Method for producing edible crosslinked porous hollow fibers and membranes by pH-induced phase separation and their uses

Abstract

Methods for producing edible crosslinked porous hollow fibers and sheet membranes suitable for producing clean meat products, hollow fibers and sheet membranes produced therefrom, and methods for using the same.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 234,796, filed August 19, 2021, the entire contents of which are hereby incorporated by reference. [Background technology]

[0002] The integrity and pore characteristics of the membrane are paramount for effective use in membrane-based bioreactors. The membrane must be self-supporting to allow for the movement of medium and nutrients through the membrane without disrupting the support structure and to allow for a larger surface area for the cultivation of adherent cells. Furthermore, to produce edible foods, the membrane must be made of generally regarded as safe (GRAS) materials. Still further, creating a membrane that is edible from both a technical standpoint (i.e., non-toxic and digestible) and a practical consumer-acceptable standpoint (i.e., having a texture and mouthfeel acceptable to consumers) has not been achieved in the art. The fabrication of such membranes, whether as flat sheets (e.g., nanoporous membranes) or fibers (e.g., hollow fibers), has been elusive. Summary of the Invention [Problem to be solved by the invention]

[0003] What is needed are high integrity membranes for use in membrane-based bioreactors that are suitable for cell culture and are edible. [Means for solving the problem]

[0004] The present inventors have developed a novel and unobvious method of producing membranes (i.e., membrane films and fibers) by pH-induced or proton-induced phase separation that have the structural integrity necessary for use in bioreactors, e.g., to produce foods for human and animal consumption. The membranes are made using GRAS materials, are self-supporting (i.e., do not collapse under their own weight or easily tear or rupture when handled or exposed to the fluid forces required by the culture conditions in a bioreactor), and are edible, both technically and practically acceptable to consumers.

[0005] In its broadest embodiment, the membranes of the present invention comprise one or more plant or animal proteins, one or more edible polysaccharides, and optionally one or more polysaccharide cross-linkers. The proteins, polysaccharides, and optional cross-linkers are co-mixed and extruded into a forming bath. The forming bath contains one or more ions (i.e., cations or anions) that result in cross-linking of the polysaccharides in the membrane. Furthermore, in some aspects of the present invention, a pH change in the forming bath results in phase separation-induced membrane formation.

[0006] The inventors have empirically learned that cross-linking of polysaccharides in membranes is often insufficient to ensure sufficient membrane integrity, especially under cell culture conditions (see examples). The inventors have further devised a method for imparting the necessary integrity to membranes. After forming the membrane in a forming bath, the membrane is exposed to an energy source, such as heat or irradiation. Without being limited by theory, the inventors believe that exposure to the energy source results in cross-linking of the polysaccharides and / or proteins in the membrane, thereby providing the necessary integrity to the membrane while maintaining the quality required for consumer acceptance.

[0007] Also, with regard to providing membranes for use in food products, prior art chemical cross-linking techniques often use toxic compounds that need to be avoided for this application, or prior art polymer modification techniques can be used to increase cross-linking sites but may face regulatory challenges.

[0008] In another embodiment, the membranes of the invention can be coated or otherwise modified with one or more agents, for example, to enhance cell adhesion and proliferation. The membranes can be coated before or after exposure to heat or irradiation.

[0009] After formation, exposure to an energy source, and optional coating, the membrane may be partially dried and / or stored, or may be subjected to further processing (e.g., by cutting to size and incorporating into a bioreactor cartridge or capsule).

[0010] Thus, the present invention relates to edible 3D nano- and microporous structures for use in membrane bioreactors (film or fiber-based), for example, to produce structured clean meat products. Culture medium passes through the membrane, growing cells on one or both sides of the membrane. Prior art hollow fiber membrane bioreactors exist for adherent cells, but require trypsin or other chemical / enzymatic processes to remove the cells. Such processes are too expensive for commercial-scale clean meat production and, furthermore, disrupt any tissue-like structure. Therefore, the present invention contemplates membranes that can be consumed with meat cells used to produce cultivated meat products. The present invention further contemplates at least partially dissolvable membranes. This aspect may be required, for example, to achieve a desired texture in the final structured meat product.

[0011] Food-based materials for adherent cell scaffolds have been described in the art. However, these material formats are not suitable for (hollow fiber) membrane bioreactors. These material formats are generally non-porous films, fiber-based mats (such as electrospinning or rotary jet spinning), or sponges (usually derived from freeze-drying, extrusion, and / or foaming processes).

[0012] Membrane bioreactors require very specific pore sizes with specific membrane geometries. Hollow fiber bioreactors (HFBRs) typically have pore sizes between 5 KDa and 0.1 µm, depending on the cell type, bioreactor design, and bioprocess.

[0013] Although the present invention contemplates hollow fibers, the general concepts of the present invention can also be applied to flat sheet (film-like) membranes. Sheet membranes are formed, for example, by casting a polymer onto a sacrificial surface, which then enters a bath designed to solidify the polymer. Hollow fibers are formed by spinning through a nozzle / spinneret into the bath. When producing hollow fibers, the bore fluid must also be precisely determined and controlled, as known to those skilled in the art. Further details regarding the production of sheet membranes and hollow fibers are provided below.

[0014] The method we have devised for making the membranes of the present invention utilizes multiple steps. Due to human nutritional and cell adhesion considerations, a high protein content is preferred. However, the molecular weight of the protein is generally too low to provide sufficient chain entanglement or structural integrity for fiber-forming properties. For this reason, an additional "carrier" polymer is added to the membrane polymer (i.e., the dope solution). As taught herein, the carrier polymer is a polysaccharide selected from one or more of, for example, alginate, cellulose, pectin, chitin, chitosan, gellan gum, xanthan gum, arabinoxylan, glucomannan, and others known to those skilled in the art.

[0015] The proteins and polysaccharides are mixed in a blend of GRAS solvents. Once one or more proteins and one or more polysaccharides are selected and the mixture is formed, they are solidified in a solidification (forming) bath to instantly or nearly instantly fix the dimensions of the cast membrane. In one embodiment, it is contemplated that the bath contains multivalent cations, such as Ca2+, Mg2+, etc. Specifically, the inventors have demonstrated that Ca2+ instantly crosslinks alginate, pectin, or other polysaccharides in the membrane. This fixes the dimensions of the fiber / sheet, achieving the desired three-dimensional target.

[0016] However, at this point, the proteins are not cross-linked; only the polysaccharides are ionically cross-linked. As described in the literature and observed in practice, ionically cross-linked polysaccharides can dissociate in cell culture media. Therefore, when used in cell culture, an additional cross-linking step is required to further enhance membrane stability and ensure membrane integrity. Because covalent cross-linking requires harsh chemicals, this approach is not preferred for edible products. The innovation of the present invention is the use of physical cross-links, which are generated via one or more energy sources, such as heat, gamma, electron beam, beta, X-ray, or UV. These are used in the food industry to kill or weaken potential pathogens, and therefore will be understood by those skilled in the art to be safe for use in food.

[0017] It is further contemplated by the present invention that an alternative approach is to use cross-linking agents for proteins that are already approved for food use, such as transglutaminase. It is still further contemplated that in addition to or instead of cross-linking the protein, the polysaccharide can be modified prior to creating the mixture to increase the potential cross-linking sites on the polymer.

[0018] The present invention further contemplates other approaches, such as dissolving the protein directly in an alcohol / water blend and solidifying the film in an acid bath. The present invention still further contemplates dissolving a plant protein isolate in an alkaline solution and then solidifying it with an organic coagulant, such as alcohol or a neutralized acid / caustic solution. For example, dissolving chitosan in 5% acetic acid and extruding it into a bath with a higher pH solidifies the polymer into the shape of fibers.

[0019] Chitosan can also be dissolved in a slightly acidic bath (about 5% acetic acid, citric acid, etc.) and then deposited / spun into a bath containing a concentration of tripolyphosphate / sodium tripolyphosphate (TPP) that preserves and / or maintains the porosity of the solidified chitosan. The bore fluid can also contain solutions similar to the bath solution.

[0020] Chemical or enzymatic cross-linking agents can also be added to the bore fluid (the fluid used in the nozzle bore when forming solid or hollow fibers; bore fluids are known to those skilled in the art) and / or the forming bath to aid in cross-linking of the plant proteins present in the polysaccharide and protein blend. Examples of cross-linking agents that can optionally be included in the bath or bore fluid are transglutaminase, tripolyphosphate, genipin (genipin is a chemical compound found in Genipa americana fruit extract), or other oxidizing enzymes known to those skilled in the art.

[0021] Another aspect of the present invention is that the dope solution (i.e., protein, polysaccharide mixture) can be impregnated with insoluble (at least in the solvent system used) fibers. These fibers can be, for example, bacterial nanocellulose, nanocellulose, or other suitable fibers. These fibers can serve two functions: first, mechanical reinforcement, resulting in increased "toughness" as defined by the stress-strain curve. The second function of these fibers would be to promote myotube alignment. During extrusion, these fibers naturally align with the hollow fibers, and the fibers at the surface of the hollow fiber membrane promote the alignment of cells grown there.

[0022] Another aspect of the present invention is the geometry and topography of the fiber itself. Preferably, the fiber has an outer diameter of about 300 to about 700 microns. Striations or grooves running parallel, substantially parallel, or essentially parallel to the fiber length can be a desired structural feature that can be incorporated into the fibers produced by the methods of the present invention. Striations or grooves along the fiber can be incorporated into the spinning process through dope solution blending and mixing, through nozzle geometry, or through turbulence in the forming bath by methods known to those skilled in the art.

[0023] It is further contemplated that another step in this method may be to increase cell adhesion on the membranes and fibers by using a desired chemical process or compound to modify the surface of the membrane or fiber or coat the membrane or fiber. Examples of suitable processes and compounds include, but are not limited to, plasma treatment, the addition of cell-binding sites through the addition of proteins, including, but not limited to, fibronectin, fibrinogen, laminin, collagen, gelatin, etc., or short peptide sequences isolated from these proteins, including, but not limited to, RGD, YIGSR, IKVAV, DGEA, PHRSN, PRARI, etc.

[0024] Coatings are contemplated that can be applied to target applications beyond cell adhesion. Heparin can increase the concentration of growth factors on the fiber surface. Compounds that aid in cell differentiation can also be applied. For example, a coating with a high lipid content can promote differentiation of appropriate cells into adipocytes.

[0025] Coatings directed to non-biological (i.e., not directly related to the growth and maintenance of desired cells) outcomes are also contemplated. Preservatives and / or antibiotics can be used to prevent spoilage or maintain a sterile environment before and during culture. Dyes, pigments, beta-carotene, and the like can be applied as coatings or directly to the fiber dope solution to achieve a desired appearance. Similarly, flavors and fragrances can be applied as coatings or directly to the fiber dope solution to achieve a desired flavor profile. Plasticizers (e.g., sugar alcohols such as sorbitol and glycerol) can also be applied as coatings or directly to the dope solution or bore fluid. Plasticizers enhance handling, minimize pore collapse, extend shelf life, and alter mouthfeel.

[0026] The invention also includes membranes (hollow fiber and sheet membranes) made by the methods of the invention.

[0027] The present invention contemplates methods for making edible crosslinked porous hollow fibers and membrane sheets, the methods comprising the steps of: a) providing i) one or more edible proteins, ii) one or more edible polysaccharides, iii) one or more solvents, and iv) a forming bath, wherein the one or more solvents or forming bath also contain one or more multivalent cations or anions; b) co-mixing the one or more edible proteins and the one or more edible polysaccharides in the one or more solvents to form a mixture; c) extruding the mixture into the forming bath to form an extruded hollow fiber, or casting the mixture onto the bath to form a membrane sheet; and d) exposing the extruded hollow fiber or membrane sheet to an energy source selected from one or more of heat and irradiation sufficient to at least partially crosslink the one or more proteins to form an edible crosslinked porous hollow fiber.

[0028] The method further contemplates that the one or more proteins are selected from the group consisting of pea, soy, wheat, pumpkin, rice, brown rice, sunflower, canola, chickpea, lentil, mung bean, navy bean, corn, oat, potato, quinoa, sorghum, and peanut.

[0029] The method further contemplates that the one or more polysaccharides are selected from the group consisting of agar, chitosan, chitin, alginate, sodium alginate, cellulose, hydroxypropyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, gellan gum, xanthan gum, pectin, tapioca, guar gum, and bean gum.

[0030] The method further contemplates that the one or more solvents are selected from the group consisting of water, acetic acid, citric acid, lactic acid, phosphoric acid, malic acid, tartaric acid, sodium hydroxide, ethanol, glycerin, and propylene glycol.

[0031] The method further contemplates that the ions are selected from the group consisting of Ca2+, Mg2+, Fe3+, Zn2+, tripolyphosphate, and trisodium citrate, and the selected ions are capable of at least allowing partial cross-linking of one or more polysaccharides.

[0032] The method further contemplates that the heat is applied at about 120°C to about 140°C, under pressure of about 0 PSI to about 20 PSI gauge, and at a relative humidity of about 50% to about 100%, for about 2 to about 60 minutes, or the fibers are immersed in a water bath at about 60°C to about 100°C at atmospheric conditions.

[0033] The method further contemplates that the irradiation is selected from the group consisting of electron beam, UV light, and gamma irradiation, that the irradiation is applied during or after the process, and that the irradiation is from about 1 to about 100 kGy or from about 10 to about 50 kGy.

[0034] The method further contemplates that the hollow fiber or membrane sheet has a porosity of from about 1% to about 90%, or from about 50% to about 80%.

[0035] It is further contemplated that the method further comprises the step of coating the edible crosslinked porous hollow fibers with a coating to enhance cell adhesion.

[0036] The method further contemplates that the coating is selected from one or more of fibronectin, fibrinogen, laminin, collagen, gelatin, or short peptide sequences isolated from these proteins.

[0037] The method further contemplates that the short peptide sequence is selected from the group consisting of RGD, YIGSR, IKVAV, DGEA, PHRSN, and PRARI.

[0038] The method further contemplates that the method further comprises modifying the outer surface of the edible crosslinked porous hollow fiber to enhance cell adhesion, wherein the surface modification is selected from one or more of plasma, corona, abrasion, etching, ablation, or sputter coating.

[0039] The method further contemplates that the protein is powdered or pulverized prior to dissolving in the solvent.

[0040] The method further contemplates that the protein is at least 70%, 80%, 90%, 95%, 98%, 99%, 99.9% pure.

[0041] The method further contemplates that the polysaccharide is at least 70%, 80%, 90%, 95%, 98%, 99%, 99.9% pure.

[0042] The method further contemplates that the ratio of protein to polysaccharide in the mixture is from about 10:1 to about 1:10, or that the ratio of protein to polysaccharide in the mixture is from about 4:1 to about 1:4. The method further contemplates that the ratio of protein to polysaccharide in the mixture is about 1:1. The method further contemplates that the ratio of protein to polysaccharide in the mixture is about 1:7 or about 7:1. In some cases, the solids ratio between protein and polysaccharide is 100:1 or about 1:100, or is exclusively 100% protein isolate.

[0043] The method further contemplates that the forming bath may comprise RO (reverse osmosis) water with dissolved calcium chloride, for example, at or about a 15 g / L concentration, although the desired concentration may be from about 4 g / L to about 20 g / L, from about 12 g / L to about 18 g / L, or from about 14 g / L to about 16 g / L. In a continuous process, the forming bath has a feed and bleed system where prepared 15 g / L calcium chloride is fed to the side of the bath and the bath is bled at the same rate.

[0044] The method further contemplates that the forming bath comprises RO water including one or more of calcium, zinc, magnesium, iron, and potassium in combination with i) one or more of water, acetic acid, citric acid, lactic acid, phosphoric acid, malic acid, tartaric acid, or ii) one or more of sodium hydroxide and potassium hydroxide.

[0045] The present invention contemplates a method for producing edible crosslinked porous hollow fibers and membrane sheets, comprising: a) providing i) one or more edible proteins, ii) one or more edible polysaccharides, iii) one or more solvents, and iv) a forming bath, the forming bath being primarily water and further comprising one or more of calcium chloride, zinc chloride, magnesium ions, potassium in combination with 1) one or more of acetic acid, citric acid, lactic acid, phosphoric acid, malic acid, tartaric acid, or other suitable acids, or 2) one or more of sodium hydroxide and potassium hydroxide, or other suitable bases; b) co-mixing the one or more edible proteins and the one or more edible polysaccharides in the one or more solvents to form a mixture; c) extruding the mixture into the forming bath to form an extruded hollow fiber or casting the mixture onto the bath to form a membrane sheet; and d) exposing the extruded hollow fiber or membrane sheet to an energy source selected from one or more of heat and irradiation sufficient to at least partially crosslink the one or more proteins to form the edible crosslinked porous hollow fiber. In this embodiment, the forming bath is supplemented with ions.

[0046] The present method further relates to and contemplates any hollow fiber or sheet membrane (ie, membrane sheet) made by the method of the present invention.

[0047] The invention further relates to clean meat, structured meat, cultured meat, lab-grown meat, cultivated meat, cell-based meat, etc. produced using the membrane or the invention, and methods of making these meats.

[0048] It is contemplated that the present invention relates to a method of making an edible crosslinked porous hollow fiber or sheet membrane, comprising the steps of: a) providing i) one or more edible proteins; ii) one or more solvents; and iii) a forming bath; wherein the one or more solvents or forming bath also contain one or more multivalent cations or anions or a buffer; b) co-mixing the one or more edible proteins in the one or more solvents to form a mixture; c) extruding the mixture into the forming bath to form an extruded hollow fiber or casting the mixture into the forming bath to form a sheet membrane; and d) exposing the extruded hollow fiber or sheet membrane to an energy source selected from one or more of heat and irradiation sufficient to at least partially crosslink the one or more proteins to form an edible crosslinked porous hollow fiber or sheet membrane.

[0049] It is further contemplated that the methods of the present invention involve providing one or more edible polysaccharides and co-mixing the one or more polysaccharides with one or more edible proteins in one or more solvents.

[0050] It is further contemplated that the methods of the present invention involve providing a plasticizer and co-mixing the plasticizer with one or more edible proteins in one or more solvents.

[0051] It is further contemplated that the methods of the present invention relate to wherein the one or more proteins are selected from the group consisting of pea, soy, wheat, pumpkin, rice, brown rice, sunflower, canola, chickpea, lentil, mung bean, navy bean, corn, oat, potato, quinoa, sorghum, and peanut.

[0052] It is further contemplated that the methods of the present invention relate to wherein the one or more polysaccharides are selected from the group consisting of agar, chitosan, chitin, alginate, sodium alginate, cellulose, hydroxypropyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, gellan gum, xanthan gum, pectin, tapioca, guar gum, and bean gum.

[0053] It is further contemplated that the methods of the present invention relate to wherein the one or more solvents are selected from the group consisting of water, acetic acid, citric acid, lactic acid, phosphoric acid, malic acid, tartaric acid, sodium hydroxide, ethanol, glycerin, and propylene glycol.

[0054] It is further contemplated that the method of the present invention relates to a forming bath comprising one or more of calcium, zinc, magnesium, iron, and potassium in combination with one or more of 1) water, acetic acid, citric acid, lactic acid, phosphoric acid, malic acid, tartaric acid, or 2) sodium hydroxide and potassium hydroxide.

[0055] It is further contemplated that the method of the present invention relates to the ion being selected from the group consisting of Ca2+, Mg2+, Fe3+, Zn2+, tripolyphosphate, and trisodium citrate, and the selected ion being capable of at least allowing partial cross-linking of one or more polysaccharides.

[0056] It is further contemplated that the method of the present invention relates to the mixture of step b) being heated.

[0057] It is further contemplated that the method of the present invention involves heating the formed hollow fiber or sheet membrane at about 70°C to about 140°C or about 120°C to 140°C under pressure of about 0 PSI to about 20 PSI gauge and a relative humidity of about 50% to about 100% for about 2 to about 60 minutes, or immersing the hollow fiber or sheet membrane in a water bath at about 60°C to about 100°C at atmospheric conditions.

[0058] It is further contemplated that the methods of the present invention involve co-mixing carried out at about 0°C to about 90°C.

[0059] It is further contemplated that the method of the present invention relates to the mixture having a pH of about 10 to about 13 and the blended bath having a pH of about 3 to about 5.

[0060] It is further contemplated that the methods of the present invention involve neutralizing the membrane after formation to a pH of about 6.8 to about 7.8.

[0061] It is further contemplated that the methods of the present invention involve neutralizing the membrane after formation to a pH of about 7.3 to about 7.5.

[0062] It is further contemplated that the methods of the present invention relate to wherein the irradiation is selected from the group consisting of electron beam, UV light, and gamma irradiation.

[0063] It is further contemplated that the methods of the invention relate to irradiation being applied during or after the process. It is further contemplated that the methods of the invention relate to irradiation being from about 1 to about 100 kGy or from about 10 to about 50 kGy.

[0064] It is further contemplated that the methods of the present invention relate to hollow fiber or sheet membranes having a porosity of from about 1% to about 90%, from about 25% to about 75%, or from about 40% to about 60%.

[0065] It is further contemplated that the methods of the present invention involve hollow fiber or sheet membranes having a porosity of from about 50% to about 80%.

[0066] It is further contemplated that the method further comprises the step of coating the edible crosslinked porous hollow fiber or sheet membrane with a coating to enhance cell adhesion.

[0067] It is further contemplated that the methods of the present invention relate to wherein the coating is selected from one or more of fibronectin, fibrinogen, laminin, collagen, gelatin, or short peptide sequences isolated from these proteins.

[0068] It is further contemplated that the methods of the present invention relate to the short peptide sequences being one or more selected from the group consisting of RGD, YIGSR, IKVAV, DGEA, PHRSN and PRARI.

[0069] It is further contemplated that the methods of the invention relate to modifying the outer surface of edible crosslinked porous hollow fibers to enhance cell adhesion. It is further contemplated that the invention relates to methods further comprising coating the edible crosslinked porous hollow fiber or sheet membrane with a plasticizer. It is further contemplated that the surface modification is selected from one or more of plasma, corona, abrasion, etching, ablation, or sputter coating.

[0070] It is further contemplated that the methods of the present invention involve the protein being powdered or pulverized prior to dissolving in the solvent.

[0071] It is further contemplated that the methods of the present invention relate to proteins that are at least 70%, 80%, 90%, 95%, 98%, 99%, 99.9% pure.

[0072] It is further contemplated that the methods of the present invention relate to the polysaccharide being at least 70%, 80%, 90%, 95%, 98%, 99%, 99.9% pure.

[0073] It is further contemplated that the methods of the invention relate to a ratio of protein to polysaccharide (protein:polysaccharide) in the mixture of about 10:1 to about 1:10, or about 1:99 to about 99:1, 98:2, 97:3, 96:4, 95:5, or 90:10. It is further contemplated that the invention relates to a ratio of protein to polysaccharide in the mixture of about 4:1 to 1:4. It is further contemplated that the invention relates to a ratio of protein to polysaccharide in the mixture of about 1:1 or 7:1.

[0074] It is further contemplated that the invention relates to a forming bath comprising one or more of calcium, zinc, magnesium, iron, and potassium in combination with one or more of i) water, acetic acid, citric acid, lactic acid, phosphoric acid, malic acid, tartaric acid, or ii) sodium hydroxide and potassium hydroxide.

[0075] It is further contemplated that the present invention relates to hollow fiber or sheet membranes made by any of the methods of the present invention.

[0076] It is contemplated that the invention relates to a method of making an edible crosslinked porous hollow fiber or sheet membrane, the method comprising the steps of: a) providing i) one or more edible proteins, ii) one or more edible polysaccharides, iii) one or more solvents, and iv) a forming bath, the forming bath comprising one or more of calcium, zinc, magnesium, iron, and potassium in combination with 1) one or more of water, acetic acid, citric acid, lactic acid, phosphoric acid, malic acid, tartaric acid, or 2) one or more of sodium hydroxide and potassium hydroxide; b) co-mixing the one or more edible proteins and the one or more edible polysaccharides in the one or more solvents to form a mixture; c) extruding the mixture into the forming bath to form an extruded hollow fiber or casting the mixture to form a sheet membrane; and d) exposing the extruded hollow fiber or sheet membrane to an energy source selected from one or more of heat and irradiation sufficient to at least partially crosslink the one or more proteins to form an edible crosslinked porous hollow fiber.

[0077] It is contemplated that the present invention relates to a method of making hollow fiber or sheet membranes in which one or more proteins, one or more polysaccharides, one or more solvents, plasticizers, and / or one or more components of the forming bath are generally recognized as safe (GRAS) by the U.S. Food and Drug Administration (FDA).

[0078] It is further contemplated that the present invention relates to the resulting membrane or hollow fiber made by any of the methods of the present invention undergoing 10-50% water exchange with glycerol for drying, wherein said drying does not result in pore collapse. [Brief explanation of the drawings]

[0079] [Figure 1] 1 is a schematic diagram of one method used to manufacture the membranes and hollow fibers of the present invention. [Figure 2] FIG. 2 is a schematic diagram of another method used to produce the membranes and hollow fibers of the present invention. [Figure 3A] FIG. 1 shows a hollow fiber membrane made by the method of the present invention. [Figure 3B] FIG. 1 shows a hollow fiber membrane made by the method of the present invention. [Figure 4A] FIG. 1 shows a scanning electron micrograph (SEM) of fibers produced by the method of the present invention. [Figure 4B] FIG. 1 shows a scanning electron micrograph (SEM) of fibers produced by the method of the present invention. [Figure 4C] Figures 1A and 1B show scanning electron micrographs (SEMs) of fibers produced by the method of the present invention. Figure 1A shows that the surface pores of the whey protein and alginate blend are found to be approximately 20 nm or about 1000 kDa. The image also shows that the streaks from the process are parallel to the length of the fiber. Figure 1B shows the surface pores of the pumpkin protein isolate and alginate blend, which have surface pores of approximately 100 nm or less. Figure 1C shows a low-resolution image of fibers made with pumpkin protein isolate. [Figure 5A] FIG. 1 shows a fiber produced by the method of the present invention. [Figure 5B] Figures 1A and 1B show fibers produced by the method of the invention. Hollow fibers of the invention can easily support the weight required in a bioreactor. (A) The fiber shown is 2 meters long. (B) Fibers produced by the method of the invention can support at least 9 grams. [Figure 6] Figure 1 shows mung bean cast film from urea and sodium hydroxide solution. The image shows mung bean dope solution cast onto glass via doctor blade technique. It can be seen that the dope solution is clear before solidification. [Figure 7] Figure 1 shows viscosity using a Brookfield (Middleboro, MA) viscometer equipped with an S64 spindle. Viscosity of 2% alginate and 10% protein isolate is shown. Each mixture had its pH adjusted to pH 11 before measurement. [Figure 8] Figure 1 shows a simple design plot in terms of quantity. This is a design of experiment using Minitab (State University of Pennsylvania) looking at water containing urea, ethanol, and sodium hydroxide. [Figure 9] Figure 9 shows the temperature sweep of 15% zein in the solvent blend form of Figure 8. This shows that solvent systems containing as little as 12.5% ​​ethanol can dissolve zein. [Figure 10] FIG. 9 shows that the solvent conditions of FIG. 8 can be used to alter the gelling properties of agarose when compared to the same agarose in water. [Figure 11] FIG. 9 shows that zein and agarose can be blended using the given solvent system of FIG. 8 within a given mixing temperature range, particularly above 40° C., without solidifying either component. [Figure 12A] Figure 1 shows images of the zein film production process consisting of a film casting step (left). [Figure 12B] FIG. 1 shows images of the zein film production process consisting of a coagulation step (right) in acetate buffer (0.2 M, pH 4.5). [Figure 13]FIG. 1 shows the cross-linking process of mung bean alginate films using a hot glycerol bath set at 120° C. for 1 hour. [Figure 14A] FIG. 10 is a graph showing the elastic modulus of the membrane (left). [Figure 14B] FIG. 10 shows a graph showing membrane strain (right). [Figure 15A] Figure 1 shows the elastic modulus of various tissues (left). [Figure 15B] FIG. 1 shows the elastic modulus of an exemplary membrane material of the present invention (right). [Figure 16] This figure shows images (1-6) of membranes fabricated according to different fabrication protocols to investigate and verify each fabrication process. AC represents "0.2 M acetate bath at pH 4.5," H represents 0.1 M HEPES buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) bath at pH 7.4, G represents "glycerol bath," HG represents "hot glycerol bath," HW represents "hot water" (autoclave), and 0 represents "no process performed." [Figure 17] 17A and 17B show the membrane elastic modulus (A: left) and the membrane breaking strain (B: right), respectively. Sample 6 was prepared according to protocol AC-0-G-HG, 5 according to protocol AC-0-0-HG, and 2 according to protocol AC-0-0-HW. [Figure 18] Figures 18A-C show the change in modulus (A, left), strain (B, center), and ultimate stress (C, right) of heat-treated (glycerol-based protocol) mung bean membranes with increasing coagulation time in an acetic acid bath. The figures show the mechanical properties of membranes coagulated for 10 min up to 3 h. [Figure 19] Figures 19A-C show the changes in modulus (A, left), strain (B, center), and final stress (C, right) with increasing glycerol-based heat treatment time for mung bean membranes. Glycerol-based heat treatment was investigated by keeping constant the duration of both the coagulation bath (10 min) and the water-glycerol exchange (10 min) and by varying the duration of the heat treatment after reaching a final temperature of 120 °C. [Figure 20]Figure 1 shows a rheological study of the heat treatment of mung bean films with glycerol.Figure 2 shows the variation of tangent delta (δ) with respect to the temperature gradient. [Figure 21] 21A-C show the changes in (A) modulus, (B) strain, and (C) ultimate stress with increasing heat treatment time based on glycerol for mung bean membranes. [Figure 22] Figure 1 shows the elastic modulus values ​​of alginate and gluten protein blends containing wheat gluten, mung bean, and zein when incubated in cell culture medium at 37° C. Mechanical tensile measurements were performed before and after 3, 10, and 21 days of incubation. [Figure 23] Figure 1 shows the strain at break values ​​of alginate and gluten protein blends containing wheat gluten, mung bean, and zein when incubated in cell culture medium at 37° C. Mechanical tensile measurements were performed before and after 3, 10, and 21 days of incubation. [Figure 24] Figure 1 shows the membrane surface area of ​​alginate protein blends containing wheat gluten, mung bean, and zein when incubated in cell culture medium at 37°C. Measurements were taken before and after 3, 10, and 21 days of incubation. [Figure 25] Figure 1 shows the membrane surface area of ​​agarose-protein blends containing wheat gluten, mung bean, and zein when incubated in cell culture medium at 37°C. Measurements were taken before and after 3, 10, and 21 days of incubation. [Figure 26] Figures 26A and B show a comparison of (A) modulus and (B) strain between brown rice-alginate blends prepared with and without transglutaminase cross-linking before and after incubation in cell culture medium at 37°C for 3, 10, and 21 days. [Figure 27]27A-F show the elastic modulus (A and B, left), breaking strain (B and E, center), and surface area (C and F, right) of protein films containing soy protein isolate (A-C, top) and mung bean (D-F, bottom). Measurements were taken before and after 3, 10, and 21 days of incubation in cell culture medium at 37°C for soy protein isolate, and before and after 5, 12, and 30 days of incubation in cell culture medium at 37°C for mung bean. [Figure 28] FIG. 1 shows scanning electron microscope images of soy protein isolate membrane surface (top) and cross section (bottom). [Figure 29] FIG. 1 shows scanning electron microscope images of mung bean protein isolate membrane surface (top) and cross section (bottom). [Figure 30] FIG. 1 shows scanning electron microscope images of zein protein isolate membrane surface (top) and cross section (bottom) and zein protein isolate & agarose membrane surface (top) and cross section (bottom). [Figure 31] FIG. 1 shows scanning electron microscope images of the surface and cross-section of zein alginate (left) and pea protein-k-carrageenan (right) films. [Figure 32] FIG. 1 shows scanning electron microscope images of the surface and cross-section of mungbean-agarose (left) and soybean-alginate (right) membranes. [Figure 33] FIG. 1 shows scanning electron microscopy of the cross section (top) and surface (bottom) of mungbean-alginate hollow fibers. [Figure 34] Figure 1 shows fluorescent cell adhesion and proliferation studies performed on zein, soybean, and mungbean TG-crosslinked mungbean membranes using the C2C12 cell line. Live (green) / dead (red) assays were performed after a 48-hour proliferation period. Micrographs show little red staining, indicating that nearly all cells are viable. [Figure 35]Figure 1 shows cell fluorescence adhesion and proliferation tests performed on mung bean membranes and chitosan membranes coated with fibronectin, collagen, and chitosan using the C2C12 cell line. Live (green) / dead (red) assays were performed after a 48-hour proliferation period. Micrographs show little red staining, indicating that nearly all cells are viable. [Figure 36] Figure 1 shows fluorescent cell adhesion and proliferation studies performed on heat-treated and non-heat-treated soybean-alginate, peanut-alginate, and zein-agarose membranes using the C2C12 cell line. Live (green) / dead (red) assays were performed after a 48-hour growth period. Micrographs show little red staining, indicating that nearly all cells are viable. [Figure 37] Figure 1 shows fluorescent cell adhesion and proliferation tests performed on soybean, fibronectin, and collagen-coated mung bean and chitosan membranes using the QM7 cell line. Live (green) / dead (red) assays were performed after a 48-hour growth period. Micrographs show little red staining, indicating that nearly all cells are viable. [Figure 38] FIG. 1 shows the effect of drying and rehydration on alginate:mungbean-based films. DETAILED DESCRIPTION OF THE INVENTION

[0080] structured meat products The present invention contemplates, for example, but not limited to, edible films comprising hollow fibers of suitable integrity for use in a bioreactor to produce structured clean meat, methods for producing structured clean meat using the same, and structured clean meat produced using the hollow fibers of the invention. Clean meat (also known in the art as "cultured meat" or "lab-grown meat") is defined in the art as meat or meat-like products (collectively referred to herein as "clean meat" or "clean meat products") that are grown from cells in a laboratory, factory, or other production facility suitable for the large-scale cultivation of cells.

[0081] A "structured meat product," "structured clean meat product," "structured cultured meat," or "structured cultured meat product" is a meat product or clean meat product that has a texture and structure similar to or reminiscent of natural meat of animal origin, such as natural meat of animal origin. The structured meat products of the present invention have a texture and structure similar to natural meat in 1) texture and appearance, 2) handleability when prepared for cooking and consumption (e.g., when sliced, ground, cooked, etc.), and 3) mouthfeel when consumed by humans. The materials and methods of the present invention, when used to make structured clean meat, achieve at least one of these criteria, two of these criteria, or all three of these criteria. The prior art is unable to produce structured meat products that adequately meet any of these criteria.

[0082] The structured meat products of the present invention meet these criteria by culturing appropriate cells (also described below) in a bioreactor (described below) containing the hollow fibers of the present invention. The hollow fibers of the present invention provide the final structured clean meat product with structure and texture that, at least in large part, provides the product with the desired appearance, handling, and mouthfeel. Additionally, the hollow fibers of the present invention help provide a suitable environment for cell growth into the structured clean meat product. In this regard, the hollow fibers of the present invention provide a suitable surface for at least the attachment of cultured cells, the elongation of the cells into morphologies resembling muscle cells or myocyte-like cells (i.e., substantially similar in structure and appearance to muscle cells), and the formation of muscle cells into myotubes or myotube-like structures (i.e., substantially similar in structure and appearance to myotubes).

[0083] Preparation of the membrane of the present invention For purposes of this invention, the term "membrane" or "membranes" is understood to refer to any porous membrane structure produced by the method of the invention, including, but not limited to, hollow fiber membranes and sheet (i.e., flat) membranes. Unless specifically indicated otherwise, references to "membrane," "hollow fiber," "hollow fiber membrane," and "sheet membrane" are understood to include any membrane structure produced by the method of the invention, regardless of shape, form, or appearance.

[0084] An exemplary manufacturing method is shown in schematic form in FIGS.

[0085] It is contemplated that the edible and / or dissolvable hollow fiber and sheet membranes of the present invention may be made from one or more of hydrocolloids (i.e., polysaccharides such as xanthan, methylcellulose, alginate, agar, pectin, gelatin, carrageenan, cellulose / gellan / guar / tara / bean / other gums, etc.), proteins (e.g., polypeptides, peptides, glycoproteins, and amino acids; e.g., various starches (corn / potato / rice / wheat / sorghum), plant isolates (e.g., soybean / zein / casein / wheat / mungbean proteins), lipids (e.g., free fatty acids, triglycerides, natural waxes, and phospholipids), alcohols (e.g., polyalcohols), carbohydrates, and other natural substances, such as alginate. Additionally, other materials that support cell attachment and cell growth may be added to the hollow fibers. It is contemplated that hollow fiber additives or coatings may be added to or coated onto the hollow fibers. For example, it is contemplated that the hollow fiber additive or coating may be one or more of the proteins, hydrogels, or other coatings known to those skilled in the art, including extracellular matrix (ECM) components and extracts isolated from plants or synthesized from simpler substances, poly-D-lysine, laminin, collagen (e.g., collagen I and collagen IV), gelatin, fibronectin, plant-based ECM materials, collagen-like, fibronectin-like, and laminin-like materials. The overall result is that the fibers of the present invention impart texture and structure to meat and meat products, giving the structured clean meat products produced by the present invention a texture, appearance, handling, and mouthfeel similar to that of actual meat.

[0086] The present inventors have recognized that soybean and mungbean protein isolates impart some of the desired properties to films produced by the methods of the present invention. The present inventors have also recognized that both soybean (Glycine max) and mungbean (Vigna radiata) are derived from the same taxonomic family, Fabaceae, which is related to legumes (i.e., peas or beans). Doyle, JJ, Leguminosae, Encyclopedia of Genetics, 2001, 1081-1085. While the present invention is not limited by theory, it is believed that other members of this family, particularly the millettioids and phaseoloids, including the genera Glycine and Vigna, behave substantially similarly to soybean and mungbean protein isolates. See Figure 39.

[0087] More specifically, hollow fibers of the present invention may comprise one or more of cellulose, chitosan, collagen, zein, alginate, agar, inulin, gluten, pectin, legume proteins, methylcellulose, gelatin, tapioca, xanthan / guar / tara / bean / other gums, proteins (e.g., polypeptides, peptides, glycoproteins, and amino acids, including, but not limited to, various forms of corn / potato / rice / wheat / sorghum starch, plant isolates, and soy / zein / casein / wheat proteins, all of which are known to those skilled in the art), lipids (e.g., free fatty acids, triglycerides, natural waxes, and phospholipids). Cellulosic polymers may include cellulose acetate butyrate, cellulose propionate, ethylcellulose, methylcellulose, nitrocellulose, and the like. More specifically, hollow fibers of the present invention may comprise a mixture of one or more legume proteins and a hydrocolloid.

[0088] In some embodiments, it is contemplated that the hollow fibers of the present invention are edible, dissolvable, or edible and dissolvable. In other words, the fibers can be edible, dissolvable, or both. Furthermore, for dissolvable fibers, there can be a range of solubilities. For example, some fibers can be readily dissolvable upon exposure to an appropriate solvent (e.g., a non-toxic solvent generally deemed safe by the U.S. Food and Drug Administration (FDA) or other organizations recognized as competent to evaluate the safety of consumable materials). Other fibers can be less readily dissolvable. In this regard, less readily dissolvable fibers can be partially dissolved after the cultured cells reach a required level of confluency, thereby leaving enough fiber to provide the structured clean meat of the present invention with the desired mouthfeel and texture, but without excess fiber that could cause the structured clean meat product of the present invention to appear indestructible or chewy. Dissolvable hollow fiber components are known to those skilled in the art. For example, alginate can be dissolved upon exposure to a Ca chelator. In some embodiments of the present invention, it is contemplated that the hollow fibers of the present invention comprise an amount of alginate that renders the fibers partially soluble, and / or that a certain percentage of the fibers in a device comprising hollow fibers of the present invention comprise alginate.

[0089] In some embodiments of the present invention, one or more cross-linking agents are contemplated for use in the hollow fibers of the present invention. Cross-linking agents, as the name implies, bond one or more of the other components of the hollow fiber to strengthen the fiber. In some embodiments of the present invention, the cross-linking agent may be a soluble component or one of the soluble components of the hollow fiber of the present invention. Exemplary cross-linking agents and cross-linking mechanisms contemplated by the present invention include, but are not limited to, covalent ester cross-linking (U.S. Pat. No. 7,247,191) and UV cross-linking (U.S. Pat. No. 8,337,598), both of which are incorporated herein by reference in their entireties. Furthermore, the use of cross-linking agents in the manufacture of hollow fibers is known to those skilled in the art. See, for example, U.S. Pat. Nos. 9,718,031; 8,337,598; 7,247,191; 6,932,859; and 6,755,900, all of which are incorporated herein in their entireties.

[0090] The membranes and fibers of the present invention are made from a blend of protein and polysaccharide. The protein to polysaccharide ratio is contemplated to be about 1:99 to about 99:1, about 1:10 to about 10:1, about 2:5 to about 5:2, about 3:7 to about 7:3, about 4:6 to about 6:4, or about 1:1, or any ratio within the stated ranges. In preferred embodiments, the protein content of the blend is higher than the polysaccharide content. In preferred embodiments, the protein content is about 90%, 95%, 98%, 99%, or more.

[0091] It is further contemplated that the membranes of the present invention may be further strengthened, i.e., endowed with increased integrity and strength, by incorporating a manufacturing process step that crosslinks proteins in the membrane. The inventors have discovered that, after formation of the membranes of the present invention, when the membranes of the present invention are exposed to an energy source at an appropriate energy level for an appropriate amount of time, the proteins at least partially crosslink, thereby endowing the membranes of the present invention with increased integrity over prior art membranes. The Exemplification section below provides examples of several membranes (i.e., hollow fiber membranes) treated with and without heat or irradiation. Hollow fibers produced without exposure to the mentioned energy source lacked integrity compared to hollow fibers produced with exposure to the energy source.

[0092] Heat can be supplied via either dry heat or moist heat. One method of the present invention utilizes temperatures of about 60°C to about 100°C at pressures of 0 psi (ambient pressure) to 20 psi or greater for about 2 to about 60 minutes at a relative humidity of about 50% to 100%. Additionally, heat can be supplied by immersing the membrane or fiber of the present invention in a water bath at about 60°C to about 100°C at atmospheric conditions.

[0093] The membranes and fibers of the present invention can also be exposed to energy via any form of radiation (e.g., electron beam, gamma, UV, etc.). The membranes and fibers of the present invention can be irradiated with about 1 to about 100 kGy, about 5 kGy to about 75 kGy, or about 10 kGy to about 50 kGy. The membranes and fibers of the present invention can be exposed to the radiation for about 0.1 minutes to about 60 minutes, about 1 minute to about 50 minutes, about 2 minutes to about 40 minutes, and about 2 minutes to about 30 minutes, and any value between the recited values.

[0094] Hollow fiber manufacturing techniques, particularly membrane manufacturing techniques, are generally known to those skilled in the art (see, for example, Vandekar, VD, Manufacturing of Hollow Fiber Membranes, Int'l J Sci&Res, 2015, 4:9, pp. 1990-1994, and references cited therein). Similar to flat-sheet membranes, known hollow fiber manufacturing methods typically involve some phase separation technique. Common methods, such as non-solvent-induced phase separation, include thermally induced phase separation, vapor-induced phase separation, heat-induced phase separation (see, for example, U.S. Pat. No. 5,444,097 to MilliporeSigma, incorporated herein by reference), or a combination thereof. However, other techniques, such as thermal extrusion and drawing, can be used for hollow fiber and membrane formation. Typically, the polymer in solution is destabilized by the removal of a non-solvent, thermal destabilization, or solvent. As described herein, dissolution of the polymers (in this case, polysaccharides and proteins) is followed by gelation or solidification through multiple cross-linking steps. The fibers can be further drawn to produce fibers with diameters less than 100 μm and wall thicknesses as thin as 10 μm.

[0095] Membrane sheets can be produced using similar phase inversion techniques, in which a liquid polymer solution enters a quenching solution and solidifies as the solvent is withdrawn, as well as other techniques known to those skilled in the art, such as, but not limited to, solvent evaporation (see, for example, U.S. Patent Application Publication No. 2020 / 0368696 to MilliporeSigma). See, for example, Gas Separation Membranes, Polymeric and Inorganic, Chapter 4, Ismail, et al., Springer, 2015, and U.S. Patent Application Publication No. 2007 / 0084788 to MilliporeSigma.

[0096] In some embodiments of the present invention, pH-induced phase separation ("pH Induced Phase Separation" or "Proton Induced Phase Separation"; Satoru Tokutomi, Kazuo Ohki, Shun-ichi, Ohnishi, Proton-induced phase separation in phosphatidylserine / phosphatidylcholine membranes, Biochimica et Biophysica Acta (BBA), Biomembranes, Volume 596, Issue 2, 28 February 1980, Pages 192-200) is used to fabricate the membranes of the present invention (i.e., hollow fiber and sheet membranes). pH-induced phase separation is exemplified in the Examples section below. While pH-controlled liquid phase separation of polymers has been studied in cell physiology (Adame-Arana, O., et al., Liquid Phase Separation Controlled by pH, 2020 Oct 20;119(8):1590-1605; Epub 2020 Sep 16), we believe that we are the first to utilize pH-induced phase separation in the fabrication of hollow fiber and sheet membranes, particularly membranes suitable for the production of clean meat or clean structured meat products. The use of pH-induced phase separation confers unexpected and surprising benefits to the membranes of the present invention, namely improved mechanical integrity, pore size, and porosity over conventional methods.

[0097] Dry spinning involves dissolving a polymer in a highly volatile solvent. The solvent / polymer mixture is heated after extrusion and evaporation of the solvent, causing the polymer to solidify.

[0098] Wet spinning is more versatile because the process involves a greater number of parameters that can be varied. The polymer / solvent mixture is extruded into a non-solvent bath where demixing and / or phase separation occurs due to the exchange of solvent and non-solvent. Between the extrusion and the non-solvent bath is a void where the formation of a hollow fiber membrane begins.

[0099] A technology that can eliminate or minimize the use of solvents is melt spinning with cold drawing (MSCS). This approach offers cost-effective manufacturing but may sacrifice structural control and potential degradation of the food material. In this technique, the material is heated for extrusion and then drawn as it cools to mechanically form pores in the hollow fiber wall. All three of these techniques have been extensively studied and are well-known in the art, where they have been thoroughly summarized (see Tan, XM. and Rodrigue, D., Polymers (Basel), 2019, Aug 5:11(8)).

[0100] Modifications of these techniques are also known to those skilled in the art. See, for example, WO 2011 / 108929 (incorporated herein by reference in its entirety), which discloses a modified wet-spinning extrusion method for producing hollow fibers composed of multiple polymers and polymer layers. The production of hollow fibers from non-synthetic materials is also known to those skilled in the art. See, for example, U.S. Patent No. 4,824,569 to Suzuki, which is incorporated herein in its entirety.

[0101] Hollow fiber membranes of the present invention for producing structured meat products In certain embodiments, it is contemplated that the macroscopic structure of the hollow fibers of the present invention promotes cell orientation along the fiber. In this regard, it is desired by the present invention that the orientation of the component molecules from which the hollow fibers are constructed be oriented parallel, essentially parallel, or predominantly parallel to the length of the hollow fiber. It is further contemplated that the component molecules create a surface texture on at least the outer surface of the hollow fiber that aids cell adhesion and cell orientation. Thus, in certain embodiments, it is contemplated that the surface texture of the hollow fibers of the present invention creates adhesion points for cell adhesion. In another embodiment, it is further contemplated that cells (particularly myocytes, myocyte-like cells, or cells having myocyte characteristics) grown on the hollow fibers of the present invention will orient and stretch along the length of the hollow fiber similar to and resembling myocytes in vivo.

[0102] Thus, the orientation of the scaffold's surface structure directly correlates with the alignment of the forming myotubes. It can be imagined as if skeletal muscle wants to form along existing structures. The fiber bundles closely mimic the skeletal muscle structure to form aligned myotubes. Therefore, the hollow fiber bioreactor not only achieves tissue-like cell density but also myotube alignment unattainable by other techniques, providing the most realistic palatability of all the techniques discussed. The alignment phenomenon can be better understood by reviewing "My mistake: Decellularized Apium graveolens Scaffold for Cell Culture and Guided Alignment of C2C12 Murine Myoblast" (Santiago Campuzano, 2020, Ph.D. thesis, University of Ottawa, pp. 58-59).

[0103] In relation to producing structured clean meat products, the hollow fibers of the present invention are contemplated to have a range of sizes suitable for the present invention. It is also contemplated that the hollow fibers of the present invention can be spaced so that cells grown on the hollow fibers achieve a density similar to that of actual meat and have minimal void space between the cells. In one embodiment, the hollow fibers of the present invention are contemplated to have an outer diameter of about 0.1 mm to about 3.0 mm, a porosity of about 0% (on a diffusion basis) to 75% porosity, and a wall thickness of about 0.008 mm to about 0.5 mm, or about 0.01 mm to about 0.2 mm, or any thickness between 0.008 mm and 0.5 mm not specifically reiterated above. The inventors have found that this size is suitable for transport of medium through the lumen of the fiber, allows adequate flow of medium through the walls of the hollow fiber, while being rigid enough to support cell growth, and provides the desired final product structure, texture, handling, and mouthfeel. However, other embodiments are contemplated (discussed below) regarding variations in fiber diameter, wall thickness, and porosity depending on the desired structured clean meat product (e.g., beef, poultry, fish, pork, etc.).

[0104] Fiber Porosity: The hollow fibers of the present invention must have a porosity that allows for adequate flow of medium through the fiber wall while providing a suitable surface for cell growth and cell support. The porosity of a hollow fiber is, in part, related to the thickness of the hollow fiber wall and the composition of the hollow fiber. If the wall is thin enough, a porosity of about 0% may be sufficient, allowing medium to diffuse through the hollow fiber wall. The porosity of hollow fibers of the present invention may be as high as 75% or 90%. Thus, the porosity range of hollow fibers of the present invention can be 0% to about 90%, about 10% to about 75%, about 30% to about 60%, or any percentage value between 0% and 75% not specifically reiterated above.

[0105] The hollow fibers of the present invention may also undergo a pore-forming process. The pore-forming mechanism is one of the following well-known techniques in the field of membrane formation: TIPS = thermally induced phase separation, NIPS = non-solvent induced phase separation, VIPS = vapor-induced phase separation, pH-induced phase separation, and MSCS = melt spinning combined with drawing (see Review on Porous Polymeric Membrane Preparation. Part II: Production Techniques with Polyethylene, Polydimethylsiloxane, Polypropylene, Polyimide, and Polytetrafluoroethylene, Xue Mei Tan, 1, 2, 2019). In all scenarios, the polymer is brought into a liquid phase either by thermal melting or chemical dissolution. From there, the polymer is extruded into a cylindrical shape and drawn onto a spindle. During the extrusion process, a bore fluid can be used to prevent the hollow fiber form from collapsing under its own weight. A pore-forming chamber, such as a water bath or atmospheric environmental chamber, can also be present between the extrusion nozzle and the unwinding spindle.

[0106] The present invention also contemplates the configuration of hollow fibers of the present invention in a bioreactor. The fiber configuration can include one or both of the fiber arrangement and spacing. The fibers can be configured in any configuration that allows for the growth of a cell population that is confluent and has minimal void space between the cells. For example, the fibers can be oriented in a square / rectangular (rows and columns) or triangular / hexagonal (honeycomb) packing mode. Thus, in one embodiment, it is contemplated that the fibers are arranged to form a regular pattern of rows and columns when viewed from the end. In another embodiment, it is contemplated that the fibers form a honeycomb pattern when viewed from the end. In another embodiment, it is contemplated that the fibers of the present invention are arranged randomly or semi-randomly. In another embodiment, it is contemplated that the hollow fibers are arranged in regular or semi-regular patterns of various densities.

[0107] Hollow fibers can have outer diameters ranging from about 0.1 mm to about 3.0 mm, from about 0.5 mm to about 2.0 mm, and from about 0.8 mm to about 1.3 mm, as well as any value between the cited values. A 1.0 mm hollow fiber allows for about 0.3 mm to about 0.5 mm of flesh growth around the outer diameter. An end diameter of approximately 1.1 mm can result in about 85 hollow fibers / cm2 of flesh.

[0108] In another embodiment, it is contemplated that the fibers have varying degrees or amounts of inter-fiber spacing. For example, rows of higher density fibers interspersed among lower density fibers can be used to create variations in the texture of the final structured clean meat product, such as is common in natural fish meat. Still further, it is contemplated that fibers of various diameters, porosities, and wall thicknesses can be used in the same hollow fiber cartridge to simulate the appearance, texture, handling, and mouthfeel of natural meat.

[0109] In all configurations, the fibers are spaced apart to allow sufficient flow of medium (and the nutrients, growth factors, etc. contained therein) to reach all of the cell mass. This, of course, is at least partially related to the medium flow rate and the porosity of the hollow fiber wall, but is also largely related to the physical distance from the surface of the hollow fiber's outer wall to the cells. In other words, medium and nutrients travel or diffuse only a limited distance through the cell mass. Currently, the maximum diffusion limit for oxygen and nutrients is believed to be 200 μm. Rouwkema, J., et al., (2009) Supply of Nutrients to Cells in Engineered Tissues, Biotechnology and Genetic Engineering Reviews, 26:1, 163-178. Therefore, the interfiber spacing should be approximately 400 μm from the outer wall of one fiber to the outer wall of the adjacent fiber. In culture conditions where medium flows through both the hollow fibers and the spaces between the fibers, the spacing can be larger. For example, the spacing can be 800 μm from the outer wall of one fiber to the outer wall of an adjacent fiber. These values ​​are for a case where the culture process relies solely on diffusion. However, the use of a pump (for example) can cause medium to flow from the hollow fibers, through the cell culture spaces between the hollow fibers, and to the housing outlet (rather than relying solely on diffusion), allowing the fibers to be spaced further apart. For example, in some embodiments, the maximum distance between fibers is about 0.05 mm (50 μm) to about 5.0 mm; about 0.1 mm to about 3.0 mm; about 0.1 mm to about 2.0 mm; about 0.1 mm to about 1.0 mm, or about 0.2 mm to about 0.5 mm, or any distance between the values ​​mentioned. While a preferred embodiment is for medium to flow from the center of the hollow fibers through the culture to the housing outlet, it is also contemplated that medium flow may be in the opposite direction, or may alternate from one direction to the other, if desired. Alternating the direction of medium flow is believed to help ensure that all cells receive an adequate supply of medium.

[0110] An embodiment of the present invention is that a degree of randomness is inherent in the distance of the hollow fibers of the present invention. The numbers given in the previous paragraph are the average inter-fiber distances for a given assembly. In some embodiments of the present invention, spacers and / or assembly techniques can be used to ensure, normalize, or control the inter-fiber distance. See, for example, Han G, Wang P, Chung TS., Highly robust thin-film composite pressure retarded osmosis (PRO) hollow fiber membranes with high power densities for renewable salinity-gradient energy generation, Environ Sci Technol. 2013 Jul 16;47(14):8070-7. Epub 2013 Jun 28 or Chun Feng Wana, Bofan Li a, Tianshi Yang a, Tai-Shung Chung, Design and fabrication of inner-selective thin-film composite (TFC) hollow fiber modules for pressure retarded osmosis (PRO), Separation and Purification Technology, 172:32-42, 2017.

[0111] If the cell density becomes too high or the cell mass becomes too thick, it becomes difficult for the medium to reach the cells furthest from the hollow fibers. The lack of medium for these cells can result in dead cells and / or dead spaces within the reactor where cells cannot grow. As a result, medium must flow through the hollow fiber cartridge to the housing outlet. That is, medium flow must be maintained at least until confluence is reached and the structured clean meat product is harvested. Based on the teachings herein, one skilled in the art will be able to calculate the exact spacing and porosity of the fibers of the present invention for a given desired structured clean meat product.

[0112] The hollow fibers of the present invention can be arranged and secured in what is referred to herein as a "hollow fiber cartridge." In one embodiment, it is contemplated that the hollow fiber cartridge is fabricated by securing the ends of the hollow fibers to end pieces in a desired arrangement. For example, each fiber has a first end and a second end. Each end is secured to an end piece, i.e., a first and a second end piece. The end pieces can be, for example, a resin or plastic known in the art to be inert and non-toxic to cells. At least one of the first or second ends of the hollow fiber is positioned within the end piece such that the internal lumen of the hollow fiber is in fluid communication with the external environment. This arrangement of the hollow fiber within the end piece therefore allows for the flow of culture medium from the external environment of the hollow fiber (i.e., outside the hollow fiber, but inside, for example, a sterile bioreactor) into the internal lumen of the hollow fiber.

[0113] Those skilled in the art understand how to assemble hollow fibers into modules or cartridges. These techniques are applicable to the hollow fibers of the present invention. Briefly, after spinning, the hollow fibers are cut to length, and the fiber ends are encased (i.e., potted) in a resin that flows around the fiber ends and solidifies. Sometimes, the fiber cross-sections can be encased in a substance (e.g., plaster of Paris or other easily removable material known to those skilled in the art) to close the fiber pores and prevent the "potting solution," i.e., liquid resin, from entering and blocking the fiber pores. See, e.g., Vandekar, V.D., Manufacturing of Hollow Fiber Membranes, Int'l J Sci&Res, 2015, 4:9, pp. 1990-1994, and references cited therein. In the present invention, once the bundle is inserted into a housing for use in producing the structured clean meat of the present invention, one or both ends of the "potted" bundle are trimmed or cut to expose the open ends of the fibers to allow for the flow of culture medium.

[0114] Still further, in some embodiments, it is contemplated that the hollow fiber cartridges of the present invention have a locking device for maintaining a desired distance between the first end piece and the second end piece, which may be necessary or preferred, for example, to more easily insert the hollow fiber cartridges of the present invention into, for example, a bioreactor housing.

[0115] Thus, in one embodiment, it is contemplated that the hollow fiber cartridge of the present invention contains a plurality of hollow fibers arranged in a desired configuration. The hollow fibers of the present invention have a first end and a second end. This configuration is maintained by securing the first and second ends of the hollow fibers to the first and second end pieces. When secured as described, the hollow fibers are arranged parallel, substantially parallel, or essentially parallel to each other. Furthermore, the first and second end pieces are arranged parallel, substantially parallel, or essentially parallel to each other. Still further, the hollow fibers of the hollow fiber cartridge of the present invention are arranged perpendicular, substantially perpendicular, or essentially perpendicular to the end pieces of the hollow fiber cartridge of the present invention. The diameter and length of the hollow fiber cartridge depend on the desired structured clean meat product and bioreactor configuration to be produced.

[0116] In one embodiment of the present invention, the hollow fibers of the hollow fiber cartridge of the present invention have a density of about 40 to about 120 / cm 2 Average density: about 60 to about 100 / cm 2 Average density: about 70 to about 90 / cm 2 or any value between those values ​​shown above but not specifically repeated.

[0117] In certain embodiments of the present invention, it is contemplated that the hollow fibers in the hollow fiber cartridge of the present invention have void space between the hollow fibers prior to the addition of cells, and the void space between the hollow fibers is between about 25% and about 75% of the total area of ​​the hollow fiber cartridge, or between about 40% and about 60% of the total area of ​​the hollow fiber cartridge, or any value between the values ​​set forth above but not specifically repeated.

[0118] In some embodiments of the present invention, it is contemplated that the hollow fiber cartridge of the present invention is designed to be removably inserted into a housing. That is, the cartridge can be inserted into the housing at the beginning of a production run and removed, i.e., harvested, at the end of the production run for any desired further processing of the structured clean meat product of the present invention. After harvesting the structured clean meat product, a new hollow fiber cartridge of the present invention can be inserted into the housing and the process repeated. In this regard, the housing for the hollow fiber cartridge of the present invention is part of a bioreactor or bioreactor system.

[0119] Reactor Configuration. The present invention is not limited to any particular reactor or reactor system configuration, so long as sufficient medium flow can be maintained through the culture and any waste products being removed. Hollow fiber reactors are typically tubular in shape, but may also be oval, flat (sheet-like), rectangular, or any other shape. In a preferred embodiment, the reactor contains an insertable / removable insert containing the hollow fibers of the present invention. After confluent cell growth (as defined herein) is reached, the insert is removed, and the product can be completed by removal of the insert ends and any further desired processing. Further processing can take the form of, for example, slicing, surface texturing, flavoring, etc. Alternatively, further meat enrichment can be performed prior to harvesting and disassembly of the device. For example, the medium can be drained from the hollow fiber device, and additives could then be pumped directly into or around the fibers.

[0120] Non-Limiting Examples of Suitable Reactor Systems. While the most suitable type of reactor system is a fed-batch system, it is contemplated that any available reactor is suitable for use with the hollow fibers and hollow fiber cartridges of the present invention. For example, the MOBIUS® system (MilliporeSigma, Bedford, Massachusetts) is an example of a commercial system that can be easily converted for use with the present invention. The bioreactor in which the structured clean meat product is produced (i.e., the reactor containing the hollow fibers of the present invention) may be inoculated with cells grown in another bioreactor. The bioreactor (a reactor suitable for cell growth (proliferation) and cell expansion) inoculating the hollow fiber device can be an existing commercially available reactor, such as a stirred tank or wave reactor. The growth / expansion bioreactor can be, for example, a stirred tank or wave reactor (known to those skilled in the art), and is contemplated to be a suspension, aggregate biomass, microcarrier culture, or other suitable reactor known to those skilled in the art. It is contemplated that production bioreactors (i.e., reactors comprising the hollow fibers of the invention) can be, for example, single use, multiple use, semi-continuous, or continuous. The invention further contemplates manifolds of multiple reactors comprising the hollow fibers of the invention.

[0121] Thus, it is contemplated that an exemplary reactor system of the present invention comprises one or more hollow fiber cartridges of the present invention, a housing sized to hold the hollow fiber cartridges; a medium source fluidly connected to one or more inlets in the housing; one or more medium outlets in the housing; and one or more pumps for supplying medium to the hollow fiber cartridge and / or removing waste medium from the hollow fiber cartridge through the medium inlets and / or outlets. Still further, the inlets are fluidly connected to the interior of the hollow fibers. Still further, the hollow fiber bioreactor may comprise an automatic controller or system.

[0122] The present invention also provides a method for producing a meat product, comprising culturing a variety of cells, for example, from 100,000 cells to 100,000,000 cells (10 5 ~108 ) into the void space between the hollow fibers in a hollow fiber reactor of the invention (Radisic, et al., Biotechnol Bioeng, 2003 May 20:82(4):403-414); culturing the cells until they achieve about 80% to about 99% confluency, 85% to about 99% confluency, about 90% to about 99% confluency, about 95% to about 99% confluency, about 98% to about 99% confluency, or about 100% confluency (or any value between the recited percentages); and removing the first and second retention devices from the first and second ends of the hollow fibers, respectively.

[0123] After seeding, the hollow fiber cartridge has culture medium supplied to the cells through one or both of the first and second ends of the hollow fibers to the interior of the hollow fibers, through the walls of the hollow fibers to the void spaces between the hollow fibers where the cells are seeded, and through one or more of the outlets to the housing. In another embodiment, it is contemplated that culture medium can flow between the fibers from both an inlet and an outlet of the device. For example, one fluid path is through the fiber wall and a second fluid path is around the fibers. It is contemplated that the device can have multiple inlets and outlets. After the cells reach confluence, any residual culture medium and waste products are washed away, and one or more of fats, flavors, colors, salts, and preservatives are injected into the interior of the hollow fibers and / or any remaining void spaces between the cells.

[0124] Fats suitable for addition to the structured clean meat products of the present invention include, but are not limited to, saturated, monounsaturated, and polyunsaturated fats, such as corn oil, canola oil, sunflower oil, and safflower oil, olive oil, peanut oil, soybean oil, flaxseed oil, sesame oil, canola oil, avocado oil, seed oil, nut oil, safflower oil and sunflower oil, palm oil, coconut oil, omega-3, fish oil, lard, butter, processed animal fat, adipose tissue, or cellular agriculture-derived fat, or a combination thereof. Synthetic fats, such as oleoresins, can also be used. In fact, any fat recognized by the Food and Drug Administration (FDA) is suitable for use in the present invention and is contemplated for use in the structured clean meat products of the present invention. The FDA food additive list includes natural substances and extracts (NATs), nutrients (NUTRs), essential oils, and / or oleoresins (solvent-free) (ESOs).

[0125] Flavors suitable for use in the structured clean meat products of the invention include, but are not limited to, any flavor listed on the FDA's food additive list, which may be listed as natural flavors (FLAV), essential oils and / or oleoresins (solvent-free) (ESO), enzymes (ENZ), natural substances and extracts (NAT), non-nutritive sweeteners (NNS), nutritive sweeteners (NUTRS), spices, other natural seasonings and flavors (SP), synthetic flavors (SY / FL), fumigants (FUM), artificial sweeteners including aspartame, sucralose, saccharin, and acesulfame potassium, and yeast extract, or combinations thereof, are contemplated for use in the structured clean meat products of the invention.

[0126] Suitable texture enhancers for use in the structured clean meat products of the invention include, but are not limited to, refined plant materials, guar gum, cellulose, hemicellulose, lignin, beta-glucan, soy, wheat, corn, or rice isolates and beet fiber, pea fiber, bamboo fiber, plant-derived fiber, plant-derived gluten, carrageenan, xanthan gum, lecithin, pectin, agar, alginates, and other natural polysaccharides, grain hulls, calcium citrate, calcium phosphate, calcium sulfate, magnesium sulfate, and salt, or any combination thereof, which may be recorded on the FDA's food additive list as solubilizers and dispersants (SDAs) and natural substances and extracts (NATs).

[0127] Nutritional additives suitable for use in the structured clean meat products of the present invention include, but are not limited to, vitamins, trace elements, bioactive compounds, endogenous antioxidants, such as vitamins A, B complex, C, D, and E, zinc, thiamine, riboflavin, selenium, iron, niacin, potassium, phosphorus, omega-3, omega-6, fatty acids, magnesium, proteins and protein extracts, amino acid salts, creatine, taurine, carnitine, carnosine, ubiquinone, glutathione, choline, glutathione, lipoic acid, spermine, anserine, linoleic acid, pantothenic acid, cholesterol, retinol, folic acid, dietary fiber, amino acids, and combinations thereof, and are contemplated for use in the structured clean meat products of the present invention. Any food additive that is Generally Regarded as Safe (GRAS) or approved by the FDA is contemplated for use in the structured clean meat products of the present invention and is incorporated herein. See, e.g., www.fda.gov / food / food-additives-petitions / food-additive-status-list.

[0128] Any natural or artificial food color that is Generally Regarded as Safe (GRAS) or approved by the FDA is contemplated for use in the structured clean meat products of the invention. See, for example: www.fda.gov / industry / color-additive-inventories / color-additive-status-list.

[0129] Prophetic Cell Types. The hollow fibers of the present invention are designed to be used to grow specific cell types suitable for producing in vitro or laboratory-grown meat and meat products, i.e., the structured clean meat of the present invention. Thus, while many different types of cells can be grown on hollow fibers (and, if desired, within hollow fiber cartridges of the present invention), the fibers were developed to be used to grow muscle cells (i.e., myocytes), or cells with muscle cell properties, or cells engineered to have muscle cell properties (collectively referred to herein as muscle cells or myocytes), to confluence and mimic the natural structure of muscle (i.e., meat). Preferably, the muscle is skeletal muscle. That is, the hollow fibers of the present invention are designed by the inventors to be suitable for growing muscle cells to obtain muscle fibers or myofibrils. Additionally, other types of cells can be grown on the hollow fibers of the present invention and within reactors containing the hollow fibers of the present invention. These cells can be grown independently or in combination with muscle cells. For example, adipocytes or cells with adipocyte characteristics or engineered to have adipocyte characteristics (collectively referred to herein as adipocytes) can be cultured with muscle cells to obtain an end product that resembles natural muscle or meat. The hollow fibers of the present invention are also suitable for containing other cells, such as fibroblasts, cells with fibroblast characteristics, or cells engineered to have fibroblast characteristics, to be co-cultured with the muscle cells of the present invention.

[0130] In particular, for co-culture of muscle cells and adipocytes, the ratio of muscle cells to adipocytes can be 99:1, 95:5, 92:8, 90:10, 88:12, 85:15, 82:18, 80:20, 75:25, or any ratio between 100:0 and 75:25 inclusive.

[0131] Cells suitable for use in the present invention can be obtained or derived from any animal from which food is currently obtained. Prominent examples are cattle, pigs, sheep, fish (e.g., fish such as tuna, salmon, cod, haddock, shark, etc.), crustaceans, and birds (e.g., chicken, turkey, duck, etc.). More exogenous cell sources can also be used, such as animals that are traditionally hunted rather than farmed (e.g., deer, elk, moose, bear, rabbit, quail, wild turkey, etc.), or combinations thereof.

[0132] Cells used in the present invention can be derived by any method suitable for producing differentiated cells with desired characteristics. For example, any procedure suitable for inducing cells with differentiated myocyte-like characteristics, adipocyte-like characteristics, etc. Such characteristics of muscle cells include, but are not necessarily limited to, having the appearance of long, tubular cells and a large complement of myosin and actin. Muscle cells also have the ability to fuse with other muscle cells to form myofibrils, the units of muscle that help give muscle, i.e., meat, its distinctive texture. Such characteristics of adipocytes (also referred to in the art as lipid cells and fat cells) include, but are not necessarily limited to, having large lipid vacuoles that can occupy more than 90% of the cell's volume. The hollow fibers of the present invention provide, at least in part, a replacement for the connective tissue (referred to in the art as "fascia") typically found in skeletal muscle.

[0133] Cells useful in the present invention include, but are not limited to, cells derived from mesenchymal stem cells or induced pluripotent stem cells (iPSCs). iPSCs are cells that have been engineered to return to their pluripotent state from which multiple cell types can be derived. In other words, iPSCs are pluripotent stem cells that can be generated directly from somatic cells. This technology was first reported in 2006 (Takahashi K, Yamanaka S, 25 August 2006, "Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors," Cell, 126(4):663-76) and has progressed since then (see, e.g., Li, et al., 30 April 2014, "Generation of pluripotent stem cells via protein transduction," Int. J. Dev. Biol., 58:21-27), including the generation of muscle cells (see, e.g., Rao, et al., 9 January 2018, "Engineering human pluripotent stem cells into a functional skeletal muscle tissue," Nat Commun., 9(1):1-12), and is well known to those skilled in the art.

[0134] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0135] When introducing elements of the disclosure or preferred embodiments thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0136] The transitional phrases "comprising," "consisting essentially of," and "consisting of" have the meanings set forth in MPEP 2111.03 (U.S. Manual of Patent Examining Procedures, 9th ed., Rev. October 2019; U.S. Patent and Trademark Office). Any claim using the transitional phrase "consisting essentially of" shall be understood to recite only essential elements of the invention, and any other elements recited in a claim depending thereon shall be understood to be non-essential to the invention recited in the claim to which it depends.

[0137] All ranges recited herein include all values ​​within the cited range (inclusive), including all integers, fractions and decimals. [Example]

[0138] General Materials and Methods: All reagents were commercially available and were used without further purification unless otherwise stated. Zein, sodium hydroxide, urea, hydroxypropyl cellulose, k-carrageenan, sodium acetate, tripolyphosphate (TPP), (HCl 37%, antibiotic-antimycotic solution (100x), and wheat gluten, fibronectin derived from bovine serum were purchased from MilliporeSigma (Burlington, MA). Bovine collagen was purchased from Corning (Corning, NY); soy protein isolate (SPI) was purchased from BulkSupplements (Henderson, NV); chitosan (derived from mushrooms) was purchased from Modernist Panty (Elliott, ME, USA); pea and peanut butter protein isolates were purchased from NorCal Organic (Crescent City, CA); mung bean, broad bean, and chickpea protein isolates were purchased from Green Boy (Redondo Beach, CA); agarose was purchased from Hispanagar (Burgos, Spain); and brown rice protein isolate was purchased from Zen. sodium alginate and MooGloo™ RM transglutaminase were purchased from Modernist Pantry (Elliott, Maine).

[0139] Cell Culture Medium Stability Testing: Membranes were cut into 1 x 3.5 inch square samples and incubated in cell culture medium containing antibiotic-antimycotic solution (2x) (known to those skilled in the art) at 37°C for up to 21 or 30 days depending on the experiment. For each membrane type, three samples were mechanically tested during each incubation time point.

[0140] viscosity: Viscosity measurements of the prepared dope solutions were performed on a Brookfield (Middleboro, Massachusetts) Viscometer DV-II+Pro using an S64 spindle.

[0141] Mechanical testing: Membrane tensile tests were performed on 1 x 3.5 or 0.5 x 3.5 inch square specimens using a Zwick Roell (Kennesaw, GA) TestControl II instrument, and data were analyzed using Zwick Roell testXpert II V3.71 software.

[0142] Lyophilization: Samples were frozen in scintillation vials in water under liquid nitrogen for 1 hour, and then dried using a Labconco (Kansas City, MO) 2.5 L freeze dryer at -80°C.

[0143] Scanning electron microscopy: Samples are coated with 3 nm of iridium, mounted on stubs, and imaged using a ThermoScientific (Waltham, MA) Quanta 200F or a JOEL (Peabody, MA) JCM 6000 scanning electron microscope (Tokyo, Japan).

[0144] Statistical analysis: Error bars are calculated as the standard error of the mean.

[0145] Rheology: Rheological analysis of the formulated dopes and films was performed on a TA Ares rheometer (New Castle, Del.) using a cone fixture.

[0146] Example 1 - Method for producing edible hollow fibers See Figures 1 and 2 for a schematic diagram of an exemplary manufacturing process for producing the membranes of the present invention.

[0147] 1. Preparation of Dope Solution a. The preparation of the dope solution requires a multi-stage mixing process.

[0148] i. First, a protein solution was prepared. This required dissolving 14% by weight of vegetable protein concentrate in a weakly alkaline buffer. The mixture was homogenized at 20,000 rpm for several minutes. Specifically, micronized vegetable protein powder was used.

[0149] The second solution contained a carrier polymer consisting of 2% alginate and 2% hydroxypropyl cellulose dissolved in the same buffer as the protein mixture, which was then dissolved in a hybridizer at 35°C for 48 hours.

[0150] iii. The protein solution and carrier polymer solution were mixed in a 1:1 ratio using an overhead stirrer, followed by 12 hours in a hybridizer at 35°C.

[0151] iv. The final mixture has a resulting concentration of 2% polysaccharides and 7% vegetable protein and is called the dope solution.

[0152] b. Complete the creation of bore solution by dissolving 15 g / L of calcium chloride in reverse osmosis (RO) water containing 0–1 g / L of transglutaminase.

[0153] 2. Stretching and solidification A pressurized vessel and a gear pump are used to force the dope solution through a coaxial orifice. There is a specific distance between the spinneret and the bath, which can be adjusted based on the rheological properties of the dope solution.

[0154] b. The setting bath (also referred to herein as the forming bath) is also 15 g / l calcium chloride, which ionically crosslinks the alginate, locking it into the 3D structure of the fibers.

[0155] 3.Crosslinking process a. In this application, ionic crosslinking of alginate may not function sufficiently to dissociate bivalent bonds with monovalent bonds created by sodium salts in the cell culture medium. Crosslinking beyond enzymatic transglutaminase crosslinking and alginate-calcium crosslinking was desired.

[0156] b. The fibers were then exposed to heat near 100°C to thermally crosslink the proteins within the fibers. Proof of concept was demonstrated via autoclaving at 121°C for 60 minutes.

[0157] Alternatively or additionally, the fibers were exposed to approximately 50 kGy (kilograys) of electron beam or gamma irradiation to physically crosslink the cellulose portion of the mixture, i.e., to crosslink the proteins. As can be determined by one of ordinary skill in the art using the teachings herein, the final dose can be from approximately 5 kGy to approximately 100 kGy, depending on the residence time of the material through the electron beam and the grade of the material.

[0158] 4. Coating process a. The fibers were passed continuously through a plasma chamber and then immersed in a solution of a 15% glycol / sorbitol (1:1) mixture in water (depending on the application, the glycol / sorbitol ratio can range from 1:14 to 14:1). This step was designed to minimize collapse of the porous structure of the hollow fibers via the plasticizer.

[0159] Figures 3A and 3B show photomicrographs of hollow fiber membranes made by the method of Example 1 (Method). Figures 4A-4C show scanning electron photomicrographs of hollow fiber membranes made by the method of this example. Figure 5A shows the length of one hollow fiber made by the method of this example. Figure 5B provides a demonstration of the tensile strength of one of the hollow fibers. Example 2 - Prophetic Example of Fiber Without Secondary Crosslinking Step a. Use the hollow fiber dope solution prepared as defined above in Example 1. This example covers three conditions. All conditions are made from the same dope solution: 1 part hydroxypropyl cellulose, 1 part alginic acid sodium salt (Sigma Aldrich, St. Louis, MO), and 7 parts pea protein isolate.

[0160] b. In the first condition, the fibers are extruded directly into a 15 g / L calcium chloride bath and immediately solidified. After 10 minutes in the bath, the fibers are rinsed with MilliQ™ water (MilliporeSigma, Bedford, MA) and then immersed in DMEM F12 medium for 72 hours. When the fibers are removed from the cell culture medium, they cannot be handled. They can no longer support their own weight outside of the solution. Most of the ionic cross-linking sites have dissociated.

[0161] c. In the second condition, the fibers are extruded directly into a 15 g / L calcium chloride bath and immediately solidified. After 10 minutes in the bath, the fibers are rinsed with MilliQ™ water and then autoclaved at 121°C for 30 minutes. After cooling to room temperature, the fibers are then immersed in DMEM / F12 (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12; ThermoFisher Scientific, Waltham, MA) medium for 72 hours. When the fibers are removed from the cell culture medium, they lose some of their integrity. Although the fibers can be removed, they are only able to self-support approximately 5 inches. Although the majority of the ionic crosslinking sites have dissociated, the thermally crosslinked proteins still play a role in enhancing the integrity of the fibers.

[0162] In the third condition, the fibers were extruded directly into a 15 g / L calcium chloride bath and immediately solidified. After 10 minutes in the bath, the fibers were rinsed with MilliQ™ water and then exposed to a single pass of 50 kGy in a benchtop electron beam reformer and immersed in DMEM / F12 medium for 72 hours. Upon removal from the cell culture medium, the fibers maintained their integrity and were able to support their own weight. Although the ionic crosslinking sites were susceptible to dissociation in cell culture medium, and some chain scission of both the alginate and cellulose backbones may be present, the physical crosslinks of the protein polymer network were resistant to dissociation in the medium.

[0163] These examples demonstrate that cross-linking of proteins by heat and / or irradiation results in improved integrity of the hollow fibers of the invention, making them suitable for use in, for example, cell culture or filtration devices. Example 3 - Preparation of dope solution 1.1. Protein Solution 1.1.2. Urea-based methods: Zein: A zein solution (15% w / v) was prepared by adding 57 g of zein powder to 300 mL of MilliQ™ water at 0° C. and under mechanical stirring. After 30 minutes, 11.25 g of urea was added to the suspension, followed by 83 mL of NaOH solution (0.4 N) (FIG. 12). The reaction was then warmed to room temperature (23° C.) and stirred for 18 hours before further use.

[0164] Zein: A zein solution (19% w / v) was prepared by adding 72 g of zein powder to 300 mL of MilliQ™ water at 0° C. and under mechanical stirring. After 30 minutes, 14.30 g of urea was added to the suspension, followed by 83 mL of NaOH solution (0.6 N) (FIG. 12). The reaction was then warmed to room temperature (23° C.) and stirred for 18 hours before further use.

[0165] Zein-hydroxypropyl cellulose blend: A 0.5% w / v hydroxypropyl cellulose (HPC) solution (0.5% w / v) was prepared by adding 1.75 g of HPC to MilliQ™ water and mixing by mechanical stirring for 18 hours. The solution was then cooled to 0°C using an ice bath, and zein (72 g) was added thereto. The suspension was allowed to stir at 0°C for an additional 20 minutes, after which 14.30 g of urea and 83 mL of NaOH solution (0.6 N) were added. The reaction was allowed to warm to room temperature (23°C) and stirred for an additional 18 hours before further use.

[0166] Soy protein isolate: A soy protein isolate (SPI) solution (20% w / v) was prepared by adding 76 g of SPI powder to 300 mL of MilliQ water under mechanical stirring. After 30 minutes, 11.25 g of urea was added to the suspension, followed by 83 mL of NaOH solution (0.4 N). The reaction was then allowed to stir for 18 hours before further use.

[0167] Pea protein isolate: A soy protein isolate (SPI) solution (20% w / v) was prepared by adding 76 g of PPI powder to 300 mL of MilliQ water under mechanical stirring. After 30 minutes, 11.25 g of urea was added to the suspension, followed by 83 mL of NaOH solution (0.4 N). The reaction was then allowed to stir for 18 hours before further use.

[0168] Mung bean: A mung bean solution (15% w / v) was prepared by adding 57 g of PPI powder to 300 mL of MilliQ™ water under mechanical stirring. After 30 minutes, 11.25 g of urea was added to the suspension, followed by 83 mL of NaOH solution (0.4 N). The reaction was then allowed to stir for 18 hours before further use. See Figure 6.

[0169] Wheat gluten: A gluten solution (15% w / v) was prepared by adding 56 g of gluten powder to 300 mL of MilliQ™ water under mechanical stirring. After 30 minutes, 11.25 g of urea was added to the suspension, followed by 83 mL of NaOH solution (0.4 N). The reaction was then allowed to stir for 16 hours before further use.

[0170] 1.1.3. Hybridization-based methods: Mungbean Alginate Blend: The mung bean protein isolate (Green Boy) and alginate (Modernist Pantry) blend are combined by weighing 45 grams of mung bean protein isolate into 252 grams of water and homogenizing at 25,000 rpm for 5 minutes. From there, 3 mL of 10 N NaOH (and optional 6 g of urea) is added, and it is homogenized for an additional 5 minutes. From there, the gel solution is placed in a homogenizer at 40°C overnight.

[0171] 1.2. Alginate-protein blend solution Alternative mung bean and alginate dope formulations: Therefore, a ranging study was conducted to find several possible blends of protein isolate and alginate. One exemplary blend and mixing method is represented by the following by weight: 0.2% alginate, 15% mung bean protein isolate, 1% ION NaOH, 2% urea (optional), 81.8% MilliQ™ water.

[0172] The first step is to wet (i.e., suspend) and disperse the protein isolate in the solution. The protein isolate is weighed and MilliQ™ water is added. A high-shear mixer, such as a homogenizer (IKA, Staufen, Germany), is set to 25,000 rpm for 5-10 minutes, or until the slurry resumes fluid-like behavior. Once dispersed, NaOH (and urea, if desired) is added to the protein and water, and the solution is then homogenized for an additional 5 minutes until a viscous gel forms. From there, an overhead mixer fitted with a propeller is set to 100-500 rpm to agitate the dissolved protein. The alginate is slowly added to the mixed solution over a 15-minute period. Once the alginate is homogenously dispersed throughout the mixture and partially dissolved, the solution is placed in a jar, capped, and placed in a hybridizer for 24 hours. See Figure 7.

[0173] i.1.2.1. Urea-based methods Zein-alginate: Zein-alginate blends with various biopolymer ratios were prepared by mixing zein solution (15% w / v) prepared according to the urea method with pre-made aqueous alginate solutions of various concentrations (2% w / v, 4% w / v, 6% w / v, and 8% w / v) under mechanical stirring for 20 minutes.

[0174] SPI-alginate blends with various biopolymer ratios were prepared by mixing SPI solution (20% w / v) prepared according to the SPI-alginate:urea method with pre-made aqueous alginate solutions of various concentrations (2% w / v, 4% w / v, 6% w / v, and 8% w / v) under mechanical stirring for 20 min.

[0175] SPI-alginate blends with various biopolymer ratios were prepared by mixing PPI solution (20% w / v) prepared according to the PPI-alginate:urea method with pre-made aqueous alginate solutions of various concentrations (2% w / v, 4% w / v, 6% w / v, and 8% w / v) under mechanical stirring for 20 min.

[0176] Mungbean-alginate blends with various biopolymer ratios were prepared by mixing mungbean solution (15% w / v) prepared according to the mungbean-alginate:urea method with pre-made aqueous alginate solutions of various concentrations (2% w / v, 4% w / v, 6% w / v, and 8% w / v) under mechanical stirring for 20 min.

[0177] Gluten-alginate blends with various biopolymer ratios were prepared by mixing gluten solution (15% w / v) prepared according to the gluten-alginate:urea method with pre-made aqueous alginate solutions of various concentrations (2% w / v, 4% w / v, 6% w / v, and 8% w / v) under mechanical stirring for 1 h.

[0178] 1.3. Protein-agarose blend solution 1.3.1. Urea-based methods Zein-agarose blends with various biopolymer ratios were prepared by mixing zein solution (15% w / v) prepared according to the urea method with pre-made aqueous agarose solutions of various concentrations (1% w / v, 2% w / v, and 4% w / v). Agarose solutions were prepared by adding each amount of agarose to 300 mL of MilliQ™ water at 60°C and stirring for 2 hours until dissolution was complete. To obtain a homogeneous blend and avoid solidifying the agarose before casting, the freshly prepared agarose solution was added to the zein solution preheated to 40°C and stirred for 20 minutes. The solution was kept at 40°C before casting.

[0179] Although investigated with zein-agarose, the formulation results and methods are expected to have similar results with other plant-based proteins. Blending agarose and corn protein (zein) is not a trivial task, as the two polymers do not utilize common solvents or dissolution temperatures. The combination of stabilizing zein above 40°C and reducing the required percentage of ethanol required to a level below approximately 20% is simultaneously achieved. Using Minitab (Pennsylvania State University) design of experiments for formulations, a solvent system containing 0.04 N sodium hydroxide, urea, and ethanol in percent w / v is investigated.

[0180] It was found that a combination of ethanol, urea, and 0.04N NaOH could dissolve zein. Surprisingly, zein could be dissolved at 10% ethanol content in the presence of 0.04N NaOH and urea. The simplex design plot is shown in Figure 8. However, zein without ethanol was not stable at temperatures below about 40°C. This finding is supported by temperature sweep rheology data. See Figure 9.

[0181] Furthermore, this solvent system, consisting of approximately 5% urea, 19% ethanol, and 76% 0.04N NaOH, was found to reduce the gelling properties of agarose. See Figure 10.

[0182] Furthermore, when agarose and zein are mixed together in this solvent system, rheological properties are observed that demonstrate the feasibility of mixing both polymers together in one system (see Figure 11).

[0183] Mung bean-agarose blends with various biopolymer ratios were prepared by mixing a mung bean solution (15% w / v) prepared according to the mung bean-agarose:urea method with various concentrations of pre-made aqueous agarose solutions (1% w / v, 2% w / v, and 4% w / v). The agarose solutions were prepared by adding the respective amounts of agarose to 300 mL of MilliQ™ water at 60°C and stirring for 2 hours until completely dissolved. To obtain a homogeneous blend and avoid solidification of the agarose before casting, the freshly prepared agarose solution was added to the mung bean solution preheated to 40°C and stirred for 20 minutes. The solution was kept at 40°C before casting.

[0184] Gluten-agarose blends with various biopolymer ratios were prepared by mixing gluten solution (15% w / v) prepared according to the gluten-agarose:urea method with pre-made aqueous agarose solutions of various concentrations (1% w / v, 2% w / v, and 4% w / v). Agarose solutions were prepared by adding the respective amount of agarose to 300 mL of MilliQ™ water at 60°C and stirring for 2 hours until completely dissolved. To obtain a homogeneous blend and avoid solidifying the agarose before casting, the freshly prepared agarose solution was added to the zein solution preheated to 40°C and stirred for 20 minutes. The solution was kept at 40°C before casting.

[0185] 1.4 Plant-based chitosan Mushroom-based chitosan was purchased from Modernist Pantry. Various concentrations of chitosan (5% w / v and 7% w / v) were dissolved in 5% acetic acid overnight via a hybridizer at 35°C. A forming bath containing 10 g / L triphenyl phosphate was used for solidification / crosslinking. Chitosan films were allowed to crosslink overnight before processing.

[0186] 1.5 K-Carrageenan K-carrageenan: K-carrageenan was heated to 90°C in MilliQ™ water at various concentrations (2% w / v, 4% w / v, and 10% w / v). At high temperature, the solution was cast onto a preheated plate and immersed in a forming bath containing 15 g / L calcium chloride. In another scenario, K-carrageenan was heated in solution with calcium chloride. Upon cooling, the solution solidified into a film.

[0187] b. Membrane preparation / formation Films were cast using either an automated film caster (BYK Drive 6 Film Caster, Leominster, MA) equipped with a 524 micron gap bar or a hand caster with a 600 micron gap. In both cases, 40 mL of dope solution was used for each film, measuring approximately 25 × 15 cm. 2 This led to membrane dimensions of area. Depending on the membrane formulation, different coagulation conditions were applied.

[0188] For hollow fibers, the dope solution was extruded through a coaxial needle purchased from Ramehart Instruments, Co. (Succasunna, NJ) Alternatively, a custom-built laboratory-scale hollow fiber spinner was used, allowing for processing of much higher viscosities (up to 100,000 centipoise (cP)).

[0189] 2.1.Protein membrane Regardless of whether they were obtained by the urea method or the hybridizer method, flat-sheet protein membranes were cast in sodium acetate buffer (0.2 M, pH 4.5) (see Figures 12A and 12B) and equilibrated in the same buffer for 10 min to a maximum of 3 h. The membranes were then washed with HEPES (0.1 M, pH 7.4) and stored in a 70 / 30% w / v ethanol / water solution.

[0190] Alternatively, zein films were stored in HEPES buffer (0.1 M, pH 7.4) containing 2x antibiotic-antimycotic solution.

[0191] 2.2. Protein-alginate blend membrane Regardless of whether they were obtained by the urea or hybridizer method, flat-sheet protein-alginate blend membranes were cast in sodium acetate buffer (0.2 M, pH 4.5) containing 15 g / L CaCl2 and equilibrated in the same buffer for 10 min to a maximum of 3 h. The membranes were then washed with HEPES (0.1 M, pH 7.4) containing 15 g / L CaCl2 and stored in a 70 / 30% w / v ethanol / water solution.

[0192] Using a doctor blade technique (as known to those skilled in the art), a mixture of alginate and mung bean protein is coated onto a PTFE sheet. The sheet is then placed in a pH 4.5 acetate buffer containing 15 g / L calcium chloride. The shift from pH 11 to pH 4.5 causes the protein to coagulate, and the calcium chloride crosslinks the alginate. The membrane is left in the buffer on the bench for 10 minutes. Once the membrane has formed and turned white (off-white), it is removed and placed in a shaking 99.5% glycerin bath for 10 minutes.

[0193] 2.3. Protein-agarose blend membrane Regardless of whether the urea or hybridizer method was used, the protein-agarose blend membrane was cast from a hot solution at 40°C into sodium acetate buffer (0.2 M, pH 4.5) and equilibrated in the same buffer for 10 min to a maximum of 3 h. The membrane was then washed with HEPES (0.1 M, pH 7.4) containing CaCl2 (15 g / L) and stored in a 70 / 30% w / v ethanol / water solution.

[0194] 3. Membrane cross-linking: 3.1 Protein-alginate cross-linked with transglutaminase (TG) Zein-Alginate-TG: Zein-alginate membranes prepared as described above were incubated for 24 hours at 4°C in MooGloo™ solution (TG) (25% w / v) purchased from Modernist Pantry (Elliott, ME), containing HEPES (0.1 M, pH 7.4) and CaCl (15 g / L). 125 mL of MooGloo™ solution was applied to each membrane. Each membrane was then washed twice with 250 mL of HEPES (0.1 M, pH 7.4) containing CaCl (15 g / L). Finally, the membranes were stored in HEPES (0.1 M, pH 7.4) containing CaCl (15 g / L) and 2x concentrated penicillin-streptavidin and antimycotic.

[0195] PPI-alginate-TG: PPI-alginate membranes prepared as described above were incubated for 24 hours at 4°C in MooGloo™ (TG) solution (25% w / v) containing HEPES (0.1 M, pH 7.4) and CaCl2 (15 g / L). 125 mL of MooGloo™ solution was applied to each membrane. Each membrane was then washed twice with 250 mL of HEPES (0.1 M, pH 7.4) containing CaCl2 (15 g / L). Finally, the membranes were stored in a 70 / 30% w / v ethanol / water solution.

[0196] Brown rice-alginate-TG: Brown rice-alginate membranes prepared as described above were incubated for 24 hours at 4°C in MooGloo™ (TG) solution (25% w / v) containing HEPES (0.1 M, pH 7.4) and CaCl2 (15 g / L). 125 mL of MooGloo™ solution was applied to each membrane. Each membrane was then washed twice with 250 mL of HEPES (0.1 M, pH 7.4) containing CaCl2 (15 g / L). Finally, the membranes were stored in a 70 / 30% w / v ethanol / water solution.

[0197] Mungbean-alginate-TG: Mungbean-alginate membranes prepared as described above were incubated for 24 hours at 4°C in MooGloo™ (TG) solution (25% w / v) containing HEPES (0.1 M, pH 7.4) and CaCl2 (15 g / L). 125 mL of MooGloo™ solution was applied to each membrane. Each membrane was then washed twice with 250 mL of HEPES (0.1 M, pH 7.4) containing CaCl2 (15 g / L). Finally, the membranes were stored in a 70 / 30% w / v ethanol / water solution.

[0198] 3.2 Thermal cross-linking with glycerol 3.2.1 Protein membranes As water is exchanged throughout the porous structure, the membrane changes from translucent to transparent. From there, the membrane is removed and placed in a third bath set at 130 °C for 10 minutes. Once the protein is crosslinked, the membrane is placed in a final bath containing HEPES buffer at pH 7.4 to ensure the scaffold is at physiological pH for biological performance.

[0199] SPI:SPI flat-sheet membranes were cast in sodium acetate buffer (0.2 M, pH 4.5) using a PTFE support sheet and equilibrated in the same buffer for 10 min to 3 h. The PTFE-supported membrane was then transferred to a glycerol bath, where the aqueous solution was exchanged with glycerol for 10 min to 3 h. The membranes were then thermally crosslinked either through a heated glycerol bath or by using an oven. In the first case, the membrane was transferred to a stirred glycerol bath at 100 °C and incubated for 10 min. Various temperature gradients were then investigated by varying the final temperature of the glycerol bath (between 110 °C and 140 °C) and the temperature increment. For oven treatment, the membranes were incubated at various temperatures ranging from 100 °C to 140 °C for various durations from 10 to 24 h.

[0200] Mungbean: Mungbean flat-sheet membranes were cast in sodium acetate buffer (0.2 M, pH 4.5) using a PTFE support sheet and equilibrated in the same buffer for 10 minutes to 3 hours. The PTFE support membrane was then transferred to a glycerol bath, and the aqueous solution was exchanged with glycerol for 10 minutes to 3 hours. The membranes were then thermally crosslinked either through a heated glycerol bath or by using an oven. In the first case, the membrane was transferred to a stirred glycerol bath at 100°C and incubated for 10 minutes. Various temperature gradients were then investigated by varying the final temperature of the glycerol bath (between 110°C and 140°C) and the temperature increment. For oven treatment, the membranes were incubated at various temperatures ranging from 100°C to 140°C for various durations from 10 to 24 hours.

[0201] Wheat gluten: Wheat gluten flat-sheet membranes were cast in sodium acetate buffer (0.2 M, pH 4.5) using a PTFE support sheet and equilibrated in the same buffer for 10 minutes up to 3 hours. The PTFE support membrane was then transferred to a glycerol bath, and the aqueous solution was exchanged with glycerol for 10 minutes to 3 hours. The membrane was then thermally crosslinked either through a heated glycerol bath or by using an oven. In the first case, the membrane was transferred to a stirred glycerol bath at 100°C and incubated for 10 minutes. Various temperature gradients were investigated by varying the final temperature of the glycerol bath (between 100°C and 140°C). For oven treatment, the membrane was incubated at various temperatures ranging from 100°C to 140°C for various durations from 10 hours to 24 hours.

[0202] 3.2.2 Protein-alginate membrane Mung bean-alginate: Mung bean-alginate flat-sheet membranes were cast in sodium acetate buffer (0.2 M, pH 4.5) containing CaCl2 (15 g / L) using a PTFE support sheet and equilibrated in the same buffer for 10 min to 3 h. The PTFE support membrane was then transferred to a glycerol bath, and the aqueous solution was exchanged with glycerol for 10 min to 3 h. The membrane was then thermally crosslinked either through a hot glycerol bath or by using an oven. In the first case, the membrane was transferred to a stirred glycerol bath at 100 °C and incubated for 10 min. Various temperature gradients were then investigated by varying the final temperature of the glycerol bath (between 110 °C and 140 °C) and the temperature increment. For oven treatment, the membrane was incubated at various temperatures ranging from 100 °C to 140 °C for various durations from 10 to 24 h. See Figure 13.

[0203] Wheat gluten-alginate: Wheat gluten-alginate flat-sheet membranes were cast using a PTFE support sheet in sodium acetate buffer (0.2 M, pH 4.5) containing CaCl2 (15 g / L) and equilibrated in the same buffer for 10 min to 3 h. The PTFE-supported membrane was then transferred to a glycerol bath, and the aqueous solution was exchanged with glycerol for 10 min to 3 h. The membranes were then thermally crosslinked either through a heated glycerol bath or by using an oven. In the first case, the membrane was transferred to a stirred glycerol bath at 100 °C and incubated for 10 min. Various temperature gradients were then investigated by varying the final temperature of the glycerol bath (between 110 °C and 140 °C) and the temperature increment. For oven treatment, the membranes were incubated at various temperatures ranging from 100 °C to 140 °C for various durations from 10 to 24 h.

[0204] Zein-alginate: Zein-alginate flat-sheet membranes were cast using a PTFE support sheet in sodium acetate buffer (0.2 M, pH 4.5) containing CaCl2 (15 g / L) and equilibrated in the same buffer for 10 min to 3 h. The PTFE-supported membrane was then transferred to a glycerol bath, and the aqueous solution was exchanged with glycerol for 10 min to 3 h. The membranes were then thermally crosslinked either through a heated glycerol bath or by using an oven. In the first case, the membrane was transferred to a stirred glycerol bath at 100 °C and incubated for 10 min. Various temperature gradients were then investigated by varying the final temperature of the glycerol bath (between 100 °C and 110 °C and between 100 °C and 140 °C). For oven treatment, the membranes were incubated at various temperatures ranging from 100 °C to 140 °C for various durations ranging from 10 to 24 h.

[0205] 4. Film coating 4.1 Bovine collagen coating (Method 1) Mung bean membranes were coated with bovine collagen to increase their affinity for cells and promote cell adhesion and proliferation. Dried 14 mm diameter mung bean membrane discs were immersed in a 3 mg / mL collagen solution at room temperature for 2 hours (20 discs per 20 mL of collagen solution). The collagen solution was then removed, and the discs were placed in 100% ethanol and stored at 4°C before use.

[0206] 4.2 Bovine collagen coating (Method 2) Mung bean membranes were coated with bovine collagen to increase their affinity for cells and promote cell adhesion and proliferation. Dried 14 mm diameter mung bean membrane discs were immersed in a 3 mg / mL collagen solution at room temperature for 2 hours (20 discs per 20 mL of collagen solution). The collagen solution was then removed, and the discs were incubated in a HEPES solution (0.1 M, pH 7.4) at 37°C for 1 hour. The HEPES solution was then removed, and the discs were stored in a 70 / 30 w / v ethanol-water solution at 4°C before use.

[0207] 4.3 Bovine fibronectin coating (Method 1) Mung bean membranes were coated with bovine fibronectin to increase their affinity for cells and promote cell adhesion and proliferation. Dried 14 mm diameter mung bean membrane disks were immersed in a 2.5 mg / mL fibronectin solution at room temperature for 2 hours (20 disks per 20 mL of fibronectin solution). The fibronectin solution was then removed, and the disks were placed in 100% ethanol and stored at 4°C before use.

[0208] 4.4 Chitosan coating Mung bean membranes were coated with chitosan to increase their affinity for cells and promote cell adhesion and proliferation. Dried 14 mm diameter mung bean membrane discs were immersed in a 1% w / v chitosan acetate solution (0.2 M, pH 4.5, 20 discs per 20 mL of chitosan solution) at room temperature for 1 hour. The chitosan solution was then removed, and the discs were placed in a 10% TPP solution and agitated for 3 hours. The discs were then washed twice with MilliQ™ water and stored at 4°C in a 70 / 30 w / v solution.

[0209] keep: Membranes can be stored in 70 / 30 ethanol / MilliQ™ w / v or HEPES containing antibiotic / antimycotic. Even when drying is possible, caution must be exercised to prevent pore collapse. Drying can be achieved using a freeze-drying device. More scalable and flexible membranes can be dried if a separate exchange bath consisting of water and 20-40% glycerin is used to exchange for HEPES. The porous structure can be dried if the membrane pores are filled with 20-40% glycerin. See Figure 38.

[0210] 5. Mechanical Testing of Membranes The mechanical properties of the membranes were characterized in tensile mode using a ZwickRoel testing machine. As shown in Figure 14, the membranes' elastic modulus covers a wide range of values, allowing our material portfolio to comprehensively address diverse hollow fiber design specifications. For example, k-carrageenan-based membranes have an elastic modulus of less than 100 kPa and are therefore suitable as substrates for muscle cell growth and differentiation (see Figure 15). Because the hollow fibers will be part of the final cultivated meat product, the texture profile of actual meat must also be considered in our material design specifications. In this regard, we designed heat-treated soy, agarose blends, and some alginate blends that fall within the elastic modulus range of 100–300 kPa, which is known to be characteristic of meat, particularly whole-cut steaks. The highest mechanical performance in terms of elastic modulus and breaking strain is achieved with pure proteins such as mung bean and zein, or alginate-protein blends. These last materials can be used as structural components that allow the hollow fibers to undergo extensive fabrication steps and maintain operating conditions when finally installed in a bioreactor.

[0211] result 5.1 Optimization of the glycerol method The final glycerol cross-linking process, which includes sequential coagulation (1), neutralization (2), glycerol-water exchange (3), and glycerol heat treatment (4) steps, was validated by examining the effects of each step, as shown in Table 1. After coagulation in acetate and neutralization in HEPES, the absence of glycerol heat treatment (Sample 1, AC-H-0-0) resulted in a mechanically unstable film with a paste consistency (see Figure 16). Similarly, replacing the glycerol treatment with autoclaving (121 °C) resulted in an unstable and brittle film (Sample 4, AC-0-0-HW). Removing the initial acetate coagulation and neutralization steps and applying only the glycerol heat treatment (Sample 3, 0-0-G-HG) also resulted in a powdery, mechanically unstable film (see Figure 16). This highlights the importance of having a coagulated protein network, which is essential for film stability. Furthermore, if coagulation occurs under neutral conditions (HEPES) rather than acidic conditions, very brittle membranes are obtained (Sample 4, 0-HG-HG). Finally, exchanging water with glycerol at room temperature before heat treatment helps avoid the formation of large bubbles due to the sudden expansion of water upon contact with a heated glycerol bath (Sample 5, AC-0-0-HG). As a result, the best membranes were obtained by coagulating the dope solution using an acetate bath, exchanging water with glycerol at room temperature, and finally thermally crosslinking the protein network using a heated glycerol bath (Sample 6, AC-0-G-HG). Compared to the other membrane samples, those obtained according to AC-0-G-HG yielded the most stable membranes with the highest Young's modulus and lowest strain, indicating a higher degree of protein crosslinking (see Figure 17).

[0212] [Table 1] Table 1: Experimental conditions for optimization of the glycerol crosslinking method. AC stands for "0.2 M acetate bath at pH 4.5", H stands for "0.1 M HEPES bath at pH 7.4", G stands for "glycerol bath", HG stands for "hot glycerol bath", HW stands for "hot water treatment" (autoclave 121 °C), 0 stands for "no step performed", Y stands for "yes", and N stands for "no".

[0213] Each step of the glycerol-based heat treatment was further optimized to improve the membrane morphology and mechanical properties. The effect of the acetate coagulation step was investigated by varying the acetate bath duration and keeping both the water-glycerol exchange (10 min) and glycerol-based heat treatment (temperature gradient: 100 °C for 10 min, ramped to 120 °C, and then 30 min at 120 °C) conditions constant. Figure 18 shows the mechanical properties of membranes coagulated from 10 min up to 3 h. No statistical differences in modulus, final strain, or final stress were observed with increasing coagulation time, indicating that coagulation was complete within the 10-min window investigated. These results suggest that 10 min is sufficient to neutralize membrane pH and, therefore, allow for a successful coagulation step. Next, the glycerol-based heat treatment was investigated by keeping the duration of both the coagulation bath (10 min) and the water-glycerol exchange (10 min) constant and varying the heat treatment duration after reaching the final temperature of 120 °C. As shown in Figure 19, increasing the heat treatment time resulted in stronger and tougher membranes, with final strain and stress values ​​doubling and tripling, respectively. After 30 minutes, the membrane's mechanical properties began to plateau, and it was also noted that there was little difference in final stress between the 30-minute and 60-minute samples. Because heat treatment appeared to have a greater effect on membrane mechanical properties, further investigations were conducted to evaluate the effect of the final temperature of the gradient. This time, rheological analysis was used to monitor changes in the membrane's physical properties. Heat treatment was performed directly in the rheometer chamber on membranes that were first solidified (10 minutes) and then subjected to a water-glycerol exchange (10 minutes). As shown in Figures 20 and 21, membranes were subjected to a thermal gradient of 4 degrees / min starting at 20°C and equilibrated at three different final temperatures: 100°C, 120°C, and 140°C. Between 50 and 60°C, the tangent (δ) began to decrease, thus suggesting the onset of the protein annealing process, leading to membrane solidification. Heat-driven protein unfolding and the formation of interchain physical crosslinks are thought to be the mechanism behind the solidification process. Interestingly, a trend in the tangent (δ) is observed upon change in the final temperature of the isothermal gradient.Lower values ​​of tangent (δ) are obtained as the gradient final temperature increases, thus suggesting that the membrane undergoes a strengthening process as the annealing temperature increases. This trend was confirmed by tensile tests performed on samples obtained from the rheological experiments. As shown in Figure 20, increases in modulus, final stress, and strain are observed as the final isothermal temperature increases.

[0214] The formation of membrane structures begins around 50-60°C and continues to form at the same rate regardless of the final isothermal temperature. However, the strength of the final resulting membrane structure appears to be affected by the final isothermal conditions. Higher isothermal temperatures result in membranes with higher elasticity (lower tangent (δ)).

[0215] 6. Stability test in cell culture medium To test the stability of the materials under cell culture conditions, films were incubated in cell culture medium at 37°C for up to 30 days, and mechanical testing was performed at various time points to investigate their integrity. k-Carrageenan and its pea protein isolate blend proved to be highly unstable in cell culture medium, dissolving completely after just one day of incubation. In contrast, the alginate and agarose blends proved to be more stable over longer incubation times. In the latter case, film performance appears to be primarily influenced by the stability of the alginate and agarose polysaccharide components. This finding is supported by the existence of two distinct stability trends depending on the nature of the polysaccharides. The alginate blend undergoes a dramatic decrease in both modulus and strain, while the zein blend experiences a more than 10-fold decrease in modulus. In contrast, the agarose blend maintains its mechanical properties almost completely throughout the entire 21-day incubation period. See Figures 22, 23, 24, and 25.

[0216] In the case of alginate blends, the gradual decline in mechanical stability was thought to be caused by decomplexation of the calcium-glutarate cross-linked polymer network. This hypothesis was supported by the pronounced swelling behavior of the membranes over incubation time, which was quantified as an increase in membrane surface area (Figure 22). In contrast, no swelling was observed in agarose blend-based membranes. The correlation between swelling and mechanical stability trends indicates that the polysaccharide network is the main structural component of the membranes that underwent collapse under culture conditions in the case of alginate.

[0217] To enhance the stability of alginate-protein blends under cell culture conditions, cross-linking of the protein components was investigated. Transglutaminase was chosen as the first cross-linking candidate to test, as it is commonly used in the food industry to prepare processed meats. Again, a decrease in both modulus and strain was observed with increasing incubation time, as shown for brown rice-alginate blends (see Figure 26).

[0218] Thermal annealing was chosen as an alternative technique to induce physical crosslinking of the protein polymer network and ultimately stabilize the membrane during cell culture. To avoid the collapse of the membrane porous structure formed by the phase inverse transition, glycerol was used as both the water exchange medium and the heat transfer vector for the annealing process. Compared to alginate blends, both thermally annealed soybean and mungbean membranes showed no decrease in modulus when incubated at 37 °C in cell culture medium. After 21 days, soybean membranes experienced an increase in modulus, nearly doubling in value. While the breaking strain (elongation at break) was unaffected, in the case of soybean, a slight decrease in surface area suggested a possible further crosslinking process over time. After 30 days of incubation, a slight decrease in the force required to cause breakage was observed for mungbean membranes. Their higher mechanical stability in cell culture conditions compared to alginate-protein blends and higher breaking strain compared to agarose-protein blends make these heat-treated pure protein materials favorable candidates for developing membranes for bioreactor applications. Please refer to Figure 27.

[0219] 7. Porosity Imaging 7.1 Flat sheet membrane The porosity of the fabricated membranes was investigated via scanning electron microscopy. As shown in Figures 28 and 29, respectively, the heat-treated soybean and mungbean protein membranes exhibit heterogeneous porosity, characterized by smaller pores in the submicron range on the surface and larger pores in the 20-50 micron range located in the cross-section. The rapid solidification process occurring at the membrane-bath solution interface during the solidification process is believed to be the origin of the thinner porosity located on the surface. In contrast, the slower solidification process occurring in the core of the membrane allows for greater phase separation, resulting in larger pores. A different scenario was observed in the case of zein and agarose-zein, where homogeneous porosity was observed throughout the membrane. Figure 30 shows that in this latter case, the phase separation process was the result of a fibrillation process, resulting in a very homogeneous pore size distribution. While the present invention is not limited by theory, it is hypothesized that both agarose and zein are known to undergo fibrillation via protein self-assembly. Similar results were observed for alginate-zein and pea-k-carrageenan films (see Figure 31), where biopolymer fibrillation was also a major step in film formation. In contrast, a skinning effect was observed for mung bean-agarose and soybean-alginate films (see Figure 32).

[0220] 7.2 Hollow fiber membranes The porosity of the hollow fibers was investigated using a scanning electron microscope. Figure 33 shows the cross section (top) and surface (bottom) of a mungbean-alginate (15%-0.2%) hollow fiber. The fiber exhibits pores in the 50 micron range or less across the entire cross section, but no skinning effect was observed. The fiber wall thickness was in the 100 micron range, a value targeted to optimize outward nutrient diffusion, taking into account theoretical diffusion typically observed in tissues with thicknesses greater than 200 microns.

[0221] 8. Cell Adhesion and Proliferation Assay The fabricated membranes were tested for cell adhesion and proliferation using the C2C12 (see Figures 34, 35, and 36) and QM7 (see Figure 37) cell lines. Generally, higher adhesion and proliferation rates were obtained with pure protein membranes, a finding supported by the presence of cells with more elongated morphology in both the C2C12 and QM7 cases. The best results were achieved when the protein membranes were coated with cell adhesion proteins such as collagen and fibronectin. In contrast, more spherical, cluster-like aggregated cells were observed in the protein-polysaccharide blends, indicating a lower affinity of the material for both the C2C12 and QM7 cell lines.

Claims

1. 1. A method for producing an edible crosslinked porous hollow fiber or sheet membrane, comprising: a) providing i) one or more edible proteins, ii) one or more solvents, and iii) a forming bath; wherein the one or more solvents or the forming bath also contain one or more multivalent cations or anions or a buffer; b) co-mixing said one or more edible proteins in said one or more solvents to form a mixture; c) extruding the mixture into the forming bath to form an extruded hollow fiber or casting the mixture into the forming bath to form a sheet membrane; d) exposing the extruded hollow fiber or sheet membrane to an energy source selected from one or more of heat and irradiation sufficient to at least partially crosslink the one or more proteins to form an edible crosslinked porous hollow fiber or sheet membrane; Including, the heat in step d) is between 70°C and 140°C and is applied under a pressure of 0 PSI to 20 PSI gauge and a relative humidity of 50% to 100% for 2 to 60 minutes, or the extruded hollow fiber or sheet membrane is immersed in a water bath at 60°C to 100°C at atmospheric conditions; The method wherein said irradiation is selected from the group consisting of electron beam, UV light and gamma irradiation.

2. 10. The method of claim 1, further comprising providing one or more edible polysaccharides, and in step b), co-mixing said one or more polysaccharides with said one or more edible proteins in said one or more solvents.

3. 10. The method of claim 1, further comprising providing a plasticizer, and in step b), co-mixing said plasticizer with said one or more edible proteins in said one or more solvents.

4. 10. The method of claim 1, wherein the one or more proteins are selected from the group consisting of pea, soy, wheat, pumpkin, rice, brown rice, sunflower, canola, chickpea, lentil, mung bean, navy bean, corn, oat, potato, quinoa, sorghum, and peanut.

5. 3. The method of claim 2, wherein the one or more polysaccharides are selected from the group consisting of agar, chitosan, chitin, alginate, sodium alginate, cellulose, hydroxypropyl cellulose, methylcellulose, hydroxypropyl methylcellulose, gellan gum, xanthan gum, pectin, tapioca, guar gum, and bean gum.

6. 10. The method of claim 1, wherein the one or more solvents are selected from the group consisting of water, acetic acid, citric acid, lactic acid, phosphoric acid, malic acid, tartaric acid, sodium hydroxide, ethanol, glycerin, and propylene glycol.

7. 10. The method of claim 1, wherein the forming bath comprises one or more of calcium, zinc, magnesium, iron, and potassium in combination with one or more of: 1) water, acetic acid, citric acid, lactic acid, phosphoric acid, malic acid, tartaric acid, or 2) sodium hydroxide and potassium hydroxide.

8. 3. The method of claim 2, wherein the one or more polyvalent cations or anions or buffers are selected from the group consisting of Ca2+, Mg2+, Fe3+, Zn2+, tripolyphosphate, and trisodium citrate, and the selected one or more polyvalent cations or anions or buffers are capable of at least allowing partial cross-linking of the one or more polysaccharides.

9. 10. The method of claim 1, wherein the mixture of step b) is heated.

10. 10. The method of claim 1, wherein the co-mixing of step b) is carried out at 0°C to 90°C.

11. The method of claim 1, wherein the mixture has a pH of 10-13 and the forming bath has a pH of 3-5.

12. 12. The method of claim 11, wherein after formation, the sheet membrane is neutralized to a pH of 6.8 to 7.

8.

13. 12. The method of claim 11, wherein after formation, the sheet membrane is neutralized to a pH of 7.3 to 7.

5.

14. The method of claim 1 , wherein the irradiation is applied during or after the process.

15. 2. The method of claim 1, wherein the irradiation is from 1 to 100 kGy or from 10 to 50 kGy.

16. 10. The method of claim 1, wherein the porous hollow fiber or sheet membrane has a porosity of 1% to 90%.

17. 10. The method of claim 1, wherein the porous hollow fiber or sheet membrane has a porosity of 50% to 80%.

18. 10. The method of claim 1, further comprising coating the edible crosslinked porous hollow fiber or sheet membrane with a coating to enhance cell adhesion.

19. 20. The method of claim 18, wherein the coating is selected from one or more of fibronectin, fibrinogen, laminin, collagen, gelatin, or short peptide sequences isolated from these proteins.

20. 20. The method of claim 19, wherein the short peptide sequences are selected from one or more of the group consisting of RGD, YIGSR, IKVAV, DGEA, PHRSN, and PRARI.

21. 10. The method of claim 1, further comprising modifying the exterior surface of the edible crosslinked porous hollow fiber to enhance cell adhesion.

22. 10. The method of claim 1, further comprising coating the edible crosslinked porous hollow fiber or sheet membrane with a plasticizer.

23. The method of claim 21, wherein the modification of the outer surface is selected from one or more of plasma, corona, abrasion, etching, ablation, or sputter coating.

24. 10. The method of claim 1, wherein the protein is powdered or pulverized before dissolving in the solvent.

25. 10. The method of claim 1, wherein the protein is at least 70% pure.

26. 3. The method of claim 2, wherein the polysaccharide is at least 70% pure.

27. 3. The method of claim 2, wherein the ratio of protein to polysaccharide in the mixture is from 10:1 to 1:10 or from 1:99 to 99:

1.

28. 3. The method of claim 2, wherein the ratio of protein to polysaccharide in the mixture is from 4:1 to 1:

4.

29. 3. The method of claim 2, wherein the ratio of protein to polysaccharide in the mixture is 1:1 or 7:

1.

30. A hollow fiber or sheet membrane made by any of the methods of claims 1 to 29.

31. 1. A method for producing an edible crosslinked porous hollow fiber or sheet membrane, comprising: a) providing i) one or more edible proteins, ii) one or more edible polysaccharides, iii) one or more solvents, and iv) a forming bath, said forming bath comprising one or more of calcium, zinc, magnesium, iron, and potassium in combination with 1) one or more of water, acetic acid, citric acid, lactic acid, phosphoric acid, malic acid, tartaric acid, or 2) one or more of sodium hydroxide and potassium hydroxide; b) co-mixing the one or more edible proteins and one or more edible polysaccharides in the one or more solvents to form a mixture; c) extruding the mixture into the forming bath to form an extruded hollow fiber or casting the mixture to form a sheet membrane; d) exposing the extruded hollow fiber or sheet membrane to an energy source selected from one or more of heat and irradiation sufficient to at least partially crosslink the one or more proteins to form edible crosslinked porous hollow fibers; Including, the heat in step d) is between 70°C and 140°C and is applied under a pressure of 0 PSI to 20 PSI gauge and a relative humidity of 50% to 100% for 2 to 60 minutes, or the extruded hollow fiber or sheet membrane is immersed in a water bath at 60°C to 100°C at atmospheric conditions; The method wherein said irradiation is selected from the group consisting of electron beam, UV light and gamma irradiation.

32. 32. A hollow fiber or sheet membrane made by the method of claim 31.

33. Any of claims 1 to 29, 31 and 32, wherein one or more proteins, one or more polysaccharides, one or more solvents, plasticizers and / or one or more components of the forming bath are generally recognized as safe (GRAS) by the U.S. Food and Drug Administration (FDA).

34. Any of claims 1 to 29, 31 and 32, wherein the obtained sheet membrane or hollow fiber undergoes 10 to 50% water exchange with glycerol in order to dry without pore collapse.

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