Hydrogel structures and methods of making and using same

Abstract

Disclosed herein are hydrogel structures and methods of making and using the same. For example, disclosed herein is a method of making a device comprising a hydrogel matrix and a first chamber in the hydrogel matrix, where the hydrogel matrix is ​​derived from a prepolymer and the first chamber is perfusable. The method can include, for example, blocking a first portion of a prepolymerized solution with a first photomask, where the prepolymerized solution includes a prepolymer, such that the prepolymerized solution includes an exposed portion and a first blocked portion. The method can further include irradiating the exposed portion of the prepolymerized solution and the first photomask with electromagnetic radiation, where the first photomask is substantially opaque to electromagnetic radiation. In some examples, the prepolymer in the exposed portion of the prepolymerized solution is photopolymerized to form a hydrogel matrix and the prepolymer in the first blocked portion is not photopolymerized to form the first chamber.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 273,222, filed October 29, 2021, and U.S. Provisional Application No. 63 / 273,224, filed October 29, 2021, both of which are incorporated by reference in their entireties herein. [Background technology]

[0002] Fabrication of current perfusable organ-on-a-chip platforms based on hydrogels requires laborious, time-consuming, and laser-based, equipment-intensive methodologies and is limited to native biological matrices. Faster, simpler methods based on synthetic hydrogels and strategies for patterning cells within organ-on-a-chip devices are needed. The compositions, devices, and methods disclosed herein address these and other needs. Summary of the Invention

[0003] In accordance with the objectives of the disclosed compositions, devices, and methods as embodied and broadly described herein, the disclosed subject matter relates to hydrogel structures, and methods of making and using same.

[0004] For example, disclosed herein is a method of making a device comprising a hydrogel matrix and a first chamber in the hydrogel matrix, where the hydrogel matrix is ​​derived from a prepolymer and the first chamber is perfusable. The method may further include, for example, blocking a first portion of a prepolymerized solution with a first photomask, where the prepolymerized solution comprises a prepolymer, such that the prepolymerized solution comprises an exposed portion and a first blocked portion. The method may further include irradiating the exposed portion of the prepolymerized solution and the first photomask with electromagnetic radiation, where the first photomask is substantially opaque to the electromagnetic radiation. In some examples, the prepolymer in the exposed portion of the prepolymerized solution photopolymerizes to form a hydrogel matrix and the prepolymer in the first blocked portion does not photopolymerize to form the first chamber.

[0005] Also disclosed herein are methods of making a device comprising a hydrogel matrix derived from a prepolymer, a first chamber in the hydrogel matrix, the first chamber being perfusable, and a second chamber in the hydrogel matrix, the second chamber being perfusable and fluidically independent from the first chamber. The method may include, for example, blocking a first portion of a prepolymerized solution with a first photomask and blocking a second portion of the prepolymerized solution with a second photomask, the prepolymerized solution including a prepolymer, such that the prepolymerized solution includes an exposed portion, a first blocked portion, and a second blocked portion. The method may further include irradiating the exposed portion of the prepolymerized solution, the first photomask, and the second photomask with electromagnetic radiation, the first photomask and the second photomask being substantially opaque to electromagnetic radiation. In some instances, the prepolymer in the exposed portion of the prepolymerized solution photopolymerizes to form a hydrogel matrix, the prepolymer in the first blocked portion does not photopolymerize to form a first chamber, and the prepolymer in the second blocked portion does not photopolymerize to form a second chamber.

[0006] In some examples, the method may further include removing the first photomask and the second photomask (if present) after irradiation.

[0007] In some examples, the method may further include rinsing the hydrogel device after irradiation to remove any remaining prepolymerization solution and / or prepolymer.

[0008] In some examples, the method may further include placing the prepolymerized solution in a mold defining a shape prior to blocking a first portion of the prepolymerized solution with a first photomask.

[0009] In some instances, the hydrogel matrix comprises a synthetic hydrogel.

[0010] In some examples, the hydrogel matrix is ​​derived from a prepolymer having a molecular weight of 0.5 to 200 kilodaltons (kDa). In some examples, the hydrogel matrix is ​​derived from a prepolymer having a molecular weight of 2 kDa to 40 kDa, 2 kDa to 25 kDa, or 2 kDa to 10 kDa.

[0011] In some instances, the hydrogel matrix is ​​derived from a branched prepolymer, hi some instances, the prepolymer has three or more branches, four or more branches, or eight or more branches.

[0012] In some instances, the hydrogel matrix is ​​derived from a prepolymer that includes polyethylene glycol or a derivative thereof.

[0013] In some examples, the hydrogel matrix is ​​derived from a prepolymer comprising poly(ethylene glycol) acrylate, poly(ethylene glycol) diacrylate (PEGDA), poly(ethylene glycol) diacrylate (PEGDMA), poly(ethylene glycol) diacrylamide (PEGDAAm), polyethylene glycol norbornene, polyethylene glycol dithiol, PEG-based peptide conjugates, cell-adhesive poly(ethylene glycol), MMP-sensitive poly(ethylene glycol), PEGylated fibrinogen, PEGylated collagen, PEGylated laminin, or a combination thereof. In some examples, the hydrogel matrix is ​​derived from a prepolymer comprising poly(ethylene glycol) vinyl sulfone, poly(ethylene glycol) acrylate, poly(ethylene glycol) maleimide, poly(ethylene glycol) norbornene, or a combination thereof. In some examples, the hydrogel matrix is ​​derived from a prepolymer comprising poly(ethylene glycol) maleimide, poly(ethylene glycol) norbornene, or a combination thereof. In some examples, the hydrogel matrix is ​​derived from a prepolymer comprising poly(ethylene glycol) norbornene.

[0014] In some examples, the prepolymerized solution includes the prepolymer in an amount of 1 wt % to 30 wt %. In some examples, the prepolymerized solution includes the prepolymer in an amount of 4 wt % to 10 wt %, or 1 wt % to 5 wt %.

[0015] In some examples, the prepolymerization solution further comprises a crosslinker and the hydrogel is further derived from the crosslinker. In some examples, the crosslinker is a multifunctional crosslinker. In some examples, the crosslinker comprises a multifunctional thiol. In some examples, the crosslinker comprises a dithiol.

[0016] In some instances, the hydrogel matrix is ​​further derivatized with one or more additional components, such as one or more additional monomers, prepolymers, ligands, chemical agents, therapeutic agents, photoinitiators, or combinations thereof. In some instances, the prepolymerization solution further comprises the one or more additional components.

[0017] In some instances, the hydrogel matrix is ​​further derived from a natural or biological prepolymer, hi some instances, the natural or biological prepolymer comprises fibrinogen, collagen, or a combination thereof.

[0018] In some examples, the prepolymerization solution further comprises a photoinitiator.

[0019] In some examples, the hydrogel matrix further comprises a chemical agent, a therapeutic agent, or a combination thereof dispersed therein. In some examples, the chemical agent and / or therapeutic agent are non-uniformly dispersed within the hydrogel matrix. In some examples, the chemical agent and / or therapeutic agent have a concentration that varies throughout the hydrogel matrix, such that the chemical agent and / or therapeutic agent has a compositional gradient throughout the hydrogel matrix. In some examples, the prepolymerization solution further comprises a chemical agent and / or therapeutic agent.

[0020] In some examples, the electromagnetic radiation includes UV radiation.

[0021] In some examples, the electromagnetic radiation includes one or more wavelengths between 10 nm and 900 nm. In some examples, the electromagnetic radiation includes one or more wavelengths between 100 nm and 900 nm or between 100 nm and 400 nm.

[0022] In some examples, the electromagnetic radiation is provided by a light source, and the light source is an artificial light source, hi some examples, the light source includes a light emitting diode (LED), a lamp, a laser, or a combination thereof.

[0023] In some examples, the exposed portions of the prepolymerized solution are photopolymerized for a time amount between 1 millisecond and 1 hour. In some examples, the exposed portions of the prepolymerized solution are photopolymerized for a time amount between 1 millisecond and 1 minute, between 1 millisecond and 10 seconds, or between 1 millisecond and 1 second.

[0024] In some instances, the hydrogel matrix exhibits swelling of 10% or less.

[0025] In some instances, the hydrogel matrix exhibits a shape fidelity of 50% or greater, 75% or greater, or 80% or greater.

[0026] In some examples, the hydrogel matrix has a storage modulus of greater than 0 Pa to 5000 Pa. In some examples, the hydrogel matrix has a storage modulus of greater than 0 Pa to 600 Pa, or greater than 0 Pa to 300 Pa.

[0027] In some examples, the hydrogel matrix is ​​configured to be stable for a time period of between 1 day and 3 months, hi some examples, the hydrogel matrix is ​​configured to be stable for a time period of between 1 and 7 days.

[0028] In some instances, the hydrogel matrix is ​​continuous.

[0029] In some instances, the hydrogel matrix is ​​monolithic.

[0030] In some instances, the hydrogel matrix is ​​porous.

[0031] In some instances, the hydrogel matrix is ​​biocompatible.

[0032] In some instances, the hydrogel matrix is ​​biodegradable.

[0033] In some instances, the hydrogel matrix comprises a photopolymerized polymer network.

[0034] In some instances, the hydrogel matrix comprises a crosslinked polymer network.

[0035] Also disclosed herein is a device produced by any of the methods disclosed herein. In some examples, the device is a microfluidic device.

[0036] Also disclosed herein are methods of use of any of the devices disclosed herein.

[0037] In some examples, the methods include using the device for diagnostics, disease modeling, regenerative medicine, drug screening, tissue modeling, or a combination thereof.

[0038] In some examples, the methods include use as a biomaterial substrate or scaffold, a cell culture substrate or platform, or a combination thereof.

[0039] In some examples, the method includes seeding the device (e.g., the first chamber and / or the second chamber) with cells and / or biomaterial and perfusing the device (e.g., the first chamber and / or the second chamber) with a solution. In some examples, the solution includes cell culture medium.

[0040] In some examples, the methods include using the device as a cell culture substrate for immune cells, B cells, lymphatic cells, dendritic cells, lung cells, intestinal cells, endothelial cells, hepatic cells, renal epithelial cells, or combinations thereof.

[0041] In some instances, the cultured cells exhibit cell viability of 50% or greater, 65% or greater, or 80% or greater after 4 days or more in the device.

[0042] In some examples, the methods include growing organoids using the device.

[0043] In some examples, the methods include growing a three-dimensional cell mass using the device.

[0044] In some examples, the methods include growing human organoids using the device.

[0045] In some examples, the method includes using the device to grow lymphoid follicular organoids, tonsillar organoids, intestinal organoids, lung organoids, or a combination thereof.

[0046] In some examples, the methods include growing human intestinal organoids using the device.

[0047] In some examples, the methods include using the device as a cell culture substrate, wherein the cultured cells exhibit cell phenotypic differentiation.

[0048] In some examples, the methods include using the device as a cell culture substrate, and the cultured cells include viable intestinal cells, differentiated epithelial cells, or combinations thereof that exhibit appropriate apical and basolateral marker localization.

[0049] In some examples, the methods include using the device as a cell culture substrate, where cultured cells three-dimensionally colonize the device.

[0050] Also disclosed herein are articles of manufacture that include any of the devices disclosed herein. In some examples, the articles include a biomaterial matrix or scaffold, a cell culture substrate or platform, or a combination thereof.

[0051] Additional advantages of the disclosed compositions, devices, and methods will be set forth in part in the description that follows, and in part will be apparent from the description. The advantages of the disclosed compositions, devices, and methods will be realized and obtained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosed compositions, devices, and methods as claimed.

[0052] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0053] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects of the disclosure and, together with the description, serve to explain the principles of the disclosure. [Brief description of the drawings]

[0054] [Figure 1] Schematic of the engineering of the multi-organ platform. [Diagram 2] Schematic diagram of immune follicles-on-a-chip. [Diagram 3] Schematic diagram of lymphoid tissue-accumulating intestinal organoids. [Figure 4] Schematic of the biomaterial-based approach to human lymphoid follicular organoids. [Diagram 5] Schematic of the biomaterial-based approach to human lymphoid follicular organoids. [Figure 6] Germinal center B cell count. [Figure 7] Schematic of the biomaterial-based approach to human lymphoid follicular organoids. [Figure 8] EMC-dependent / independent spreading of stromal CD40L cells and FDCs. [Figure 9] EMC-dependent / independent survival of stromal CD40L cells and FDCs. [Figure 10]Effect of polymer wt% on time stability and hydrogel stiffness. [Figure 11] Imaging of primary human B cell survival over time. [Figure 12] Comparison of primary human B cell viability in tonsils and PBMCs. [Figure 13] Tonsillar primary human B cell viability. [Figure 14] Primary human B cell viability in PBMCs. [Figure 15] Schematic of the microfluidic device for lymphoid follicle-on-chip engineering. [Figure 16] Image and geometric fidelity of various microfluidic devices for lymphoid follicle-on-chip engineering. [Figure 17] Schematic of the synthesis of thiol-norbornene photoclickable PEG hydrogels. [Figure 18] Cell viability in various hydrogels. [Figure 19] Degradation of hydrogels over time. [Figure 20] Cell viability in various hydrogels. [Figure 21] Schematic diagram of the development of lymphoid tissue-enriched intestinal organoids to study vaccine-mediated immunity. [Figure 22] PEG-4MAL density and adhesion peptide controls. [Figure 23] HIO viability and development validation of hiPSCs in PEG-4MAL. [Figure 24] HIO viability and development validation of hiPSCs in PEG-4MAL. [Diagram 25] Schematic diagram investigating the effect of PEG-4MAL density on HIO generation. [Figure 26] Images of HIOs in different hydrogels. [Figure 27] Images of intestinal cells from hiPSCs grown in hydrogels. [Figure 28]Image of the intestinal organoid-on-a-chip microchip system comprising an elastomeric device with a central hydrogel chamber for subsequent organoid culture and perfusion. [Figure 29] Characterization of composite PEG-4aNB hydrogel bulk and PEG-4MAL microgels containing human B cells. [Diagram 30] Schematic diagram of HIO and its uses. [Diagram 31] Schematic diagram of the synthetic hydrogel photopolymerization mechanism. [Diagram 32] Schematic of photopatterning PEG-4NB hydrogels for the fabrication of perfusable mini-intestinal structures using UV light and a photomask. [Diagram 33] Effect of crosslinker and weight percent on hydrogel swelling. [Diagram 34] Effect of PEG-4NB molecular weight on hydrogel swelling. [Diagram 35] Effect of temperature on hydrogel swelling. [Diagram 36] Shape fidelity of hydrogels. [Figure 37] 1 is an image of an exemplary device having a complex shape. [Figure 38] Schematic of seeding of HIOs in a gut-on-a-chip device. [Figure 39] Image showing the importance of medium perfusion on cell viability. [Diagram 40] Images showing the importance of medium perfusion on cell viability and device coverage. [Diagram 41] Images showing HIO colony formation growth in hydrogel devices. [Diagram 42] Image of Bullseye's hydrogel device. [Diagram 43] Image of Bullseye Design's hydrogel device. [Diagram 44] Image of Bullseye Design's hydrogel device. [Diagram 45] Fluorescence over time of the bullseye design hydrogel device. [Diagram 46]Schematic of tangential flow in a bull's-eye design hydrogel device. [Figure 47] Schematic of orthogonal flow in a hydrogel device using a bull's-eye design. [Figure 48] Image showing tangential flow in a bull's-eye design hydrogel device. [Figure 49] Images showing orthogonal flow in a bull's-eye design hydrogel device [Figure 50] MFI for hydrogel devices in bull's-eye design using tangential or orthogonal flow. [Figure 51] B cell viability in hydrogel devices of different compositions. [Figure 52] GCB in hydrogel devices of different compositions. [Diagram 53] Mean fluorescence intensity in hydrogel devices of different compositions. [Figure 54] Cell proliferation in hydrogel devices of different compositions. [Figure 55] Images of cell proliferation and clustering over time in PEG-4MAL-based hydrogel devices. [Figure 56] PEG-4NB: Images of cell proliferation and clustering over time in PEG-4NB-based hydrogel devices with a fibrin / collagen ratio of 1:0. [Figure 57] PEG-4NB: Images of cell proliferation and clustering over time in PEG-4NB-based hydrogel devices with a fibrin / collagen ratio of 3:1. [Figure 58] PEG-4NB: Images of cell proliferation and clustering over time in PEG-4NB-based hydrogel devices with a fibrin / collagen ratio of 1:1. [Figure 59] B cell viability in PEG-4NB hydrogel devices with various amounts of FC. [Figure 60] B cell viability in PEG-4NB hydrogel devices with various amounts of FC. [Figure 61] B cell viability in PEG-4NB hydrogel devices with various amounts of FC. [Figure 62] B cell viability in PEG-4NB hydrogel devices with various amounts of FC. [Figure 63] Schematic of an exemplary hydrogel device. [Figure 64] Images showing human B and T cell migration induced by a CXCL12 gradient within a hydrogel device. [Figure 65] Schematic of an exemplary hydrogel device. [Figure 66] Images showing cell differentiation in hydrogel devices containing PEG-4NB, fibrinogen, and collagen versus hydrogel devices containing PEG-4NB. [Figure 67] Schematic of patterning of native-based matrices using the PALM microbeam Zeiss microscope as a dissection tool. [Figure 68] Immunostaining of intestine on a chip after 4 days of culture. [Figure 69] Immunostaining of intestine on a chip after 4 days of culture. [Figure 70] Immunostaining of intestine on a chip after 4 days of culture. [Figure 71] Images showing colonization of hydrogel devices. [Figure 72] Images showing colonization of hydrogel devices. [Figure 73] Images showing colonization of hydrogel devices. [Figure 74] Images showing colonization of hydrogel devices. [Figure 75] Images showing colonization of hydrogel devices. [Figure 76] Images showing colonization of hydrogel devices at the first time point. [Figure 77] Image showing colonization of the hydrogel device at a second time point, a later time point than in FIG. 76. [Figure 78]Image showing colonization of hydrogel devices at a third time point, a later time point than in FIG. 77. [Figure 79] Image showing colonization of hydrogel devices at the fourth time point, a later time point than in Figure 78. [Figure 80] Image showing colonization of hydrogel devices at time point 5, a later time point than in Figure 79. [Figure 81] Image showing colonization of hydrogel devices at time point 6, a later time point than Figure 80. [Figure 82] Image showing colonization of hydrogel devices at time point 7, a later time point than in Figure 81. [Figure 83] Image showing colonization of hydrogel devices at time point 8, a later time point than in Figure 82. [Figure 84] Immunostaining of hiPSC-derived spheroids in the tubular intestine on a chip device after 7 days of culture. [Figure 85] Immunostaining of hiPSC-derived spheroids in the tubular intestine on a chip device after 7 days of culture. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0055] The compositions, devices, and methods described herein can be understood more readily by reference to the following detailed description of certain aspects of the disclosed subject matter and the examples included therein.

[0056] Before the present compositions, devices, and methods are disclosed and described, it is to be understood that the aspects described below are not limited to particular synthetic methods or to particular reagents, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting.

[0057] Also, throughout this specification, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the art to which the disclosed subject matter pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

[0058] General definition In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings.

[0059] Throughout the description and claims of this specification, the word "comprise" and other forms of that word, such as "comprising" and "comprises," are intended to include, but are not limited to, other additives, components, elements, or steps.

[0060] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "composition" includes mixtures of two or more such compositions, reference to an "agent" includes mixtures of two or more such agents, reference to a "component" includes mixtures of two or more such components, etc.

[0061] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and is meant to include instances in which the event or circumstance occurs and instances in which it does not occur.

[0062] As used herein, ranges may be expressed as from "about" one particular value and / or to "about" another particular value. "About" means within 5% of that value, for example, within 4, 3, 2, or 1% of that value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations by use of the antecedent "about," it is understood that the particular value forms another embodiment. It will be further understood that each endpoint of the range is significant both in relation to the other endpoint and independently of the other endpoint.

[0063] "Exemplary" means "an example of" and is not intended to convey an indication of a preferred or ideal embodiment. "Etc." is used in a limiting sense, but rather for descriptive purposes.

[0064] Values ​​may be expressed herein as "average" values. "Average" generally refers to a statistical average value.

[0065] By "substantially" is meant within 5%, for example, within 4%, 3%, 2%, or 1%.

[0066] "Exemplary" means "an example of" and is not intended to convey an indication of a preferred or ideal embodiment. "Etc." is used in a limiting sense, but rather for descriptive purposes.

[0067] It is understood that throughout this specification, the identifiers "first" and "second" are used merely to aid in distinguishing various components and steps of the disclosed subject matter. The identifiers "first" and "second" are not intended to imply any particular order, quantity, priority, or importance to the components or steps modified by these terms.

[0068] References in this specification and the concluding claims to parts by weight of a particular element or component in a composition indicate the weight relationship between the element or component and any other element or component in the composition or article for which the parts by weight are expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight of component Y, X and Y are present in a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are included in the compound.

[0069] Weight percentages (wt %) of components are based on the total weight of the formulation or composition in which the component is included, unless stated to the contrary.

[0070] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C, or combinations thereof" includes at least one of A, B, C, AB, AC, BC, or ABC, as well as BA, CA, CB, CBA, BCA, ACB, BAC, or CAB, if order is important in the particular context.

[0071] Continuing with this example, explicitly included are combinations containing one or more repeats of an item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc. One of ordinary skill in the art will understand that, unless otherwise clear from the context, there is typically no limit to the number of items or terms in any combination.

[0072] As used herein, "subject" refers to an individual. Thus, "subject" can include domestic animals (e.g., cats, dogs, etc.), livestock (e.g., cows, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, etc.), and birds. "Subject" can also include mammals, such as primates or humans. Thus, a subject can be a human or veterinary patient. The term "patient" refers to a subject under the care of a clinician, e.g., a physician.

[0073] "Biocompatible" and "biologically compatible," as used herein, generally refer to compounds and / or compositions that, together with any metabolic or degradation products thereof, are generally non-toxic to and do not cause significant adverse effects to normal cells and tissues when the cells and tissues are incubated (e.g., cultured) in their presence.

[0074] As used herein, the term "biodegradable" refers to materials or substances that undergo physical dissolution and / or chemical degradation under physiological conditions.

[0075] As used herein, "antimicrobial" refers to the ability to treat or control (e.g., reduce, prevent, treat, or eliminate) the growth of microorganisms at any concentration. Similarly, the terms "antibacterial," "antifungal," and "antiviral" refer to the ability to treat or control the growth of bacteria, fungi, and viruses, respectively, at any concentration.

[0076] As used herein, "reduce" or other forms of the word, such as "reducing" or "reduction," refers to a decrease in an event or characteristic (e.g., microbial population / infection). It is understood that the reduction is typically relative to some standard or expected value. For example, "reducing a microbial infection" means reducing the spread of a microbial infection compared to a standard or control.

[0077] As used herein, "prevent" or other forms of the word, such as "preventing" or "prevention," refers to stopping a particular event or characteristic, stabilizing or delaying the onset or progression of a particular event or characteristic, or minimizing the likelihood of a particular event or characteristic occurring. "Prevent" is typically more absolute than, for example, "reduce," and does not require a comparison to a control. As used herein, something can be reduced but not prevented, but something that is reduced can be prevented. Similarly, something can be prevented, resulting in a net reduction, but something that is prevented can also be reduced.

[0078] As used herein, "treat" or other forms of the word, such as "treated" or "treatment," refers to the administration of a composition or the implementation of a method to reduce, prevent, inhibit, or eliminate a particular characteristic or event (e.g., microbial growth or survival). The term "control" is used synonymously with the term "treat."

[0079] The term "anti-cancer" refers to the ability of any concentration to treat or control cell proliferation and / or tumor growth.

[0080] The term "therapeutically effective" refers to the amount of a composition used sufficient to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration does not necessarily require elimination, but only a reduction or alteration.

[0081] The term "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0082] Chemical Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0083] The organic moieties referred to in defining variable positions within the general formulae described herein (e.g., the term "halogen") are collective names for the individual substituents encompassed by the organic moiety. The prefixes Cn-Cm before a group or moiety indicate in each case the possible number of carbon atoms in the following group or moiety.

[0084] As used herein, the term "ion" refers to any molecule, molecular portion, molecular cluster, molecular complex, moiety, or atom that can be made to contain a charge (positive, negative, or both simultaneously within one molecule, molecular cluster, molecular complex, or moiety (e.g., a zwitterion)) or a charge. Methods for generating a charge on a molecule, molecular portion, molecular cluster, molecular complex, moiety, or atom are disclosed herein and can be accomplished by methods known in the art, such as protonation, deprotonation, oxidation, reduction, alkylation, acetylation, esterification, deesterification, hydrolysis, and the like.

[0085] The term "anion" is a type of ion and is included within the meaning of the term "ion." An "anion" is any molecule, part of a molecule (e.g., a zwitterion), cluster of molecules, molecular complex, moiety, or atom that contains or can be made to contain a net negative charge. The term "anion precursor" is used herein specifically to refer to a molecule that can be converted to an anion via a chemical reaction (e.g., deprotonation).

[0086] The term "cation" is a type of ion and is included within the meaning of the term "ion." A "cation" is any molecule, part of a molecule (e.g., a zwitterion), cluster of molecules, molecular complex, moiety, or atom that contains or can be made to contain a net positive charge. The term "cation precursor" is used herein specifically to refer to a molecule that can be converted to a cation via a chemical reaction (e.g., protonation or alkylation).

[0087] As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Exemplary substituents include, for example, those described below. The permissible substituents can be one or more and can be the same or different for appropriate organic compounds. For purposes of this disclosure, heteroatoms such as nitrogen can have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valence of the heteroatom. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. The terms "substituted" or "substituted with" also include the implicit proviso that such substitution is in accordance with the permissible valences of the substituted atom and substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation by rearrangement, cyclization, elimination, and the like.

[0088] "Z 1 ","Z 2 ","Z 3 " and "Z 4 " is used herein as a generic symbol to represent various specific substituents. These symbols can be any substituent, including but not limited to those disclosed herein, and if one instance is defined as a specific substituent, in another instance it may be defined as some other substituent.

[0089] The term "aliphatic," as used herein, refers to non-aromatic hydrocarbon groups, including branched and unbranched alkyl, alkenyl, or alkynyl groups.

[0090] As used herein, the term "alkyl" refers to a saturated, straight-chain or branched saturated hydrocarbon moiety. Unless otherwise specified, C1-C 24 (For example, C1~C 22 , C1~C 20 , C1~C 18 , C1~C 16 , C1~C 14 , C1~C 12 , C1~C 10, C1-C8, C1-C6, or C1-C4) alkyl groups are contemplated. Examples of alkyl groups include methyl, ethyl, propyl, 1-methyl-ethyl, butyl, 1-methyl-propyl, 2-methyl-propyl, 1,1-dimethyl-ethyl, pentyl, 1-methyl-butyl, 2-methyl-butyl, 3-methyl-butyl, 2,2-dimethyl-propyl, 1-ethyl-propyl, hexyl, 1,1-dimethyl-propyl, 1,2-dimethyl-propyl, 1-methyl-pentyl, 2-methyl-pentyl, 3-methyl-pentyl, 4-methyl-pentyl, 1,1-dimethyl-propyl ...propyl, 2-methyl-propyl, 2-methyl-propyl, 2-methyl-propyl, 2-methyl-propyl, 2-methyl-propyl, 2-methyl-propyl, 2-methyl-propyl, 2-methyl-propyl, 2-methyl-propyl, 2-methyl-propyl, 2-methyl-propyl, 2-methyl-propyl, 2-methyl-propyl, 2-methyl-propyl, 2-methyl-propyl, 2-methyl-propyl, 2-methyl-propyl, 2-methyl-propyl, Examples of alkyl groups include 1,2-ethyl-butyl, 1,2-dimethyl-butyl, 1,3-dimethyl-butyl, 2,2-dimethyl-butyl, 2,3-dimethyl-butyl, 3,3-dimethyl-butyl, 1-ethyl-butyl, 2-ethyl-butyl, 1,1,2-trimethyl-propyl, 1,2,2-trimethyl-propyl, 1-ethyl-1-methyl-propyl, 1-ethyl-2-methyl-propyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, etc. Alkyl substituents can be unsubstituted or substituted with one or more chemical moieties. Alkyl groups may be substituted with one or more groups, including, but not limited to, hydroxyl, halogen, acyl, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, cyano, carboxylic acid, ester, ether, ketone, nitro, phosphonyl, silyl, sulfoxo, sulfonyl, sulfone, sulfoxide, or thiol, provided that the substituents are sterically compatible and the rules of chemical bonding and strain energy are satisfied, as described below.

[0091] Throughout this specification, "alkyl" is used generally to refer to both unsubstituted and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substituent(s) on the alkyl group. For example, the term "halogenated alkyl" or "haloalkyl" specifically refers to an alkyl group substituted with one or more halides (halogens: e.g., fluorine, chlorine, bromine, or iodine). The term "alkoxyalkyl" specifically refers to an alkyl group substituted with one or more alkoxy groups, as described below. The term "alkylamino" specifically refers to an alkyl group substituted with one or more amino groups, as described below. If "alkyl" is used in one instance and a specific term, such as "alkylalcohol," is used in another instance, this does not mean that the term "alkyl" does not refer to the specific term, such as "alkylalcohol."

[0092] This practice is also used for other groups described herein. That is, a term such as "cycloalkyl" refers to both unsubstituted and substituted cycloalkyl moieties, although the substituted moieties may be further specifically identified herein. For example, a particular substituted cycloalkyl may be referred to as, for example, an "alkylcycloalkyl." Similarly, a substituted alkoxy may be specifically referred to as, for example, a "halogenated alkoxy," a particular substituted alkenyl may be, for example, an "alkenylalcohol," and the like. Again, the practice of using a general term such as "cycloalkyl" and a specific term such as "alkylcycloalkyl" does not imply that the general term does not also include the specific term.

[0093] As used herein, the term "alkenyl" refers to an unsaturated, straight-chain, or branched hydrocarbon moiety containing a double bond. Unless otherwise specified, C2-C 24 (For example, C2~C 22 , C2~C 20 , C2~C 18 , C2~C 16 , C2~C 14、C2~C 12 、C2~C 10, C2-C8, C2-C6, or C2-C4) alkenyl groups are contemplated. The alkenyl group may contain two or more unsaturated bonds.Examples include ethenyl, 1-propenyl, 2-propenyl, 1-methylthenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-1-butenyl, 2-methyl-1-butenyl, 3-methyl-1-butenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl. , 3-methyl-3-butenyl, 1,1-dimethyl-2-propenyl, 1,2-dimethyl-1-propenyl, 1,2-dimethyl-2-propenyl, 1-ethyl-1-propenyl, 1-ethyl-2-propenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-1-pentenyl, 2-methyl-1-pentenyl, 3-methyl-1-pentenyl, 4-methyl-1-pentenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, 1-methyl 1-methyl-3-pentenyl, 2-methyl-3-pentenyl, 3-methyl-3-pentenyl, 4-methyl-3-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1,1-dimethyl-2-butenyl, 1,1-dimethyl-3-butenyl, 1,2-dimethyl-1-butenyl, 1,2-dimethyl-2-butenyl, 1,2-dimethyl-3-butenyl, 1,3-dimethyl-1-butenyl, 1,3-dimethyl-2-butenyl, 1,3-dimethyl-3-butenyl, 2,2-dimethyl-3-butenyl dimethyl-1-butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 3,3-dimethyl-1-butenyl, 3,3-dimethyl-2-butenyl, 1-ethyl-1-butenyl, 1-ethyl-2-butenyl, 1-ethyl-3-butenyl, 2-ethyl-1-butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, 1,1,2-trimethyl-2-propenyl, 1-ethyl-1-methyl-2-propenyl, 1-ethyl-2-methyl-1-propenyl, and 1-ethyl-2-methyl-2-propenyl.The term "vinyl" refers to a group having the structure -CH=CH2, 1-propenyl refers to a group having the structure -CH=CH-CH3, and 2-propenyl refers to a group having the structure -CH2-CH=CH2. (Z. 1 Z 2 )C=C(Z 3 Z 4 Asymmetric structures such as alkene, alkoxy ...

[0094] As used herein, the term "alkynyl" refers to a straight or branched chain hydrocarbon moiety containing a triple bond. Unless otherwise specified, C2-C 24 (For example, C2~C 24 , C2~C 20 , C2~C 18 , C2~C 16 , C2~C 14 , C2~C 12 , C2~C 10, C2-C8, C2-C6, or C2-C4) alkenyl groups are contemplated. The alkynyl group may contain one or more unsaturated bonds. Examples include C2-C6-alkynyl, such as ethynyl, 1-propynyl, 2-propynyl (or propargynyl), 1-butynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 3-methyl-1-butynyl, 1-methyl-2-butynyl, 1-methyl-3-butynyl, 2-methyl-3-butynyl, 1,1-dimethyl-2-propynyl, 1-ethyl-2-propynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 3-methyl-1-pentynyl, Alkynyl substituents include 4-methyl-1-pentynyl, 1-methyl-2-pentynyl, 4-methyl-2-pentynyl, 1-methyl-3-pentynyl, 2-methyl-3-pentynyl, 1-methyl-4-pentynyl, 2-methyl-4-pentynyl, 3-methyl-4-pentynyl, 1,1-dimethyl-2-butynyl, 1,1-dimethyl-3-butynyl, 1,2-dimethyl-3-butynyl, 2,2-dimethyl-3-butynyl, 3,3-dimethyl-1-butynyl, 1-ethyl-2-butynyl, 1-ethyl-3-butynyl, 2-ethyl-3-butynyl, and 1-ethyl-1-methyl-2-propynyl. Alkynyl substituents can be unsubstituted or substituted with one or more chemical moieties. Examples of suitable substituents include, for example, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below.

[0095] As used herein, the term "aryl," as well as derivative terms such as aryloxy, refers to a group containing a monovalent aromatic carbocyclic group of 3 to 50 carbon atoms. An aryl group can contain a single ring or multiple fused rings. In some embodiments, an aryl group is a C6-C 10The term "aryl" includes aryl groups. Examples of aryl groups include, but are not limited to, benzene, phenyl, biphenyl, naphthyl, tetrahydronaphthyl, phenylcyclopropyl, phenoxybenzene, and indanyl. The term "aryl" also includes "heteroaryl," which is defined as a group that contains an aromatic group with at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. The term "non-heteroaryl," which is also included in the term "aryl," defines a group that contains an aromatic group that does not contain a heteroatom. Aryl substituents may be unsubstituted or substituted with one or more chemical moieties. Examples of suitable substituents include, for example, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described herein. The term "biaryl" is a specific type of aryl group and is included in the definition of aryl. Biaryl refers to two aryl groups bonded together through a fused ring structure, as in naphthalene, or through one or more carbon-carbon bonds, as in biphenyl.

[0096] As used herein, the term "cycloalkyl" refers to a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. The term "heterocycloalkyl" refers to a cycloalkyl group as defined above, in which at least one of the carbon atoms of the ring is replaced with a heteroatom, such as nitrogen, oxygen, sulfur, or phosphorus. Cycloalkyl and heterocycloalkyl groups can be substituted or unsubstituted. Cycloalkyl and heterocycloalkyl groups can be substituted with one or more groups, including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described herein.

[0097] The term "cycloalkenyl" as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms and containing at least one double bond, i.e., C=C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like. The term "heterocycloalkenyl" is a type of cycloalkenyl group as defined above and is included in the meaning of the term "cycloalkenyl", in which at least one of the ring carbon atoms is replaced with a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. Cycloalkenyl and heterocycloalkenyl groups can be substituted or unsubstituted. The cycloalkenyl and heterocycloalkenyl groups may be optionally substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described herein.

[0098] The term "cyclic group" is used herein to refer to either aryl groups, non-aryl groups (i.e., cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups), or both. Cyclic groups have one or more ring systems (e.g., monocyclic, bicyclic, tricyclic, polycyclic, etc.) that can be substituted or unsubstituted. Cyclic groups can contain one or more aryl groups, one or more non-aryl groups, or one or more aryl groups and one or more non-aryl groups.

[0099] As used herein, the term "acyl" refers to a group of the formula -C(O)Z 1 In the formula, Z 1may be hydrogen, hydroxyl, alkoxy, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl groups as described above. As used herein, the term "acyl" may be used interchangeably with "carbonyl." Throughout this specification, "C(O)" or "CO" is an abbreviation for C=O.

[0100] As used herein, the term “acetal” refers to a compound of the formula (Z 1 Z 2 )C(=OZ 3 )(=OZ 4 ) wherein Z 1 , Z 2 , Z 3 , and Z 4 can be independently hydrogen, halogen, hydroxyl, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl groups as described above.

[0101] As used herein, the term "alkanol" refers to an alkanol having the formula Z 1 OH, wherein Z 1 can be an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

[0102] As used herein, the term "alkoxy" as used herein refers to an alkyl group attached through a single, terminal ether bond, i.e., an "alkoxy" group has the formula Z 1 -O-, where Z 1 is unsubstituted or substituted alkyl as defined above. Unless otherwise specified, Z 1 C1~C 24 (For example, C1~C 22 , C1~C 20 , C1~C 18 , C1~C 16 , C1~C 14, C1~C 12 , C1~C 10 , C1-C8, C1-C6, or C1-C4) alkyl groups are contemplated. Examples include methoxy, ethoxy, propoxy, 1-methyl-ethoxy, butoxy, 1-methyl-propoxy, 2-methyl-propoxy, 1,1-dimethyl-ethoxy, pentoxy, 1-methyl-butyloxy, 2-methyl-butoxy, 3-methyl-butoxy, 2,2-dimethyl-propoxy, 1-ethyl-propoxy, hexoxy, 1,1-dimethyl-propoxy, 1,2-dimethyl-propoxy, 1-methyl-pentoxy, 2-methyl-pentoxy, 3-methyl-butoxy, 2,2-dimethyl-propoxy, 1-ethyl-propoxy, hexoxy, 1,1-dimethyl-propoxy, 1,2-dimethyl-propoxy, 1-methyl-pentoxy, 2-methyl-pentoxy, 3-methyl-butoxy, 2,2-dimethyl-propoxy, 1-ethyl-propoxy, hexoxy, 1,1-dimethyl-propoxy, 1,2-dimethyl-propoxy, 1-methyl-pentoxy, 2-methyl-pentoxy, 3-methyl-butoxy, 2,2-dimethyl-propoxy, 1-ethyl-propoxy, 2-methyl-pentoxy, 3-methyl-butoxy, 2,2-dimethyl-propoxy, 1-ethyl-propoxy, 2-methyl-but ... Examples of aryloxy groups include 1,1-pentoxy, 4-methyl-penoxy, 1,1-dimethyl-butoxy, 1,2-dimethyl-butoxy, 1,3-dimethyl-butoxy, 2,2-dimethyl-butoxy, 2,3-dimethyl-butoxy, 3,3-dimethyl-butoxy, 1-ethyl-butoxy, 2-ethyl-butoxy, 1,1,2-trimethyl-propoxy, 1,2,2-trimethyl-propoxy, 1-ethyl-1-methyl-propoxy, and 1-2-methyl-propoxy.

[0103] The term "aldehyde" as used herein is represented by the formula -C(O)H. Throughout this specification, "C(O)" is an abbreviation for C=O.

[0104] As used herein, the term "amine" or "amino" refers to a group of the formula -NZ 1 Z 2 Z 3 In the formula, Z 1 , Z 2 , and Z 3 may each be a substituent as described herein, for example, hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

[0105] As used herein, the term "amide" or "amido" refers to a group having the formula -C(O)NZ 1 Z 2In the formula, Z 1 and Z 2 may each be a substituent as described herein, for example, hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

[0106] As used herein, the term "anhydride" refers to a compound of formula Z 1 C(O)OC(O)Z 2 In the formula, Z 1 and Z 2 can be independently an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

[0107] The term "cyclic anhydride" as used herein is represented by the formula: [ka] In the formula, Z 1 can be an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

[0108] The term "azide" as used herein is represented by the formula -N=N=N.

[0109] The term "carboxylic acid" as used herein is represented by the formula --C(O)OH.

[0110] As used herein, a "carboxylate" or "carboxyl" group is represented by the formula --C(O)O.

[0111] As used herein, a "carbonate" group has the formula Z 1 O.C.O.Z. 2 It is represented by:

[0112] The term "cyano" as used herein is represented by the formula --CN.

[0113] As used herein, the term "ester" refers to an ester of the formula -OC(O)Z 1 or -C(O)OZ 1 In the formula, Z 1 can be an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

[0114] As used herein, the term "ether" refers to a group of the formula Z 1 OZ 2 In the formula, Z 1 and Z 2 can be independently an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

[0115] The term "epoxy" or "epoxide" as used herein refers to a cyclic ether having a three atom ring and can be represented by the following formula: [ka] In the formula, Z 1 , Z 2 , Z 3 , and Z 4 can be independently an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

[0116] As used herein, the term "ketone" refers to a compound of the formula Z 1 C(O)Z 2 In the formula, Z 1 and Z 2can be independently an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group.

[0117] The terms "halide" or "halogen" or "halo" as used herein refer to fluorine, chlorine, bromine, and iodine.

[0118] The term "hydroxyl" as used herein is represented by the formula --OH.

[0119] The term "nitro" as used herein is represented by the formula --NO.sub.2.

[0120] The term "phosphonyl" as used herein refers to a group of the formula -P(O)(OZ 1 )2, wherein Z 1 can be hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl groups as described above.

[0121] As used herein, the term "silyl" refers to a group of the formula -SiZ 1 Z 2 Z 3 In the formula, Z 1 , Z 2 , and Z 3 may independently be hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl groups as described above.

[0122] The term "sulfonyl" or "sulfone" as used herein refers to a group of the formula -S(O)Z 1 is used to refer to a sulfo-oxo group represented by the formula 1can be hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl groups as described above.

[0123] As used herein, the term "sulfide" includes groups of the formula -S-.

[0124] The term "thiol" as used herein is represented by the formula --SH.

[0125] "R 1 ", "R 2 ", "R 3 ", "R 4 " etc., where n is an integer, as used herein, can independently have one or more of the groups listed above. For example, R 1 When is a straight chain alkyl group, one of the hydrogen atoms of the alkyl group can be optionally replaced with a hydroxyl group, an alkoxy group, an amine group, an alkyl group, a halide, etc. Depending on the group selected, the first group can be incorporated into the second group, or alternatively, the first group can be pendant (i.e., attached) to the second group. For example, in the phrase "an alkyl group comprising an amino group," the amino group can be incorporated into the backbone of the alkyl group. Alternatively, the amino group can be attached to the backbone of the alkyl group. The nature of the group(s) selected will determine whether the first group is embedded in or attached to the second group.

[0126] Unless otherwise noted, formulas in which chemical bonds are shown only as solid lines and not as wedges or dashed lines contemplate each possible stereoisomer or mixture of stereoisomers (e.g., each enantiomer, each diastereomer, each meso compound, racemic mixtures, or scalemic mixtures).

[0127] "Phase", as used herein, generally refers to a region of a material having a substantially uniform composition that is a different physically separate part of a heterogeneous system. The term "phase" does not mean that the materials that make up the phase are chemically pure substances, but simply that the chemical and / or physical properties of the materials that make up the phase are essentially uniform throughout the material, and that these chemical and / or physical properties are significantly different from the chemical and / or physical properties of another phase within the material. Examples of physical properties include density, thickness, aspect ratio, specific surface area, porosity, and dimensions. Examples of chemical properties include chemical composition.

[0128] The terms "olefinically unsaturated group" or "ethylenically unsaturated group" are used broadly herein and are intended to encompass any group that contains a carbon-carbon double bond group (>C=C< group). Exemplary ethylenically unsaturated groups include, but are not limited to, (meth)acrylate, (meth)acrylamide, (meth)acryloyl, allyl, vinyl, styrenyl, or other >C=C< containing groups.

[0129] "Polymer" means a material formed by polymerizing one or more monomers.

[0130] The term "(co)polymer" includes homopolymers, copolymers, or mixtures thereof.

[0131] The term "(meth)acrylic..." includes "acrylic...", "methacrylic..." or mixtures thereof.

[0132] As used herein, the "molecular weight" of a polymeric material (including monomeric or macromonomer materials) refers to the molecular weight, unless otherwise specified or test conditions dictate otherwise. 1 It refers to the number average molecular weight as measured by H NMR spectroscopy.

[0133] Compositions, Devices, and Methods Disclosed herein are hydrogel structures and methods of making and using same.

[0134] For example, disclosed herein is a method of making a device comprising a hydrogel matrix and a first chamber in the hydrogel matrix, where the hydrogel matrix is ​​derived from a prepolymer and the first chamber is perfusable. The method may include, for example, blocking a first portion of a prepolymerized solution with a first photomask, such that the prepolymerized solution includes an exposed portion and a first blocked portion. The prepolymerized solution may include a prepolymer. The method may further include irradiating the exposed portion of the prepolymerized solution and the first photomask with electromagnetic radiation, where the first photomask is substantially opaque to the electromagnetic radiation. The prepolymer in the exposed portion of the prepolymerized solution photopolymerizes to form a hydrogel matrix and the prepolymer in the first blocked portion does not photopolymerize to form the first chamber.

[0135] Also disclosed herein are methods of making a device comprising a hydrogel matrix derived from a prepolymer, a first chamber in the hydrogel matrix, the first chamber being perfusable, and a second chamber in the hydrogel matrix, the second chamber being perfusable and fluidically independent from the first chamber. The method may include, for example, blocking a first portion of a prepolymerized solution with a first photomask and blocking a second portion of the prepolymerized solution with a second photomask, the prepolymerized solution including a prepolymer, such that the prepolymerized solution includes an exposed portion, a first blocked portion, and a second blocked portion. The method may further include irradiating the exposed portion of the prepolymerized solution, the first photomask, and the second photomask with electromagnetic radiation, the first photomask and the second photomask being substantially opaque to electromagnetic radiation. The prepolymer in the exposed portion of the prepolymerized solution photopolymerizes to form a hydrogel matrix, the prepolymer in the first blocked portion does not photopolymerize and forms a first chamber, and the prepolymer in the second blocked portion does not photopolymerize and forms a second chamber.

[0136] As used herein, a "chamber" generally refers to a volume that is at least partially surrounded, and in some instances completely surrounded, by a hydrogel matrix. The chamber can be, for example, hollow. In some instances, the chamber can be at least partially filled with a substance.

[0137] In some examples, the first chamber can be a first elongated chamber. In some examples, the first chamber can form a first continuous channel within the hydrogel matrix. In some examples, the first continuous channel can branch.

[0138] In some examples, the second chamber can be a second elongated chamber. In some examples, the second chamber can form a second continuous channel within the hydrogel matrix. In some examples, the second continuous channel can branch.

[0139] In some examples, the method may further include removing the first photomask and the second photomask (if present) after irradiation.

[0140] In some examples, the method may further include rinsing the hydrogel device after irradiation to remove any remaining prepolymerization solution and / or prepolymer.

[0141] In some examples, the method may further include placing the prepolymerized solution in a mold defining a shape prior to blocking a first portion of the prepolymerized solution with a first photomask.

[0142] The hydrogel matrix may comprise any suitable hydrogel matrix, in some instances, the hydrogel matrix may be selected based on the intended use of the device, the desired properties of the hydrogel matrix, or a combination thereof.

[0143] In some examples, the hydrogel matrix comprises a synthetic hydrogel. The synthetic hydrogel may, for example, comprise a network of crosslinked hydrophilic polymers. Suitable hydrophilic polymers include polyalkylene glycol polymers, polyalkylene oxide homopolymers, such as polypropylene glycol, polyoxyethylated polyols, their copolymers and their block copolymers, as well as poly(oxyethylated polyols), poly(olefin alcohols), poly(vinylpyrrolidone), poly(hydroxypropylmethacrylamide), poly(α-hydroxy acids), poly(vinyl alcohols), polyphosphazenes, polyoxazolines, poly(N-acryloylmorpholines), and copolymers, terpolymers, and mixtures thereof.

[0144] The prepolymer can include any suitable prepolymer. In some instances, the prepolymer can be selected based on the intended use of the device, the desired properties of the hydrogel matrix, or a combination thereof.

[0145] The term prepolymer is used herein to refer to a polymer having reactive groups available for bond-forming reactions to crosslink (inter- and / or intra-molecular crosslinks). It is not meant to imply that a prepolymer is not yet a polymer (e.g., a monomer or polymer precursor). Rather, "prepolymer" refers to a starting polymer that contains multiple crosslinkable groups and can be cured (e.g., crosslinked) to obtain a crosslinked polymer having a higher molecular weight than the starting polymer.

[0146] The prepolymers have reactive groups available for bond formation, i.e., the prepolymers can be crosslinked when reactive groups on separate prepolymers or on the same prepolymer form bonds with reactive groups of a crosslinking agent, such as a multifunctional crosslinking agent.

[0147] Examples of suitable reactive groups on the prepolymer include nucleophilic or electrophilic groups. Specific examples of nucleophilic reactive groups include thiols (sulfides), amines, azides, nitrites, alcohols (alkoxides), peroxides, carboxylic acids (carboxylates), thiocarboxylic acids (thiocarbonates), sulfonic acids (sulfoxides), and phosphonic acids (phosphates), with the deprotonated form of the reactive group being listed in parentheses. Enolates may also be suitable nucleophilic reactive groups. Specific examples of electrophilic reactive groups may include ketones, aldehydes, alkenes, acyl halides, acrylates, carboxylic acids, esters, hemiacetals, acetals, hemiketals, ketals, orthoesters, amides, imines, imides, azo compounds, cyanates, thiocyanates, nitrates, nitriles, nitrites, thials, phosphines, and phosphodiesters. Other suitable reactive groups can be unsaturated moieties, such as alkenes, alkynes, dienes, nitriles, azides, carbonyls, or imines.

[0148] In some examples, the prepolymer includes photosensitive groups, such as photosensitive end groups.

[0149] In some examples, the prepolymer comprises a photosensitive prepolymer. The photosensitive prepolymer may comprise any suitable material. For example, the photosensitive prepolymer can include poly(ethylene glycol) diacrylate (PEGDA), poly(ethylene glycol) dimethacrylate (PEGDMA), poly(ethylene glycol) diacrylamide (PEGDAAm), gelatin methacrylate (GelMA), collagen methacrylate, silk methacrylate, hyaluronic acid methacrylate, chondroitin sulfate methacrylate, elastin methacrylate, cellulose acrylate, dextran methacrylate, heparin methacrylate, NIPAAm methacrylate, chitosan methacrylate, polyethylene glycol norbornene, polyethylene glycol dithiol, thiolated gelatin, thiolated silk, PEG-based peptide conjugates, cell adhesive poly(ethylene glycol), MMP-sensitive poly(ethylene glycol), PEGylated fibrinogen, PEGylated collagen, PEGylated laminin, or combinations thereof.

[0150] In some examples, the hydrogel matrix is ​​derived from a prepolymer comprising polyethylene glycol or a derivative thereof. In some examples, the hydrogel matrix is ​​derived from a prepolymer comprising poly(ethylene glycol) acrylate, poly(ethylene glycol) diacrylate (PEGDA), poly(ethylene glycol) diacrylate (PEGDMA), poly(ethylene glycol) diacrylamide (PEGDAAm), polyethylene glycol norbornene, polyethylene glycol dithiol, PEG-based peptide conjugates, cell-adhesive poly(ethylene glycol), MMP-sensitive poly(ethylene glycol), PEGylated fibrinogen, PEGylated collagen, PEGylated laminin, or a combination thereof. In some examples, the hydrogel matrix is ​​derived from a prepolymer comprising poly(ethylene glycol) vinyl sulfone, poly(ethylene glycol) acrylate, poly(ethylene glycol) maleimide, poly(ethylene glycol) norbornene, PEGylated fibrinogen, PEGylated collagen, PEGylated laminin, or a combination thereof. In some examples, the hydrogel matrix is ​​derived from a prepolymer comprising poly(ethylene glycol)vinylsulfone, poly(ethylene glycol)acrylate, poly(ethylene glycol)maleimide, poly(ethylene glycol)norbornene, or a combination thereof. In some examples, the hydrogel matrix is ​​derived from a prepolymer comprising poly(ethylene glycol)maleimide, poly(ethylene glycol)norbornene, or a combination thereof. In some examples, the hydrogel matrix is ​​derived from a prepolymer comprising poly(ethylene glycol)norbornene.

[0151] In some examples, the hydrogel matrix has a molecular weight of 0.5 kilodaltons (kDa) or more (e.g., 1 kDa or more, 1.5 kDa or more, 2 kDa or more, 2.5 kDa or more, 3 kDa or more, 3.5 kDa or more, 4 kDa or more, 4.5 kDa or more, 5 kDa or more, 6 kDa or more, 7 kDa or more, 8 kDa or more, 9 kDa or more, 10 kDa or more, 15 kDa or more, 20 kDa or more, 25 kDa or more, 30 kDa or more). or greater, 35 kDa or greater, 40 kDa or greater, 45 kDa or greater, 50 kDa or greater, 60 kDa or greater, 70 kDa or greater, 80 kDa or greater, 90 kDa or greater, 100 kDa or greater, 110 kDa or greater, 120 kDa or greater, 130 kDa or greater, 140 kDa or greater, 150 kDa or greater, 160 kDa or greater, 170 kDa or greater, 180 kDa or greater, or 190 kDa or greater. In some examples, the prepolymer is 200 kDa or less (e.g., 190 kDa or less, 180 kDa or less, 170 kDa or less, 160 kDa or less, 150 kDa or less, 140 kDa or less, 130 kDa or less, 120 kDa or less, 110 kDa or less, 100 kDa or less, 90 kDa or less, 80 kDa or less, 70 kDa or less, 60 kDa or less, 50 kDa or less, 45 kDa or less). , 40 kDa or less, 35 kDa or less, 30 kDa or less, 25 kDa or less, 20 kDa or less, 15 kDa or less, 10 kDa or less, 9 kDa or less, 8 kDa or less, 7 kDa or less, 6 kDa or less, 5 kDa or less, 4.5 kDa or less, 4 kDa or less, 3.5 kDa or less, 3 kDa or less, 2.5 kDa or less, 2 kDa or less, 1.5 kDa or less, or 1 kDa or less. The molecular weight of the prepolymer can range from any of the minimum values ​​recited above to any of the maximum values ​​recited above.For example, the prepolymer may be of any of a variety of molecular weights ranging from 0.5 to 200 kilodaltons (kDa) (e.g., 0.5 to 100 kDa, 100 to 200 kDa, 0.5 to 50 kDa, 50 to 100 kDa, 100 to 150 kDa, 150 to 200 kDa, 0.5 to 175 kDa, 0.5 to 150 kDa, 0.5 to 125 kDa, 0.5 to 75 kDa, 0.5 to 25 kDa, 0.5 to 10 kDa, 1 to 200 kDa, 2.5 to 200 kDa, 5 to 200 kDa, 10 to 200 kDa, 25 to 20 The molecular weight of the antibody may be 0 kDa, 50 to 200 kDa, 75 to 200 kDa, 125 to 200 kDa, 1 to 175 kDa, 1 to 150 kDa, 2 to 150 kDa, 5 to 100 kDa, 10 to 100 kDa, 20 to 100 kDa, 20 to 80 kDa, 20 to 60 kDa, 1 kDa to 50 kDa, 5 to 50 kDa, 10 to 50 kDa, 2 kDa to 40 kDa, 20 kDa to 40 kDa, 40 kDa to 60 kDa, 2 kDa to 25 kDa, or 2 kDa to 10 kDa.

[0152] In some examples, the prepolymer may include a branched or multi-armed prepolymer. As used herein, a multi-armed prepolymer refers to a prepolymer having a central core to which at least two prepolymers are covalently attached. Generally, all prepolymers attached to the core are the same, but in some cases different prepolymers can be used. A multi-armed prepolymer may have two or more arms (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more). For example, the prepolymer may include a branched prepolymer having two or more branches (e.g., three or more branches, four or more branches, five or more branches, six or more branches, seven or more branches, eight or more branches, nine or more branches, or ten or more branches).

[0153] In some examples, the prepolymerized solution comprises prepolymer in an amount of 1 wt% or more (e.g., 2 wt% or more, 3 wt% or more, 4 wt% or more, 5 wt% or more, 6 wt% or more, 7 wt% or more, 8 wt% or more, 9 wt% or more, 10 wt% or more, 11 wt% or more, 12 wt% or more, 13 wt% or more, 14 wt% or more, 15 wt% or more, 16 wt% or more, 17 wt% or more, 18 wt% or more, 19 wt% or more, 20 wt% or more, 21 wt% or more, 22 wt% or more, 23 wt% or more, 24 wt% or more, 25 wt% or more, 26 wt% or more, 27 wt% or more, 28 wt% or more, or 29 wt% or more). In some examples, the prepolymerization solution includes prepolymer in an amount of 30% by weight or less (e.g., 29% by weight or less, 28% by weight or less, 27% by weight or less, 26% by weight or less, 25% by weight or less, 24% by weight or less, 23% by weight or less, 22% by weight or less, 21% by weight or less, 20% by weight or less, 19% by weight or less, 18% by weight or less, 17% by weight or less, 16% by weight or less, 15% by weight or less, 14% by weight or less, 13% by weight or less, 12% by weight or less, 11% by weight or less, 10% by weight or less, 9% by weight or less, 8% by weight or less, 7% by weight or less, 6% by weight or less, 5% by weight or less, 4% by weight or less, 3% by weight or less, or 2% by weight or less). The amount of prepolymer in the prepolymerization solution can range from any of the minimum values ​​described above to any of the maximum values ​​described above. For example, the prepolymerization solution may contain the prepolymer in an amount of 1 wt% to 30 wt% (e.g., 1 wt% to 15 wt%, 15 wt% to 30 wt%, 1 wt% to 5 wt%, 5 wt% to 10 wt%, 10 wt% to 15 wt%, 15 wt% to 20 wt%, 20 wt% to 25 wt%, 25 wt% to 30 wt%, 1 wt% to 25 wt%, 1 wt% to 20 wt%, 1 wt% to 10 wt%, 5 wt% to 30 wt%, 10 wt% to 30 wt%, 20 wt% to 30 wt%, 2 wt% to 29 wt%, 5 wt% to 25 wt%, 10 wt% to 20 wt%, or 4 wt% to 10 wt%).

[0154] In some examples, the prepolymerization solution further comprises a crosslinker. In some examples, the hydrogel is further derived from the crosslinker. The crosslinker can be any suitable crosslinker. In some examples, the crosslinker can be selected based on the prepolymer, the intended use of the device, the desired properties of the hydrogel matrix, or a combination thereof.

[0155] In some examples, the crosslinker is a multifunctional crosslinker. The multifunctional crosslinker has reactive groups available for bond formation, i.e., the multifunctional crosslinker can crosslink reactive groups of the prepolymer. Examples of reactive groups on suitable multifunctional crosslinkers include nucleophilic groups or electrophilic groups. The reactive groups of the multifunctional crosslinker can be complementary to the reactive groups of the prepolymer. For example, if the reactive groups of the prepolymer include electrophilic reactive groups, the multifunctional crosslinker can include nucleophilic reactive groups.

[0156] In some examples, the multifunctional crosslinker can include two or more reactive groups (e.g., three or more, four or more, or five or more). In some examples, the multifunctional crosslinker can include six or fewer reactive groups (e.g., five or fewer, four or fewer, or three or fewer). The number of reactive groups in the multifunctional crosslinker can range from any of the minimum values ​​listed above to any of the maximum values ​​listed above, for example, 2 to 6 (e.g., 2 to 4, 4 to 6, 3 to 5, 2 to 3, 3 to 4, 4 to 5, or 5 to 6).

[0157] In some examples, the multifunctional crosslinker can include a multifunctional thiol. In some examples, the crosslinker includes a dithiol.

[0158] In some examples, the prepolymer includes a poly(ethylene glycol) norbornene, such as a branched or multi-armed poly(ethylene glycol) norbornene, and the crosslinker includes a thiol, such as a multifunctional thiol. For example, the photopolymerization can include thiol-norbornene photopolymerization.

[0159] In some instances, the hydrogel matrix is ​​further derived from one or more additional components, such as one or more additional monomers, prepolymers, ligands, chemical agents, therapeutic agents, photoinitiators, or combinations thereof. In some instances, the prepolymerization solution further comprises the one or more additional components.

[0160] In some instances, the hydrogel matrix is ​​further derived from natural or biological components, such as natural or biological prepolymers, hi some instances, the natural or biological prepolymers include fibrinogen, collagen, or combinations thereof.

[0161] In some examples, the prepolymerization solution further comprises a photoinitiator.

[0162] In some examples, the hydrogel matrix further comprises a chemical agent, a therapeutic agent, or a combination thereof dispersed therein. In some examples, the prepolymerized solution further comprises a chemical agent and / or a therapeutic agent. The therapeutic agent may include, for example, an anti-cancer agent, an anti-inflammatory agent, an anti-microbial agent, or a combination thereof. As used herein, an anti-microbial agent includes, for example, an antibacterial agent, an anti-fungal agent, and an anti-viral agent.

[0163] In some instances, the chemical and / or therapeutic agent is non-uniformly dispersed within the hydrogel matrix. For example, the chemical and / or therapeutic agent may have a concentration that varies throughout the hydrogel matrix, such that the chemical and / or therapeutic agent has a compositional gradient throughout the hydrogel matrix. The compositional gradient may be, for example, a linear gradient, a step gradient, an exponential gradient, a logarithmic gradient, or the like, or a combination thereof.

[0164] The electromagnetic radiation can include any suitable electromagnetic radiation. In some examples, the electromagnetic radiation can be selected based on the crosslinker, the prepolymer, the intended use of the device, the desired properties of the hydrogel matrix, or a combination thereof.

[0165] In some examples, the electromagnetic radiation may include light, hi some examples, the electromagnetic radiation includes UV radiation.

[0166] In some examples, electromagnetic radiation includes one or more wavelengths of 10 nanometers (nm) or greater (e.g., 15 nm or greater, 20 nm or greater, 25 nm or greater, 30 nm or greater, 35 nm or greater, 40 nm or greater, 45 nm or greater, 50 nm or greater, 60 nm or greater, 70 nm or greater, 80 nm or greater, 90 nm or greater, 100 nm or greater, 125 nm or greater, 150 nm or greater, 175 nm or greater, 200 nm or greater, 225 nm or greater, 250 nm or greater, 275 nm or greater, 300 nm or greater, 325 nm or greater, 350 nm or greater, 375 nm or greater, 400 nm or greater, 450 nm or greater, 500 nm or greater, 550 nm or greater, 600 nm or greater, 650 nm or greater, 700 nm or greater, 750 nm or greater, 800 nm or greater, or 850 nm or greater). In some examples, the electromagnetic radiation includes one or more wavelengths of 900 nm or less (e.g., 850 nm or less, 800 nm or less, 750 nm or less, 700 nm or less, 650 nm or less, 600 nm or less, 550 nm or less, 500 nm or less, 450 nm or less, 400 nm or less, 375 nm or less, 350 nm or less, 325 nm or less, 300 nm or less, 275 nm or less, 250 nm or less, 225 nm or less, 200 nm or less, 175 nm or less, 150 nm or less, 125 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, 60 nm or less, 50 nm or less, 45 nm or less, 40 nm or less, 35 nm or less, 30 nm or less, 25 nm or less, 20 nm or less, or 15 nm or less). The one or more wavelength(s) of the electromagnetic radiation can range from any of the minimum values ​​mentioned above to any of the maximum values ​​mentioned above.For example, the electromagnetic radiation may be in the range of 10 nm to 900 nm (e.g., 10 nm to 450 nm, 450 nm to 900 nm, 10 nm to 300 nm, 300 nm to 600 nm, 600 nm to 900 nm, 10 nm to 800 nm, 10 nm to 700 nm, 10 nm to 600 nm, 10 nm to 500 nm, 10 nm to 400 nm, 10 nm to 200 nm, 10 nm to 100 nm, 25 nm to 900 nm , 50 nm to 900 nm, 100 nm to 900 nm, 200 nm to 900 nm, 300 nm to 900 nm, 400 nm to 900 nm, 500 nm to 900 nm, 700 nm to 900 nm, 800 nm to 900 nm, 25 nm to 850 nm, 100 nm to 900 nm, 100 nm to 400 nm, 400 nm to 900 nm, or 400 nm to 750 nm).

[0167] In some examples, the electromagnetic radiation is provided by a light source. The light source can be any type of light source. Examples of suitable light sources include natural light sources (e.g., sunlight) and artificial light sources (e.g., incandescent light bulbs, light emitting diodes, gas discharge lamps, arc lamps, lasers, etc.). In some examples, the light source is an artificial light source. In some examples, the light source includes a light emitting diode (LED), a lamp, a laser, or a combination thereof.

[0168] The exposed portions of the prepolymerized solution, the first photomask, and the second photomask (if present) are exposed for 1 millisecond or more (e.g., 2 milliseconds or more, 3 milliseconds or more, 4 milliseconds or more, 5 milliseconds or more, 10 milliseconds or more, 15 milliseconds or more, 20 milliseconds or more, 25 milliseconds or more, 30 milliseconds or more, 35 milliseconds or more, 40 milliseconds or more, 50 milliseconds or more, 60 milliseconds or more, 70 milliseconds or more, 80 milliseconds or more, 90 milliseconds or more, 100 milliseconds or more, 125 milliseconds or more, 150 milliseconds or more, 175 milliseconds or more, 200 milliseconds or more, 225 milliseconds or more, 250 milliseconds or more, 300 milliseconds or more, 350 ...0 milliseconds or more, 400 milliseconds or more, 400 milliseconds or more, 400 milliseconds or more, 400 milliseconds or more, 400 milliseconds or more, 400 milliseconds or more, 400 milliseconds or more, 400 milliseconds or more, 400 millisecond milliseconds or more, 400 milliseconds or more, 450 milliseconds or more, 500 milliseconds or more, 600 milliseconds or more, 700 milliseconds or more, 800 milliseconds or more, 900 milliseconds or more, 1 second or more, 2 seconds or more, 3 seconds or more, 4 seconds or more, 5 seconds or more, 10 seconds or more, 15 seconds or more, 20 seconds or more, 25 seconds or more, 30 seconds or more, 35 seconds or more, 40 seconds or more, 45 seconds or more, 50 seconds or more, 55 seconds or more, 1 minute or more, 2 minutes or more, 3 minutes or more, 4 minutes or more, 5 minutes or more, 10 minutes or more, 15 minutes or more, 20 minutes or more, 25 minutes or more, 30 minutes or more, 35 minutes or more, 40 minutes or more, 45 minutes or more, 50 minutes or more, or 55 minutes or more).In some examples, the exposed portion of the prepolymerized solution, the first photomask, and the second photomask (if present) are exposed for 1 hour or less (e.g., 55 minutes or less, 50 minutes or less, 45 minutes or less, 40 minutes or less, 35 minutes or less, 30 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, 4 minutes or less, 3 minutes or less, 2 minutes or less, 1 minute or less, 55 seconds or less, 50 seconds or less, 45 seconds or less, 40 seconds or less, 35 seconds or less, 30 seconds or less, 25 seconds or less, 20 seconds or less, 15 seconds or less, 10 seconds or less, 5 seconds or less, 4 seconds or less, 3 seconds or less, 2 seconds or less, 1 second or less, 900 milliseconds or less, 800 milliseconds or less, 700 milliseconds or less, The prepolymerized solution may be irradiated for an amount of time of up to 600 milliseconds, up to 500 milliseconds, up to 450 milliseconds, up to 400 milliseconds, up to 350 milliseconds, up to 300 milliseconds, up to 250 milliseconds, up to 225 milliseconds, up to 200 milliseconds, up to 175 milliseconds, up to 150 milliseconds, up to 125 milliseconds, up to 100 milliseconds, up to 90 milliseconds, up to 80 milliseconds, up to 70 milliseconds, up to 60 milliseconds, up to 50 milliseconds, up to 40 milliseconds, up to 35 milliseconds, up to 30 milliseconds, up to 25 milliseconds, up to 20 milliseconds, up to 15 milliseconds, up to 10 milliseconds, up to 5 milliseconds, up to 4 milliseconds, up to 3 milliseconds, or up to 2 milliseconds). The time for which the exposed portion of the prepolymerized solution, the first photomask, and the second photomask (if present) are irradiated may range from any of the minimum values ​​described above to any of the maximum values ​​described above. For example, the exposed portions of the prepolymerized solution, the first photomask, and the second photomask (if present) may be irradiated for an amount of time from 1 millisecond to 1 hour (e.g., 1 millisecond to 1 second, 1 second to 1 minute, 1 minute to 1 hour, 1 millisecond to 30 minutes, 1 millisecond to 15 minutes, 1 millisecond to 10 minutes, 1 millisecond to 5 minutes, 1 millisecond to 1 minute, 1 millisecond to 30 seconds, 1 millisecond to 15 seconds, 1 millisecond to 10 seconds, or 1 millisecond to 5 seconds).

[0169] In some examples, the hydrogel matrix exhibits swelling of 10% or less (e.g., 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less) in physiological saline. Swelling can be determined, for example, based on the weight change of the hydrogel before and after exposure to or immersion in physiological saline.

[0170] In some examples, the hydrogel matrix exhibits a shape fidelity of 50% or more (e.g., 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more). Shape fidelity can be measured based on optical changes in the geometry of features of the hydrogel matrix before and after exposure to or immersion in a solvent, such as saline.

[0171] In some examples, the hydrogel matrix has a viscosity of more than 0 Pa (1 Pa or more, 2 Pa or more, 3 Pa or more, 4 Pa ​​or more, 5 Pa or more, 10 Pa or more, 15 Pa or more, 20 Pa or more, 25 Pa or more, 30 Pa or more, 35 Pa or more, 40 Pa or more, 45 Pa or more, 50 Pa or more, 60 Pa or more, 70 Pa or more, 80 Pa or more, 90 Pa or more, 100 Pa or more, 125 Pa or more, 150 Pa or more, 175 Pa or more, 200 Pa or more, 225 Pa or more, 25 The storage modulus is 0 Pa or more, 300 Pa or more, 350 Pa or more, 400 Pa or more, 450 Pa or more, 500 Pa or more, 550 Pa or more, 600 Pa or more, 700 Pa or more, 800 Pa or more, 900 Pa or more, 1000 Pa or more, 1250 Pa or more, 1500 Pa or more, 1750 Pa or more, 2000 Pa or more, 2250 Pa or more, 2500 Pa or more, 3000 Pa or more, 3500 Pa or more, 4000 Pa or more, or 4500 Pa or more. In some examples, the hydrogel matrix has a compressibility of 5000 Pa or less (e.g., 4500 Pa or less, 4000 Pa or less, 3500 Pa or less, 3000 Pa or less, 2500 Pa or less, 2250 Pa or less, 2000 Pa or less, 1750 Pa or less, 1500 Pa or less, 1250 Pa or less, 1000 Pa or less, 900 Pa or less, 800 Pa or less, 700 Pa or less, 600 Pa or less, 550 Pa or less, 500 Pa or less, 450 Pa or less, 400 Pa or less, 350 Pa or less, a or less, 300 Pa or less, 250 Pa or less, 225 Pa or less, 200 Pa or less, 175 Pa or less, 150 Pa or less, 125 Pa or less, 100 Pa or less, 90 Pa or less, 80 Pa or less, 70 Pa or less, 60 Pa or less, 50 Pa or less, 45 Pa or less, 40 Pa or less, 35 Pa or less, 30 Pa or less, 25 Pa or less, 20 Pa or less, 15 Pa or less, 10 Pa or less, 5 Pa or less, 4 Pa ​​or less, 3 Pa or less, 2 Pa or less, or 1 Pa or less). The storage modulus of the hydrogel matrix can range from any of the minimum values ​​recited above to any of the maximum values ​​recited above.For example, the hydrogel matrix may have a pressure of greater than 0 Pa to 5000 Pa (e.g., greater than 0 to 2500 Pa, 2500 to 5000 Pa, greater than 0 to 1000 Pa, 1000 to 2000 Pa, 2000 to 3000 Pa, 3000 to 4000 Pa, 4000 to 5000 Pa, greater than 0 to 4000 Pa, greater than 0 to 3000 Pa, greater than 0 to 2000 Pa, greater than 0 to 800 Pa, greater than 0 to 600 Pa, greater than 0 to 500 Pa, greater than 0 to 400 Pa, greater than 0 to 300 Pa, greater than 0 to 200 Pa, greater than 0 to 100 Pa, greater than 0 to 50 Pa, greater than 0 to 25 Pa, greater than 0 to 10 Pa, 1 to 5000 Pa, 5 to 5000 Pa, 10 to 5000 Pa, 25 to 5000 Pa, 50 to 5000 Pa, 100 to 5000 Pa, 200 to 5000 Pa, 300 to 5000 Pa, 400 to 5000 Pa, 500 to 5000 Pa, 600 to 5000 Pa, 800 to 5000 Pa, 2000 to 5000 Pa, 3000 to 5000 Pa, 5 to 4500 Pa, 10 to 4000 Pa, or 10 to 1000 Pa).

[0172] The hydrogel matrix can be, for example, stable for a certain amount of time. For example, the hydrogel can be stable for a certain amount of time after exposure to or immersion in a solvent, such as saline. As used herein, "stable" means that 10% or less by weight of the hydrogel matrix (e.g., 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less) degrades over a selected period of time.

[0173] In some examples, the hydrogel matrix is ​​stable for an amount of time of 1 day or more (e.g., 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 1 week or more, 1.5 weeks or more, 2 weeks or more, 2.5 weeks or more, 3 weeks or more, 3.5 weeks or more, 1 month or more, 1.5 months or more, 2 months or more, or 2.5 months or more). In some examples, the hydrogel matrix is ​​stable for an amount of time of 3 months or less (e.g., 2.5 months or less, 2 months or less, 1.5 months or less, 1 month or less, 3.5 weeks or less, 3 weeks or less, 2.5 weeks or less, 2 weeks or less, 1.5 weeks or less, 1 week or less, 6 days or less, 5 days or less, 4 days or less, 3 days or less, or 2 days or less). The amount of time the hydrogel matrix is ​​stable can range from any of the minimum values ​​above to any of the maximum values ​​above. For example, the hydrogel matrix may be stable for an amount of time between 1 day and 3 months (e.g., 1 day to 1.5 months, 1.5 months to 3 months, 1 day to 1 week, 1 week to 1 month, 1 month to 3 months, 1 day to 2 months, 1 day to 1 month, 1 day to 3 weeks, 1 day to 2 weeks, 1 day to 5 days, 2 days to 3 months, 3 days to 3 months, 4 days to 3 months, 5 days to 3 months, 6 days to 3 months, 1 week to 3 months, 2 weeks to 3 months, 3 weeks to 3 months, 2 days to 2.5 months, or 5 days to 2 months).

[0174] In some instances, the hydrogel matrix is ​​continuous. "Continuous," as used herein, generally refers to a phase in which all points within the phase are directly connected in three dimensions, such that for any two points within the continuous phase, a path exists in three-dimensional space that connects the two points without leaving the phase.

[0175] In some instances, the hydrogel matrix is ​​monolithic.

[0176] In some instances, the hydrogel matrix is ​​porous.

[0177] In some instances, the hydrogel matrix is ​​biocompatible.

[0178] In some instances, the hydrogel matrix is ​​biodegradable.

[0179] In some instances, the hydrogel matrix comprises a photopolymerized polymer network. In some instances, the hydrogel matrix comprises a crosslinked polymer network.

[0180] In some instances, the hydrogel matrix comprises a photopolymerized polymer network derived from a photosensitive polymer. In some instances, the hydrogel matrix comprises a crosslinked polymer network derived from a photosensitive polymer.

[0181] Also disclosed herein is a device made by any of the methods disclosed herein.For example, also disclosed herein is a device made by the methods disclosed herein, the device comprising a hydrogel matrix and a first chamber in the hydrogel matrix, the hydrogel matrix being derived from a prepolymer, and the first chamber being perfusable.Also disclosed herein is a device made by the methods disclosed herein, the device comprising a hydrogel matrix derived from a prepolymer, a first chamber in the hydrogel matrix (the first chamber being perfusable), and a second chamber in the hydrogel matrix (the second chamber being perfusable and fluidically independent from the first chamber).

[0182] In some examples, the hydrogel matrix, the first chamber, the second chamber, or a combination thereof can have complex shapes, chemical gradients, or patterned cells.

[0183] In some examples, the device is a microfluidic device.

[0184] In some instances, the hydrogel structure is implantable into a subject.

[0185] Also disclosed herein are methods of use of any of the devices disclosed herein, e.g., also disclosed herein are methods of use of any of the devices made by any of the methods disclosed herein.

[0186] The methods may include, for example, using the device for diagnostics, disease modeling, regenerative medicine, drug screening, tissue modeling, or a combination thereof.

[0187] In some examples, the methods may include using the device as a biomaterial substrate or scaffold, a cell culture substrate or platform, or a combination thereof.

[0188] In some examples, the methods include using the device as a biomaterial substrate or scaffold and / or cell culture substrate or platform for surface-functionalizing proteins, peptides, biomolecules, or combinations thereof.

[0189] In some examples, the method includes seeding the device (e.g., the first chamber and / or the second chamber) with cells and / or biomaterials and perfusing the device (e.g., the first chamber and / or the second chamber) with a solvent or solution. In some examples, the solvent or solution can include cell culture medium, saline, proteins, peptides, sugars, ions, nucleic acids (e.g., DNA, RNA), oligonucleotides, metabolites, exosomes, bacteria, viruses, and / or other biological or chemical molecules.

[0190] In some examples, the methods include using the device as a cell culture substrate for any cell, such as an engineered cell. For example, the methods can include using the device as a cell culture substrate for any cell, such as an engineered cell, that displays a protein or other biomolecule.

[0191] In some examples, the methods include using the device as a cell culture substrate for immune cells, B cells, lymphatic cells, dendritic cells, lung cells, intestinal cells, endothelial cells, hepatic cells, renal epithelial cells, or combinations thereof.

[0192] In some examples, the methods include using the device as a cell culture substrate, and the cultured cells exhibit a cell viability of 50% or more (e.g., 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more) after 4 days or more (e.g., 5 days or more, 6 days or more, 1 week or more, 1.5 weeks or more, 2 weeks or more, 2.5 weeks or more, 3 weeks or more, 3.5 weeks or more, 1 month or more, 1.5 months or more, 2 months or more, or 2.5 months or more) on the device.

[0193] In some examples, the methods include using the device as a cell culture substrate, wherein the cultured cells exhibit cell phenotypic differentiation.

[0194] In some examples, the methods include using the device as a cell culture substrate, and the cultured cells include viable intestinal cells that exhibit appropriate apical and basolatetal marker localization, differentiated epithelial cells, or a combination thereof.

[0195] In some examples, the methods include using the device as a cell culture substrate, where cultured cells colonize the device in a multi-dimensional (eg, three-dimensional) manner.

[0196] In some examples, the methods include growing a three-dimensional cell mass using the device.

[0197] In some examples, the method includes growing organoids using the device. In some examples, the method includes growing lymphoid follicular organoids, tonsillar organoids, intestinal organoids, lung organoids, or combinations thereof using the device.

[0198] In some examples, the method includes growing human organoids using the device. In some examples, the method includes growing human intestinal organoids using the device.

[0199] Also disclosed herein are articles of manufacture that include any of the devices disclosed herein. For example, also disclosed herein are articles of manufacture that include any of the devices made by any of the methods disclosed herein. The articles can include, for example, a biomaterial matrix or scaffold, a cell culture substrate or platform, or a combination thereof.

[0200] Several embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0201] The following examples are intended to further illustrate certain aspects of the systems and methods described herein, and are not intended to limit the scope of the claims. EXAMPLES

[0202] The following examples are described below to illustrate the methods and results of the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention that are apparent to those skilled in the art.

[0203] Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless otherwise specified, parts are parts by weight, temperature is °C or is ambient temperature, and pressure is at or near atmospheric. Numerous variations and combinations of measurement conditions, e.g., component concentrations, temperature, pressure, and other measurement ranges and conditions, can be used to optimize the described processes.

[0204] Example 1 Disclosed herein is a rapid and facile light-based approach to generate complex hydrogel structures for tissue / organ-on-chip models.

[0205] In some examples, the methods disclosed herein include a rapid and simple light-based approach to generate complex hydrogel structures for use in tissue / organ-on-chip models for diagnostic, disease modeling, and drug screening applications. Currently, complex tissue-on-chip models, such as perfusable gut-on-chip, involve laborious, time-consuming, and equipment-intensive methods that use matrix laser ablation techniques. This approach is limited to natural biological matrices (e.g., Matrigel), further limiting its application. The methods disclosed herein employ light-induced polymerization and photomasking of synthetic hydrogels to generate complex patterns including perfusable channels. This approach reduces preparation time from hours to minutes and uses relatively simple instrumentation. In addition, the ability to use synthetic hydrogels rather than natural matrices allows for great flexibility and reduces regulatory burden.

[0206] Advantages of the methods disclosed herein include, but are not limited to, rapidity (minutes instead of hours), simplicity (simple exposure and equipment), and use of synthetic matrices.

[0207] Example 2 Disclosed herein is a rapid and simple method for patterning hydrogels to generate complex scaffolds.

[0208] In some examples, the methods disclosed herein involve a rapid and facile approach to generate complex hydrogel structures for use in tissue / organ-on-chip models for diagnostics, disease modeling, regenerative medicine, and drug screening applications. Currently, complex tissue models involve laborious, time-consuming, and equipment-intensive methods using laser-based patterning or matrix ablation. The strategies disclosed herein employ spatial patterning of multiple cell types and other non-cellular entities within a synthetic hybrid hydrogel system for the generation of complex patterns, including biomolecular and chemical gradients. In this approach, one or more types of cells are encapsulated in microgels and co-encapsulated with another set of cells in a bulk hydrogel matrix, with gel compositions designed for tunable degradation and biophysical properties. This approach reduces preparation time from hours to minutes and requires simple instrumentation. In addition, the ability to use this approach for complex patterning approaches such as laser patterning allows for great flexibility and reduces regulatory burden.

[0209] Advantages of the methods disclosed herein include, but are not limited to, speed (minutes instead of hours), simplicity (simple exposure and equipment), and use of synthetic matrices.

[0210] Example 3 - Deciphering specificity between antibodies and vaccines using enriched human lymphoid, intestinal, and pulmonary organoids For targeted interventions in cancer, infectious diseases, and organ transplantation, it is desirable to construct a multi-organ platform that recapitulates and couples the lymphatic system and tissue-resident immunity to recapitulate the immunological mechanisms of pathology and drug-induced responses (Figure 1).

[0211] Described herein are compositions, devices, and methods for generating human lymphoid-on-a-chip systems and interconnecting them with intestinal and lung organ model systems to better understand mucosal lymphoid-immune crosstalk during infection and vaccination. Biomimetic immune follicles-on-a-chip can recapitulate germinal center (GC)-like cell populations and functionality (Figure 2). Lymphoid tissues can be integrated with immune-competent intestinal organoids (Figure 3) and other mucosal organ systems such as the lung.

[0212] Follicle-on-a-chip It is desirable to engineer immune follicles-on-a-chip that recapitulate the phenotype, characteristics, and decision-making of human B cells (Figure 2).

[0213] Experimental approaches include defining lymphoid microenvironments, engineering immune follicles-on-chip, and assessing B cell activation. The immune follicle-on-chip device can be a microfluidic-based system that includes hydrogel features that can support B cell processes. The device can include gradients of hydrogel-encapsulated immune and stromal cells. B cell activation can be assessed using omics, sequencing, biochemical assays, and flow cytometry.

[0214] A biomaterial-based approach to human lymphoid follicular organoids. A schematic of this approach is shown in Figures 4, 5, and 7.

[0215] Lymphoid cell survival and activation. Germinal center responses were determined from tonsil / PBMC in organoids (Figure 6). Conditions were optimized for tonsil / PBMC organoids.

[0216] Cytokines included IL4 (2d) and IL21 (2d). Cell types were 1) PBMC, engineered CD40LL, and FDC (human), and 2) tonsil, engineered CD40LL, and FDC (human). Endpoints included D4 and D8 (N=3-5). Preparations included PBS, H1N1 antigen only, TLR7 / 8 agonist and H1N1, and TLR3 agonist and H1N1.

[0217] PEG-4MAL Organoid Characterization The effect of ECM and hydrogel weight % on PEG-4MAL-based organoids was evaluated. ECM-dependent / independent proliferation and survival of stromal CD40L cells and FDCs was established (Figures 8-9). The effect of polymer weight % on temporal stability and hydrogel stiffness was quantified (Figure 10).

[0218] Primary human B cell viability To evaluate primary human B cell viability, PBMCs and tonsils were compared (Figures 11 to 14). At D8, CD19 hi The number of + B cells was observed twice as much in PBMC organoids as in tonsillar organoids (Figure 12). At D8, PBMC-organoids have more GC B cells than tonsillar organoids (Figures 13-14). At D8, PDMC-organoids show the effect of TLR agonists (Figures 13-14).

[0219] For lymphoid follicle on-chip engineering - PEG-4MAL lymphoid follicle on-chip engineering, it was desired to develop a microfluidic device with high fidelity, e.g., greater than 85% of the desired features were retained (Figures 15-16). The challenge was that the PEG-4MAL macromers gel quickly and therefore may not be suitable for inclusion in a large-scale device.

[0220] Bioengineered gradient devices for lymphoid follicle-on-chip. Due to the challenges presented above with PEG-4MAL, thiol-norbornene (thiol-ene) photoclickable PEG hydrogels were investigated (Figure 17). These compositions were found to gel within 10-15 seconds of UV exposure. Unlike PEG-vinylsulfone and acrylates, the thiol-norbornene bioconjugate chemistry was found to be compatible with high viability of primary B cells comparable to PEG-maleimide (Figure 18).

[0221] A schematic diagram of lymphoid follicle on-chip manipulation is shown in Figure 15. It is desirable to generate more than 300 microgels per minute using the microfluidic device. Furthermore, microgel degradation can be obtained within 1-7 days in a user-defined manner (Figure 19). At least 50% viability of immune cells was obtained in the microgels (Figure 20).

[0222] Lymphoid tissue-accumulated intestinal organoids It would be desirable to develop lymphoid tissue-enriched intestinal organoids to study vaccine-mediated immunity (Figure 21).

[0223] Experimental approaches involve generating lymphoid tissue-enriched intestinal organoids, validating the organoids, and evaluating vaccine responses (e.g., influenza vaccine response). Tubular mini-guts and integrated lymphoid tissues were established. Designer hydrogels were integrated into a perfusable platform to generate a hybrid microchip system. B-cell follicles in lymph nodes and immune-competent immune microenvironments representing the first line of defense were engineered. The microbiome was enriched. Organoids were validated using state-of-the-art omics, sequencing, and microscopy.

[0224] Human intestinal organoid PEG-4MAL density and adhesion peptide control were investigated (Figure 22). HIO viability and development were examined for hiPSCs in PEG-4MAL (Figures 23 and 24).

[0225] PEG-4MAL density regulates HIO formation. The effect of PEG-4MAL density on HIO formation was investigated and validated in HiPSCs (Figure 25). Polarized distribution of apical EZRIN and basolateral β-catenin, as well as expression of ZO-1 and ECAD in the apical junctional complex, were observed (Figure 26).

[0226] To transfer the technology to a gut-on-a-chip, a perfusable microfluidic system is required, which is difficult to achieve with PEG-4MAL. The fast gelation of PEG-4MAL requires laser dissociation to form a perfusable microfluidic system, which hampers facile fabrication.

[0227] Characterizing intestinal cells from hiPSCs grown in hydrogels. Intestinal cells from hiPSCs grown in hydrogels were characterized (Figure 27). Spheroid vs. HIO (d14) structures showed high variability after 24 hours of growth in 4% PEG-4NB and 4% PEG-4MAL (Figure 27). The effect of the presence of immune cells on differentiation has also been investigated.

[0228] Fabrication of Intestinal Organoid-on-a-Chip Device The intestinal organoid-on-a-chip microchip system comprises an elastomeric device with a central hydrogel chamber for subsequent organoid culture and perfusion (Figure 28). Optimization of fabrication parameters to obtain a crosslinked intestinal hydrogel system with high shape fidelity and controlled swelling has been investigated.

[0229] Optimization of swelling properties and incorporation of hiPSC-derived intestinal cells are being investigated, as well as the performance of intestinal organoids when cultured with immune cells.

[0230] Integrated organs-on-chips For example, it may be desirable to develop lymphoid and intestinal integrated organ-on-a-chip models, which in some instances may be extended to other mucosal organ systems, such as the lung.

[0231] Establishing and Characterizing Hybrid Bulk and Microgels Composite PEG-4aNB hydrogel bulk and PEG-4MAL microgels containing human B cells were characterized (Figure 29).

[0232] Example 4 - A rapid and simple light-based approach to generate complex hydrogel structures for organ-on-chip models Human intestinal organoids (HIOs) represent an excellent tissue source for intestinal disease and tissue modeling (Figure 30), however HIO induction in a three-dimensional matrix without perfusion may limit their development and functionality, resulting in a closed architecture with reduced growth and homeostasis.

[0233] Fabrication of current perfusable gut-on-a-chip platforms based on hydrogels requires laborious, time-consuming, and laser-based, equipment-intensive methodologies and is limited to natural biological matrices (e.g., Matrigel).

[0234] Described herein is a rapid and simple light-based approach to generate complex hydrogel structures for use in gut-on-a-chip models. The method is highly flexible and reduces regulatory burdens by reducing preparation time from hours to seconds, using simple instrumentation, and allowing the use of synthetic hydrogels rather than natural matrices.

[0235] Methods. A schematic of the synthetic hydrogel photopolymerization mechanism is shown in Figure 31. A schematic of photopatterning PEG-4NB hydrogels for the fabrication of perfusable mini-intestinal structures using UV light and a photomask is shown in Figure 32. Photopatterning of complex structures in synthetic hydrogels, including perfusable channels for cell culture and medium perfusion, can be achieved in less than 1 second.

[0236] Photopatterned hydrogels with controlled swelling and shape fidelity. Hydrogel swelling can be difficult to control. Swelling can be an important parameter for maintaining high shape fidelity in hydrogel features. The effect of crosslinker and weight % (Figure 33), PEG-4NB molecular weight (Figure 34), and temperature (Figure 35) on swelling was investigated. The hydrogel preparations result in reduced swelling (8±2%) leading to high shape fidelity (83±8%) of photopatterned features with different shapes (Figure 36). Different photomask designs can be used to create complex shapes (Figure 37).

[0237] Seeding the gut-on-a-chip with HIOs. A schematic diagram of seeding the gut-on-a-chip with HIOs is shown in Figure 38. Single cells from day 28 HIOs were used to seed within the device (5-10 × 10 6 cells / mL).

[0238] Single cells from HIOs are cultured in the gut-on-a-chip. Medium perfusion can be important for cell viability over time (Figures 39-40). Medium perfusion improves cell viability and device surface coverage % over time (Figure 40).

[0239] The gut-on-a-chip device allows for long-term culture of HIOs, which grow colony-forming hydrogel surfaces (Figures 41 and 71).

[0240] Conclusion: These results demonstrated that the gut-on-a-chip system is a suitable and accessible platform for the development of relevant intestinal organoids as well as long-term culture systems. Further experiments can focus on the assessment of cell type distribution and intestinal function studies.

[0241] Example 5 Establishing a chemokine gradient within a polymeric PEG-4NB network for cell migration. A bull's-eye design was created in which gels were placed in the two innermost rings and flow was circulated in the outermost ring (Figure 42-Figure 45). The design allows flow to flow around the gel without disturbing the gel. Flow can traverse the device without backflowing into the inlet. A PEG-4NB gel (7.5% (w / v), 100% DDT) was used within the bull's-eye design.

[0242] Establishing the in situ stability of new PEG-4NB in ​​microfluidics for 4 days. Gels were placed in the two innermost rings. A comparison between the use of tangential and cross-flow patterns was performed (Figures 46-50). The gel was PEG-4NB (7.5% (w / v); VPM:DTT 80:20). A BSA gradient was tagged throughout the gel for 48 hours. The gradient travels the foil diameter of the device. The experiment established the in situ stability of PEG-4NB in ​​microfluidics for 4 days.

[0243] B cell maturation in PEG-4NB bulk gel. Experiments were performed with the intent of demonstrating at least 50% viability of B cells, with 50% or more of the cells acquiring a phenotype characteristic of GC B cells, and at least 10% of the cells acquiring a memory or plasma cell phenotype in the initial culture. The experimental design included 400k PBMC, 40k CD40LL, and 40k FDC / HK cells in 40 μL of gel; 50% VPM, 0.3 mM GFOGER, and 0.7 mM RDG; media: IL4 (D0-D2), H1N1 antigen (D0-D2), IL21 (D2-D12), and nBAFF (D0-D12). CD40LL and FDC / HK cells are present as engineered cells or through soluble or bead-based functionalization of proteins, peptides, or other biomolecules.

[0244] The results showed that: PEG-4NB supported greater than 50% B cell viability and proliferation, human PBMC B cells were able to differentiate in PEG-4NB cells (greater than 50% differentiated into GC B cells), and PEG-4NB results were within 10-50% of the PEG-4MAL results (Figures 51-54).

[0245] Incorporation of fibrinogen / collagen into PEG-4NB. The composition contained PEG-4MAL / 4NB (7.5% (w / v)), REDV (3.0 mM), VPM, and DTT (VPM:DTT-75:25). The composition further contained fibrinogen (4 mg / ml), collagen (1 mg / ml), and / or thrombin (2 u / ml).

[0246] The composition further comprises PEG-4NB:fibrin / collagen in a volume ratio of 1:0, 3:1, or 1:1.

[0247] The addition of a secondary polymer network was initially explored in BUMBLE-B. PEG-4NB with fibrin and collagen remains injectable and supports cell proliferation and clustering. This was demonstrated through the B-cell model cell line LY3 ​​clustering in the presence of CD40LL (Figures 55-58).

[0248] B cell maturation in PEG-4NB+FC gels in devices. The number of live B cells was increased in PEG-4NB hydrogels with fibrinogen and collagen (FC) (Figures 59-62). Based on these results, the use of PEG-4NB+FC will be used in experiments in lymphoid follicle-like on-chip. Fibrinogen and collagen are examples, but other devices containing synthetic gel-ECM protein blends can be used, for example with other synthetic or natural polymers used in place of fibrinogen and / or collagen.

[0249] Demonstration of cell migration in the device. The device contains PEG-4NB (5% (w / v)), REDV (3.0 mM), VPM, and DTT (VPM:DTT=80:20). The composition further contained fibrinogen (4 mg / ml), collagen (1 mg / ml), and / or thrombin (2 u / ml). A schematic diagram of the device is shown in Figure 63. Human B and T cell migration induced by a CXCL12 gradient over 4 days (Figure 64).

[0250] Immune cell differentiation. Cell differentiation was compared in devices containing PEG-4NB (FIG. 65) compared to those containing PEG-4NB, fibrinogen, and collagen. Results showed that immune cell differentiation was achieved as distinct phenotypic compartments in devices containing PEG-4NB, fibrinogen, and collagen (FIG. 66).

[0251] Example 6 - A rapid and simple light-based approach to generate complex hydrogel structures for organ-on-chip models Inflammatory bowel disease (IBD) is the term for two conditions (Crohn's disease and ulcerative colitis) that are characterized by chronic inflammation of the digestive tract. IBD affects more than six million people worldwide. The exact cause of IBD is unknown.

[0252] Human intestinal organoids (HIOs) represent an excellent tissue source for IBD and tissue modeling (Figure 30).

[0253] Conventional human intestinal organoid induction is performed in Matrigel (a trade name for a solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells produced by Corning Life Sciences) and in static conditions. This may limit the development and function of the final organoids, such as resulting in a closed architecture with reduced growth and homeostasis. This indicates the need for a perfusion system to obtain a more functional and relevant intestinal model.

[0254] Currently, the fabrication of perfusable intestine-on-a-chip platforms using hydrogels involves laborious, time-consuming, and laser-based, equipment-intensive methodologies and is limited to natural biological matrices (e.g., Matrigel). For example, the fabrication of current perfusable intestine-on-a-chip platforms using hydrogels is based on the patterning of natural-based matrices using a PALM microbeam Zeiss microscope as a dissection tool (Figure 67) (Nikolaev M, Mitrofanova et al. Homeostatic mini-intestines through scaffold-guided organoid morphogenesis. Nature. 2020 Sep; 585(7826):574-578).

[0255] Herein, we describe a method for photopatterning PEG-4NB hydrogels using UV light and a photomask (Figure 32). In particular, an unpolymerized polymer solution is covered with a photomask of the desired design and then irradiated with UV light. The portions of the polymer that are covered by the features of the photomask remain uncrosslinked, while the polymer that is exposed to UV light is crosslinked. The uncrosslinked polymer can be washed away to obtain complex structures patterned within the hydrogel, including perfusion channels for cell culture and medium perfusion (Figure 37). This technique reduces preparation time from hours to seconds and uses very simple instrumentation. In addition, the ability to use synthetic polymers in these methods provides flexibility and reduces regulatory burden compared to methods that use natural matrices.

[0256] One of the major challenges was to control the swelling of the hydrogel in order to maintain high shape fidelity of the developed features. For this, different polymer formulations were analyzed. The effects of crosslinker, polymer concentration and molecular weight, as well as temperature were studied (Figures 33-36). A polymer formulation was obtained that reduced swelling and provided excellent shape fidelity: 10 wt% PEG-4NB5kDa crosslinked with DTT. This hydrogel formulation reduces swelling and leads to high shape fidelity (83±8%) of photopatterned features with different geometries.

[0257] The next step was to culture single cells obtained from the HIO in the "gut-on-a-chip" device. Single cells from day 28 HIO were used to seed within the device (5–10 × 10 6 After 3 days of culture, it was observed that devices maintained under constant perfusion exhibited greater surface coverage by cells seeded within the lumen with superior viability compared to those cultured under static conditions (Figure 40). In summary, medium perfusion improves cell viability and device surface coverage over time.

[0258] Barrier function was demonstrated with at least 80% device coverage by viable hiPSC intestinal cells exhibiting appropriate apical and basolateral marker localization, the presence of differentiated epithelial cells (Paneth, Goblet, and Colonocytes), fatty acid adsorption, and at least 90% FITC-dextran exclusion.

[0259] Immunostaining of the gut-on-a-chip after 4 days of culture is shown in Figures 68 to 70. After 4 days of culture, some CDX2-positive cells, a hindgut marker, were also observed in the cells grown on the device.

[0260] These devices also allowed for long-term culture of cells / HIOs for up to 7 days, as cells began to three-dimensionally colonize the device, demonstrating full coverage of the available surface within the device lumen (Figures 41 and 71-72).

[0261] Another interesting approach is to seed intestinal spheroids instead of single cells from HIOs in the device. Seeding of spheroids derived from hiPSCs in tubular intestines on chip devices after 24 h of culture is shown in Figure 73-Figure 74. In this case, the formation of 3D complex structures was observed after 6 days of culture (Figure 75). Initial attachment, coverage, and 3D complex structure formation were observed after 6 days of culture (red arrows, Figure 76-Figure 83).

[0262] Immunostaining of hiPSC-derived spheroids in the tubular gut-on-a-chip device after 7 days of culture results are shown in Figures 84-85. Positive staining for E-cadherin and CDX2 markers suggests the presence of organized epithelial cell regions.

[0263] The results demonstrate that the method described herein is a powerful tool for accessible, convenient, and rapid hydrogel patterning.The gut-on-a-chip system is a suitable platform for the development of associated intestinal organoids as well as long-term culture systems.

[0264] Other advantages which are self-evident and inherent to the present invention will be apparent to those skilled in the art. It will be understood that certain features and subcombinations are useful and may be employed without reference to other features and subcombinations. This is contemplated by and is within the scope of the claims. Since many possible embodiments can be made of the present invention without departing from its scope, it should be understood that all matter herein described or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense.

[0265] The compositions, devices, and methods of the appended claims are not limited in scope by the specific compositions, devices, and methods described herein, but are intended as illustrations of some aspects of the claims, and any methods that are functionally equivalent are intended to be within the scope of the claims. Various modifications of the compositions, devices, and methods in addition to those shown and described herein are intended to be within the scope of the appended claims. Furthermore, although only certain representative compositional elements, system elements, and method steps disclosed herein are specifically described, other combinations of compositional elements, device elements, and method steps are also intended to be within the scope of the appended claims even if not specifically recited. Thus, although combinations of steps, elements, components, or elements may be explicitly referred to herein, other combinations of steps, elements, components, and elements are included even if not explicitly stated.

Claims

1. A method of fabricating a device comprising a hydrogel matrix and a first chamber within the hydrogel matrix, wherein the hydrogel matrix is derived from a prepolymer, the first chamber is perfusable, and the method comprises: blocking a first portion of a prepolymerization solution with a first photomask, wherein the prepolymerization solution comprises the prepolymer, such that the prepolymerization solution comprises an exposed portion and a first blocked portion; irradiating the exposed portion of the prepolymerization solution and the first photomask with electromagnetic radiation, wherein the first photomask is substantially impermeable to the electromagnetic radiation; comprising: wherein the prepolymer within the exposed portion of the prepolymerization solution photopolymerizes to form the hydrogel matrix, and the prepolymer within the first blocked portion does not photopolymerize, forming the first chamber.

2. A method of fabricating a device comprising a hydrogel matrix derived from a prepolymer, a first chamber within the hydrogel matrix, wherein the first chamber is perfusable, and a second chamber within the hydrogel matrix, wherein the second chamber is perfusable and fluidically independent from the first chamber, the method comprising: the method comprising: blocking a first portion of a prepolymerization solution with a first photomask and blocking a second portion of the prepolymerization solution with a second photomask, wherein the prepolymerization solution comprises the prepolymer, such that the prepolymerization solution comprises an exposed portion, a first blocked portion, and a second blocked portion; irradiating the exposed portion of the prepolymerization solution, the first photomask, and the second photomask with electromagnetic radiation, wherein the first photomask and the second photomask are substantially impermeable to the electromagnetic radiation; wherein the prepolymer within the exposed portion of the prepolymerization solution photopolymerizes to form the hydrogel matrix, the prepolymer within the first blocked portion does not photopolymerize, forming the first chamber, and the prepolymer within the second blocked portion does not photopolymerize, forming the second chamber.

3. The method according to claim 1 or 2, further comprising removing the first photomask and the second photomask (if present) after irradiation.

4. The method according to claim 1 or 2, further comprising flushing the hydrogel device after irradiation to remove any remaining prepolymerization solution and / or prepolymer.

5. The method according to claim 1 or 2, further comprising disposing the prepolymerization solution in a molding die that defines a shape before the first portion of the prepolymerization solution is blocked by the first photomask.

6. The method according to claim 1 or 2, wherein the hydrogel matrix comprises a synthetic hydrogel.

7. The method according to claim 1 or 2, wherein the prepolymerization solution further comprises a crosslinking agent, and the hydrogel is further derived from the crosslinking agent.

8. The method according to claim 1 or 2, wherein the hydrogel matrix is further derived from one or more additional components, such as one or more additional monomers, prepolymers, ligands, chemical agents, therapeutic agents, photoinitiators, or combinations thereof.

9. The method according to claim 8, wherein the prepolymerization solution further comprises the one or more additional components.

10. The method according to claim 8, wherein the hydrogel matrix is further derived from a natural or biological prepolymer.

11. The method according to claim 10, wherein the natural or biological prepolymer comprises fibrinogen, collagen, or combinations thereof.

12. The method according to claim 1 or 2, wherein the prepolymerization solution further comprises a photoinitiator.

13. The hydrogel matrix further comprises a chemical agent, a therapeutic agent, or a combination thereof dispersed therein, wherein the chemical agent and / or the therapeutic agent has a concentration that varies throughout the hydrogel matrix, such that the chemical agent and / or the therapeutic agent has a compositional gradient throughout the hydrogel matrix. The method according to claim 1 or 2.

14. The method according to claim 1 or 2, wherein the electromagnetic radiation comprises UV radiation.

15. The method according to claim 1 or 2, wherein the exposed portion of the prepolymerization solution photopolymerizes in a time amount of 1 millisecond to 1 hour.

16. The hydrogel matrix exhibits a swelling of 10% or less; the hydrogel matrix exhibits a shape fidelity of 50% or more; the hydrogel matrix has a storage modulus of more than 0 Pa to 5000 Pa; or a combination thereof, the method according to claim 1 or 2.

17. A device produced by the method according to claim 1 or 2, wherein the device is a microfluidic device.

18. A method of using the device according to claim 17 for diagnosis, disease modeling, regenerative medicine, drug screening, tissue modeling, or a combination thereof.

19. A method of using the device according to claim 17 as a biomaterial substrate or scaffold, a cell culture substrate or platform, or a combination thereof.

20. The method according to claim 19, wherein the method includes growing organoids and / or three-dimensional cell aggregates using the device.

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