Use of thiol-containing compounds to improve viability of a material following encapsulation within radical-mediated crosslinked hydrogels

Abstract

Embodiments of the present disclosure generally relate to compositions, articles, and processes for improving viability of a material, for example, a biological material or a synthetic material, of interest. In an embodiment, a composition is provided. The composition includes a hydrogel comprising, in polymerized form, one or more photoreactive monomers, the hydrogel encapsulating: a material of interest; and a thiol-containing compound, the thiol-containing compound not chemically bonded to the hydrogel, the thiol-containing compound different from the material of interest.

Description

PCT Patent Application Attorney Docket No.: UWYO / 0122PC Title: Use of Thiol-Containing Compounds to Improve Viability of a Material Following Encapsulation within Radical-Mediated Crosslinked Hydrogels Inventors: Alan Stenquist; John Oakey; Cassidy Enloe GOVERNMENT RIGHTS

[0001] This invention was made with government support under Grant Number 1254608 awarded by the National Science Foundation. The government has certain rights in the invention. CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims benefit of and priority to U.S. Provisional Patent Application No. 63 / 655,479, filed on June 3, 2024, which is incorporated herein by reference in its entirety. FIELD

[0003] Embodiments of the present disclosure generally relate to compositions, articles, and processes for improving viability of a material, for example, a biological material or a synthetic material, of interest. BACKGROUND

[0004] Hydrogels that crosslink through radical-initiated polymerization require free radicals to be generated from an endogenous or exogenous source. Free radicals not participating in the crosslinking reaction react with molecular oxygen dissolved in the hydrogel forming solution, forming extremely cytotoxic and deleterious reactive oxygen species (ROS) upon reaction with oxygen. When the hydrogels are crosslinked in the presence of a material of interest (for example, cells or biomolecules)—by encapsulation, for instance—these ROS or free radicals damage the material of interest.

[0005] Modern biomaterial fabrication techniques for tissue engineering or regenerative medicine increasingly include microgel fabrication or bioprinting. While these conventional techniques allow excellent spatiotemporal control over gelation and therefore fabrication resolution, the conventional techniques also increase the surface area to volume ratio of hydrogel solutions during gelation. This increase in surface area increases the oxygen flux to the hydrogel volume, thus increasing the amount of generated ROS and therefore ROS- induced damage. As the length scale of formed features decrease, these effects arePCT Patent Application Attorney Docket No.: UWYO / 0122PC exaggerated and the cytotoxicity of ROS increases to the point where cell viability (or viability of a material of interest) is no longer possible.

[0006] There is a need for new compositions, articles, and processes for improving viability of a material, for example, a biological material or a synthetic material, of interest. SUMMARY

[0007] Embodiments of the present disclosure generally relate to compositions, articles, and processes for improving viability of a material, for example, a biological material or a synthetic material, of interest. To the inventors’ knowledge, there are no compositions or articles that include a hydrogel or hydrogel network that encapsulates a material of interest and a thiol-containing compound not chemically bonded to the hydrogel or hydrogel network. Unlike conventional technologies, embodiments of the present disclosure enable increased viability or preservation of various materials of interest from the deleterious effects of, for example, reactive oxygen species. Such materials of interest may include a biological material, a biologically-derived material, a semi-synthetic material, a synthetic materials, or combinations thereof.

[0008] In an embodiment, a composition is provided. The composition includes a hydrogel comprising, in polymerized form, one or more photoreactive monomers, the hydrogel encapsulating: a material of interest; and a thiol-containing compound, the thiol- containing compound not chemically bonded to the hydrogel, the thiol-containing compound different from the material of interest.

[0009] In another embodiment, a drug delivery composition is provided. The drug delivery composition includes a hydrogel comprising, in polymerized form, one or more photoreactive monomers, the hydrogel encapsulating: a drug comprising a material of interest; and a thiol-containing compound, the thiol-containing compound not chemically bonded to the hydrogel, the thiol-containing compound different from the material of interest.

[0010] In another embodiment, a tissue scaffold is provided. The tissue scaffold includes a plurality of hydrogel particles, each hydrogel particle comprising, in polymerized form, one or more photoreactive monomers, each hydrogel particle encapsulating: a material of interest; and a thiol-containing compound not chemically bonded to the hydrogel, the thiol-containing compound different from the material of interest.PCT Patent Application Attorney Docket No.: UWYO / 0122PC

[0011] In another embodiment, a three-dimensional printed article is provided. The three-dimensional printed article includes a fluidic channel. The three-dimensional printed article further includes a hydrogel structure disposed in the fluidic channel. The three- dimensional printed article further includes one or more hydrogel features coupled to and extending above the hydrogel structure, the one or more hydrogel features comprising, in polymerized form, one or more photoreactive monomers, the one or more hydrogel features encapsulating: a material of interest; and a thiol-containing compound, the thiol-containing compound not chemically bonded to the hydrogel feature or the hydrogel structure, the thiol- containing compound different from the material of interest. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] So that the manner in which the above recited features of the present disclosure may be understood in detail, a more particular description of the disclosure, briefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only exemplary embodiments and are therefore not to be considered limiting of its scope, may admit to other equally effective embodiments.

[0013] FIG. 1A shows a schematic of cellular encapsulation in poly(ethylene glycol) diacrylate (PEGDA) microgel “posts” using digital light mediated photolithography.

[0014] FIG.1B shows single cells encapsulated in features (microgel posts) of varying diameter to assess dimensional effects on viability.

[0015] FIG. 1C shows a schematic of an example device for forming a hydrogel- encapsulated (or other material(s) of interest) according to at least one embodiment of the present disclosure.

[0016] FIG.1D is an image showing an example hydrogel-encapsulated cell (or other material of interest) according to at least one embodiment of the present disclosure.

[0017] FIG. 1E is an image showing example hydrogel-encapsulated cell (or other material of interest) according to at least one embodiment of the present disclosure.

[0018] FIG.2A shows data for viability of cells bulk encapsulated in PEGDA of varying molecular weights within 96 well plates under argon inert gas atmosphere.

[0019] FIG. 2B shows data for viability of single cells microencapsulated in 125 µm depth × 250 µm width PEGDA posts of varying molecular weight.PCT Patent Application Attorney Docket No.: UWYO / 0122PC

[0020] FIG.2C shows data for varying stoichiometric ratios of acryl groups in 10,000 kilodalton (kD) PEGDA.

[0021] FIG. 3A is a plot of percent cell viability (left y-axis) and percent increase in reactive oxygen species (ROS) (right y-axis) after microgel fabrication vs. microgel width. The open bars correspond to the cell viability data and the shaded bars corresponds to the ROS data.

[0022] FIG. 3B is a brightfield image of single cells inside of fabricated microgel features (dimensions of each feature: 30 µm diameter × 100 µm height).

[0023] FIG. 3C is a brightfield image of single cells inside of fabricated microgel features (dimensions of each feature: 100 µm diameter × 100 µm height).

[0024] FIG. 3D is a plot of percent cell viability (left y-axis) and percent increase in ROS (right y-axis) after microgel fabrication vs. the channel height in which the microgel feature was fabricated. Cell viability corresponds to the open bars and ROS corresponds to the shaded bars.

[0025] FIG. 3E is a brightfield image of single cells inside of fabricated microgel features (dimensions of each feature: 250 µm diameter × 35 µm height).

[0026] FIG.3F is a live / dead fluorescent image of the single cells inside of fabricated microgel features shown in FIG. 3E, demonstrating viable cells, modified to accentuate stain.

[0027] FIG.4A is a schematic of computer-modeled ROS accumulation in a 35-micron depth microgel under ambient conditions. The legend on the right side of the figure shows the amount of ROS, in mol / m3, with darker shading corresponding to a lower amount of ROS and lighter shading corresponding to a higher amount of ROS.

[0028] FIG. 4B is the same schematic shown in FIG. 4A, but under inert gas purge conditions. The legend on the right side of the figure shows the amount of ROS, in mol / m3, with darker shading corresponding to a lower amount of ROS and lighter shading corresponding to a higher amount of ROS.

[0029] FIG. 4C shows a sensitivity analysis of ROS in 3 µm vertical increments for microgels fabricated in a 35-µm height channel under ambient conditions.

[0030] FIG. 4D shows a sensitivity analysis of ROS in 3 µm vertical increments for microgels fabricated in a 35-µm height channel under inert gas purge conditions.PCT Patent Application Attorney Docket No.: UWYO / 0122PC

[0031] FIG.5A is a plot of percent cell viability, after microgel fabrication, of a cell in microgels (microgel dimensions: 250 µm diameter × 25 µm height) under ambient atmospheric conditions (Air / O2) and under inert gas (nitrogen, N2) purge conditions.

[0032] FIG.5B is a plot of percent cell viability, after microgel fabrication, of a cell in microgels (microgel dimensions: 30 µm diameter × 150 µm height channel) under ambient atmospheric conditions (Air / O2) and under inert gas (N2) purge conditions.

[0033] FIG.5C is a brightfield image of single Madin-Darby Canine Kidney (MDCK) epithelial cells inside of fabricated microgel PEGDA posts (dimensions: 30 µm diameter × 100 µm height) polymerized in a nitrogen-purged PDMS microfluidic channel.

[0034] FIG.5D is a live / dead color combined image (reproduced in grayscale) of single MDCK epithelial cells inside of fabricated microgel PEGDA posts (dimensions: 30 µm diameter × 100 µm height) polymerized in a nitrogen-purged PDMS microfluidic channel.

[0035] FIG. 6A is data for % cell viability (% cells alive) versus chamber depth for gelatin methacrylate (GelMA) microgels (feature size: 50 µm) polymerized around a single cell in PDMS microchannels of varying depths (45 µm, 100 µm, and 150 µm), indicating that GelMA suffers from similar cytotoxicity to PEGDA with similar size limitations.

[0036] FIG. 6B is data for % cell viability (% cells alive) versus chamber depth for GelMA microgels (feature size: 150 µm) polymerized around a single cell in PDMS microchannels of varying depths (45 µm, 100 µm, and 150 µm), indicating that GelMA suffers from similar cytotoxicity to PEGDA with similar size limitations.

[0037] FIG. 7A is a plot of cell viability (%) versus feature height (in µm) for cells polymerized in poly(ethylene glycol) norbornene (PEGNB) features.

[0038] FIG.7B is a plot of cell viability (%) versus feature diameter (in µm) for cells polymerized in PEGNB features.

[0039] FIG.8A is a plot of % cell viability of cells polymerized in PEGDA features and a mixed mode PEGNB-PEGDA features, indicating that the PEGNB-PEGDA features effectively rescues cell viability in microgels.

[0040] FIG.8B is a plot of % cell viability of cells polymerized without the presence of a free thiol-containing compound or thiol-containing linker (control), in the presence of a free thiol-containing compound (40 mM glutathione), or in the presence of thiol-containing linker (20 mM PEG-dithiol).PCT Patent Application Attorney Docket No.: UWYO / 0122PC

[0041] To facilitate understanding, identical reference numerals have been used, where possible, to designate identical elements that are common to the figures. It is contemplated that elements and features of one embodiment may be beneficially incorporated in other embodiments without further recitation. DETAILED DESCRIPTION

[0042] Embodiments of the present disclosure generally relate to compositions, articles, and processes for improving viability of a material, for example, a biological material or a synthetic material, of interest. As used herein, a “composition” may include component(s) of the composition, reaction product(s) of two or more components of the composition, and / or a remainder balance of remaining starting component(s), or combinations thereof.

[0043] Hydrogels that crosslink through radical-initiated polymerization require free radicals to be generated from an endogenous or exogenous source. In the presence of ambient oxygen, generated radicals react with oxygen to produce reactive oxygen species (ROS). When crosslinked in the presence of a material of interest (cells or biomolecules)— for encapsulation, for instance—these ROS or free radicals damage the material of interest (for example, bioactive components, biomolecules, or living cells). Herein, the inventors found that the addition of a thiol-containing compound (such as glutathione or cysteine), a thiol-containing polymer (such as dithiolated polyethylene glycol), or combinations thereof to the hydrogel forming solution may prevent, or at least mitigate, cell death and damage to sensitive bioactive compounds or other materials of interest. The thiol-containing compounds may accomplish this by reacting with cytotoxic ROS.

[0044] The inventors show that a thiol-containing compound, a thiol-containing polymer, or combinations thereof may be added to hydrogel forming solutions in order to reduce biomolecule damage and cell death during radical-initiated gelation. Once electron radicals are introduced to hydrogel forming solutions, either from an appropriate photoinitiator, chemical reaction, or external source, a crosslinking reaction proceeds. Free radicals not participating in the crosslinking reaction react with molecular oxygen dissolved in the hydrogel forming solution, forming extremely cytotoxic ROS. This ROS burst, a byproduct of the radial initiated crosslinking reaction, severely attenuates cell viability by damaging cell membranes and oxidizes and damages sensitive compounds such as growth factors, drugs, peptides, and other bioactive compounds. However, functional thiol groups on the thiol-containing compound, the thiol-containing polymer, or both oxidize to thiylPCT Patent Application Attorney Docket No.: UWYO / 0122PC radicals, thus eliminating superfluous free radicals, thus preventing radicals from oxidizing biological components. Direct electron transfer from ROS to thiol radicals is favorable, allowing any ROS present to be converted into less toxic thiyl radicals. The stability of thiyl radical limits electron transfer to bioactive components and thus limits toxicity. The addition of thiol-containing compound, thiol-containing polymer, or combinations thereof effectively rescues cell viability under conditions that would otherwise cause lethal damage to a cell.

[0045] Modern biomaterial fabrication techniques for tissue engineering or regenerative medicine increasingly include microgel fabrication or bioprinting. While these conventional techniques allow excellent spatiotemporal control over gelation and therefore fabrication resolution, the conventional techniques also increase the surface area to volume ratio of hydrogel solutions during gelation. This increase in surface area increases the oxygen flux to the hydrogel volume, thus increasing the amount of generated ROS and therefore ROS- induced damage. As the length scale of formed features decrease, these effects are exaggerated and the cytotoxicity of ROS increases to the point where cell viability is no longer possible. Use of thiol-containing compounds protects a material of interest from the ROS toxicity or ROS induced degradation, allowing for extremely high cell viability in the harshest of polymerization conditions (or extremely high viability of a material of interest). While potentially lacking the spatiotemporal control over photopolymerization, chemical reactions may also be used to produce the free radicals utilized to initiate the polymerization of the polymer. In this context, thiol-containing compounds may also serve to scavenge excess electrons and limit unwanted oxidation of sensitive components in the pre-polymer solution (the hydrogel forming solution). By scavenging ROS with thiol-containing compounds, unwanted oxidation and damage of a material of interest such as a bioactive compound is mitigated, expanding the utility and scope of radical-crosslinked hydrogels.

[0046] Embodiments described herein generally relate to compositions for improving viability or stability of a material of interest. The composition may include a hydrogel encapsulating: a material of interest; and a thiol-containing compound, the thiol-containing compound not chemically bonded to the hydrogel. The thiol-containing compound is different from the material of interest. When the thiol-containing compound is not chemically bonded to the hydrogel, the thiol-containing compound may be characterized as “free flowing” inside or within the hydrogel. For example, and in some embodiments, thePCT Patent Application Attorney Docket No.: UWYO / 0122PC thiol-containing compound is not chemically (covalently) bonded to the polymerized monomer or polymer network of the hydrogel. That is, the polymerized monomer or polymer network of the hydrogel may be formed without chemically (covalently) bonding to the thiol-containing compound.

[0047] The hydrogel may be formed by a radical-initiated polymerization of one or more photoreactive monomers. The hydrogel may be a polymer network. The hydrogel may be porous and / or degradable. The pores of the hydrogel may be large enough to release an encapsulated component such as the material of interest (for example, a cell), the thiol- containing compound, or both. Alternatively, the pores of the hydrogel may be large enough to release only the thiol-containing compound or materials that are similar in size to the thiol-containing compound.

[0048] The hydrogel may be in the form of a hydrogel droplet or particle. Alternatively, the hydrogel may be in the form of a post (for example, hydrogel feature 103) attached to a channel or other surface of a microfluidic device.

[0049] Materials of interest may include a biological material, a biologically-derived material, a synthetic material, or combinations thereof. A biological material may be derived from a living organism, in its natural state or its modified state, and / or synthesized. The biological material, also referred to as a biological material of interest may include any suitable biological material such as at least one or more of the following: a cell, a peptide, a polypeptide, a protein, an enzyme, an antibody, a globular protein, a hormone, an antibiotic, a nucleic acid, a nucleotide, a lipid, a polylipid, a fat, a monosaccharide, a polysaccharide, a carbohydrate, a natural product, a derivative thereof, or combinations thereof. Biologically-derived materials include materials that are, for example, derived from or produced from a biological material, as well as biological materials that have been modified. Such biologically derived materials may include, for example, a biologic, a vaccine, an agricultural product, therapeutic agent, a diagnostic agent, an agent used for research purposes, a pharmaceutical such as a protein-based pharmaceutical, a derivative thereof, or combinations thereof, among others. The inventor contemplates that certain examples of biological materials and biologically-derived materials may fall within or outside both categories; however, such biological materials and biologically-derived materials may be included in the compositions described herein. Other illustrative, but non-limiting, examples of biological material and / or biologically-derived material may include a nucleic acid-basedPCT Patent Application Attorney Docket No.: UWYO / 0122PC biologic, a nucleic acid-based therapeutic, a nucleic acid-based vaccine such as an mRNA vaccine; protein-based biologics, protein-based therapeutics, and protein-based diagnostics (for example, vaccines, antibodies, enzymes, et cetera). Other biological materials and / or biologically-derived materials are contemplated. One or more materials of interest may be present.

[0050] Synthetic compounds may include synthetic (for example, chemically synthesized) counterparts of biological materials, biologically-derived materials, or combinations thereof. Synthetic compounds may also include pharmaceuticals and drugs, such as antibiotics.

[0051] Materials of interest present in compositions of the present disclosure may be chemically bonded to the hydrogel. Alternatively, materials of interest present in compositions of the present disclosure may not be chemically bonded to the hydrogel.

[0052] The thiol-containing compound may include compounds represented by Formula (I): (I), wherein: R of Formula (I) is an unsubstituted hydrocarbyl, a substituted hydrocarbyl, or a functional comprising at least one element from Group 13-17 of the periodic table of the elements.

[0053] An “unsubstituted hydrocarbyl” refers to a group that consists of hydrogen and carbon atoms only. Non-limiting examples of unsubstituted hydrocarbyl include an alkyl group having any suitable number of carbon atoms such as from 1 to 300 carbon atoms, such as from 1 to 100 carbon atoms, such as from 1 to 40 carbon atoms, such as from 1 to 20 carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, and tert-butyl, pentyl, hexyl, heptyl, octyl, ethyl-2-hexyl, isooctyl, nonyl, n-decyl, isodecyl, or isomers thereof; a cycloaliphatic group having from 3 to 20 carbon atoms such as, for example, cyclopentyl or cyclohexyl; an aromatic group having from 6 to 20 carbon atoms such as, for example, phenyl or naphthyl; or any combination thereof.

[0054] A “substituted hydrocarbyl” refers to an unsubstituted hydrocarbyl in which at least one hydrogen of the unsubstituted hydrocarbyl has been substituted with at least one heteroatom or heteroatom-containing group, such as one or more elements from Group 13- 17 of the periodic table of the elements, such as one or more elements from Group 13-17 of the periodic table of the elements, such as halogen (F, Cl, Br, or I), O, N, Se, Te, P, As, Sb,PCT Patent Application Attorney Docket No.: UWYO / 0122PC S, B, Si, Ge, Sn, Pb, and the like, such as C(O)R*, C(C)NR*2, C(O)OR*, NR*2, OR*, SeR*, TeR*, PR*2, AsR*2, SbR*2, SR*, SOx(where x = 2 or 3), BR*2, SiR*3, GeR*3, SnR*3, PbR*3, and the like, where R* is, independently, hydrogen or unsubstituted hydrocarbyl, or where at least one heteroatom has been inserted within the unsubstituted hydrocarbyl.

[0055] Substituted hydrocarbyls may include glycols, polyethylene glycols, combinations thereof, among others.

[0056] Unless specified to the contrary or the context clearly indicates, when a compound is described herein such that a particular isomer, enantiomer or diastereomer of the compound is not specified, for example, in a formula or in a chemical name, that description is intended to include each isomer, diastereomer, and enantiomer of the compound described individual or in any suitable combination.

[0057] The thiol-containing compound may include one thiol functional group or a plurality of thiol functional groups such as two, three, four, or more thiol functional groups.

[0058] The thiol-containing compound may include glutathione, cysteine, N- acetylcysteine, homocysteine, dithiothreitol, lipoic acid, a thiolated polymer (for example, a monothiolated polyethylene glycol), a derivative thereof, or combinations thereof.

[0059] The thiol-containing compound may be derived from a prothiol. A prothiol is a compound that becomes a thiol-containing compound in the presence of a reaction component or a reaction conditions (UV light, temperature, etc.). An illustrative, but non- limiting, example of a prothiol may include lipoic acid. Lipoic acid may be converted to a dithiol under various conditions or in the presence of various reactants. For example, the prothiol may be converted to a thiol-containing compound during photopolymerization.

[0060] The hydrogel of compositions described herein may be formed from a hydrogel forming solution. Hydrogel forming solutions may include one or more polymer precursors (also referred to herein as photoreactive monomers) that that produce radicals during radical-initiated polymerization of the one or more polymer precursors. Hydrogel forming solutions may further include one or more linkers (for example, PEG-dithiol linkers), one or more photoinitiators, solvent(s), other component(s). Hydrogel forming solutions may further include one or more thiol-containing compounds.

[0061] The one or more photoreactive monomers used to form the hydrogel contain photoreactive functional groups chemically attached to, e.g., polyethylene glycol (PEG), gelatin, or combinations thereof. Illustrative, but non-limiting, examples of photoreactivePCT Patent Application Attorney Docket No.: UWYO / 0122PC functional groups include an alkene functional group, an acrylate functional group, a methacrylate functional group, an acid functional group, a thiol functional group, or combinations thereof. Upon irradiation, the photoreactive monomers (with or without co- reactants, such as linkers described below) may react to form a hydrogel, for example, hydrogel feature 103 (a post), hydrogel particle (polymerized hydrogel droplet made using polymerization control device 150), or other structure. The hydrogel may comprise, consist essentially of, or consist of a polymer network. The polymer network may be porous, degradable, or combinations thereof.

[0062] Photoreactive monomers may include PEG-based monomers, such as polyethylene glycol norbornene (PEGNB), polyethylene glycol diacrylate (PEGDA), PEG methacrylate, polyethylene glycol di-photodegradable acrylate (PEGdiDPA), derivatives thereof, or combinations thereof.

[0063] Photoreactive monomers may include non-PEG-based monomers such as acrylates, acids (e.g., lactic acid, hyaluronic acid), gelatin, collagen, or combinations thereof. For example, polylactic acid (PLA), acrylated hyaluronic acid, gelatin methacrylate (GelMA), derivatives thereof, and combinations thereof may be used.

[0064] Illustrative, but nonlimiting, examples of photoreactive monomers may include polyethylene glycol norbornene (PEGNB), polyethylene glycol diacrylate (PEGDA), gelatin methacrylate (GelMA), PEG methacrylate, polylactic acid (PLA), polyethylene glycol di-photodegradable acrylate (PEGdiDPA), derivatives thereof, or combinations thereof, such as PEGNB, PEGDA, GelMA, or combinations thereof. Mixtures of one or more photoreactive monomers, for example, a mixture of PEGNB and PEGDA, may also be used, as well as mixtures that include non-PEG-based photolabile hydrogels such as gelatin methacrylate and / or photolabile hyaluronic acid.

[0065] Photoreactive monomers may be branched (e.g., ~20k 4-arm PEGNB and ~40k 8-arm PEGNB) or unbranched. Other PEG-based derivatives having varied reactive functional groups are also contemplated. The molecular weight and shape (e.g., number of arms on PEGNB) of the one or more photoreactive monomers, among other characteristics, may be changed. Changing the molecular weight and shape of the photoreactive monomers (as well as the linker) may enable the tuning of various properties of the hydrogel.

[0066] Molecular conformation of the photoreactive monomers may be varied to, for example, impart desired material properties to the hydrogel microenvironment. ForPCT Patent Application Attorney Docket No.: UWYO / 0122PC example, 1-arm molecular structures to 12-arm molecular structures may be used, such as 4-arm, 8-arm, or 12-arm molecular structures, such as 4-arm PEGNB, 8-arm PEGNB, 12- arm PEGNB, or combinations thereof.

[0067] A molecular weight of the one or more photoreactive monomers may be in a range from about 100 Da to about 75,000 Da, such as from about 250 Da to about 50,000 Da, such as from about 5,000 Da to about 50,000 Da, such as from about 10,000 Da to about 45,000 Da, such as from about 15,000 Da to about 40,000 Da, such as from about 20,000 Da to about 35,000 Da, such as from about 25,000 Da to about 30,000 Da. Illustrative, but non-limiting, examples of the molecular weight of the photoreactive monomer may be in a range from about 250 Da to about 15,000 Da, 10,000 Da, such as from about 500 Da to about 12,000 Da, such as from about 1,000 Da to about 11,000 Da, such as from about 2,000 Da to about 10,000 Da, such as from about 4,000 Da to about 8,000 Da. In some examples, the molecular weight of the one or more photoreactive monomers may be about 30,000 Da or less. A molecular weight of the one or more photoreactive monomers may range from MW1to MW2where each of MW1to MW2(in Da) is, independently, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1,000, about 1,500, about 2,000, about 2,500, about 3,000, about 3,500, about 4,000, about 4,500, about 5,000, about 5,500, about 6,000, about 6,500, about 7,000, about 7,500, about 8,000, about 8,500, about 9,000, about 9,500, about 10,000, about 10,500, about 11,000, about 11,500, about 12,000, about 12,500, about 13,000, about 13,500, about 14,000, about 14,500, about 15,000, about 15,500, about 16,000, about 16,500, about 17,000, about 17,500, about 18,000, about 18,500, about 19,000, about 19,500, about 20,000, about 20,500, about 21,000, about 21,500, about 22,000, about 22,500, about 23,000, about 23,500, about 24,000, about 24,500, about 25,000, about 25,500, about 26,000, about 26,500, about 27,000, about 27,500, about 28,000, about 28,500, about 29,000, about 29,500, about 30,000, about 30,500, about 31,000, about 31,500, about 32,000, about 32,500, about 33,000, about 33,500, about 34,000, about 34,500, about 35,000, about 35,500, about 36,000, about 36,500, about 37,000, about 37,500, about 38,000, about 38,500, about 39,000, about 39,500, about 40,000, about 40,500, about 41,000, about 41,500, about 42,000, about 42,500, about 43,000, about 43,500, about 44,000, about 44,500, about 45,000, about 45,500, about 46,000, about 46,500, about 47,000, about 47,500, about 48,000, about 48,500, about 49,000, about 49,500, or about 50,000, as long as MW1< MW2.PCT Patent Application Attorney Docket No.: UWYO / 0122PC Higher or lower molecular weights of the one or more photoreactive monomers are contemplated. The molecular weight of the photoreactive monomer refers to the number average molecular weight (Mn). The Mn is the Mn provided by the manufacturer of the photoreactive monomer.

[0068] Suitable organic and / or aqueous solvents may be utilized as a portion of the hydrogel forming solution. Such organic and / or aqueous solvents may include water, distilled water, saline, phosphate buffered saline, appropriate biologically compatible liquid, or combinations thereof.

[0069] A concentration of the one or more photoreactive monomers in the hydrogel forming solution may be in a range from about 1 wt% to about 99 wt%, such as from about 2 wt% to about 90 wt%, such as from about 3 wt% to about 85 wt%, such as from about 4 wt% to about 80 wt%, such as from about 5 wt% to about 75 wt%, such as from about 10 wt% to about 70 wt%, such as from about 15 wt% to about 65 wt%, such as from about 20 wt% to about 60 wt%, such as from about 25 wt% to about 55 wt%, such as from about 30 wt% to about 50 wt%, such as from about 35 wt% to about 45 wt%, based on a total weight percent of the components of the hydrogel forming solution, the total wt% of the hydrogel forming solution is 100 wt%.

[0070] The hydrogel forming solution may optionally include one or more linkers, such as a dithiol linker, such as a polyethylene glycol-dithiol (PEG-dithiol) linker, a derivative thereof, or combinations thereof. PEG-dithiol is a PEG having two thiol groups. The linkers are also photoreactive monomers. The linkers are different from the thiol-containing compound.

[0071] When a dithiol linker is utilized, the photoreactive monomer(s) react with the thiol-containing monomer(s) via, e.g., a step-growth polymerization reaction occurring between the ene portion of the monomers and the thiol of the thiol-containing monomer.

[0072] A molecular weight of the one or more linkers (e.g., the PEG-dithiol linker) may be in a range from about 500 Da to about 10,000 Da, such as from about 1,000 Da to about 9,500 Da, such as from about 1,500 Da to about 9,000 Da, such as from about 2,000 Da to about 8,500 Da, such as from about 2,500 Da to about 8,000 Da, such as from about 3,000 Da to about 7,500 Da, such as from about 3,500 Da to about 7,000 Da, such as from about 4,000 Da to about 6,500 Da, such as from about 4,500 Da to about 6,000 Da, such as from about 5,000 Da to about 5,500 Da. In some examples, the molecular weight of the linkerPCT Patent Application Attorney Docket No.: UWYO / 0122PC may be in a range about 6,000 Da or less, such as from about 500 Da to about 6,000 Da, such as from about 1,000 Da to about 5,000 Da, such as from about 1,500 Da to about 4,500 Da, such as from about 2,000 Da to about 4,000 Da, such as from about 2,500 Da to about 3,500 Da. In some embodiments, the molecular weight of the one or more linkers may range from MW3to MW4where each of MW3to MW4(in Da) is, independently, about 500, about 600, about 700, about 800, about 900, about 1,000, about 1,500, about 2,000, about 2,500, about 3,000, about 3,500, about 4,000, about 4,500, about 5,000, about 5,500, about 6,000, about 6,500, about 7,000, about 7,500, about 8,000, about 8,500, about 9,000, about 9,500, or about 10,000, as long as MW3< MW4. The molecular weight of the linker refers to the number average molecular weight (Mn). The Mn is the Mn provided by the manufacturer of the linker. Higher or lower molecular weights of the one or more linkers are contemplated. Illustrative, but non-limiting, examples of PEG-dithiol linkers include 1.5k PEG-dithiol, 3.5k PEG-dithiol, 5k PEG-dithiol, or combinations thereof.

[0073] A concentration of the one or more linkers (e.g., PEG-dithiol) in the hydrogel forming solution may be in a range from about 1 mM to about 50 mM, such as from about 5 mM to about 45 mM, such as from about 10 mM to about 40 mM, such as from about 15 mM to about 35 mM, such as from about 20 mM to about 30 mM.

[0074] The hydrogel forming solution may further include one or more photoinitiators. Illustrative, but non-limiting, examples of photoinitiators may include lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP) photoinitiator, 2-hydroxy-2-methyl propiophenone (e.g., Irgacure™ 1173, Darocur™ 1173), and combinations thereof. A concentration of the one or more photoinitiators in the hydrogel forming solution may be in a range from about 0.0001 wt% to about 1 wt%, such as from about 0.001 wt% to about 0.9 wt%, such as from about 0.01 wt% to about 0.5 wt%, such as from about 0.05 wt% to about 0.1 wt%, based on the total wt% of the components of the hydrogel forming solution.

[0075] The hydrogel forming solution may further include one or more thiol-containing compounds such as those thiol-containing compounds described herein. A concentration of the one or more thiol-containing compounds present in the hydrogel forming solution may be in a range from about 1 mM to about 200 mM, such as from about 2 mM to about 175 mM, such as from about 3 mM to about 150 mM, such as from about 5 mM to about 100 mM, such as from about 10 mM to about 70 mM, such as from about 20 mM to about 60 mM, such as from about 25 mM to about 55 mM, such as from about 30 mM to about 50PCT Patent Application Attorney Docket No.: UWYO / 0122PC mM, such as from about 35 mM to about 45 mM, such as about 40 mM. Alternatively, a concentration of the one or more thiol-containing compounds present in the hydrogel forming solution may be in a range from about 1 µM to about 100 mM, such as from about 100 µM to about 80 mM, such as from about 500 µM to about 60 mM, such as from about 1 mM to about 50 mM, such as from about 5 mM to about 40 mM, such as from about 10 mM to about 30 mM. Alternatively, a concentration of the one or more thiol-containing compounds present in the hydrogel forming solution may be in a range from about 1 µM to about 10 mM, such as from about 10 µM to about 1 mM, such as from about 50 µM to about 800 µM, such as from about 100 µM to about 600 µM, such as from about 200 µM to about 500 µM, such as from about 300 µM to about 500 µM.

[0076] As described herein, the thiol-containing compound does not become part of the hydrogel network. That is, the thiol-containing compound is not chemically bonded to the hydrogel. During photopolymerization, the thiol-containing compounds react with ROS that would be deleterious to the material of interest, such as cells.

[0077] The hydrogel forming solution may further include one or more materials of interest such as those materials of interest described herein, such as a single cell. A concentration of the one or more materials of interest present in the hydrogel forming solution may be in a range from about 3% w / v to about 90% w / v, such as from about 10% w / v to about 60% w / v, such as from about 15% w / v to about 50% w / v, such as from about 20% w / v to about 40% w / v. Alternatively, a concentration of the one or more materials of interest present in the hydrogel forming solution may be in a range from about 1 mM to about 200 mM, such as from about 2 mM to about 175 mM, such as from about 3 mM to about 150 mM, such as from about 5 mM to about 100 mM, such as from about 10 mM to about 70 mM, such as from about 20 mM to about 60 mM, such as from about 25 mM to about 55 mM, such as from about 30 mM to about 50 mM, such as from about 35 mM to about 45 mM, such as about 40 mM. Alternatively, a concentration of the one or more materials of interest present in the hydrogel forming solution may be in a range from about 1 µM to about 100 mM, such as from about 100 µM to about 80 mM, such as from about 500 µM to about 60 mM, such as from about 1 mM to about 50 mM, such as from about 5 mM to about 40 mM, such as from about 10 mM to about 30 mM Alternatively, a concentration of the one or more materials of interest present in the hydrogel forming solution may be in a range from about 1 µM to about 10 mM, such as from about 10 µM toPCT Patent Application Attorney Docket No.: UWYO / 0122PC about 1 mM, such as from about 50 µM to about 800 µM, such as from about 100 µM to about 600 µM, such as from about 200 µM to about 500 µM, such as from about 300 µM to about 500 µM

[0078] In some embodiments, which may be combined with other embodiments, a concentration of the one or more materials of interest present in the hydrogel forming solution is such that a single cell is encapsulated within a hydrogel (such as a hydrogel post or a hydrogel particle).

[0079] Any suitable molar ratio of the material of interest to the thiol-containing compound may be utilized. For example, a molar ratio of the material of interest to the thiol- containing compound may be less than 10,000:1, such as less than 1,000:1, such as less than 500:1, such as in a range from about 1:1 to about 100:1, such as from about 1.1:1 to about 20:1, such as from about 1.2:1 to about 10:1, such as from about 1.4:1 to about 5:1, such as from about 1.5:1 to about 2.5:1, such as from about 1.5:1 to about 2:1, such as about 1.5:1 (material of interest:thiol-containing compound).

[0080] In another embodiment is provided a drug delivery composition. The drug delivery composition includes a hydrogel such as those hydrogels described above. The hydrogel may be a polymer network such as porous polymer network and / or degradable polymer network. The porous polymer network and / or degradable polymer network may be formed by a radical-initiated polymerization of component(s) present in the hydrogel forming solution as described herein.

[0081] The hydrogel may encapsulate a thiol-containing compound and a drug or drug formulation. the drug or drug formulation encapsulated is different from the thiol-containing compound. The thiol-containing compound is not chemically bonded to the hydrogel or to the polymer network. Such a drug delivery composition is adapted to provide sustained release of the drug or the drug formulation.

[0082] The drug or drug formulation may include a material of interest such as a biological material, a biologically-derived material, a synthetic material, or combinations thereof, such as those described above. Materials of interest present in drug delivery compositions of the present disclosure may be chemically bonded to the hydrogel. Alternatively, materials of interest present in drug delivery compositions of the present disclosure may not be chemically bonded to the hydrogel.PCT Patent Application Attorney Docket No.: UWYO / 0122PC

[0083] Thiol-containing compounds of the drug delivery composition include those described above. The hydrogel of the drug delivery compositions may be formed from one or more photoreactive monomers that produce radicals during radical-initiated polymerization such as those described above, such as PEGNB, PEGDA, GelMA, or combinations thereof. PEG-dithiol may be used as a linker.

[0084] The hydrogel of the compositions described herein or drug delivery compositions described herein may be in the form of a droplet or a plurality of droplets. The droplet or plurality of droplets may be in the form of an oil-in-water droplet or a particle. Alternatively, the hydrogel of the compositions described herein or drug delivery compositions described herein may be in the form of a feature or a plurality of features, such as a post or plurality of posts, attached to and extending above a channel or other surface of a microfluidic device. Such droplets and features, as opposed to a bulk hydrogel, allows for the design of different droplets with different properties to be present in a single composition.

[0085] Embodiments described herein also relate to a tissue scaffold. The tissue scaffold includes a plurality of hydrogel particles. The hydrogel particles may be in the form of those hydrogels described herein. Each of the hydrogel particles include a polymer network that may be formed by a radical-initiated polymerization of component(s) present in the hydrogel forming solution as described herein. The hydrogel particles encapsulate a material of interest and a thiol-containing compound in an interior of the plurality of hydrogel particles. The thiol-containing compound encapsulated within the hydrogel particles is not chemically bonded to the polymer network of the hydrogel particles.

[0086] While the hydrogel particles may act as a scaffold, the tissue scaffold optionally includes an additional a natural or synthetic biomaterial serving as a scaffold. Natural biomaterials may include collagen, gelatin, chitosan, alginate, hyaluronic acid, or combinations thereof. Synthetic biomaterials may include: PEG; modified PEG; poly(α- hydroxy esters) such as poly(glycolic acid) (PGA), poly(lactic acid) (PLA), their copolymers (PLGA), or combinations thereof; poly(N-isopropyl acrylamide) (PNIPAM); Pluronic block copolymers; or combinations thereof. Pluronic block copolymers are triblock copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO).

[0087] These hydrogel particles, also referred to as granular particles, provide various advantages over a bulk hydrogel. For example, the porosity as a result of accumulation ofPCT Patent Application Attorney Docket No.: UWYO / 0122PC microgels is advantageous for cellular and vascular invasion, which is in contrast to the fibrous encapsulation of bulk counterparts. In addition, hydrogel particles may be designed with different properties for tissue repair, including the ability for injection and delivery to a desired site of a tissue, porosity for improved cell recruitment and / or invasion, and modularity through the combination of different types of particles to create multifunctional treatment systems. In contrast, bulk hydrogels are not highly tunable.

[0088] The plurality of hydrogel particles may be porous or degradable. The plurality of porous and / or degradable hydrogel particles of tissue scaffolds described herein may be formed as described herein.

[0089] The thiol-containing compound present in tissue scaffolds of the present disclosure is encapsulated within the hydrogel particle, but is not chemically bonded to the hydrogel. Suitable thiol-containing compounds that are not chemically bonded to the hydrogel include those described above.

[0090] The material of interest present in tissue scaffolds described herein may include a biological material, a biologically-derived material, a synthetic material, or combinations thereof, such as those described herein. Materials of interest present in tissue scaffolds described herein of the present disclosure may be chemically bonded to the hydrogel particles. Alternatively, materials of interest present in tissue scaffolds described herein of the present disclosure may not be chemically bonded to the hydrogel particles.

[0091] The tissue scaffold may be injected / implanted into a desired location (site of injury, defect area, etc.).

[0092] In another embodiment is provided a three-dimensional (3D) printed article such as the article shown in FIG. 1A (article 100) described below. The 3D printed article includes a fluidic channel. As described herein, the 3D printed article may further include a hydrogel structure disposed in the fluidic channel. The hydrogel structure may be at least partially dehydrated or at least partially hydrated.

[0093] The 3D printed article may further include one or more hydrogel features (such as hydrogel features 103 or posts) coupled to and extending above the hydrogel structure. The one or more hydrogel features may be formed by a radical-initiated polymerization. The one or more hydrogel features of the 3D printed article may be at least partially dehydrated or at least partially hydrated. The one or more hydrogel features of the 3D printed article may encapsulate a material of interest and a thiol-containing compound. Suitable materialsPCT Patent Application Attorney Docket No.: UWYO / 0122PC of interest and thiol-containing compounds useful with the 3D printed article are described herein. Suitable thiol-containing compounds include those described herein. The thiol- containing compound present in the 3D printed article is not chemically bonded to the hydrogel feature or to the hydrogel structure.

[0094] The one or more hydrogel features of the 3D printed article may be formed by radical-initiated polymerization of component(s) present in the hydrogel forming solution as described herein.

[0095] The one or more hydrogel features of the 3D printed article may be porous, degradable or combinations thereof. The pores of the one or more hydrogel features may be large enough to release an encapsulated component such as the material of interest (for example, a cell), the thiol-containing compound, or both. Alternatively, the pores of the hydrogel may be large enough to release only the thiol-containing compound or materials that are similar in size to the thiol-containing compound.

[0096] Hydrogels such as droplets, particles, features, etc. described herein may be microscale (1 µm to 999 µm) in size, though other sizes are contemplated. The hydrogels may be highly tunable, and their properties may be engineered for various applications such as in endogenous tissue repair. Design of the hydrogels may be based on: the injectability of the hydrogels, the porosity such as packing density, size, and / or shape; the encapsulated material of interest; inter-hydrogel (for example, inter-particle) interactions between hydrogels; degradation ability; etc.

[0097] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use embodiments of the present disclosure, and are not intended to limit the scope of embodiments of the present disclosure. Efforts have been made to ensure accuracy with respect to numbers used but some experimental errors and deviations should be accounted for. Examples

[0098] In the examples, cells were tested as the material of interest. As described herein, the material of interest may be any suitable biological material, a biologically-derived material, a synthetic material, or combinations thereof. Example 1: Cytocompatibility of Microengineered PEGDA, GelMA, and PEGNB Hydrogels 1.1. IntroductionPCT Patent Application Attorney Docket No.: UWYO / 0122PC

[0099] Tissue, from the French word tisser, meaning to weave, describes the diverse collections of similar cells and their extracellular matrices (ECM) that work in concert to perform essential functions for a living organism. Understanding and recreating these adroit fabrications is a mission of tissue engineering, a field that has enabled myriad biotherapeutic technologies. Tissue engineering has revolutionized regenerative medicine, enhanced understanding of cellular pathologies, and proved invaluable in developing diagnosis and treatment methods for disease. At the core of tissue engineering techniques is the ability to recapitulate the natural complexity of native tissue constructs. The individual cells comprising a tissue interact with both each other and their unique microenvironment (niche) within the ECM directing vital cellular processes including migration, differentiation, and cell fate—ultimately determining the organization and function of tissue architecture. For example, a longstanding tissue engineering challenge has been the differentiation of chondrocytes for articular cartilage repair. The phenotypic characteristics of chondrocytes are dictated by precise microenvironmental cues on a cellular scale, and in order to produce cells in therapeutic quantities, these conditions must be recreated or cells will de- differentiate into unwanted phenotypes. To engineer functional tissues with physiologically accurate characteristics, there is a need to design biomaterials that mimic their endogenous counterparts at the level of the microenvironment.

[0100] An aspect of microengineered tissues are the biomaterials from which they are constructed, as they must combine features of high spatial and temporal resolution with cytocompatibility to create biologically functional environments on a micrometer scale. A conventional material for microengineered tissue is photoactive hydrogel. This class of materials mimics the water swollen matrix of endogenous ECM. Incorporating photopolymerizable moieties such as acryl groups enables facile local spatiotemporal manipulation that cannot occur with thermally or chemically crosslinked gels. Using this technique, a number of systems have been employed to enable micro modeling of a two- dimensional (2D) scaffold, producing modular “bottom-up” three-dimensional (3D) structures assembled like building blocks, or 3D print constructs using cytocompatible “bioinks”.

[0101] Despite these achievements, conventional microengineered tissues have consisted of hundreds or thousands of cells encapsulated within hydrogel width on the scale of hundreds to thousands microns, too large a scale to measure individual cell behaviors.PCT Patent Application Attorney Docket No.: UWYO / 0122PC Though advanced photopatterning techniques such as stereolithography and 2 photon laser patterning may generate features on the scale of a single micrometer, the application of these techniques in patterning live cells involves additional considerations.

[0102] Fabrication of microengineered hydrogel tissue constructs may be aided through the use of polydimethylsiloxane (PDMS)-based microfluidic devices. PDMS is used in bioengineering applications as it is inexpensive and easily fabricated from a photolithographically patterned master mold. Its high oxygen permeability makes it particularly suited to cell culture applications, and also enables high resolution patterning using oxygen inhibition of photopolymerization reactions. However, the ubiquitous nature of oxygen, coupled with the use of convenient oxygen permeable materials like PDMS, complicates the application of common photopolymerizable hydrogels due to generation of cytotoxic reactive ROS. While acrylated materials like PEGDA and gelatin methacrylate (GelMA) have been shown to be cytocompatible, enhanced oxygen transport over short length scales calls into question their efficacy as biomaterials for microengineering applications at cellular length scales, and has yet to be fully quantified. As microfabrication technologies continue to advance to cell-scale resolutions, there is a need to understand the impact of enhanced diffusion, ROS, and other factors on the viability of cells encapsulated in photopolymerizable materials.

[0103] To this end, and in some examples, cells were encapsulated in PEGDA, PEGNB, and / or GelMA in miniaturized hydrogels. PEG hydrogels are utilized due, for example, to their good biocompatibility, hydrophilic nature, and customizable chemistry that allows for integration of numerous bioactive compounds onto inert backbones spanning a range of sizes and shapes. To assess PEGDA as a potential biomaterial for microengineered tissue, cells were encapsulated at progressively decreasing length scales over a range of molecular weights.

[0104] Projection stereolithography via a digital light projecting device (DLP) was employed to enable the facile polymerization of PEGDA at multiple length scales (FIG. 1A), and showed a decrease in viability as length scales and molecular weight decreases, respectively. The experimental findings were then corroborated using a finite element reaction diffusion model to predict the concentration of peroxy-radicals within the polymerized area. The rapid decrease of cell viability in acrylate-based gels at single cell resolutions suggests considerable oxygen radical effects at the length scales necessary forPCT Patent Application Attorney Docket No.: UWYO / 0122PC microengineering the ECM. However, it was determined that these effects may be mitigated via, for example, inert gas purging during polymerization. Based on the findings, PEGDA may still have merit for use in microengineering applications, but when considering biomaterials for constructing tissues, size matters.

[0105] It was also determined that toxicity may be decreased by utilizing a thiol- containing compound that is not chemically (covalently) bonded to the hydrogel. For example, and in some embodiments, the thiol-containing compound may be characterized as “free flowing” inside or within the hydrogel. For example, the thiol-containing compound is not chemically (covalently) bonded to the polymerized monomer or polymer network of the hydrogel. That is, the polymerized monomer or polymer network of the hydrogel may be formed without chemically (covalently) bonding to the thiol-containing compound.

[0106] The “clear box” in FIG. 1A is oxygen permeable and served the same function as oil surrounding the hydrogel droplets. That is, the “posts” or “features” are akin to a 2D representation of oil-in-water droplets. Table 1 shows selected elements of an article 100 and selected elements utilized to form the article 100. Table 1

[0107] A procedure for forming the device shown in FIG. 1A is provided in the Examples section. Briefly, the PDMS microchamber 107 may be prepared by pouring PDMS onto a silicon wafer mold with microfluidic channels photopatterned on its surface, curing the mold, removing the PDMS microchamber from the silicon wafer, and punching an inlet and outlet into the PDMS microchamber 107. The PDMS microchamber 107, having a fluidic channel, may then be plasma bonded to glass slide 111. The glass slide 111, having the PDMS microchamber 107 disposed thereon, may be placed on automated stage 109. The height of the PDMS microchamber controls the height of the hydrogel feature 103.PCT Patent Application Attorney Docket No.: UWYO / 0122PC

[0108] To prepare the hydrogel features 103, shown as posts in FIG. 1A, a microchamber 105 may be filled with hydrogel forming solution via inlet tubing 101a. The hydrogel forming solution may additionally contain the material of interest, for example, cells. An ultraviolet (UV) light source 113, positioned under the stage, may then be used to expose the hydrogel forming solution to UV light 115. Software may be used to control feature width and geometry in an suitable pattern such as circle, pentagon, and star shapes. Exposure to UV light 115 patterns the hydrogel features 103. In this example, the hydrogel features are attached to the glass slide. In other examples, the hydrogel features may be coupled to and extend from a hydrogel structure. After patterning, tubings 101a / 101b, microchamber 105, and PDMS microchamber 107 may be removed, leaving the hydrogel features 103 (posts) on the glass slide 111. In this example, the microfluidic device includes PDMS microchamber 107 and the glass slide 111.

[0109] The “height” of the hydrogel features 103 (for example, posts) may be limited by oxygen. After a critical diameter from the center of the post, oxygen inhibits the polymerization reaction as described in the Examples section. Similarly, and as described herein, when forming hydrogel particles (droplets), the size of the hydrogel particles may be limited by oxygen. After a critical diameter from the center of the hydrogel particle, oxygen inhibits the polymerization reaction. The hydrogel feature, whether in the form of a post, particle, or some other form, serves to prevent invasion of the oxygen into the hydrogel feature and damage the material of interest. By preventing invasion of oxygen, ROS does not damage the material of interest.

[0110] FIG. 1B shows single cells encapsulated in hydrogel features 103 (microgel posts) of varying diameters—30 µm, 75 µm, and 100 µm to assess dimensional effects on viability. As shown, the single cells are encapsulated inside the posts.

[0111] FIG. 1C is a schematic of an example device for forming a hydrogel- encapsulated cell (or other material of interest) according to at least one embodiment of the present disclosure. Such hydrogel-encapsulated cells (or other materials of interest) produced may be in the form of microparticles. Polymerization control device 150 may be used for continuous production of hydrogel-encapsulated cells or other materials of interest.

[0112] Polymerization control device 150 includes a microfluidic device 151 having a fluidic channel 153. The fluidic channel 153 may have a diameter of micrometers (µm) to millimeters (mm). For example, the fluidic channel 153 may have a diameter from about 1PCT Patent Application Attorney Docket No.: UWYO / 0122PC µm to about 2 mm and / or a depth of about 1 µm to about 2 mm. One or more portions of the fluidic channel 153 may be in the form of loops, discussed below. The fluidic channel 153 includes a mixing area 162a where a hydrogel forming solution, discussed below, may be mixed with a material of interest (for example, a cell) and an oil, and a polymerization area 162b where monomers of the hydrogel forming solution polymerize to form hydrogels that encapsulate the material of interest.

[0113] As stated above, portions of the fluidic channel 153 may be in the form of loops. The loops enable control over, for example, the kinetics of mixing, the kinetics of polymerization, the exposure time for polymerization, and / or the gelation of the hydrogels. That is, the loops may enable uniform processing of microparticles. Other morphologies or shapes besides, or in addition to, loops are contemplated to enable processing of the microparticles. Such morphologies or shapes include spirals or other tortuous paths. That is, any suitable morphology or shape that extends the length of the fluidic channel 153 in, for example, the mixing area 162a and / or the polymerization area 162b would have the same or similar effect of controlling the exposure time so that the desired cross-linking may be achieved on a microfluidic chip with high-throughput droplet production capabilities.

[0114] The microfluidic device 151 has an opening 160 for introducing a hydrogel forming solution to the fluidic channel 153. The hydrogel forming solution may include a photoinitiator, a photoreactive monomer (PEGDA, PEGNB, GelMA, PEG-dithiol cross- linker, etc.), a reaction component, a solvent, or combinations thereof. A thiol-containing compound (for example, glutathione, cysteine, a thiolated polymer, etc.) may form part of the hydrogel forming solution or it may be added separately to the fluidic channel 153 via opening 160 or a separate opening. A material of interest, such as a cell in a buffer, may form part of the hydrogel forming solution or may be introduced to the fluidic channel 153 via opening 160 or a separate opening.

[0115] The microfluidic device 151 includes another opening 158 for introducing a suspension fluid to the fluidic channel 153. The suspension fluid may be an oil, such as a fluorocarbon oil. The oil may serve to pinch off the material and hydrogel forming solution into droplets and carry the droplets through the microfluidic device 151. Openings 158 and 160 are coupled to the fluidic channel 153. As shown, tubings are coupled to the individual openings 158, 160 to allow introduction of the oil, material of interest, thiol-containing compound, hydrogel forming solution, and / or other reaction components to the fluidicPCT Patent Application Attorney Docket No.: UWYO / 0122PC channel 153 of the microfluidic device 151. However, it is contemplated that introduction of the oil, material of interest, thiol-containing compound, hydrogel forming solution, and / or other reaction components to the microfluidic device 151 may be performed in other suitable ways, such as direct connecting Leuer lock type devices, snap-together microfluidic assemblies, and syringe-like devices, without departing from the scope of the present disclosure.

[0116] Although two openings are described, more or less openings may be used to introduce the oil, material of interest, thiol-containing compound, hydrogel forming solution, and / or other reaction components to the microfluidic device 151. The inset identified as 153a is a pictorial representation of the fluidic channel 153 showing droplets 154 (also known as oil-in-water droplets) in suspension fluid (for example, the oil). The aqueous portion of the oil-in water droplets (for example, droplets 154) may include a material of interest, a photoreactive monomer, a photoinitiator, a thiol-containing compound, other reaction component, or combinations thereof.

[0117] The fluidic channel 153 includes the polymerization area 162b. At the polymerization area 162b, monomers and / or reaction components of the droplets 154 polymerize to form, for example, a hydrogel particle 156, that suspends, encapsulates, retains, or otherwise holds a cell, a one or more cells, and / or another material(s) of interest. The hydrogel may be a hydrogel network comprising a polymer network made from the polymerization of one or more photoreactive monomers.

[0118] As shown, the fluidic channel 153 of the polymerization area 162b includes a suitable number of loops (and / or other suitable shape) to enable, for example, sufficient polymerization of the monomers and other reaction components as well as sufficient gelation of the hydrogels.

[0119] The polymerization control device 150 further includes a polymerization control device 155 optically and / or mechanically coupled to at least a portion of the fluidic channel 153. The polymerization control device 155 may be adapted to or configured to cause a polymerization reaction when the desired materials are within the polymerization area 162b. The polymerization control device 150 may include a UV-light source(s), such as a UV lamp, UV light source concentrated via lenses, and / or microscope objective, or laser, that polymerizes the one or photoreactive monomers and / or reaction components to form the hydrogel. Coupling of the polymerization control device 155 may take any suitable forms.PCT Patent Application Attorney Docket No.: UWYO / 0122PC For example, the microfluidic device 151 may be placed on top of, below, or otherwise adjacent to, the polymerization control device 155. The UV light source may be located in a stand-alone unit outside of the microfluidic device 151.

[0120] FIGS.1D and 1E are exemplary images of the polymerized hydrogels within the fluidic channel 153 of the polymerization area 162b. A portion of the image shows cells in a hydrogel droplet. After polymerization, the hydrogel particles 156 (for example, the hydrogel-encapsulated material of interest(s)) move toward the fluidic channel exit 164 where the hydrogel particle 156 may be collected via any suitable collection unit 172, for example, flask, centrifuge tube, reservoir, vessel, or the like. Other materials (byproducts, suspension fluid, unreacted materials, etc.) may exit the fluidic channel exit 164 along with the hydrogel-encapsulated cell(s). Accordingly, the hydrogel-encapsulated cell(s) or compositions comprising the hydrogel encapsulated cell(s) may be purified, or otherwise isolated, from the other materials exiting the microfluidic device 151. Although FIG. 1E shows that there are multiple materials of interest inside an individual hydrogel particle 156, only one material of interest, such as a single cell, may be inside the individual hydrogel particle 156.

[0121] Movement of the various materials (for example, suspension fluid, material of interest, photoreactive monomers, photoinitiators, aqueous solvents, and / or reaction components, etc.) from the one or more openings 158, 160 to the fluidic channel exit 164 may be controlled by any suitable method, such as capillary action, laminar flow, temperature, a pumping mechanism (for example, a syringe pump, pressure pump, or piezoelectric pump), electrodes, and the like. Such elements controlling the movement may be placed at either opposing ends of the device, opposite ends, or along various regions along a length of the fluidic channel 153. 1.2. Materials and Methods 1.2.1. Device Preparation

[0122] Sylgard 184 Polydimethylsiloxane (PDMS) (Dow Corning) was poured onto silicon wafers molds with straight microfluidic channels of varying depth photopatterned onto their surface. The molds were vacuumed to remove air bubbles and cured at 70°C for 3 hours. Microfluidic devices were removed from the molds using a scalpel to cut around the features. The PDMS microfluidic devices were punched with an inlet and outlet hole, then plasma bonded with a PDC-002 plasma cleaner (Herrick) to No. 1.5 glass slides.PCT Patent Application Attorney Docket No.: UWYO / 0122PC Nitrogen purge chambers were constructed in an analogous manner and were plasma bonded to the top of devices directly over the microfluidic channel. Completed devices were placed on a 37°C hot plate until use. 1.2.2. Cell Preparation

[0123] Madin-Darby Canine Kidney (MDCK) cells (ATCC) at 80% confluency were trypsinized for 12 minutes, then 4 ml of Dubelco’s Modified Eagle Medium (DMEM, Genesee) containing 10% fetal bovine serum (FBS, Gibco) was added to neutralize the trypsin. That volume was then spun down at 200 revolutions per minute (rpm) for 5 minutes, the media decanted, and then resuspended in 1ml of phosphate-buffered saline (PBS, Fisher) containing 3% FBS. 1.2.3. Synthesis of Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) Photoinitiator

[0124] The initiator species LAP was synthesized according to the following procedure. 3.0 g of 2,4,6-trimethylbenzoyl chloride (Sigma Aldrich, USA) was added dropwise to a 250 mL round-bottom flask containing an equimolar amount of dimethyl phenylphosphonite and stirred at about room temperature under nitrogen for 8 to 24 hours. A four-fold excess of lithium bromide (LiBr, Sigma Aldrich, USA) dissolved in 100 mL of methyl ethyl ketone (MEK, Sigma Aldrich, USA) was added to the round-bottom flask and the resulting mixture was heated to 50° C for 10 minutes. White crystalline salts were formed upon cooling to about room temperature over a period of about 8 to 24 hours. Product crystals were filtered on a Buchner funnel and rinsed with ice-cold MEK, then placed under vacuum until a constant weight was achieved. LAP was confirmed via 400 MHz proton nuclear magnetic resonance (NMR) using a suitable deuterated solvent such as deuterated toluene (toluene-d8). 1.2.4. Hydrogel Forming Solution Preparation

[0125] The following hydrogel forming solutions included the biological material of interest, which in these examples, are MDCK cells.

[0126] PEGDA hydrogel forming solution.2,000 kilodalton (kD), 3,400 kD, 6,000 kD, and / or 10,000 kD molecular weight PEGDA (Sigma) was added to 1x PBS at 20 wt%, and vortexed for 5 min. LAP photoinitiator was synthesized, added to 1x PBS at 1 wt%, and vortexed for 2 min. MDCK cells at a concentration of 105 cells per µL in PBS were obtained and kept at a temperature of 37°C via water bath until ready for use. Aliquots of PEGDAPCT Patent Application Attorney Docket No.: UWYO / 0122PC (for example, 10,000 kD) and LAP were added to the cell solution to achieve 10 wt% and 0.014 wt% by total mass, respectively, then vortexed gently for 10 seconds.

[0127] Mixed PEGNB / PEGDA hydrogel forming solution. PEGDA (3,500 kD molecular weight) was added to 1x PBS at 40 wt%, and vortexed. LAP photoinitiator was synthesized and added to 1x PBS at 4 wt% and vortexed. MDCK cells were obtained at a concentration of 105 cells per ml in PBS and kept at a temperature of 37°C until ready for use. PEGDA and LAP were added to the solution of PBS with cells at 8.2 wt% and 0.3 wt%, respectively, by total mass, and then vortexed gently.

[0128] 1,500 kD molecular weight of PEG-dithiol was added to PEGNB (Sigma) at 20 mmol and vortexed. LAP photoinitiator was synthesized, added to 1x PBS at 4 wt%, and vortexed. MDCK cells were obtained at a concentration of 105 cells per ml in PBS and kept at a temperature of 37°C until ready for use. PEGNB and LAP were added to the solution of PBS with cells at 17 wt% and 0.01 wt%, respectively, by total mass, and then vortexed gently.

[0129] When a thiol-containing compound is utilized, and that thiol-containing compound is one that is not chemically bonded to the resultant hydrogel, the thiol-containing compound may be added directly to the hydrogel forming solution in any suitable amount such as those described herein such as in a range from about 1 µM to about 200 mM. For example, 1 µL to 1,000 µL of a thiol-containing compound such as glutathione may be added to the hydrogel forming solution, and then vortexed gently. 1.2.5. Microgel Fabrication

[0130] The microgel fabrication apparatus included a Polygon 400 digital micromirror device (DMD) (Mightex) attached to the rear aperture of an IX 81 inverted microscope (Olympus) with a Retiga 2000R camera (Q Image). MetaMorph software (Molecular Devices) was used to control image visualization and stage automation.

[0131] A hydrogel forming solution (for example, that described in Section 1.2.4) was immediately added to a pre-warmed microfluidic channel device and placed back on a 37°C hotplate for 10 min or until cells settled to the bottom of the channel. The microfluidic channel device was then placed on the microscope stage and a 20× LUCPlan FLN objective (Olympus) was used to focus at the bottom of the channel. Tygon tubing connected to a nitrogen source was inserted into the inlet of the purge chamber and pressurized to 7 psi. Mightex software was used to create a circular exposure area of desired size and the filePCT Patent Application Attorney Docket No.: UWYO / 0122PC loaded to the Polygon. The microscope stage was manually controlled to position cells in the center of the exposure area and exposed with 365 nm light at an intensity of 335 mW / cm2for 3 seconds. This was repeated for as many cells as could be exposed in 4 minutes. At the end of the 4-minute period the device was removed from the stage and flushed with 0.5 mL of LIVE / DEAD solution (Fisher) warmed to 37°C at standard concentration in PBS. This protocol was repeated in triplicate for channels of varying height at a constant feature size (for example, 250 µm), and features of varying diameter at a constant channel height (for example, 125 µm). It was then repeated in its entirety without the nitrogen purge. The “features” refer to the “posts” shown in FIG. 1A. The microgel can be fabricated by any suitable 3D printing method. Any suitable hydrogel forming solution may be utilized such as those described herein.

[0132] FIG.1C shows the oil-in-water encapsulation process to form microdroplets (for example, hydrogel particles 156). Here, the hydrogel particles encapsulate the cells and the thiol-containing compound.

[0133] In general, the process for cell encapsulation in a post is not fundamentally different from the process for cell encapsulation in hydrogel particles. A difference is continuous flow of cells and the hydrogel forming solution for the cell encapsulation in hydrogel particles as opposed to a closed reaction for cell encapsulation in a post. 1.2.6. Viability Analysis

[0134] Devices were placed in a Galaxy 170 S incubation chamber (New Brunswick) immediately after flushing and incubated for 20 minutes. After incubation they were removed and imaged using an IX 71 inverted microscope (Olympus). An X-Cite 120 LEDmini fluorescent illumination system (Excelitas Technologies) was used to provide white light, which was filtered through a FITC or Texas Red filter cube for visualizing fluorescence of live and dead stains, respectively. Images of encapsulated cells were captured in brightfield, FITC and Texas Red. Image J software was used to analyze viability of images using the color combining feature. Data was plotted in IGOR (Wavemetrics). 1.2.7. COMSOL Modeling

[0135] A reaction diffusion model of PEGDA microgels was created using COMSOL Multiphysics software. Cytotoxic reactive oxygen species (ROS) generated during microgel polymerization were modeled for features of varying width and height, and the data correlated with experimental viability results. The model was simplified based onPCT Patent Application Attorney Docket No.: UWYO / 0122PC assumptions made through experimental observation and analogous use in literature. These include constant UV intensity throughout the exposure depth given high intensity and shallow channel depth, diffusion of chemical species throughout the modeled area both during and after the reaction resulting in heterogeneous polymerization rates, oxygen diffusion, and ROS consumption, as well as a higher conversion rate of monomer due to higher than reported kD values for LAP photoinitiator. Oxygen flux through the PEGDA solution has been shown to be primarily dependent on its viscosity, and thus considerably impacted by the PEG molecular weight. Oxygen flux for the 10,000 kD PEGDA model was fit to experimental observations by determining the zone of polymerization inhibition for posts fabricated in a 30µm depth PDMS channel. The coefficient governing oxygen diffusion resulting from viscosity effects (Cab) was adjusted until the model reflected the experimentally determined zone of inhibition. Modeled PEGDA polymerization inhibition was determined by a local monomer conversion rate of 1% or less as specified in literature.

[0136] Selected transport and kinetic parameters as well as equations used in the reaction-diffusion model for the oxygen inhibited photopolymerization of PEGDA particles within droplets in a microfluidic device are shown in Table 2. As shown in Table 2, a second photoinitiator, 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone (I2959), may be utilized to form hydrogels described herein. Table 2PCT Patent Application Attorney Docket No.: UWYO / 0122PC

[0137] Various Reactions (Equations R1-R7) and Equations S1-S9 may be used for modeling, including the following:

[0138] 1. Reactions. ^ ^^^^^^^2^^∗ [Eq. R1]^^∗ ^ ^^ →^^^^∗ [Eq. R2]^^∗ ^ ^^∗ →^^^^ [Eq. R4]^ ^^∗^^^∗→^^^ [Eq. R5]^^∗ ^ ^^^ଶ^¾ೀ^మ^^^^^^ [Eq. R6] ^^^∗ ^ ^^ ^¾ೀ^మଶ ^^^^^^ [Eq. R7]

[0139] 2. Kinetics Equations.PCT Patent Application Attorney Docket No.: UWYO / 0122PC^^ ൌ^ெ^ ^ெ^^ െ 1 [Eq. S7]wherein: ^^^^ is photoinitiator; ^^∗is primary radical; ^^ is acrylate functional group; ^^∗is propogating radical; ^^ଶis oxygen; and ^^ is extent of conversion.

[0140] 3. Mass Transport Equations. Wilke-Chang Equation:wherein: ^^^is diffusivity of small molecule in liquid phase system; ^^ is absolute temperature (K); ^^ௌ^is solvent association coefficient; ^^ௌ^is solvent molecular weight; ^^^is molar volume at the normal boiling point of solute.1.2.8. Statistical Analysis

[0141] All data represent mean ± standard deviations from at least three independent experiments. Student’s t-test was used to analyze the statistical significance of the data. Values with a p-value less than 0.05 were considered statistically significant. 1.3. Non-limiting Results and Discussion

[0142] A system of experiments was designed to investigate the relationship between length scales and cell viability for microengineered PEGDA, PEGNB, and GelMA features (microgels). Attributes of these cylindrical environments were varied to decouple relevant variables, and results are reported as a function of cell viability. The concentration of PEGDA, PEGNB, and GelMA was tuned to yield gelation across all molecular weights at a constant mass fraction. The quantity of LAP photoinitiator was also tuned to yield polymerization across the range of PEGDA, PEGNB, and GelMA molecular weights and feature sizes. Because photoinitiators are inherently cytotoxic and produce damaging free radicals upon UV exposure, the LAP concentration was systematically decreased to the minimum mass fraction that yielded gelation at a constant UV intensity in the shortest microfluidic channel depth used (25 µm).PCT Patent Application Attorney Docket No.: UWYO / 0122PC

[0143] “Gelation” refers to a stable microfeature that retains its original shape once the polymerization reaction has completed. This metric was chosen because lower LAP concentrations have been shown to correspond with higher viabilities for cells encapsulated in PEGDA, PEGNB, and GelMA droplets, and a shorter channel depth corresponds to faster oxygen transport, shown to be proportional to the inhibition of PEGDA, PEGNB, and GelMA polymerization. Finding the minimum concentration of LAP to fabricate features in the shortest depth channel ensured polymerization across the range of feature heights while maintaining the maximum cell viability possible. 1.3.1. Digital Microenvironment Fabrication

[0144] To assist in the creation of microengineered niches of precise size and shape, maskless photolithographic fabrication technique using a Polygon 400 digital micromirror device (DMD) was employed. DMD based photolithography is superior to other commonly used methods. Here, conventional methods that rely on printed photomasks placed in front of an aperture, where the user disassembling the microscope and reattaching a new mask each time the projection is changed. Conventional methods are slow, impractical for creating heterogeneous features, lacks resolution, and is detrimental to equipment (particularly as the photomask may melt).

[0145] The Polygon 400 uses digital light processing (DLP) to microproject a desired fabrication design through the objective of a microscope, and has been employed in a number of biomaterial applications including micropatterning photosensitive PEG hydrogels and creating 3D scaffolds for cell seeding. DMD technology is a micro-electro- mechanical system that includes of over one million individually adjustable micromirrors that may be dynamically positioned on or off via a digital user interface to create arbitrary projection designs with user specified exposure times and intensities. It was found that dynamically adjusting the intensity and exposure time further minimizes the amount of cytotoxic LAP photoinitiator utilized to achieve crosslinking of the PEGDA, PEGNB, and GelMA microenvironments, and thereby minimized the production of free radical species generated during UV exposure. This provided a more sensitive analysis of the cellular response to ROS generation over short length scales (25-250 µm). It also ensured the structural homogeneity of all features through tight control of exposure parameters across experiments. The use of DMD technology for encapsulating cells is significant as it may be easily modified to produce stereolithographically printed 3D tissues. DMD enablesPCT Patent Application Attorney Docket No.: UWYO / 0122PC multimaterial bioprinting and presents an exciting possibility for on-demand microengineered tissue structures. 1.3.2. Effect of PEGDA Molecular Weight on Cell Viability

[0146] The molecular weight of PEGDA, PEGNB, and GelMA may play a major role in the kinetics of photopolymerization, but little information exists regarding its impact on cell viability—particularly in microfabricated features on the length scales of individual cells. The implication of mechanical cues in cellular behavior underscores the importance of this understanding when constructing microenvironments of varying physical properties tailored to specific cellular functions.

[0147] A recent study of microfluidic droplets illustrates that the viscosity of PEGDA hydrogel solution significantly affects the diffusive properties of oxygen on micrometer length scales—showing decreasing diffusive flux of oxygen with increasing PEGDA solution viscosity. Using this information coupled with the correlation between microscale ROS generation and cell viability, it was hypothesized that high molecular weight PEGDA, PEGNB, and GelMA with greater viscosity may have slower oxygen transport properties, and as a result generates less cytotoxic ROS during polymerization. To probe this hypothesis, the viability of cells encapsulated in PEGDA, PEGNB, and GelMA at both bulk and microscale resolutions was investigated.

[0148] A bulk polymerization system was designed to eliminate oxygen diffusion from the surroundings. This enabled the isolation of PEGDA molecular weight as the sole variable impacting cell viability. Here, cells suspended in three PEGDA solutions of differing molecular weights were polymerized within a plastic 96-well plate inside a pressurized purge chamber filled with argon to displace oxygen. Results shown in FIG.2A demonstrated equal viability (or not significantly different viability) across the range of molecular weights when oxygen is purged from the system.

[0149] Oxygen flux was reintroduced as a variable by repeating this experiment at microscale resolutions and ambient atmospheric conditions. Single cells were encapsulated at the bottom of a 125 µm deep microfluidic channel in 250 µm diameter cylindrical posts fabricated of four different molecular weight PEGDA solutions. The results shown in FIG. 2B show decreasing cell viability with decreasing molecular weight, indicating that oxygen causes reduced viability in cell microenvironments of varying PEGDA molecular weights. 1.3.3. Effect of Acryl Concentration on Cell ViabilityPCT Patent Application Attorney Docket No.: UWYO / 0122PC

[0150] To further test whether oxygen causes reduced viability in cell microenvironments of varying PEGDA molecular weights, 400 kD PEG monoacrylate (PEGMA) was added to PEGDA 10,000 kD and encapsulated cells using the same microfluidic system. The comparably low molecular weight of PEGMA (400 kD) enabled investigation of differing acryl group stoichiometry without appreciably changing the overall viscosity of the polymer solution, and thereby holding oxygen flux constant. The addition of more reactive acryl groups has the effect of increasing the reaction rate according to photopolymerization kinetics—a function of the acryl group concentration. Aggressive kinetic properties may be another variable that adversely affects encapsulated cell viability. Here, it was hypothesized that adding a low molecular weight PEGMA (400 kD) to PEGDA (10,000 kD) may provide a sensitive analysis of viability as a function of kinetics.

[0151] The results shown in FIG. 2C indicated no change in viability with increasing acryl group stoichiometry. This result suggested that reaction kinetics may not appreciably impact cell viability for PEGDA used in tissue engineering applications, and may provide further evidence that oxygen flux may be the primary contributor to the negative viability effects observed with decreasing molecular weight. 1.3.4. Effect of Microfabricated Feature Width on Cell Viability

[0152] The unintuitive nature of molecular weight effects on cell viability highlight the importance of length scale when examining microengineered feature phenomena. To build a more complete picture of this relationship, the effect of microenvironment width on cell viability in PDMS microfluidic channels was examined. Single cells were encapsulated in PEGDA (10,000 kD) microgel cylinders (“post” features) at the bottom of a 150 µm deep channel and varied the diameter of the post features between 250 µm and 30 µm (FIG.3B and FIG. 3C). This experiment was designed to probe the effects of radial oxygen flux on cell viability.

[0153] 10,000 kD PEGDA was used because of its viability characteristics—intended to maximize the sensitivity of the system. The 150 µm channel height ensured diffusive effects were a function primarily of post width, not height. The results shown in FIG. 3A indicated a trend of decreasing viability with decreasing microenvironment diameter.

[0154] To explain this behavior, these experimental findings were compared to a COMSOL reaction diffusion model. Modeling the average concentration of ROS species generated within a 15 µm × 15 µm circular area occupied by the cell yielded data showingPCT Patent Application Attorney Docket No.: UWYO / 0122PC that the drop in viability is accompanied by a rise in ROS concentration, with higher average ROS concentration corresponding to lower experimental viability (FIG.3A). This behavior demonstrated the same relationship between enhanced oxygen transport, higher ROS concentration local to the cell, and lower cell viability. Larger diameter microgels provided resistance to oxygen flux during polymerization, generating lower concentrations of radical species around the encapsulated cell and thereby less cytotoxic conditions. 1.3.5. Effect of microenvironment height on cell viability

[0155] Next the effect of changing channel height on cell viability in microgels of constant width was investigated. Cells were encapsulated at the bottom of PEGDA 10,000 microgel cylinders with a diameter of 250 µm (FIG. 3E and FIG. 3F) and varied the microfluidic channel height between 25 µm and 150 µm. An aim of this experiment was to investigate how vertical oxygen flux and accompanying ROS generation impacts cell viability in the microengineered features. The live / dead fluorescent image shown in FIG.3F of the image of FIG.3E demonstrated viable cells as single cells inside the microgel features. The results indicated constant cell viability of 100% with decreasing height until a critical height of about 25 µm, where viability drops to 0% (FIG.3D). While similar ROS levels of ~0.2 mol / m3are present in both width and depth, the decreased height of features in the 27 µm channel position the cells closer to a larger ROS flux from the oxygen diffusing in through the top of the PDMS device. This result indicates that more oxygen diffusing in through the top of the device causes more pronounced cell death than when oxygen is diffusing though the sides of the feature.

[0156] A COMSOL reaction diffusion model to determine the generation of ROS across a 12 µm × 12 µm surface area occupied by the cell within channels of varying height. This data showed a 66% increase in ROS generation at the site of the cell in the 25 µm depth channel, and a 10% increase in the 35 µm depth channel.

[0157] These results indicated that changing channel depth may not appreciably impact vertical oxygen transport though microgel features during fabrication until a height of about 35 µm, at which point decreasing channel depth was accompanied by rapid increase in oxygen flux available to be converted to ROS. This behavior was consistent with established oxygen permeability properties of PDMS. Depletion of oxygen in the microgel during polymerization induces rapid oxygen flux through the PDMS channel top, generating high levels of ROS near the microgel-channel interface. Much slower rates of oxygen and ROSPCT Patent Application Attorney Docket No.: UWYO / 0122PC transport though water within the pore network of the polymerizing hydrogel may cause the formation of well-defined areas of high and low ROS concentration, leading to the sudden drop in cell viability and accompanying jump in ROS concentration observed between the 35 µm and 25 µm depth channels. 1.3.6 Prediction of Peroxy Radical Concentration using COMSOL

[0158] To further understand the behavior of ROS in the microgel system, a 2D axisymmetric steady-state model was developed for free radical hydrogel photopolymerization. This 2D axisymmetric steady state model describes spatial and temporal concentration of chemical species within oil-in-water droplets to discrete areas of photolithographically patterned microgels within a PEGDA hydrogel forming solution (or other hydrogel forming solution). Briefly, the model was based on the interaction of two distinct regions within a PDMS microfluidic channel, a cylindrical area of PEGDA hydrogel forming solution exposed to UV light, and the surrounding unexposed hydrogel forming solution. The UV light source is shown at the bottom of FIGS.4A and 4B.

[0159] This model demonstrated the production of elevated concentrations of ROS near the interface of the PDMS device top (e.g., top of PDMS microchamber 107 in FIG. 1A) and the cylindrical UV exposure area as shown in FIG. 4A / 4B. The PDMS device top in FIG.4A / 4B is the side opposite the UV light and the PDMS device top in FIG. 1A is the top of the PDMS microchamber 107.

[0160] A free radical polymerization step sequence was modeled where the photolysis of LAP photoinitiator generates radical species that result in the preferential conversion of soluble oxygen to ROS. The ready diffusion of oxygen though PDMS enables the rapid replenishment of oxygen near this interface, and results in the generation of locally high concentrations of ROS.

[0161] FIG.4B shows modeled ROS concentration in an inert-gas purged environment. Without an external oxygen source, the vertical ROS concentration does not appreciably vary. The model showed that a small gradient of ROS may be present near the edge of the microgel due to reaction of dissolved oxygen and resulting oxygen flux through the surrounding hydrogel forming solution. The model also indicated that the dramatically slower diffusion rate of oxygen through PEGDA hydrogel forming solution compared to PDMS results in less ROS generated.PCT Patent Application Attorney Docket No.: UWYO / 0122PC

[0162] A sensitivity analysis was performed on the ROS concentration in a microgel feature modeled in 3 µm vertical increments for a 35 µm height channel under ambient atmospheric conditions (plot shown in FIG.4C) and inert gas purged channel (plot shown in FIG. 4D). The model indicated that the slow diffusion rate of ROS results in static concentrations across the microgel on experimental timescales (less than 1 hour). ROS concentrations within the microgel increase vertically from channel bottom to top under ambient conditions, and remain at their baseline across the height of the purged scenario. A locally higher ROS concentration is observed near the edge of the microgel in both ambient and purged cases. This may be the result of dissolved oxygen in the hydrogel forming solution as discussed above. The magnitude of this concentration gradient steadily decreases with increasing height and disappears entirely near the channel top for the ambient model. This behavior may be due to higher ROS concentrations throughout the microgel due to enhanced oxygen transport through the PDMS channel top.

[0163] Referring back to FIGS. 3A-3F, viability results suggested less sensitivity of encapsulated cells to ROS for varying microgel heights (FIG.3D) than diameters (FIG.3A), as modeled by average ROS production across a 15 µm diameter circular area approximating the space occupied by the cell within the microgel. To investigate this, the ROS concentrations at different heights were examined. It was found that there was not a significant increase until between 12 µm and 15 µm above the glass channel bottom. Given that the average diameter of MDCK cells is approximately 15 µm, and that the MDCK cells were allowed to fully settle to the bottom of the channel before encapsulation, the concentration of ROS local to the cells was near the baseline across the majority of cell surface area. Only the top of the assumed cylindrical cell was exposed to elevated ROS concentrations. This contrasts with the larger cell surface area exposed to radially elevated ROS concentration for decreasing microgel diameter. This data indicated that a difference in cell exposure area to ROS accounts for superior viability of cells at a comparatively higher ROS concentration in the 35 µm channel height. 1.3.7. N2 purging to rescue cell viability

[0164] To enable tissue printing at single cell resolutions with PEGDA, there is a need to overcome the adverse effects of ROS at these length scales. To accomplish this, single cells were encapsulated in PEGDA posts of diameter=35 µm, height=100 µm, and diameter=250 µm height=25 µm within a PDMS microfluidic channel purged with nitrogen.PCT Patent Application Attorney Docket No.: UWYO / 0122PC These microenvironment sizes were selected to evaluate viability under conditions with significant ROS accumulation from oxygen flux originating from the surrounding hydrogel forming solution in the case of post diameter restriction, and the PDMS channel top in the case of post height restriction.

[0165] Results shown in FIGS.5A-5D contrast the viability of cells encapsulated under standard atmospheric conditions (Air / O2) to cells encapsulated within an analogous PDMS channel under nitrogen (N2) purge. Cells in diameter-restricted features (FIG. 5B) demonstrated a 76 ± 6% increase in viability over ambient conditions, while those in height- restricted features (FIG. 5A) showed an 88 ± 10% increase. Cells in diameter restricted microgels stained for viability are shown in brightfield (FIG. 5C) and a live / dead color combined image reproduced in grayscale (FIG.5D). The dramatic increase in viability under nitrogen purge conditions further confirmed that the conversion of oxygen to ROS is responsible for compromised cell viability. It also demonstrated the ability of inert gas purging to enable superior synthetic tissues fabrication by providing a route for high viability printing at cellular resolutions. 1.3.8. GelMA Cytotoxicity

[0166] GelMA is denatured collagen chemically modified with acryl moieties that allow for photopolymerization into a hydrogel through free radicals provided by the decay of a photoinitiator. Broadly biocompatible, with robust cell adhesion and the ability to be degraded through endogenous cellular mechanisms, GelMA has found some success in tissue engineering applications and as a printable bioink. Despite GelMA’s widely accepted biocompatibility, it has been noted that radicals produced during the photopolymerization process decrease cell viability on the bulk scale. For example, in bulk 12 mm disks, cell viability may be reduced by about 25%.

[0167] Because GelMA is polymerized through a similar radical-initiated process as PEGDA, it was investigated whether GelMA microgels impart similar ROS stress and reduced cell viability at the same scales as the PEGDA microgels. Single cells were encapsulated in 5% w / w GelMA microgels in PDMS microchannels using the Polygon 400 system in a similar manner as described herein with cells in PEGDA microgels. 1.3.8.A. Effect of GelMA Microenvironment Size on Cell Viability

[0168] Similar to the results with PEGDA encapsulation of cells, it was determined that the distance from the edge of the polymerization interface determined cell viability. WhenPCT Patent Application Attorney Docket No.: UWYO / 0122PC a 50 µm microgel was polymerized around a single cell in microchannels of varying depths, a significant reduction in cell viability was observed in cells in shallower, 45 µm deep PDMS microchannels compared to 100 µm and 150 µm deep PDMS microchannels (FIG. 6A). Increasing the feature size to a 150 µm diameter microgel led to an increase in viability observed in all microchannel depths (FIG.6B). The 50 µm device produces more ROS than a 150 µm device leading to the lower cell viability for the 50 µm device. This may be a function of how far oxygen can diffuse during the polymerization reaction. In FIGS.6A and 6B, the “chamber” corresponds to PDMS microchamber 107 in FIG.1A.

[0169] As with PEGDA, it is likely that there is locally high ROS concentration at the edge of polymerized GelMA features and that increasing the distance from the edge of this area of concentrated ROS increases cell survival. Wider GelMA features increase the distance between the cell and highly concentrated ROS at the edge of the microgel with radially reduced ROS towards the cell at the center of the microgel. In deeper PDMS microchannels, there is a larger distance between the ROS rich microgel top, and the cell at the bottom of the microgel. 1.3.9. PEGNB Rescue

[0170] PEGNB is an alternative PEG-based hydrogel to PEGDA in which a norbornene functional group reacts with thiol-containing linkers such as PEG-dithiol. During polymerization, the norbornene groups on PEGNB macromers are bridged together forming a homogenous network (Scheme I) as opposed to PEGDA bridged. This step-growth polymerization reaction produces less radicals in the initiation step than its chain growth counterparts, and its linear crosslinking produces a more homogenous polymer network that reduces network contractions that are potentially deleterious to cell viability. As fewer radicals are produced, it was hypothesized that cell viability would increase as compared to PEGDA microgels produced under similar conditions.

[0171] Scheme I shows a proposed and non-limiting mechanism for PEGNB polymerizing through a radical initiated step-growth reaction. First, in I-1, the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) (A) absorbs light (hv) leading to bond cleavage and the formation of reactive radicals (B) and (C). Next, in I-2, two aldehyde radicals (C) react with the PEG-dithiol (D) to form two aldehydes (E) and a PEG-dithiol radical (F). Next, in I-3, the PEG-dithiol radical (F) reacts with two hydrogel PEGNB (G, where R=portion of PEGNB polymer) to form adduct (H) as a diradical. In I-4, diradicalPCT Patent Application Attorney Docket No.: UWYO / 0122PC (H) reacts with PEG-dithiol (D) to form the product PEGNB crosslinked with PEG-dithiol(I-4) Scheme I

[0172] FIGS.7A and 7B show plots of cell viability (%) versus feature height (in µm) and feature diameter (in µm), respectively, for cells polymerized in PEGNB features. In printing PEGNB microgels around single cells in PDMS microchannels, excellent cell viability was observed. Feature height (FIG.7A) and feature width (FIG.7A) did not impact cell viability as with PEGDA or GelMA microgels. Feature height also had no impact on cell viability with identical cell survival regardless of the device depth (FIG.7A). Overall, the data shown in FIGS. 7A and 7B indicated that cells have extremely high viability in PEGNB features compared to cells polymerized in PEGDA features or GelMA features.PCT Patent Application Attorney Docket No.: UWYO / 0122PC

[0173] Despite the superior cell viability observed with PEGNB hydrogels, it was also found that pure PEGNB hydrogels may have poor resolution and feature stability when printed at single cell resolutions. Efforts to overcome poor feature stability and resolution led to the development of a mixed mode PEGNB-PEGDA gel. Mixed mode PEGNB- PEGDA gel refers to a hydrogel made from a hydrogel forming solution that includes both PEGNB and PEGDA.

[0174] It was observed that the addition of PEGNB to PEGDA also considerably rescues cell viability over pure PEGDA gels as shown in FIGS. 8A and 8B. In FIG. 8A, “mixed” refers to the PEGNB-PEGDA gel. These findings indicated that PEGNB may possess significant advantages in cytocompatibility over PEGDA for high resolution bioprinting applications, and that a mixed mode PEGNB-PEGDA gel may maintain this cytocompatibility while providing enhanced structural integrity of features printed at single cell length scales.

[0175] For FIG.8B, the hydrogel was 10% PEGDA 700. A small portion of PEG-dithiol will covalently incorporate into the PEGDA network, but as PEGDA 700 polymerizes approximately 10x faster than PEG-dithiols, this is a minimal contribution to the gel. It is believed that the PEG-dithiol not chemically bound to / incorporated into the hydrogel may act as a thiol-containing compound described herein to mitigate the cytotoxic effects of ROS and rescue cell viability.

[0176] PEGDA gels with 20 mM of PEG-dithiol linker (thiol-linker in FIG.8B) show a rescue of viability similar to the PEGNB-PEGDA mixed mode gel whereas PEGNB- PEGDA microgels that do not contain any PEG-dithiol linker show similar poor viability as PEGDA gels. It was also found that adding 40 mM glutathione (GSH) to the hydrogel forming solution rescued cell viability in 27 µm deep channels which are conditions the inventors previously found to be extremely lethal to cells. Other thiol-containing compounds may be used to rescue cell viability.

[0177] GSH is a tripeptide produced by cells to deal with the free radicals produced by metabolism, contains a single oxidizable thiol. Reduced is present in millimolar quantities in cells and possesses strong radical scavenging and neutralizing abilities. While produced by cells to deal with the inevitable radicals produced by cellular biochemical processes, GSH present in solution protects cells from external ROS stress.PCT Patent Application Attorney Docket No.: UWYO / 0122PC

[0178] Scheme II shows a proposed and non-limiting radical scavenging mechanism of thiols through a thiyl intermediate that effectively removes ROS buildup during the polymerization reaction. In II-1, a representative superoxide anion (O2•–) reacts with glutathione (GSH) to produce an intermediate species (GS•••O2H). Next, in II-2, intermediate (GSO•–) reacts with glutathione, removing a proton and forming the stable intermediate GSOH and glutathione radical (GS•).(II-2) Scheme II

[0179] To elucidate the mechanisms behind the rescue of cell viability that the mixed mode PEGNB-PEGDA gel provided, it was hypothesized that the dithiolated-PEG linker used in the polymerization of PEGNB scavenged the ROS generated and thus limited the damage to cells. Upon absorption of a radical electron, a thiol group undergoes a reduction to a thiyl intermediate; thiyl intermediates have a greater affinity for radical electrons than molecular oxygen, likely preventing the accumulation of more toxic ROS intermediates. Direct electron transfer from superoxides to thiols to form thiyl intermediates is favorable, allowing for the removal of ROS from the polymerization area (Scheme II). The longer half- life of thiyl radicals compared to ROS may limit cellular toxicity. 1.4. Non-limiting Conclusions

[0180] The study presented herein investigated the cytocompatibility of microfabricated hydrogels formed from PEGDA, PEGNB, GelMA, or combinations thereof. Molecular weights of PEGDA, PEGNB, and GelMA, acryl concentration, and exposure to oxygen were systematically examined as culprits of compromised viability for cells encapsulated in microfabricated PEGDA, PEGNB, and GelMA microgel “posts”.

[0181] The presence of oxygen was determined to be a primary factor contributing to compromised cell viability likely due to the accumulation of cytotoxic ROS during polymerization. It was determined that microgel feature height and width may play a significant role in cell viability due to scale-dependent oxygen flux, and this data was correlated with ROS concentrations within PEGDA, PEGNB, and GelMA microgel posts via a COMSOL reaction diffusion model.PCT Patent Application Attorney Docket No.: UWYO / 0122PC

[0182] To overcome cytotoxic ROS effects at spatial resolutions on the order of single cells with PEGDA, a nitrogen-purged PDMS microfluidic channel was implemented to mitigate atmospheric oxygen flux through the channel during polymerization.

[0183] Cells encapsulated in GelMA microgels were constrained by similar limitations with both feature size and height for viability. In addition, it was found that thiolated-PEG (for example, a PEG-dithiol linker) and a thiol-containing compound (for example, glutathione) may be used to mitigate the cytotoxic effects of ROS and rescue cell viability, without affecting feature integrity.

[0184] Using these techniques, embodiments of the present disclosure enable fabrication of high viability microgels on a single-cell scale with acrylated materials and alkene materials. It is anticipated that these capabilities will enable the future engineering of superior synthetic tissues through high resolution cellular printing with exceptional viability. Embodiments Listing

[0185] The present disclosure provides, among others, the following embodiments, each of which may be considered as optionally including any alternate embodiments:

[0186] Embodiment A1. A composition, comprising: a hydrogel comprising, in polymerized form, one or more photoreactive monomers, the hydrogel encapsulating: a material of interest; and a thiol-containing compound, the thiol-containing compound not chemically bonded to the hydrogel, the thiol-containing compound different from the material of interest.

[0187] Embodiment A2. The composition according to Embodiment A1, wherein: the hydrogel is a polymer network; and / or the hydrogel is porous, degradable, or combinations thereof.

[0188] Embodiment A3. The composition according to any one of Embodiments A1- A2, wherein the hydrogel is formed by radical-initiated polymerization of the one or more photoreactive monomers.

[0189] Embodiment A4. The composition according to any one of Embodiments A1- A3, wherein the one or more photoreactive monomers comprises an alkene functional group,PCT Patent Application Attorney Docket No.: UWYO / 0122PC an acrylate functional group, a methacrylate functional group, an acid functional group, or combinations thereof.

[0190] Embodiment A5. The composition according to any one of Embodiments A1- A4, wherein the one or more photoreactive monomers comprises poly(ethylene glycol) norbornene, poly(ethylene glycol) diacrylate, gelatin methacrylate, or combinations thereof.

[0191] Embodiment A6. The composition according to any one of Embodiments A1- A5, wherein the hydrogel further comprises, in polymerized form, one or more thiol linkers, the one or more thiol linkers different from the thiol-containing compound.

[0192] Embodiment A7. The composition according to any one of Embodiments A1- A6, wherein the hydrogel is: (a) in the form of a droplet or a plurality of droplets; or (b) in the form of a post attached to a channel or other surface of a microfluidic device.

[0193] Embodiment A8. The composition according to any one of Embodiments A1- A7, wherein the thiol-containing compound is represented by Formula (I): (I), wherein: R of Formula (I) is an unsubstituted hydrocarbyl, a substituted hydrocarbyl, or a functional comprising at least one element from Group 13-17 of the periodic table of the elements.

[0194] Embodiment A9. The composition according to any one of Embodiments A1- A8, wherein the thiol-containing compound comprises glutathione, cysteine, N- acetylcysteine, homocysteine, dithiothreitol, lipoic acid, or combinations thereof.

[0195] Embodiment A10. The composition according to any one of Embodiments A1- A9, wherein the thiol-containing compound comprises a prothiol, a compound derived from a prothiol, or combinations thereof.

[0196] Embodiment A11. The composition according to any one of Embodiments A1- A10, wherein the material of interest comprises a biological material, a biologically-derived material, a synthetic material, or combinations thereof, such as a cell, such as a single cell.

[0197] Embodiment A12. The composition according to any one of Embodiments A1- A11, wherein a molar ratio of the material of interest to the thiol-containing compound is less than 10,000:1, such as less than 1,000:1, such as less than 500:1, such as in a range from about 1:1 to about 100:1, such as from about 1.1:1 to about 20:1, such as from about 1.2:1 to about 10:1, such as from about 1.4:1 to about 5:1, such as from about 1.5:1 to about 2.5:1,PCT Patent Application Attorney Docket No.: UWYO / 0122PC such as from about 1.5:1 to about 2:1, such as about 1.5:1 (material of interest:thiol- containing compound).

[0198] Embodiment A13. A drug delivery composition, comprising: a hydrogel comprising, in polymerized form, one or more photoreactive monomers, the hydrogel encapsulating: a drug comprising a material of interest; and a thiol-containing compound, the thiol-containing compound not chemically bonded to the hydrogel, the thiol-containing compound different from the material of interest.

[0199] Embodiment A14. The drug delivery composition according to Embodiment A13, wherein the drug delivery composition provides sustained release of the drug.

[0200] Embodiment A15. The drug delivery composition according to any one of Embodiments A13-A14, wherein: the hydrogel is a polymer network; and / or the hydrogel is porous, degradable, or combinations thereof.

[0201] Embodiment A16. The drug delivery composition according to any one of Embodiments A13-A15, wherein the hydrogel is formed by radical-initiated polymerization of the one or more photoreactive monomers.

[0202] Embodiment A17. The drug delivery composition according to any one of Embodiments A13-A16, wherein the one or more photoreactive monomers comprises an alkene functional group, an acrylate functional group, a methacrylate functional group, an acid functional group, or combinations thereof.

[0203] Embodiment A18. The drug delivery composition according to any one of Embodiments A13-A17, wherein the one or more photoreactive monomers comprises poly(ethylene glycol) norbornene, poly(ethylene glycol) diacrylate, gelatin methacrylate, or combinations thereof.

[0204] Embodiment A19. The drug delivery composition according to any one of Embodiments A13-A18, wherein the hydrogel further comprises, in polymerized form, one or more thiol linkers, the one or more thiol linkers different from the thiol-containing compound.

[0205] Embodiment A20. The drug delivery composition according to any one of Embodiments A13-A19, wherein the thiol-containing compound is represented by Formula (I):PCT Patent Application Attorney Docket No.: UWYO / 0122PC (I), wherein: R of Formula (I) is an unsubstituted hydrocarbyl, a substituted hydrocarbyl, or a functional comprising at least one element from Group 13-17 of the periodic table of the elements.

[0206] Embodiment A21. The drug delivery composition according to any one of Embodiments A13-A20, wherein the thiol-containing compound comprises glutathione, cysteine, N-acetylcysteine, homocysteine, dithiothreitol, lipoic acid, or combinations thereof.

[0207] Embodiment A22. The drug delivery composition according to any one of Embodiments A13-A21, wherein the thiol-containing compound comprises a prothiol, a compound derived from a prothiol, or combinations thereof.

[0208] Embodiment A23. The drug delivery composition according to any one of Embodiments A13-A22, wherein the material of interest comprises a biological material, a biologically-derived material, a synthetic material, or combinations thereof, such as a cell, such as a single cell.

[0209] Embodiment A24. The drug delivery composition according to any one of Embodiments A13-A23, wherein a molar ratio of the material of interest to the thiol- containing compound is less than 10,000:1, such as less than 1,000:1, such as less than 500:1, such as in a range from about 1:1 to about 100:1, such as from about 1.1:1 to about 20:1, such as from about 1.2:1 to about 10:1, such as from about 1.4:1 to about 5:1, such as from about 1.5:1 to about 2.5:1, such as from about 1.5:1 to about 2:1, such as about 1.5:1 (material of interest:thiol-containing compound).

[0210] Embodiment A25. A tissue scaffold, comprising: a plurality of hydrogel particles, each hydrogel particle comprising, in polymerized form, one or more photoreactive monomers, each hydrogel particle encapsulating: a material of interest; and a thiol-containing compound not chemically bonded to the hydrogel, the thiol-containing compound different from the material of interest.

[0211] Embodiment A26. The tissue scaffold according to Embodiment A25, wherein each hydrogel particle is porous, degradable, or both.PCT Patent Application Attorney Docket No.: UWYO / 0122PC

[0212] Embodiment A27. The tissue scaffold according to any one of Embodiments A25-A26, wherein each hydrogel particle is formed by radical-initiated polymerization of the one or more photoreactive monomers.

[0213] Embodiment A28. The tissue scaffold according to any one of Embodiments A25-A27, wherein the one or more photoreactive monomers comprises an alkene functional group, an acrylate functional group, a methacrylate functional group, an acid functional group, or combinations thereof.

[0214] Embodiment A29. The tissue scaffold according to any one of Embodiments A25-A28, wherein the one or more photoreactive monomers comprises poly(ethylene glycol) norbornene, poly(ethylene glycol) diacrylate, gelatin methacrylate, or combinations thereof.

[0215] Embodiment A30. The tissue scaffold according to any one of Embodiments A25-A29, wherein each hydrogel particle further comprises, in polymerized form, one or more thiol linkers, the one or more thiol linkers different from the thiol-containing compound.

[0216] Embodiment A31. The tissue scaffold according to any one of Embodiments A25-A30, wherein the thiol-containing compound is represented by Formula (I): (I),

[0217] wherein: R of Formula (I) is an unsubstituted hydrocarbyl, a substituted hydrocarbyl, or a functional comprising at least one element from Group 13-17 of the periodic table of the elements.

[0218] Embodiment A32. The tissue scaffold according to any one of Embodiments A25-A31, wherein the thiol-containing compound comprises glutathione, cysteine, N- acetylcysteine, homocysteine, dithiothreitol, lipoic acid, or combinations thereof.

[0219] Embodiment A33. The tissue scaffold according to any one of Embodiments A25-A32, wherein the thiol-containing compound comprises a prothiol, a compound derived from a prothiol, or combinations thereof.

[0220] Embodiment A34. The tissue scaffold according to any one of Embodiments A25-A33, wherein the material of interest comprises a biological material, a biologically- derived material, a synthetic material, or combinations thereof, such as a cell, such as a single cell.PCT Patent Application Attorney Docket No.: UWYO / 0122PC

[0221] Embodiment A35. The tissue scaffold according to any one of Embodiments A25-A34, wherein a molar ratio of the material of interest to the thiol-containing compound is less than 10,000:1, such as less than 1,000:1, such as less than 500:1, such as in a range from about 1:1 to about 100:1, such as from about 1.1:1 to about 20:1, such as from about 1.2:1 to about 10:1, such as from about 1.4:1 to about 5:1, such as from about 1.5:1 to about 2.5:1, such as from about 1.5:1 to about 2:1, such as about 1.5:1 (material of interest:thiol- containing compound).

[0222] Embodiment A36. A three-dimensional printed article, comprising: a fluidic channel; a hydrogel structure disposed in the fluidic channel; and one or more hydrogel features coupled to and extending above the hydrogel structure, the one or more hydrogel features comprising, in polymerized form, one or more photoreactive monomers, the one or more hydrogel features encapsulating: a material of interest; and a thiol-containing compound, the thiol-containing compound not chemically bonded to the hydrogel feature or the hydrogel structure, the thiol-containing compound different from the material of interest.

[0223] Embodiment A37. The three-dimensional printed article according to Embodiment A36, wherein: each hydrogel feature of the one or more hydrogel features is a polymer network; and / or each hydrogel feature of the one or more hydrogel features is porous, degradable, or combinations thereof.

[0224] Embodiment A38. The three-dimensional printed article according to any one of Embodiments A36-A37, wherein each hydrogel feature of the one or more hydrogel features is formed by radical-initiated polymerization of the one or more photoreactive monomers.

[0225] Embodiment A39. The three-dimensional printed article according to any one of Embodiments A36-A38, wherein the one or more photoreactive monomers comprises an alkene functional group, an acrylate functional group, a methacrylate functional group, an acid functional group, or combinations thereof.

[0226] Embodiment A40. The three-dimensional printed article according to any one of Embodiments A36-A39, wherein the one or more photoreactive monomers comprises poly(ethylene glycol) norbornene, poly(ethylene glycol) diacrylate, gelatin methacrylate, or combinations thereof.PCT Patent Application Attorney Docket No.: UWYO / 0122PC

[0227] Embodiment A41. The three-dimensional printed article according to any one of Embodiments A36-A40, wherein each hydrogel feature of the one or more hydrogel features further comprises, in polymerized form, one or more thiol linkers, the one or more thiol linkers different from the thiol-containing compound.

[0228] Embodiment A42. The three-dimensional printed article according to any one of Embodiments A36-A41, wherein the thiol-containing compound is represented by Formula (I): (I),

[0229] wherein: R of Formula (I) is an unsubstituted hydrocarbyl, a substituted hydrocarbyl, or a functional comprising at least one element from Group 13-17 of the periodic table of the elements.

[0230] Embodiment A43. The three-dimensional printed article according to any one of Embodiments A36-A42, wherein the thiol-containing compound comprises glutathione, cysteine, N-acetylcysteine, homocysteine, dithiothreitol, lipoic acid, or combinations thereof.

[0231] Embodiment A44. The three-dimensional printed article according to any one of Embodiments A36-A43, wherein the thiol-containing compound comprises a prothiol, a compound derived from a prothiol, or combinations thereof.

[0232] Embodiment A45. The three-dimensional printed article according to any one of Embodiments A36-A44, wherein the material of interest comprises a biological material, a biologically-derived material, a synthetic material, or combinations thereof, such as a cell, such as a single cell.

[0233] Embodiment A46. The three-dimensional printed article according to any one of Embodiments A36-A45, wherein a molar ratio of the material of interest to the thiol- containing compound is less than 10,000:1, such as less than 1,000:1, such as less than 500:1, such as in a range from about 1:1 to about 100:1, such as from about 1.1:1 to about 20:1, such as from about 1.2:1 to about 10:1, such as from about 1.4:1 to about 5:1, such as from about 1.5:1 to about 2.5:1, such as from about 1.5:1 to about 2:1, such as about 1.5:1 (material of interest:thiol-containing compound).

[0234] Embodiment B1. A composition, comprising: a hydrogel formed by radical-initiated polymerization, the hydrogel encapsulating:PCT Patent Application Attorney Docket No.: UWYO / 0122PC a material of interest; and a thiol-containing compound, the thiol-containing compound lacking a covalent bond to the hydrogel.

[0235] Embodiment B2. The composition of Embodiment B1, wherein the hydrogel is porous and / or degradable.

[0236] Embodiment B3. A drug delivery composition, comprising: a hydrogel comprising a porous polymer network and / or degradable polymer network formed by a radical-initiated polymerization, the hydrogel encapsulating: a thiol-containing compound, the thiol-containing compound lacking a covalent bond to the hydrogel; and a drug comprising a material of interest, wherein the drug delivery composition provides sustained release of the drug.

[0237] Embodiment B4. The composition of any one of Embodiments B1-B3, wherein the hydrogel is formed from one or more polymer precursors that produce radicals during radical-initiated polymerization of the one or more polymer precursors.

[0238] Embodiment B5. The composition of Embodiment B4, wherein the one or more polymer precursors comprises an acrylate group, a methacrylate group, or combinations thereof.

[0239] Embodiment B6. The composition of any one of Embodiments B4-B5 wherein the one or more polymer precursors comprises poly(ethylene glycol) diacrylate (PEGDA), gelatin methacrylate, or combinations thereof.

[0240] Embodiment B7. The composition of any one of Embodiments B1-B6, wherein the hydrogel is: (a) in the form of a droplet or a plurality of droplets; or (b) in the form of a post attached to a channel or other surface of a microfluidic device.

[0241] Embodiment B8. A tissue scaffold, comprising: a plurality of porous and / or degradable hydrogel particles, the hydrogel particles comprising a polymer network formed by a radical-initiated polymerization, the hydrogel particles encapsulating a material of interest and a thiol-containing compound in an interior of the plurality of porous and / or degradable hydrogel particles, the thiol-containing compound lacking a covalent bond to the polymer network of the hydrogel particles.

[0242] Embodiment B9. The tissue scaffold of Embodiment B8, wherein the plurality of porous and / or degradable hydrogel particles are formed from one or more polymerPCT Patent Application Attorney Docket No.: UWYO / 0122PC precursors that produce radicals during the radical-initiated polymerization of the one or more polymer precursors.

[0243] Embodiment B10. The tissue scaffold of Embodiment B9, wherein the one or more polymer precursors comprises an acrylate group, a methacrylate group, or combinations thereof.

[0244] Embodiment B11. The tissue scaffold of any one of Embodiments B9-10, wherein the one or more polymer precursors comprises poly(ethylene glycol) diacrylate (PEGDA), gelatin methacrylate, or combinations thereof.

[0245] Embodiment B12. The composition or tissue scaffold of any one of Embodiments B1-B11, wherein the thiol-containing compound is represented by Formula (I): (I), wherein: R of Formula (I) is an unsubstituted hydrocarbyl, a substituted hydrocarbyl, or a functional comprising at least one element from Group 13-17 of the periodic table of the elements.

[0246] Embodiment B13. The composition or tissue scaffold of any one of Embodiments B1-B12, wherein the thiol-containing compound comprises glutathione, cysteine, N-acetylcysteine, homocysteine, dithiothreitol, lipoic acid, a derivative thereof, or combinations thereof.

[0247] Embodiment B14. The composition or tissue scaffold of any one of Embodiments B1-B12, wherein the thiol-containing compound comprises glutathione.

[0248] Embodiment B15. The composition or tissue scaffold of any one of Embodiments B1-B13, wherein the thiol-containing compound is derived from a prothiol.

[0249] Embodiment B16. The composition or tissue scaffold of any one of Embodiments B1-B14, wherein the material of interest comprises a biological material, a biologically-derived material, a synthetic material, or combinations thereof.

[0250] Embodiment B17. The composition or tissue scaffold of any one of Embodiments B1-B15, wherein the material of interest comprises a cell.

[0251] Embodiment B18. A three-dimensional printed article, comprising: a fluidic channel; a hydrogel structure disposed in the fluidic channel; andPCT Patent Application Attorney Docket No.: UWYO / 0122PC one or more hydrogel features coupled to and extending above the hydrogel structure, the one or more hydrogel features formed by a radical-initiated polymerization, the one or more hydrogel features encapsulating: a material of interest; and a thiol-containing compound, the thiol-containing compound lacking a covalent bond to the hydrogel feature or the hydrogel structure.

[0252] Embodiment B19. The three-dimensional printed article of Embodiment B18, wherein the one or more hydrogel features are formed from one or more polymer precursors that produce radicals during a radical-initiated polymerization of the one or more polymer precursors.

[0253] Embodiment B20. The three-dimensional printed article of any one of Embodiments B18-B19, wherein the one or more polymer precursors comprises an acrylate group, a methacrylate group, or combinations thereof.

[0254] Embodiment B21. The three-dimensional printed article of any one of Embodiments B18-B20, wherein the one or more polymer precursors comprises poly(ethylene glycol) diacrylate (PEGDA), gelatin methacrylate, or combinations thereof.

[0255] Embodiment B22. The three-dimensional printed article of any one of Embodiments B18-B21, wherein the thiol-containing compound is represented by Formula (I): (I), wherein: R of Formula (I) is an unsubstituted hydrocarbyl, a substituted hydrocarbyl, or a functional comprising at least one element from Group 13-17 of the periodic table of the elements.

[0256] Embodiment B23. The three-dimensional printed article of any one of Embodiments B18-B22, wherein the thiol-containing compound comprises glutathione, cysteine, N-acetylcysteine, homocysteine, dithiothreitol, lipoic acid, a derivative thereof, or combinations thereof.

[0257] Embodiment B24. The three-dimensional printed article of any one of Embodiments B18-B23, wherein the thiol-containing compound is derived from a prothiol.

[0258] Embodiment B25. The three-dimensional printed article of any one of Embodiments B18-B24, wherein the material of interest comprises a biological material, a biologically-derived material, a synthetic material, or combinations thereof.PCT Patent Application Attorney Docket No.: UWYO / 0122PC

[0259] Embodiment B26. The three-dimensional printed article of any one of Embodiments B18-B25, wherein the one or more hydrogel features is porous, degradable, or combinations thereof.

[0260] All documents described herein are incorporated by reference herein, including any priority documents and / or testing procedures to the extent they are not inconsistent with this text. As is apparent from the foregoing general description and the specific embodiments, while forms of the embodiments have been illustrated and described, various modifications may be made without departing from the spirit and scope of the present disclosure. Accordingly, it is not intended that the present disclosure be limited thereby. Likewise, the term “comprising” is considered synonymous with the term “including.” Likewise whenever a composition, an element, a group of elements, or a method is preceded with the transitional phrase “comprising,” it is understood that we also contemplate the same composition, method, or group of elements with transitional phrases “consisting essentially of,” “consisting of,” “selected from the group of consisting of,” or “Is” preceding the recitation of the composition, element, elements, or method, and vice versa, such as the terms “comprising,” “consisting essentially of,” “consisting of” also include the product of the combinations of elements listed after the term.

[0261] In the foregoing, reference is made to embodiments of the disclosure. However, it should be understood that the disclosure is not limited to specific described embodiments. Instead, any combination of the following features and elements, whether related to different embodiments or not, is contemplated to implement and practice the disclosure. Furthermore, although embodiments of the disclosure may achieve advantages over other possible solutions and / or over the prior art, whether or not a particular advantage is achieved by a given embodiment is not limiting of the disclosure. Thus, the foregoing aspects, features, embodiments, and advantages are merely illustrative and are not considered elements or limitations of the appended claims except where explicitly recited in a claim(s). Likewise, reference to “the disclosure” shall not be construed as a generalization of any inventive subject matter disclosed herein and shall not be considered to be an element or limitation of the appended claims except where explicitly recited in a claim(s).

[0262] For purposes of this present disclosure, and unless otherwise specified, all numerical values within the detailed description and the claims herein are modified by “about” or “approximately” the indicated value, and consider experimental error andPCT Patent Application Attorney Docket No.: UWYO / 0122PC variations that would be expected by a person having ordinary skill in the art. For the sake of brevity, only certain ranges are explicitly disclosed herein. However, ranges from any lower limit may be combined with any upper limit to recite a range not explicitly recited, as well as, ranges from any lower limit may be combined with any other lower limit to recite a range not explicitly recited, in the same way, ranges from any upper limit may be combined with any other upper limit to recite a range not explicitly recited. For example, the recitation of the numerical range 1 to 5 includes the subranges 1 to 4, 1.5 to 4.5, 1 to 2, among other subranges. As another example, the recitation of the numerical ranges 1 to 5, such as 2 to 4, includes the subranges 1 to 4 and 2 to 5, among other subranges. Additionally, within a range includes every point or individual value between its end points even though not explicitly recited. For example, the recitation of the numerical range 1 to 5 includes the numbers 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, among other numbers. Thus, every point or individual value may serve as its own lower or upper limit combined with any other point or individual value or any other lower or upper limit, to recite a range not explicitly recited.

[0263] As used herein, the indefinite article “a” or “an” shall mean “at least one” unless specified to the contrary or the context clearly indicates otherwise. For example, embodiments comprising “a thiol-containing compound” include embodiments comprising one, two, or more thiol-containing compounds, unless specified to the contrary or the context clearly indicates only one thiol-containing compound is included.

[0264] While the foregoing is directed to embodiments of the present disclosure, other and further aspects of the disclosure may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.

Claims

PCT Patent Application Attorney Docket No.: UWYO / 0122PC Claims What is claimed is:

1. A composition, comprising: a hydrogel comprising, in polymerized form, one or more photoreactive monomers, the hydrogel encapsulating: a material of interest; and a thiol-containing compound, the thiol-containing compound not chemically bonded to the hydrogel, the thiol-containing compound different from the material of interest.

2. The composition according to claim 1, wherein: the hydrogel is a polymer network; and / or the hydrogel is porous, degradable, or combinations thereof.

3. The composition according to claim 1, wherein the hydrogel is formed by radical- initiated polymerization of the one or more photoreactive monomers.

4. The composition according to claim 1, wherein the one or more photoreactive monomers comprises an alkene functional group, an acrylate functional group, a methacrylate functional group, an acid functional group, or combinations thereof.

5. The composition according to claim 1, wherein the one or more photoreactive monomers comprises poly(ethylene glycol) norbornene, poly(ethylene glycol) diacrylate, gelatin methacrylate, or combinations thereof.

6. The composition according to claim 1, wherein the hydrogel further comprises, in polymerized form, one or more thiol linkers, the one or more thiol linkers different from the thiol-containing compound.

7. The composition according to claim 1, wherein the hydrogel is: (a) in the form of a droplet or a plurality of droplets; or (b) in the form of a post attached to a channel or other surface of a microfluidicPCT Patent Application Attorney Docket No.: UWYO / 0122PC device.

8. The composition according to claim 1, wherein the thiol-containing compound is represented by Formula (I): (I), wherein: R of Formula (I) is an unsubstituted hydrocarbyl, a substituted hydrocarbyl, or a functional comprising at least one element from Group 13-17 of the periodic table of the elements.

9. The composition according to claim 1, wherein the thiol-containing compound comprises glutathione, cysteine, N-acetylcysteine, homocysteine, dithiothreitol, lipoic acid, or combinations thereof.

10. The composition according to claim 1, wherein the thiol-containing compound comprises a prothiol, a compound derived from a prothiol, or combinations thereof.

11. The composition according to claim 1, wherein the material of interest comprises a biological material, a biologically-derived material, a synthetic material, or combinations thereof.

12. A drug delivery composition, comprising: a hydrogel comprising, in polymerized form, one or more photoreactive monomers, the hydrogel encapsulating: a drug comprising a material of interest; and a thiol-containing compound, the thiol-containing compound not chemically bonded to the hydrogel, the thiol-containing compound different from the material of interest.

13. The drug delivery composition according to claim 12, wherein: the hydrogel is a polymer network; and / or the hydrogel is porous, degradable, or combinations thereof.PCT Patent Application Attorney Docket No.: UWYO / 0122PC 14. The drug delivery composition according to claim 13, wherein the one or more photoreactive monomers comprises an alkene functional group, an acrylate functional group, a methacrylate functional group, an acid functional group, or combinations thereof.

15. The drug delivery composition according to claim 13, wherein the hydrogel further comprises, in polymerized form, one or more thiol linkers, the one or more thiol linkers different from the thiol-containing compound.

16. The drug delivery composition according to claim 13, wherein the thiol-containing compound is represented by Formula (I): (I), wherein: R of Formula (I) is an unsubstituted hydrocarbyl, a substituted hydrocarbyl, or a functional comprising at least one element from Group 13-17 of the periodic table of the elements.

17. The drug delivery composition according to claim 13, wherein the thiol-containing compound comprises glutathione, cysteine, N-acetylcysteine, homocysteine, dithiothreitol, lipoic acid, or combinations thereof.

18. A tissue scaffold, comprising: a plurality of hydrogel particles, each hydrogel particle comprising a hydrogel network, the hydrogel network comprising, in polymerized form, one or more photoreactive monomers, each hydrogel particle encapsulating: a material of interest; and a thiol-containing compound not chemically bonded to the hydrogel network, the thiol-containing compound different from the material of interest.

19. The tissue scaffold according to claim 18, wherein the one or more photoreactive monomers comprises an alkene functional group, an acrylate functional group, a methacrylate functional group, an acid functional group, or combinations thereof.PCT Patent Application Attorney Docket No.: UWYO / 0122PC 20. The tissue scaffold according to claim 18, wherein: each hydrogel particle further comprises, in polymerized form, one or more thiol linkers, the one or more thiol linkers different from the thiol-containing compound; the thiol-containing compound is represented by Formula (I): (I), wherein: R of Formula (I) is an unsubstituted hydrocarbyl, a substituted hydrocarbyl, or a functional comprising at least one element from Group 13-17 of the periodic table of the elements; or a combination thereof.

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