WO2023239640A1 - Biopolymer-based tissue scaffolds and apparatus and method for producing the same - Google Patents

Abstract

A biopolymer-based tissue scaffold on which stem cells, muscle cells, or cultivated meat are growable can include at least one protein, at least one starch, at least one polysaccharide, and at least one crosslinking agent. The protein, the starch, the polysaccharide, and the crosslinking agent meet generally recognized as safe (GRAS) standards. Processes for making the scaffolds and apparatuses for making and/or using the scaffolds can facilitate stem cell growth, muscle cell growth and/or cultivated meat growth in a way that results in the scaffold decomposing or being consumed during the cultivated meat or muscle cell growth process.

Description

BIOPOLYMER-BASED TISSUE SCAFFOLDS AND APPARATUS AND METHOD FOR PRODUCING THE SAME

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63/349,607, which was filed on June 7, 2022. The entirety of this application is incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [0002] This invention was made with government support under Hatch Act Project No. PEN04708 awarded by the United States Department of Agriculture. The Government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] Embodiments relate to biopolymer-based tissue scaffolds for cell cultured meat applications. Embodiments further relate to methods of producing a biopolymer-based tissue scaffold for cell cultured meat applications and apparatuses that can implement such methods. The biopolymer-based tissue scaffolds preferably meet generally recognized as safe (GRAS) requirements.

BACKGROUND OF THE INVENTION

[0004] Meat and livestock play a role so fundamental in human civilization that meat production and consumption are seen as the natural way of things. Meat is a nutrient dense food and is a significant source of proteins, essential amino acids, vitamins (specifically vitamin Bl 2), minerals, and fats. Additionally, meat consumption has strong connections to religious, cultural, and traditional practices that hold great importance in human society. The global demand for

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SUBSTITUTE SHEET ( RULE 26) meat is growing; over the past 50 years meat production has more than tripled. This demand is currently being met by conventional animal agriculture, which has large environmental impacts such as increased greenhouse gas emissions, deforestation, degradation of wildlife habitats, and eutrophication of water ways. It also raises concerns about food safety and security, public health, and the ethical treatment of animals. Cell cultivated meat (CM) has the potential to provide a significant supply of animal protein and can help enhance global food security while offering human health, environmental, and animal welfare benefits.

[0005] CM involves proliferation of stem cells acquired from an animal in bioreactors (cultivators) in the presence of oxygen-rich cell culture medium that contains basic nutrients such as amino acids, glucose, vitamins, inorganic salts, protein supplements, and growth factors. This proliferation and growth of cells takes place on structural supports called scaffolds, whose role is to mimic the natural extra cellular matrix present in tissues. A successful scaffold ensures the efficient transport of oxygen, nutrients, and waste products to and from the cells, controlling the growing tissue’s geometry and cell type distribution and contributing structure to the final product. One of the most used techniques for producing scaffolds is the fabrication of non-woven meshes containing fibers ranging from the microscale to the nanoscale via electrospinning. Electrospinning is a spinning process that uses electrostatic forces to produce fibrous scaffolds from biocompatible polymers. Electrospinning is versatile, easy to use, and cost effective, resulting in fibers with high surface area to volume ratios and tunable porosities which are highly beneficial for successful cell proliferation, differentiation, and migration. SUMMARY

[0006] Currently, all successful scaffolds for 3D skeletal muscle formation are animal-derived, and, to our knowledge, no commercially available microcarriers have been specifically designed for the cultivated meat industry. For cultivated meat specifically, scaffolds must be composed of biopolymers that can degrade into safe biomolecules. It would be advantageous if the scaffold was part of the cultivated meat or degraded over time, leaving behind just the meat. To progress towards a greener and more sustainable environment, meat production should involve renewable, cost effective, and abundantly available materials. Starch is the second most abundant polymer on earth and is biodegradable, cost-effective, and nontoxic Scaffolds primarily comprising starch could provide a sustainable and edible substrate for cultivated cell growth.

[0007] Biomaterials such as proteins and peptides are biocompatible and their structural and functional properties in combination with other biopolymers have motivated interest in developing biomaterials containing proteins and peptides. Proteins can also be non-cytotoxic and can increase cell adhesion. Whey protein isolate (WPI) has excellent gas barrier properties at low and intermediate relative humidity and is water soluble, which makes it an attractive candidate for biodegradable nanofiber structures. Glycomacropeptide’s (GMP) unique set of amino acids makes it a sought-after ingredient with nutraceutical properties. It exhibits solubility and emulsifying properties over a wide pH range. The incorporation of bioactive proteins and/or peptides into polymer scaffolds could improve their functionality as extra-cellular matrix (ECM) analogs for enhanced cell proliferation, adhesion, and differentiation.

[0008] Smooth, uniform and bead-free nanofibers are always desired in cell growth applications since beads do not possess the high surface area to volume ratio and porosity that nanofibers have. Relative humidity (RH) plays an important role in fiber morphology, yet the effect of RH on electrospinning, specifically electrospinning of biopolymers has been scarcely studied.

[0009] Ultimately, there is a need for sustainable, environment friendly, organic, and edible scaffolds for cultivated meat applications.

[0010] In an exemplary embodiment, a method of forming a biopolymer-based tissue scaffold on which stem cells, muscle cells or cultivated meat are grown comprises mixing biopolymer components and a solvent to form a polymer mixture, wherein the biopolymer components comprise at least one protein, at least one starch, at least one polysaccharide, and at least one crosslinking agent; spinning the polymer mixture to form nanofibers; and exposing the nanofibers to crosslinking conditions such that the at least one crosslinking agent is activated thereby forming the tissue scaffold, wherein the biopolymer components and solvent meet generally recognized as safe (GRAS) standards.

[0011] In some embodiments, the biopolymer components can be included in a biopolymer component mixture before being included in a solvent to form the polymer mixture. In other embodiments, the biopolymer components can be included in the solvent to form the polymer mixture.

[0012] The biopolymer components can be included to make up pre-selected concentrations within the overall mixture of the biopolymer components included in the polymer mixture. For example, the at least one protein may be present in an amount ranging from 1-50 weight percent on a biopolymer basis (wt.% or wt%), the at least one starch can be present as a major component of the mixture of biopolymer components (e.g. in an amount ranging from 50 wt% to 95 wt% or ranging from 50 wt% to over 95 wt%), the at least one polysaccharide can be present as a minor component in the biopolymer component mixture (e.g. in an amount ranging from over 0 wt% to 20 wt%), and at least one crosslinking agent can be present as another minor component of the biopolymer component mixture (e.g. in an amount ranging from greater than 0 wt% to 10 wt%) in some embodiments. When the polymer mixture is formed, the total of the biopolymer components in the polymer mixture can make up 40 wt% to 20 wt% of the polymer mixture and the solvent can make up 60 wt% to 80 wt% of the polymer mixture in some embodiments.

[0013] In some embodiments, the method further comprises feeding the tissue scaffold to a cell growth device comprising a cell culture medium, wherein the cell growth device is configured to facilitate the growth of stem cells, muscle cells or cultivated meat on the tissue scaffold. In some embodiments, the stem cells that may be used can be either embryonic stem cells, muscle stem cells, or muscle satellite cells.

[0014] In some embodiments, the tissue scaffold is configured to decompose during the growth of stem cells, muscle cells or cultivated meat on the tissue scaffold.

[0015] In some embodiments, spinning the polymer mixture to form nanofibers comprises electrospinning.

[0016] In some embodiments, the nanofibers are aligned.

[0017] In some embodiments, the crosslinking conditions comprise ultraviolet (UV) light exposure. The UV light can be in a UV bandwidth (e.g. 280 nm to 185 nm).

[0018] In some embodiments, the protein is whey protein or glycomacropeptide, the starch is a type of octenyl succinylated starch (OS starch), and the polysaccharide is pullulan.

[0019] In an exemplary embodiment, a biopolymer-based tissue scaffold on which stem cells, muscle cells or cultivated meat are grown comprises at least one protein, at least one starch, at least one polysaccharide, and at least one crosslinking agent, wherein the protein, the starch, the polysaccharide, and the crosslinking agent meet generally recognized as safe (GRAS) standards.

[0020] In some embodiments, the at least one protein is whey protein or glycomacropeptide.

[0021] In some embodiments, the at least one starch is OS starch.

[0022] In some embodiments, the at least one polysaccharide is pullulan.

[0023] In some embodiments, the protein is whey protein or glycomacropeptide, the starch is OS starch, and the polysaccharide is pullulan.

[0024] In some embodiments, the crosslinking agent is selected from the group consisting of phosphate, sodium hydroxide, sodium benzoate, sodium citrate, and citric acid.

[0025] In some embodiments, the tissue scaffold further comprises at least one plasticizer.

[0026] In some embodiments, the at least one plasticizer is sorbitol.

[0027] Embodiments of an apparatus for forming and/or utilizing a biopolymer-based tissue scaffold on which stem cells, muscle cells are growable or cultivated meat is growable is also provided. The apparatus can include a mixing device positioned to mix biopolymer components and a solvent to form a polymer mixture, wherein the biopolymer components comprise at least one protein, at least one starch, at least one polysaccharide, and at least one crosslinking agent. A spinner device can be positioned to spin the polymer mixture formed via the mixing device to form unidirectionally aligned nanofibers.

[0028] Embodiments of the apparatus can be configured so that the spinning device includes at least one crosslinking device configured to expose the nanofibers to one or more crosslinking conditions such that the at least one crosslinking agent is activatable to crosslink the nanofibers and form the tissue scaffold. Alternatively, a crosslinking device can be positioned to receive the nanofibers and expose the nanofibers to one or more crosslinking conditions for a crosslinking time period such that the at least one crosslinking agent is activatable to crosslink the nanofibers and form the tissue scaffold.

[0029] Embodiments of the apparatus can also include a cell growth device positioned to receive stem cells, muscle cells or cultivated meat and the tissue scaffold for growing the stem cells, muscle cells or cultivate meat on the tissue scaffold. The cell growth device can be positioned to receive a cell culture medium to facilitate the growing of the stem cells, muscle cells or cultivate meat on the tissue scaffold. The cell growth device can be configured such that the tissue scaffold is consumed or decomposes as the stem cells, muscle cells or cultivated meat grows.

[0030] Embodiments of the apparatus can be configured to implement an embodiment of our process or method for forming a tissue scaffold and/or using the tissue scaffold for growing meat cells or cultivated meat. Embodiments of the apparatus and process can be configured to form an embodiment of our tissue scaffold as well.

[0031] Other details, objects, and advantages of our process for biopolymer-based tissue scaffold utilization, our apparatus for utilization of biopolymer-based tissue scaffolds, and methods of making and using the same will become apparent as the following description of certain exemplary embodiments thereof proceeds.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The above and other objects, aspects, features, advantages, and possible applications of embodiments of the present innovation will be more apparent from the following more particular description thereof, presented in conjunction with the following drawings. Like reference numbers used in the drawings may identify like components.

[0033] FIG. 1 shows an exemplary method of forming an exemplary biopolymer-based tissue scaffold. [0034] FIG. 2 shows an exemplary rotating drum collector system.

[0035] FIG. 3 shows a contour plot of surface tension (mN/m) for polymer mixtures containing glycomacropeptide.

[0036] FIG. 4 shows a contour plot of surface tension (mN/m) for polymer mixtures containing whey protein isolate.

[0037] FIG. 5 shows a contour plot of apparent viscosity at 100 s'¹ (Pa.s) for polymer mixtures containing glycomacropeptide.

[0038] FIG. 6 shows a contour plot of apparent viscosity at 100 s'¹ (Pa.s) for polymer mixtures containing whey protein isolate.

[0039] FIG. 7 shows a contour plot of tip viscosity (Pa s) for polymer mixtures containing glycomacropeptide.

[0040] FIG. 8 shows a contour plot of tip viscosity (Pa.s) for polymer mixtures containing whey protein isolate.

[0041] FIG. 9 shows a contour plot of elastic modulus (Pa) for polymer mixtures containing glycomacropeptide.

[0042] FIG. 10 shows a contour plot of elastic modulus (Pa) for polymer mixtures containing whey protein isolate.

[0043] FIG. 11 shows a graphical representation of elastic modulus vs. strain percentage.

[0044] FIG. 12 shows a contour plot of phase angle (°) for polymer mixtures containing glycomacropeptide.

[0045] FIG. 13 shows a contour plot of phase angle (°) for polymer mixtures containing whey protein isolate. [0046] FIG. 14 shows a contour plot of cohesive energy (Pa) for polymer mixtures containing glycomacropeptide.

[0047] FIG. 15 shows a contour plot of cohesive energy (Pa) for polymer mixtures containing whey protein isolate.

[0048] FIG. 16 shows a graphical representation of timescale vs % of glycomacropeptide.

[0049] FIG. 17 shows a graphical representation of timescale vs % of whey protein isolate.

[0050] FIG. 18 shows beading in nanofibers electrospun from polymer mixtures.

[0051] FIG. 19 shows a scanning electron microscope (SEM) image of electrospun nanofibers containing whey protein isolate at 2% relative humidity.

[0052] FIG. 20 shows an SEM image of electrospun nanofibers containing whey protein isolate at 13% relative humidity.

[0053] FIG. 21 shows SEM images of nanofibers electrospun from polymer mixtures containing glycomacropeptide on a collector rotating at various speeds.

[0054] FIG. 22 shows SEM images of nanofibers electrospun from polymer mixtures containing whey protein isolate on a collector rotating at various speeds.

[0055] FIG. 23 shows SEM images of BP-SB5 at various stages.

[0056] FIG. 24 shows SEM images of BP-SB1 at various stages.

[0057] FIG. 25 shows an SEM image of BP-SB2.5 after 10 days in DI water.

[0058] FIG. 26 shows an SEM image of nGMP before UV exposure.

[0059] FIG. 27 shows an SEM image of nGMP-30.

[0060] FIG. 28 shows an SEM image of nGMP-30.

[0061] FIG. 29 shows an SEM image of nGMP-60 after 14.5 hours in DI water.

[0062] FIG. 30 shows an SEM image of nGMP-120 after 14.5 hours in DI water. [0063] FIG. 31 is a block diagram of an exemplary apparatus for forming and/or utilizing a biopolymer-based tissue scaffold.

DETAILED DESCRIPTION

[0064] The following description is of exemplary embodiments and methods of use that are presently contemplated for carrying out the present invention. This description is not to be taken in a limiting sense, but is made merely for the purpose of describing the general principles and features of various aspects of the present invention. The scope of the present invention is not limited by this description.

[0065] Referring to FIG. 1, embodiments relate to a method of forming a biopolymer-based tissue scaffold. The method comprises mixing biopolymer components via a mixing device to form a polymer mixture. The biopolymer components include at least one protein, at least one starch, and at least one polysaccharide. In some embodiments, the biopolymer components may further comprise at least one crosslinking agent and/or at least one plasticizer.

[0066] It is contemplated that the mixing device may be any suitable mixing device. For example, the biopolymer components of the polymer mixture may be placed in a vessel suitable for mixing the components. More specifically, the biopolymer components of the polymer mixture may be placed in an agitator. It is contemplated that, heat and/or pressure may be provided to assist in mixing of the components. For example, at least one heater or heating medium can be applied to the vessel undergoing mixing to heat the vessel and its contents being mixed and/or the vessel can be pressurized to increase the pressure of the contents in the vessel (e.g. application of an inert gas into the enclosed vessel to increase its pressure, etc.).

[0067] In exemplary embodiments, the at least one protein may be selected from the group consisting of whey protein, glycomacropeptide, caseins, RGD proteins, or mixtures thereof. In other embodiments, the one or more proteins can include whey protein, glycomacropeptide, caseins, RGD proteins, or mixtures thereof. It is contemplated that the at least one protein may be present in an amount ranging from 1-50 weight percent (wt.% or wt%) of the biopolymer components, preferably in an amount ranging from 10-33 wt.% of the biopolymer components in the biopolymer component mixture. Other embodiments may utilize other concentrations of protein in the mixture.

[0068] In exemplary embodiments, the at least one starch may be selected from the group consisting of OS starch, water soluble starches (e.g., hydroxypropyl starches), or mixtures thereof. In other embodiments, the at least one starch can include an OS starch, water soluble starches, or mixtures thereof. It is contemplated that the at least one starch may be the major component by weight of the biopolymer component mixture (e.g., at a weight percentage greater than the weight percentages of other biopolymer components in the biopolymer component mixture) in some embodiments.

[0069] In exemplary embodiments, the at least one polysaccharide may be selected from the group consisting of pullulan, guar gum, or mixtures thereof. In other embodiments, the at least one polysaccharide can include pullulan, guar gum, or mixtures thereof. It is contemplated that the at least one polysaccharide may be a minor component by weight of the biopolymer components in the biopolymer component mixture (e.g., at a weight percentage less than the weight percentage of the at least one starch) in some embodiments.

[0070] In exemplary embodiments, the at least one crosslinking agent may be selected from the group consisting of phosphate, sodium hydroxide, sodium benzoate, sodium citrate, citric acid, riboflavin, vitamin k (e.g., 2-methyl-l,4-naphthoquinone (3-) derivatives) or mixtures thereof. In other embodiments, the crosslinking agent can include phosphate, sodium hydroxide, sodium benzoate, sodium citrate, citric acid, riboflavin, vitamin k (e.g., 2-m ethyl- 1,4-naphthoquinone (3- ) derivatives), or mixtures thereof. The cross-linking agent(s) can make up a minor portion of the biopolymer component mixture in some embodiments (e.g. make up less than 10 weight percent of the mixture of biopolymer components, make up less than 25 weight percent of the biopolymer component mixture, make up less than 5 weight percent of the mixture of biopolymer components, be between greater than 0 weight precent of the mixture of biopolymer components and less than 5 weight percent of the mixture of biopolymer components, etc.). [0071] In exemplary embodiments, the at least one plasticizer may be selected from the group consisting of sorbitol, xylitol, mannitol, maltitol, lactitol, glycerol, erythritol, isomalt, or any other suitable sugar alcohols and polyols, or mixtures thereof. In other embodiments, the one or more plasticizers can include sorbitol, xylitol, mannitol, maltitol, lactitol, glycerol, erythritol, isomalt, or any other suitable sugar alcohols and polyols, or mixtures thereof. It is contemplated that the at least one plasticizer may be present in an amount ranging from 0-20 wt.% of the mixture of biopolymer components, or biopolymer component mixture, in some embodiments. [0072] It is contemplated that the mixture of biopolymer components can be placed into a solvent, contained in a solvent, or can be mixed within a solvent to form a biopolymer component/solvent mixture, which can also be referred to as a polymer mixture. In exemplary embodiments, the solvent may be selected from the group consisting of water, or mixtures thereof. In other embodiments, the solvent can be water or include water. It is contemplated that the solvent may be present in an amount ranging from 60-80 wt.% of the weight of the biopolymer component/solvent mixture (or polymer mixture) in some embodiments.

[0073] It is understood that the polymer mixture (e.g., the biopolymer components and the solvent) can include generally recognized as safe (GRAS) substances. [0074] The polymer mixture may be a three-component mixture (e.g., one protein, one starch, and one polysaccharide). It is further contemplated that the polymer mixture may be a four- component mixture (e.g., one protein, one starch, one polysaccharide, and one crosslinking agent). It is further contemplated that the polymer mixture may be a five-component mixture (e.g., one protein, one starch, one polysaccharide, one crosslinking agent, and one plasticizer). Yet other embodiments can utilize more than five components.

[0075] As shown in FIG. 1, after the polymer mixture if formed via the mixing occurring for a pre-selected period of time for forming the polymer mixture, the polymer mixture can then be spun (e.g. undergo spinning) to form nanofibers via a spinning device. The nanofibers may have diameters ranging from 50-5000 nm, preferably diameters ranging from 100-2000 nm.

[0076] It is contemplated that spinning device may be any suitable spinning device. For example, the spinning device may be an electrospinner or other suitable spinning device. It is contemplated that temperature and/or pressure may be controlled to provide a desired spinning of the polymer mixture for a pre-determined spinning time. It is contemplated that the nanofibers may be considered a tissue scaffold.

[0077] In a preferred embodiment, the nanofibers are bead-free or include bead-free nanofibers. [0078] In a preferred embodiment, the relative humidity of the spinning process may be in a range of less than 50% relative humidity to greater than 0% relative humidity. For example, in some embodiments, the relative humidity can be less than 50%, preferably less than 30%, and more preferably less than 10%.

[0079] It is contemplated that the spinning device spins the polymer mixture to create aligned nanofibers (e.g. a fabric). It is understood that nanofibers aligned in a unidirectional fashion can contribute to topographical cues in the tissue scaffold and functions of stem cells. Specifically, unidirectional growth of cells contributes to the overall texture of the food product.

[0080] It is contemplated that the polymer mixture may be water soluble. A water-soluble polymer mixture can be advantageous for spinning (e.g., electrospinning) the polymer mixture for a pre-determined or pre-selected spinning time period to align the nanofibers in a desired orientation or in a pre-determined or pre-selected alignment.

[0081] As shown in FIG. 1, the method may further comprise exposing the nanofibers to crosslinking conditions (e.g., conditions that promote crosslinking), such that the nanofibers form a crosslinked material or become crosslinked. It is contemplated that the crosslinking conditions may activate the at least one crosslinking agent of the polymer mixture. It is further contemplated that the crosslinking conditions may include temperature, pressure, and/or UV light exposure (e.g., photo-crosslinking) over a desired period of time. The crosslinking conditions may be controlled to assist in providing a desired level of crosslinking formation within the nanofibers. It is contemplated that the crosslinked material may be considered a tissue scaffold or a biopolymer-based tissue scaffold.

[0082] As shown in FIG. 1, the method can further include feeding the tissue scaffold (e.g., the nanofibers or the crosslinked material) to a cell growth device to grow stem cells, muscle cells or cultivated meat thereon. For example, the cell growth device may be a reactor (e.g. a profusion reactor, etc.). The stem cells, muscle cells or cultivated meat may grow on the tissue scaffold for a pre-determined residence time. It is contemplated that as the stem cells, muscle cells or cultivated meat grow on the tissue scaffold, the tissue scaffold may be consumed via the cell growth process. For example, the tissue scaffold may be consumed via the cell growth process, or the cell growth process may utilize the tissue scaffold in the process such that the tissue scaffold is later consumer/decomposed via the cell growing process (e.g. stem cells, the muscle cells or cultivated meat can consume the tissue scaffold as it grows in the reactor or other type of cell growth device).

[0083] It is contemplated that the cell growth device may comprise a cell culture medium such that the tissue scaffold may be exposed to the medium to promote cell growth on the tissue scaffold. The cell culture medium can be oxygen-rich and contain basic nutrients such as amino acids, glucose, vitamins, inorganic salts, protein supplements, and growth factors. The cell culture medium may be liquid (e.g., water) or include a liquid and comprise a starting material for cell growth.

[0084] It is contemplated that the tissue scaffold may be water resistant. A water-resistant tissue scaffold can be advantageous in facilitating the cell growth process without dissolving in the cell culture medium. We have found that biopolymers are often hydrophilic and that crosslinking that can be provided via embodiments of our method can allow the formed tissue scaffold to become water-resistant.

[0085] Embodiments of the above-described method and process for forming a biopolymerbased tissue scaffold can be further appreciated from the below discussed examples.

[0086] FIG. 31 illustrates an apparatus for forming and/or utilizing an embodiment of our biopolymer-based tissue scaffold. The apparatus can be utilized to grow stem cells, cultivated meat and/or muscle cells as discussed above. In some embodiments, the stem cells that may be used and/or grown can be either embryonic stem cells, muscle stem cells, or muscle satellite cells.

[0087] For example, the apparatus 1 can include a mixer that receives a feed of biopolymer components and also receives a solvent for being mixed in the mixer for a pre-determined mixing time period to form the polymer mixture. The mixing can be performed via the mixer in a batch process, semi-batch process, or a continuous process in different embodiments.

[0088] The formed polymer mixture can be fed to a spinner device (e g. electrospinner, etc.) for undergoing spinning for a pre-selected spinning time period. The feeding of the mixture can be provided via a conduit or other conveying device to provide the formed polymer mixture to the spinner for spinning of the polymer mixture to form the nanofibers. The nanofibers can be fibers having a diameter of between 5 micrometers and a diameter that is greater than or equal to 1 nanometer in some embodiments. For example, in some embodiments the nanofibers can have a diameter in the range of 50 nm to 5000 nm, a diameter in a range of 400 nm to 500 nm, a diameter in a range of 50 nm to 1,000 nm, or in another suitable range for a particular set of design criteria.

[0089] The formed nanofibers can be exposed to crosslinking conditions to activate the crosslinking agent (when utilized) to form the tissue scaffold. The crosslinking conditions can be provided via application of heat, pressure, at least one cross-link promoting gas (e.g. ozone or other gas that can contribute to formation of oxygen radicals that may help facilitate crosslinking or promote cross-linking), and/or UV light at the spinner device or by feeding the formed nanofibers to a crosslinking device to provide the crosslinking conditions. In some embodiments, the crosslinking device can be an oven or heater that can also be pressurized and/or include UV lights for applying UV lights and/or pressure and/or receiving a cross-linking promoting gas (e.g. ozone gas, an oxygen containing gas, etc.) while the nanofibers are heated to form the tissue scaffold. Applying such cross-linking promoting conditions can also be performed in a way that can also sterilize the formed tissue scaffold (e.g. application of UV, heat, and/or other conditions to promote crosslinking and also sterilize the formed tissue scaffold). The formed tissue scaffold can then be fed to a cell growth device for growing cultivated meat and/or meat cells as discussed above.

[0090] In other embodiments, the spinning device can be heated, pressurized and/or have UV lights turned on to apply the UV light to the formed nanofibers while they reside in the spinner device. In such embodiments, the formed tissue scaffold can be output from the spinner device or conveyed from the spinning device to a cell growth device directly as shown in broken line in FIG. 31.

[0091] The cell growth device can receive the formed tissue scaffolds and also receive stem cells or muscle cells from a source of cells (e.g. a source of stem cells or muscle cells, a source of embryonic stem cells, muscle stem cells, and/or muscle satellite cells, etc.) and a cell culture medium from a source of cell culture medium. The sources of stem cells or muscle cells and cell culture medium can be storage tanks, storage vessels, or other devices that can be in fluid communication with the cell growth device to feed those materials onto the tissue scaffold or adjacent the tissue scaffold in the cell growth device, for example. The cell growth device can be structured as a reactor (e g. a profusion reactor, etc.) in some embodiments and can be heated and/or pressurized in some embodiments to facilitate muscle cell growth and/or cultivated meat growth.

[0092] The cell growth device can be utilized to facilitate the stem cells, muscle cells or cultivated meat growing on the tissue scaffold for a pre-determined residence time. As the stem cells, muscle cells or cultivated meat grow on the tissue scaffold, the tissue scaffold may be consumed via the cell growth process occurring in the reactor. For example, the tissue scaffold may be consumed via the cell growth process, or the cell growth process may utilize the tissue scaffold in the process such that the tissue scaffold is later consumer/decomposed via the cell growing process (e.g. the stem cells, muscle cells or cultivated meat can consume the tissue scaffold as it grows in the reactor or other type of cell growth device).

[0093] As noted above, the cell culture medium can be composed so that the tissue scaffold may be exposed to the medium to promote cell growth on the tissue scaffold in the cell growth device. The cell culture medium can be oxygen-rich and contain basic nutrients such as amino acids, glucose, vitamins, inorganic salts, protein supplements, and growth factors. The cell culture medium may be liquid (e.g., water) or include a liquid and comprise a starting material for cell growth.

[0094] The cell growth device can then output the grown stem cells, muscle cells or cultivated meat. Alternatively, the grown stem cells, meat or muscle cells can be removed by other removal means (e.g. a meat removal device, etc.) for subsequent processing (e.g. cutting the meat and/or packaging the material for transport and/or testing the grown meat cells, etc.).

[0095] Cultivated meat (CM) can be an animal-based meat that is produced in vitro from animal cells. This process does not have to require the slaughter of animals - rather, it can employ the use of proliferation and multiplication of stem cells isolated from the source animal. Because of reduced human-animal contact, CM can prevent the spread of animal-borne diseases and epidemic zoonoses. Controlled conditions during cell proliferations enable the manipulation of texture, taste, and nutritional profdes on a specific level. The quantity and quality of fat in the meat can be controlled, thereby reducing the risk of cardiovascular diseases. Relative to conventional beef production, CM is projected to cause significantly less eutrophication and greenhouse gas emissions and utilize much less land.

[0096] The general process can involve target cell procurement from the animal, preparation of production cells, biomass production, product collection, and food processing. Most cells used in CM are anchorage dependent, meaning they require a foundation, i.e., scaffold, to attach to and grow. Scaffolds can help promote cell growth by helping the transport of oxygen, nutrients, and waste products to and from the cells, controlling the growing tissue’s geometry and cell type distribution, and contributing to the final structure of the product. In general, cells may need a three-dimensional (3D) environment with biochemical and biophysical cues, governed by the biochemistry and mechanical properties of the surrounding extra cellular matrix (ECM). Scaffolds for cell growth can possess an appropriate level of porosity, sufficient surface area, and appropriate chemistry that allow for adequate cell migration, proliferation, and adhesion, and most importantly, be non-toxic. For CM specifically, scaffolds must also be food-safe and edible. There are many ways to produce scaffolds e g., recombinant technology (customizing DNA fragments with the use of enzymes), fermentation, bioprinting, electrospinning, etc.

[0097] Scaffolds can be structured as extracellular matrices (ECM) that support three- dimensional tissue formation. They can be designed to (i) promote cell-biomaterial interactions, cell adhesion, and ECM deposition, (ii) permit sufficient transport of gases, nutrients, and regulatory factors to allow cell survival, proliferation, and differentiation, (iii) biodegrade at a controllable rate that approximates the rate of tissue regeneration and (iv) invoke a minimal degree of toxicity or inflammation in vivo. Macromolecular polymers are the primary materials used in scaffolds for tissue engineering and cell culture applications. The selection of materials depends on the type of scaffold required based on the application. For porous solid-state scaffolds, linear aliphatic polyesters are desired because they do not dissolve or melt under aqueous in vitro tissue culture conditions. Natural polymers like proteins and polysaccharides (collagen, gelatin, silk fibroin, chitosan, alginate) have also been used either alone or in composite mixtures with other natural or synthetic materials. For hydrogel scaffolds (which are attractive to fill irregularly shaped defects), poly(ethylene glycol) (PEG), collagen, fibrin, alginates, poly(N-isopropylacrylamide) and linear poly(acrylic acid) have been extensively studied. The main interaction between cells and scaffolds is through ligands on the material surface. Scaffolds fabricated via natural materials may contain these ligands in the form of RGD (arginine-glycine-aspartic acid) binding sequences (cell attachment sites of adhesive extracellular matrices, blood, and cell surface proteins), which are very beneficial to cell migration and proliferation.

[0098] The structural design of a scaffold can help provide optimal cell growth. Scaffolds can be formed to have an interconnected pore structure and high porosity to enable cellular penetration and sufficient transport of nutrients and oxygen to the cells. This porous structure can also enable the diffusion of waste products out of the scaffolds.

[0099] Scaffolds can be fabricated via several techniques. Some methods include particle leaching, phase separation, melt molding, fiber bonding, emulsion freeze drying, solution casting, freeze drying, and 3D printing. However, since many biologically functional molecules and cells interact at a nanoscale level, nanofibrous scaffolds can come closest to mimicking the natural extracellular matrix. To construct nanofibrous scaffolds, spinning can be utilized to spin non-woven fibers to form or facilitate forming of a scaffold.

[00100] Electrospinning is a technology that can utilize electrical forces to produce polymer fibers in the range of nanometers to micrometers (e.g. nanofibers). Electrospun nanofibers can possess several advantages such as large surface area, increased porosity, high electrical charge holding ability, high permeability, and adjustable pore size and surface area. [00101] Electrospinning can be provided by use of a high voltage power supply, a grounded collector, a pump, syringe, and needle in some embodiments. An electric field can be generated between the needle and the collector, and as the electrical forces on the polymer droplet approach a critical value, the drop begins to deform into a Taylor cone. When the electrical forces overcome the surface tension of the droplet, a charged jet emits out of the Taylor cone, which undergoes an unstable whipping motion between the needle tip and the oppositely charged collector. This results in the solvent being evaporated, leaving a polymer fiber behind. [00102] Cohesive energy (E_c), which is defined as the measure of polarity and binding energy of the polymers, is another parameter that can be used to assess the electrospinnability of a solution. Solutions with high E_c produce smooth, continuous fibers and solutions with low E_c tend to electrospray (dispersal of charged polymer droplets rather than drawing of filaments). Strong nanofibers with high E_c also detach by peeling smoothly away from the substrate while weak fibers with low E_c break prematurely. The E_c of a polymer solution is a function of the elastic modulus (S') and critical strain (y_cr), and can be calculated using the formula: E_c = There are two broad types of electrospinning - dry and wet. Dry

electrospinning involves collecting the electrospun fibers on a dry collector. This method can be useful when the solvent used is volatile, like water. Wet electrospinning employs a solid collector immersed in a coagulation bath. This method is favorable when the solvent in the spinning dope is not readily volatile, such as dimethyl sulfoxide (DMSO). When the fibers are collected in the coagulation bath, the nonvolatile solvent is extracted, and the fibers are precipitated. Therefore, the coagulation bath can be a poor solvent for the polymer in the spinning dope. The fibers produced via the wet electrospinning process can have diameters more in the micro-scale range than in the nano-scale range. Overall, the wet spinning process is less flexible than dry electrospinning and involves additional steps when compared to dry electrospinning (e.g. wet spinning can require use of a coagulation bath and fiber precipitation). [00103] Naturally occurring connective tissue organizes muscle fibers into aligned bundles. These bundles are oriented in the direction of contraction and undergo changes upon application of heat while cooking, producing the characteristic meat texture. For CM to mimic conventional meat, the cells can grow in an organized fashion. We have found that this can be provided or facilitated by utilization of a tissue scaffold composed of unidirectionally aligned nanofibers or consisting of unidirectionally aligned nanofibers, which can help improve cell growth and differentiation and guided tissue formation. Additionally, myoblasts aligned unidirectionally prior to differentiation can enable aligned myotubes to be engineered. Aligned nanofibers can be produced by use of a rotating collector. A rotating collector can be included in a spinning device and can be positioned to provide extra force to the electrospinning process in addition to the high shear and elongation forces that help orient the chains and align the fibers in the axis direction. The type of target/collector and its rotating speed can determine the alignment of the fibers.

[00104] Electrospun nanofibers can be smooth or beaded. Solution properties such as viscosity, conductivity, and surface tension are the main factors in determining the extent of beading in nanofibers. Higher viscosity leads to fibers with fewer beads and higher net charge density to bead-free and thinner fibers. Since liquids tend to minimize their surface area due to the Rayleigh instability, higher surface tension leads to beaded fibers. A relationship between the timescale of fiber beading and solution viscosity, surface tension, and jet radius has been established, where the timescale is directly proportional to solution viscosity and jet radius, and inversely proportional to solution surface tension. Ambient factors such as temperature and relative humidity can also influence fiber morphology. While beading in nanofibers may be desirable in highly selective applications where adhesion properties are enhanced by the beads, drug delivery as well as in air and water filters, smooth, uniform and bead free fibers can be more preferred for tissue engineering and cultured meat applications.

[00105] The effect of humidity on electrospun nanofibers is poorly understood, and sometimes contradictory effects have been observed depending on the type of polymer, polymer- solvent combination, molecular weight, polymer hydrophilicity, and size of electrospun structure. For a hydrophilic polymer such as starch, high humidity levels can lead to water absorption by the jet that can result in delayed solidification either due to slower evaporation or plasticizing of the polymer. In this case, the jet thins until capillary instability occurs, which results in beaded fibers.

[00106] Electrospinning can be performed on both synthetic and natural polymers. Natural polymers exhibit better biocompatibility, biodegradability, easy digestibility, and low immunogenicity when compared to synthetic polymers. Biopolymers are polymers from natural sources made up of monomeric units bonded together by covalent bonds to form larger molecules. Their unique nontoxicity, edibility, biodegradability, and renewability has made them attractive candidates for the food industry. Additionally, biopolymers have found an increased application in the production of advanced scaffolds and substrates for cell cultures. Biopolymers, which are highly biomimetic in nature, and can provide cells with an environment similar in morphology and chemical structure to the natural ECM. We have found electrospinning of biopolymers to be specifically interesting due to the numerous advantages of nano scaled morphologies like high surface to volume ratio, porosity, ability to interact with cells at a molecular level, biomimetism, etc.

[00107] Polysaccharides are polymers of monosaccharides that can be found naturally in many organisms. They can possess excellent biological properties such as biocompatibility, biodegradability, hydrophilicity, and promote enhanced cell adhesion and proliferation. Their biological functions mainly depend on their chemical structure, chemical composition, molecular weight, and ionic character of the molecule. Electrospinning of natural polysaccharides including those of algal nature (e.g., alginate), plant origin (e.g., cellulose, starch), microbial origin (e.g., dextran), and animal origin (e.g., chitosan) can be successfully performed.

[00108] Chitosan is a sugar-based polymer obtained from chitin via deacetylation which has exceptional qualities that can make it an attractive green biomaterial for biomedical applications. However, the electrospinning of chitosan can be very challenging due to its tendency to form highly viscous solutions in the entangled regime. While this requires the use of solvents with high toxi cities such as 1,1, 1,3, 3, 3 hexafluoro-2-propanol (HFP) and triflouroacetic acid (TFA), other safer alternatives have been attempted like the use of acetic acid, or with the application of alkali treatment. Alginate is a biopolymer harvested from the cell walls of brown algae. It possesses many valuable properties like biocompatibility, biodegradability, high hygroscopicity, antimicrobial properties, and high ion adsorption which makes it extremely sought-after in the biomedical field. The rigid inter- and intramolecular hydrogen bonding network in alginates makes their electrospinning challenging. Another abundantly available polymer is cellulose, which has been evaluated for biomedical tissue engineering applications and could also potentially be used for CM. However, chitosan, alginate and cellulose are not degradable by cultivated cells, which can limit their application in the CM industry.

[00109] Kefiran is a branched polysaccharide that has probiotic properties that can enhance the growth of favorable microflora while simultaneously inhibiting cancer cells and bacteria. Kefiran has been successfully electrospun alone and has been used to enhance the electrospinnability of chitosan in composite blends. Kefiran being a relatively newly investigated polysaccharide requires many more studies to reveal its true potential. Cyclodextrins, derived from the enzymatic conversion of starch, are another example of electrospinnable polysaccharides. They are naturally occurring, water-soluble, and non-toxic. Although the successful electrospinning of cyclodextrins has been described by several researchers, the lack of evidence for their prolonged stability in various conditions requires more studies on enhancing this quality. The last two polysaccharides of interest are starch and pullulan.

[00110] Proteins and peptides are involved in the structure of many essential elements of the body such as enzymes, hormones, and immunoglobulins. They are responsible for enzyme catalysis, signal transduction, metabolic processes, immunogenic defense mechanisms, and gene regulation. The use of protein-based nanostructures has the potential to preserve the bioactivity of bioactive compounds like vitamins, nutraceuticals, and anti-inflammatories in health food products, which has always been a challenge due to their interaction with harsh environmental factors like gastric fluids, low pH, high concentrations of salts and ionic strengths. Peptides, specifically cell-binding peptides are sought after for scaffold design because of their ability to improve cell migration, growth, and differentiation. The inclusion of proteins and/or peptides can also result in the enrichment of the biomaterial with pro-adhesive sequences and affects its surface-wettability and charge. Functionalizing with bioactive peptides can be used to engineer cell-material interactions and to mimic the signaling environment involved in cell growth. The incorporation of proteins in scaffolds aids in multiple signal cascades that mediate the traction force involved in cell movement. This control of spatial and temporal signaling is especially important for cell migration and network formation.

[00111] Starch is a carbohydrate naturally found in many grains and vegetables like wheat, maize, com, potatoes, peas, mung beans, tapioca, rice, pulses, etc. It is regenerated from carbon dioxide and water by photosynthesis in plants. Because of its low cost, easy digestibility, biodegradability, and abundance, we believe it is a promising candidate for developing sustainable materials. Starch is mainly composed of two types of molecules - linear amylose and branched amylopectin. Starch can contain different proportions of amylose and amylopectin depending on the source. For example, native corn starch contains 27% amylose and 73% amylopectin, whereas tapioca starch contains approximately 15% amylose and 85% amylopectin. [00112] The retrograding propensity of native starch make it a challenging polymer to work with in the field of electrospinning. When starch is heated in the presence of water and subsequently cooled, the disrupted amylose and amylopectin chains can gradually reassociate into a different ordered structure in a process termed retrogradation and is usually accompanied by a number of changes in its physical properties like turbidity of pastes, gel formation, and increased viscosity. Regardless, a number of researchers have demonstrated the fabrication of micro and nano-scale fibers via electrospinning of starch through the wet electrospinning process. However, the long fiber drying time (> 6 hours) after the precipitation makes wet electrospinning a time consuming and inefficient process.

[00113] The poor solubility of native starch in water at room temperature is due to its strong hydrogen bonding between starch chains within the starch semicrystalline structure. We have found that one way to combat this challenge while still ensuring the use of starch and green solvents is to utilize modified starch.

[00114] There are 4 basic types of starch modification systems - physical, chemical, enzymatic, and genetic. The three hydroxyl groups at positions C2, C3 and C6 on the glucose monomers of starch can be chemically modified through esterification, etherification, and oxidation. Such modifications in starch can result in increased cold water dispersibility, resistance to retrogradation, and can alter gelatinization, swelling and solubility properties. [00115] One chemical that can be used for chemical modification of starch is octenyl succinic anhydride (OSA). When esterified with OSA, the normally hydrophilic starch gains a hydrophobic element in the form of octenyl groups, resulting in molecules with an amphiphilic character. Amphiphilic polymers are useful in many applications such as encapsulation, emulsification, gels, and film production. OSA starch has been used as a substitute for gum Arabic, fats, and proteins.

[00116] OS starch can be synthesized by suspending granular starch in distilled water and adding OSA dropwise with continuous stirring while maintaining the pH at 8 with the addition of NaOH. The reaction is carried out between 25°C and 35°C until the pH of the mixture stabilizes. After that, the mixture is filtered and/or centrifuged, followed by washing with water and acetone/ethanol. Then, the product is dried and ground. The extent of substitutions of the anhydroglucose units in the starch molecule is referred to as the degree of substitution (DS) and is determined by titration. The extent of substitution depends on various factors such as the type of native starch chosen, pH, amount of OSA used, temperature and reaction time. Even though this traditional method results in modified starch with low solubility in water, it is still greater than native starch since the modification reduces retrogradation. Nevertheless, several variations to the standard method such as mechanical treatment, debranching pretreatment, chemical pretreatment, hydrothermal pretreatment, and heat-moisture pretreatment have been introduced to increase the solubility of modified starches. These variations also result in a higher degree of substitution when compared to the traditional method under the same conditions. [00117] OSA starches can be capable of lowering surface tension/interfacial tension at the air/water interface. This attribute is of particular interest during electrospinning since the morphology of electrospun fibers is highly dependent on the surface tension of the polymer solution. Higher surface tension correlates to beaded nanofibers driving the surface area per unit mass smaller by changing jets to spheres. OS starches are also colorless and tasteless in solution, which makes them attractive candidates for use as emulsifiers and stabilizers in food and cosmetic industries.

[00118] OSA starch is a low-cost fat free biodegradable ingredient. With its resistance to digestion, we believe its application can be advantageous in the preparation of healthier foods to cater to the world’s current obesity, diabetes, and cardiovascular issues. Modification of native starch with OS is said to increase its slowly digested starch (SDS) and resistant starch (RS), which are beneficial to human nutrition.

[00119] Pullulan is an exopolysaccharide produced by a yeast like fungus named Aureobasidium pullulans in the form of amorphous slime on the surface of microbial cells. The formation of glycolipids in fungal mycelial cells leads to accumulation of pullulan on the outer surface of cells. It is a linear and unbranched polymer with maltotriose repeating units connected by alpha — (1 — 6) glycosidic bonds. Sometimes partial acid hydrolysis gives rise to rare forms of pullulan with panose and isopanose as repeating units. Pullulan is highly soluble in water and insoluble in alcohol. Pullulan is non-ionic without any toxicity, mutagenicity, carcinogenicity, and is edible, odorless, and tasteless. Due to these unique properties, pullulan has found use in food, biomedical, and tissue engineering applications.

[00120] Pullulan is a slowly digestible carbohydrate by human beings and can help maintain blood glucose levels. Pullulan also has the potential to increase Lactobacillus and Bifidobacterium activity, which is a desirable nutritional trait. It is stable over a broad range of pH and has excellent adhesive properties. Pullulan’s (1 —> 6) linkages allows it to mimic synthetic polymers in many aspects such as biocompatibility, biodegradability, and human and environment compatibility. These linkages also provide pullulan with enhanced solubility and structural flexibility which result in excellent film and fiber forming capabilities. Pullulan’s biocompatibility and high hydration capacity makes it an attractive candidate for scaffolds. [00121] Native ECM is mainly composed of fibrous proteins (various types of collagen and elastin), adhesive glycoproteins (fibronectin, laminin, and vitronectin), proteoglycans (PGs), and glycosaminoglycans (GAGs). Functionalizing of polymer scaffolds can involve the incorporation of proteins and/or peptides to enable the fabrication of biocompatible biomaterials that mimic natural ECM. This can be performed in a few ways. One way is to prepare functionalized monomers before polymerization and electrospinning. Another way is to graft peptides onto inert polymers before electrospinning. A third way is to functionalize the surface of already electrospun fibers with bioactive peptides. The best approach to utilize for a particular application may be selected based on the desired physical, chemical, and biological properties of the scaffold to be formed. In embodiments of our process as discussed in examples herein, a protein was homogenized with OS starch and pullulan into a polymer solution prior to electrospinning.

[00122] Interactions between starches and proteins can determine texture, mechanical properties, nutrition, and digestibility of the final product. The relationship between starches and proteins is mainly non-covalent, involving hydrogen bonding, hydrophobic interactions, electrostatic forces and ionic interactions. The electrostatic interactions between proteins and polysaccharides involve positively charged -NH3⁺ groups interacting with the negative charges carried by the carboxyl groups of polysaccharides. These interactions are affected by the intrinsic properties of the polymers like solubility, net charge, size, and weight ratios.

[00123] Whey is residue that remains after recovery of the curd from the clotting of milk with proteolytic enzymes or acid. Whey protein is the group of proteins isolated from whey. The proteins are usually beta-lactoglobulin, alpha-lactalbumin, glycomacropeptide, immunoglobulins, and bovine serum albumin. Using enzymatic or fermentation processes, whey proteins can be transformed into peptides that can exert beneficial physiological effects in vivo. Whey protein isolate (WPI), which contains over 90% protein, is obtained by microfiltration or ion exchange chromatography of pasteurized whey. Due to its excellent functional properties and high water solubility, WPI has been used by various researchers to make edible films, hydrogels, and porous scaffolds.

[00124] Whey protein isolate has been successfully electrospun in combination with other polymers. Additionally, the thermal stability of these fibers can be increased via heat treatment of these blend fibers at temperatures above the gelation temperature of the protein. Prolonged heat treatment of these fibers resulted in whey protein crosslinks which rendered them less soluble in water. Ultra-thin bead free nanofibers fabricated from WPI and guar gum blends displayed improved stability at higher temperatures. Continuous and uniform WPI pullulan nanofibers with varying concentration ratios of WPI. In the examples discussed herein, WPI was chosen as one of the proteins to be electrospun along with OS starch and pullulan due to its abundance, bioactivity, functional properties, and excellent water solubility.

[00125] Glycomacropeptide (GMP) is a hydrophilic water-soluble molecule which makes up about 12% w/w of whey protein. GMP is rich in branched chain amino acids and due to its absence of phenylalanine, it can to be used to fulfil the nutritional profile of people suffering from phenylketonuria, a genetic disorder that prevents people from metabolizing phenylalanine.

Bioactive compounds with different solubilities were successfully incorporated into lactoferrin-

GMP hydrogels, which show a great potential for controlled delivery systems.

[00126] To the best of our knowledge, there have been no studies demonstrating the inclusion of isolated GMP in electrospun nanofibers. In the examples discussed herein, GMP was combined with OS starch and pullulan at different concentrations to fabricate electrospun nanofibers. GMP, with its 106 amino acid profile, contains side chains that may function to enhance the degree of cell proliferation, differentiation, and adhesion for cultivated meat applications. Although GMP is currently sourced from animal milk protein, there is future opportunity for a vegan counterpart to be utilized instead, like the animal protein made via fermentation by Perfect Day, Inc.

[00127] Solution conductivity can affect the Taylor cone formation and determining fiber diameter during electrospinning. Droplets in a solution with low conductivity may not possess sufficient charge density required to form a Taylor cone, thereby restricting electrospinning. When conductivity is increased, the jet carries more charges, causing increased repulsion of charges at the surface and resulting in decreased fiber diameter. One way to control solution conductivity is by the addition of salt in the solvent. The addition of salt affects the electrospinning process by increasing the number of ions in the polymer solution, which results in the increase of surface charge density of the fluid and the electrostatic force generated by the applied electric field. It also increases the conductivity of the polymer solution, which results in the decrease in tangential electrical field along the surface of the fluid. In addition, the minimum voltage required to electrospin fibers can also be reduced by increasing the conductivity of the polymer solution. [00128] Sodium citrate, the sodium salt of citric acid, is used as a food additive as a flavor enhancer or preservative. It is water soluble and has a high ionic strength. This is one type of salt that can be added to a solvent to facilitate improved electrospinning performance in some embodiments.

EXAMPLES

[00129] Example 1. Experiments were conducted in an attempt to incorporate whey protein isolate (WPI) and glycomacropeptide (GMP) into electrospun OSA-starch-pullulan composite nanofibers. While maintaining a constant overall polymer concentration at 30% (w/w of polymer/solvent), the concentration of protein (WPI or GMP) was varied, and fibers were electrospun at predetermined electrospinning parameters. The rheological properties, surface tension and conductivities of the solutions were measured. The electrospun nanofibers were characterized for fiber morphology and diameter using scanning electron microscopy and image analysis.

[00130] It was hypothesized that the timescale of fiber beading will be lower for solutions with higher protein content compared to solutions with lower protein content, i.e., solutions with lower protein content will display fibers with comparatively higher uniformity and lesser beading. The timescale was estimated using the relationship: T O |j//y], where T is the timescale of fiber beading, [i is the solution viscosity, and y is the solution surface tension.

[00131] The influence of relative humidity on spinnability of OSA-starch-pullulan-protein nanofibers was also determined. In particular, polymer solutions were electrospun at predetermined electrospinning parameters at varying humidity levels. Fibers were characterized for uniformity of morphology and extent of beading using scanning electron microscopy. [00132] It was hypothesized that the beaded morphology of fibers will increase as the relative humidity during the electrospinning process is increased.

[00133] Finally, a relationship between RPM of a rotating drum collector system and the extent of alignment of the nanofibers was established using scanning electron microscopy and image analysis.

Materials

[00134] Purity Gum Ultra (PGU) (M_w - 12.4 x 10⁴ g/mol, MC - 5.2%, DS - 0.008, lot number LKI6890) was provided by Ingredion (Bridgewater, NJ). Food grade pullulan (M_w - 10- 20 x 10⁴ g/mol, MC - 6.15%) was obtained from Hayashibara Biochemical Laboratories Inc.

(Okayama, Japan). Whey Protein Isolate (WPI) (90% protein content, lot number 17030701) was purchased from Nutricost (Vineyard, UT). Glycomacropeptide (GMP) (lot number JE 0018-20- 440) was obtained from Agropur Ingredients (La Crosse, WI). Sodium citrate was purchased from VWR International (Radnor, PA). DI water was used to prepare all solutions.

Methods

[00135] A mixture design with PGU, pullulan, and protein was created using Minitab® with the overall polymer concentration set constant at 30% (w/w of polymer/solvent). This concentration was determined after a series of trials which revealed that a concentration higher than 30% resulted in a very high dope viscosity which caused frequent needle clogging, thereby hindering the electrospinning process, and a concentration much lower than 30% resulted in insufficient dope viscosity that resulted in electrospraying. The lower limits were chosen by determining the minimum required concentration of each of the components that would still result in an electrospinnable dope. The upper limits were determined by keeping in mind the overall polymer concentration, minimizing the use of pullulan, and maximizing the use of protein while ensuring the majority of the mixture was composed of starch. All concentrations were on w/w basis of polymer/solvent.

Table 1 : Polymer Concentration Limits

Table 2: Mixture Design

* G = GMP

** W = WPI

[00136] Polymer mixtures were prepared in 0.05M sodium citrate (w/w: solids/solvent) according to Table 2. For GMP samples, solids and solvent were heated together in a boiling water bath on a hot plate with continuous stirring for 3 hours, then cooled to room temperature (20°C). For WPI samples, PGU and pullulan were first heated together with the solvent in a boiling water bath on a hot plate with continuous stirring for 3 hours, then cooled to room temperature. WPI was then added and set for stirring without heat for 24 hours. This was done to avoid denaturation of protein (which begins at 70°C) and to ensure complete dissolution of protein in the polymer mixture without heat. All further characterizations and experiments were performed immediately after dope preparation.

[00137] The conductivity of the samples was measured at room temperature (20°C) using an EXTECH conductivity meter (EC100, FLIR systems, Inc., Waltham, MA, USA). The surface tension of the mixtures was measured using an automated tensiometer (Rame-Hart model 260, Sucasunna, NJ, USA) using pendant drop mode.

[00138] The apparent shear viscosity of the mixtures was measured using a strain- controlled rheometer (ARES, TA Instrument, New Castle, DE, USA) with cone and plate geometry (50 mm), a gap of 0.043 mm and cone angle of 0.04 radians. Apparent shear viscosity vs shear rate from 0.1 s'¹ to 100 s'¹ was collected at room temperature (20°C). Shear viscosities at 100 s'¹ were reported for all the mixtures.

[00139] Dynamic measurements were conducted to measure the viscoelasticity of the mixtures according to Balik & Argin, (2020). Measurements were carried out using cone and plate geometry (50 mm), a gap of 0.043 mm and a cone angle of 0.04 radians. An amplitude sweep test was performed for each sample at a constant frequency of 2 Hz and at 25 °C with an increasing shear strain from 0.1% to 1000% The averages of the G’ and 8 which corresponded to 1-10% strain were reported.

[00140] The data from the shear viscosity values were fit to the power law model to calculate the flow behavior index “n”. The maximum shear rate was calculated using equation (1). The tip viscosity was calculated using the power law model equation (2).

[00141] R is the inside radius of the syringe tip, Q is the volumetric flow rate (mL h'¹), and n is the flow behavior index obtained by the power law model equation.

[00142] r] is the tip viscosity and K is the flow consistency coefficient.

[00143] To determine the strength of the internal structure, the cohesive energies of the polymer solutions were calculated by using the critical strain (intersection of the linear and nonlinear response) and G' according to equation (3).

E_c = X G' X y² _r (Eq. 3)

[00144] G' is the elastic modulus at 1 - 10% strain, and y_cr is the critical strain.

[00145] The prepared mixtures were electrospun via a dry electrospinning technique using 10 mL syringes fitted with 22-gauge needles. The electrospinning setup consisted of a high voltage power supply (ES40P, Gamma High Voltage Research, Inc., Ormond Beach, FL), a syringe pump (81,620, Hamilton Company, Reno, NV), and a piece of aluminum foil as the collector. A hot plate was used to keep the internal temperature of the chamber at 30°C to maintain the relative humidity at ca 0.1%. Electrospinning was performed under the predetermined conditions shown in Table 3.

Table 3: Predetermined Electrospinning Conditions

[00146] The voltage was adjusted between 10 and 15 kV until a Taylor cone and fiber jet was observed. Upon electrospinning, the fiber mats were dried for 48 hours in a 50°C oven.

[00147] A humidity study (Table 4) was performed by reducing the relative humidity (RH) periodically by introducing heat into the electrospinning chamber via a hot plate with a temperature controller. A fan was installed in the chamber to ensure even distribution of heat. A lower concentration of polymer (26% solids) was chosen to eliminate needle clogging due to the application of heat during the electrospinning process. Electrospinning was performed under the predetermined conditions shown in Table 3.

Table 4: Humidity Study Conditions

* The GMP sample solidified and clogged the needle at temperatures above 40°C, therefore, the humidity test for GMP was limited to three RH levels.

[00148] Aligned nanofibers were fabricated via a needle syringe and a home-made rotating drum collector system (FIG. 2), (Model no. 4554-00, Cole-Parmer Instrument Co., Barrington, IL). The polymer sample was loaded into a 10 mL syringe fitted with a 22-gauge needle. The electrospinning setup consisted of a high voltage power supply (ES40P, Gamma High Voltage Research, Inc., Ormond Beach, FL), a syringe pump (81,620, Hamilton Company, Reno, NV). The rotating collector was covered with aluminum foil for ease of fiber removal. Electrospinning was performed at 20°C and 12% RH for the WPI sample and at 23 °C and 65% RH for the GMP sample under the predetermined conditions shown in Table 3.

[00149] The collector rotational speed was varied to investigate the relationship between degree of alignment and collector RPM. Upon electrospinning, the fiber mats were maintained in a 50°C oven until further analysis.

[00150] Fiber mats were characterized using a Phenom G2 Pro scanning electron microscope (SEM, Phenom-World, Eindhoven, the Netherlands) at an accelerating voltage of 5 keV. The support was a standard SEM aluminum stub covered with a double-sided carbon tape. The samples were sputter coated with iridium at a thickness of 2 - 5 nm depending on the density of the fiber mats with a sputter rate of 0.17 - 0.21 nm/sec (Leica microsystems, Wetzlar, Germany). Open software ImageJ with the diameter plugin was used for analyzing and measuring fiber diameters from the SEM images. Open software Fiji was used to measure the directionality of the aligned fibers. [00151] Mixture regression with a stepwise model (a quadratic model containing linear terms and two-way interactions with a 95% confidence interval) was used to analyze the effects of component proportions on the various responses (Minitab 21 Statistical Software, State College, PA). A stepwise regression can identify a useful subset of the predictors by eliminating variables in the model that have P-values greater than the alpha value and including all variables in the model that have P-values less than or equal to the alpha value for inclusion (0.15). Due to the dependence between the components in this mixture design, regression statistics were used to establish significance between component proportions and responses.

[00152] The conductivity and surface tension of all the polymer solutions were measured prior to electrospinning, and the values are provided in Table 5

Table 5: Conductivity and Surface Tension Measurements of Polymer Solutions

[00153] The conductivity for WPI solutions ranged from 3.42 to 3.67 mS/cm and the conductivity for GMP solutions ranged from 3.15 to 4.04 mS/cm. The high conductivity levels for both sets of polymers can be attributed to two factors - (i) the addition of sodium citrate in the solvent, and (ii) the presence of charged amino acids in the WPI molecules and the GMP peptide. We found that the polymer mixtures with changing component concentrations do not have a statistically significant effect on the conductivity of either the WPI or GMP solutions. Even though the amount of charged protein/peptide is varied within the polymer mixtures, the fact that the conductivity values remain independent of their concentrations indicates that the dominant factor in determining the conductivity is likely the ionic strength of the solvent containing 0.05M sodium citrate. These high conductivity values are expected to reduce nanofiber diameter and increase bead-free morphology

[00154] The surface tension (y) of a polymer solution was found to depend on the type of polymer and solvent used. We found that the component concentrations in the polymer mixtures had a statistically significant effect on the surface tension (p < 0.05). Specifically, for GMP solutions, the linear terms and the quadratic term pullulan*protein had a statistically significant effect on y, and for WPI solutions, all linear terms and the quadratic terms pul lul an* protein and PGU*protein have a significant effect on y. The contour plots for the surface tensions for GMP polymer mixtures and WPI mixtures (FIGS. 3 and 4, respectively) show that the surface tension decreases as protein concentration increases. This is because proteins are surfactants and are known to contribute to reduced surface tensions. Also, the y of GMP samples was found to be consistently lower than its WPT counterpart. This is because whey proteins are less surface active than caseins, due to their globular structure. Additionally, OSA starches have surface tension reducing capabilities, which also contribute to y reduction (as evidenced by the decrease in y with increasing OSA % in FIGS. 3 and 4). The emulsifying properties of OSA starches increase with increase in degree of substitution.

[00155] Rheological properties of fluids are very important to process optimization and understanding the material behavior. One of the main factors driving solution viscosity is varying the polymer concentration. The viscosity of the solution is generally considered a dominant parameter determining fiber diameter and the electrospinnability of polymer solutions. Too low a viscosity will result in interruption of the polymeric filaments, electrospray, or beaded nanofibers, whereas a too high viscosity may render it impossible to extrude the polymer out of the needle. The minimum required viscosity for electrospinning depends on molecular weight of the polymers used, nature of the solvent, and overall polymer concentration. Generally, the polymer concentration needs to be above the entanglement concentration (C_e), below which nanofibers are not produced. However, this is not an absolute condition and in some configurations the polymer concentration can be below or at C_e and still facilitate nanofiber production. Entanglement refers to the spherical structure formed by the crosslinking points produced in or in between the polymer chains, making the polymer unable to move unencumbered. The C_e of PGU was 6.43% (w/v) and pullulan was 6.08% (w/v), and the minimum electrospinnable concentrations 4.7 times and 2.3 times C_e for PGU and pullulan, respectively. It has been determined that the lowest pullulan addition levels for achieving good nanofibers from 12%, 15%, and 20% aqueous OS starch dispersions were 6%, 5%, and 3% respectively, which indicates that a relatively small addition of pullulan significantly improves molecular entanglement and viscoelastic properties required for electrospinning. Pullulan has excellent fiber forming capabilities due to its ability to enhance molecular entanglement, thereby improving the electrospinnability of otherwise non-fiber forming polymers. The use of PGU levels from 16% to 24% and pullulan levels from 4% to 6% are employed - this ensures that a polymer solution with the lowest concentrations of both PGU and pullulan (16% and 4% respectively) is still electrospinnable.

[001561 Table 6 provides the measured rheological properties of the polymer solutions.

Table 6: Rheological Properties of Polymer Solutions

* FB = Few beads; SB = Several beads; MS = Mostly Smooth; NBO = No beads observed [00157] The shear viscosity vs. shear rate of the polymer solutions was measured and apparent viscosities at 100 s'¹ reported. It was observed that the solutions exhibited nonNewtonian behaviors, i.e., their viscosities depend on shear rate. FIGS. 5 and 6 show that solution viscosity decreases with increase in protein content and that the effect of linear components and PGU*protein quadratic component is statistically significant on viscosity values for both GMP and WPI mixtures. This reduction in viscosity with increasing protein concentrations can be attributed to the hydration properties of the PGU-pullulan-protein blends and interactions between them and the water molecules. Since protein is part of a mixture design with a constant polymer concentration in this study, any variation in protein concentration automatically means a variation in the other components (OSA starch and pullulan). OSA starch and pullulan have extended, stretched out conformations, which favor the formation of entanglements, subsequently favoring increased viscosity. WPI is a globular protein, and hence exhibits poor molecular entanglement. Therefore, when there is an increase in WPI concentration in any mixture, it results in a decrease in OSA starch and/or pullulan concentration, which leads to decreased molecular entanglement, subsequently contributing to a decrease in viscosity. GMP is a linear molecule, however, it’s molecular weight (-7,500 Da) is much lower than that of OSA starch/pullulan, thereby causing a reduction in solution viscosity. However, due to its fibrous structure promoting increased entanglements when compared to WPI, solutions with GMP have higher viscosities than solutions with WPI. A similar effect can be observed in the tip viscosity as well, and a comparable trend can be seen in FIGS. 7 and 8, where tip viscosity decreases with increasing protein concentration in the polymer blend.

[00158] To draw a comparison with the conditions in the electrospinning process, elastic modulus (G') and phase angle (<5) values were determined from the response of fiber forming solutions to % strain and the average of G' values under 1-10% strain was reported. Elastic modulus is defined as the stress applied to the material divided by the strain. Simply put, a stiffer material will have a higher elastic modulus than a flexible material. Statistical analysis shows that the effect of all linear components is statistically significant on the G' values for solutions containing GMP and the effect of all linear components and the quadratic component PGU*protein is significant for solutions containing WPT. As seen in FIGS. 9 and 10, G' values decrease with increasing protein concentrations. This is potentially due to the high degree of entanglements provided by the OSA starch and pullulan, giving rise to temporary crosslinks. Additionally, OSA contributes hydrophobic and steric characteristics to OSA starch which makes it attractive as a stabilizer. Due to Rayleigh instability, liquids tend to minimize their surface area by virtue of their surface tension. During the electrospinning process, this instability translates to the formation of beaded fibers, due to the reduction in cohesiveness of the polymer solution. Since cohesive energy is a function of O', higher G' values mean higher cohesive energies, which in turn indicate that those solutions have a lesser chance of being affected by the Rayleigh instability and higher chance of producing smooth, continuous fibers. The G’ vs % strain data for solutions with the highest and lowest amounts of GMP and WPI provides an idea of the stability of the solutions during the electrospinning process. The mixtures with no protein (GO and WO) show high G’ values that stay linear for a long range of % strain, indicating a more stable electrospinning system. In contrast, the mixtures with highest % of protein (GIO and W10) show lower G’ values that are linear for a much shorter range of % strain, which imply that these composite solutions get less stable with the addition of protein (FIG. 11).

[00159] The phase angle (G”/G’, 6) of a material ranges from 0 to 90 degrees. Elastic materials exhibit a low value of 5 while viscous materials a high value of 5. 6s for both GMP and WPI mixtures are above 70° (Table 6), indicating that the solutions behave in a more viscous manner. Although the P-values we determined from conducted evaluation work show that the component mixtures can have a significant effect on the 8 (linear components and all quadratic combinations for GMP, and linear components and quadratic component PGU*protein for WPI),

FIGS. 12 and 13 illustrate just how small the variation in 5 is with changing component concentrations. For GMP mixtures, the 5 of most of the design space lies very close to 80°. For WPI mixtures, even though the range is comparatively larger, the 5 of the whole design space still lies between approximately 78° and 84°. This is most likely because the polymer mixtures all have a constant solids concentration at 30%.

[00160] The cohesive energies of GMP and WPI containing samples were calculated and Cohesive energy (E_c) is a measure of binding energy of the polymers. A higher E_c indicates a more stable internal system since it requires more amount of energy to destabilize it.

Additionally, high Ec facilitates the electrospinning of smooth fibers, whereas low E_c could lead to broken fibers or electrospray. The P-values we determined for mixtures containing GMP in our testing, the linear components and the quadratic component pullulan*PGU indicate that they can have a statistically significant effect on the E_c, and for mixtures containing WPI, the linear components and quadratic components PGU*protein and pullulan*protein have a statistically significant effect on the E_c. The E_cs of both systems decrease with increasing amount of protein (FIGS. 14 and 15). This could be due to the hydrogen bonding between the carbohydrate polymers and/or the electrostatic repulsion between the charged amino acids. Additionally, as discussed earlier, E_c is a function of G', therefore systems with higher G' values have higher E_cs and vice versa. GMP mixtures also have much higher E_cs than WPI mixtures, as a result of GMP’s increased entanglements due to its fibrous structure.

[00161] There are three main groups of parameters that affect electrospun nanofiber morphology and diameter - (i) solution parameters, (ii) process parameters, and (iii) ambient parameters. In this example, the process and ambient parameters were kept constant to investigate the effect of changing solution properties on nanofiber morphology. While these are three main parameters, other parameters can also have an effect on the spun fibers as well and may also be accounted for to obtain spun fibers. Viscosity and surface tension (y) are important attributes that determine electrospun nanofiber morphology and diameter. In this case however, even though the polymer concentrations have a significant effect on the viscosity and y of the solutions, they do not have a significant effect on the overall fiber diameter, most likely because the overall solids concentration and needle-to-collector distance were kept constant for all mixtures in our evaluation work. Of course, other embodiments can utilize different needle to collector distances.

[00162] For the GMP mixtures we evaluated, the average fiber diameters ranged from 459 - 565 nm, and for WPI mixtures, the average fiber diameters ranged from 461 - 526 nm, which fall within the suitable range of 3 - 5000 nm, which can provide a relevant physiological structure for tendon-like ECM environments. Additionally, the fiber diameters from this study are comparable to the diameters of the only component of the ECM that can get to a size similar to electrospun fibers - collagen fibrils, whose fibrous structures have diameters varying from 50 to 500 nm. Generally, spinning dopes with y values between 35 and 55 mN/m are considered to be solutions suitable for electrospinning. The y values from this work mostly fall within this range, which helps explain the successful electrospinning of all the polymer mixtures.

[00163] Viscoelastic properties of a solution resist deformation changes in shape and support the formation of electrospun fibers. Electrospun fibers can be smooth or beaded, and their morphology is largely dependent on solution properties such as viscosity, surface tension, and conductivity. When the viscosity of a solution is increased, the polymer beads are larger, the average distance between them is longer and the diameter of the obtained fibers is increased. Additionally, the shape of the beads changes from spherical to spindle like, eventually resulting in smooth, bead-free fibers. The composite mixtures in this study exhibit a trend of decreasing viscosities with increasing protein concentration. Due to this, WPI fibers in this study show increased beading (elaborated in the next section). GMP-containing solutions have comparatively higher shear viscosities than those with WPI, resulting in smoother and less- beaded fibers.

[00164] To determine the effect of protein inclusion on fiber beading, the timescale of fiber beading (T) was calculated by calculating a ratio of the solution viscosity and y. It was hypothesized that solutions with higher protein concentration will have a lower T, resulting in more frequent beading when compared to solutions with lower protein concentrations.

[00165] As seen in FIGS. 16 and 17, as protein % increases, the timescale of fiber beading generally decreases. This implies that solutions with higher protein levels will be susceptible to quicker beading when compared to solutions with lower protein levels. This phenomenon can be attributed to the decrease in solution viscosity relative to the small change in y with increase in protein concentration which results in bead formation. Higher viscosities result in limited Rayleigh instability and varicose instability, consequently increasing the whipping instability, resulting in the formation of smooth fibers. Lower surface tension favors the formation of fibers without beads. Because GMP-containing solutions comparatively have lower y and higher viscosity than WPI solutions, the timescales of GMP-containing solutions are a slightly greater than those of WPLcontaining solutions, which indicates that smoother fibers are obtainable from GMP-containing solutions. The morphology of the fibers is provided in Table 6. A comparison of level of beading in (A) 9% GMP, (B) 4% GMP, (C) 1% GMP, (D) 9% WPI, (E) 4% WPI, and and (F) 1% WPI containing nanofibers is provided in FIG. 18. The circles in FIG. 18 encompass headings in the fibers.

[00166] Ambient parameters such as temperature and relative humidity (RH) play a key role in the electrospinning process. However, the effect of RH on the morphology of electrospun nanofibers has been seldom studied. In the case of water-soluble polymers, high ambient RH leads to water absorption of the jet, which in turn leads to delayed solidification due to slower evaporation. This results in the jet thinning until it is subjected to capillary instability, leading to the formation of bead on string fibers. It has been concluded that the bead-on-string morphology of nanofibers is dependent on solution jet solidification rate and capillary breakup of the viscoelastic fluid. In this present study, two polymer solutions - one containing WPI and one containing GMP were subjected to various humidity levels to determine the effect of RH on fiber morphology. For each sample and RH level, 5 representative SEM images were taken at a constant magnification, and the number of beads were counted and averaged to provide quantitative data (Table 7).

Table 7: Approximate Number of Beads on GMP and WPI Nanofibers

[00167] Table 7 shows that the number of beads increases with increase in RH. For example, at 13% RH, WPI-containing fibers had 16 beads, whereas at 42% RH, they had 54 beads. Increased RH leads to the increase in the number of water vapor molecules in the air, resulting in decreased charges on the jet due to molecular polarization. Consequently, this leads to the electrical forces being overcome by the surface tension, resulting in beads.

[00168] It should also be noted that GMP-containing fibers exhibited less beads than WPI- containing fibers at comparable RH levels. This could be because GMP-containing solutions have a higher viscosity than WPI-containing solutions at the same protein content (see Table 6), and higher viscosity favors formation of fibers without beads. It could also be because the GMP- containing sample had a slightly greater percentage of pullulan when compared to the WPI- containing sample, however, it is unlikely that the effect of that increased pullulan concentration is significant on fiber morphology. A sample comparison of WPI-containing nanofibers at 2% and 13% RH is provided in FIGS. 19 and 20.

[00169] Nanofibers oriented in a unidirectional manner were electrospun from both protein-containing solutions using a rotating drum collector system. Polymer solutions containing 16% PGU, 6% pullulan, and 8% protein (S2) were selected. Four (4) different rotational speeds - 3600, 4200, 4900 and 5600 RPM were used to evaluate the alignment of the fibers. The solution containing GMP proved to be challenging to electrospin at the same ambient conditions (12% RH, 20°C) and process conditions (14 kV) as WPI, therefore, the RH and temperature was increased to approximately 65% and 23°C, respectively, and the voltage was increased to 18 kV to facilitate the process. Additionally, the GMP-containing sample needed to be electrospun for a longer time (35 minutes vs 10 mins) to achieve a similar level of fiber mat density. Five (5) representative images were taken from each sample and analyzed using the directionality plugin in Fiji®. The data is fitted by a Gaussian function, and a ‘goodness’ fit and standard deviation (< ) is generated for each image. This data is provided in Table 8.

Table 8: Alignment Data for Mixtures Containing GMP and WPI at Different Collector Rotation

Speeds

[00170] The cr at each collector speed tells us how narrow the fiber angle histograms are, which translates to the degree of alignment of the nanofibers. Table 8 shows that for both GMP- and WPI-containing fibers, the highest rotation speed (5600) resulted in fibers with the smallest a of alignment. The overall data indicates that a reasonable level of alignment can be achieved with composite starch-pullulan-protein blends at speeds at or above 3600 RPM. The rotational speed can be dependent on the type of collector employed and other suitable rotational speeds can also be utilized. The observed entanglements in both GMP- and WPI-containing nanofibers could be due to the accumulation of charge on the fiber mat due to the presence of charged proteins, which could lead to straying of the jet. The beading observed in the GMP-containing fibers is due to the increased humidity levels during the electrospinning process, as discussed above. FIGS. 21 and 22 show SEM images of the aligned nanofibers. [00171] The electrospinning of OSA starch-pullulan-protein composite mixtures was successfully demonstrated in this example. GMP was effectively incorporated as part of an electrospinning dope and fabricated into nanofibers. The incorporation of proteins in polysaccharide scaffolds may provide increased functionality and improve cell adhesion, migration, proliferation, and differentiation. Nanofibers with average diameters in the range of ~ 450 nm to 570 nm were obtained. GMP-containing nanofibers showed lesser beading than those with WPI, potentially due to the higher viscosity of GMP-containing solutions. The component concentrations did not have a significant effect on the overall fiber diameter, most likely because of the constant solids concentration or due to a narrow design space. Increasing RH levels resulted in increased levels of beading in both protein samples. The GMP-containing solution required the adjustment of process and ambient parameters to electrospin aligned fibers. Aligned fibers of good degrees of alignment were obtained from both protein samples at the selected speeds.

[00172] Example 2. Experiments were also conducted in an attempt to electrospin and photo-crosslink OSA-starch-pullulan-GMP nanofibers. The quantity of a photo-crosslinker (e.g., sodium benzoate) in these experiments was varied to minimize the chemicals involved. Finally, experiments were conducted to electrospin and photo-crosslink OSA-starch-pullulan to see if GMP was necessary for crosslinking.

[00173] For Example 2, the biopolymer consisted of octenyl succinylated anhydride (OSA) starch, glycomacropeptide (GMP), and pullulan (PUL). The OSA starch was provided by Ingredion and the version used in the research was PURITY GUM® ULTRA (PGU) (Lot Number: LKL6890). [00174] Sorbitol was used for a plasticizing effect and was provided by Amresco® (Lot

Number: 1122C224). Sodium benzoate was used as a photo-crosslinker and was provided by

Alfa Aesar (Lot Number: 10203291). Sodium citrate (0.05M) in deionized water was used as a solvent.

Methods

[00175] The biopolymers, plasticizer, and crosslinker were all calculated and weighed out on dry weight basis. The biopolymer content was limited to 24% (w/w) of the total weight of the sample, where PGU was 15% (w/w), PUL 5% (w/w) and GMP 4% (w/w). Sorbitol was calculated on dry biopolymer basis, where it was 12.5% (w/w). Sodium benzoate (SB) was calculated on dry (biopolymer + sorbitol) basis, where it was varied at 5% (w/w), 2.5(w/w) & 1% (w/w). The weight of the solvent was calculated by subtracting the total dry weight (biopolymers + crosslinker + plasticizer) from the calculated total weight of the sample. Three samples were made and named BP-SB5, BP-SB2.5 and BP-SB1 respectively, where BP stands for biopolymer.

[00176] To determine if GMP is necessary for crosslinking, PGU was weighed out at 19% (w/w) and PUL was kept at 5% (w/w). The calculations were made on the same dry basis and SB was fixed at 5% (w/w) on dry (sorbitol + biopolymer) and sorbitol was kept at 12.5% (w/w) on dry biopolymer basis. The sample was named nGMP.

Table 9: Composition Percentages

[00177] The remaining sodium citrate solution weight was added to their respective dry biopolymer mix and was put in a boiling water bath for 2 hours with continuous stirring. After the bath, the mixture was allowed to cool down and then sorbitol and sodium benzoate were added to the mixture and stirred until homogenous.

[00178] The mixtures were then electrospun at room temperature (~20°C) till a thick mat was formed which could be peeled from the aluminum foil collector. Electrospinning parameters are shown in Table 10.

Table 10: Electrospinning Parameters

[00179] The sample obtained was put in a desiccator for 12 hours, then moved and conditioned over a saturated Magnesium Nitrate solution to yield a relative humidity of 57% in an airtight box for 24 hours.

[00180] After that, the sample was irradiated in an UV-Ozone oven (Model Number: T10X10/OES) provided by UVOCS® for a specific period. All the samples had an unirradiated control version. The UV-Ozone oven functions at two wavelength peaks: 254 nm and 185 nm.

Table 11 : Sample Irradiation Duration

[00181] The irradiated and non-irradiated samples were all subjected to water solubility test, where the fibers were submerged in deionized (DI) water.

[00182] The samples which didn’t disintegrate in hydrated conditions were subsequently extracted from the water and were then given a 60% aqueous ethanol followed by an 100% acetone wash. The ethanol and acetone washes were necessary to dehydrate the sample, after that the samples were dried in a desiccator for few hours before being inspected under a Scanning Electron Microscope (SEM). The SEM used was a Phenom G2 Pro which has an accelerating voltage of 5 keV. The samples were looked under the SEM using a standard aluminum stub with double sided carbon tape. [00183] The control samples which weren’t irradiated disintegrated once they encountered DI water but the one which was exposed to the irradiation survived the water solubility test. FIG. 23 accounts for the sample BP-SB5 through the various stages of its water solubility test. The survival of the sample after 24 hours and 10 days in DI water shows that certain degree of crosslinking was achieved because the controlled version disintegrated at once in DI water.

[00184] The two variations of SB were 1% (w/w) and 2.5% (w/w) respectively where other components were fixed, and the amount of solvent used was recalculated accordingly. The controlled versions of BP-SB2.5 and BP-SB1 disintegrated once they encountered DI water, but the irradiated ones survived the water insolubility test (see FIG. 24).

[00185] Lowering the concentration of SB to 1% (w/w) was sufficient to get the nanofibers crosslinked and pass the water insolubility test.

[00186] There were four versions of nGMP, which varied on their irradiation exposure duration.

Table 12: Exposure Duration of nGMP Samples

[00187] The samples nGMP-0 and nGMP-30 did not survive the water solubility test and disintegrated at once in contact with DI water. The samples nGMP-60 and nGMP-120 managed to survive the solubility test but it was noticed that there were bead in those nanofibers (see

FIGS. 26-30).

[00188] It can be said that samples nGMP-0 and nGMP-30 did not survive the water solubility test because of not getting crosslinked to a certain degree to survive the hydrated conditions. It can be concluded that nGMP-60 and nGMP-120 were crosslinked to a certain degree to maintain its structural morphology but apparently not to the same degree as fibers containing GMP.

[00189] The experiments showed that PGU-PUL-GMP can be electrospun and photocrosslinked at varying concentration levels of SB. It was also observed that PGU-PUL can be electrospun and photo-crosslinked provided if the exposure period was increased, it was also seen that addition of GMP did aid in producing bead free nanofibers.

[00190] It should be understood that modifications to the embodiments disclosed herein can be made to meet a particular set of design criteria. For instance, the number of or configuration of components or parameters may be used to meet a particular objective.

[00191] It will be apparent to those skilled in the art that numerous modifications and variations of the described examples and embodiments are possible in light of the above teachings of the disclosure. The disclosed examples and embodiments are presented for purposes of illustration only. Other alternative embodiments may include some or all of the features of the various embodiments disclosed herein. For instance, it is contemplated that a particular feature described, either individually or as part of an embodiment, can be combined with other individually described features, or parts of other embodiments. The elements and acts of the various embodiments described herein can therefore be combined to provide further embodiments. [00192] It is the intent to cover all such modifications and alternative embodiments as may come within the true scope of this invention, which is to be given the full breadth thereof.

Additionally, the disclosure of a range of values is a disclosure of every numerical value within that range, including the end points. Thus, while certain exemplary embodiments of the apparatus and process for biopolymer scaffold formation and/or utilization and methods of making and using the same have been discussed and illustrated herein, it is to be distinctly understood that the invention is not limited thereto but may be otherwise variously embodied and practiced within the scope of the following claims.

Claims

What is claimed is:

1. A method of forming a biopolymer-based tissue scaffold on which stem cells, muscle cells, or cultivated meat are grown, the method comprising: mixing biopolymer components and a solvent to form a polymer mixture, wherein the biopolymer components comprise at least one protein, at least one starch, at least one polysaccharide, and at least one crosslinking agent; spinning the polymer mixture to form nanofibers; and exposing the nanofibers to crosslinking conditions such that the at least one crosslinking agent is activated thereby forming the tissue scaffold.

2. The method of claim 1, further comprising: feeding the tissue scaffold to a cell growth device comprising a cell culture medium, wherein the cell growth device is configured to facilitate the growth of stem cells, muscle cells, or cultivated meat on the tissue scaffold.

3. The method of claim 2, wherein the tissue scaffold is configured to decompose or be consumed during the growth of stem cells, muscle cells, or cultivated meat on the tissue scaffold.

4. The method of claim 1, wherein spinning the polymer mixture to form nanofibers comprises electrospinning.

5. The method of claim 1, wherein the nanofibers are aligned via the spinning.

6. The method of claim 1, wherein the crosslinking conditions comprise ultraviolet (UV) light exposure.

7. The method of claim 1, wherein the protein is whey protein or glycomacropeptide, the starch is octenyl succinylated starch, and the polysaccharide is pullulan.

8. The method of claim 1, wherein the biopolymer components and the solvent meet generally recognized as safe (GRAS) standards.

9. A biopolymer-based tissue scaffold on which stem cells, muscle cells, or cultivated meat are grown, comprising: a plurality of crosslinked unidirectionally aligned nanofibers, the nanofibers comprising: at least one protein, at least one starch, at least one polysaccharide, and at least one crosslinking agent, wherein the protein, the starch, the polysaccharide, and the crosslinking agent meet generally recognized as safe (GRAS) standards.

9. The biopolymer-based tissue scaffold of claim 8, wherein the at least one protein is whey protein or glycomacropeptide.

10. The biopolymer-based tissue scaffold of claim 8, wherein the at least one starch is OS starch.

11. The biopolymer-based tissue scaffold of claim 8, wherein the at least one polysaccharide is pullulan.

12. The biopolymer-based tissue scaffold of claim 8, wherein the protein is whey protein or glycomacropeptide, the starch is octenyl succinylated starch, and the polysaccharide is pullulan.

13. The biopolymer-based tissue scaffold of claim 8, wherein the crosslinking agent is selected from the group consisting of phosphate, sodium hydroxide, sodium benzoate, sodium citrate, and citric acid.

14. The biopolymer-based tissue scaffold of claim 8, further comprising at least one plasticizer.

15. The biopolymer-based tissue scaffold of claim 8, wherein the at least one plasticizer is sorbitol.

16. An apparatus for forming and/or utilizing a biopolymer-based tissue scaffold on which stem cells are growable, muscle cells are growable or cultivated meat is growable, the apparatus comprising: a mixing device positioned to mix biopolymer components and a solvent to form a polymer mixture, wherein the biopolymer components comprise at least one protein, at least one starch, at least one polysaccharide, and at least one crosslinking agent; and a spinner device positioned to spin the polymer mixture formed via the mixing device to form unidirectionally aligned nanofibers.

17. The apparatus of claim 16, wherein the spinning device includes at least one crosslinking device configured to expose the nanofibers to one or more crosslinking conditions such that the at least one crosslinking agent is activatable to crosslink the nanofibers and form the tissue scaffold.

18. The apparatus of claim 16, comprising a crosslinking device positioned to receive the nanofibers and expose the nanofibers to one or more crosslinking conditions for a crosslinking time period such that the at least one crosslinking agent is activatable to crosslink the nanofibers and form the tissue scaffold.

19. The apparatus of claim 17 or claim 18, comprising: a cell growth device positioned to receive stem cells, muscle cells, or cultivated meat and the tissue scaffold for growing the stem cells, muscle cells, or cultivate meat on the tissue scaffold.

20. The apparatus of claim 19, wherein the cell growth device is positioned to receive a cell culture medium to facilitate the growing of the stem cells, muscle cells, or cultivate meat on the tissue scaffold.

21. The cell growth device configured such that the tissue scaffold is consumed or decomposes as the stem cells, muscle cells, or cultivated meat grows.