mV, -20 mV, -21 mV, -22 mV, -23 mV, -24 mV, -25 mV, -26 mV, -27 mV, -28 mV, -29 mV, -30 mV, -31 mV, -32 mV, -33 mV, -34 mV, -35 mV, -36 mV, -37 mV, -38 mV, -39 mV, -40 mV, - 41 mV, -42 mV, -43 mV, -44 mV, -45 mV, -46 mV, -47 mV, -48 mV, -49 mV, -50 mV.

17. The method of claim 15, wherein the predetermined zeta potential of the hydrogel particle cell mimics is about -15 mV, -16 mV, -17 mV, -18 mV, -19 mV, -20 mV, -21 mV, -22 mV, -23 mV, -24 mV, -25 mV, -26 mV, -27 mV, -28 mV, -29 mV, -30 mV, -31 mV, -32 mV, - 33 mV, -34 mV, -35 mV, -36 mV, -37 mV, -38 mV, -39 mV, or -40 mV in deionized water.

18. The method of claim 15, wherein the predetermined zeta potential of the hydrogel particle cell mimics is about -1 mV, -2 mV, -3 mV, -4 mV, -5 mV, -6 mV, or -7 mV at pH 4.

19. The method of claim 15, wherein the predetermined zeta potential of the hydrogel particle cell mimics is about -2 mV, -3 mV, -4 mV, -5 mV, -6 mV, -7 mV, -8 mV, -9 mV, -10 mV, -11 mV, -12 mV, -13 mV, -14 mV, -15 mV, -16 mV, -17 mV, -18 mV, -19 mV, -20 mV, - 21 mV, -22 mV, -23 mV, -24 mV, or -25 mV at pH 7 or pH 10.

20. A method for measuring a zeta potential of a population of biological particles, comprising: (i) inserting into an ELS instrument a population of hydrogel particle cell mimics comprising a predetermined zeta potential, (ii) measuring electrophoretic mobility (EPM) of the population of hydrogel particle cell mimics using the ELS instrument, (iii) repeating (i)-(ii) for a panel of populations of hydrogel particle cell mimics, each population comprising a distinct zeta potential, (iv) generating a calibration curve from the EPM and predetermined zeta potential for the panel, (v) measuring EPM of the population of biological particles, and (vi) determining the zeta potential of the population of biological particles based on a comparison of the EPM measurement to the calibration curve.

21. The method of claim 20, wherein the hydrogel particle cell mimics in the populations of steps (i)-(iii) are substantially the same size as the biological particles.

22. The method of claim 20, wherein the population of hydrogel particle cell mimics have an average diameter of about 0.1 μm to about 1 μm, about 1 μm to about 10 μm, or about 10 μm to about 100 μm.

23. The method of claim 20, wherein the population of hydrogel particle cell mimics has a net positive surface charge at acidic pH, neutral pH, or basic pH.

24. The method of claim 23, wherein the predetermined zeta potential of the population of hydrogel particle cell mimics is at least about +2 mV, +3 mV, +4 mV, +5 mV, +6 mV, +7 mV, +8 mV, +9 mV, +10 mV, +11 mV, +12 mV, +13 mV, +14 mV, +15 mV, +16 mV, +17 mV, +18 mV, +19 mV, +20 mV, +21 mV, +22 mV, +23 mV, +24 mV, +25 mV, +26 mV, +27 mV, +28 mV, +29 mV, +30 mV, +31 mV, +32 mV, +33 mV, +34 mV, +35 mV, +36 mV, +37 mV, +38mV, +39 mV, +40 mV, +41 mV, +42 mV, +43 mV, +44 mV, +45 mV, +46 mV, +47 mV, +48 mV, +49 mV, +50 mV.

25. The method of claim 23, wherein the predetermined zeta potential of the predetermined zeta potential of the hydrogel particle cell mimics is about +2 mV, +3 mV, +4 mV, +5 mV, +6 mV, +7 mV, +8 mV, +9 mV, +10 mV, +11 mV, +12 mV, +13 mV, +14 mV, +15 mV, +16 mV, +17 mV, +18 mV, +19 mV, +20 mV, +21 mV, +22 mV, +23 mV, +24 mV, +25 mV, +26 mV, +27 mV, +28 mV, +29 mV, +30 mV, +31 mV, +32 mV, +33 mV, +34 mV, +35 mV, +36 mV, +37 mV, +38 mV, +39 mV, +40 mV, +41 mV, +42 mV, +43 mV, +44 mV, +45 mV, +46 mV, +47 mV, +48 mV, +49 mV, +50 mV in deionized water.

26. The method of claim 23, wherein the predetermined zeta potential of the population of hydrogel particle cell mimics is about +7 mV, +8 mV, +9 mV, +10 mV, +11 mV, +12 mV, +13 mV, +14 mV, +15 mV, +16 mV, +17 mV, +18 mV, +19 mV, +20 mV, +21 mV, +22 mV, +23 mV, +24 mV, or +25 mV at pH 4, pH 7, or pH 10.

27. The method of claim 20, wherein the population of hydrogel particle cell mimics has a net negative surface charge at acidic pH, neutral pH, or basic pH.

28. The method of claim 27, wherein the predetermined zeta potential of the population of hydrogel particle cell mimics is at least about -1 mV, -2 mV, -3 mV, -4 mV, -5 mV, -6 mV, -7 mV, -8 mV, -9 mV, -10 mV, -11 mV, -12 mV, -13 mV, -14 mV, -15 mV, -16 mV, -17 mV, -18 mV, -19 mV, -20 mV, -21 mV, -22 mV, -23 mV, -24 mV, -25 mV, -26 mV, -27 mV, -28 mV, - 29 mV, -30 mV, -31 mV, -32 mV, -33 mV, -34 mV, -35 mV, -36 mV, -37 mV, -38 mV, -39 mV, -40 mV, -41 mV, -42 mV, -43 mV, -44 mV, -45 mV, -46 mV, -47 mV, -48 mV, -49 mV, or -50 mV.

29. The method of claim 27, wherein the predetermined zeta potential of the population of hydrogel particle cell mimics is about -15 mV, -16 mV, -17 mV, -18 mV, -19 mV, -20 mV, -21 mV, -22 mV, -23 mV, -24 mV, -25 mV, -26 mV, -27 mV, -28 mV, -29 mV, -30 mV, -31 mV, -32 mV, -33 mV, -34 mV, -35 mV, -36 mV, -37 mV, -38 mV, -39 mV, or -40 mV in deionized water.

30. The method of claim 27, wherein the predetermined zeta potential of the population of hydrogel particle cell mimics is about -1 mV, -2 mV, -3 mV, -4 mV, -5 mV, -6 mV, or -7 mV at pH 4.

31. The method of claim 27, wherein the predetermined zeta potential of the population of hydrogel particle cell mimics is about -2 mV, -3 mV, -4 mV, -5 mV, -6 mV, -7 mV, -8 mV, -9 mV, -10 mV, -11 mV, -12 mV, -13 mV, -14 mV, -15 mV, -16 mV, -17 mV, -18 mV, -19 mV, - 20 mV, -21 mV, -22 mV, -23 mV, -24 mV, or -25 mV at pH 7 or pH 10.

32. The method of claim 20, wherein the population of biological particles comprises biological cells.

33. The method of claim 20, wherein the population of biological particles comprises pre- apoptotic cells.

34. The method of claim 33, wherein the pre-apoptotic cells comprise surface-exposed phosphatidylserine.

35. The method of claim 20, wherein the population of biological particles comprises extracellular vesicles.

36. The method of claim 32, wherein the biological particles or the biological cells comprise a surface label.

37. The method of claim 20, wherein the hydrogel particle cell mimics comprise a surface label.

38. The method of claim 37, wherein the surface label comprises a biomarker.

39. The method of claim 38, wherein the biomarker is a nucleic acid, protein, peptide, carbohydrate, or combination thereof.

40. The method of claim 20, wherein the populations of hydrogel particle cell mimics in the populations of steps (i)-(iii) are at substantially the same concentration as the population of biological particles.

41. The method of claim 20, wherein the hydrogel particle cell mimics comprise a neutral monomer and a charged monomer.

42. The method of claim 41, wherein the charged monomer comprises a cationic monomer that has a positive charge.

43. The method of claim 42, wherein the cationic monomer comprises (i) a tertiary amine salt thereof, or (ii) an aminium or salt thereof.

44. The method of claim 42, wherein the cationic monomer comprises a methacrylate moiety and a quaternary ammonium group.

45. The method of claim 44, wherein the quaternary ammonium group comprises trimethylammonium, tetraethylammonium, tetrapropylammonium, or tetrabutylammonium.

46. The method of claim 42, wherein the cationic monomer comprises 3- (methacryloylamino)propyl]trimethylammonium, 3-Acrylamidopropyl)trimethylammonium chloride (APTAC), or a combination thereof.

47. The method of claim 41, wherein the charged monomer comprises an anionic monomer that has a negative charge.

48. The method of claim 47, wherein the anionic monomer comprises an anionic ionizable functional group selected from the group consisting of carboxylate, phosphate, sulfate, sulfonate, phenolate, thiolate, enolate, alkoxide, and phosphonate.

49. The method of claim 47, wherein the anionic monomer comprises a methacrylate moiety and a succinate group; or a methacrylate moiety and a carboxylate group.

50. The method of claim 47, wherein the anionic monomer is selected from mono-2- (methacryloyloxy)ethyl succinate and acrylate.

51. The method of claim 41, wherein the neutral monomer comprises hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinylpyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glycerol methacrylate (GMA), 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, glycerol 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-phenyl acrylamide, N-phenyl methacrylamide, N-benzyl acrylamide, N-benzyl methacrylamide, N,N-dibenzyl acrylamide, N,N-dibenzyl methacrylamide, N-diphenylmethyl acrylamide, N-(4-methylphenyl)methyl acrylamide, N-1-naphthyl acrylamide, N-4-nitrophenyl acrylamide, N-(2-phenylethyl)acrylamide, N-triphenylmethyl acrylamide, N-(4-hydroxyphenyl)acrylamide, N,N-methylphenyl acrylamide, N,N-phenyl phenylethyl acrylamide, N-diphenylmethyl methacrylamide, N-(4-methyl phenyl)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'-phenyl phenylethyl methacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridine, or a combination thereof.

52. The method of claim 41, comprising a w / w ratio of the neutral monomer and the charged monomer, wherein the w / w ratio is about 1:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, or 50:

1.

53. The method of claim 20, wherein the ELS instrument is a zeta potential analyzer.

54. The method of claim 20, wherein the population of hydrogel particle cell mimics comprise another property that is substantially similar to the biological cells or biological particles, said other property selected from the group consisting of: volume, FSC, SSC, background fluorescence; fluorescence profile; shared surface marker, and refractive index.

55. A charged cell mimic comprising a hydrogel particle comprising a neutral monomer and a charged monomer, wherein the hydrogel particle comprises a predetermined zeta potential and wherein the predetermined zeta potential is substantially similar to a zeta potential of a target biological cell.

56. The charged cell mimic claim 55, comprising a w / w ratio of the neutral monomer and the charged monomer, wherein the w / w ratio is about 1:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, or 50:

1.

57. The charged cell mimic of claim 55, wherein the hydrogel particle has a diameter of about 0.1 μm to about 1 μm, about 1 μm to about 10 μm, or about 10 μm to about 100 μm.

58. The charged cell mimic of claim 55, wherein the hydrogel particle comprises a cationic monomer and has a net positive surface charge at acidic pH, neutral pH, or basic pH.

59. The charged cell mimic of claim 58, wherein the predetermined zeta potential of the hydrogel particle is at least about +2 mV, +3 mV, +4 mV, +5 mV, +6 mV, +7 mV, +8 mV, +9 mV, +10 mV, +11 mV, +12 mV, +13 mV, +14 mV, +15 mV, +16 mV, +17 mV, +18 mV, +19 mV, +20 mV, +21 mV, +22 mV, +23 mV, +24 mV, +25 mV, +26 mV, +27 mV, +28 mV, +29 mV, +30 mV, +31 mV, +32 mV, +33 mV, +34 mV, +35 mV, +36 mV, +37 mV, +38 mV, +39 mV, +40 mV, +41 mV, +42 mV, +43 mV, +44 mV, +45 mV, +46 mV, +47 mV, +48 mV, +49 mV, or +50 mV.

60. The charged cell mimic of claim 58, wherein the predetermined zeta potential of the hydrogel particle is about +2 mV, +3 mV, +4 mV, +5 mV, +6 mV, +7 mV, +8 mV, +9 mV, +10 mV, +11 mV, +12 mV, +13 mV, +14 mV, +15 mV, +16 mV, +17 mV, +18 mV, +19 mV, +20 mV, +21 mV, +22 mV, +23 mV, +24 mV, +25 mV, +26 mV, +27 mV, +28 mV, +29 mV, +30 mV, +31 mV, +32 mV, +33 mV, +34 mV, +35 mV, +36 mV, +37 mV, +38 mV, +39 mV, +40 mV, +41 mV, +42 mV, +43 mV, +44 mV, +45 mV, +46 mV, +47 mV, +48 mV, +49 mV, or +50 mV in deionized water.

61. The charged cell mimic of claim 58, wherein the predetermined zeta potential of the hydrogel particle is about +7 mV, +8 mV, +9 mV, +10 mV, +11 mV, +12 mV, +13 mV, +14 mV, +15 mV, +16 mV, +17 mV, +18 mV, +19 mV, +20 mV, +21 mV, +22 mV, +23 mV, +24 mV, or +25 mV at pH 4, pH 7, or pH 10.

62. The charged cell mimic of claim 58, wherein the cationic monomer comprises (i) a tertiary amine salt, or (ii) an aminium or salt thereof.

63. The charged cell mimic of claim 58, wherein the cationic monomer comprises a methacrylate moiety and a quaternary ammonium group.

64. The charged cell mimic of claim 63, wherein the quaternary ammonium group comprises trimethylammonium, tetraethylammonium, tetrapropylammonium, or tetrabutylammonium.

65. The charged cell mimic of claim 58, wherein the cationic monomer is selected from the group consisting of 3-(methacryloylamino)propyl]trimethylammonium, 3- Acrylamidopropyl)trimethylammonium chloride, and 3-Acrylamidopropyl)trimethylammonium chloride (APTAC).

66. The charged cell mimic of claim 55, wherein the hydrogel particle comprises an anionic monomer and have a net negative surface charge at acidic pH, neutral pH, or basic pH.

67. The charged cell mimic of claim 66, wherein the predetermined zeta potential of the hydrogel particle is at least about -1 mV, -2 mV, -3 mV, -4 mV, -5 mV, -6 mV, -7 mV, -8 mV, -9 mV, -10 mV, -11 mV, -12 mV, -13 mV, -14 mV, -15 mV, -16 mV, -17 mV, -18 mV, -19 mV, - 20 mV, -21 mV, -22 mV, -23 mV, -24 mV, -25 mV, -26 mV, -27 mV, -28 mV, -29 mV, -30 mV, -31 mV, -32 mV, -33 mV, -34 mV, -35 mV, -36 mV, -37 mV, -38 mV, -39 mV, -40 mV, -41 mV, -42 mV, -43 mV, -44 mV, -45 mV, -46 mV, -47 mV, -48 mV, -49 mV, or -50 mV.

68. The charged cell mimic of claim 66, wherein the predetermined zeta potential of the hydrogel particle is about -15 mV, -16 mV, -17 mV, -18 mV, -19 mV, -20 mV, -21 mV, -22 mV, -23 mV, -24 mV, -25 mV, -26 mV, -27 mV, -28 mV, -29 mV, -30 mV, -31 mV, -32 mV, -33 mV, -34 mV, -35 mV, -36 mV, -37 mV, -38 mV, -39 mV, or -40 mV in deionized water.

69. The charged cell mimic of claim 66, wherein the predetermined zeta potential of the hydrogel particle is about -1 mV, -2 mV, -3 mV, -4 mV, -5 mV, -6 mV, or -7 mV at pH 4.

70. The charged cell mimic of claim 66, wherein the predetermined zeta potential of the hydrogel particle is about -2 mV, -3 mV, -4 mV, -5 mV, -6 mV, -7 mV, -8 mV, -9 mV, -10 mV, -11 mV, -12 mV, -13 mV, -14 mV, -15 mV, -16 mV, -17 mV, -18 mV, -19 mV, -20 mV, -21 mV, -22 mV, -23 mV, -24 mV, or -25 mV at pH 7 or pH 10.

71. The charged cell mimic of claim 66, wherein the anionic monomer comprises an anionic ionizable functional group selected from the group consisting of carboxylate, phosphate, sulfate, sulfonate, phenolate, thiolate, enolate, alkoxide, and phosphonate.

72. The charged cell mimic of 66, wherein the anionic monomer comprises a methacrylate moiety and a succinate group; or a methacrylate moiety and a carboxylate group.

73. The method of claim 66, wherein the anionic monomer is selected from the group consisting of mono-2-(methacryloyloxy)ethyl succinate (MMES) and acrylate.

74. The charged cell mimic of claim 66, wherein the anionic monomer comprises mono-2- (methacryloyloxy)ethyl succinate (MMES).

75. The charged cell mimic of claim 55, wherein the neutral monomer comprises hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinylpyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glycerol methacrylate (GMA), 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, glycerol 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-phenyl acrylamide, N-phenyl methacrylamide, N-benzylacrylamide, N-benzyl methacrylamide, N,N-dibenzyl acrylamide, N,N-dibenzyl methacrylamide, N-diphenylmethyl acrylamide, N-(4-methylphenyl)methyl acrylamide, N-1-naphthyl acrylamide, N-4-nitrophenyl acrylamide, N-(2-phenylethyl)acrylamide, N-triphenylmethyl acrylamide, N-(4- hydroxyphenyl)acrylamide, N,N-methylphenyl acrylamide, N,N-phenyl phenylethyl acrylamide, N-diphenylmethyl methacrylamide, N-(4-methyl phenyl)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'-phenyl phenylethyl methacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridine, or a combination thereof.

76. The charged cell mimic of claim 55, wherein the target biological cell is an apoptotic cell or a pre-apoptotic cell.

77. The charged cell mimic of claim 55, wherein the target biological cell comprises a biomarker.

78. The charged cell mimic of claim 55, wherein the hydrogel particle comprises the biomarker.

79. The charged cell mimic of claim 55, comprising an antibody or antigen binding fragment thereof linked to the hydrogel particle, the antibody or antigen binding fragment thereof selected from the group consisting of: an anti-CD3 antibody or antigen binding fragment thereof, an anti- CD8 antibody or antigen binding fragment thereof, and an anti-CD28 antibody or antigen binding fragment thereof.

80. The charged cell mimic of claim 55, wherein the hydrogel particle is substantially the same size as the target biological cell.

81. The charged cell mimic of claim 55, wherein the hydrogel particle comprises a substantially similar optical property to the target biological cell.

82. The charged cell mimic of claim 81, wherein the substantially similar optical property is selected from forward side scatter, side scatter, autofluorescence, and a combination thereof.

83. The charged cell mimic of claim 55, wherein the target biological cell is pre-apoptotic.

84. The charged cell mimic of claim 83, wherein the target biological cell comprises surface- exposed phosphatidylserine.

85. The charged cell mimic of claim 77, wherein the biomarker is a nucleic acid, protein, peptide, carbohydrate, or combination thereof.

86. The charged cell mimic of claim 55, wherein the hydrogel particle comprises another property that is substantially similar to the target biological cell, said other property selected from the group consisting of: volume, FSC, SSC, background fluorescence; fluorescence profile; shared surface marker, and refractive index.

87. A charged cell mimic comprising a hydrogel particle comprising a neutral monomer, a charged monomer, and an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen binding fragment thereof is selected from the group consisting of an anti-CD3 antibody or fragment thereof, an anti-CD8 antibody or fragment thereof, and an anti-CD28 antibody or fragment thereof, wherein the hydrogel particle comprises a predetermined zeta potential, and wherein the predetermined zeta potential is substantially similar to a zeta potential of a target biological cell.

88. The charged cell mimic of claim 87, wherein the hydrogel particle comprises a main body, the main body comprising a plurality of macropores.

89. A method of inducing an immune cell response, comprising contacting an immune cell with the charged cell mimic of claim 55,wherein the immune cell response comprises activation and / or expansion of the immune cell.

90. A method of inducing an immune cell response, comprising contacting an immune cell with a charged cell mimic, wherein the charged cell mimic comprises a neutral monomer, a charged monomer, and an antibody or an antigen-binding fragment thereof, wherein the hydrogel particle comprises a predetermined zeta potential, wherein the predetermined zeta potential is substantially similar to a zeta potential of the immune cell, and wherein the immune cell response comprises activation and / or expansion of the immune cell.

91. The method of claim 90, wherein the antibody or antigen binding fragment thereof is selected from the group consisting of: an anti-CD3 antibody or fragment thereof, an anti-CD8 antibody or fragment thereof, and an anti-CD28 antibody or fragment thereof.

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