Flexible fluidic percolated networks and uses thereof

Abstract

Compositions, methods and kits for forming a structure having a network of perfusable channels are disclosed. A composition includes sacrificial fibers configured to be degraded by a degrading agent, each sacrificial fiber having an anisotropic shape, and a structural material substantially stable with respect to the degrading agent. The structural material is configured to inducibly undergo a state change, the state change causing the composition to become more solid. The sacrificial fibers are mixed with the structural material at a concentration of an overall volume of the composition sufficient to allow formation of the structure having the network of perfusable channels upon degrading of at least a portion of the sacrificial fibers by the degrading agent. The composition enables rapid formation of perfusable channels in arbitrary structures through percolation which may be useful in tissue engineering, battery technology, or construction.

Description

Flexible Fluidic Percolated Networks and Uses ThereofRELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 562,022, filed on March 6, 2024. The entire teachings of the above application are incorporated herein by reference.GOVERNMENT SUPPORT

[0002] This invention was made with government support under R01 EB00262 and R01 EB033821 from National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health and under EEC-647837 from the National Science Foundation Engineering Research Center for Cellular Metamaterials. The government has certain rights in the invention.BACKGROUND

[0003] Percolation theory endeavors to probabilistically predict the threshold at which random distributed voids or pores in a substrate will connect such that a fluid is able to flow from one surface of the substrate to the opposite one. Study of percolation has led to advancements in a range of applications, for example, filter design, hydraulic engineering, tissue engineering, and fluid and gas extraction in natural materials. In recent years, engineers have utilized these insights to develop materials that leverage the advantages of anisotropic elements, e.g., cylinders, ellipsoids, or sphereocylinders, to create electrical and material networks that may more efficiently connect relative to isotropic bulk materials alone. Such anisotropic elements may allow a decrease in overall tortuosity of the network formed, thereby increasing network efficiency, and may also decrease the volume to surface area ratio inherent in the material, leading to increased exchange at material surfaces or boundaries.SUMMARY

[0004] The embodiments disclosed herein may be helpful for creating a mixture comprising a bulk material and anisotropic fibers distributed therein, whereby degrading of the distributed anisotropic fibers may create a material having a connected sacrificial fluidic network.

[0005] In an example embodiment, a composition for forming a structure having a network of perfusable channels includes sacrificial fibers configured to be degraded by a degrading agent, each sacrificial fiber of the sacrificial fibers having an anisotropic shape. The compositionfurther includes a structural material substantially stable with respect to the degrading agent. The structural material is configured to inducibly undergo a state change, the state change causing the composition to become more solid. The sacrificial fibers are mixed with the structural material at a concentration of an overall volume of the composition sufficient to allow formation of the structure having the network of perfusable channels upon degrading of at least a portion of the sacrificial fibers by the degrading agent.

[0006] The sacrificial fibers can include a polymer based on alginate, or a precursor or derivative thereof.

[0007] The sacrificial fibers can be composed of crosslinked polymers. The degrading agent can be a chelator that disrupts crosslinking of polymers. For example, the crosslinked polymers of the sacrificial fibers may include metal ions, e.g., Al3+, Ca2+, Ba2+, Cu2+, Fe2+, Fe2+, Mg2+, Sr2+, Co2+, and Zn2+, and the chelator may bind to the metal ions, thereby degrading the sacrificial fibers.

[0008] The sacrificial fibers can be cylindrical or spherocylindrical. As described herein, the sacrificial fibers have an anisotropic shape. The aspect ratio, e.g., width versus length, of the sacrificial fibers is not unity.

[0009] The sacrificial fibers can include a smooth surface or a patterned surface. A surface of the sacrificial fibers may be used to create perfusable channels with desired features, e.g., surface area or flow turbulence.

[0010] The sacrificial fibers can be of different sizes or aspect ratios. The sizes and the aspect ratios of the sacrificial fibers may be determined based on a desired parameter, e.g., perfusion rate or network density, of the network of perfusable channels.

[0011] The sacrificial fibers can include a cell adherent material or can be coated with a cell adherent material. For example, the sacrificial fibers may include arginine-glycine-aspartate (RGD) alginate. The sacrificial fibers including the cell adherent material may encapsulate cells that can be released upon degradation of the sacrificial fibers. As another example, the sacrificial fibers coated with the cell adherent material may be pre-coated with cells. Mixing the cell-coated sacrificial fibers with the structural material may enable the cells to coat or to fill the perfusable channels made by degrading of the sacrificial fibers.

[0012] The concentration of the sacrificial fibers in the composition can be determined by parameters of the sacrificial fibers and by parameters of the structural material. The parameters of the sacrificial materials may include fiber length and fiber diameter. The parameters of the structural material may include structural integrity of the structural material.

[0013] The structural material can include a pre-hydrogel configured to inducibly undergo the state change from the pre-hydrogel to a hydrogel. The structural material may be a prehydrogel including or derived from collagen, agarose, fibrin, dextran, or polyethylene glycol.

[0014] The structural material can include graphite or metal. The structural material can be configured to conduct electricity and may be useful for applications including batteries or supercapacitors.

[0015] The degrading agent can be an enzyme. The degrading agent may be configured enzymatically degrade the sacrificial fibers, which may include decrosslinking or cleaving of polymers or monomers of the sacrificial fibers. The enzyme can be alginate lyase and the sacrificial fibers are composed of alginate polymers

[0016] The structural material can be configured to remodel after undergoing the state change. The structure is configured to retain at least a portion of the network of perfusable channels after remodeling of the structural material. For example, the structural material may be mechanical remodeled, e.g., compacted or stretched. The structural material can be configured to relax or contract after causing the sacrificial fibers to degrade.

[0017] The composition can include the degrading agent in a substantially inactive state. The degrading agent in the substantially inactive state may be activated at a desired time. For example, the degrading agent may be an enzyme that is activated by increasing a temperature of the composition or changing a pH of the composition. In another example, the degrading agent may be a chelator, e.g., ethylenediaminetetraacetic acid (EDTA). An affinity of the chelator towards a metal ion in a crosslinked polymer may be increased by changes in pH. The degrading agent may be photoactivatable chelator that is a photo-caged molecule. The photoactivatable chelator may be activated when exposed to light.

[0018] The composition can further include cells. The composition may include individual cells or one or more cultures of one or more cell types each. The structural material may include the cells.

[0019] In another example embodiment, a method for forming a structure having a network of perfusable channels includes dispensing a composition in a desired shape. The composition includes: (i) sacrificial fibers configured to be degraded by a degrading agent, each sacrificial fiber configured having an anisotropic shape, and (ii) a structural material substantially stable with respect to the degrading agent, the structural material configured to inducibly undergo a state change. The sacrificial fibers are mixed with the structural material in the composition. The method further includes inducing the state change of the structural material, the state causing thecomposition to solidify in the desired shape. The method still further includes, with the degrading agent, causing selective degrading of at least a portion of the sacrificial fibers to form a structure having a network of perfusable channels.

[0020] The method can include adding the degrading agent in the composition. The degrading agent may degrade the sacrificial fibers over time. The degrading agent added can be in a substantially inactive state prior to dispensing the composition. Causing selective degrading can include activating the degrading agent. Activating the degrading agent can include changing a temperature or pH of the composition, adding an activating agent, or exposing the degrading agent to light.

[0021] The method can further include fabricating the sacrificial fibers prior to preparing the composition. For example, the sacrificial fibers may be alginate fibers composed of crosslinked alginate. Fabricating the sacrificial fibers may include dispensing alginate, crosslinking the alginate, and cutting the alginate into the alginate fibers.

[0022] Dispensing the composition can include pipetting, extruding, aliquoting, pouring, or compacting the composition into the desired shape. For example, the composition may be dispensed into a mold. As another example, the composition may be dispensed into a water-in- oil solution to form spherical constructions. The composition may be further compacted or manipulated prior to, during, or after undergoing the state change.

[0023] The method can further include aligning the sacrificial fibers in the composition prior to inducing the state change. Such embodiments may be used to create perfusable channels with greater directed alignment. Aligning the sacrificial fibers in the composition can include shear aligning the sacrificial fibers during the dispensing of the composition.

[0024] Inducing the state change of the composition can include changing a temperature of the composition, changing a chemical environment of the composition, exposing photosensitive molecules to light, or introducing a solidifying agent to the composition.

[0025] The method can further include allowing the structural material to remodel after causing selective degrading of at least a portion of the sacrificial fibers. For example, the structural material may relax or contract to change sizes of the network of perfusable channels.

[0026] The method can further include preparing the composition and, optionally, storing the composition prepared prior to dispensing the composition. Preparing the composition may include mixing the sacrificial fibers and the structural materials at a desired concentration. The composition may be stored for a period of time, e.g., days, weeks, months, or other periods of time, prior to use. Additionally, the sacrificial fibers and the structural material may be storedseparately until time of use and preparing the composition may include mixing the sacrificial fibers and structural material when needed.

[0027] The method can further include manipulating the composition prior to causing selective degrading of at least a portion of the sacrificial fibers. For example, the composition may be compacted, wherein the sacrificial fibers resist the compacting and retains a volume within the composition.

[0028] The method can further include adding a secondary structural material to the structure having the network of perfusable channels. The secondary structural material may fill at least a portion of the perfusable network. Examples of suitable secondary structural materials include, but are not limited to, a hydrogel with a substantially different stiffness, or an electrically conducting hydrogel polymer for neural stimulation.

[0029] The method can further include embedding the composition or the structure with cells.

[0030] The method can further include allowing the cells to migrate in the composition or the structure based on a preferred 3-dimenionsal (3-D) environment. The perfusable channels formed by the selective degrading of at least a portion of the sacrificial fibers may form a surface at an interface between the structural material and a void of the perfusable channels left by the sacrificial fibers. The surface may be a preferential 3-D environment for some cell types, for example, cells possessing an apical-basal polarity. Endothelial cells may be such a cell type and may migrate to the surface. Other cell types, for example, fibroblasts, cardiomyocytes, or hepatocytes, may show preference to other 3-D environments, and may remain within the composition or the structure.

[0031] In another example embodiment, a kit for forming a structure having a network of perfusable channels includes sacrificial fibers, each sacrificial fiber having an anisotropic shape, and a degrading agent configured to cause the sacrificial fibers to degrade. The degrading agent is substantially inert with respect to a structural material of the structure. When the sacrificial fibers are dispersed within the structural material, the degrading agent causes at least a portion of the sacrificial fibers to degrade to form the structure having the network of perfusable channels.

[0032] The kit can further include the structural material, wherein the structural material is inducibly configured to undergo a state change. The sacrificial fibers and structural material can be mixed together. For example, the sacrificial fibers and the structural material may be mixed at specific concentrations.

[0033] The degrading agent can be combined with the sacrificial fibers or the structural material. The kit can further include a solidifying agent separated from the sacrificial fibers and the structural material. The solidifying agent can be configured to induce the structural material to undergo the state change to cause the composition to become more solid.

[0034] Embodiments disclosed in the present invention may include one or more of the following properties or features. The embodiments may allow triggered degradation of anisotropic elements. The anisotropic elements may be rapidly degraded shortly after the bulk or supporting material has set or crosslinked or may resist compaction from the bulk material, thereby keeping future voids and percolated networks open until degradation and flow is desired. Furthermore, a pre-degraded network may morph with the overall material but still form a final percolated network. The degradation process may also be utilized with soft or flexible structural materials, including low weight percentage gels, soft gels, or natural materials. In some embodiments, the degradation process may be compatible with live cells. The anisotropic properties of the elements may enable shear alignment of fibers to create a more efficient fluidic network. Varying sizes or dimensions of the anisotropic elements, for example, the diameter, length, or aspect ratios, may be helpful in generating a network with desired characteristics. Degradation of the anisotropic elements may be triggered before or after the network is formed. In some embodiments, a percolated network may be reinforced by a reagent or cells in the bulk material, the bulk material subsequently undergoing degradation to leave undegraded fibers and the percolated reinforced network.

[0035] Embodiments may further enable introducing smooth, pseudo-2-dimensional structures within a volume or generating low volume, high surface area fluidic networks.BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0037] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.

[0038] FIG. 1 schematically illustrates a process for fabricating fibers and for a pipettable technique for creating a scalable fluidic network through percolation according to an example embodiment. The process includes (i) spinning alginate fibers in a bath of CaCh and cutting thealginate fibers spun into a desired length, (ii) aliquoting the fibers pipetted with a desired hydrogel mixture with or without cells, (iii) distributing the fibers volumetrically within a crosslinked hydrogel, and (iv) creating a fluidic network of connected voids after the fibers are degraded.

[0039] FIG. 2A illustrates a schematic of an example fiber segment establishing fluidic connections to another fiber segment.

[0040] FIGS. 2B and 2C illustrate brightfield images of example inter-vessel junctions in a fibrin hydrogel after alginate has been degraded.

[0041] FIG. 3 schematically illustrates an example percolation through a bulk material achieved by selective degrading of fiber cylinders distributed in a bulk material.

[0042] FIG. 4 schematically illustrates a process for allowing sacrificial elements to adapt and compact with a bulk hydrogel that naturally compacts or is actively remodeled, according to example embodiments.

[0043] FIGS. 5 A and 5B illustrate microscopy images of example wet spun alginate fibers.

[0044] FIG. 6A illustrates a schematic of an example device for cutting alginate fibers.

[0045] FIG. 6B illustrates an example embodiment of the device of FIG. 6 A.

[0046] FIG. 7 illustrates a microscopy image of cut alginate fibers, according to example embodiments.

[0047] FIGS. 8A and 8B illustrate plots of the distribution of sizes of fabricated alginate fibers, according to example embodiments.

[0048] FIGS. 9 A and 9B illustrate schematics of alginate fibers suspended in a solution in a pipette, according to example embodiments.

[0049] FIGS. 10A-10F illustrate brightfield images of example mixtures of fibers and fibrin gel, where mixtures include fibers of different diameters, lengths, and filler volumes.

[0050] FIG. 11 illustrates a schematic showing a water-in-oil emulsion technique for fabricating large hydrogel spheres, according to example embodiments. The inset shows a magnified view of an oil-hydrogel boundary.

[0051] FIG. 12A illustrates a rendering of an example spherical hydrogel encapsulating alginate fibers that can be selectively degraded when desired.

[0052] FIG. 12B illustrates a maximum projection image of a middle section of an example spherical agarose gel, similar to the spherical hydrogel of FIG. 12 A.

[0053] FIG. 13 A illustrates a rendering of an example spherical gel with a perfusable network.

[0054] FIG. 13B illustrates an image of an example spherical hydrogel, similar to the spherical hydrogel of FIG. 13 A, perfused with beads to show a connected fluidic network.

[0055] FIG. 13C illustrates a maximum projection image of the spherical hydrogel of FIG. 13B.

[0056] FIG. 14A illustrates a rendering of an example gel with a perfusable network within a fluidic device.

[0057] FIG. 14B illustrates an extended cross-section image of an example gel, similar to the gel of FIG. 14 A, perfused with beads to show a connected fluidic network.

[0058] FIGS. 15A and 15B schematically illustrate a needle extruding a mixture of hydrogel precursor with alginate fibers, the alginate fibers being shear aligned with the fluid, according to example embodiments.

[0059] FIG. 16A illustrates a rendering of an example gel with a shear-aligned perfusable network.

[0060] FIGS. 16B and 16C illustrate cross-section images of example gels with a shear- aligned perfusable network, similar to the gel of FIG. 16 A, perfused with beads to show a connected fluidic network.

[0061] FIG. 17A illustrates a rendering of an example gel with two large cylindrical channels fabricated by needles in a microfluidic device.

[0062] FIG. 17B illustrates an extended cross-section image of an example gel with two large cylindrical channels, similar to the gel of FIG. 17A, perfused to show a connected fluidic network.

[0063] FIGS. 18A-18C illustrate microscopy images of voids that may form a connected fluidic network in gels including different materials, according to example embodiments.

[0064] FIG. 19 illustrates a confocal image of a large-scale percolated fibrin hydrogel with microvascular sized degradable fibers, according to an example embodiment.

[0065] FIG. 20 illustrates a temporally-coded confocal slice of large bead perfusion in a two- channel device, according to an example embodiment.

[0066] FIG. 21 illustrates a schematic of an example microfluidic device including a gel that may include a perfusable network, including indicators for a central position and a peripheral position of the gel.

[0067] FIG. 22 A illustrates a doppler image of an example fibrin only gel.

[0068] FIG. 22B illustrates a doppler image of an example gel with a percolated network.

[0069] FIG. 23 illustrates a plot of normalized mean flux acquired using doppler imaging for different compositions of fibers in gels, including the gels of FIGS. 22A and 22B, according to example embodiments.

[0070] FIG. 24 illustrates a diagram of an example hypoxia sensor for assessing mass transport with individual cells.

[0071] FIGS. 25 A and 25B illustrate maximum projections of confocal images of a hypoxia reporter, similar to the hypoxia reporter of FIG. 24, in example gels with or without a perfusable network, respectively, at a central position of a microfluidic device similar to the microfluidic device of FIG. 21.

[0072] FIGS. 25C and 25D illustrate maximum projections of confocal images of a hypoxia reporter, similar to the hypoxia reporter of FIG. 24, in example gels with or without a perfusable network, respectively, at a peripheral position of a microfluidic device similar to the microfluidic device of FIG. 21.

[0073] FIG. 26 illustrates a violin plot of hypoxia reporter expression at different locations of the gels of FIGS. 25A-D.

[0074] FIG. 27 illustrates a plot of numbers of cells expressing a hypoxia reporter at different locations of the gels of FIGS. 25A-D.

[0075] FIG. 28A illustrates an example embodiment of a microfluidic device for creating a hypoxia gradient.

[0076] FIG. 28B illustrates a schematic of an example microfluidic device with a hypoxia gradient similar to the microfluidic device of FIG. 28 A.

[0077] FIGS. 29A and 29B illustrate microscopy images of cells in the fluidic device of FIG. 28A under normoxia conditions, according to example embodiments.

[0078] FIGS. 30A and 30B illustrate microscopy images of cells in the fluidic device of FIG. 28A under hypoxia conditions, according to example embodiments.

[0079] FIG. 31 illustrates a graph of intensity of a hypoxia marker versus oxygen saturation, according to an example embodiment.

[0080] FIG. 32A illustrates an extended cross-section image of an example gel showing a region with a lack of voids.

[0081] FIG. 32B illustrates a maximum projection of microscopy images of cell nuclei and a hypoxia reporter in the region of FIG. 32A according to an example embodiment.

[0082] FIG. 33A illustrates an extended cross-section images of an example gel showing a region with voids.

[0083] FIG. 33B illustrates a maximum projection of microscopy images of cell nuclei and a hypoxia reporter in the region of FIG. 33A according to an example embodiment.

[0084] FIG. 34 illustrates a violin plot of expression of a hypoxia reporter in the regions of FIG. 32A and 32B.

[0085] FIG. 35 illustrates a graph depicting a relationship between volume of functional parenchyma versus fiber volume fraction according to an example embodiment.

[0086] FIG. 36 illustrates a graph depicting a relationship between permeability of a material versus fiber volume fraction according to an example embodiment.

[0087] FIG. 37 schematically illustrates of a process of formation of a hydrogel including embedded cells, perfusion, and endothelialization, according to an example embodiment.

[0088] FIGS. 38A-38C illustrate fluorescence images of perfusion in hydrogels, similar to the hydrogel of FIG. 37, with or without perfusable channels, according to example embodiments.

[0089] FIGS. 39A-39C illustrate fluorescence images of endothelial cells in hydrogels, similar to the hydrogel of FIG. 37, with or without perfusable channels, according to example embodiments. An inset of a magnified region of each respective gel is also included.

[0090] FIGS. 40A-40C illustrate depth-coded max projection images of the zoomed-in inset of FIGS. 39A-39C, respectively.

[0091] FIGS. 41A and 41B illustrate minimum projection images of voids in example gels thresholded to create a binary image of voids formed using fibers of different parameters.

[0092] FIGS. 42A and 42B illustrate maximum projection images of epithelial cells in example gels with voids thresholded to create a binary image of endothelial cells, wherein the gel with voids is formed using fibers of different parameters.

[0093] FIG. 43 illustrates a graph of coverage percentage of the projected void areas of FIGS. 41A-42B by endothelial cells.

[0094] FIG. 44A illustrates a schematic of a liver triculture in a gel without perfusable channels according to an example embodiment.

[0095] FIG. 44B illustrates an extended fluorescence image of an example liver triculture and gel similar to the liver tri culture and the gel of FIG. 44 A.

[0096] FIG. 44C illustrates a fluorescence image of an example liver triculture and gel similar to the liver tri culture and the gel of FIG. 44 A with arrows indicating fibroblasts.

[0097] FIG. 45 A illustrates a schematic of a liver triculture in a gel with perfusable channels according to an example embodiment.

[0098] FIG. 45B illustrates an extended fluorescence image of an example liver triculture and gel similar to the liver tri culture and the gel of FIG. 45 A.

[0099] FIG. 45C illustrates a fluorescence image of an example liver triculture and gel similar to the liver tri culture and the gel of FIG. 45 A with arrows indicating fibroblasts.

[0100] FIG. 46A illustrates a schematic of an example cardiac triculture in a gel without perfusable channels.

[0101] FIG. 46B illustrates an extended fluorescence image of an example cardiac triculture and gel similar to the cardiac tri culture and the gel of FIG. 46 A.

[0102] FIG. 46C illustrates a fluorescence image of an example cardiac triculture and gel similar to the cardiac tri culture and the gel of FIG. 46 A with arrows indicating fibroblasts.

[0103] FIG. 47A illustrates a schematic of an example cardiac triculture in a gel with perfusable channels.

[0104] FIG. 47B illustrates an extended fluorescence images of an example cardiac triculture and gel similar to the cardiac tri culture and the gel of FIG. 47 A.

[0105] FIG. 47C illustrates a fluorescence image of an example cardiac triculture and gel similar to the cardiac tri culture and the gel of FIG. 47 A with arrows indicating fibroblasts.

[0106] FIG. 48A illustrates a schematic of an example culture of parenchymal cells and fibroblasts in a gel with perfusable channels.

[0107] FIG. 48B illustrates an extended fluorescence image of an example culture of parenchymal cells and fibroblasts similar to the culture and the gel of FIG. 48 A.

[0108] FIG. 49A illustrates a schematic of an example culture of stromal cells and fibroblasts in a gel with perfusable channels.

[0109] FIG. 49B illustrates an extended fluorescence image of an example culture of stromal cells and fibroblasts similar to the culture and the gel of FIG. 49A.

[0110] FIG. 50 illustrates a schematic of a general approach for culturing cells in a material having selectively degradable fibers according to an example embodiment.

[0111] FIG. 51 illustrates a schematic of relative time to perfusion and resolution of voids in a gel versus a construct size of the gel according to an example embodiment.

[0112] FIG. 52 illustrates an example embodiment of a kit including sacrificial fibers and a degrading agent configured to degrade the sacrificial fibers.DETAILED DESCRIPTION

[0113] A description of example embodiments follows.

[0114] Creating a network with anisotropic elements may be achieved, for example, by placing distinct elements and developing connections by surface-surface contacts or by forming voids by utilizing disparate materials that may react differently to similar stimuli. However, current methods may struggle to create a material with a flexible fluidic percolated network and anisotropic elements that is configured to undergo triggerable and orthogonal degradation. Such a material could, prior to degradation of the anisotropic elements, retain its overall shape and inhibit infiltration of the percolated network by surrounding bulk material and, following degradation, provide a highly dissipative or high flux exchange network. Applications for such materials may include without limitation tissue engineering, super-capacitor and flow battery batteries, and materials research.

[0115] Disclosed herein are embodiments of methods, compositions, and materials whereby fiber segments are distributed in an arbitrary bulk material, the fiber segments subsequently triggered to degrade to create a connected sacrificial fluidic network. Fiber degradation may be triggered at a desired time, and if a fiber volume density is high enough, voids left by degraded fibers may create a perfusable fluidic network through percolation. The embodiments described herein may be particularly suited towards combining rapidly degradable elements in a bulk material. For example, in an embodiment for tissue engineering, alginate fiber cylinders may be combined with fibrous hydrogels (i.e., fibrin, collagen) or with bulk-type hydrogels (agarose, dextran, PEG). This overall process may also work with non-aqueous materials as long as the material that makes up the fiber segments may be selectively degraded at a desired time. Such a system may allow for rapid creation of a perfusable network with pipettable, injectable, or sprayable, and thus automatable, fibers. Due to the anisotropic nature of the fiber cylinders, a volume taken up by fluidic network may be reduced and a resulting structure may be more resilient compared to isotropic degradable gels achieving percolation while concurrently increasing the surface area available for exchange. Embodiments of the present invention may be used alone; however, such embodiments may be compatible with many different engineering techniques already developed, e.g., 3D printing, material casting, electrode slurry deposition, film deposition, and photopatterning.

[0116] As used herein, the terms “degrade” and “degrading” refer to a reversible or irreversible process of breaking down a material. For example, in some embodiments, a crosslinked polymer may be degraded by a chelator, wherein the degrading includes decrosslinking of the crosslinked polymer. In such an embodiment, the process is reversible as the polymer may undergo crosslinking again. In some embodiments, a material may be degraded byan enzymatic process, wherein the degrading includes to cleave the material into smaller units in an irreversible process. In other embodiments, reversible or irreversible degrading may be caused by a change in temperature, light, or other agents. In some embodiments, degrading may include as non-limiting examples disintegrating, dissolving, de-crosslinking, or liquifying. A “degrading agent”, as used herein, refers to an element or means causing a material to degrade as described, such as, but not limited to, a chemical agent, an enzyme, heat, and light.

[0117] Products and applications including the embodiments described herein may be useful in a number of scientific fields or disciplines. For instance, biomedical products for the embodiments may include the anisotropic fibers, which may be aliquoted into individual use containers and later pipetted or dispensed into tissue engineering workflows, including without limitation 3-dimensional (3D) printing, molding hydrogels, and automated processes with pipetting robots. The fibers may also be super-critically dried to retain a shape while being more shelf stable. Further biomedical applications may include adding the fibers to a 3D printing gel ink sold as a product to 3D printing companies and promoted as printing a microvasculature that can be shear aligned or not. The fibers may also be included in a structural material with a freeform geometry, for example, a free-floating gel sphere, to create a perfusable sphere containing adherent or non-adherent cells within the structural material for biopharmaceutical applications. The cells may produce secreted proteins that are especially difficult to produce in bacteria due to, for example, post-translational modifications, such as in human insulin that have disulfide bonds, or large sizes, for example, adeno-associated virus (AAV) for therapies. The anisotropic fibers may also be added to fibrin glues for wound healing to increase the rate of vascularization, which may passively promote microvasculature reconnection after amputation reattachment. Because the degradation is triggerable, the fibrin may be allowed to compact fully in the wound and, once compaction is slowed, the fibers may be degraded to reveal a fluidic network. The fibers may further be used to scale up tissue and organ manufacturing in a laboratory setting and may resist compaction in vivo to keep a fluidic network open during initial stages of engraftment. Further applications of the fibers may include laboratory grown meat manufacturing for thicker meats, which are currently limited by vascularization and lack of fluidic networks to feed a 3D tissue.

[0118] Examples of applications for the embodiments in electrical products may include porous electrodes, which may be useful in supercapacitors or batteries (or flow batteries) by enabling a larger volumetric electrode that still has fluidic access to high surface areas of the electrode material. The triggerable degradation would be advantageous for manufacturing theelectrodes cleanly and cheaply. In some embodiments, batteries may be processed in the exact same way and the degradation agent may be added along with the electrolyte after the battery or supercapacitor is already fully assembled.

[0119] Further examples of applications for embodiments in building material products may include porous concrete. The porous concrete may be created by incorporating degradable fibers into a concrete precursor and, after the concrete has set, the fibers could be degraded to allow water to follow through percolated networks down into the ground to recharge ground water. Such an embodiment may be especially helpful in flood-prone areas that have a lot of surface area covered by roads and sidewalks.

[0120] Additional further embodiments may include, for example, water filters or heat sinks.

[0121] With respect to biomedical applications and tissue engineering, efficient distributed mass transport is an indispensable prerequisite to life, enabling any cell within multicellular organisms to access necessary oxygen and nutrients in order to grow to sizes exceeding the diffusion limit. To enable mass transport in highly metabolic tissues such as the liver, brain, and muscles, each cell in the tissue resides within approximately 200 pm of a capillary blood vessel. When blood supply is interrupted, the tissue may become ischemic within 20 minutes resulting in irreversible damage.

[0122] Creating thick, highly cellularized tissues may be useful for therapeutic applications. However, a major barrier to generating engineered tissue of clinically relevant size is the lack of a strategy to rapidly generate perfusable fluidic networks of appropriate caliber (10-150 pm diameter) throughout the tissue, while still operating in the temperature, pH and aqueous confines dictated by living, cellularized tissues. Biological 3D printing may be suited for creating repeatable large scale hollow structures (>200 pm), including those with physiological cell densities, but has yet to achieve the capillary scale networks required for effective tissue nutrient exchange. Light-based methods recently have achieved smaller length scales with high cell numbers, but the serial nature of 3D printing nonetheless further increases assembly time as the vasculature becomes smaller and scale of the engineered tissue increases. In contrast, vasculogenic self-assembly may be employed to create capillary sized structures (5-40 pm), including from endothelial coated alginate beads. However, these methods take days to become perfusable - too slow for highly metabolic tissues. As a result, rapid generation of appropriatesized vasculature within large tissue constructs remains a significant challenge.

[0123] Disclosed here is a system and method, termed SPAN - Sacrificial Percolation of Anisotropic Networks, that utilizes anisotropic alginate cylinders as pipettable sacrificial unitsthat can be suspended into a bulk material, for example, a hydrogel pre-polymer, spontaneously forming a percolated network. Following gelation of the surrounding material and degradation of the alginate fibers, a perfusable network of interconnected cylindrical voids is left behind by overlapping fibers. Furthermore, when the volume percentage occupied by the cylinders exceeds the percolation threshold and reaches a percentage that establishes a distributed continuous network, perfusion can be circulated throughout a three-dimensional space rather than a single percolated fluidic path. This technique is inherently scalable to arbitrary construct sizes and flexible to many applications.

[0124] As demonstrated herein, anisotropic sacrificial building blocks may be used to create aligned or unaligned percolated networks with established tissue engineering techniques such as needle casting, suspended culture, and shear aligned networks with 3D extrusion printing. The process may be compatible with a variety of hydrogels commonly used in cell culture. This process may be applied to rapidly establish perfusion to support the function of dense, multi-cell type laden constructs. Furthermore, the perfusable networks may guide tissue self-organization, whereby endothelial cells preferentially line the void surfaces while the stromal and parenchymal cells largely remain in the 3D matrix. The result is the generation of a perfusable vascularized network, all while maintaining perfusion across the entire tissue.

[0125] In a specific embodiment, a method for creating a structure having perfusable networks begins by creating the anisotropic fibers. Low viscosity alginate, e.g., alginate at 1-2% weight by volume (w / v) is crosslinked with 20 grams / liter CaCh. Alternative ions that may be used for crosslinking include Al3+, Ca2+, Ba2+, Cu2+, Fe2+, Fe2+, Mg2+, Sr2+, Co2+, and Zn2+. The fibers may be manufactured at a desired length, diameter, and aspect ratio, and the fibers are aliquoted into individual tubes at a desired concentration. In some embodiments, the fibers may be of different lengths, diameters, or aspect ratios. The fibers are pipetted to mix the fibers with a pre-gel mixture, with or without cells. The gel is formed in a desired configuration and set using a solidifying agent. With certain gels, additional steps may be necessary to prevent the alginate fibers from degrading prematurely. For instance, in fibrin-based gels, the sodium citrate used with fibrinogen to prevent clotting may chelate calcium ions used to crosslink the alginate. Phosphate buffered saline (PBS) may also chelate the calcium ions and may be replaced with HEPES buffered saline with added CaCh. Medias that use bicarbonate buffer may also chelate calcium ions. After the bulk hydrogel is crosslinked or set, alginate microfiber cylinders may be rapidly degraded using ethylenediaminetetraacetic acid (EDTA) as a chelator, enzymatically with alginate lyase, or slowly chelated by interacting with bicarbonate buffers in most cellculture medias. In some embodiments, the alginate fibers may be degraded at a later time to prevent compaction of the hydrogel from disrupting the perfusable networks and the degradation may be triggered after compaction slows. Alginate lyase may also be encapsulated in polymers to slow release of the enzyme. This may be particularly advantageous in an in vivo injection or implantation setting. Once alginate is degraded or de-crosslinked, voids form in place of the alginate fibers. If two of the alginate fibers degraded were in contact, or sufficiently close together in bulk hydrogels that can slightly degrade or relax when only a thin gel structure remains, the voids may connect fluidically. If the alginate fiber density in the mixture is sufficiently high enough for contacts between fibers to span the distance between the fluidic input and output, percolation may occur and a perfusable network may form.

[0126] Many gels are available that may be used as the bulk material. Commonly used hydrogels may include physically crosslinked gels (collagen, agarose, gelatin, hyaluronic acid), thermo-reversible physically crosslinked (poly(N-isopropylacrylamide) (PNIPAAm), poloxamer 407), ionically crosslinked (alginate, chitosan), enzymatically crosslinked (fibrin, gelatin, and other natural protein-based gels along with synthetic polymers modified with enzyme-reactive groups), and natural or synthetic polymers modified with chemically-reactive groups (i.e., methacrylate, thiol) to either form covalent chemical crosslinking or photocrosslinking when combined with an appropriate photoinitiator (i.e., lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP)). Additionally, some compounds, e.g., hyaluronic acid, polyethylene glycol (PEG), and gelatin, may be modified to crosslink by physical, enzymatic, or chemical means depending on formulation. New synthetic hydrogels are constantly being developed that may be adapted towards the embodiments described herein.

[0127] Alternatively, if the alginate fibers are initially distributed in the bulk hydrogel below the percolation threshold, but the bulk hydrogel may compact or be remodeled by cells and the bulk gel is free floating or non-attached, compaction of the bulk hydrogel may bring the fibers into contact. Fiber degradation may be triggered at any time during compaction. For example, fibers may resist the compaction until compaction slows and then be degraded to leave behind a percolation network that is connected to the outside of the gel, degrade immediately so the voids may be compacted and remodeled with the gel, or remain in the gel even after compaction has reduced. Leaving the fibers in the gel may serve as a resistant boundary to maturing cells, such as cardiomyocytes beating around the circumference of the fiber.Degradable fibers create scalable fluidic networks through percolation

[0128] In a specific embodiment of the present invention, degradable anisotropic fibers are used to develop simple, robust system capable of rapidly establishing a perfusable fluidic network to support cells in various engineered tissues. Alginate fibers are used as a sacrificial element due to their facile degradability and ease of mixing with various prehydrogel mixtures, both with and without cells. The versatility of the alginate fibers as the sacrificial element may be useful towards ensuring broad applicability through their compatibility with various hydrogel formulations and existing tissue engineering platforms. The combination of the alginate fibers may be dispensed to form a composite construct upon solidification, after which the alginate may be rapidly degraded to support perfusion. Although many scalable techniques exist for production of alginate fibers, off-the-shelf alginate was selected for ease of adoption.

[0129] FIG. 1 schematically illustrates a process 100 for fabricating fibers 106 and for a pipettable technique for creating a scalable fluidic network 120 through percolation according to an example embodiment. The process includes (i) spinning alginate fibers in a bath of CaCh and cutting the alginate fibers spun into a desired length, (ii) aliquoting the fibers pipetted with a desired hydrogel mixture with or without cells, (iii) distributing the fibers volumetrically within a crosslinked hydrogel, and (iv) creating a fluidic network of connected voids after the fibers are degraded. The fibers 106 are configured to be degradable by a degrading agent, for example, alginate fibers in the presence of a chelator or alginate lyase. Fabrication of the fibers 106 includes use of a syringe pump 102 connected to a blunt ended needle 103 immersed in a spinning bath 104 of calcium chloride to create the fibers 106. In a particular embodiment, the fibers, e.g., 106, may range in diameter from 10 to 150 / m. Additional characterization of the fibers is provided herein with respect to FIGS. 5A-10F. The fibers 106 are allowed to crosslink in the spinning bath 104 before being manually cut to specific lengths, e.g., between 250 pm to 1 mm, to form fiber segments 118. The fiber segments 118 may be sterilized and stored in aliquots 108 at high concentration until used. Long term storage of the aliquots 108 in a microcentrifuge tube 109 format may allow for the addition a prehydrogel mixture 110 with or without cells, for example, adding the prehydrogel mixtures using a pipette tip 111, when desired to form a composition 112. The composition 112 may be dispensed, e.g., using another pipette tip 114, wherein the hydrogel may be crosslinked to form a solidified hydrogel 116 with a desired geometry. The fiber segments 118 may be degraded immediately or at a later time to create a perfusable network 120 with anisotropic voids in the shape of the sacrificial fiber segments 118.

[0130] Storing the fiber fragments 118 at high concentrations in aliquots, e.g. the aliquots 108, may be helpful for creating compositions, e.g., the composition 112, with sufficientconcentrations of the fiber fragments 118 to create a perfusable network, e.g., 120, within a solidified bulk material, e.g., the solidified hydrogel 116. An example method for creating the aliquots with a high concentration of the fiber fragments 118 may include pipetting an aliquot with a low concentration of the fiber fragments 118. The fiber fragments are held in the pipette tip and allowed to settle, similar to the pipette tips described herein with reference to FIGS. 9 A and 9B. A portion of the aliquot with a high concentration of the fiber fragments 118 (i.e., the portion in which the fiber fragments have settled, is dispended into a storage container, e.g., a microcentrifuge tube, for long term storage.

[0131] Some embodiments of the present invention may include a selectively degradable composition, for example, the composition 112. The selectively degradable composition includes sacrificial fibers, e.g., the fiber segments 118, configured to be degraded by a degrading agent. Each sacrificial fiber of the sacrificial fibers may, similar to the fiber segments 118, have an anisotropic shape. The selectively degradable composition further includes a structural material stable with respect the degrading agent. The structural material is configured to inducibly undergo a state change that causes the composition to become more solid. For example, the structural material may be the prehydrogel mixture 110 that undergoes crosslinking to become the solidified hydrogel 116. In some embodiments, the sacrificial fibers may include alginate polymers crosslinked with calcium ions, wherein the degrading agent may include a calcium chelator or alginate lyase and wherein the structural material is substantially stable with respect to the calcium chelator or the alginate lyase. For a specific example embodiment of the selectively degradable composition, the sacrificial fibers may be mixed with the structural material at a concentration between 25% to 75% of an overall volume of the composition.

[0132] An example embodiment of a method for forming a structure having a network of perfusable channels is described herein. The method includes dispensing a composition 112 in a desired shape. The composition 112 may be configured to be selective degradable, the composition including: (i) sacrificial fibers 118 configured to be degraded by a degrading agent, each sacrificial fiber configured having an anisotropic shape, and (ii) a structural material, e.g., the prehydrogel mixture 110, substantially stable with respect to the degrading agent, the structural material configured to inducibly undergo a state change. The method further includes inducing the state change of the structural material, the state causing the composition to solidify in the desired shape, e.g., the solidified hydrogel 116. The method still further includes, with the degrading agent, causing selective degrading of at least a portion of the sacrificial fibers 118 toform a structure having a network of perfusable channels, for example, the perfusable network 120.

[0133] FIG. 2A illustrates a schematic of an example fiber segment establishing fluidic connections to another fiber segment. Establishing a percolated network, for example, the perfusable channels 120 of FIG. 1, requires fluidic integration of individual anisotropic voids. By introducing a calcium chelator such as ethylenediaminetetraacetic acid (EDTA) or alginate lyase (AL), an enzyme that rapidly degrades alginate, a contact area between two alginate fibers 218-1, 218-2 may forms an open inter-vessel junction 222a that connects the two remaining cylindrical voids in the gel. A probability of these junctions, e.g., the inter-vessel junction 222a, fluidically connecting increases as a volume fraction of the alginate fibers increases. Dispersing the fibers within a structural material, for example, the two alginate fibers 218-1, 218-2 in the solidified hydrogel 116 of FIG. 1, may enable fluidic network to become volumetrically dispersed to drive sufficient mass transport to support living tissue.

[0134] Additionally, as illustrated in FIG. 2A, the two alginate fibers 218-1, 218-2 include smooth surfaces. In some embodiments, fiber segments may include patterned surfaces. The patterned surfaces of the fiber segments may introduce patterned surfaces on voids of a perfusable network, e.g., the perfusable network 120 described herein with reference to FIG. 1, left behind after degradation of the fiber segments, which may be useful for creating different flow patterns or modifying cell adherence to surfaces of the voids.

[0135] FIGS. 2B and 2C illustrate brightfield images of example inter-vessel junctions in a fibrin hydrogel after alginate has been degraded. The FIGS. 2B and 2C are acquired for fibrin gels stained with crystal violet with voids left over after alginate fibers are degraded by EDTA. FIG. 2B illustrates an open inter-vessel junction 222b at an intersection of cylindrical voids 224- 1, 224-2 in a gel 226 formed by degrading sacrificial fibers. FIG. 2C illustrates additional openvessel junctions, as indicated by white arrows, that are fluidically connected. Black arrows indicate voids that may not be connected fluidically. The scalebar for FIG. 2C is 100 pm.

[0136] As described herein with reference to FIGS. 2A-2C, a degrading agent may be introduced to degrade fiber segments embedded within a bulk material. In some embodiments, the degrading agent may be introduced after solidifying of the bulk gel. For example, EDTA, a calcium chelator, may be introduced to the solidified bulk gel to cause de-crosslinking of alginate fibers. In other embodiments, a degrading agent may be added to a composition of a bulk gel and fiber fragments in a substantially inactive state. The degrading agent may later be activated, an activated state of the degrading agent causing degradation of the fiber segments.Examples of such degrading agents, which may be added in a substantially inactive state and later activated, include an enzyme activated by changes in temperature or pH, e.g., alginate lyase, or a photoactivatable chelator.

[0137] FIG. 3 schematically illustrates an example percolation through a bulk material 316 achieved by selective degrading of fiber cylinders, e.g., 318-1, distributed in the bulk material 316. The bulk material 316 may be similar to the solidified hydrogel 116 of FIG. 1. Similar features are designated with like reference numbers but increased by 200. Percolation through the bulk material 316 may be achieved by distribution of fiber cylinders, e.g., 318-1, 318-2, before the bulk material 316 is set or crosslinked, followed by selective degradation of the fiber cylinders, e.g., 318-1, in which voids left over from fiber degradation connect and span a distance from a fluidic input through a bulk material 316 and to the other side. It is necessary that a number of the fiber cylinders, e.g., 318-1, 318-2, is sufficiently high enough to probabilistically span the desired distance give diameters, lengths, and aspect ratios of the fiber cylinders, e.g., 318-1,318-2. Percolation paths 330 may form that proceed from a fluidic boundary 328-1 to another fluidic boundary 328-2 if fiber-fiber contacts are made at each area showing overlapping fiber cylinders, e.g., 318-1, 318-2. The bulk material 316 itself may have low relative permeability. Isolated fiber cylinders, e.g. 318-2, may be isolated from others and are not part of a percolated network 320. The bulk material 316 may also attach to a fluid impermeable boundary 328-3, which may force a fluid entering the bulk material 316 from a fluidic input, for example, from the fluidic boundary 328-1, to an output on a specific side, for example, the another fluidic boundary 328-2. Fluidic access may also surround bulk materials.

[0138] In some example embodiments, the percolated network 320 may be filled at least in part with a secondary structural material. The secondary structural material may be configured to undergo a phase change to become more solid, similar to the bulk material 316. The secondary structural material may further be different from the bulk material such that conditions causing the phase change or degradation of the secondary structural material are different from conditions causing the phase change or degradation of the bulk material 316.

[0139] FIG. 4 schematically illustrates a process for allowing sacrificial elements, e.g., fiber segments 418, to adapt and compact with a bulk hydrogel 416 that naturally compacts or is actively remodeled, according to example embodiments. The bulk material 416 may be similar to the solidified hydrogel 116 of FIG. 1. Similar features are designated with like reference numbers but increased by 300. Active remodeling of the bulk hydrogel 416 may be achieved, for example, by cells in a free floating or untethered tissue construct. In some embodiments, the bulkhydrogel 416, for example, a floating sphere of a hydrogel with a low density of the fiber segments 418, the fiber segments being cylinders composed of alginate, may initially have a low probability of being in contact. For instance, (1) compacted bulk hydrogel 432-1 may decrease in volume while the fiber segments 418 remain largely unchanged in volume. The fiber segments 418 may be brought into contact with each other during compaction, the fiber segments 418 themselves resisting compaction and retaining a similar diameter and length. Some of the fiber segments 418 may be exposed to the environment outside of the bulk hydrogel 416. (2) Once compaction slows or ceases, the fiber segments 418 may be degraded, leaving behind fluidic conduits connecting an outside environment to a percolated network 420-2 inside the bulk hydrogel 416 with voids approximately the same size as the fiber segments 418 before degradation. (3) Degradation may also be triggered mid-compaction, which will form percolated network 420-2 with voids that may be compacted, or reduced in size, along with the bulk hydrogel 416. (4) The percolated network 420-3 formed with a compacted hydrogel 432-2 using such a technique may include voids that are significantly smaller than the fiber segments 418.

[0140] As illustrated in FIG. 1, alginate fibers may be fabricated by dispensing from a syringe pump. In some embodiments, a specific process for fiber fabrication may include the following. Low viscosity alginic acid sodium salt may be mixed with ultrapure water at 2% w / v, heated to 60°C for 2 hours, and then left at 4°C overnight. Rehydrated alginate may be used within 1 week. To fabricate fibers, alginate is drawn into a 2-5 mL syringe and left to come to room temperature. The syringe is connected to EVA plastic tubing via lock adapters with a blunt ended needle connected to the end. Various gauges may be used to achieve desired alginate fiber diameters. Dispensing speed may be determined by a syringe pump. A crystallization dish is mounted on a vertical motor controlled by a microcontroller and motor shield, filled with 20 g / L calcium chloride dihydrate and spun at a speed of -150 revs / min. Once the dish is up to speed, the blunt needle is inserted (-1 cm from the dish wall) and the shear force from the gelation bath extrudes the fiber. After fabrication, the fiber is left to sit in the gelation solution for at least 20 minutes before cutting and the transferred to a solution of 10 mM CaC12.

[0141] FIGS. 5A and 5B illustrate microscopy images of example wet spun alginate fibers. FIG. 5 A illustrates fibers fabricated with a diameter size of 10 pm, including an inset showing the fibers at higher magnification. FIG. 5B illustrates fibers fabricated with a diameter of 55 pm.

[0142] To cut the alginate fibers, the gelled alginate fiber coils are laid perpendicularly on an array of razor blades with fixed spacing determined by custom cut microscope slides or polyimide sheets that are held together with a stainless-steel binder clip, and then cut by rolling asyringe along the individual blades. Once cut, large pieces of fiber and fibers annealed to each other from the cutting process are removed. Fibers are kept in 5 mM CaC12 in water at 4°C until aliquoted.

[0143] FIG. 6A illustrates a schematic of an example device 634a for cutting alginate fibers. As described herein, the device 634a includes an array of razor blades, e.g., 636a, which may be positioned at pre-determined intervals. Fibers 618 are placed perpendicularly on the array of razor blades, e.g., 636a, of the device 634a to be cut.

[0144] FIG. 6B illustrates example embodiments of the device of FIG. 6 A. The devices 634b, 634c 634d include an array of razor blades, e.g., 636b, held together by a holder, e.g., a binder clip 638. The razor blades of the devices 634b, 634c, 634d are arrayed with spacings of 250 «m (634b), 500 pm (634c), and 1 mm (634d).

[0145] FIG. 7 illustrates a microscopy image of cut alginate fibers, according to example embodiments. Fibers 718-1, 718-2, 718-3, 718-4 may be cut to a desired size based upon the spacing of razors in a fiber cutting device, e.g., the devices 634b, 634c, 634d described herein with reference to FIG. 6B.

[0146] In some embodiments, cutting fibers into fiber segments may result in slight deviations from a desired fiber segment size. The table below presents desired lengths versus actual fiber lengths achieved for cutting alginate fibers using devices similar to the devices 634b, 634c, 634d described herein with reference to FIG. 6B.

[0147] FIGS. 8A and 8B illustrate plots of the distribution of sizes of fabricated alginate fibers, according to example embodiments. FIG. 8A plots the distributions of diameters of two batches of alginate fibers with a target fabrication diameter of 60 pm. FIG. 8B plots the distributions of diameters of two batches of alginate fibers with a target fabrication diameter of 120 pm.

[0148] If desired, fiber segments may be sterilized. CaCh solution is aspirated using a 40 pm cell strainer placed on 50 mL conical tube and inverted. Sterile filtered ethanol with 5 mM CaCh is added to resuspend the fiber segments and left overnight at 4°C. The ethanol was aspirated, and fibers were washed three times with sterile filtered HEPES buffered saline (HBS) with 5mM CaCh. HBS may be adapted from a Cold Spring Harbor Protocol [1] by adding 5 mM CaCh and without sodium bicarbonate or other non-HEPES buffers because such solutions may chelate calcium from ionically crosslinked alginate.

[0149] Cut fiber segments may be aliquoted for storage or for later use. Dilute cut alginate fibers are pipetted into open ended 1 mL serological pipettes and left to settle out at the bottom of the pipette tip. After settling for around 30 minutes, fixed volumes of concentrated fibers are dispensed into 1.5 mL microcentrifuge tubes. 12.5 pL and 25 pL aliquots are most used in the experiments described herein. Aliquots are stored at 4°C and wrapped in parafilm to prevent evaporation until used. Large orifice pipette tips may be used when mixing pre-gel solutions or pipetting fibers.

[0150] FIGS. 9A and 9B illustrate schematics of alginate fibers, e.g., 918-1, 918-2, suspended in a solution in a pipette 940-1, 940-2, according to example embodiments. The alginate fibers, e.g., 918-1 in an aliquot 908-1 may be pipetted up into the pipette 940-1, which may be a serological pipette, as illustrated in FIG. 9A. FIG. 9B illustrates the pipette 940-2 with an aliquot 908-2 wherein the alginate fibers, e.g., 918-2 have been allowed to settle at the bottom of the serological pipette 940-2 and to become more close-packed.

[0151] FIGS. 10A-10F illustrate brightfield images of example mixtures of fibers and fibrin gel, where mixtures include fibers of different diameters, lengths, and filler volumes. The fibers may be tunable within microvascular ranges. Illustrated are fibers of diameter 120 pm, length 1 mm, and filler volume 50% (FIG. 10A); diameter 60 pm, length 500 pm, and filler volume 50% (FIG. 10B); diameter 30 pm, length 250 pm, and filler volume 50% (FIG. 10C); diameter 120 pm, length 1 mm, and filler volume 25% (FIG. 10D); diameter 60 pm, length 500 pm, and filler volume 25% (FIG. 10E); and diameter 30 pm, length 250 pm, and filler volume 25% (FIG. 10F).

[0152] Degradation of alginate fibers may be achieved using alginate lyase (AL). The enzyme is specific to degradation of alginate, specifically M-block sugars, while not affecting cell viability over long periods of time. EDTA may be used in addition to AL to increase a rate of degradation. While EDTA may be a more commonly used reagent to degrade alginate gels, prolonged exposure to EDTA is lethal to cells. Additionally, because the AL acts enzymatically, it may be included in a prehydrogel mix in a state of substantially slow enzymatic activity. In such an embodiment, the enzyme, evenly distributed throughout the construct, acts to degrade the alginate when the temperature is raised to 37°C.

[0153] Filler volumes may be adjusted to vary a density of fibers within a bulk hydrogel. For some of the embodiments described herein, 25-50% filler volumes are utilized. Filler volumesranging between 25%-50% are able to facilitate creation of disperse fluid flows while retaining bulk gel structure within commonly used soft hydrogels and while leaving adequate volume to include bulk hydrogel precursors and high numbers of cells. Filler volumes of up to 75% may be used. However, at this proportion some hydrogels (i.e., 2.5 mg / mL fibrin) begin to fail due resulting weak structure after degradation. However, increasing stiffness and weight percentage of a bulk gel allows for high filler volumes. Additionally, pipetting mixtures using filler volumes above 75% becomes difficult and resulting gels become inconsistent. Potential ranges of filler volumes for creating a structure having a perfusable network may depend upon the type of bulk material and the parameters, e.g., size, diameter, or length, of the fibers.

[0154] Void fidelity relative to sacrificial alginate fibers is highly dependent on choice of bulk gel. Fibrin stress relaxes after crosslinking and voids may expand around 5 pm after 12 hours post crosslinking and degradation. In contrast, no detectable difference was found within 1% agarose between fiber and void diameters after degradation.

[0155] The embodiments described herein involving tissue constructs were performed over a maximum of 5 days. However, in some embodiments using the same material system in a similar device with an increased number of fibroblasts, tissues appeared stable for two weeks without issue. Without cells added, many hydrogels presented can be stored for months at 4°C.

[0156] Additionally, some embodiments of the present invention may include a kit for creating a structure having perfusable channels.

[0157] FIG. 52 illustrates an example kit 5599 including sacrificial fibers 5518 and a degrading agent 5519 configured to degrade the sacrificial fibers 5518. The sacrificial fibers 5518 may be in an aliquot, for example, the aliquot 108 described herein with respect to FIG. 1, and may be mixed with a given structural material. The degrading agent 5519 may be substantially inert with respect with the structural material such that, when the sacrificial fibers 5518 are dispersed within the given structural material, the degrading agent 5519 may cause selective degradation of the sacrificial fibers 5518 to form a structure having perfusable channels.Sacrificial percolated anisotropic networks in current fabrication techniques and biomaterials

[0158] A strength of the process for generating perfusable networks in bulk material using sacrificial fibers may lie in the scalability to any construct size without compromising resolution of the microvasculature. Several example embodiments of fabricating structures having such perfusable networks are described herein.

[0159] In some embodiments, structural material may include a bulk hydrogel, e.g., the prehydrogel 110 or the solidified hydrogel 116 described herein with respect to FIG. 1, including agarose gel, collagen gel, or fibrin gel. In some embodiments using an agarose gel, low gelling temperature agarose is heated in HBS with 5 mM CaCh until uniform. Agarose is then mixed with HBS with 5 mM CaCh and alginate fibers then immediately put on ice to rapidly gel. In other embodiments, 500 pg / mL of alginate lyase is added last to other mixed prehydrogel components and the mixture was added immediately to oil emulsion.

[0160] In some embodiments using a collagen gel, a collagen prehydrogel may be prepared as described in [2], except HBS with 5 mM CaCh is used in place of Dulbecco’s phosphate- buff ered saline with calcium and magnesium (DPBS++), and lOx low glucose Dulbecco’s Modified Eagle’s Medium (DMEM) in liquid form. Briefly, Type I bovine collagen is brought to a neutral pH on ice, then crosslinked for 1 hour in a 37 °C tissue culture incubator with 5% CO2, after which 500 pg / mL of alginate lyase is added overnight.

[0161] In some embodiments using a fibrin gel, fibrinogen and thrombin solutions are made by mixing HBS with CaCh with fibrinogen to neutralize sodium citrate in the rehydrated fibrinogen to prevent premature degradation of the alginate fibers. HBS w / 5 mM CaCh, alginate fibers, and thrombin are added to the fibrinogen mixture. After the addition of thrombin, the solution is quickly injected into a tissue chamber, and the tissue chamber is repeatedly rotated over approximately 1 minute while the solution crosslinks to ensure even distribution of alginate fibers.

[0162] In some embodiments of the present invention, a water-in-oil emulsion system may be used to produce large spherical hydrogels with embedded alginate fibers. In such embodiments, fabricating hydrogels of different sizes require only pipetting a larger volume of a mixture, for example, the composition 112 described herein with respect to FIG. 1.

[0163] FIG. 11 illustrates a water-in-oil emulsion technique for fabricating large hydrogel spheres, e.g., 1142, according to example embodiments. The inset shows a magnified view of an oil-hydrogel boundary. This water-in-oil emulsion technique may be used to produce the large hydrogels, e.g. 1142, with embedded alginate fibers (not shown, but similar with respect to the fibers 118 embedded in the solidified hydrogel 116 described herein with respect to FIG. 1). Silicone oil 1146 is mixed with polyethylene glycol (PEG)-12 dimethicone 1144 with the emulsifier (PEG-12 dimethicone 1144) making up 0.01% v / v. Aqueous gel solutions are pipetted directly into the solution and left to gel. A pre-gel mixture, e.g., the composition 112, described herein with respect to FIG. 1, is then pipetted directly into the emulsion solution contained in astandard tissue culture plate. The hydrogel is allowed to crosslink and is then moved to a buffered solution or cell media. Alginate lyase is included at a concentration to drive fiber degradation in approximately 30 minutes, although increasing the concentration of alginate lyase may result in even faster times to fiber degradation.

[0164] FIG. 51 illustrates relative time to perfusion and resolution of voids in a gel 5416a, 5416b, 5416c versus a construct size of the gel 5416a, 5416b, 5416c according to an embodiment. As described herein, a degrading agent for fibers embedded within a gel, for example, the gel 5416a, 5416b, 5416c may be alginate lyase and may be distributed throughout a gel. Thereby, fabricating hydrogels of different sizes may require around the same amount of time, and time to achieve perfusion may be similar for differently sized gels, e.g., the gel 5416a, 5416b, 5416c.

[0165] FIG. 12A illustrates a rendering of an example spherical hydrogel 1242a encapsulating alginate fibers, e.g., 1218, that can be selectively degraded when desired. In the presently described embodiments, the agarose spheres, e.g., 1242a, containing alginate fibers, e.g., 1218a, that were 60 pm in diameter and 1 mm long, are crosslinked for 5 minutes and transferred to a well of PBS containing 20 mM EDTA for 15 minutes. The spheres are perfused with 500 nm fluorescent beads for 15 minutes, fixed, and cut into 500 pm sections, e.g., 1248.

[0166] FIG. 12B illustrates a maximum projection image of a middle section of an example spherical agarose gel 1242b, similar to the spherical hydrogel of FIG. 12A. Confocal images of a middle section, e.g., 1248 of FIG. 12 A, show voids 1220 filled with fluorescent beads, suggesting that the spherical agarose gel 1242b is successfully perfused through the voids 1220.

[0167] FIGS. 13A-18C provide additional exemplification of embodiments of forming a structure having a network of perfusable channels through the use of degradable fiber segments. In some embodiments, beads fluorescent beads are perfused through a hydrogel to show the network of perfusable channels. Agarose gels were perfused are 500 nm fluorescent beads while collagen and fibrin gels are perfused with 2 pm fluorescent beads and either imaged during perfusion or fixed in place by photocrosslinking dextran-methacrylate (dextran-MA) mixed with the fluorescent beads. Imaging is acquired using a laser scanning confocal microscope.

[0168] FIG. 13 A illustrates a rendering of an example spherical gel 1342a with a perfusable network 1320a. In some embodiments, the spherical gel 1342a may be generated using a common fiber / matrix prepolymer of 1% weight by volume (w / v) agarose as a bulk gel. Additionally, in some embodiments, the spherical gel 1342a may be generated using the water-in-oil emulsion technique described herein. In further embodiments, perfusion may be enhanced in spheroid droplet cultures using orbital shaker culture baths.

[0169] FIG. 13B illustrates a image of an example spherical hydrogel 1342b, similar to the spherical hydrogel 1342a rendered in FIG. 13 A, perfused with beads to show a connected fluidic network 1320b. FIG. 13B includes a 3-D image stack of confocal microscopy images of the spherical hydrogel 1342b, wherein magenta beads embedded within the spherical hydrogel 1342b show the spherical shape and perfused beads show the connected fluidic network 1320b in white.

[0170] FIG. 13C illustrates a maximum projection image of the spherical hydrogel of FIG. 13B. The maximum projection image is acquired from the top of the spherical hydrogel 1342b, showing a connected fluidic network 1320b using perfused fluorescent beads.

[0171] FIG. 14A illustrates a rendering of an example gel 1442a with a perfusable network 1420a within a fluidic device 1450. Structures formed within the fluidic device 1450, e.g., the gel 1442a, may be perfused within the fluidic device 1450.

[0172] Fluidic devices, e.g., the fluidic device 1450 may be created using polydimethylsiloxane (PDMS). Molds for the fluidic devices are constructed using stereolithography. PDMS is cured at a 1 : 10 mixing ratio overnight at 60°C in the molds, and individual devices are cut and plasma-bonded to glass slides. To enhance extracellular matrix (ECM) bonding to PDMS, a surface inside a tissue chamber of the fluidic devices is functionalized with 0.01% poly-l-lysine and 1% glutaraldehyde following plasma-activation and washed overnight in de-ionized (DI) water. When seeding the fluidic devices with cells, the fluidic devices are soaked in 70% ethanol (EtOH) and dried. Devices are sterilized using ultraviolet (UV) light for 15 min before cell seeding

[0173] FIG. 14B illustrates an extended cross-section image of an example gel 1442b, similar to the gel 1442a rendered in FIG. 14 A, perfused with beads to show a connected fluidic network 1420b. FIG. 14B further includes cross-section views along orthogonal axes, i.e., the Y- X and X-Z views, indicating three-dimensional distribution of the connected fluidic network 1420b.

[0174] FIGS. 15A and 15B schematically illustrate a needle 1552 extruding a mixture 1512 of hydrogel precursor 1510 with alginate fibers, e.g. 1518, the alginate fibers being shear aligned with the fluid, according to example embodiments. The mixture 1512 may be similar to the composition 112 of FIG. 1 and similar features are designated with like reference numbers but increased by 1400. FIG. 15A shows the needle 1552 extruding the alginate fibers, e.g., 1518dispersed throughout a bulk material, e.g., the hydrogel precursor 1510 to shear align individual alginate fibers, e.g., 1518. As the mixture 1512 is extruded from the needle 1552, the mixture 1512 lays down onto a substrate 1554, which may be a 3D printing stage, a wound bed, or other in vivo tissue. FIG. 15B shows a solidified hydrogel 1516, which may be formed from the mixture 1512 of FIG. 15A after undergoing gelation, after degradation of the alginate fibers 1518 is triggered. In the solidified hydrogel 1516, an aligned fluidic network 1520 is exposed, allowing perfusion through the solidified hydrogel 1516.

[0175] FIG. 16A illustrates a rendering of an example gel 1642a with a shear-aligned perfusable network 1620a within a confined structure 1656. The voids of the perfusable network 1620a may be created in the gel 1642a by shear-aligning fibers using the process described herein with respect to FIGS. 15A and 15B. Furthermore, the shear-aligned perfusable network 1620a is made by flowing a composite material, e.g., the composition 112 described in reference to FIG. 1, through the confined structure 1656 before polymerization or crosslinking.

[0176] FIGS. 16B and 16C illustrate cross-section images of example gels 1642b with a shear-aligned perfusable network 1620b, similar to the gel 1642a rendered in FIG. 16 A, perfused with beads to show a connected fluidic network. The gel 1642b is confined within a 750 pm diameter tubing.

[0177] FIG. 17A illustrates a rendering of an example gel 1742a with two large cylindrical channels 1758a-l, 1758a-2 fabricated by needles in a microfluidic device 1750. The gel 1742a is made by sacrificially casting a composition, for example, the composition 112 described herein with reference to FIG. 1, around acupuncture needles. The acupuncture needles may be removed after solidifying the composition to form the two large cylindrical channels 1758a-l, 1758a-2. Degrading of fibers within the gel 1742a reveals a perfusable network 1720a. The gel 1742a may be connected to a larger microfluidic lumen of the microfluidic device 1750.

[0178] FIG. 17B illustrates an extended cross-section image of an example gel 1742b with two large cylindrical channels 1758b- 1 , 1758b-2, similar to the gel 1742a rendered in FIG. 17A, perfused to show a connected fluidic network 1720b. Additional cross-section images along the Y-Z plane and the X-Z plane are also shown.

[0179] FIGS. 18A-18C illustrate microscopy images of voids that may form a connected fluidic network in gels including different materials, according to example embodiments. The voids in FIGS. 18A-18C are perfused with fluorescent beads and shown in white. FIG. 18A illustrates the connected fluidic network in an agarose gel, FIG. 18B illustrates the connectedfluidic network in a collagen gel, and FIG. 18C illustrates the connected fluidic network in a fibrin gel.

[0180] FIG. 19 illustrates an example confocal image of a large-scale percolated fibrin hydrogel with microvascular sized degradable fibers. The percolated fibrin hydrogel may be similar to the gel 1442a rendered in FIG. 14 A. Additionally, the percolated fibrin hydrogel of FIG. 19 is of size 1 cm x 1 cm x 750 pm and shows dispersed voids created by degradation of 120 pm alginate fibers at a 50% filler volume.

[0181] FIG. 20 illustrates a temporally-coded confocal slice of large bead perfusion in a two- channel device, according to an example embodiment. The two-channel device of FIG. 20 may be similar to the gel 1742b with the two large cylindrical channels 1758b- 1 , 1758b-2 of FIG.17B and is created by casting a gel around needles, the needles removed after the gel is solidified to form large channels. 6 pm fluorescent beads are flowed into the gel though a channel of the large channels. Temporal coding shows positions of beads in time windows of approximately 500 ms across a period of 10 seconds. FIG. 20 shows the fluorescent beads moving to the left within the needle cast channel (top) down and to the right into a fiber created void (bottom) in the device.SPAN enhances mass transport to support living cells within a construct

[0182] Embodiments of the present invention may be helpful to achieve adequate oxygen / mass transport to cells in a cell culture. A number of parameters may play a role in creating a construct or a structure having a perfusable network, including filler volume (25 - 50%), fiber diameters (250 pm - 1 mm), and fiber lengths (60 - 120 pm), and example embodiments of constructs having degradable fibers of different parameters within fibrin gels have been evaluated and described herein. Embodiments including fibers up to 2 mm long were explored; however, the embodiments presented herein are constrained to fibers of up to 1 mm in length to maintain uniform pipettability of the pre-gel solution, e.g., the composition 112 described herein with reference to FIG. 1, using a standard wide-orifice pipette tip. In some embodiments with fibers with 120 pm in diameter, consistent percolation may be achieved using a filler volume of 25%, a percentage reminiscent of vasculature volume percentages observed in cell dense in vivo tissues (15-30%).

[0183] FIG. 21 illustrates a schematic of an example microfluidic device 2150 including a gel 2142 that may include a perfusable network, including indicators for a central position 2162 and a peripheral position 2164 of the gel 2142. The microfluidic device 2150 may be used to evaluate flow through the gel 2142, which may be embodiments of gels having a perfusablenetwork made using different filler volumes of alginate fibers, fiber diameters, and fiber lengths. The microfluidic device 2150 further includes media wells 2160-1, 2160-2 that may contain a medium, e.g., a liquid, that may be configured to flow from one media well of the media wells 2160-1, 2160-2 to another media well. Confocal imaging is acquired for the microfluidic device, as described herein with respect to FIGS. 25A-27, at the central section 2162 and the peripheral section 2164 of the gel.

[0184] Flow, or fluid flux, through a hydrogel may be measured using Doppler imaging. In some embodiments, Doppler imaging may be performed using a PDMS fluidic device fabricated and surface treated as described herein. The PDMS fluidic device may be similar to the fluidic device 2150 described herein with respect to FIG. 21 and may include fibrin gels assembled encapsulating alginate fibers of varying length and filler volumes. The fibers are degraded and 2% milk is perfused overnight at 4°C. The following day, the PDMS fluidic devices are imaged using a laser Doppler imager with a pressure head difference between wells of the PDMS fluidic device, e.g., the media wells 2160-1, 2160-2 as described herein with respect to FIG. 21, of 9.25 mmH20.

[0185] FIG. 22 A illustrates an example Doppler image of a fibrin only gel. The fibrin only gel does not have a percolated network.

[0186] FIG. 22B illustrates an example Doppler image of a gel with a percolated network. Doppler imaging of the gel with the percolated network reveals enhanced fluid flux through a construct (the gel) with respect to the fibrin only gel of FIG. 22 A.

[0187] FIG. 23 illustrates an example plot of normalized mean flux acquired using Doppler imaging for different compositions of fibers in gels, including the gels of FIGS. 22A (2366) and 22B (2368). Doppler imaging shows in FIG. 23 that fluid flux through a construct is enhanced when filler volume is increased to 50%. Furthermore, while shorter fibers may advantageously provide more compact packing within a volume, computational models have shown that this advantage of increased density is outweighed by a necessity for fluidic connections to be established between each void within the system. Consistent with these predictions, embodiments including the longest pipettable cylinder unit (1mm) provided the largest average fluid flux among tested fibrin gels. Because the largest average fluid was achieved with embodiments using 120 pm diameter fibers that arelmm long at a filler volume of 50%, embodiments of this configuration are used to determine if increased mass transport provided by a perfused network is sufficient to prevent hypoxia within dense, cell-laden engineered tissue constructs, a common surrogate used to infer adequate oxygen delivery to the tissue.

[0188] A hypoxia reporter, similar to one reported in [3], may be used to evaluate an ability of percolation networks to support viable tissue. A construct of an example embodiment of the hypoxia reporter may couple UnaG, an eel fluorophore with a chromophore capable of oxygenindependent maturation to a PEST degron. This may promote rapid degradation of any low-level leak in the circuit and enables detection of transient changes in a degree of oxygenation.

[0189] FIG. 24 illustrates a diagram of an example hypoxia sensor for assessing mass transport with individual cells. The diagram of FIG. 24 includes a genetic construct including the combined fluorescent protein-degron (UnaG-PEST) construct described herein. Furthermore, the hypoxia sensor may be driven by a minimum cytomegalovirus (CMV) promotor and five hypoxia response elements (HRE). As such, cellular stabilization of native hypoxia inducible factor 1 (HIF-1) in hypoxic environments may drive construct expression. Under normoxic conditions, the fluorescent protein is minimally expressed while under hypoxic conditions (approximately less than 5 % oxygen) the UnaG protein accumulates within a cytoplasm of a cell. The genetic construct is enclosed within two long terminal repeat (LTR) segments. Using this construct, a human embryonic kidney (HEK)293FT-HRE cell line is developed. Behavior of the HEK293FT-HRE cell line under hypoxic conditions may be evaluated and calibrated in a microfluidic device described herein with respect to FIGS. 28A-31.

[0190] In some embodiments, for culturing HEK-293FT cells, HEK-293FT cells are cultured in lx DMEM with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. In some embodiments, generation of HRE-HEK cells may include plating 500,000 HEK293FT cells into a single well of a 6 well plate. 4 hours later, 1 pg of pLenti-HRE-dUnaG, 700ng of pCMVR8.74, 200 ng of pMD2.G VSVG, and 100 ng of p Ad Vantage™ are co-transfected into each well using an NaCl-polyethylenimine transfection. 72 hours following transfection, supernatant is collected, filtered through at 0.45 pm filter, and concentrated using the PEG-it™ viral precipitating agent. The viral pellet is resuspended in 100 pL PBS and stored at -80°C. HEK 293 FT cells are plated in a 6-well at a density of 100,000 cells per well. The next day, 50 pL of resuspended viral particles are added to each well of the 6-well and allowed to incubate overnight. Media is changed 12 hours after addition of viral particles. Forty-eight hours after viral transduction, cells are treated with media containing 1 pg / mL puromycin for two days. Cells surviving selection are tested for function (hypoxia driven fluorophore expression and calibration as described herein with respect to FIGS. 28A-31) and immediately cryopreserved.

[0191] HEK293 cells may rapidly degrade fibrin gels. As such, in some embodiments, HEK293 may be embedded into collagen gels, for example, at a concentration of 25 millioncells / mL, with or without alginate fibers. In some embodiments, the alginate fibers may measure 120 gm in diameter and 1 mm in length and may be added at a 50% filler volume. The constructs including the HEK293 cells and the collagen gels, with or without the alginate fibers, are cultured for two days in a standard cell culture rocker to drive fluid flow. Live imaging is conducted in two different areas, e.g., the center position 2162 and the peripheral position 2164 as described herein with respect to FIG. 21, assessing hypoxia in a very center of the construct or at a periphery near a source of cell culture media, respectively.

[0192] In some embodiments, to encapsulate HEK293FT-HRE cells within a collagen gel, the HEK293FT-HRE cells are growth arrested with 10 pg / mL mitomycin C in DMEM growth media and washed five times in stock DMEM. HRE-HEKs are lifted from tissue culture plates using TrypLE™ Express, centrifuged, and resuspended at a concentration of 250 million cells / mL in DMEM+. To create cell-containing collagen hydrogels, collagen hydrogels are assembled as described herein with HRE-UnaG HEKs and lx DMEM+ added immediately after collagen solution is brought to a neutral pH. Solutions of cells and collagen hydrogel are then quickly injected into a tissue chamber, and the tissue chamber is manually flipped every 5 seconds for approximately 1 minute while the solutions crosslink to ensure even distribution of the cells and alginate fibers. Alginate lyase is added 1 hour after tissue assembly. The solution is placed on a programable rocker that pauses for 3 minutes per side at angles of + / - 40° from level and left for 24 hours. Media is exchanged daily.

[0193] In further embodiments, hypoxia in a tissue sample may be quantified. After two days of culture, the tissue chambers including HRE-UnaG HEKs cells are live stained with a nucleic acid stain for 15 mins. A pressure head is created between media wells to increase flow through the gel. Media is then exchanged from DMEM+ to L-15 Leibovitz media without phenol red. Images are captured. For confocal image stacks, surfaces may be created surrounding individual cells within 3D stack using thresholded fluorescence signals from HRE-HEK cells. A maximum intensity within individual cells is calculated along with a total number of cells expressing the HRE signal above a detectable threshold applied to all conditions.

[0194] FIGS. 25 A and 25B illustrate maximum projections of confocal images of a hypoxia reporter, similar to the hypoxia reporter of FIG. 24, in example gels with or without a perfusable network, respectively, at a central position of a microfluidic device similar to the microfluidic device of FIG. 21. The gel with a perfusable network are created using alginate fibers measuring 120 pm in diameter and 1 mm in length and may be added at a 50% filler volume. White spots in FIGS. 25A and 25B indicate expression of the hypoxia reporter. The gels (bulk collagen gels)may enable adequate mass transport to allow HEK cells to survive. FIG. 25 A indicates significant expression of the hypoxia indicator by the HEK cells in the central position of the gel without a perfusable network. In comparison, FIG. 25B indicates lower expression of the hypoxia reporter by the HEK cells in the central position of the gel with the perfusable network.

[0195] FIGS. 25C and 25D illustrate maximum projections of confocal images of a hypoxia reporter, similar to the hypoxia reporter of FIG. 24, in example gels with or without a perfusable network, respectively, at a peripheral position of a microfluidic device similar to the microfluidic device of FIG. 21. The gel with a perfusable network is created using alginate fibers measuring 120 pm in diameter and 1 mm in length and may be added at a 50% filler volume. White spots in FIGS. 25C and 25D indicate expression of the hypoxia reporter and dotted white lines indicate an edge of a media well, similar to the media well 2160-2 described herein with respect to FIG. 21. Significant expression of the hypoxia reporter is seen in the peripheral position of the gel without the perfusable network (FIG. 25C), with the exception of a strip within around 300 pm of the media well, and less expression of the hypoxia reporter is present in the peripheral position of the gel with the perfusable network (FIG. 25D). The scalebar of FIGS. 25A-25D indicate a length of 200 pm.

[0196] FIG. 26 illustrates a violin plot of hypoxia reporter expression at different locations of the gels of FIGS. 25A-D. For both the central position and the peripheral position, lower maximum intensities of HRE-UnaG, the hypoxia reporter, are observed for cells in the gels with the percolated network, as shown by Center / Well 120pm sections of the plot. The 120 pm denotes a diameter of fiber segments used to create the percolated network. In FIG. 26, ns indicates not significant and “***” indicates statistical significance at a -value of / ?<0.001.

[0197] FIG. 27 illustrates a plot of numbers of cells expressing a hypoxia reporter at different locations of the gels of FIGS. 25A-D. For both the central position and the peripheral position, fewer cells expressing the hypoxia reporter are observed for cells in the gels with the percolated network, as shown by Center / Well 120pm sections of the plot. In FIG. 27, ns indicates not significant, indicates statistical significance at a -value of <0.05, and “****” indicates statistical significance at a -value of / ?<0.0001.

[0198] FIG. 28A illustrates an example embodiment of a microfluidic device 2850a for creating a hypoxia gradient. The microfluidic device 2850a may be used to evaluate an HEK293FT-HRE cell line configured to detect hypoxic conditions as described herein with respect to FIG. 24. The microfluidic device 2850a includes a cell channel with normoxia 2876 and a cell channel with oxygen gradients 2874a. The microfluidic device 2850a creates theoxygen gradient in the cell channel with oxygen gradients 2874a using an oxygen scavenging channel 2866a configured to create a hypoxic environment. A selectively permeable barrier between the oxygen scavenging channel 2866a and the cell channel with oxygen gradients 2874a allows oxygen to diffuse into the hypoxic oxygen scavenging channel 2866a to create the oxygen gradients. The hypoxic conditions of the oxygen scavenging channel are created using pyrogallol, introduced at a pyrogallol inlet 2868, and sodium hydroxide (NaOH) to create a basic pH environment, introduced at an NaOH inlet 2870. The pyrogallol and the NaOH may be extracted from the microfluidic device at an oxygen scavenger outlet 2872.

[0199] Additionally, the cell channel with oxygen gradients 2874a and the cell channel with normoxia 2876 include cell inlets 2878a, 2880, respectively, that may be used for seeding cells and cell outlets 2882a, 2884, respectively, that may be used to generate a flow direction. The cell outlets 2882a may include multiple outlet ports that may be used to sort cells.

[0200] FIG. 28B illustrates a schematic of an example microfluidic device 2850b with a hypoxia gradient similar to the microfluidic device 2850a of FIG. 28 A. The microfluidic 2850b includes a cell channel with oxygen gradients 2874b positioned adjacently to an oxygen scavenging channel 2866b. The cell channel with oxygen gradients 2874b includes a cell inlet 2878b that may be used to seed cells, e.g., the HEK293FT-HRE cells, and cell outlets 2882b, wherein the cell outlets 2882b may include multiple outlet ports. An inset of the cell channel with oxygen gradients 2874b shows hypoxia conditions 2886-1, 2886-2, 2886-3 with respect to the oxygen scavenging channel 2866b. FIG. 28B further illustrates the oxygen gradient distribution 2888 as a function of distance from the oxygen scavenging channel 2866b.

[0201] FIGS. 29A and 29B illustrate microscopy images of cells in the fluidic device 2850a of FIG. 28 A under normoxia conditions, according to example embodiments. HEK298FT-HRE cells are seeded into the cell channel with normoxia 2876 and imaged. FIG. 29A is a brightfield microscopy image of the cells in cell channel with normoxia 2876 and FIG. 29B is an epifluorescence image of the cells in the cell channel with normoxia conditions 2876.

[0202] FIGS. 30A and 30B illustrate microscopy images of cells in the fluidic device 2850a of FIG. 28 A under hypoxia conditions, according to example embodiments. HEK298FT-HRE cells are seeded into the cell channel with oxygen gradients 2874a and imaged. The position of an oxygen scavenger channel, i.e., the oxygen scavenger channel 2866a, is labeled on each image. FIG. 29A is a brightfield microscopy image of the cells in cell channel with oxygen gradients 2874a and FIG. 29B is an epifluorescence image of the cells in the cell channel with oxygen gradients 2874a.

[0203] Brightfi eld microscopy imaging reveals a darker region of cells close to the oxygen scavenging channel for cells in the cell channel with oxygen gradients 2874a (FIG. 30A) when compared to cells in the cell channel with normoxia 2876 (FIG. 29A). Epifluorescence imaging shows greater expression of a fluorescent hypoxia reporter in cells in the cell channel with oxygen gradients 2874a (FIG. 30B) when compared to cells in the cell channel with normoxia 2876 (FIG. 29B).

[0204] FIG. 31 illustrates a graph of intensity of a hypoxia marker versus oxygen saturation, according to an example embodiment. The graph of FIG. 31 may be determined using the microfluidic channel 2850a described herein with respect to FIG. 28A and using methods from previous studies [4,5], FIG. 31 shows that expression of fluorescent makers associated with hypoxia increases as dissolved oxygen within a medium decreases.

[0205] With respect to FIGS. 25A-27, variance in levels of hypoxia in samples prepared under identical conditions may be observed. Densities of fibers, and therefore perfusion, within a given region may vary locally and may contribute towards the variance in levels of hypoxia observed.

[0206] FIG. 32A illustrates an extended cross-section image of an example gel showing a region with a lack of voids. The gel is created using alginate fibers measuring 120 pm in diameter and 1 mm in length and may be added at a 50% filler volume, similar to the gel with the perfusable network illustrated in FIGS. 25B and 25D.

[0207] FIG. 32B illustrates a maximum projection of microscopy images of cell nuclei and a hypoxia reporter in the region of FIG. 32A, according to an example embodiment.

[0208] FIG. 33A illustrates an extended cross-section images of an example gel showing a region with voids. The gel of FIG. 33 A is created using alginate fibers measuring 120 pm in diameter and 1 mm in length and may be added at a 50% filler volume, the same parameters as those used for the gel in FIG. 32A. Regions in black indicate a presence of the voids left after degradation of the alginate fibers.

[0209] FIG. 33B illustrates a maximum projection of microscopy images of cell nuclei and a hypoxia reporter in the region of FIG. 33A, according to an example embodiment. The region of FIG. 33 A, which includes the voids, shows lower expression of the hypoxia reporter when compared to the region of FIG. 32A, which does not include voids.

[0210] FIG. 34 illustrates a violin plot of expression of a hypoxia reporter in the regions of FIG. 32A and 32B. Higher maximum intensities of expression of the hypoxia are observable for Region 1 (the region of FIG. 32A) than for Region 2 (the region of FIG. 33A). As describedherein, the gels used for imaging of Region 1 and Region 2 are created using the same bulk material and conditions.

[0211] FIG. 35 illustrates a graph depicting a relationship between volume of functional parenchyma versus fiber volume fraction according to an example embodiment. High fiber volume fraction may decrease a volume of functional parenchyma, which may thereby decrease a volume of functional cells and reduce structural integrity of a gel.

[0212] FIG. 36 illustrates a graph depicting a relationship permeability of a material versus fiber volume fraction according to an example embodiment. The percolation threshold, as shown on the graph of FIG. 36, may indicate a fiber volume fraction needed to successfully create a network of perfusable channels after degradation of fiber segments within a bulk material. Increasing fiber volume fraction beyond the percolation threshold may increase permeability, but excessive fiber volume fraction may affect structural integrity of the bulk material.SPAN for Generating Endothelial Cell-Lined Vessel Networks

[0213] Engineered tissues may require not only a fluidic network to sustain them but also a functional vasculature lined with an endothelial cell monolayer to provide active regulation of transport between vessel lumens and the tissue. Traditional tissue engineering approaches may rely on introducing endothelial cells into a construct via an inlet channel to coat fabricated channels. Such an approach may prove effective in printed luminal structures (approximately 200 pm - 1 mm) when compared to the diameter of endothelial cells in suspension (approximately 20 - 50 pm). However, as conduits decrease in diameter closer to diameters of individual cells, complete lumen occlusion may become more frequent as multiple cells may obstruct a narrowing passage. Moreover, a highly distributed network may be difficult to uniformly coat, especially when fluidic junctions between voids is smaller than a diameter of individual voids, as is the case with fluidic networks described herein.

[0214] Rather than introducing endothelial cells through an inlet channel to populate percolated networks, endothelial cells possess a natural propensity to line and populate lumens that may be leveraged to create such endothelial cell-lined constructs. For example, distributing endothelial cells in a bulk gel, for example, the solidified hydrogel 116 described herein with respect to FIG. 1, may allow the endothelial cells to find and populate an existing perfused network without interfering with an ability of the perfused networks to continue to perfuse a construct.

[0215] FIG. 37 schematically illustrates of a process of formation of a hydrogel 3716-1, 3716-2, 3716-3 including embedded cells, e.g., 3790, perfusion, and endothelialization,according to an example embodiment. The hydrogel 3716-1 includes the embedded cells, e.g., 3790, and fiber, e.g., 3718. The hydrogel 3716-2 may be created after degradation of the fiber, e.g., 3718, of the hydrogel 3716-1 and may include perfusable channels, e.g., 3720. The hydrogel 3716-3 may be created from the hydrogel 3716-2 after allowing the embedded cells, e.g., 3790, to migrate. In some embodiments, wherein the embedded cells, e.g., 3790, are endothelial cells, the embedded cells, e.g., 3790, may migrate to existing voids, e.g., the perfusable channels 3720, within the hydrogel 3716-3.

[0216] In some embodiments, the example schematic illustrated in FIG. 37 may be implemented in the microfluidic device 2150 described herein with reference to FIG. 21. In some embodiments of the example schematic of FIG. 37, human dermal microvascular endothelial cells (HMVECs) may be cultured in an endothelial growth media and used at passages 4-7. In further embodiments, fibrin hydrogels containing HMVECs are formed with or without alginate fibers. HMVECs are encapsulated with or without alginate fibers of 60 pm or 120 pm added at a 25% filler volume in a 5 mg / mL fibrin gel as described herein. Alginate lyase (250 pg / mL) is added 30 mins after tissue assembly and left for 24 hours. Devices are continually rocked on a cell culture rocker with tilt angles of 30° from level with daily media changes. After 5 days, devices are fixed, stained, and then imaged.

[0217] To stain the image, in some embodiments, fluorophore conjugated anti-ulex europaeus I (UEA-I) lectin is used at 1 :500 dilution, a fluorophore conjugated phalloidin and 4',6-diamidino-2-phenylindole (DAPI) are used. Anti-arginase 1 antibodies are used followed with anti-rabbit secondary antibodies with a fluorophore. Prior to cell staining, fixed tissues are blocked and permeabilized with 3% BSA. False color is applied to both the UEA lectin, Phalloidin, and 2 pm fluorescent polystyrene beads as magenta, magenta, and cyan, respectively.

[0218] In further embodiments, after acquiring confocal imaging of constructs including HMVECs, confocal stack images of each tissue construct from the HMVEC encapsulation experiment are analyzed using ImageJ2 [6] to determine a percent area coverage of the endothelial cells on voids. Images are cropped to exclude portions of the images that included the PDMS device and media wells. A sub-stack is created to exclude image slices within a reflectance channel that show reflection off a glass coverslip during confocal image acquisition and slices where endothelial signals are not robustly defined. Endothelial and reflectance channels are split into individual images and processed separately. A five-pixel median filter is applied to the reflectance channel slices, then a minimum projection is applied, followed by Huang auto-thresholding [7], A three-pixel median filter is applied to the endothelial channel,followed by sum projection and Huang auto-thresholding. Respective endothelial and reflectance channels are then multiplied together, and the resulting image is compared to the reflectance minimum projection to ascertain a perfect area coverage of the endothelial cells within fiber created voids.

[0219] FIGS. 38A-38C illustrate fluorescence images of perfusion in hydrogels, similar to the hydrogel of FIG. 37, with or without perfusable channels, according to example embodiments. The perfusable channels are indicated by perfused 2 pm fluorescent polystyrene beads as described herein and a cyan color is applied to the image. Extended cross-sections for Y-Z and X-Z planes are also presented. FIG. 38A illustrates a gel formed with only cells embedded within fibrin. FIG. 38B illustrates a gel formed with cells and alginate fibers of 60 pm diameter embedded within fibrin. FIG. 38C illustrates a gel formed with cells and alginate fibers of 120 pm diameter embedded within fibrin. FIGS. 38B and 38C indicate a presence of the perfusable channels within the respective fibrin gels. The gel seeded with only cells (FIG. 38 A) is unable to establish a perfused network.

[0220] FIGS. 39A-39C illustrate fluorescence images of endothelial cells in hydrogels, similar to the hydrogel of FIG. 37, with or without perfusable channels, according to example embodiments. An inset of a magnified region of each respective gel is also included. As described herein, the endothelial cells, specifically HMVECs (or labeled in the image as hMVECs) are colored in magenta. Extended cross-sections for Y-Z and X-Z planes are also presented. FIG. 39A, corresponding to FIG. 38A, illustrates a gel with only cells embedded within fibrin. The endothelial cells are dispersed within the gel and some multicellular structures may be present. FIG. 39B, corresponding to FIG. 38B, illustrates a gel formed with cells and alginate fibers of 60 pm diameter embedded within fibrin. FIG. 39C, corresponding to FIG. 39C, illustrates a gel formed with cells and alginate fibers of 120 pm diameter embedded within fibrin. hMVEC staining in FIGS. 39B and 39C indicate preferential lining of voids by endothelial cells. Continued perfusion is supported in the gels of FIGS. 39B and 39C over several days. High-magnification images, as shown in the insets of each respective image of FIGS. 39A-39C, are also acquired for the fibrin gel using reflectance microscopy along with F- actin of the endothelial cells in the center of each device. While the endothelial cells of FIGS. 39B and 39C did not fully cover exposed fibrin within voids, analysis of entire constructs for three repeats of each fiber condition, similar to the gels of FIGS. 39B and 39C, reveals significant coverage and are further described herein with respect to FIGS. 41A-43.

[0221] FIGS. 40A-40C illustrate depth-coded max projection images of the zoomed-in inset of FIGS. 39A-39C, respectively. Inter-vessel junctions between 60 pm and 120 pm voids remain intact and fully endothelialized in FIGS. 40B and 40C, respectively. These findings may suggest that surfaces created after degradation of fibers, for example, cylindrical alginate fibers, may be a preferential substrate for HMVECs to cover without a need for specialized growth factors or support cells.

[0222] In FIGS. 38A-39C, the scalebar represents a length of 1 mm. In the insets of FIGS. 39A-C and in FIGS. 40A-C, the scalebar represents a length of 50 pm.

[0223] FIGS. 41A and 41B illustrate minimum projection images of voids in example gels thresholded to create a binary image of voids formed using fibers of different parameters. FIG. 41 A illustrates the voids in a gel formed with fibers of 60 pm diameter added at 25% filler volume, similar to the gels of FIGS. 38B, 39B, and 40B. FIG. 41B illustrates the voids in a gel formed with fibers of 120 pm diameter added at 25% filler volume, similar to the gels of FIGS. 38C, 39C, and 40C.

[0224] FIGS. 42A and 42B illustrate maximum projection images of epithelial cells in example gels with voids thresholded to create a binary image of endothelial cells, wherein the gel with voids is formed using fibers of different parameters. FIG. 41 A illustrates the epithelial cells in a gel with voids formed with fibers of 60 pm diameter added at 25% filler volume, similar to the gels of FIGS. 38B, 39B, and 40B. FIG. 41B illustrates the epithelial cell in a gel with voids formed with fibers of 120 pm diameter added at 25% filler volume, similar to the gels of FIGS. 38C, 39C, and 40C.

[0225] FIG. 43 illustrates a graph of coverage percentage of endothelial cells within projected voids as determined by the minimum and maximum projection images of FIGS. 41 A- 42B. In some embodiments, the endothelial coverage may be computed by dividing the area of the endothelial cells by the area of the projected voids as determined by the maximum and minimum thresholded projection images, respectively. Based on three repeats of gels using each type of fiber, 73% of the 60 pm voids are covered in an endothelialized monolayer and 51% of the 120 pm voids are covered in an endothelialized monolayer.SPAN supports perfusion and organization in engineered tissues

[0226] The assembly of an endothelialized fluidic network may be useful for certain applications. In further embodiments of a structure having a percolated network, cell cultures may include multiple cell types. For example, in some embodiments, the multiple cell types may be a model liver tissue and may include HepG2 cells (an immortalized hepatocyte cell line),primary human dermal fibroblasts as stromal cells (HDFs), or primary human umbilical vein endothelial cells (HUVECs) and the multiple cell types may be embedded within a construct configured to have or to form a perfusable network. In other embodiments, the multiple cell types may be a model cardiac tissue and may include induced pluripotent stem cell (iPSC) cardiomyocytes, HUVECs, or HDFs. Similar to previously described embodiments, the cells may be uniformly distributed within the construct and cultured for several days. Control constructs may lack alginate fibers or lack endothelial cells.

[0227] FIG. 50 illustrates a schematic of a general approach for culturing cells in a material having selectively degradable fibers, e.g. 5318, according to an example embodiment. The material may be a gel 5316a, 5316b, 5316c, for example, a hydrogel. The gel 5316a includes cells, e.g., 5392a, and degradable fibers, e.g., 5318, which may be alginate fibers. The degradable fibers, e.g., 5318, may be configured to degrade over a first period of time 5396. The first period of time 5396 may be short. For example, alginate fibers may be degraded by alginate lyase over a period of minutes. The gel 5316b following degrading of the degradable fibers includes perfusable voids, e.g., 5320, and the cells, e.g., 5392a. The cells, e.g., 5392a, may be allowed to culture in the gel 5316b over a second period of time 5398. The second period of time may be longer, for example, hours or days. The gel 5316c, after allowing culturing, may include the cells, e.g., 5392b, that may have proliferated and may have preferentially migrated due to a presence of the perfusable voids, e.g., 5320.

[0228] In embodiments of a construct of liver tri cultures, HFDs are growth arrested with 10 pg / mL mitomycin C in a fibroblast growth medium on the day of seeding and washed five times in a fibroblast growth medium basal media. HDFs, HepG2s, and HUVECs, the HUVECs transfected with a fluorescent reporter gene, are lifted from tissue culture plates using recombinant enzyme replacing animal trypsin, centrifuged, and resuspended at a concentration of 20 M / mL in fibroblast growth medium for HDFs, 50 M / mL in Eagle’s minimum essential medium (EMEM+) (HepG2s), or 100 M / mL in an endothelial growth medium (HUVECs). Cells are then encapsulated with or without alginate fibers (50% filler volume) in a 5 mg / mL fibrin gel as described herein at final concentrations of 1 M / mL (HDFs), 5 M / mL (HepG2s), and for the tri culture condition - lOM / mL (HUVECs). Biculture tissues are cultured in EMEM+. Tri culture tissues are cultured in a composite media consisting of half EMEM+ and half endothelial growth medium with 2x growth factors and fetal bovine serum so that appropriate levels growth factors are available when the two medias are mixed. Alginate lyase (250 pg / mL) is added 30 mins after tissue assembly and the construction is allowed to rest for 24 hours. Tissues are cultured for 5days on a programmable rocker that paused for 3 minutes per side at angles of +- 40° from level to drive flow through the gel between media wells. Media is changed daily. After 5 days devices are fixed, stained, and then imaged by a confocal microscope. False color is applied to the fluorophores of the HUVECs and stained hepatocytes as magenta, and green, respectively.

[0229] FIG. 44 A illustrates a schematic of a liver tri culture a gel 4416a, 4416b without perfusable channels according to an example embodiment illustrates an example schematic of a liver tri culture in gels 4416a, 4416b without perfusable channels. The gel 4416a includes cells immediately after gel solidification, the cells including hepatocytes, e.g., 4492a, which may be HepG2 cells, endothelial cells, e.g., 4493a, which may be HUVECs, and fibroblasts 4494a, which may be HDFs. After five days, the gel 4416b may include cultures and multicellular constructs of the hepatocytes (e.g., 4492b), the endothelial cells (e.g., 4493b), and the fibroblasts (e.g., 4494b).

[0230] FIG. 44B illustrates an extended fluorescence image of an example liver triculture and gel similar to the liver tri culture and the gel of FIG. 44A. The gel of FIG. 44B is a control construct lacking alginate fibers and the fluorescence image and extended cross-sections of FIG. 44B indicate HUVECs and HepG2 cells are dispersed within the gel as magenta and green clusters, respectively.

[0231] FIG. 44C illustrates a fluorescence image of an example liver triculture and gel similar to the liver tri culture and the gel of FIG. 44 A with arrows indicating fibroblasts. In FIG. 44C, fibroblasts, e.g., HDFs, are a cell-type without specific cell makers and are shown by exclusion of other cell stains.

[0232] FIG. 45 A illustrates a schematic of a liver tri culture in a gel 4516a, 4516b with perfusable channels, e.g., 4520a, according to an example embodiment. The gel 4516a includes cells immediately after gel solidification and alginate degradation, the cells including hepatocytes, e.g., 4592a, endothelial cells, e.g., 4593a, and fibroblasts 4594a. After five days, the gel 4516b may include cultures and multicellular constructs of the hepatocytes (e.g., 4592b), the endothelial cells (e.g., 4593b), and the fibroblasts (e.g., 4594b). Additionally, the endothelial cells, e.g., 4593b, may migrate to percolated networks (the perfusable channels, e.g., 4520a) while the hepatocytes, e.g., 4592b, and the fibroblasts, e.g., 4594b, may remain in the gel.

[0233] FIG. 45B illustrates an extended fluorescence image of an example liver triculture and gel similar to the liver tri culture and the gel of FIG. 45 A. The gel of FIG. 45B is a construct with a percolated network (a perfusable channel, e.g., 4520b) created using sacrificial alginatefibers. Epithelial cells (magenta) have migrated to and lined voids of the percolated network. Hepatocytes (green) remain in the gel.

[0234] FIG. 45C illustrates a fluorescence image of an example liver triculture and gel similar to the liver tri culture and the gel of FIG. 45 A with arrows indicating fibroblasts. In FIG. 45C, fibroblasts, e.g., HDFs, are a cell-type without specific cell makers and are shown by exclusion of other cell stains. Despite the presence of a percolated network (a perfusable channel, e.g., 4520c), as indicated by the black voids, the fibroblasts remain in the gel.

[0235] In embodiments of a construct of cardiac tri cultures, HDFs are growth arrested as described hereinabove. HDFs, iPSC cardiomyocytes (iPSC-CMs), and HUVECs, the HUVECs transfected with a fluorescent reporter gene, are lifted from tissue culture plates using recombinant enzyme replacing animal trypsin, centrifuged, and resuspended at a concentration of 20 M / mL in a fibroblast growth medium (HDFs), 100 M / mL in RPMI+ (iPSC-CMs), or 100 M / mL in an endothelial growth medium (HUVECs). Cells are encapsulated in a composite gel consisting of 5 mg / mL fibrin gel as described herein and 10% Matrigel® (Coming ®) with final concentrations of 1 M / mL (HDFs), 5 M / mL (CMs), and for the tri culture condition - lOM / mL (HUVECs). Biculture tissues are cultured in a maintenance growth medium containing high- glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, 1% nonessential amino acids, 1% GlutaMAX™ (stable alternative for L-glutamine; Thermo Fisher Scientific). Triculture tissues are cultured in a composite media consisting of half cardiomyocyte maintenance growth media and half endothelial growth medium with 2x growth factors and fetal bovine serum from both. 5 pM Rho kinase inhibitor Y- 27632 and aprotinin (0.016 mg / mL) are added to the media. The growth medium is replaced every day. Aprotinin is increased to 0.033 mg / mL after the first day. Y-27632 is removed 2 days following tissue seeding. Alginate lyase (250 pg / mL) is added 30 mins after tissue assembly and left for 24 hours. Tissues are cultured for 5 days on a programmable rocker that paused for 3 minutes per side at angles of +- 40° from level. Media is changed daily. After 3 days (triculture) or 5 days (biculture) devices are fixed, stained, and then imaged by a confocal microscope. False color was applied to the fluorophores of the HUVECs and a green fluorescent protein (GFP-titin) expressed by the cardiomyocytes as magenta and green, respectively.

[0236] FIG. 46 A illustrates a schematic of an example cardiac tri culture in a gel 4616a,4616b without perfusable channels. The gel 4616a includes cells immediately after gel solidification, the cells including cardiomyocytes, e.g., 4692a, which may be iPSC-CM cells, endothelial cells, e.g., 4693a, which may be HUVECs, and fibroblasts 4694a, which may beHDFs. After three days, the gel 4616b may include cultures and multicellular constructs of the cardiomyocytes (e.g., 4692b), the endothelial cells (e.g., 4693b), and the fibroblasts (e.g., 4694b).

[0237] FIG. 46B illustrates an extended fluorescence image of an example cardiac triculture and gel similar to the cardiac tri culture and the gel of FIG. 46A. The gel of FIG. 46B is a control construct lacking alginate fibers and the fluorescence image and extended cross-sections of FIG. 44B indicate HUVECs and cardiomyocyte (CM) cells are dispersed within the gel as magenta and green clusters, respectively.

[0238] FIG. 46C FIG. 46C illustrates a fluorescence image of an example cardiac triculture and gel similar to the cardiac tri culture and the gel of FIG. 46 A with arrows indicating fibroblasts. In FIG. 44C, fibroblasts, e.g., HDFs, are a cell-type without specific cell makers and are shown by exclusion of other cell stains.

[0239] FIG. 47 A illustrates a schematic of an example cardiac tri culture in a gel 4716a,4716b with perfusable channels, e.g., 4720a. The gel 4716a includes cells immediately after gel solidification and fiber degradation, the cells including cardiomyocytes, e.g., 4792a, endothelial cells, e.g., 4793a, and fibroblasts 4794a. After three days, the gel 4716b may include cultures and multicellular constructs of the cardiomyocytes (e.g., 4792b), the endothelial cells (e.g., 4793b), and the fibroblasts (e.g., 4794b). Additionally, the endothelial cells, e.g., 4793b, may migrate to percolated networks (the perfusable channels, e.g., 4720a) while the cardiomyocytes, e.g., 4792b, and the fibroblasts, e.g., 4794b, may remain in the gel.

[0240] FIG. 47B illustrates an extended fluorescence images of an example cardiac triculture and gel similar to the cardiac tri culture and the gel of FIG. 47A. The gel of FIG. 47B is a construct with a percolated network (a perfusable channel, e.g., 4720b) created using sacrificial alginate fibers. Epithelial cells (magenta) have migrated to and lined voids of the percolated network. Cardiomyocytes (green) remain in the gel.

[0241] FIG. 47C illustrates a fluorescence image of an example cardiac triculture and gel similar to the cardiac tri culture and the gel of FIG. 47 A with arrows indicating fibroblasts. In FIG. 47C, fibroblasts, e.g., HDFs, are a cell-type without specific cell makers and are shown by exclusion of other cell stains. Despite the presence of a percolated network (a perfusable channel, e.g., 4720c), as indicated by the black voids, the fibroblasts remain in the gel.

[0242] In some embodiments, cell bicultures may demonstrate that fibroblasts with hepatocytes (liver tissue construct) or cardiomyocytes (cardiac tissue construct) remain in a gel even in the absence of endothelial cells.

[0243] FIG. 48A illustrates a schematic of an example culture of parenchymal cells (hepatocytes), e.g., 4892a, 4892b, and fibroblasts, e.g., 4894a, 4894b, in a gel 4816a, 4816b with perfusable channels, e.g., 4820a. The gel 4416a includes cells immediately after gel solidification and fiber degradation, the cells including the hepatocytes, e.g., 4892a, which may be HepG2 cells, and the fibroblasts, e.g., 4894a, which may be HDFs. After five days, the gel 4816b may include cultures and multicellular constructs of the hepatocytes (e.g., 4892b) and the fibroblasts (e.g., 4894b).

[0244] FIG. 48B illustrates an extended fluorescence image of an example culture of parenchymal cells and fibroblasts similar to the culture and the gel of FIG. 48 A. The gel of FIG. 48B is a construct with a percolated network (a perfusable channel, e.g., 4820b) created sacrificial using alginate fibers. Even in the absence of epithelial cells, parenchymal cells, e.g., hepatocytes (green), may not migrate to voids of the percolated network.

[0245] FIG. 49A illustrates a schematic of an example cardiac biculture of stromal cells (cardiomyocytes), e.g., 4992a, 4992b, and fibroblasts, e.g., 4994a, 4994b, in a gel 4916a, 4916b with perfusable channels, e.g., 4920a. The gel 4916a includes cells immediately after gel solidification and fiber degradation, the cells including the cardiomyocytes, e.g., 4992a, which may be iPSC-CM cells, and the fibroblasts, e.g., 4994a, which may be HDFs. After five days, the gel 4916b may include cultures and multicellular constructs of the cardiomyocytes (e.g., 4992b) and the fibroblasts (e.g., 4994b).

[0246] FIG. 49B illustrates an extended fluorescence image of an example culture of stromal cells and fibroblasts similar to the culture and the gel of FIG. 49A. The gel of FIG. 49B is a construct with a percolated network (a perfusable channel, e.g., 4920b) created using sacrificial alginate fibers. Even in the absence of epithelial cells, stromal cells, e.g., cardiomyocytes (green), may not migrate to voids of the percolated network.

[0247] Collectively, FIGS. 44A-49B and the materials illustrated therein suggest that SPAN may facilitate rapid assembly of constructs that may not only support perfusion requirements for engineered tissues, but also provide architectural cues enabling endothelial cells to form a vasculature in the presence of stromal and parenchymal cell populations.

[0248] Generation of large volume tissue constructs may have been limited by a lack of approaches to generate perfusable vasculature required to provide adequate transport of nutrients and oxygen to cells throughout the construct. Recent advances in 3D printing may demonstrate a possibility of generating perfusable networks with arbitrary architectures but, thus far, may be limited to larger (>200 pm diameter) lumens, which may still be an order of magnitude largerthan those found in natural tissues (<10 pm). Because transport efficiency may be limited by surface area for diffusion between vascular lumens and bulk tissue, smaller and more dense vessel networks may be needed. While laser-induced degradation of materials may generate smaller lumens and further improvements in 3D printing may eventually achieve the desired resolutions, such serial fabrication strategies in general may not escape the tradeoff between resolution and build-time. For assembly of cellularly dense living tissues, rapid perfusion may be required to avoid tissue necrosis and failure. A pipettable one-pot, two-step approach - percolated assembly and dissolution - has been demonstrated herein that requires a fixed time (minutes) to achieve construction and perfusion, irrespective of construct size. These distinct and controllable steps may allow for fiber degradation timing to be tuned to a specific bulk material used. The pipettability of SPAN systems may also allow access to many enclosed systems used in tissue engineering such as microfluidic devices and bioreactors that are otherwise inaccessible to 3D printers, along with standard tissue culture formats such as 96-well plates. The alginate fibers used and described herein have been focused on lumen diameters spanning a range between those available through either vasculogenic self-assembly or 3D printing. However, other embodiments of fabricating alginate fibers using microfluidic device may create pure alginate fibers with diameters down to approximately 2 pm and electrospun down to 90 nm. Embodiments of the present invention may address a long-recognized need for generating perfusable constructs of arbitrary size.

[0249] The embodiments described herein demonstrate an ability to generate and adequately perfuse constructs densely packed with cells. It has previously been demonstrated that the diffusion distance of oxygen in native tissues can range from 200-500 pm. Similar diffusion distances may be observed when cells incorporating a hypoxia-sensitive reporter a densely packed (~25 million cells / mL) in a solid gel, for example, as described herein with respect to FIGS. 25A-25D and FIGS. 28A-31. In contrast, hypoxia may be relieved in SPAN gels, where densities of perfused networks may be sufficient to deliver oxygen throughout a percolated network. Given a general compatibility of this approach to develop percolated networks in a variety of forms, biomaterials, and fabrication processes, SPAN may be applied to a wide range of tissue engineering applications.

[0250] The embodiments described herein, in addition to rapidly constructing paths for perfusion within a construct, may be used to generate engineered vascular networks lined with endothelial cells. The primary approach currently to build living vascular networks may be through a process of vasculogenic self-assembly where endothelial cells distributed in a bulk gelare able to spontaneously find each other, form an interconnected network of cells, and then degrade the gel within their vicinity to form a dense perfusable vascular network. Alternatively, in vivo studies have shown that a presence of microvascular scale channels within a gelatin hydrogel is sufficient to rescue ischemic injury through host angiogenesis. However, such processes may take days to weeks and may not be compatible with requirements for perfusing newly assembled constructs quickly. By seeding endothelial cells into bulk gels of the embodiments described herein, the endothelial cells may preferentially exit the bulk gel and may line an inner surface of voids in the bulk gels, rapidly generating a scalably endothelializable network of lumens. Because densities of voids are designed to be high (for perfusing the construct), endothelial cells in the bulk appear to encounter the voids quickly and assemble into vasculature within days. However, unlike self-assembly approaches that may require timeconsuming cell-mediated tissue vascularization, the embodiments described herein are perfused within minutes of assembly such that a tissue is adequately supported while endothelial cells seek and line the voids to form a functional vasculature. Embodiments described herein for generating vascularized lumens that be used to build liver and cardiac tissues in minutes my present further potential utilities of this approach.Additional exemplification

[0251] While the examples described herein have pertained to a particular application in tissue engineering, it should be understood by one skilled in the art that the selectively degradable composition with anisotropic elements and method of forming a structure having a network of perfusable channels are applicable to a wide range of applications.

[0252] General variations of the embodiments described herein may include different methods of fiber production. For example, fibers may be produced by electro spinning, although the concentration of the alginate gel may need to be increased. Fibers may also be made using a droplet generator by making individual plugs of varying lengths at a T-junction. Fibers may be made from a thermos-responsive gel instead such as gelatin, Pluronic 127, or others, which may lead to a decreased ability to trigger fiber degradation. Degradation may occur through photodegradation using photodegradable fiber cylinders.

[0253] Additionally, fiber networks may be used as a hand-off structure. The fibers may be distributed in a gel or other bulk material that is itself degradable. The bulk material may have a chemical, metal particles, or biological material distributed throughout the gel. That material may then be attracted or preferentially deposited on the fibers, therefore creating a shell structure. The bulk material may then be degraded to leave the network structure of the coatedfiber network, where the fibers act as a reinforcement to the shell that might otherwise collapse. This network may then be transferred into another substrate, where the fibers could then be triggered to degrade, leaving a hollow network structure, which could then be refilled with some other material if desired. This multistep approach and triggered degradation may be used in electronics or flow cell batteries, or for vascular implants.

[0254] In some embodiments for biomedical engineering, another material, for example, arginine-glycine-aspartate (RGD) alginate may be used to either fully produce the fibers or coat the outside of the fibers. This could allow for attachment of adherent cells, such as endothelial cells. The cells may create a monolayer of cells in normal tissue culture conditions, then the cell coated fibers could be added to a tissue engineering construct or injected with another hydrogel in vivo to rapidly create a perfusable endothelialized vascular network. Such embodiments may enable implantation of tissue constructs or create a bridging vascular network to reconnect native vasculature caused by injury, ischemia, or amputation. Fibers may also be coated by an individual cell type (i.e., blood endothelial cells) and then combined with fibers coated with another cell type (i.e., lymphatic endothelial cells) to promote network separation.

[0255] In some embodiments for wound healing applications, fibers may be incorporated into a pre-gel solution with fibrinogen or other hydrogels used in wound healing, where the solution could be injected into a deep wound bed where blood vessels have been disrupted. The psuedo-2D surface of the fibrin surrounding the alginate fibers may be preferred by cells with an apical-basal polarity, and thus the voids created by the fibers may be used for more rapid revascularization in the wound, and possibly prevent ischemia.

[0256] The triggerable nature of the fibers may allow the fibers to be degraded either immediately following gelation of the bulk hydrogel, or degradation could be delayed by hours or days to allow the vasculature to possibly connect to the native vasculature before blood would be able to flow, thus possibly preventing clotting. Delaying degradation of the fibers may also allow the tissue construct or injectable solution to compact in vivo (since both are usually low in cell density), keeping space take up by the alginate fiber open to eventually become a perfusable void. The delayed degradation may also create a semi-static environment for endothelial cells to coat the pseudo-2D surface of the voids and establish apical-basal polarity.

[0257] Fibers may resist compaction within tissues and spheres, allowing the voids to be kept open. Compaction may lead to a higher probability that the fiber cylinders within a gel come into contact, increasing the likelihood of percolation. Creation of percolated networks may also enable the creation of in vitro tissue constructs involving difficult-to-combine cell types,such as endothelial cells and cardiac cells. Due to the constant contraction of cardiomyocytes, culturing cardiomyocytes in a traditional microfluidic device may be difficult without the cells ripping the gel off of walls of the microfluidic device eventually. The mechanical perturbations have also been shown complicate vasculogenesis for endothelial cells, since the nascent vascular networks are physically weak and the media compositions of the different cell-types are incompatible in some situations. The fibers may be helpful in overcoming these limitations by resisting physical deformation while the endothelial cells create a monolayer on the pseudo-2D surface of the gel-fiber interface until the tissue construct is sufficiently compacted.

[0258] In some embodiments, cells may be encapsulated within a hydrogel sphere that have a fluidic network created by the percolated fibers. Cells encapsulated in hydrogel may be useful for long-term production of secreted proteins, such as human insulin, which may be difficult to produce in bacterial bioreactors due to post translational modifications necessary for human therapeutic use. The fibers may enable culturing of cells for production of compounds with benefits towards a range of applications, for example, human or animal health, without the need for endothelialization, which may allow for greater transport between the cells encapsulated and external fluids.

[0259] In other embodiments, fibers in a bulk hydrogel may be shear aligned through techniques such as 3D printing. Shear alignment of fibers may provide aligned vasculature or alignment cues for cardiomyocytes or other tissues with aligned structure. In some embodiments, the fibers may be sprayed with another material, e.g., a spray wound healing patch that is degradable. In other embodiments, the surface of the fibers may be altered. For example, longitudinally aligned ridges may be fabricated on the outside of cylinders to act as guidance cues for cells. In additional embodiments, ex vivo tissue may be explanted and placed in a gelfiber mixture to fluidically connect the tissue to the gel when the alginate fibers are degraded.

[0260] Example embodiments of variations of the present invention for battery or supercapacitor applications may include fabrication of anisotropic pores. Anisotropic pores may be created in graphite or other anode or cathode materials along with a binder for batteries or a highly porous electrode for supercapacitors. High flow rate and surface area may allow for a rapid exchange of ions, for example, lithium. The present invention may provide the ability to deposit a slurry of electrode material along with anisotropic fiber segments, which would be degraded after an electrode had been fabricated or when an electrolyte is added to assembled battery or supercapacitor. The fibers are also flexible and may be molded or deposited with abulk electrode material. This would leave behind a porous percolation network where the ions may more easily flow and access surface area within a larger volume electrode.

[0261] Some example embodiments of the present invention may be incorporated into a flow battery. Electrodes including or created using such an embodiment may take up a majority of the volume of the battery or supercapacitor, wherein the electrodes would be separated by a thin filter membrane and the electrolyte is distributed throughout the pores left by degraded fibers. Batteries manufactured using such an approach may provide greater efficiency, higher energy and improved power density.

[0262] In some embodiments, due to the low cost of fabricating alginate fibers, the alginate fibers may be used in gel used to absorb toxic chemicals or heavy metals from wastewater solutions. In other embodiments, the anisotropic nature of fibers may increase a surface area to volume ratio, which may allow the fibers to filter out a solution on their own. Close packing of the fibers may act as a mesh and filter a substrate with higher flow rates than closely packed spheres. Triggerable degradation may be used to flow out residue after the filter is used up.

[0263] Some embodiments of the present invention may include ultra-high flow marker nibs. In such embodiments, fibers are embedded into a nib material, such as polyethylene or polyester fiber. Degrading the fibers would increase capillarity of a fluidic network. Embedding fibers in other materials may also be useful for forming overall nibs with lower porosity.

[0264] In some embodiments, anisotropy and shear alignment of fibers may enable directed porosity of a material, for example, concrete, to increase strength of the material in a desired direction, such as in the making of concrete beams. In other embodiments, degradable anisotropic fiber segments may be added to concrete or to other building materials used in sidewalks or roadway materials to increase porosity while reducing the porosity percentage of the material. These materials may be used in place of nonporous concrete. Such embodiments may allow for rainwater to percolate through the material to recharge ground water in areas with large amounts of man-made surfaces. Similar embodiments may also be used in driveways in non-cold environments, or in the prevention of flooding in areas with high ground coverage.

[0265] In some embodiments, degradable fiber segments may be used in textile yarn, the fiber segments configured to be later be triggered to degraded, to reduce waste. In other embodiments, degradable fibers may be used in high exchange rate water filters, wherein degradable fibers are added along with activated charcoal particles or fibers as they are fabricated. The high surface area of the degraded fiber voids may allow for high exchange and filtering while allowing for lower fluidic resistance and potentially smaller filter sizes.Additional applications for embodiments of the present invention may include water desalination filters, spray on seaweed masks for custom spa treatments, or heat exchangers.

[0266] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[0267] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.References[1] HEPES-buffered saline (2006). Cold Spring Harb Protoc 2006, pdb.rec8786.10.1101 / pdb.rec8786.[2] Doyle, A.D. (2016). Generation of 3D Collagen Gels with Controlled Diverse Architectures. CP Cell Biology 72. 10.1002 / cpcb.9.[3] Erapaneedi R, Belousov VV, Schafers M, Kiefer F. A novel family of fluorescent hypoxia sensors reveal strong heterogeneity in tumor hypoxia at the cellular level. EMBO J. 2016 Jan 4;35(1): 102-13. doi: 10.15252 / embj .201592775[4] Hsu, H.-H., Ko, P.-L., Peng, C.-C., Cheng, Y.-J., Wu, H.-M., and Tung, Y.-C. (2023). Studying sprouting angiogenesis under combination of oxygen gradients and co-culture of fibroblasts using microfluidic cell culture model. Materials Today Bio 21, 100703.10.1016 / j .mtbio.2023.100703.[5] Wu, H.-M., Lee, T.-A., Ko, P.-L., Liao, W.-H., Hsieh, T.-H., and Tung, Y.-C. (2019).Widefield frequency domain fluorescence lifetime imaging microscopy (FD-FLIM) for accurate measurement of oxygen gradients within microfluidic devices. Analyst 144, 3494-3504. 10.1039 / C9AN00143C.[6] Rueden, C.T., Schindelin, J., Hiner, M.C., DeZonia, B.E., Walter, A.E., Arena, E.T., and Eliceiri, K.W. (2017). ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics 18, 529. 10.1186 / sl2859-017-1934-z.[7] Huang, L.-K., and Wang, M.-J.J. (1995). Image thresholding by minimizing the measures of fuzziness. Pattern Recognition 28, 41-51. 10.1016 / 003 l-3203(94)E0043-K.

Claims

CLAIMSWhat is claimed is:

1. A composition for forming a structure having a network of perfusable channels, the composition comprising: sacrificial fibers configured to be degraded by a degrading agent, each sacrificial fiber of the sacrificial fibers having an anisotropic shape; and a structural material substantially stable with respect to the degrading agent, the structural material being configured to inducibly undergo a state change, the state change causing the composition to become more solid; wherein the sacrificial fibers are mixed with the structural material at a concentration of an overall volume of the composition sufficient to allow formation of the structure having the network of perfusable channels upon degrading of at least a portion of the sacrificial fibers by the degrading agent.

2. The composition of claim 1, wherein the sacrificial fibers include a polymer based on alginate, or a precursor or derivative thereof.

3. The composition of claim 1, wherein the sacrificial fibers are composed of crosslinked polymers.

4. The composition of claim 3, wherein the degrading agent is a chelator that disrupts crosslinking of polymers.

5. The composition of claim 1, wherein the sacrificial fibers are cylindrical or spherocylindrical.

6. The composition of claim 1, wherein the sacrificial fibers include a smooth surface or a patterned surface.

7. The composition of claim 1, wherein the sacrificial fibers are of different sizes or aspect ratios.

8. The composition of claim 1, wherein the sacrificial fibers include a cell adherent material or are coated with a cell adherent material.

9. The composition of claim 1, wherein the concentration of the sacrificial fibers in the composition is determined by parameters of the sacrificial fiber including fiber lengths and fiber diameters and by parameters of the structural material including structural integrity.

10. The composition of claim 1, wherein the structural material includes a pre-hydrogel configured to inducibly undergo the state change from the pre-hydrogel to a hydrogel.

11. The composition of claim 10, wherein the structural material is a pre-hydrogel including or derived from collagen, agarose, fibrin, dextran, or polyethylene glycol.

12. The composition of claim 1, wherein the structural material includes graphite or metal and wherein the structural material is configured to conduct electricity.

13. The composition of claim 1, wherein the degrading agent is an enzyme.

14. The composition of claim 13, wherein the enzyme is alginate lyase and wherein the sacrificial fibers are composed of alginate polymers.

15. The composition of claim 1, wherein the structural material is configured to remodel after undergoing the state change, further wherein the structure is configured to retain at least a portion of the network of perfusable channels after remodeling of the structural material.

16. The composition of any one of claims 1-15, wherein the composition includes the degrading agent in a substantially inactive state.

17. The composition of any one of claims 1-15, further comprising cells.

18. The composition of claim 17, wherein the structural material includes the cells.

19. A method of forming a structure having a network of perfusable channels, the method comprising: dispensing a composition in a desired shape, the composition including: (i) sacrificial fibers configured to be degraded by a degrading agent, each sacrificial fiber having an anisotropic shape, and (ii) a structural material substantially stable with respect to the degrading agent, the structural material being configured to inducibly undergo a state change, wherein the sacrificial fibers are mixed with the structural material;inducing the state change of the structural material, the state change causing the composition to solidify in the desired shape; and with the degrading agent, causing selective degrading of at least a portion of the sacrificial fibers to form the structure having the network of perfusable channels.

20. The method of claim 19, further comprising adding the degrading agent to the composition.

21. The method of claim 20, wherein the degrading agent added is in a substantially inactive state prior to dispensing the composition, and wherein causing selective degrading includes activating the degrading agent.

22. The method of claim 21, wherein activating the degrading agent includes changing a temperature or pH of the composition, adding an activating agent, or exposing the degrading agent to light.

23. The method of claim 19, further comprising fabricating the sacrificial fibers prior to preparing the composition.

24. The method of claim 19, wherein dispensing the composition includes pipetting, extruding, aliquoting, pouring, or compacting the composition into the desired shape.

25. The method of claim 19, further comprising aligning the sacrificial fibers in the composition prior to inducing the state change.

26. The method of claim 25, wherein aligning the sacrificial fibers in the composition includes shear aligning the sacrificial fibers during the dispensing of the composition.

27. The method of claim 19, wherein inducing the state change of the composition includes changing a temperature of the composition, changing a chemical environment of the composition, exposing photosensitive molecules to light, or introducing a solidifying agent to the composition.

28. The method of claim 19, further comprising allowing the structural material to remodel after causing selective degrading of at least a portion of the sacrificial fibers.

29. The method of claim 19, further comprising preparing the composition and, optionally, storing the composition prepared prior to dispensing the composition.

30. The method of claim 19, further comprising manipulating the composition prior to causing selective degrading of at least a portion of the sacrificial fibers.

31. The method of claim 19, further comprising adding a secondary structural material to the structure having the network of perfusable channels, the secondary structural material filling at least a portion of the perfusable channels.

32. The method of any one of claims 19-31, further comprising embedding the composition or the structure with cells.

33. The method of claim 32, further comprising allowing the cells to migrate in the composition or the structure based on a preferred 3 -dimensional environment.

34. A kit for forming a structure having a network of perfusable channels, the kit comprising: sacrificial fibers, each sacrificial fiber having an anisotropic shape; and a degrading agent configured to cause the sacrificial fibers to degrade, wherein the degrading agent is substantially inert with respect to a structural material of the structure; wherein, when the sacrificial fibers are dispersed within the structural material, the degrading agent causes at least a portion of the sacrificial fibers to degrade to form the structure having the network of perfusable channels.

35. The kit of claim 34, further comprising the structural material, wherein the structural material is inducibly configured to undergo a state change.

36. The kit of claim 35, wherein the sacrificial fibers and structural material are mixed together.

37. The kit of claim 34, wherein the sacrificial fibers include a cell adherent material or are coated with a cell adherent material.

38. The kit of any one of claims 34-37, wherein the degrading agent is combined with the sacrificial fibers or the structural material.

39. The kit of any one of claims 34-37, further comprising a solidifying agent separated from the sacrificial fibers and the structural material, the solidifying agent configured to inducethe structural material to undergo the state change, the state change causing the composition to become more solid.

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