Edible fiber scaffolds and methods, systems, and devices for generating and using the same

Abstract

Disclosed herein are edible fiber scaffolds, methods of generating an edible fiber scaffold, devices for generating modified edible fiber scaffolds, and systems for producing strands of cultured meat tissue. The edible fiber scaffolds may comprise, for example, soy protein isolate (SPI) and at least one additive, e.g., glycerol or pectin. The glycerol or pectin may facilitate advantageous physical properties that mimic natural meat products. This disclosure also provides methods, systems, and devices for facilitating the culture of cells on the edible fiber scaffolds.

Description

T002744, T002850, and T002851Aty. Dkt. No. 166118.01557EDIBLE FIBER SCAFFOLDS AND METHODS, SYSTEMS, AND DEVICES FOR GENERATING AND USING THE SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application No. 63 / 688,687 filed August 29, 2024, U.S. Provisional Patent Application No. 63 / 688,688 filed August 29, 2024, and U.S. Provisional Patent Application No. 63 / 763,059 filed February 25, 2025. The entire contents of each of which are hereby incorporated by reference.FEDERAL FUNDING

[0002] This invention was made with government support under grant number 2021-69012- 35978 awarded by the USDA. The government has certain rights in the invention.BACKGROUND

[0003] Conventional animal agriculture for the production of meat (muscle and / or fat tissue) is linked to numerous drawbacks such as environmental degradation, zoonic disease emergence, antimicrobial resistance, and animal welfare concerns. To provide the world with alternatives to animal products having reduced negative impacts on animals and the environment, there is increasing interest in producing cultured (in vitro) tissue such as meat.

[0004] Cultured meat (also called in vitro, cultivated, and / or lab grown meat) is prepared using tissue and bioengineering techniques in vitro as an alternative to traditional animal agriculture. By directly growing meat (muscle and fat tissue) in vitro, energy and nutrients may be more efficiently focused in the produced product. The time frame to generate cultured meat tissues in vitro is faster than traditional animal agriculture and may only require weeks as opposed to months or years for pork and beef, for example. Moreover, tight control over cell biology during tissue cultivation, as well as the production process, allows for the fine tuning of nutritional parameters by engineering muscle or fat cells to produce vital nutrients that would otherwise not be found (or found only at low concentrations) in conventional meat. Thus, cultured meat production systems may offer healthier, more efficient, and more environmentally friendly alternatives to animal -derived meats.Atty. Dkt. No. 166118.01557

[0005] Scaling the production of cultivated meats in a cost-effective manner is an ongoing challenge and presents a significant challenge to making cultivated meat technology affordable for consumers (e.g., in supermarkets). Existing methods and systems for producing cultivated meats face significant technological barriers which currently prevent the cost-effective production of dense, structured meats such as chops and steaks which comprise over 50% of the US meat market by sales. While research continues to engineer structured meats in vitro in sizes less than 1 cm, unstructured meats such as chicken nuggets have been produced at industrial scales and are sold in select restaurants, including the recent FDA / USDA approvals in the US for Upside Foods and Good Meats. Even if current bioreactor technology used in pharmaceutical and enzyme production is used for bio-production scale up using stirred tank bioreactors, this process is too expensive in energy and materials to justify production of structured meats at a scale desired to impact consumers. Thus, innovative solutions are needed for the large-scale production of cultured meat tissue. The present disclosure provides technical solutions for this need.SUMMARY

[0006] In an aspect of the current disclosure, edible fiber scaffolds are provided. In some embodiments, the edible fiber scaffolds comprise about 10% (weight) soy protein isolate (SPI) to about 30% (weight) SPI and at least one scaffold additive selected from pectin and glycerol.

[0007] In an aspect of the current disclosure, methods of generating an edible fiber scaffold are provided. In some embodiments, the methods comprise (a) preparing an edible fiber dope, wherein the edible fiber dope comprises about 10% (weight / volume) soy protein isolate (SPI) to about 30% (weight / volume) SPI and at least one scaffold additive; (b) extruding the edible fiber dope to form an extruded edible fiber dope; and (c) contacting the extruded edible fiber dope with a coagulation solution to generate the edible fiber scaffold.

[0008] In an aspect of the current disclosure, compositions comprising the edible fiber scaffold generated by the disclosed methods are provided.

[0009] In an aspect of the current disclosure, strands of cultured meat tissue are provided. In some embodiments, the strands of cultured meat tissue comprise an edible fiber scaffold comprising about 10% (weight) soy protein isolate (SPI) to about 30% (weight) SPI and at least one scaffold additive selected from pectin and glycerol and a plurality of cells grown on the edible fiber scaffold.Atty. Dkt. No. 166118.01557

[0010] In an aspect of the current disclosure, food products are provided. In some embodiments, the food products comprise the compositions comprising edible fiber scaffolds of this disclosure and / or the strands of cultured meat tissue of this disclosure.

[0011] In an aspect of the current disclosure, devices for generating a modified edible fiber scaffold are provided. In some embodiments, the devices comprise (a) a movement mechanism to extend an edible fiber scaffold into the device, wherein the movement mechanism is controlled by at least one controller; (b) a first chamber housing a component for treating the edible scaffold; (c) a second chamber housing a wetting system.

[0012] In an aspect of the current disclosure, systems for producing strands of cultured meat tissue are provided. In some embodiments, the systems comprise: (a) an edible fiber scaffold; (b) a movement mechanism configured to move the scaffold into a culture medium contact area in a bioreactor or vessel to put the edible fiber scaffold in contact with a culture medium; (c) a seeding mechanism configured to seed cells onto the scaffold; and (d) a cell growth area inside the bioreactor or vessel configured to grow the cells seeded on the scaffold to increase the number and / or density of the cells on the scaffold to form a strand of meat tissue.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention.

[0014] FIG. 1 depicts an overview of a system and process for producing cultured meat tissue in accordance with certain aspects of the disclosure.

[0015] FIG. 2 depicts an overview of a system and process for producing fibers and fibers having cells thereon in accordance with certain aspects of the disclosure.

[0016] FIG. 3 depicts a flow chart of steps that may be involved in producing the cultured meat tissue, in accordance with certain aspects of the present disclosure.

[0017] FIG. 4 depicts a flow chart of steps that may be involved in producing seeded fiber and / or the cultured meat tissue, in accordance with certain aspects of the present disclosure.Atty. Dkt. No. 166118.01557

[0018] FIG. 5 is a diagram showing components and steps of a fiber seeding process.

[0019] FIG. 6 is a diagram showing insertion of a seeded fiber into a bioreactor.

[0020] FIG. 7 is a diagram showing an example retrieval of a seeded fiber from a bioreactor.

[0021] FIG. 8 is a diagram showing an example continuous fiber seeding and / or culture process and components.

[0022] FIG. 9 depicts a flow chart of steps that may be involved in producing seeded fiber and / or the cultured meat tissue, in accordance with certain aspects of the present disclosure.

[0023] FIG. 10 depicts a flow chart of steps that may be involved in producing seeded fiber and / or the cultured meat tissue, in accordance with an example batch process of the present disclosure.

[0024] FIG. 11 depicts a flow chart of steps that may be involved in producing seeded fiber and / or the cultured meat tissue, in accordance with an example continuous process of the present disclosure.

[0025] FIG. 12 depicts an example fiber feeding and plasma treating mechanism in accordance with certain aspects of the disclosure.

[0026] FIG. 13 depicts an example motorized syringe pump in accordance with certain aspects of the disclosure.

[0027] FIG. 14 depicts an example of a mechanical system to coordinate the motion of a fiber feeding system relative to a bioreactor.

[0028] FIG. 15 depicts an example control system used for controlling a prototype system showing stepper motor driver integrated circuits and MOSFETs for heating sections of the conveyor belt along with the film thereon.

[0029] FIGs. 16A, 16B, 16C, and 16D show initial testing pf plasma coating showed significant improvement for GFP-C2C12 cell attachment on: (A) cotton and (B) silk fibers (n=4 for each fiber type, Cl -4 and SI -4). However, the proportion of cells attached relevant to the number of cells delivered to the fibers was small (<50% on average of the 20,000 cells delivered). These levels were not adequate to coat the fibers and support cell growth and proliferation in the time frames relevant for cultivated meat applications, thus, alternative fibers and coatings were pursued. Scale bars = 100 um. All images taken at 4x. GFP-C2C12 cells shown in green in (A) and (B). Fiber diameters were as follows: C-l = 1 mm, C-2 = 1.5 mm, C-3 = 2 mm, C-4 = 2.5 mm, SI = 1 mm, S-2 = 1.5 mm, S-3 = 2 mm, S-4 = 2.5 mm. All fibers were cut to 2.5 cm long. Non-GFP C2C12Atty. Dkt. No. 166118.01557 cell attachment in (C) and (D) was analyzed using unpaired two-tailed T-tests with Tukey’s post- hoc comparison. Additional one-way ANOVA testing showed that fiber diameter did not impact cell attachment.

[0030] FIG. 17A-L shows initial pretreatment results in increased cell attachment at 24 h. Pretreatment resulted in even and cell attachment on the silk and cotton fibers. Fibronectin coating did not significantly improve initial cell attachment on the pretreated fibers. Compared to the minimal attachment on untreated silk (FIG. 3A) and cotton (FIG. 3B) fibers, cells adhered throughout the fibers and covered the individual fiber strands. Scale bar = 1 OOum. All images taken at 4x.

[0031] FIGs. 18A, 18B, 18C, 18D, and 18E show C2C12 cells were viable over several weeks in culture on pretreated cotton and silk fibers. (A) Alamar blue assay showed cells were viable cotton, and silk fibers over 10 weeks in culture. (B) Pretreatment of the cotton and silk fibers with boiling and nonionic detergents did not compromise fiber stress at failure. (C) Over 8 weeks in culture, cotton and silk fibers seeded with cells had significantly lower stress at failure compared to fibers in cell culture conditions (37oC, in culture medium, 5% CO2) without cells. Statistical analysis of fiber mechanical properties by unpaired two-tailed T-tests with Tukey’s post hoc correction. (D, E) Details of cells on fibers shown via ECM imaging. Pretreatments of fibers consisted of washes in nonionic detergent and boiling in water. One-way ANOVA was used to evaluate differences in cell attachment between fiber types. Values shown are mean standard deviation. * = p<0.05; ** = p<0.01.

[0032] FIGs. 19A, 19B, 19C, 19D, 19E, 19F, 19G, 19H, 191, 19J, 19K, and 19L show cells progressively coated the pretreated non-degummed and degummed silk fibers with secreted extracellular matrix (ECM) over 8 weeks in culture. Non-degummed silk refers to fibers from commercial silk pretreated with the nonionic detergent and boiling in water. Degummed silk refers to commercial silk degummed following standard protocols (Rockwood et al., 2011) and with nonionic detergent and boiling in water. Compared to acellular fibers (A, B, E, F), cell-seeded silk fibers were mostly coated with ECM at 5 weeks in culture (C, D, G, H; note fibrous morphology is mostly covered by sheets of ECM). At 8 weeks the fiber morphology was no longer visible, and the fiber surface took on a smooth appearance from the secreted ECM (I-L; sheets of ECM are obscuring the individual fibrils that make up the larger silk fiber). All scale bars are listed on individual images.Atty. Dkt. No. 166118.01557

[0033] FIGs. 20A, 20B, 20C, 20D, 20E, 20F, 20G, and 20H show cells progressively coated the pretreated (washed with nonionic detergent and boiled in water) cotton fibers with secreted extracellular matrix (ECM) as they grew over 8 weeks in culture. Compared to acellular fibers (A, B), cell-seeded cotton fibers were mostly coated with ECM at 5 weeks in culture (C, D; note fibrous morphology is mostly covered by sheets of ECM). At 8 weeks the fiber morphology was no longer visible, and the fiber surface took on a smooth appearance from the secreted ECM (I-L; sheets of ECM are obscuring the individual fibrils that make up the larger cotton fiber). All scale bars are listed on individual images.

[0034] FIG. 21 A-0 shows initial screening of edible commercial fibers. After approximately 10 days in culture the mungbean, wheat gluten and soy fibers disintegrated, thus, were not pursued further. Green fluorescent protein(GFP)-C2C12 cells are shown in green. Scale bars = 100 um.

[0035] FIG. 22 shows mechanical testing of edible fiber material properties. The initial fiber assessments found that only the cotton and silk fibers had mechanical properties that supported long-term cell culture for cultivated meat applications.

[0036] FIG. 23 A-0 shows initial fiber screening for silk. Cells did not adhere well to silk within 24 h, thus the need for pretreatment. Note the significant autofluorescence that allows for clear visualization of the fiber structure and morphology, but still permits cell visualization (white arrows in A, D, G, K; cells are visible as brighter punctate spots on the fibers). After 24 hours minimal cells are adhered to the silk fibers). Scale bar = 100 um. All images taken at 4x.

[0037] FIG. 24A-O shows initial fiber screening results for cotton. Cells did not adhere well to cotton within 24 h, determining the need for pretreatment. Note the significant autofluorescence that allows for clear visualization of the fiber structure and morphology, but still permits cell visualization (white arrows showing both individual cells and clusters, 3 G, H I, N, O). Scale bar = 100 um. All images taken at 4x.

[0038] FIG. 25A, 25B, 25C, and 25D shows (A) Western Blot (WB) analysis of C2C12 cells grown on pretreated cotton, non-pretreated silk, and pretreated degummed silk show cells remain viable after 5 weeks in culture, and produce myosin heavy chain (MHC) throughout the experiment, indicating ongoing myogenesis. P-actin levels show cell viability higher on pretreated silk fibers (darker bands) compared to cotton and non-pretreated silk fibers (lighter bands). (B) At 5 weeks in culture, cell attachment was significantly higher on pretreated degummed silk, compared to pretreated commercial silk and cotton fibers. By 8 weeks in culture, cell attachmentAtty. Dkt. No. 166118.01557 remained significantly higher on pretreated degummed silk compared to pretreated commercial silk, but was no longer significantly higher than cell attachment on pretreated cotton fibers.[00391 FIG. 26A-F shows fluorescent images of cells grown in pretreated silk and cotton fibers over 8 weeks to show that C2C12 cells grow and differentiate within the fibers. In both cotton and silk fibers, early myotube formation is evident at 3 weeks in culture (A and D, white arrows). Cell number increases from week 3 to week 4 (DAPI staining in B shows much higher cell numbers in cotton fibers between weeks 3 and 4). White arrows indicate continued myotube formation at 4 weeks (B, E). By 8 weeks there are large numbers of cells on both the cotton (C) and silk (F) fibers. MF20 production at 3, 4, and 8 weeks (shown in red) suggests ongoing myogenic differentiation. Despite the strong autofluorescence from the silk and cotton scaffolds, cells and myotubes are visible at all time points. All scale bars = 100 um. All images taken at 20x.

[0040] FIG. 27A-F shows bovine satellite cells cultured on cotton (A, B, C) and degummed silk (D, E, F) at week 4. MF20 (red) is expressed in bovine satellite cells adhered to both cotton and degummed silk. The expression confirms the cells used here are myogenic stem cells. Note the solid blue autofluorescence in the myotubes, with punctate blue staining indicating cell nuclei. White arrows (C, D, E, F) indicate multinucleated myotube formation. Blue=cell nuclei, red=MF20. Scale bars = 200 um.

[0041] FIG. 28 shows an image of Isolated Pork Fascicle.

[0042] FIG. 29 shows ultimate tensile strength (UTS) of Skeletal Muscle Tissues Across Common Meat Species.

[0043] FIG. 30 shows Young's Modulus of Skeletal Muscle Tissues Across Common Meat Species.

[0044] FIG. 31 shows Maximum Strain of Skeletal Muscle Tissues Across Common Meat Species.

[0045] FIG. 32 shows Pectin and Glycerol Concentration Effects on Fiber Formation. Check marks indicate pectin-glycerol combinations that successfully produced fibers, while Xs represent formulations where fiber formation was unsuccessful.

[0046] FIG. 33 shows mechanical Comparison of Prototype Soy Protein Isolate Fibers to Pork Fascicles.

[0047] FIG. 34 shows 3D Surface Comparison of Mechanics of Prototype Soy Protein Prototypes.Atty. Dkt. No. 166118.01557

[0048] FIG. 35 shows Young’s Modulus of Stored SPI Fibers Over Time.

[0049] FIG. 36 shows UTS of Stored SPI Fibers Over Time.

[0050] FIG. 37 shows Maximum Strain of Stored SPI Fibers Over Time.

[0051] FIG. 38 shows Detected Exogenous DNA Over Time.

[0052] FIG. 39 shows a single Layer of Bound SPI Fibers.

[0053] FIG. 40 shows a bundle of SPI Fibers.

[0054] FIG. 41 shows incorporation of cells and cell-derived materials into SPI fibers, (a) Presto Blue assay showing viable and metabolically active muscle and fat cells growing on the surface of SPI fibers after 1 week of culture, (b) 4X image of primary muscle cells stained with Cell Tracker Green growing on the surface of SPI fibers 5 hours after seeding, (c) 40X image of differentiated fat cells stained with BODIPY growing on the surface of SPI fibers after accumulating lipids.

[0055] FIG. 42 shows an overview of the manufacture of meat using protein fibers.

[0056] FIG. 43 shows flowcharts of exemplifying embodiments of scaling meat fiber manufacturing processes without (A) and with (B) cells.

[0057] FIG. 44 shows a flowchart of an exemplifying process of a meat fiber manufacturing process.

[0058] FIG. 45 shows a detailed diagram of a minimalist approach of a meat fiber manufacturing process.

[0059] FIG. 46 shows a flowchart of an exemplifying process of a meat fiber manufacturing process.

[0060] FIG. 47 shows manual fiber drawing from spinneret in coagulant. The scale bar in the bottom right corner of each image equals 10 mm.

[0061] FIG. 48 shows an example of an automatic collection spool.

[0062] FIG. 49 shows an example of trough style bath for coagulation (A) and accumulated wet- spun fibers on spool (B).

[0063] FIG. 50 shows enhanced vats with a driven idler pulley (A), pulling fiber through coagulant (B), the accumulation of fibers (C).

[0064] FIG. 51 shows a coagulate-then-rinse wet-spinner setup with driven pulleys and takeup spool.

[0065] FIG. 52 shows accumulated wet-spun fibers on spool without binder. The scale bar in the bottom right corner of each image equals 1 mm.Atty. Dkt. No. 166118.01557

[0066] FIG. 53 shows accumulated wet-spun fibers without binder removed from spool.

[0067] FIG. 54 shows a visible cross-section of accumulated fibers. The scale bar in the bottom right corner of each image equals 1 mm.

[0068] FIG. 55 shows an overview of seeding method for manufacturing tissues.

[0069] FIG. 56 shows a flowchart for the seeding process.

[0070] FIG. 57 shows a concept diagram of a method of seeding a plasma- treated fiber bundle. The black, thick lines represent hydrophilic fibers; the circles represent cells; and the circle represent a high surface area which attracts fluids leading to the natural formation of menisci which holds cells against fibers.

[0071] FIG. 58 shows sample embodiments of methods of adhering cells to threads using protein coating and plasma treatments.

[0072] FIG. 59 shows a stain of live vs dead C2C12 cells on plasma-treated cotton where green cells are colored green and red cells are colored red. The scale bar in the bottom right corner of the image equals 1 mm.

[0073] FIG. 60 shows cell proliferation of C2C12s on plasma treated fibers using a metabolic assay.

[0074] FIG. 61 shows comparative immunostaining of C2C12s with pre-soaking vs plasma coated cotton on day 3.

[0075] FIG. 62 shows comparative immunostaining of C2C12s with pre-soaking vs plasma coated cotton on day 7.

[0076] FIG. 63 shows comparative immunostaining of C2C12s with pre-soaking vs plasma coated silk on day 3.

[0077] FIG. 64 shows comparative immunostaining of C2C12s with pre-soaking vs plasma coated silk on day 7.

[0078] FIG. 65 shows the results of ANOVA testing of C2C12s on plasma treated scaffolds across days 3, 7, and 10 as illustrated in FIGs. 7-10 for cotton and silk.

[0079] FIG. 66 shows the results of ANOVA testing of C2C12s on plasma treated scaffolds across days 14, 21, and 28.

[0080] FIG. 67 shows cell proliferation of IBSCs on plasma treated fibers using a metabolic assay.Atty. Dkt. No. 166118.01557

[0081] FIG. 68 shows comparative immunostaining of iBSCs with pre-soaking vs plasma coated cotton on day 3.

[0082] FIG. 69 shows comparative immunostaining of iBSCs with pre-soaking vs plasma coated cotton on day 28.

[0083] FIG. 70 shows comparative immunostaining of iBSCs with pre-soaking vs plasma coated silk on day 3.

[0084] FIG. 71 shows comparative immunostaining of iBSCs with pre-soaking vs plasma coated silk on day 28.

[0085] FIG. 72 shows the results of ANOVA testing of iBSCs on plasma treated scaffolds across days 3, 7, and 10 as illustrated in FIGs. 14 and 16 for cotton and silk, respectively.

[0086] FIG. 73 shows the results of ANOVA testing of iBSCs on plasma treated scaffolds across days 14, 21, and 28 as illustrated in FIGs. 15 and 17 for cotton and silk, respectively.

[0087] FIG. 74 shows the distribution of fiber volume via the measurement of wetting capacity of a cotton fiber post plasma treatment.

[0088] FIG. 75 shows the optimization of the minimum attachment time of DF-1 cells on treated cotton thread.

[0089] FIG. 76 shows a comparative analysis of DF-1 cell culture on cotton cellulose thread versus tissue culture plastic (TCP).

[0090] FIG. 77 shows a comparison of cell attachment on Fetal Bovine Serum (FBS) treated, plasma treated, and untreated threads.

[0091] FIG. 78 shows the attached cell number vs the seeding density illustrating the cellular capture of treated fibers.

[0092] FIG. 79 shows a model drawing of one embodiment of the disclosed devices wherein two inlets are shown in the second chamber (wetting chamber).

[0093] FIG. 80 shows experimental data demonstrating that plasma treatment is effective at sterilizing fibers.

[0094] FIG. 81 shows an analysis of liquid absorbed per unit length of fiber.

[0095] FIG. 82 shows an analysis of seeding efficiency over time using a vertical seeding method.

[0096] FIG. 83 shows an analysis of cell viability after seeding on an exemplary fiber.Atty. Dkt. No. 166118.01557

[0097] FIG. 84 shows that plasma treating a fiber is as effective at enhancing cell attachment as FBS adsorption.

[0098] FIG. 85 shows experiments demonstrating the mean volume of liquid absorbed per unit of candidate fiber and also shows that plasma treated fiber wicks water as compared to untreated fiber which repels water.

[0099] FIG. 86 shows an embodiment of the disclosed devices functioning to wet a fiber proceeding through the device.DETAILED DESCRIPTION

[0100] Disclosed herein are, inter alia, edible fiber scaffolds, methods of generating an edible fiber scaffold, devices for generating modified edible fiber scaffolds, and systems for producing strands of cultured meat tissue.Edible Fiber Scaffold

[0101] In an aspect of this disclosure, edible fiber scaffolds are provided. In some embodiments, the edible fiber scaffolds comprise about 10% (weight) soy protein isolate (SPI) to about 30% (weight) SPI and at least one scaffold additive selected from pectin and glycerol. The edible fiber scaffolds may variously be referred to as “fibers” or “scaffolds” depending on context.

[0102] The edible fiber scaffold may comprise about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30% or more SPI by weight, or any subrange or value therein. The edible fiber scaffold may comprise about 25% SPI.

[0103] The at least one scaffold additive may include, but is not limited to, pectin, glycerol, pea protein, wheat gluten, glutenin, gliadin, mycoprotein, rice protein, potato protein, sweet potato protein, mung bean protein, fava bean protein, chickpea protein, lentil protein, hemp protein, quinoa protein, oat protein, sunflower seed protein, pumpkin seed protein.

[0104] Dope and binder solvents may include urea, alkali, salts, co-solvents, reducing agents, cysteine, glutathione, sodium metabisulfite, sodium thiosulfate, buffer, formic acid, denaturants, chaotropic salts, lipids. For example, dope and binder additives may include carrageenan, xanthan gum, alginic acid, konjac gum, agarose, locust bean gum, guar gum, pea fiber, bamboo fiber, oat fiber, potato starch, corn starch, tapioca starch, rice starch, beet juice, coconut oil, sunflower oil,Atty. Dkt. No. 166118.01557 canola oil, palm oil, lecithin, emulsifiers, lipids, yeast, yeast extracts, fungal extracts, miso extracts, liquid aminos, soy sauce, seaweed, kelp powder, tomato paste, powder, onion powder, garlic powder, vegetable broth, smoke flavoring, glutamates, hydrolyzed vegetable protein, herbs and spices, maltodextrin, cellulose derivatives. For example, coagulants may include citric acid, sodium sulfate, acetic acid, ormic acid, gluconic acid, glucono delta-1 actone, hydrochloric acid, buffer (ph < 11), kosmotropic salts, cosolvents, sodium chloride, zinc chloride, emulsions, structured fluids, slurries, enzymes, lipids. For example, rinse may include water, buffers, alcohols, solvents, urea, alkali, salts, co-solvents, reducing agents, cysteine, glutathione, sodium metabisulfite, sodium thiosulfate, buffer, formic acid, denaturants, chaotropic salts, lipids, albumin, lentils / legumes, mycelium and derivatives, hemp seed and derivatives.

[0105] The edible fiber scaffold may comprise pectin, by weight, about 0.01% to about 2%, or any subrange or value therein, e.g., about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2%.

[0106] The edible fiber scaffold may comprise glycerol, by weight, about 0.01% to about 16%, or any subrange or value therein, e.g., about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14% about 15%, about 16%, or more.

[0107] The edible fiber compositions may comprise sodium sulfite, by percent weight / weight of SPI in the edible fiber scaffold, e.g., about 0.01% to about 2.1%, or any subrange or value therein, e.g., about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, or about 2.1%.Atty. Dkt. No. 166118.01557

[0108] The edible fiber scaffolds may further comprise a plurality of cells, e.g., invertebrate cells or vertebrate cells. The plurality of cells may be any suitable cells, e.g., cells used in the production of cultured meats, e.g., muscle or adipose cells, or precursors of muscle or adipose cells. The cells may further comprise cells that generate connective tissue or precursors of cells that generate connective tissue. The cells may comprise precursor cells, pluripotent stem cells, mesenchymal stem cells, satellite cells, myoblast cells, myofibroblast cells, fibroblasts cells, endothelial cells, adipose cells, hepatocytes, cardiomyocytes, or combinations thereof.

[0109] The plurality of cells may be derived from any suitable source, e.g., from animals that are traditionally farmed, e.g., porcine, galline, bovine cells. However, any suitable animal cell may be used, e.g., cells from aquatic organisms, e.g., mollusks, gastropods, teleosts, chondrichthyes, cnidarians, etc., or insects.

[0110] The cells may be bovine, porcine, or galline satellite cells or stromal vascular cells (FIG. FIGs. 40A, B, and C).[01U] The edible fiber scaffold may be present with cells in a suitable culture medium, e.g., B8, RPMI, DMEM, etc. The selection of a culture medium is routine in the art.

[0112] The edible fiber scaffold may be “bundled” to form a composition comprising the edible fiber scaffold wrapped and / or layered on top of itself or multiple edible fiber scaffolds (e.g., as in FIGs. 39 and 40 or FIG. 42). The bundled edible scaffold fibers may be adhered to one another by the intrinsic tackiness or stickiness of the edible fiber scaffolds produced by the wet-spinning process.

[0113] The edible fiber scaffolds may be “woven” into a thread by a twisting process (e.g., as in FIGs. 42-45) with or without cells.

[0114] The edible fiber scaffolds may be adhered using at least one binder, e.g., transglutaminase, gelatin, collagen, fibrin, elastin, animal protein, silk fibroin, seaweed gel, alginate, carrageenan, agarose, algal protein, fungal protein, chitosan, bacterial gel, gellan gum, plant protein, leaf protein, soy protein, wheat gluten, glutenin, gliadin, zein, mung bean protein, rice protein, potato protein, sweet potato protein, fava bean protein, chickpea protein, rapeseed protein, lentil protein, hemp protein, quinoa protein, oat protein, sunflower seed protein, pumpkin seed protein, heparin, heparin sulfate, pullulan, amylose, dextran, starch, cellulose, hemicellulose, lignin, glucomannan, chondroitin sulfate, chitin, polynucleotides, polysaccharide, a glycosaminoglycan, natural polyesters, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic)Atty. Dkt. No. 166118.01557 acid, polycaprolactone, polyhydroxyalkanoates, bioesters, or a polymer material made of a monomer selected from the group consisting of: polylactic acid, polyglycolic acid, poly(lactic-co- glycolic) acid, polyhydroxyalkanoates, monosaccharides, glucose, fructose, mannose, galactose or any derivatives or variants thereof, glycosamines, amino sugars, amino acids and derivatives thereof, nucleotides and derivatives thereof, and fatty acids and derivatives thereof, genipin, albumin, riboflavin A, plasticizers, PEG, quercetin, or tannic acid.

[0115] The edible fiber scaffold may further be “treated” to improve properties of the scaffold, e.g., the ability of cells to adhere to the scaffold. Treating the edible fiber scaffold may comprise exposing the scaffold to e.g., cold plasma (FIGs. 55 and 56), flame, corona discharge, ion beam, electron beam, ionizing radiation, UV, ozone, chemical etching, laser treatment, enzymatic modification, grafting polymerization, surface polymerization, peroxides, or supercritical fluids.Methods of generating an edible fiber scaffold

[0116] In an aspect of the current disclosure, methods of generating an edible fiber scaffold are provided. In some embodiments, the methods comprise (a) preparing an edible fiber dope, wherein the edible fiber dope comprises about 10% (weight / volume) soy protein isolate (SPI) to about 30% (weight / volume) SPI and at least one scaffold additive; (b) extruding the edible fiber dope to form an extruded edible fiber dope; and (c) contacting the extruded edible fiber dope with a coagulation solution to generate the edible fiber scaffold.

[0117] As used herein, “edible fiber dope” refers to a pre-coagulated liquid or semi-solid composition which is used in a coagulation process to generate an edible fiber composition. In some embodiments, the “edible fiber dope” itself is not edible. The edible fiber dope may not be edible, but the resulting coagulated fiber may be edible. In other embodiments, the methods are performed using a “fiber dope” and the resulting fiber, after coagulation, is a “fiber.” The “fiber dope” or “fiber” may or may not be edible. Edibility may refer to being edible by a human, an animal, or a human and / or an animal.

[0118] General methods for wet spinning of soy protein isolates are known in the art and can be found, e.g., in Properties of fibers produced from soy protein isolate by extrusion and wetspinning, Journal of the American Oil Chemists' Society 1995, 72 (12), 1453-1460, which is incorporated by reference herein in its entirety.

[0119] The edible fiber dope may comprise at least one additive including, but not limited to, pectin, glycerol, pea protein, wheat gluten, glutenin, gliadin, mycoprotein, rice protein, potatoAtty. Dkt. No. 166118.01557 protein, sweet potato protein, mung bean protein, fava bean protein, chickpea protein, lentil protein, hemp protein, quinoa protein, oat protein, sunflower seed protein, pumpkin seed protein. For example, dope and binder solvents may include urea, alkali, salts, co-solvents, reducing agents, cysteine, glutathione, sodium metabisulfite, sodium thiosulfate, buffer, formic acid, denaturants, chaotropic salts, lipids. For example, dope and binder additives may include carrageenan, xanthan gum, alginic acid, konjac gum, agarose, locust bean gum, guar gum, pea fiber, bamboo fiber, oat fiber, potato starch, corn starch, tapioca starch, rice starch, beet juice, coconut oil, sunflower oil, canola oil, palm oil, lecithin, emulsifiers, lipids, yeast, yeast extracts, fungal extracts, miso extracts, liquid aminos, soy sauce, seaweed, kelp powder, tomato paste, powder, onion powder, garlic powder, vegetable broth, smoke flavoring, glutamates, hydrolyzed vegetable protein, herbs and spices, maltodextrin, cellulose derivatives. For example, coagulants may include citric acid, sodium sulfate, acetic acid, ormic acid, gluconic acid, glucono delta-1 actone, hydrochloric acid, buffer (ph < 11), kosmotropic salts, cosolvents, sodium chloride, zinc chloride, emulsions, structured fluids, slurries, enzymes, lipids. For example, rinse may include water, buffers, alcohols, solvents, urea, alkali, salts, co-solvents, reducing agents, cysteine, glutathione, sodium metabisulfite, sodium thiosulfate, buffer, formic acid, denaturants, chaotropic salts, or lipids.

[0120] The edible fiber dope may comprise pectin, by weight, about 0.01% to about 2%, or any subrange or value therein, e.g., about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2%.

[0121] The edible fiber dope may comprise glycerol, by weight, about 0.01% to about 16%, or any subrange or value therein, e.g., about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14% about 15%, about 16%, or more.Atty. Dkt. No. 166118.01557

[0122] The edible fiber dope may comprise sodium sulfite, by percent weight / weight of SPT in the edible fiber scaffold, e.g., about 0.01% to about 2.1%, or any subrange or value therein, e.g., about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, or about 2.1%.

[0123] The method may further comprise treating the edible fiber scaffold with, e.g., cold plasma, flame, corona discharge, ion beam, electron beam, ionizing radiation, UV, ozone, chemical etching, laser treatment, enzymatic modification, grafting polymerization, surface polymerization, peroxides, or supercritical fluids.

[0124] The treatment may, in some embodiments, increase surface energy, surface charge, hydrophilicity, and / or capillary action relative to the scaffold before it is treated, e g., with cold plasma.

[0125] The methods may further comprise seeding a plurality of cells on the edible fiber scaffold. “Seeding” as used herein, comprises contacting or applying the plurality of cells onto the edible fiber scaffold, e.g., by soaking the edible fiber scaffold in a medium comprising cells. The methods may comprise multi-extrusion, e.g., co-extrusion of the dope with cells, tri-extrusion, etc. In some embodiments, cells may be one of the at least one additives to the dope.

[0126] Seeding the plurality of cells may comprise activating a movement mechanism to move an edible fiber scaffold into contact with a culture medium, optionally, wherein the culture medium is inside a bioreactor or vessel. Seeding cells may further comprise seeding cells onto the scaffold in a seeding area and may further comprise growing the cells to increase the number and / or density of the cells on the edible fiber scaffold.

[0127] The plurality of cells may comprise any suitable cell, e.g., precursor cells, pluripotent stem cells, mesenchymal stem cells, satellite cells, myoblast cells, myofibroblast cells, fibroblasts cells, endothelial cells, adipose cells, hepatocytes, cardiomyocytes, or combinations thereof. In some embodiments, the plurality of cells comprises muscle cells, muscle progenitors, adipose cells, adipose progenitors, or combinations of the foregoing.

[0128] The plurality of cells may comprise invertebrate cells, vertebrate cells, e.g., porcine, galline, or bovine cells.Atty. Dkt. No. 166118.01557

[0129] The method may further comprise arranging two or more edible fiber scaffolds to form an edible scaffold bundle. The edible fiber scaffold may be arranged in the bundle in any suitable formation, e.g., stacked, or woven in a thread.

[0130] Coagulating may comprise the addition of a coagulant. Examples of the coagulant may include, but are not limited to, any one or more of urea, e.g., 8M urea, polyvalent cation salts, glycerol, citric acid, sodium sulfate, acetic acid, formic acid, polysaccharides, di saccharides, sucrose, maltose, lactose, monosaccharides, fructose, glucose, mannose, galactose, xylose, glucuronic acid, mannuronic acid, gluconic acid, glucono delta-lactone, polyethylene glycol, hydrochloric acid, buffer (ph < 11), kosmotropic salts, nonsolvents, cosolvents, acids, bases, monovalent salts, emulsions, structured fluids, slurries, enzymes, crosslinkers, complexing agents, and lipids.

[0131] In some aspects, at least one step in the method is followed by a rinsing step. The rinsing step may comprise the addition of a rinse, and the rinse may be selected from the group consisting of water, buffers, alcohols, solvents, urea, alkali, salts, co-solvents, reducing agents, cysteine, glutathione, sodium metabisulfite, sodium thiosulfate, buffer, formic acid, denaturants, chaotropic salts, and lipids.

[0132] In some aspects, the method is conducted on a scaffold, and the scaffold may comprise a material selected from the group consisting of gelatin, seaweed gel, collagen, plant protein, soy protein, wheat gluten, zein, animal protein, silk, amylose, algal protein, fungal protein, dextran, alginate, chitosan, starch, heparin, heparin sulfate, pullulan, cellulose, hemicellulose, glucomannan, agar, chondroitin sulfate, chitin, polynucleotides, polysaccharide, a glycosaminoglycan, natural polyesters, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic) acid, and polycaprolactone. As used herein, the term “scaffold” may refer to a structure or matrix used for providing the foundation of growth.

[0133] In another aspect, the present disclosure provides a meat product produced by any one of the methods or systems disclosed herein. In some aspects, a bundle of the edible fiber scaffolds has a diameter of about 0.3 mm to about 1.2 mm. A cross-section of the bundles may reveal a packing arrangement resulting from the method of producing the bundled edible fiber scaffolds. A “cross-section” may refer to a surface or shape that is exposed by making a straight cut through a composition, especially at right angles to an axis. One such possible packing arrangement is a Voronoi packing arrangement. In a Voronoi packing arrangement, each thread is associated withAtty. Dkt. No. 166118.01557 a particle, and the distance of any point within the thread to the particle is smaller than the distance of that point to any other particle. In 2D, Voronoi packing arrangements often comprise hexagons because they are the most efficient way to pack shapes in a plane. Hexagonal shapes have at least two advantages: they ensure that no empty space is left between shapes (or in the case of the present disclosure, threads or fibrous bundles), and they offer the highest ratio between surface area and perimeter. Therefore, in some aspects, a cross-section of the bundle comprises a Voronoi packing arrangement. The Voronoi packing arrangement may be an arrangement of individual fiber cross-sections.

[0134] In another aspect, the present disclosure provides a meat product comprising at least one bundle of the edible fiber scaffolds, wherein the bundle comprises at least two threads, wherein the at least two threads may, optionally, be seeded with cells. In some aspects, the bundle has a diameter of about 0.05 mm to about 5 mm. The threads may have diameters of about 0.3 mm to about 1.2 mm. In some aspects, the bundle comprises a binder, and the binder may comprise transglutaminase, gelatin, collagen, fibrin, elastin, animal protein, silk fibroin, seaweed gel, alginate, carrageenan, agarose, algal protein, fungal protein, chitosan, bacterial gel, gellan gum, plant protein, leaf protein, soy protein, wheat gluten, glutenin, gliadin, zein, mung bean protein, rice protein, potato protein, sweet potato protein, fava bean protein, chickpea protein, rapeseed protein, lentil protein, hemp protein, quinoa protein, oat protein, sunflower seed protein, pumpkin seed protein, heparin, heparin sulfate, pullulan, amylose, dextran, starch, cellulose, hemicellulose, lignin, glucomannan, chondroitin sulfate, chitin, polynucleotides, polysaccharide, a glycosaminoglycan, natural polyesters, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic) acid, polycaprolactone, polyhydroxyalkanoates, bioesters, or a polymer material made of a monomer selected from the group consisting of: polylactic acid, polyglycolic acid, poly(lactic-co- glycolic) acid, polyhydroxyalkanoates, monosaccharides, glucose, fructose, mannose, galactose or any derivatives or variants thereof, glycosamines, aminosugars, aminoacids and derivatives thereof, nucleotides and derivatives thereof, and fatty acids and derivatives thereof.

[0135] The disclosure addresses a range of factors and variables in the process that can be exploited for cultivated meat goals, building upon this core technology, including:

[0136] (a) Materials - different options for the scaffolds for the process can be considered, with edible and safe materials as the goal; the scaffold materials can be provided as prespooled rolls to incorporate into the system as part of a supply chain or the scaffolds can be generated in situ asAtty. Dkt. No. 166118.01557 part of the system; various compositions or composites of the scaffold materials can be considered, to tailor mechanical, stability and nutrition / digestibility goals; the scaffolds can be pre-treated to contain / sequester key growth factors or other media ingredients (e.g., flavor or taste components, antibiotics / antifungals to minimize use when needed) to support the cells growing on the scaffolds, to optimize the processes involved, to help reduce costs, and to optimize consumer acceptance downstream; patterned or unpatterned scaffolds can be used depending on the needs to promote cell interactions and / or alignment; scaffolds can be solid systems as well as porous to house cells on or in the materials; sterilization of the scaffolds can be done in bulk prior to loading into the system, sterilized in place in the device or combinations of the above. When scaffold production occurs in situ, scaffold diameter can be controlled along scaffold length.

[0137] (b) Cells - cells to seed the system can be grown in separate bioreactors and then used here, or can be grown in concert with this system as feeder streams to directly interface, to generate on-demand production streams, or added as prepared pastes or strips to the systems; cells can be added at different points, in different time-dependent sequence and in combinations in the system - cell deposition can occur at the onset to coat the scaffolds in the system, such as by deposition, spray, 3D printing, coating via solutions, and submersion approaches; cells can be single cell types (e.g., muscle, fat, connective tissue) or mixtures of cells depending on the goals; cell growth conditions (time, temperature, atmosphere, media composition) can be manipulated to optimize cell-scaffold outcomes related to texture, nutrition, taste, etc.); cells can be added as-is or with a gelation solution (e.g., alginate, starches, other polymers) to maintain location on scaffold for downstream processing.

[0138] (c) Treatments - scaffolds can be treated by exposure to chemical and physical agents to enhance and diminish specific properties such as scaffold hydration, elastic modulus, sterility, hydrophobicity, handleability, and tensile strength, to name a few. For example, a scaffold can be exposed to ionizing radiation and ionized gas to increase the percentage of cell-adhesive surface chemistry such as amines. Treatments can occur before and after cell seeding. Treatments may occur after seeding with cells and be designed to enhance retention of cells. Such treatments might include environmental controls such as culture conditions of 37°C, 95% humidity, and 5% CO2, or chemical and physical agents such as gelators and crosslinking treatments such as aldehydes. Chemical treatment agents may be applied to scaffolds using methods such as dripping, extrusion, sprayingjetting, misting, electrophoresis, and partial or complete submersion.Atty. Dkt. No. 166118.01557

[0139] (d) Media - the media can be applied in various ways to support cell growth and / or differentiation. This can include: sequestering some of the components within the materials used, to optimize efficiency and utility and reduce costs; media can be applied as a bath within which the scaffolds progress to support cell growth, media can be sprayed on to support the cells as in a car-wash like scenario, media can be mixed with the cell suspension and applied with the cells, media can be regionalized on the scaffolds to support selective cell growth and outcomes in different regions of the scaffolds, similar to a treatment - as in a silk screening process, which is often used on woven scaffolds produced at industrial scales; finally, media can also include antibiotics and / or antifungal components.

[0140] (e) Downsteam Processing - the cell-laden scaffolds can be used in different ways: directly in a folding, spinning, rolling, cutting, braiding, weaving, finishing operation to generate 3D outcomes for subsequent storage, cooking, or packaging for food production or for other downsteam operations (e.g., to provide other companies to use in ingredients); can be spooled up as output and stored for future processing; can be combined with other materials and ingredients as in composite systems to achieve specific food outcomes (e.g., texture, taste, nutritional composition); can be used in further downstream processing steps such as in curing, aging, salting, cooking, pickling or other steps to optimize specific food-related goals; can be milled into smaller pieces for use as ingredients in less structured meat goals (e.g., sausages, burgers, etc.).

[0141] (f) Analytics and Process Control - the system can be integrated with online monitoring and process control to optimize cell outcomes, including imaging to track cell growth and differentiation with adjustments of ingredients and scaffold movement rate to assure consistent quality control at the end of the process; the system can be designed to be continuous or noncontinuous depending on the outcome goals; the entire system can be designed to be computer controlled to avoid any human-handling or potential contamination; the system can be run on- demand or run to generate cell-scaffold supplies for storage or distribution; the system can be designed to be contamination-free and the system can have a fdtration and sterilization system in place to keep contamination minimized; the system can be designed for operation in a cleanroom; the system can be designed for remote operation; the system can be designed for operational resiliency and robustness by incorporating redundancy in process controls; the system can be miniaturized to (for example) output specialty materials suited for research or scaled up to produce cell -containing scaffolds at industrial or commodity scale; finally, the system can be designed toAtty. Dkt. No. 166118.01557 accommodate shifts in throughput such as sudden lack of resource availability, and demand volatility.[01421 Referring now to the drawings, and with specific references to Figures 1-15, in one embodiment, muscle or fat cells are seeded (optionally continuously) onto a scaffold scaffold where the cells proliferate and / or differentiate as the scaffold moves through a bioreactor to form strands of meat tissue.

[0143] In one embodiment the chemistry of the cell culture media within the bioreactor may vary from one end of the bioreactor to the other end. For instance, the composition of the cell culture media at one end of the bioreactor may be different than the composition of the cell culture media at the opposite end of the bioreactor. The difference in the composition of the cell culture media may be such that it drives or encourages the cells to grow at or near one end of the bioreactor while at or near the other end of the bioreactor the cells are encouraged to differentiate.

[0144] A plurality of strands of meat tissue may be twisted together to form a bundle of meat tissue. Preferably the bundle of meat tissue has a structural hierarchy, density, texture and other properties similar to that of animal grown meat. The strand of meat tissue may include both strands of meat cells and / or strands of adipose cells and / or strands having mixtures of meat cells and adipose cells.

[0145] In one embodiment a plurality of strands of meat tissue and / or a plurality of bundles of meat tissue (formed by twisting together strands of meat tissue as described) maybe weaved or knitted into a structured meat tissue. The structured meat tissue may include meat cells and / or adipose cells. In one embodiment the structured meat tissue is a two-dimensional sheet or a three- dimensional meat tissue structure which may subsequently be stacked, folded, and / or rolled onto a spool, or otherwise formed into a three-dimensional assembly. In other embodiments the structured meat tissue may be formed directly into a three-dimensional assembly.

[0146] In vivo, animal skeletal muscles are striated and packed into dense arrangements of scaffold bundles where the underlying structural hierarchy is key to mechanical features. In meat, these features translate to the specific texture and mouthfeel obtained when biting into a whole muscle cut of meat (e.g., steak). Thus, tissue density and structural organization are key outcomes for food texture and consumer acceptance. In the present disclosure, we describe muscle cell-laden, edible scaffolds as an initial step toward meat / muscle-like tissue with structural attributes to mimic native skeletal muscle, for instance for utility in cultivated meat applications. Suitable scaffoldAtty. Dkt. No. 166118.01557 candidates include silk, cellulose / cotton, wheat gluten, zein, mungbean noodle, and soy protein with silk and cellulose / cotton, being preferred. Various coatings and pretreatments may be provided or performed on the scaffold (plasma coating, fibronectin, transglutaminase, alginate) for instance to improve initial cell attachment and proliferation. As described herein, scaffolds were seeded with C2C12 mouse skeletal muscle myoblasts or bovine satellite muscle cells (BSCs) and cultured for up to 8 weeks. Cell survival was high on both scaffold candidates, with extensive cell coating of the scaffolds and extracellular matrix secretion. Cell differentiation and myotube formation were observed at longer culture times. Mechanical testing showed the silk and cotton scaffolds had sufficient mechanical properties for further exploration toward cultivated meat goals. Overall, cells adhered to and coated the scaffolds effectively, properties making them suitable for cultivated meat applications.

[0147] Preferred scaffolds for use in accordance with the systems and methods described herein exhibit any or all of the following properties:

[0148] Availability: the scaffold format should be readily commercially available in large quantities or such that manufacturing could be readily scaled up

[0149] Edibility: raw materials should have GRAS (Generally Recognized as Safe) status for human consumption, or generally be edible or otherwise suitable for human or animal consumption.

[0150] Cost: sourcing or manufacturing the scaffolds should be at a price point that allows them to be used for food-related or medicinal goals.

[0151] Mechanical Properties: ideally the scaffolds are strong enough to be handled and loaded before, between, and / or after bioreactor components (about 1 kPa to about 5 MPa or, e.g., 3 kPa to 40 kPa, UTS range).

[0152] Cell Culture Support: preferably the scaffolds should support cell viability at greater than 80%, with or without coatings for improved adhesion of cells, as needed.

[0153] As shown in Figure 2, cell-coated scaffolds are provided for cultivated meat applications. These cell-coated scaffolds may include meat and / or adipose cells and the scaffold may be selected from a number of commercially available and inexpensive scaffold candidates based on edibility and cell culture compatibility. As an initial proof-of-concept, scaffolds were coated with murine C2C12 cells and cultured for 8 weeks. Cotton (cellulose) and silk protein scaffolds were selected due to their mechanical properties and resistance to degradation under standard cell cultureAtty. Dkt. No. 166118.01557 conditions. C2C12 and bovine satellite cells were then grown on the scaffolds up to 8 weeks and cell attachment, morphology, and differentiation were quantified.[01541 The systems and methods described herein yield a cultured meat that is comparable to conventional animal-sourced meat in nutrition, taste, texture, smell, mouthfeel and sensory properties so as to satisfy consumer preferences that drive the ever-increasing global demand for meat.

[0155] Cultivated meat (also called cellular agriculture, in vitro, cultured, clean, or lab grown meat) aims to produce skeletal muscle and adipose tissues naturally found in animal meats by using in vitro tissue, bioengineering, and bioprocessing techniques. By directly growing meat (muscle and fat) in vitro, energy and nutrients can be more efficiently focused on the protein- enriched outcome, thus bypassing the low feed-to-food energy conversion ratio of calories fed to protein obtained when raising livestock. The time frame to generate cultivated meat tissues in vitro at commercial scale is expected to be significantly faster compared to animal agriculture, requiring days to weeks versus weeks to months, or longer, depending on the target species. For example, cultivated meat may only require a few weeks to reach “maturity”, as opposed to months or years for pork and beef [9-11], The tight control over the cell biology during tissue cultivation, as well as the overall production process, allows for the fine tuning of parameters such as nutritional content, where muscle and fat cells can be fed appropriate ingredients for direct uptake, or bioengineered to produce vital nutrients that would otherwise not be present (or only at low concentrations) in conventional animal meat

[0012] , There are also opportunities to improve food safety at all levels of production in the approaches with cultivated meat, including by reducing the extensive use of antibiotics in livestock, as well as opportunities to avoid contamination. Taken together, multiple factors suggest that cultivated meat production systems can offer healthy, efficient, and environmentally compatible options when compared to traditional animal-sourced livestock meats

[0013] ,

[0156] A major current challenge for cultivated meat is scalability

[0014] , Small-scale production increases the price of cultivated meat, making such products prohibitively expensive for most consumers, or for commercial use, which also limits their potential to positively impact climate change by reducing resource and energy usage. The methods and systems described herein provide, scalable, replicable, and automated processes for cultivated meat production, storage, and distribution, towards viable mass-scale availability

[0015] , To address these challenges, a system thatAtty. Dkt. No. 166118.01557 enables the large-scale construction of tissue engineered muscle and fat for human consumption is proposed.[01571 The present disclosure describes a scaffold-based scaffolding to support cells towards tissue engineered muscle outcomes, and cultivated meat. The scaffold-based scaffolding may be used, for instance, in creating tissue, including with techniques such as braiding, knitting and weaving.

[0158] Commercially available and edible scaffolds, such as cotton, silk, soy protein, mungbean, and durum wheat semolina products (pasta), among others, may be used for tissue engineering scaffolds for cultivated meat applications as disclosed herein. Each of these polymers or materials is commercially available at relatively low costs and in large quantities, which are helpful for eventual large-scale commercial production of cultivated meat via cell culture. Additionally, all of these materials are edible or food safe, or may be provided in food safe forms, although some many require additional processing steps for digestibility or optimization for food- related needs. In the systems and processes described here, these scaffolds may benefit from coatings to maximize cell interactions, and several animal and non-animal derived coatings are described herein. A scalable pre-treatment procedure is also described here to improve cell attachment to the scaffolds without the use of coatings such as laminin and fibronectin [31,32], A simple, rapid, and cost-effective method of preparing commercial scaffolds for use in cultivated meat applications is described here as a first stage of a more complicated process to achieve complex, structured meat-like products via textile engineering.

[0159] Some cell-laden scaffolds have been explored in the context of tissue engineering [30,33— 36], That said, the present disclosure provides edible muscle cell-coated scaffolds suitable for cultivated meat production. The scaffolds may be used to support cell attachment, growth, proliferation, and differentiation. As described below, muscle cell-coated scaffolds were cultured in vitro using a model mammalian cell (murine C2C12s) as well as with a more food-relevant cell (primary bovine muscle satellite cells) to demonstrate effectiveness for cultivated meat-production goals.

[0160] Among other things, a cultivated meat production process and related systems are disclosed that utilize a scaffold, such as a ID or near one dimensional scaffold such a as thread as a scaffold and as a medium of growth, expansion, and / or differentiation of cells in a cultured meat manufacturing process. The disclosure provides a simplified production process with oneAtty. Dkt. No. 166118.01557 dimensional or near one dimensional (ID) cell culture and a reduction in energy and other process control costs that plague known bioreactor systems. A one dimensional seeded-scaffold production line may be paired with a bioreactor or vessel (Figure 1) to addresses scalability challenges by utilizing an edible substrate as the medium for cell growth, expansion and / or differentiation, thereby minimizing medium exchange and optimizing medium utility, providing for efficient cell passaging, and reducing energy consumption. This solution offers a scalable, self-contained (maximum safety and limited risk of contamination), and versatile method, aiming to produce high volumes of dense, structured meat. Further, the system is scalable and allows for the use of modular units under controlled conditions. Inputs, such as spools of edible scaffold to feed into the process can be prepared separately, or may be commercially available, with or without entrained growth factors and other expensive ingredients to minimize costs. Cells can be pregrown in smaller bioreactors or vessels to suitable scales for spray or deposition onto the scaffold systems. Downstream collection of the cell-bearing scaffold can capture the products for further processing into foods (e.g., twisting multiple cell bearing scaffolds together into a larger thread, rope or meat tissue, weaving cell bearing scaffolds, threads, or ropes into structured meat tissues, stacking, folding, cutting or punching out forms, etc.).

[0161] As described, the processes may be carried out in a bioreactor, such as a stirred suspension tank bioreactor or a hollow scaffold bioreactor having hollow scaffold membranes. Other types of bioreactors apparent to those skilled in the art may also be used and are within the scope of the present disclosure such as, but not limited to, rotating wall vessel bioreactors (RWVBs) and packed bed bioreactors. Alternatively, the bioreactor may be a simple bioreactor, providing for adequate sterility and environmental (e.g., temperature control) conditions, or even a simple vessel may be suitable in some instances.

[0162] A pioneering method is provided involving cell-coated scaffolds which are preferably edible and may be provided in the form of scaffolds, enabling a unique approach to creating and forming structured meat. Scaffolds may be edible so that they may be suitable for human or animal consumption and / or they may also serve as a nutrient or food source for the cells themselves. In one example, the scaffold may be stored in a spool. The scaffolds can also be prepared to include any desired components (e.g., growth factors) to provide all, any, or some of the ingredients to drive cell growth and expansion and / or differentiation, including, or in addition to, the basic sugars and salts in the media used for cell growth and expansion and / or differentiation.Atty. Dkt. No. 166118.01557

[0163] Seeding, expanding, differentiating, and / or processing the cells into a foodstuff on a continuous scaffold (which may be a thread of fiber, e.g., the edible fiber scaffolds of this disclosure) minimizes fluid usage (for instance culture media) and creation of waste, processing time, and the risk of contamination, all while maximizing cell viability and growth. As used herein, expansion refers to cell growth in terms of increase in cellular size, weight, volume, population density, and / or cells dividing and increasing in number.

[0164] The cell-coated scaffolds described herein can be spooled, twisted into larger fibers or twisted into roper or yarn, rolled, laminated, wrinkled, woven, braided folded, manipulated and / or combined with additional elements to produce stratified meat, offering flexibility in production and storage. For instance, both (i) the scaffold production process and / or the (ii) cell delivery and growth process on the scaffold may be performed in batch processes or continuous modes, either together or separately.

[0165] The processes may be self-contained, modular, scalable, and capable of automation, facilitating streamlined operations and reducing potential points for contamination in the process.

[0166] Animal skeletal muscles have a dense arrangement of fiber bundles, giving meat its unique texture and mouthfeel. The approach described herein contemplates a new bioreactor design that applies principles from textile production processes to produce large volumes of cellrich tissue in a continuous or batch heterogeneous analogs to desirable cuts of meat having desirable size, shape, texture, visual appearance and taste like or similar to animal-sourced meat.

[0167] Scaffold production and the related manufacture of cell-coated scaffolds can be scaled similar to other manufacturing processes, for instance roll-to-roll manufacturing.

[0168] Cells may adhere to or be incorporated in a scaffold, for instance where the scaffold is a continuous fiber, after which the cells function as passengers throughout the meat production process. When intended for consumption as human or animal food, the cells may not be required to survive or be alive at or near the end of the manufacturing process, only to expand and / or differentiate during the foodstuff manufacturing process, at which point the cells provide the desired density, nutrients, flavors, textures and taste similar to an animal meat. In certain cases, the cells do not need to remain alive and viable in the final cell-bearing structures described below, for instance for use in medicinal applications.

[0169] Various combinations and ranges of scaffolds (textures, digestibility, mechanics, nutrition, thickness, tensile strength, surface morphology, etc.), and / or cells (e g., muscle, fat,Atty. Dkt. No. 166118.01557 connective tissue, etc.) may be utilized to form the core cell-bearing strands of meat tissue, and used as muscle, fat or connective tissue building blocks to assemble into complex, desirable meat architectures.

[0170] Continuous or Batch Production: The production system may operate as a standalone batch process and / or in continuous process that circumvents many of the resource constraints posed by traditional cell culture (e.g. avoiding mass transfer challenges and the associated process control costs, as well as reducing risk of contamination). The scaffold-based system and methods described herein provide a relatively low-cost, high-volume solution that may be capable of generating cultivated meats with structural hierarchy similar to that found in vivo.

[0171] In the system design of the cultivated meat production described herein, the steps or scales progress from the continuous or batch production of seeded, fiber scaffolds through cell seeding, growth, expansion, and, when needed, differentiation, to produce threads bearing cells that can be formed into dense, structured meats. In a preferred embodiment, the fibers with the cells are rolled up or “spooled” to form a roll of meat and scaffold, or twisted, woven or braided to form a structure from which desired cuts of meat can be removed. A thread may be spooled around a central spindle, or a thread may be spooled around itself without a central spool.

[0172] A variety of advantages may be achieved by expanding cells on edible fibers as substrates serving as scaffolds including: (i) certain parts of the techniques may be adapted from methods in the fiber and thread making industry, making economic mass production possible, (ii) the utilization of 1 -dimensional and edible scaffolds enables effective nutrient and oxygen transfer in the cell culture system, as cell-bearing fibers may be fully or partially surrounded by culture media, avoiding complications inherent to perfusion systems for 3D cell growth for foods, (iii) a sealed, continuous process may minimize fluid usage (for instance culture media), process time, and the risk of contamination, while at the same time maximizing cell viability, (iv) the technique facilitates fabrication of cell / scaffold constructs with cell alignment (e.g., due to the mechanical draw on the scaffolds, surface features provided on the scaffolds, or stretching the scaffolds), controlled microstructure, mechanical properties, and cellular distribution, which plays an important role in the engineering of structured tissues like meat (e.g., marbling, texture, cut, organoleptics).

[0173] Further advantages of the system and methods may include: (i) economic mass production; (ii) effective nutrition and / or oxygen transfer, circumventing complications of 3D cellAtty. Dkt. No. 166118.01557 growth; (iii) minimization of waste and maximization of efficiency; (iv) controlled fabrication of cell and cell / scaffold constructs, ensuring the desired characteristics like cell density, marbling, texture, taste, and / or organoleptics.

[0174] For instance, in the event of contamination, a contaminated bioreactor may be shut down, cleaned and / or sterilized and the limited amount of scaffold and cells within that bioreactor may be removed as waste or recycled. The systems and methods described here may be configured in a way that limits the amount of loss due to a contamination event, for instance because the amount of scaffold and cells being treated at a given time could be less as compared to a traditional large bioreactor.

[0175] Potential sources of affordable, edible scaffold materials may include animal or nonanimal derived materials. For example, many cost-effective materials for engineering tissue scaffolds are animal derived. Non-animal derived materials such as wheat gluten, chitosan, zein, starch, soy, may also be used as scaffolding and fiber to be used in the systems. The materials are preferably edible and may be stand-alone fibers or they may be coated fibers, either of which may be conveniently stored on a spool prior to use.

[0176] Additional materials for the scaffold may include gelatin, seaweed gel, collagen, plant protein, animal protein, algal protein, fungal protein, dextran, alginate, chitosan, starch, heparin, heparin sulfate, pullulan, cellulose, hemicellulose, glucomannan, agar, chondroitin sulfate, chitin, polynucleotides, polysaccharide, a glycosaminoglycan, natural polyesters, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic) acid, polycaprolactone, polyhydroxyalkanoates, and / or a bioester. The scaffold may be a synthetic polymer or any variant thereof, including with combinations with the foregoing materials. Examples of synthetic polymers include, but are not limited to, synthetic polypeptides for instance from microorganisms or prepared by synthetic chemical techniques, bioesters including those extracted from microorganisms or prepared by chemical synthesis from their monomers such as polylactic acid, polyglycolic acid, poly(lactic-co- glycolic) acid, polyhydroxyalkanoates, or any other synthetic polymer prepared from generally recognized natural monomers such as monosaccharides (such as glucose, fructose, mannose, galactose or any derivatives or variants such as glycosamines, aminosugars, and the like, aminoacids and derivatives thereof, nucleotides derivatives thereof, fatty acids and derivatives thereof, and / or combinations of the foregoing. Combinations of the natural and synthetic materials described above are expressly contemplated by this disclosure.Atty. Dkt. No. 166118.01557

[0177] Suitable additional materials for the scaffold may include flavoring, flavor enhancer, colorant, color enhancer, salt, acidity regulator, buffer, thickener, emulsifiers, stabilizer, solubilizer, nutritional enhancer, probiotic, prebiotic, saponin, antioxidant, essential fatty acid, mineral, light scattering agents, ingredients for altering the appearance, or the texture, or any combinations thereof.

[0178] In another embodiment, the scaffold further comprises one or more bioactive ligands. The bioactive ligands may interact with the cells or bind biomolecules that interact with biomolecules of the cells that may be distributed within the scaffold. The bioactive ligands may be helpful to direct cell differentiation or induce cells to form a tissue or a cultured meat. Certain ligands may enhance cell adhesion, growth, and / or proliferation of the cells. The identity of the bioactive ligands may depend upon the identity of the target cells, and some examples of suitable bioactive ligands include: carboxyl, amine, phenol, guanidine, thiol, indole, imidazole, hydroxyl, sulfate, norbornene, maleimide, laminin, fibrinogen, adhesion molecules such as inmuglobulins, cadherins, selectins, and integrins, as well as fibronectins, poly-L-omithine, collagen, vitronectins, lectin, poly-ornithine, poly-L-lysine, poly-D-lysine, poly-s-lysine, peptides, cyclic peptides, RGD- containing peptides (Arg-Gly-Asp), RGDS-containing peptides (Arg-Gly-Asp-Ser), or any other compound or substance effective to promote cell adhesion to the scaffold.

[0179] Cells suitable for growth in accordance with this disclosure may include a variety of living cells, preferably muscle cells, and optionally, further comprising a plurality of precursor cells, satellite cells, myoblast cells, myofibroblast cells, fibroblasts cells, endothelial cells, adipose cells, hepatocytes, cardiomyocytes and / or combinations thereof. In certain embodiments the cells are non-human cells. In certain embodiments, the cells are from an animal source and preferred animal sources may be porcine, bovine, ovine, horse, dog, cat, avian, reptile, amphibian, cephalopod, crustacean, or any combinations thereof. The cells may also be derived from seafood such as fish (e.g., salmon, tuna, etc.), shellfish (e.g., clams, mussels, and oysters); crustaceans (e.g., lobsters, shrimp, prawns, and crayfish), and echinoderms (e.g., sea urchins and sea cucumbers). In some embodiments, the cells may be engineered, for instance to produce vital nutrients such as proteins and essential fatty acids. In addition, transgenic cells may be used to decrease the time needed for cell differentiation. In some aspects, media formulations may include transgenic components to drive cell differentiation. For example, tetracycline-responsive promoters inserted into transgenic cells may be activated by including tetracycline in the culture medium, resulting inAtty. Dkt. No. 166118.01557 forced expression of myogenic or adipogenic genes in edible cell lines (e g., chicken fibroblasts, bovine satellite cells, etc.).[01801 Non-limiting examples of precursor cells include stem cells such as pluripotent stem cells (PSCs), mesenchymal stem cells (MSCs), muscle-derived stem cells (MDSCs), satellite cells, adipose-derived stem cells (ADSCs) and adipogenic precursor cells. In addition, transdifferentiated cells may also be utilized. Other precursor cells may include, but are not limited to, dedifferentiated cells and fibroblasts.

[0181] A series of rollers or another suitable mechanism to apply a motive force to the scaffold may be used to direct and move a scaffold along a path so that the various treatments can be applied to the scaffold and cells. A scaffold, which may be a thread or fiber, and may be edible, and may be extruded, for instance a liquid polymer extruded onto an extrusion conveyor which moves the scaffold to a drying stage or into a coagulation bath. The drying stage may include the application of heat by a heater or blower and / or low humidity to transform the liquid or wet scaffold into a dried or dryer scaffold. The scaffold may be peeled or removed from the extrusion conveyor and then crosslinked, for instance via a chemical process, for instance with a food-safe crosslinking agent and / or radiation treatment such as ultraviolet light. The scaffold may then be fed into a bioreactor or vessel where rollers direct the scaffold into a media bath and cells are seeded and optionally adhered to the scaffold.

[0182] The fiber may be poured or extruded as a liquid in the form of a protein and solvent, optionally including a thickener such as cornstarch, polysaccharides such as alginate, as well as other coloring agents, preservatives, nutrients, antibiotics or sterilizers, and / or fillers. The scaffold may also include or be made of polymers or polymer precursors that may be later crosslinked, for instance with ultraviolet light or crosslinking agents as described. Suitable solvents may include aqueous solutions, organic solvents, culture media, or combinations thereof. Organic solvents may include ethanol, dioxane, acetone, acetic acid, alcohol, isopropyl alcohol, acetonitrile, dimethylsulfoxide, trifluoracetic acid or any other suitable organic solvent, in pure form or diluted as a co-solvent in an aqueous solution.

[0183] Optionally, the scaffold may be poured into a mold or use other manufacturing processes, for instance to facilitate accurate and ease of movement and manipulation of the scaffold along the intended treatment pathway.Atty. Dkt. No. 166118.01557

[0184] Figure 1 shows an exemplary scaffold seeding bioreactor where a scaffold, in this example an edible fiber, is input into the bioreactor where it may be directed and moved along by a series of rollers. In certain embodiments the scaffold may partially or optionally be moved along by a conveyor, such as a conveyor belt. The rollers or conveyor cause the scaffold to enter a hydration area where the scaffold is exposed to, or, preferably, immersed, in a cell culture media, which hydrates the scaffold. The scaffold is also moved to a seeding area which may be within or outside of the cell culture media. In one embodiment a cell suspension is released into the seeding area and the cells are allowed to settle onto the scaffold, for instance due to gravity. In an adhesion area, the cells may be allowed to adhere to the scaffold and may begin to grow and expand, dividing and multiplying to increase their number. The adhesion area may be a separate tank (not shown) that allows for a different culture medium to encourage cell adhesion and / or expansion, thereby forming a scaffold seeded with cells, or the adhesion area may be adjacent to or the same as the seeding area in the original bioreactor or vessel. The cells grow, for instance on the surface and / or in the pore of the scaffold. After the cells have reached a desired degree of coverage on the scaffold, density, or other indicator of readiness, the rollers or conveyor then direct and move the seeded scaffold to an extraction area, and the scaffold seeded with cells may be output from the bioreactor.

[0185] In an embodiment of the disclosed system for the production of cultured tissue the system may include a first bioreactor. The first bioreactor may include an internal chamber containing culture medium, an inlet for feeding a scaffold into the internal chamber, and a cell inlet for feeding cells or a cell slurry into the internal chamber and onto the scaffold. The cells may be precursor cells and they may proliferate and differentiate on a surface of the scaffold in the culture medium to provide a cell-bearing scaffold composed of cells attached to the scaffold. The first bioreactor may further include an outlet through which the cell-bearing scaffold emerges from the first bioreactor. The cell-bearing scaffold may be further used in the production of the cultured tissue. Additionally, in some embodiments, the first bioreactor of this paragraph is the only bioreactor, while in other embodiments there are additional bioreactors involved in the system and methods, as described herein.

[0186] The cultured tissue may be cultured whole muscle meat suitable for consumption and having a structural organization and hierarchy that mimics natural whole muscle meat. The system may employ principles from textile engineering to generate the cultured tissue, whereby fibers of muscle and fat are first cultured in vitro on a scaffold and then spooled, rolled, folded and / orAtty. Dkt. No. 166118.01557 stacked, as well as twisted, woven or braided into larger, macroscale two-dimensional (2D) or three-dimensional (3D) tissue constructs. This process imparts strength in the resulting cultured tissue and provides a structural organization and hierarchy similar to that of skeletal muscle tissue. In some embodiments, the cultured tissue may have a density and / or stiffness that approaches, matches, or surpasses that of native bovine muscle (about 12 kilopascals (kPa)). The cultured tissue may further exhibit marbling of fat tissue that resembles fat marbling in whole muscle meat.

[0187] The system may include one or more bioreactors or bioreactor stations which operate to produce the cultured tissue. The system may be run in separate unit operations, or as a continuous, manual or robotically-controlled, and automated process. For the continuous, automatic process, the output from each stage / bioreactor may be fed directly into the next, allowing for minimal human intervention, sterility, and reduced risk of cell contamination. One or more computer controllers may be in communication with the bioreactors for automating and controlling the operations thereof. In some embodiments, the system may include a first bioreactor, optionally a second bioreactor downstream of the first bioreactor, and, optionally, a third bioreactor downstream of the second bioreactor.

[0188] Inputs into the first bioreactor may include the scaffold, the cells, and culture medium, and the output of the first bioreactor may be the cell-bearing or cell-seeded scaffold, with a strand of cell tissue as an optional output. Input and the output of the second bioreactor may be cellbearing or cell-seeded scaffold and optionally a strand of cell tissue. The input and the output of a third bioreactor may include cell-bearing scaffold including a strand of cultured tissue. The scaffold and cells and / or tissue may be spooled into a roll or other structure prior to input in the second or third bioreactor. In alternative embodiments, more or fewer bioreactors may be used for the production of the cultured tissue, with the above operations of the bioreactors delegated in various different ways.

[0189] In some embodiments, the scaffolds disclosed here with cultured cells may be harvested and formed directly into a cultured meat product following incubation in a bioreactor or vessel for an appropriate time to allow cells to attach, grow, and proliferate to a desired degree or density (e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, aboutl2 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hrs, or more, orAtty. Dkt. No. 166118.01557 any time period within about 1 hour and about 24 hours, inclusive of the endpoints. An appropriate time may comprise about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or more or any time period within about 1 day to about 14 days, inclusive of the endpoints, etc.). Once the cells have divided and sufficiently proliferated on the scaffold, the scaffolds and cells may be allowed or encouraged to fuse to form a cultured meat product. In some embodiments a scaffold is sufficiently covered with the cells when it is, e.g., greater than an amount such as about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%, or even about 100% covered (as well as any ranges beginning and ending with these percentages).

[0190] In certain embodiments, once sufficiently covered with cells, a fiber of scaffold may be spooled or rolled up to form a spool of scaffold and cells. A plurality of strands of meat tissue or fiber loaded with cells may also be twisted together to form a thread or yarn or rope-like material. Additional layers or threads may be added and this material maybe woven or braided to form a structured meat tissue. The tissue formed of the scaffold and cells may be harvested, or, in some embodiments, further growth and / or differentiation of the cells is encouraged, for instance, in a bioreactor or vessel, to increase the volume and / or density, or other characteristics of the cultured meat tissue. Once the cells have divided and sufficiently proliferated, the scaffolds and cells may be harvested, for instance by cutting or punching out desired cuts of meat, grinding, or otherwise. In some embodiments the scaffold may be dissolved, or, in other embodiments, or in addition, the scaffold may serve as a nutrient source for the cells such that the scaffold is partially or fully or near fully eliminated (e g. about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%, or even about 100%, as well as any ranges beginning and ending with these percentages).

[0191] Figure 1 shows multiple stages of an exemplary scaffold production process: (A) a bioreactor stage, (B) a muscle fiber twisting stage, and (C) a weaving or knitting for structured meat tissue assembly stage.

[0192] Once assembled the cultured meat depicted in stage C may be used to support further cell expansion and / or differentiation within a bioreactor or other vessel or tank, to increase the number and / or density of the muscles, thereby producing a meat that is preferably of a desired density and structure. In such a method, a structured meat tissue may be placed into an environment such as a bioreactor, vessel, or tank with a cell medium that facilitates cell expansion, thereby increasing the number and density of the cells such that the cells proliferate to confluency or to a desired cellAtty. Dkt. No. 166118.01557 density to form a desired tissue. The cells in the tissue may be encouraged to differentiate into a tissue that closely resemble or is indistinguishable from animal muscle, including, for instance, muscle cells and fat cells yielding a portion of meat. In some embodiments, a single culture medium may be used for both proliferation and differentiation while in other embodiments two or more different culture mediums may be used, for instance one for proliferation and another for differentiation. Edible meat can then be harvested, for instance by cutting or stamping the meat from the meat structure.

[0193] The bioreactor may include a body having an internal chamber containing a culture medium, one or more scaffold inlets for feeding the scaffold into the internal chamber, and one or more cell inlets for feeding the cells into the internal chamber. The cell inlet may feed the cells into a cell feeder and the cell feeder may deliver the cells to the scaffold. In some aspects, the scaffold and the cells may be fed into the first bioreactor via the same inlet. The first bioreactor may further include one or more outlets through which the cell-bearing scaffold emerges from the first bioreactor. In some aspects, the first bioreactor may include one or more translucent sections to allow observation of the internal chamber and monitoring of the cell seeding / differentiation processes. As the scaffold translates from the inlet to the outlet, the cells may attach to and proliferate on the surface of the scaffold, and differentiate into mature cells on the surface of the scaffold. Different bioreactors may be used for different cell types (different cell sources or different muscle cells and fat cells and combinations thereof) with appropriate media conditions for each. The scaffold lines with different cell types may be combined in later stages of the process with continuous cultivation for expansion and differentiation with sufficient residence time in the system to optimize tissue outcomes (e.g., myotubes for muscle, fat droplets for fat, extracellular matrix depositions representative of those found in meat).

[0194] Turning to Figure 3, a method for producing the seeded fibers and cultured tissue is shown. The method first involves activating a movement mechanism to move a fiber scaffold into a seeding area in a bioreactor or vessel; in the seeding area the scaffold may be immersed in a media bath; then cells are seeded onto the scaffold in the seeding area, for instance by dropping the cells or a cell slurry and allowing the cells to settle on the scaffold. The next step is optional and involves adhering the cells to the scaffold in an adhesion area of the bioreactor or vessel. From there, the next step involves growing the cells to increase the number and density of the cells on the portion of the scaffold to form a threadlike meat tissue. The next step involves extracting theAtty. Dkt. No. 166118.01557 scaffold seeded with the cells and / or the meat tissue from the bioreactor or vessel and then optionally twisting or spooling a plurality of scaffold fibers having the cells and / or that are the threadlike meat tissue to form a larger fiber or yam or rope-like structure, the scaffold having the cells either alone or in the form of a larger fiber, yarn or rope like structure may optionally then be immersed in a culture medium within a second bioreactor or vessel to allow for further cell growth, expansion, and / or differentiation. In one embodiment, the method continues with further differentiating the cells. An additional optional step may involve braiding or weaving a plurality of the fiber scaffolds, larger fiber yam, rope or rope-like structures to form a structured meat tissue, they're twisting or braiding or weaving steps may involve the use of multiple different types of fibers having different or the same types of cells, for instance a fiber bearing meat cells may be twisted or braided together with a fiber bearing adipose cells. Optionally the method may also include removing for instance dissolving a portion of the original fiber scaffold. The method may then include harvesting the resulting meat tissue, for instance by cutting or stamping out from the spooled roll the desired portions of meat in desired shapes and sizes.

[0195] Figure 4 depicts a flow chart of steps that may be involved in producing seeded fiber and / or the cultured meat tissue, in accordance with certain aspects of the present disclosure. As shown in Figure 4 the steps include an upstream step which may include fiber formation or delivery. This is followed by a fiber input step where the fiber is provided to the system and subsequently the fiber may be subjected to one or more treatments as described herein. Subsequently the cells may be input to the system and add it as a coating or adherent to the fiber and then the combined cell coated fiber may then be treated as described herein. Subsequently the seated fiber may be provided as an output for later downstream use.

[0196] Figure 5 is a diagram showing components and steps of a fiber seeding process and includes a fiber supply spool which may be used to supply fiber to the system. As shown the fiber maybe fed through a set of rollers which may include one or more pinch rollers or wheels to move the fiber along in the process. Subsequently the fiber may be subjected to a treatment such as a plasma treatment and then these cells may be seeded upon the fiber.

[0197] Figures 6 and 7 show a fiber insertion and retrieval method and components for a batch process in accordance with an example of the disclosure. Figure 6 is a diagram showing insertion of a seeded fiber into a bioreactor, where the seeded fiber feeder is provided with a mechanism to allow it to move laterally relative to a bioreactor with a media bath. As shown the seated fiber mayAtty. Dkt. No. 166118.01557 be draped over a series of stationary dowels which allow the bioreactor to hold more seeded fiber in the media bath. It can be appreciated that as the fiber feeder moves relative to the bioreactor more and more of the fiber is released from the feeder into the bioreactor.

[0198] Figure 7 is a diagram showing an example retrieval of a seeded fiber from a bioreactor, which may use the same feeder or a different feeder as is shown in Figure 6. In figure 7 the feeder takes up the seeded fiber retrieving it from the bioreactor and media bath and the feeder or retriever, as the case may be, may also move laterally relative to the bioreactor to facilitate the best removal of the seeded fiber from the bioreactor.

[0199] Figure 8 is a diagram showing an example continuous fiber seeding and / or culture process and components. In contrast to Figures 6 and 7 where the seeded fiber feeder is in motion, in Figure 8 the seated fiber feeder is stationary and a number of conveyor rods move within or as part of a bioreactor, allowing the draped seeded fiber to move laterally away from the stationary feeder.

[0200] Figure 9 depicts a flow chart of steps that may be involved in producing seeded fiber and / or the cultured meat tissue, in accordance with certain aspects of the present disclosure. As shown in Figure 9 a fiber is input into the system, for instance a silk fiber, and this fiber may then be exposed to one or more treatments, for instance the treatment may involve exposure to cold nitrogen plasma. A cell suspension, for instance cells in media, may then be deposited onto the treated fiber. The method may also include treating the cell coated fiber, for instance by incubating the cell coated fiber for an at 37°C in an atmosphere of 5% carbon dioxide and optionally also providing exposure to 0.1% glutaraldehyde. The seated fiber may then be output and used for further downstream purposes.

[0201] Figure 10 depicts a flow chart of steps that may be involved in producing seeded fiber and / or the cultured meat tissue, in accordance with an example batch process of the present disclosure. The method begins with activating a movement mechanism to feed sterile fiber from a spool into a treatment subsystem such as a bioreactor and / or media bath. One or more treatment subsystems may then be activated to apply a cold treatment to a portion of the fiber for instance at 20°C. A movement mechanism which may be the same as that described above in this paragraph may then advance a portion of the fiber to a cell seeding subsystem and one or more cell seeding subsystems may be activated to apply a suspension of cells and media to the fiber, for instance at 37°C in an atmosphere of 5% carbon dioxide. The same or a different movement mechanism mayAtty. Dkt. No. 166118.01557 then be activated to suspend the seeded fiber across a series of horizontal tines or gals inside a bioreactor for instance a watertight chamber and chamber subsystems are activated to maintain internal conditions at 37°C, 5% carbon dioxide atmosphere and 95% relative humidity. These conditions may then be maintained while the suspended cells adhere to the fiber. Appropriate bioreactor subsystems may also be activated to fill the bioreactor or chamber with media and circulate that media at 37°C and an atmosphere of 5% carbon dioxide and these chamber conditions may be maintained to allow the cells to proliferate (i.e. grow and or divide) on the fibers while submerged in the media. A subsystem may then be activated to drain the bioreactor or chamber of media thereby exposing the seeded fiber having an increased biomass of cells. A subsystem may then be activated to crosslink the cells for instance by providing the chamber with glutaraldehyde for instance at 0.10-0.15% followed by an excess of a pH neutral buffer. My movement mechanism may then be activated to wind this cell loaded fiber onto a spool for further downstream processing or use.

[0202] Figure 11 depicts a flow chart of steps that may be involved in producing seeded fiber and / or the cultured meat tissue, in accordance with an example continuous process of the present disclosure. As shown in Figure 11, a movement mechanism is activated to advance fiber from a spool across rollers or dowels and through a series of subsystems. One or more treatment subsystems may treat the fiber for instance by exposing the fiber to cold nitrogen plasma at 20°C for approximately 15 minutes. Hey cell seating subsystem may then apply a suspension of cells and media to the passing fiber preferably at 37°C and a 5% carbon dioxide atmosphere for about one minute or less. The fiber is then moved through culture subsystems that maintain 37°C and 5% carbon dioxide atmosphere at 95% relative humidity while the cells adhere to the fiber for a period of about two hours. The fiber may then be moved across rollers into a bioreactors bath of cell growth media which may be maintained at a temperature of 37°C and an atmosphere of 5% carbon dioxide to facilitate proliferation of the cells for instance spending approximately two days circulating across rollers and / or with media being circulated around the cell laden fibers. The fiber may then be moved out of the bioreactor and passed through another treatment subsystem for instance to cross link the cells by applying glyceraldehyde at approximately 0.1-0.15%, for instance, at 40°C for approximately 15 minutes. The fiber may then spend approximately 15 minutes in a bath of PH neutral buffer to wash away any unreacted aldehyde residue and subsequently may be cooked and dried by heating in a convection oven at approximately 85°C forAtty. Dkt. No. 166118.01557 about 30 minutes. The cooked, dried fiber may then be wound onto a spool for downstream use and or further processing.[02031 Figure 12 depicts an example fiber feeding and plasma treating mechanism in accordance with certain aspects of the disclosure. As shown in Figure 12 a series of pinch rollers and / or wheels may be driven by appropriate motors and gearing to cause a fiber to move between the rollers and / or wheels. A plasma generator may be provided to provide a plasma treatment to the fiber as it passes by the plasma generator through a plasma treatment cell.

[0204] Figure 13 depicts an example motorized syringe pump in accordance with certain aspects of the disclosure, where a syringe may be depressed by rotary motion of an attached lead screw. The lead screw may be driven by an appropriate mechanical mechanism such as a belted transmission and motor.

[0205] Figure 14 depicts an example of a mechanical system to coordinate the motion of a fiber feeding system relative to a bioreactor. As shown the mechanical system may be used to cause movement in three different dimensions.

[0206] Figure 15 depicts an example control system used for controlling a prototype system showing stepper motor driver integrated circuits and MOSFETs for heating sections of the conveyor belt along with the film thereon. The control system depicted in Figure 15 includes a number of drivers for stepper motors for instance to control movement or actuation of various components. These components may be controlled by a central processor or collection of processors including a number of thermistors and MOSFETs. Along with movement of the fiber this system may control the application of cells, the temperature of the cell culture and media bath as well as the temperature of the ambient air in different parts of the system.

[0207] The transit time and culture medium in the bioreactors may be tuned or adjusted in real time based on system diagnostics and feedback and / or based on manual control to provide a desired degree of coverage of differentiated cells on the scaffold. In some embodiments, confluence (or a desired degree of coverage of differentiated cells on the surface of the scaffold) may be identified when at least 70-80% of a surface area of the scaffold is coated with the mature cells. For example, the cells 24 may be cultured to a confluence of at least 75% surface area coverage in the first bioreactor. Cell differentiation may be indicated by the expression of myosin heavy chain (MHC) in muscle cells, and by the accumulation of lipid in fat cells.Atty. Dkt. No. 166118.01557

[0208] In some aspects, a time for the cells to attach to and reach confluence on the scaffold may range from 12 to 48 hours, and a time for cell differentiation into the mature cells may range from 7 to 21 days or even up to 28, 35 or 42 days (e.g., a time may comprise about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, about 31 days, about 32 days, about 33 days, about 34 days, about 35 days, about 36 days, about 37 days, about 38 days, about 39 days, about 40 days, about 41 days, about 42 days, or more, or any time period within about 1 day to about 42 days, inclusive of the endpoints, etc.). In some embodiments, cell growth may be continued until a surface of the scaffold is at least 70% or at least 80% covered by differentiated cells. In some embodiments, cell differentiation of at least 90% may be achieved in the first bioreactor. Cell growth and differentiation may continue once the scaffold having cells seeded or grown thereon are spooled or rolled into a desired form, and may be halted by refrigeration or freezing for instance for storage and or transport. It may not be necessary for the cells to be alive once the cultured tissue is formed; like animal meat, cultured tissue cells may be dead after harvest and may be cooked prior to consumption.

[0209] Factors such as, but not limited to, the rate of translation of the scaffold through the first bioreactor and the composition of the culture medium may be tuned and or adjusted to provide a desired level of cell coverage or confluence on the scaffold and / or to control cell differentiation. For example, cell proliferation to differentiation may be driven by a shift in media composition, nutrients, growth factors, etc. As a non-limiting example, satellite cells may be proliferated in a growth factor-rich proliferation media, and triggered for differentiation in a growth factor-poor differentiation media. Optionally, the concentration of the growth factor may change, for instance, decreasing along the length of the internal chamber from the end at which the scaffold enters to the end from which the scaffold and cells exit.

[0210] The bioreactors described herein may include a first roller run by a motor which may provide a feed of the scaffold into the inlet and into the internal chamber of the bioreactor. The feed of the scaffold may be continuous, for instance a slow continuous movement, or discontinuous. In some embodiments, the first roller may be mounted on an outside or inside of the first bioreactor or at or near a proximal end of the first bioreactor. A cover may be placed overAtty. Dkt. No. 166118.01557 the first roller to maintain sterility of the scaffold. The entire process may be performed in a clean or sterile room. A second roller also optionally run by a motor may spool the cell-bearing scaffold that emerges from the outlet of the first bioreactor. The second roller may be mounted inside or outside of the first bioreactor at or near a distal end of the first bioreactor. The second roller having the spooled cell-bearing scaffold may be removed, shipped, and / or stored if desired. Alternatively, the second spool may be moved to a second bioreactor and immersed or put into contact with culture medium so that the cells may be permitted to continue to grow and / or to differentiate on the spool.

[0211] Within the internal chamber, one or more guides, in the form of rollers or rods may extend into the culture medium for keeping the scaffold submerged inside of the culture medium and preserving cell viability. As the guides may keep the scaffold submerged in the culture media, the internal chamber may not need to be completely filled with culture media and smaller volumes of culture media may be used. In one embodiment, the guides may be posts having rotating sponges on their ends. The internal chamber may also include a media divider to keep different media separated into different compartments. For instance, two different cell proliferation media may be used, or a cell proliferation and a cell differentiation media may be separated in two compartments of the internal chamber. A media divider may not be included in some embodiments which use a single, combination proliferation and / or differentiation culture medium (see below). Culture media may be replenished via one or more inlet ports and outlet ports. Sterility of the developing cellbearing scaffold may be maintained by the closed system of the internal chamber. In one aspect, the first bioreactor may have a slide-on or retractable lid. In some embodiments, all or a portion of the body of the first bioreactor may be composed of a translucent material, such as polycarbonate, allowing for visual inspection. Pumps may be provided to move the media within the bioreactor.

[0212] In certain embodiments, a second bioreactor may be provided to house a spool of scaffold with cells seeded or grown on the scaffold. The second bioreactor may be provided with a media to submerge or contact the scaffold and allow further cell growth and / or differentiation. The second bioreactor may be sized to house one spool or multiple spools. Inlet ports and outlet ports may be provided to replenish media. Motors and movement actuators may be provided to move the spools and pumps may be provided to move the media within the second bioreactor. All or a portion of the bioreactors or vessels described herein may be composed of a translucent material, such as polycarbonate, allowing for visual inspection.Atty. Dkt. No. 166118.01557

[0213] The scaffold may include one or more coatings to provide desirable properties such as those mentioned above, and / or to improve cell attachment to the scaffold. Various cost-effective biopolymers or complex extracts from natural sources may be used as coating materials. In some embodiments, extracellular matrix proteins and / or chemi cal / synthetic coatings may be used as coatings to improve cell attachment to the scaffold and mimic in vivo cell behavior. Other types of coating materials may include commercially available products such as, but not limited to, fibronectin, laminin, vitronectin, collagen, cadherin, elastin, hyaluronic acid, poly-D-lysine, poly- L-lysine, poly-L-omithine, concanavalin A, soy, and other adhesive, non-toxic chemicals. Conconavalin A, laminin, and hyaluronic acid may be obtained from animal-free origins, and have been shown to enhance muscle cell attachment to various biomaterials. The scaffold may have a gel coating. In some embodiments, the coating component may be integrated into the continuous bioreactor system to produce the coated scaffold on demand. In some embodiments, the scaffold itself may have the components described in this paragraph integrated into the scaffold itself.

[0214] The scaffold may be food grade and may be edible for humans and / or animals. Additionally, and / or optionally, the scaffold may be made from materials intended to be consumed by the cells as the cells grow and expand and / or differentiate. Additionally, and / or optionally, the scaffold may be made from a material that is selectively dissolvable so that during any or all of the steps of expansion, differentiation, or after differentiation, the scaffold may be dissolved and washed away or otherwise removed. When intended for medicinal applications the scaffold fiber may have additional or alternative properties such as increased strength and / or durability. Additionally, and / or optionally, the scaffold may be any combination of two or more of the properties described herein.

[0215] The scaffold may be treated to make it hydrophilic and more adherent for cells. Most cells carry a negative or weak negative charge on the cell surface; imparting a positive or weak positive charge to the scaffold helps the cells adhere to the scaffold. The scaffold may be cold treated, for instance with cold plasma (e.g. nitrogen) to impart a positive or weak positive charge.

[0216] A matrix, for instance in the form of a gel, may also be applied to the scaffold once the cells have been seeded. For instance, a liquid gel that is later solidified may help ensure the cells adhere to the scaffold. Growth factors, nutrients and other desirable components, as described herein, may be provided in the matrix to encourage cell growth, division, and / or differentiation.Atty. Dkt. No. 166118.01557

[0217] An aqueous alginate solution may be used to form the scaffold and / or the matrix for instance by exposing the alginate to a cationic agent, for instance with divalent cations, such as calcium ions, in a coagulation or gelling bath. Later, a scavenger may be added to capture the cationic agent, such as citrate ethylenedi aminetetraacetic acid (EDTA), thereby dissolving the alginate. Thus, the cells could be grown up on an alginate or similar scaffold or in an alginate or similar gel, and subsequently, after suitable cell growth, the scaffold or gel could be dissolved, or partially dissolved. In a similar fashion the scaffold or gel could be starch based, and the cells could be grown up on or in a starch based scaffold or gel and then after suitable cell growth the scaffold or gel could be dissolved or broken down, for instance through an enzymatic digestion, for instance with an amylase.

[0218] Optionally, features or processes may be provided to the cell population to include functionalization (e g., surface chemistry, cross-linking, physical etching, morphology patterns to guide cell organization, controlled porosity, etc.).

[0219] Suitable cell culture media for use in the bioreactor include those known in the art as a solution containing nutrients to support cell survival, growth and / or proliferation. Examples of suitable nutrient rich culture media may include Dulbecco’s Modified Eagle Medium (DMEM) and variants thereof. A single combination proliferation and / or differentiation culture medium may be used for both cell proliferation and / or differentiation in the first bioreactor. A simultaneous proliferation and / or differentiation culture medium may be beneficial for rapid cell attachment and differentiation, while also minimizing the amount of medium needed within the chamber. An exemplary combination proliferation-differentiation culture medium for muscle cells may be Dulbecco’s Modified Eagle Medium (DMEM) with fetal bovine serum (FBS), insulin-like growth factor 1 (IGF-1), and insulin. For example, a suitable proliferation-differentiation medium which achieves both high cell density and efficient myogenesis of the cells may be DMEM, 10% FBS, IGF-1 (1 nanogram (ng) / milliliter (mL), 10 ng / mL, or 100 ng / mL) and 10 micrograms (|lg) / mL insulin. In one particular embodiment, the combination proliferation-differentiation culture medium may be DMEM, 10% FBS, 100 ng / mL IGF-1, and 10 pg / mL insulin. In other embodiments, two different media formulations for cell proliferation and cell differentiation may be used within two different compartments of the first bioreactor, or between a first bioreactor and a second bioreactor.Atty. Dkt. No. 166118.01557

[0220] In one embodiment, bovine satellite cells may be continuously seeded onto the scaffold (with or without coatings). Bovine satellite cells may be cultured in growth media with growth factors (e.g., DMEM with Glutamax, 20% FBS, and 1% antibiotic-antimycotic, and 1 ng / mL human fibroblast growth factor 2 (FGF-2)). To differentiate satellite cells into mature myotubes, cells may be cultured to confluence and triggered for differentiation by a low growth factor environment. For example, the culture medium may shift from a growth factor-rich proliferation media to a growth factor-poor differentiation media.

[0221] Bovine fat cells may also be coated onto the scaffold and cultured in growth media (e.g., DMEM with Glutamax, 20% FBS, 1% antibiotic-antimycotic). To differentiate adipogenic precursor cells into mature adipocytes, cells may be cultured to a desired confluence (e.g., 75%), and the media may then be supplemented with free fatty acid solution. An exemplary free fatty acid solution may be 50 millimolar (mM) free fatty acid solutions containing elaidic acid, erucic acid, myristoleic acid, oleic acid, palmitoleic acid, phytanic acid, and pristanic acid. To verify lipid accumulation, Oil Red O (ORO) may be used to stain differentiated cells.

[0222] Various parameters of the system may be controlled and / or programmed via the computer controller (or controlled manually) to optimize features such as cell proliferation and / or differentiation, cell attachment to the scaffold, and the composition, density, bite, and texture of the cultured tissue. For example, a time frame for proliferation and differentiation of the precursor cells in the first bioreactor (or the transit time in the first bioreactor) may be controlled to reach target percentages for differentiation and degree of cell attachment on the scaffold. Other controlled parameters may include the rotation of the spools upon which the scaffold is loaded, the packing density of the cultured tissue, and the composition of the cultured tissue including the cell types, scaffold composition, and the ratio of muscle cells and fat cells in the cultured tissue product. As noted above, the structural hierarchy and marbling of the cultured tissue construct may be tunable by changing the ratio of muscle cell fibers and fat cell fibers. Wamer-Bratzler shear force test may be used to assess the texture and tenderness of the cultured tissue product.

[0223] The bioreactors or vessels within which the cells are seeded and expanded preferably provide consistent function and operation, including reproducible outcomes, in terms of scaffold coverage with cells and / or tissue outcomes. In one embodiment, a target scale of 1 m long fibers is provided. In some embodiments, the fibers are about 1 cm to about 10 m long, or any value or subrange therein, inclusive of the endpoints. Preferably, the time from seeding cells to expansionAtty. Dkt. No. 166118.01557 to tissue and / or meat and, optionally differentiation into meat and / or higher quality meat is kept to a minimum, for instance a range of thereby reducing operating costs, reducing opportunity for contamination, and increasing food production safety. The bioreactors or vessels may include environmental controls, for to control ambient temperature and / or humidity, including, for instance, refrigeration, air conditioning, freezing, warming and / or heating, including capability to warm or cool the liquid culture media. The environmental control may include a cell culture incubator and components to provide regular culture media changes. One or more fdtration assemblies may be provided to filter the liquid culture media and / or ambient air to improve and / or maintain sterility.

[0224] The system may operate in a continuous process including fibers from start to finish, with cells and scaffold from spools as inputs and cell or tissue loaded scaffold as outputs. Alternatively, the system may operate in a batch process, for instance, where a cartridge system enables easy changing of differentiated tissue scaffolds for new scaffolds.

[0225] In some embodiments, a step may involve embedding the cells with a hydrogel or a binder. Suitable hydrogels or binders include, but are not limited to, food safe compounds such as alginate, cellulose, gelatin, starch, hyaluronic acid, fibrin, carrageenan, guar gum, inulin, konjac, oat bran, pectin, locust bean gum, xanthan gum, soy protein, wheat gluten, zein protein, silk fibroin, pullulan, cellulose derivatives and combinations thereof. In some embodiments, the hydrogel or binder is alginate which is a material used as a fat replacer in the food industry. For instance, harvested tissue or meat may be put in contact with an alginate solution at a specified volumetric ratio. In one embodiment, a slow gelling alginate solution may be prepared by adding calcium carbonate and glucono delta-lactone (GDL) powders to an alginate solution, and the slow gelling alginate solution may be combined with the harvested tissue or meat may be put in contact with at a 1 :1 volumetric ratio with the alginate solution.

[0226] In some aspects, the method may include cross-linking the tissue or meat cells. The crosslinking may be carried out using a suitable protein-protein cross-linking enzyme such as, but not limited to, a transglutaminase, a tyrosinase, a peroxidase, and / or a laccase. In some aspects, crosslinking the cells includes enzymatically cross-linking the cells using transglutaminase. For example, cross-linking the cells may involve bathing the cells in a solution of transglutaminase. Helper proteins may be added during the cross-linking. In some embodiments, the helper proteins may be selected from casein and gelatin. Chemical crosslinking can also be particularly withAtty. Dkt. No. 166118.01557 reactants or catalysts that are food safe, such as EDC-NHS reactions between acid and amine groups. Photochemical crosslinking can also be utilized with food safe photosensitizers .[02271 Alternatively, or additionally, other types of immobilization, entrapment, or crosslinking agents may be used with the cells, such as, but not limited to, polymers functionalized with aldehyde groups, genipin, phenolic compounds, and combinations thereof. Suitable polymers functionalized with aldehyde groups include, but are not limited to, periodate oxidized pectin, dextran, chitosan, Arabic gum, sucrose, raffinose, stachyose, cyclodextrin, and starch. Suitable phenolic compounds include, but are not limited to, caffeic acid, chlorogenic acid, caftaric acid, quercetin, and rutin derived from plants such as grapes and coffee.

[0228] The tissue or meat cells may be supplemented at various stages to tune the sensorial characteristics (e.g., density, texture, color, and flavor) and / or the nutritional attributes of the meat. For example, supplementation with additives such as, but not limited to, flavorants, colorants, texturizers, vitamins, minerals, amino acids, proteins / peptides, and fatty acids is encompassed by the present disclosure. In some embodiments, tunable control of nutrition, health, and or fat content may be implemented. The fatty acid composition of the cultured meat may be tailored via cell selection and / or cell feeding strategies, such as by supplementing fatty acids into the culture media during in vitro culture. Genetic interventions may also serve to bolster the nutrition of the cultured meat. For example, in one embodiment, omega 3 desaturases may be expressed or pathways to produce lipophilic nutrients (e.g., beta carotene, vitamin A) may be activated in the cells. This may be beneficial to the consumer as certain nutrients are more bioavailable when consumed in food versus a micronutrient supplement. Additionally, the texture of the meat may be tunable based on variables such as the fat content, the presence and concentration of hydrogel and / or binder (e.g., alginate), cross-linker selection and levels, as well as the inclusion of helper proteins (e.g., casein, gelatin, etc.) during cross-linking. In one embodiment, the cells may be supplemented with methylated branched fatty acids to impart a "mutton" flavor in the cells. Additionally, the relative extracellular matrix production and fat production levels may be optimized to achieve the texture, taste, and / or nutritional outcomes desired.

[0229] In certain cases, the cultured tissue may be particularly useful for introducing nutrients into subjects. For example, a nutrient that are challenging for uptake in humans can be supplemented into the cells here and the resulting cultured tissue may enable greater uptake of those nutrients than if they were administered outside of the cultured tissue.Atty. Dkt. No. 166118.01557

[0230] Other additives that add no proven nutritional or therapeutic value are also contemplated. In some cases, additives can be purely aesthetic. For example, one additive could be gold nanoparticles, which could be added at any stage of the process and taken up into the cells, thereby producing "gold fat". As another example, various materials from the nutraceutical industry can be incorporated into the cells at various stages of the process, including but not limited to silver compositions which are believed by some to have antimicrobial properties, or properties beneficial to the gut bacteria or human or animal microbiome.

[0231] In some cases, the systems and methods described herein can provide advantageous ability to administer growth factors to the cells, in the interest of providing them with more optimal receipt and uptake of the growth factors.

[0232] Certain growth factors may advantageously be administered to cells for the purpose of aiding and / or enhancing their development in the systems and methods disclosed herein.

[0233] In some cases, some of the growth factors can be incorporated into the scaffold.

[0234] A potentially useful additive for the present disclosure is a fatty acid. Without wishing to be bound by any particular theory, it is believed that cell lines that are generally lacking in the production of a desirable fatty acid can be supplemented in the development process to include more of the desirable fatty acid. Again, without wishing to be bound by any particular theory, it is believed that cultivated and / or in vitro cells have historically been lacking in certain fatty acids.

[0235] The most abundantly missing fatty acid is linoleic acid (18:2), as mammals take this from the diet rather than synthesizing it themselves. There can also be a lack of arachidonic acid (20:4), as it is made from 18:2. The lack of 20:4 can be both good and bad, as it's been reported to be important for flavor but also a precursor molecule to pro-inflammatory compounds (eicosanoids).

[0236] In vitro mammalian cells are also typically lacking in omega 3 fatty acids as these are also dietary. These would be alpha-linolenic acid (18:3), eicosapentaenoic acid (EP A, 20:5), and docosahexaenoic acid (DHA, 22:6). Omega 3s are generally less abundant in terrestrial (land) animals so a lack of them is not that detrimental. However, they can make up a significant proportion of fish lipids, so it may be important to have for fish meats.

[0237] In a general sense, when multiple different fatty acids are supplemented, the supplementing concentrations can be chosen to provide desirable end concentrations.

[0238] One specific avenue for introducing growth factors and / or agents and / or additive into the inventive compositions is by way of adding the growth factors and / or agents and / or additives intoAtty. Dkt. No. 166118.01557 the culture media at the time that those culture media are made and / or when those culture media are introduced into their respective tanks with the cells.[02391 The culture media can be tailored for proliferation and differentiation, as well for other steps of the process. In addition, the various steps can use more than one culture media. For example, one culture media could be used for a first part of a proliferation process and the cells can be transferred to a different culture media for a later part of the proliferation process.

[0240] The culture media is typically comprised of a basal medium containing basic nutrients (sugars / carbohydrates, lipids / fatty acids, amino acids, vitamins, minerals, salts, water) combined with a growth factor or complex component containing proteins, peptides, hormones, or other bioactive compounds responsible for directing cell behavior, (e.g., to promote proliferation / cell survival, or to promote adipogenesis, or to suppress other cell pathways such as osteogenesis)

[0241] Basal media will likely be best optimized for the specific adipogenic progenitor cell that is used, but may be similar to or based off of existing formulations such as Dulbecco’s modified eagle medium (DMEM), Ham’s F12, Ham’s MCDB 131, Iscove’s modfied Dulbecco’s medium (IMDM).

[0242] Growth factors for proliferation media formulations may include non-exhaustively : serum albumins, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), fetuin, bone morphogenic proteins (BMPs, e.g., BMP4), Wnt proteins, leukemia inhibitor factor (LIF), hydrocortisone, testosterone, progesterone, estrogen, hypoxia inducible factors (HIFs), stem cell growth factor-P (SCGF-P), tumor necrosis factor alpha (TNFa), interleukin- 1 beta (IL-ip) and other interleukins (ILs), transforming growth factor beta (TGF ), insulin-like growth factor 1 (IGF-1), insulin, transferrin, myoglobin, vitronectin, truncated vitronectin peptides, laminin, laminin peptides (e.g., 511-E8), fibronectin, collagen, gelatin, trypsin inhibitors.

[0243] Growth factors may be added specifically for cell differentiation may promote differentiation and may overlap with ingredients in the basal medium formulation.

[0244] In some embodiments, the cells may be genetically modified. For example, the cells may be genetically modified to improve their growth and / or lipid accumulation for more efficient scale up and improved taste or other qualities. In cases where improved growth is desired, genetic modifications such as those described in WO 2018 / 208628, which is incorporated herein in its entirety by reference, may be usable. A skilled artisan will recognize the variety of techniques thatAtty. Dkt. No. 166118.01557 allow suitable genetic manipulation, including but not limited to, gene editing technologies such as CRISPR gene editing. With respect to the specific genetic modifications, there are a host of categories that are more conventional genetic modifications (e.g., nutritional content, flavor, etc.) and some that are less conventional (e.g., enhancing surface proteins to facilitate the crosslinking or adhesion described herein).

[0245] The culturing process of the present disclosure may be compatible with one dimensional or two dimensional (2D) culture strategies. In some embodiments, the cells may be cultured in thin layers on the scaffold (e.g. a thread) and then, e.g., upon reaching a desired cell density, the scaffold and the cells thereon may be spooled, twisted together, woven or braided and the scaffold and cells may be immersed in a culture medium and the cells may be grown and / or differentiated to yield a spooled or rolled meat tissue. For instance, the cells may be grown to confluency (or to a desired density or coverage / number of cells on the scaffold) and differentiated into the meat cells on the scaffold, including while in the roll. Harvesting or collecting the cells from the ID substrate followed by aggregating the harvested cells may provide the cultured tissue. In some embodiments, the ID substrate may by edible and incorporated into the final food product, such that the cells do not need to be detached from the ID substrate.

[0246] In one embodiment the culturing process utilizes a movement mechanism comprising at least one of a roller, belt, and / or conveyor and preferably at least one of a plurality of rollers, belts, and / or conveyors. In several instances, the present disclosure describes the movement of a portion of the scaffold, but as one skilled in the art would appreciate, the portion of the scaffold may be, and preferably is, a part of a longer scaffold fiber. The entire scaffold may initially be provided as a roll or spool of scaffold for ease of storage and movement, similar to, for instance, a spool of wire or thread. Similarly, once cells are loaded onto the scaffold, the scaffold and cells may be rolled or spooled, facilitating convenient storage and movement. Thus, movement of a portion of the scaffold moves the scaffold along and progressively pulls the scaffold off of the spool. The movement mechanism may define a pathway that moves a portion of the scaffold in sequence, for instance through a series of optional treatments, for instance, first putting the portion of the scaffold in contact with a culture medium, then allowing for seeding of cells onto the portion of the scaffold, then allowing for adhering the cells to the portion of the scaffold, then allowing for growing the cells on the portion of the scaffold to increase the number and / or density of the cells on the portion of the scaffold. The movement mechanism may then move the portion of the scaffold onto a secondAtty. Dkt. No. 166118.01557 roll, sometimes referred to herein as a strand of meat tissue when tissue has begun to be formed on the portion of the scaffold. The strand of meat tissue may be twisted to form a bundle or thread of meat tissue and these threads of meat tissue and or the original strands of meat tissue may be woven or braided together to form a structured meat tissue.

[0247] In certain aspects the process utilizes continuous or discontinuous movement of the scaffold.

[0248] The system and methods may also include the use of a cutting mechanism to cut the scaffold, for instance when a predetermined or desired amount of scaffold seeded with cells has been created, spooled, twisted into a larger fiber, etc. As indicated, the tissue may then be put in contact with a culture medium, which may be the same or different than the culture medium used during seeding and initial growth of the cells. For example, the tissue may be placed in a bioreactor or vessel to soak the tissue spool or roll in the culture medium.

[0249] The purity of the water may be important for the media and elsewhere in the processes described. In some cases, the system includes on-site water purification and / or sterilization. If the culture media is provided in powder form, the method may include hydrating the powder to form a culture media. The culture media can be sterile filtered at any step of the method. Various contemplated modifications to the culture media include, but are not limited to, bulk mixed powder including all of the non-water components of the culture media, separately maintained ingredients that are mixed on demand to make culture media, growth factors or artificial mimics thereof can be present in the culture media, or a combination thereof.

[0250] When different reactors are used for proliferation and differentiation or for stepwise combinations of proliferation and differentiation and then subsequent proliferation and differentiation or simply subsequent differentiation then the cells and scaffold may be moved from one reactor to the another. Different or the same culture media may be used in the different reactors. It should be appreciated that the different reactors may be the same type of reactor, but merely with a different culture media inside of it, though different styles or sizes of reactors are also contemplated.

[0251] In some cases, it may be advantageous to do one type of culture for proliferation and a different type of culture for differentiation. For example, if may be useful to conduct proliferation directly on a scaffold following cell seeding, but to conduct differentiation on a spool of scaffold and cells, such as the ones described elsewhere herein.Atty. Dkt. No. 166118.01557

[0252] Is it contemplated that the scaffold loaded with cells itself may be an end product, so the system and method might end there in certain circumstances. In other instances, the process continues so that the scaffold and cells are spooled and further growth and / or differentiation and / or treatment of the cells is performed.

[0253] In some cases, the processing / harvesting decisions can be based on monitoring of individual cells. Without wishing to be bound by any particular theory, it is believed that analytical (e.g., optical, etc.) techniques that interrogate individual cells can provide meaningful distinctions between cells that are ripe for processing / harvesting versus cells that are not yet ready. For example, individual optical interrogation of cells revealing underlying lipid accumulation, cell maturation and / or differentiation can be performed, followed by comparing that interrogation to a benchmark threshold for cells known to have a desired measurable property. These techniques can include active techniques, where the property or properties of the cells are directly measured, and more passive techniques, where the property or properties of the cells are more indirectly observed.

[0254] In some cases, the processing / harvesting decisions can be based on monitoring of populations of cells. Without wishing to be bound by any particular theory, it is believed that analytical (e.g., chemical, optical, electrical, etc.) techniques that interrogate populations of cells can provide meaningful distinctions between the ripeness of those populations for processing / harvesting. For example, populations of cells can have optical measurements taken (e.g., absorption, scattering measurements, etc.) or electrical measurements taken (e.g., impedance, capacitance, etc ), which can represent important meaningful properties of the population of cells, for instance, cell density, maturation, and / or differentiation. These techniques can include active techniques, where the property or properties of the population of cells are measured directly, and more passive techniques, where the property or properties of the population of cells are more indirectly observed.

[0255] The processing / harvesting decisions can be based on cell growth. In some cases, growth can be determined in terms of growth of individual cells (i.e., cell size and / or mass and / or density). In some cases, growth can be determined in terms of growth of the number of cells (i.e., cell count, or cell count per area).

[0256] The processing and / or harvesting decisions can be based on a degree of cell differentiation. As described elsewhere herein, the system and method can include steps relatingAtty. Dkt. No. 166118.01557 to differentiation of certain cell types into mature meat or adipose cells and / or precursors of meat or adipose cells.[02571 The monitoring can be user-originated, such that a user can specifically request that the system or method observe a given cell or a given population of cells at a specific time.

[0258] The monitoring can be automated, such that the monitoring occurs at a predetermined time or that a computer program selected a time based on a given set of criteria. Skilled artisans in the automation arts will recognize a host of options for automating the monitoring of the individual cells or populations of cells.

[0259] In some cases, the monitoring involves a destructive sampling process, where one or more cells are removed from the system or method in order to make a representative measurement.

[0260] For manual destructive sampling processes, traditional sampling techniques can be used, such as physical removal of cells or populations of cells. A user can arbitrarily select the cells for sampling or the user can be directed in some fashion to select specific cells and / or a specific location. In cases where specific direction is given, the system can include labels that direct the user to a proper sampling location. In some cases, these labels can be digital labels or they can be printed labels.

[0261] For automatic destructive sampling processes, a variety of sampling complexities can be pursued. On the simpler end of the spectrum, automated sampling can involve routinely retrieving cells from a specific location in the system / method and simply acquiring whatever sample happens to be occupying that location at the given time. This is a good bulk sampling technique to be used in cases where the overall number of cells required for sampling is adequately small when compared with the overall cell population size. On the more complicated end of the spectrum, automated sampling can involve complex decision trees and / or machine-learning-derived algorithms for selecting cells or populations of cells for interrogation. Examples of suitable selection algorithms include, but are not limited to, random sampling techniques, weighted sampling techniques, and the like.

[0262] In some cases, the monitoring involves a non-destructive sampling process, where the cells are not disturbed to a degree that their growth, differentiation, and / or proliferation remain generally unaltered.

[0263] For manual, non-destructive sampling processes, the user can manually move an analytical device into a location for interrogating a cell or population of cells. A user can arbitrarilyAtty. Dkt. No. 166118.01557 select the cells for interrogation or the user can be directed in some fashion to select specific cells and / or a specific location. As with the destructive techniques, the systems and methods can utilize labels to direct a user to a proper location. Such labels can be digital or analog.

[0264] In some cases, the monitoring is performed on adhered cells. In these cases, the monitoring systems are configured to interrogate a location where the scaffold is positioned during processing. Because the systems and methods are typically deployed in a reproducible fashion, the scaffolds will typically be located in predictable locations.

[0265] In some cases, the monitoring can include monitoring the culture media. This analyzing of the culture media can be used for making harvesting decisions or can inform control of other aspects of the method.

[0266] In certain cases, the culture media can be monitored to determine a concentration of ammonia. The concentration of ammonia can be used as a feedback for manual or automated control of the system.

[0267] In certain cases, the ammonia can be extracted and processed into an end product. For example, ammonia can be extracted and processed into fertilizer using methods known in the art.

[0268] In one case, the culture media can be monitored to determine a concentration of lactic acid in the culture media. The concentration of lactic acid can be used as a feedback for manual or automated control of the system.

[0269] In general, waste product that are not useful as recycled source materials in the system and method described herein can be monitored and removed from the system. If those waste products are capable of being transformed into a useful product, then the system can include those facilities.

[0270] In certain cases, the lactic acid can be extracted and processed into an end product. For example, lactic acid can be extracted and processed into poly(lactic acid) using methods known in the art.

[0271] In one case, harvesting decisions can be phenotypic.

[0272] The harvesting decisions can be supplemented with various feedback loops, relaying information about the process as it is underway.

[0273] In certain cases, the secretome of the cells can be used to make a harvesting decision. In some cases, the level of adipokine surrounding the cells can be used as a measure of readiness for harvest. Similarly, the level of adiponectin could be utilized for harvesting decisions.Atty. Dkt. No. 166118.01557

[0274] The cultured meat products described here are edible and intended for consumption by human beings, non-human animals, and both, for instance as food products. In other embodiments, the cultured meat products may be used as animal feed such as feed for livestock, aquaculture, or for domestic pets.

[0275] At a high level, the materials produced by the systems and methods described herein can generally be incorporated into edible products.

[0276] At a high level, the materials produced by the systems and methods described herein can generally be incorporated into consumer products or medicinal products.

[0277] In one specific example, the materials produced by the systems and methods described herein can be included in cosmetics, such as make-up, skin creams, lotions, and the like.

[0278] In another specific example, the materials produced by the systems and methods described herein can be included in soaps, detergents, or other generally emulsifying compositions.

[0279] In another specific example, the materials produced by the systems and methods described herein can be included in industrial compositions, such as lubricants used in a variety of industries, liquids useful in extraction industries like the oil and gas industry, and the like.

[0280] In another specific example, the materials produced by the systems and methods described herein can be included in sustainability systems for environmental purposes, such as animal feeds, aquaculture compositions, and the like.

[0281] In another specific example, the materials produced by the systems and methods described herein can be included in end-use compositions that include some proportion of material that is produced from the systems and methods described herein and some proportion from naturally occurring sources. For instance, the cultured tissue described herein can be combined with lean meat from a natural source to make ground meat having an artificially enhanced content.

[0282] The raw material sources can be generally conventional material storage and supply technologies as would be appropriate for cell agriculture applications. Preferably the materials are sterile when they are eventually introduced into the system for executing the methods described above. Another optional feature of the raw material sources is temperature control. Particularly for the cellular material, the temperature control is important. Similarly, the conduits and other means of transport for the various material flows also require sterility and optionally include temperature controls.Atty. Dkt. No. 166118.01557

[0283] The raw material sources can have quality control facilities. The raw material sources can have cellular testing facilities (e g., PCR, morphology assessment, genetic testing, etc.). The raw material sources can have appropriate facilities to confirm sterility, such as sampling and testing facilities as would be understood by those having ordinary skill in the art.

[0284] The raw material sources can be adapted to sterilize the raw materials, using techniques known in the art.

[0285] At least one portion of the raw material sources is adapted to provide a supply of cells for use in methods herein. With commercially provided cells, the supplier would provide information regarding the quality control of the materials, such as genetic information, instructions, etc.

[0286] In some cases, the system includes an entire sub-facility dedicated to producing scaffold for use in the methods described herein.

[0287] The raw material sources can take any physical form that meets the requirements of the applications described, including but not limited to, storage tanks, hoppers, reservoirs, silos, and other means of storing raw materials on-site for use in the methods described herein.

[0288] Receiving raw materials into the raw materials sources can be handled in generally conventional ways, with the understanding that the ordering and delivery of cells is an emerging field.

[0289] Without wishing to be bound by any particular theory, it is believed that the technology for preparing cells such as the ones described elsewhere herein may be more accessible to the commercial market, so the system is adapted to receive commercial-scale delivery of cells as raw material for the methods described herein. This sub-facility can include all of the various processing systems that are required for producing the scaffold as described herein.

[0290] A distinct or joined sub-facility may be dedicated to seeding the scaffold with cells and causing cell growth and differentiation. In some cases, the systems may be paired together.

[0291] In a general sense, the system can be divided into raw material production facilities, where any of cells, cell slurries, scaffold, cell media and other reagents are produced, and end product production facilities, where the desired cultured tissue is produced.

[0292] The system may be adapted to receive commercial-scale quantities of material, so the system includes receiving docks and pipe infrastructure to receive trailer trucks and tanker trucksAtty. Dkt. No. 166118.01557 of raw materials. Similarly, rail, air, and sea transport features are contemplated. As outlined above, pipelines are also a viable option for receiving raw material.[02931 The raw materials can be recycled materials, including recycled agricultural materials and other recycled materials that can be useful in the processes described herein. In some cases, the raw materials are recycled from the system or method described herein.

[0294] The raw materials are delivered from the raw material sources into the first bioreactor. The means for delivery are conventional, preferably with sterility maintained.

[0295] Material can be transferred between bioreactors using conventional delivery means, preferably with sterility maintained.

[0296] Waste streams can be moved in conventional ways. However, the systems and methods described herein make improved use of waste. For example, most of the waste streams in this system and method can be recycled within the system.

[0297] In some cases, the culture media within a given bioreactor is replaced with every usage. In some cases, the culture media is retained for some length of time before being replaced. In some cases, the culture media is slowly regenerated over time by supplementing the portions of the media that are removed in the process.

[0298] The system may include culture media filters, which filter culture media for reintroduction into the bioreactors.

[0299] The system may include an analytical device, such as optical spectrophotometer, a gas chromatography system, a mass spectrometry system, other analytical devices known in the art to useful for assessing quality control of liquid compositions such as culture media, or a combination thereof. This analytical device can be used to assess new or used culture media to determine if it is appropriate for further use.

[0300] In some cases, the analytical device is used to search for contaminants, such as undesirable bacterial growth or harsh chemical solvents. If contamination is identified, then the culture media can be drained or routed to waste or to a refining facility for removal of the contaminants.

[0301] In some cases, the analytical device is used to confirm that certain desirable components are present. For example, the analytical device can confirm the presence or amount of one of the growth factors discussed above. If the analytical device identifies one or more desirableAtty. Dkt. No. 166118.01557 components are missing from a new or used culture media, then the culture media can be supplemented with the missing desirable components.[03021 Insome cases, the analytical device inspects for both contaminants and desirable components and directs the system to make the necessary remedial corrections, if needed.

[0303] In an aspect, the facility is equipped with the necessary scanning and tracking devices that would be required to keep track of the sourcing of the various components that go into the methods described herein. Without wishing to be bound by any particular theory, one of the powerful advantages that may be provided with cellular agriculture is the reduction in transportation costs that are required to transport meat from places where the animals are harvests to the consumer. In the case of the inventive system and methods, the system can be located nearer to the consumer, so the bulk of the shipping cost and environmental impact is related to raw material shipping.

[0304] The system includes the necessary computing facilities to generate and / or modify blockchains in ways that are understood by those in the art to be useful for tracking the authenticity or sourcing of food products. Again, without wishing to be bound by any particular theory, it is believed that there may be advantage to providing consumers with evidence of how little shipping was required to produce a given food product. By using blockchain sourcing authentication techniques (or other methods known in the art to provide similar capabilities), the culture tissue produced by the methods described herein can have corresponding entries into a blockchain regarding the cultured tissue's provenance.

[0305] In addition to the digital provenance capability, the system and method described herein has the full capability to acquire and record any data that is necessary for complying with food regulatory authorities. Logs of processing parameters, test results, and other information that is relevant to regulators can be created and saved. The logs can be made manually or automatically.

[0306] In some cases, the digital records can be utilized to improve the system and method. In some cases, the improvements can be based on measured properties of the products, but in other cases, the improvements can be based on user feedback. There is inherently a lag between the facility producing a product and consumers enjoying it, so if there is a wave of product that has a particularly positive or negative customer response, then it would be possible to look up specific information from the run that led to that customer response for the purpose of intentionally repeating or not repeating it.Atty. Dkt. No. 166118.01557

[0307] The data can include both information about the cell source material and the other formulation information from the process. If a certain cell line, process, and set of formulations produces a superior product, then the formula can be retrieved for reproduction.

[0308] Data can be collected from multiple facilities, thereby providing a global data set, from which broader conclusions can be drawn. For example, if all plants around the world show an inefficiency in the use of a given reagent, then this flags the problem as likely being related to the portions of the system or method that are deployed in all facilities.

[0309] The present disclosure describes a continuous process for producing an edible, highly structured composite material designed to mimic the properties of meat. The composite may preferably be comprised of one-dimensional substrates, such as fibers or threads (which can be braided or twisted), embedded within a binder material. Both the substrates and the binder may be made from edible materials, often plant-based proteins, and can be seeded with various cell types including myoblasts, fibroblasts, adipocytes, and stem cells. Control of the composite's structure is precise: fibers may be wound around a rotating spindle while the binder is added, with an elastic roller used to compress the layers, removing excess binder and increasing fiber packing density. Said process results in a low-void (less than 5%), high- water-content (at least 30% by weight) composite with tailored mechanical properties.

[0310] The mechanical properties of both the fibers and the binder may be engineered to mimic those of skeletal muscle fascicles and perimysium, respectively, in both raw and cooked states for various meat types (chicken, pork, beef). The fibers may typically be 0.02-1 mm thick and may be longer than 1 mm, and can be aligned isotropically or anisotropically, even in multiple directions, and arranged in a non-crimp configuration. This allows for high relative fiber content and precise control over fiber spacing (usually less than 1 mm). The result is a scalable approach to producing structured meat alternatives that closely resemble real meat in terms of bulk mechanical properties, thermal behavior, and cooking characteristics. The modular, simple processes involved (such as wet-spinning and fiber bundling) contribute to its potential for large-scale production.

[0311] Other characteristics and benefits of the processes and methods described herein include, but are not limited to, a continuous process for production of an edible composite comprised of 1- dimensional substrates embedded within a binder; 1 -dimensional substrates comprising fibers or threads; threads can be braided or twisted fibers; 1 -dimensional substrates can be seeded with cells; 1-dimensional substrates within composite may be surrounded by a binder material; all materialsAtty. Dkt. No. 166118.01557 may be edible; final composite is at least 30% water by weight; fibers can be wet-spun from protein-rich dopes using coagulation baths; fibers can possess mechanical properties such as elastic modulus, ultimate tensile strength, and elongation at break that mimic the mechanical properties of chicken, porcine, and bovine skeletal muscle fascicles in cooked and uncooked states (for chicken(0.05 -0.3 MPa raw, 0.3 MPa cooked), pork(0.2- 0.6 MPa raw, 0.5- 1 MPa cooked); binder can possess mechanical properties such as elastic modulus, ultimate tensile strength, and elongation at break that mimic the mechanical properties of chicken, porcine, and bovine skeletal muscle perimysium in cooked and uncooked states; 1-dimensional substrate can be added to itself by being wrapped around a rotating object such as a spindle; binder can be added while 1- dimensional substrate is being wrapped around a rotating object or immediately prior; an elastic roller can be used to press or iron 1-dimensional substrate against itself and the rotating substrate to remove non-gelled binder and increase packing density of fiber; void space within composite material is low (for example, less than 5%); spacing between 1 -dimensional substrates can be variable or uniform; alignment of material can be isotropic or anisotropic; the composite can feature multidirectional fiber alignment; 1 -dimensional substrates within the composite may be aligned in various directions or unidirectional; and 1 -dimensional substrates can be arranged in a non-crimp configuration, allowing for multiple orientations and high relative fiber content.

[0312] Further, technical characteristics of the fibers and binders include, but are not limited to, fibers between 0.02 mm and 1 mm in thickness, longer than 1 mm; spacing between fibers in a composite is typically less than 1mm; fiber materials may include plant-based proteins such as zein, soy, chickpea, pea, and rice proteins; fiber materials can include GRAS substances; fiber materials can be edible; binder materials may include plant-based proteins such as zein, soy, chickpea, pea, and rice proteins; binder materials can include GRAS substances; binder materials can be edible; binder materials can include enzymes and other factors for crosslinking; and included cell types may be myoblasts, fibroblasts, adipocytes, and stem cells.

[0313] In one aspect, the present disclosure provides a method for meat production, the method comprising extruding a polymer-rich dope, coagulating the polymer-rich dope to form a gelled fiber, winding two or more of the gelled fibers to form a thread, seeding the thread to provide a seeded thread, bundling at least two seeded threads into a fibrous bundle, harvesting the fibrous bundles as a meat product. As used herein, the term “meat production” may refer to means of expanding, creating, growing, or otherwise increasing the size and / or growth of cells to be used asAtty. Dkt. No. 166118.01557 meat or meat substitutes. As used herein, the term “extruding” may refer to the thrusting or forcing out of a composition which may include the shaping of the composition through a die. As used herein, the term “polymer-rich dope” may refer to a homogeneous thermodynamically stable polymer solution formed by a polymer or a copolymer, solvent or a solvent mixture, and / or additive(s) (i.e., the additive can be a second polymer, a non-solvent, organic or inorganic particles of different sizes, etc.). It is also possible to add a non-solvent to the polymer solution to such an extent that all the components are still miscible. The polymer-rich dope may comprise at least one binder, and the at least one binder selected from the group consisting of gelatin, collagen, fibrin, elastin, animal protein, silk fibroin, seaweed gel, alginate, carrageenan, agarose, algal protein, fungal protein, chitosan, bacterial gel, gellan gum, plant protein, leaf protein, soy protein, wheat gluten, glutenin, gliadin, zein, mung bean protein, rice protein, potato protein, sweet potato protein, fava bean protein, chickpea protein, rapeseed protein, lentil protein, hemp protein, quinoa protein, oat protein, sunflower seed protein, pumpkin seed protein, heparin, heparin sulfate, pullulan, amylose, dextran, starch, cellulose, hemicellulose, lignin, glucomannan, chondroitin sulfate, chitin, polynucleotides, polysaccharide, a glycosaminoglycan, natural polyesters, polyacrylic acid, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic) acid, polycaprolactone, polyhydroxyalkanoates, bioesters, or a polymer material made of a monomer selected from the group consisting of: polylactic acid, polyglycolic acid, poly(lactic-co-glycolic) acid, polyhydroxyalkanoates, monosaccharides, glucose, fructose, mannose, galactose or any derivatives or variants thereof, glycosamines, aminosugars, aminoacids and derivatives thereof, nucleotides and derivatives thereof, and fatty acids and derivatives thereof. The polymer-rich dope may comprise at least one solvent, and the at least one solvent may be selected from the group consisting of urea, alkali, salts, co-solvents, reducing agents, cysteine, glutathione, sodium metabisulfite, sodium thiosulfate, buffer, formic acid, denaturants, chaotropic salts, and lipids. “Buffer” may refer to an aqueous solution comprising a weak acid and its conjugate base (or vice versa) that allows for minimal pH change when adding a strong acid or a strong base adds to the buffer. Buffers may be adjusted to maintain a specific pH; therefore, buffer may be used interchangeably with “pH’d buffer” or “pH buffer”. The polymer-rich dope may comprise at least one additive, and the at least one additive may be selected from the group consisting of carrageenan, xanthan gum, alginic acid, konjac gum, agarose, locust bean gum, guar gum, pea fiber, bamboo fiber, oat fiber, potato starch, com starch, tapioca starch, rice starch, beet juice,Atty. Dkt. No. 166118.01557 coconut oil, sunflower oil, canola oil, palm oil, lecithin, emulsifiers, lipids, yeast, yeast extracts, fungal extracts, miso extracts, liquid aminos, soy sauce, seaweed, kelp powder, tomato paste, powder, onion powder, garlic powder, vegetable broth, smoke flavoring, glutamates, hydrolyzed vegetable protein, herbs and spices, maltodextrin, and cellulose derivatives. As used herein, the term “coagulating” may refer to the process of changing a fluid to a solid or semisolid state. As used herein, the term “form” may refer to producing a composition to a final or semi-final state and may include shaping. As used herein, the term “gelled fiber” may refer to the end product of coagulating the polymer-rich dope. As used herein, the term “winding” may refer to a twisting movement or course and may include a spiraling movement or course. As used herein, the term “seeding” may refer to the process of embedding or otherwise introducing a composition to another composition at hand. In some aspects, the thread is seeded with cells. Cells may be selected from the group consisting of muscle cells, muscle precursor cells, pluripotent stem cells, mesenchymal stem cells, satellite cells, myoblast cells, myofibroblast cells, fibroblasts cells, endothelial cells, adipose cells, hepatocytes, cardiomyocytes, and / or combinations thereof. In some aspects, the thread is seeded with acellular materials. For example, the spinning, binder addition, and bundling (such as winding onto a spool) without cells may be scaled to involve the spinning of two separate fibers to then be combined during binder addition then bundled. Examples of acellular materials that may be seeded in the methods herein include, but are not limited to, vitamins, minerals, nutraceuticals, pharmaceuticals, antibiotics, buffers, salts, proteins, polysaccharides, lipids, essential oils, dyes, lubricants, surfactants, acids, bases, solvents, enzymes, growth factors, and extracellular matrices. As used herein, the term “a thread” may refer to a narrow strand. As used herein, the term “provide” may refer to producing a composition to a final or semi-final state and may include shaping. As used herein, the term “seeded thread” may refer to the end product of seeding the thread. As used herein, the term “bundling” may refer to tying or rolling together a plurality of compositions together. As used herein, the term “fibrous bundle” may refer to the end product of bundling at least two seeded threads. As used herein, the term “harvesting” may refer to the collection of a final product. In some aspects, harvesting comprises spooling. As used herein, the term “spooling” may refer to winding onto a spool, or cylindrical platform. The method may further comprise wet spinning the gelled fiber prior to step (c). As used herein, the term “wet spinning” may refer to a form of solution spinning where polymer powder is dissolved in a suitable solvent and the polymer solution is extruded through spinneret into a solvent-non solvent mixtureAtty. Dkt. No. 166118.01557(coagulant). Accordingly, the disclosed devices may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more “spinnerets” for extrusion of the dope into a coagulant. The spinnerets may comprise outlets with a cross-sectional diameter of about 10 am to about 1000 pm.

[0314] Due to mutual diffusion of solvent and non-solvent, polymer solution coagulates to form fibers. In some aspects, the thread is pulled into a treatment system. As used herein, the term “pulled” may refer to mechanic forcing of a composition through a physical space. As used herein, the term “treatment system” may refer to a means of subjecting a composition to one or a series of solutions or conditions. In some aspects, the thread is further seeded with media, and the seeded thread may be incubated for a period. As used herein, the term “media” may refer to any suitable media for growing, expanding, or otherwise sustaining or maintaining live cells. As used herein, the term “incubated” may refer to subjecting living cells to conditions for a period of time. As used herein, the term “period” may refer to a desired duration.

[0315] In some aspects, the method further comprises adding a binder solution to the fibrous thread. As used herein, the term “adding” may refer to contacting or otherwise exposing a solution to the composition. As used herein, the term “binder solution” may refer to the solution necessary to bind fibrous threads. Examples of the binding solution include but are not limited to gelatin, collagen, fibrin, elastin, animal protein, silk fibroin, seaweed gel, alginate, carrageenan, agarose, algal protein, fungal protein, chitosan, bacterial gel, gellan gum, plant protein, leaf protein, soy protein, wheat gluten, glutenin, gliadin, zein, mung bean protein, rice protein, potato protein, sweet potato protein, fava bean protein, chickpea protein, rapeseed protein, lentil protein, hemp protein, quinoa protein, oat protein, sunflower seed protein, pumpkin seed protein, heparin, heparin sulfate, pullulan, amylose, dextran, starch, cellulose, hemicellulose, lignin, glucomannan, chondroitin sulfate, chitin, polynucleotides, polysaccharide, a glycosaminoglycan, natural polyesters, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic) acid, polycaprolactone, polyhydroxyalkanoates bioesters, or a polymer material made of a monomer selected from the group consisting of: polylactic acid, polyglycolic acid, poly(lactic-co-glycolic) acid, polyhydroxyalkanoates, monosaccharides, glucose, fructose, mannose, galactose or any derivatives or variants thereof, glycosamines, aminosugars, aminoacids and derivatives thereof, nucleotides and derivatives thereof, and fatty acids and derivatives thereof. In some aspects, the fibrous bundle is exposed to a treatment to gel the binder solution. As used herein, the term “treatment” may refer to any means which is suitable to gel the binder solution. As used herein,Atty. Dkt. No. 166118.01557 the term “gel” may refer to setting or becoming more solid. In some aspects, winding comprises adding a binder solution.[03161 Insome aspects, bundling the at least two seeded threads comprises compressing the at least seeded two threads.

[0317] In some aspects, the at least two seeded threads are seeded with at least two different cell types, and, In some aspects, the at least two different cell types comprise meat cells and fat cells.

[0318] In some aspects, the gelled fibers are incubated in a bioreactor or vessel for a period, the processes may be carried out in a bioreactor, such as a stirred suspension tank bioreactor or a hollow fiber bioreactor having hollow fiber membranes. Other types of bioreactors apparent to those skilled in the art may also be used and are within the scope of the present disclosure such as, but not limited to, rotating wall vessel bioreactors (RWVBs) and packed bed bioreactors. Alternatively, the bioreactor may be a simple bioreactor, providing for adequate sterility and environmental (e.g., temperature control) conditions, or even a simple vessel may be suitable in some instances. In some aspects, the cells are differentiated prior to seeding.

[0319] In some aspects, the cells are grown inside a bioreactor or vessel. The bioreactor or vessel may comprise a plurality of rollers within the bioreactor or vessel wherein the rollers are configured to accommodate a plurality of threads at least partially or fully around the plurality of rollers and the rollers are positioned so that activating a rotational force against at least one of the plurality of rollers moves the threads and applies a tension on a portion of the threads, and at least one or more of the rollers individually serves to at least one of support, apply motive force to, and / or direct the thread.

[0320] Coagulating may comprise the addition of a coagulant. Examples of the coagulant may include, but are not limited to, any one or more of polyvalent cation salts, glycerol, citric acid, sodium sulfate, acetic acid, formic acid, polysaccharides, disaccharides, sucrose, maltose, lactose, monosaccharides, fructose, glucose, mannose, galactose, xylose, glucuronic acid, mannuronic acid, gluconic acid, glucono delta-1 act one, polyethylene glycol, hydrochloric acid, buffer (ph < 11), kosmotropic salts, nonsolvents, cosolvents, acids, bases, monovalent salts, emulsions, structured fluids, slurries, enzymes, crosslinkers, complexing agents, and lipids.

[0321] In some aspects, at least one step in the method is followed by a rinsing step. The rinsing step may comprise the addition of a rinse, and the rinse may be selected from the group consisting of water, buffers, alcohols, solvents, urea, alkali, salts, co-solvents, reducing agents, cysteine,Atty. Dkt. No. 166118.01557 glutathione, sodium metabisulfite, sodium thiosulfate, buffer, formic acid, denaturants, chaotropic salts, and lipids.[03221 Insome aspects, the method is conducted on a scaffold, and the scaffold may comprise a material selected from the group consisting of gelatin, seaweed gel, collagen, plant protein, soy protein, wheat gluten, zein, animal protein, silk, amylose, algal protein, fungal protein, dextran, alginate, chitosan, starch, heparin, heparin sulfate, pullulan, cellulose, hemicellulose, glucomannan, agar, chondroitin sulfate, chitin, polynucleotides, polysaccharide, a glycosaminoglycan, natural polyesters, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic) acid, and polycaprolactone. As used herein, the term “scaffold” may refer to a structure or matrix used for providing the foundation of growth.

[0323] In another aspect, the present disclosure provides a meat product produced by any one of the methods or systems disclosed herein. In some aspects, the fibrous bundle of the meat product has a diameter of about 0.3 mm to about 1.2 mm. A cross-section of the fibrous bundles making up the meat product may reveal a packing arrangement resulting from the method of producing the meat product. A “cross-section” may refer to a surface or shape that is exposed by making a straight cut through a composition, especially at right angles to an axis. One such possible packing arrangement is a Voronoi packing arrangement. In a Voronoi packing arrangement, each thread is associated with a particle, and the distance of any point within the thread to the particle is smaller than the distance of that point to any other particle. In 2D, Voronoi packing arrangements often comprise hexagons because they are the most efficient way to pack shapes in a plane. Hexagonal shapes have at least two advantages: they ensure that no empty space is left between shapes (or in the case of the present disclosure, threads or fibrous bundles), and they offer the highest ratio between surface area and perimeter. Therefore, in some aspects, a cross-section of the fibrous bundle comprises a Voronoi packing arrangement. The Voronoi packing arrangement may be an arrangement of individual fiber cross-sections.

[0324] In another aspect, the present disclosure provides a meat product comprising a fibrous bundle, wherein the fibrous bundle comprises at least two threads, wherein the at least two threads are seeded with cells. In some aspects, the fibrous bundle has a diameter of about 0.3 mm to about 1.2 mm. The threads may have diameters of about 0.3 mm to about 1.2 mm. In some aspects, the fibrous bundle comprises a binder, and the binder may comprise gelatin, collagen, fibrin, elastin, animal protein, silk fibroin, seaweed gel, alginate, carrageenan, agarose, algal protein, fungalAtty. Dkt. No. 166118.01557 protein, chitosan, bacterial gel, gellan gum, plant protein, leaf protein, soy protein, wheat gluten, glutenin, gliadin, zein, mung bean protein, rice protein, potato protein, sweet potato protein, fava bean protein, chickpea protein, rapeseed protein, lentil protein, hemp protein, quinoa protein, oat protein, sunflower seed protein, pumpkin seed protein, heparin, heparin sulfate, pullulan, amylose, dextran, starch, cellulose, hemicellulose, lignin, glucomannan, chondroitin sulfate, chitin, polynucleotides, polysaccharide, a glycosaminoglycan, natural polyesters, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic) acid, polycaprolactone, polyhydroxyalkanoates, bioesters, or a polymer material made of a monomer selected from the group consisting of: polylactic acid, polyglycolic acid, poly(lactic-co-glycolic) acid, polyhydroxyalkanoates, monosaccharides, glucose, fructose, mannose, galactose or any derivatives or variants thereof, glycosamines, aminosugars, aminoacids and derivatives thereof, nucleotides and derivatives thereof, and fatty acids and derivatives thereof.

[0325] In some aspects, the cells are selected from the group consisting of muscle cells, muscle precursor cells, pluripotent stem cells, mesenchymal stem cells, satellite cells, myoblast cells, myofibroblast cells, fibroblasts cells, endothelial cells, adipose cells, hepatocytes, cardiomyocytes, and / or combinations thereof.

[0326] The polymer-rich dope may comprise at least one binder, and the at least one binder selected from the group consisting of gelatin, collagen, fibrin, elastin, animal protein, silk fibroin, seaweed gel, alginate, carrageenan, agarose, algal protein, fungal protein, chitosan, bacterial gel, gellan gum, plant protein, leaf protein, soy protein, wheat gluten, glutenin, gliadin, zein, mung bean protein, rice protein, potato protein, sweet potato protein, fava bean protein, chickpea protein, rapeseed protein, lentil protein, hemp protein, quinoa protein, oat protein, sunflower seed protein, pumpkin seed protein, heparin, heparin sulfate, pullulan, amylose, dextran, starch, cellulose, hemicellulose, lignin, glucomannan, chondroitin sulfate, chitin, polynucleotides, polysaccharide, a glycosaminoglycan, natural polyesters, polyacrylic acid, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic) acid, polycaprolactone, polyhydroxyalkanoates, bioesters, or a polymer material made of a monomer selected from the group consisting of: polylactic acid, polyglycolic acid, poly(lactic-co-glycolic) acid, polyhydroxyalkanoates, monosaccharides, glucose, fructose, mannose, galactose or any derivatives or variants thereof, glycosamines, aminosugars, aminoacids and derivatives thereof, nucleotides and derivatives thereof, and fatty acids and derivatives thereof. The polymer-rich dope may comprise at least one solvent, and the at least one solvent may beAtty. Dkt. No. 166118.01557 selected from the group consisting of urea, alkali, salts, co-solvents, reducing agents, cysteine, glutathione, sodium metabisulfite, sodium thiosulfate, buffer, formic acid, denaturants, chaotropic salts, and lipids. The polymer-rich dope may comprise at least one additive, and the at least one additive may be selected from the group consisting of carrageenan, xanthan gum, alginic acid, konjac gum, agarose, locust bean gum, guar gum, pea fiber, bamboo fiber, oat fiber, potato starch, corn starch, tapioca starch, rice starch, beet juice, coconut oil, sunflower oil, canola oil, palm oil, lecithin, emulsifiers, lipids, yeast, yeast extracts, fungal extracts, miso extracts, liquid aminos, soy sauce, seaweed, kelp powder, tomato paste, powder, onion powder, garlic powder, vegetable broth, smoke flavoring, glutamates, hydrolyzed vegetable protein, herbs and spices, maltodextrin, and cellulose derivatives.

[0327] In some aspects, the cells comprise at least two different cell types. The at least two different cell types may be selected from the group consisting of muscle cells, precursor cells, pluripotent stem cells, mesenchymal stem cells, satellite cells, myoblast cells, myofibroblast cells, fibroblasts cells, endothelial cells, adipose cells, hepatocytes, and cardiomyocytes. In some aspects, the at least two different cell types comprise meat cells and fat cells.

[0328] In some aspects, the fibrous bundle is woven. As used herein, the term “woven” may refer to a formation of composition or threads through the process of interlacing a plurality of threads by interlacing threads passing in one direction with others at a right angle to them.

[0329] In another aspect, the present disclosure provides a system for producing meat tissue comprising an extruder to extrude a polymer-rich dope; a winding mechanism; a bundling mechanism; a vessel or bioreactor to grow cells; and a cell media within the vessel or bioreactor, the cell media being a growth and / or differentiation media to further grow and / or differentiate cells and to optionally increase the number and / or density of the cells. As used herein, the term “extruder” may refer to a means to extrude as defined herein. As used herein, the term “winding mechanism” may refer to a means to wind as defined herein. As used herein, the term “binding mechanism” may refer to a means to bind as defined herein. Cell media may include media that leads to growth or differentiation of the cells (“growth and / or differentiation media”). As used herein, the term “soak” may refer to the process of making or allowing a composition to become thoroughly wet by immersing it in liquid which may be a solution, for example. In some aspects, the vessel or bioreactor comprises a plurality of rollers within the vessel or bioreactor wherein the rollers are configured to accommodate a plurality of wraps of a plurality of threads at least partiallyAtty. Dkt. No. 166118.01557 or fully around the plurality of rollers and the rollers are positioned so that activating a rotational force against at least one of the plurality of rollers moves the plurality of threads and applies a tension on a portion of the plurality of threads, and at least one or more of the rollers individually serves to support, apply motive force to, and / or direct the plurality of threads; wherein the plurality of threads is wound and comprises coagulated polymer-rich dope and the cells. The at least one of the rollers may be positioned to cause the plurality of threads to be in contact with the culture media in the vessel or bioreactor and / or to soak the plurality of threads in the culture media to grow the cells to increase the number and / or density of the cells in the plurality of threads. In some aspects, the system further comprises a coagulation bath, and the coagulation bath may comprise a coagulant for gelling or solidifying the polymer-rich dope. As used herein, the term “coagulation bath” may refer to a volume of coagulant.

[0330] In some aspects, the system further comprises one or more additional vessels or bioreactors to grow and / or differentiate the cells. The one or more of the one or more additional vessels or bioreactors may comprise a plurality of rollers within the additional vessels or bioreactors and the rollers may be configured to accommodate a plurality of wraps of the plurality of threads at least partially or fully around the plurality of rollers and the rollers are positioned so that activating a rotational force against at least one of the plurality of rollers moves the plurality of threads and applies a tension on a portion of the plurality of threads.

[0331] In some aspects, the at least one of the rollers is positioned to cause the plurality of threads to be in contact with the cell media in the vessel or bioreactor. In some aspects, the system further comprises a spooler to spool the plurality of threads. As used herein, the term “spooler” may refer to a means to spool as defined herein. In some aspects, the system further comprises a harvesting mechanism to cut or stamp out a portion of meat tissue. As used herein, the term “harvesting mechanism” may refer to a means to harvest as defined herein. As used herein, the term “cut or stamp” may refer to the isolation of a desired product via physical separation from undesired byproducts or extraneous products and may include harvesting a particular shape of the product. As used herein, the term “meat tissue” may refer to the final or semi-final desired meat product desired. In some aspects, the bioreactor or vessel comprises an entry opening where the plurality of threads enters the bioreactor or vessel and an exit opening where the plurality of threads exits the bioreactor or vessel.Atty. Dkt. No. 166118.01557

[0332] The present disclosure also provides a continuous process for efficiently seeding scaffolds with cells or delivering particulate / suspension payloads to porous materials. The approach may be in the following two steps: first, the scaffold undergoes a treatment to enhance its surface wettability, followed by exposure to a wetting fluid carrying a suspension payload. The approach at least enhances wettability which encourages rapid adsorption of the wetting fluid and its ingress into pores, delivering the payload deeper into the scaffold. By restricting the volume of the introduced wetting fluid, the method confines the payload against pores and crevices via surface tension, promoting retention within the scaffold.

[0333] The versatility of the method is evident in its applicability to various scaffold types (for example: fibers, films, particles, textiles) and materials (for example: proteins, polysaccharides, synthetic polymers), which can be GRAS (Generally Recognized as Safe) and edible. The wettability enhancement treatments span a wide range, from plasma and radiation to chemical and enzymatic processes. The payloads can include cells, small particles, or even non-solvent fluids. Notably, the process has demonstrated high efficiency in cell seeding, with rapid and uniform cell distribution throughout the scaffold, even in its interior. This fast, scalable approach not only improves seeding speed and uniformity compared to traditional methods but also promotes quicker cell adhesion and spreading. Its potential for continuous processing, such as seeding rolls of thread, positions it as a promising technology for large-scale tissue engineering and advanced material manufacturing applications.

[0334] With an increase in payload concentration in the wetting fluid, this method can enhance retention of the payload in the scaffold. The scaffold may be small particles, a fiber or bundle of fibers, a film, a textile, or a porous solid. The scaffold may be a protein thread composed of smaller fibers. The wetting fluid may be a cell culture medium. The suspension payload may be cells, small particles, or non-solvent fluids such as oils mixed into the wetting fluid. The treatment to enhance wettability may comprise plasma, flame, corona discharge, ion beam, electron beam, ionizing radiation, UV / ozone, chemical etching, laser, enzymes, grafting polymerization, or supercritical fluids such as CO2. The process can be used to deliver cells to continuous materials such as rolls of thread in a continuous fashion. Scaffold materials can be crosslinked or selfassembled. Residual ions, chemical modifications, and surface charges from treatment may enhance retention of payload against scaffolding surface. Treatment and wetting may drive payload cell suspensions to adhere and spread faster than on untreated scaffolding.Atty. Dkt. No. 166118.01557

[0335] Scaffolds may typically be fibers, but can be films, particles such as microcarriers, or woven fibers in the form of textiles. Fiber scaffolds may typically be between 0.02 mm and 3 mm in thickness, longer than 10 mm. Cell types may include myoblasts, fibroblasts, adipocytes, and stem cells. Scaffold treatment may be an atmospheric or cold plasma. Scaffold materials may include fibrin, collagen, extracellular matrices, silk, cellulose, alginate, PNIPAAM, zein, soy, chickpea, pea, and rice proteins. Scaffold materials can include GRAS substances and / or edible. Scaffold materials can include enzymes and other factors for crosslinking. Scaffold materials can include animal and non-animal derived proteins

[0336] The approach provides numerous improvements over prior art including but not limited to enhanced payload delivery, versatile scaffold options, customizable wetting fluids and payloads, diverse treatment options, continuous processing, post-wetting flexibility, enhanced retention mechanisms, improved cell behavior, scalability, simplicity, versatility beyond bioengineering, continuous or batch production, and automation. Using wettability enhancement of scaffolds followed by controlled introduction of a wetting fluid carrying a suspension payload ensures deeper penetration into pores and crevices and encourages payload retention within the scaffold structure. The process accommodates a wide range of scaffold materials including small particles, fibers or fiber bundles, films, textiles, or porous solids. Notably, it can work with protein threads composed of smaller fibers, and scaffolds can be GRAS (Generally Recognized As Safe) and edible, suitable for food applications. Wetting fluids can be tailored to the application, such as using cell culture media for cellular payloads. The suspension payload may consist of cells, small particles, or even non-solvent fluids like oils mixed into the wetting fluid. Wettability enhancement can be achieved through various means such as plasma, flame, corona discharge, ion beam, electron beam, ionizing radiation, UV / ozone, chemical etching, laser treatment, enzymatic modification, grafting polymerization, or supercritical fluids like CO2. The technology enables delivery of payloads such as cells to continuous materials like rolls of thread in an uninterrupted manner, significantly improving throughput. After initial wetting, the scaffold can undergo subsequent processes such as rinsing in dilute buffers, crosslinking, or heating while retaining a substantial portion of the introduced payload. Restricting the volume of introduced wetting fluid enhances payload delivery by confining it (via surface tension) against the scaffolding surface. Additionally, residual ions, chemical modifications, and surface charges from the wettability treatment may further improve payload retention. In the case of cell suspensions, the treatment andAtty. Dkt. No. 166118.01557 wetting process may drive payload cells to adhere and spread faster compared to untreated scaffolding. The wettability enhancement and seeding process can be scaled horizontally like many other continuous manufacturing processes, such as those used in textile or paper production. By enhancing wettability and controlling wetting fluid introduction, the method reduces the complexity often associated with traditional seeding processes, protecting the payload from damage and improving overall efficiency. While well-suited for cellular agriculture and tissue engineering, this approach can be adapted for a range of applications where controlled delivery of a payload into a porous structure is required. Each subcomponent can operate as a modular, isolated batch process in a stepwise progression from scaffold preparation to seeded scaffold collection, or in continuous mode, offering flexibility in production and storage. The process is streamlined, modular, scalable, and capable of automation, facilitating efficient operations and reducing potential points for contamination.

[0337] In one aspect, the present disclosure provides a method preparing a scaffold, the method comprising treating the scaffold with a treatment to provide a treated scaffold and exposing the treated scaffold to a wetting system to provide a wetted scaffold. The wetting system may comprise a payload. As used herein, the term “payload” may refer to a desired composition with an intended delivery destination. The payload may comprise at least one of cells, small particles, small molecules, dissolved molecules, suspended molecules, and / or non-solvent fluids. The payload may be mixed into the wetting system. The payload may comprise cell culture media. As used herein, the term “scaffold” may refer to a structure or matrix. As used herein, the term “treating” may refer to means of exposing the scaffold to a treatment. The treatment may involve at least one member selected from the group consisting of plasma, flame, corona discharge, ion beam, electron beam, ionizing radiation, UV, ozone, chemical etching, laser treatment, enzymatic modification, grafting polymerization, surface polymerization, peroxides, and supercritical fluids. In some preferred embodiments, the treatment is cold plasma. As used herein, the term “treated scaffold” may refer to the scaffold following treatment or being treated. As used herein, the term “exposing” may refer to the introduction of a system or solution to a composition which may include a scaffold which may, for example, be introduced to a wetting system. The wetting system may comprise cell culture media. The wetting system may be continuous. As used herein, “continuous wetting” may refer to uninterrupted displacement of gas with a liquid to maintain contact with a solid surface, substrate, or scaffold. As used herein, the term “wetted scaffold” may refer to a treated scaffold followingAtty. Dkt. No. 166118.01557 exposure to a wetting system. As used herein, the term “introducing” may refer to contacting or otherwise exposing a composition to another composition.[03381 Insome aspects, the wetting system comprises a wetting fluid. As used herein, the term “wetting fluid” may refer to aa liquid or gas intended to wet a target composition, for example a scaffold. In some aspects, the payload comprises the wetting fluid.

[0339] In some aspects, the method further comprises a post-wetting modification. As used herein, a “post-wetting modification” may refer to further manipulation to the scaffold following exposure to the wetting system. In some aspects, the post-wetting modification comprises at least one of rinsing in dilute buffers, crosslinking, and / or heating.

[0340] In some aspects, the treated scaffold has increased surface energy, surface charge, hydrophilicity, and / or capillary action relative to the scaffold before it is treated. Wetting has the effect of passivating the increased surface energy provided by the treatment. Therefore, in some aspects, the wetted scaffold has decreased surface tension, hydrophilicity, and / or capillary action relative to the scaffold before it is wetted. Surface tension, hydrophilicity, and / or capillary action may combine to wick fluid and payload along a treated fiber which may, thus, improve the ability to seed said treated fiber. In some aspects, an increase in capillary action draws the payload into the scaffold.

[0341] In some aspects, the scaffold is a thread. As used herein, the term “thread” may refer to a narrow cylindrical fiber or strand. In some aspects, the thread comprises gelatin, collagen, fibrin, elastin, animal protein, silk fibroin, seaweed gel, alginate, carrageenan, agarose, algal protein, fungal protein, chitosan, bacterial gel, gellan gum, plant protein, leaf protein, soy protein, wheat gluten, glutenin, gliadin, zein, mung bean protein, rice protein, potato protein, sweet potato protein, fava bean protein, chickpea protein, rapeseed protein, lentil protein, hemp protein, quinoa protein, oat protein, sunflower seed protein, pumpkin seed protein, heparin, heparin sulfate, pullulan, amylose, dextran, starch, cellulose, hemicellulose, lignin, glucomannan, chondroitin sulfate, chitin, polynucleotides, polysaccharide, a glycosaminoglycan, natural polyesters, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic) acid, polycaprolactone, polyhydroxyalkanoates, bioesters, or a polymer material made of a monomer selected from the group consisting of: polylactic acid, polyglycolic acid, poly(lactic-co-glycolic) acid, polyhydroxyalkanoates, monosaccharides, glucose, fructose, mannose, galactose or any derivatives or variants thereof, glycosamines, aminosugars, aminoacids and derivatives thereof, nucleotides and derivativesAtty. Dkt. No. 166118.01557 thereof, and fatty acids and derivatives thereof, cotton, flax, wool, hemp, jute, sisal, or combinations thereof. In some aspects, the scaffold is porous. As used herein, the term “porous” may refer to a state of having minute spaces or holes through which liquid or air may pass. In some aspects, the scaffold is comprised of small particles, fibers, fiber bundles, films, textiles, protein threads, edible materials, porous solids, suitable for food applications, edible, or combinations thereof. In some aspects, the scaffold is three-dimensional. The presently disclosed methods may be suitable for seeding scaffolds larger than methods disclosed in the art. Therefore, in some aspects, the scaffold is larger than 1 cm3. In some aspects, the scaffold comprises fibers, and wherein the fibers are able to absorb about 50 pL of fluid per cm length of scaffold. Absorbing may include the taking up or embodying of a payload or cells.

[0342] The presently disclosed methods may decrease the necessary time to incubate compositions in a wetting fluid or payload solution. Therefore, in some aspects, the payload is introduced for less than or equal to about one hour, and, in some aspects, the payload is introduced for less than or equal to about 30 minutes. In some aspects, the scaffold is treated for less than or equal to about 60 seconds.

[0343] Delivering a payload comprising cells using the presently disclosed methods may result in cells adhering faster due to increased surface contact, which can be forced by limiting the volume of fluid, driving fluid menisci (and cells) closer to fiber surfaces. Additionally, the wicking of cell suspension into bundles of fibers (threads, braids, fabric, etc.) can transport cells deeper into thread and seed them more uniformly. Therefore, in some aspects, the payload comprises a plurality of cells, and, in some aspects, the plurality of cells is selected from the group consisting of muscle cells, muscle precursor cells, smooth muscle cells, keratinocytes, melanocytes, macrophages, t-cells, b-cells, dendritic cells, neuronal cells, endothelial cells, epithelial cells, chondrocytes, osteoblasts, mammary epitheilial cells, parietal cells, podocytes, mesangial cells, renal tubule cells, pericytes, ovarian cells, pluripotent stem cells, mesenchymal stem cells, satellite cells, myoblast cells, myofibroblast cells, fibroblasts cells, endothelial cells, adipose cells, hepatocytes, cardiomyocytes, insect cells, algal cells, fungal cells, reptilian cells, amphibian cells, avian cells, fish cells, mollusc cells, lobster cells, crayfish cells, mammalian cells, and / or combinations thereof. These methods may preferably be utilized in the production of meat tissue. In some aspects, the plurality of cells comprises at least two different cell types, and, in some aspects, the at least two cell types is selected from the group consisting of muscle cells, muscleAtty. Dkt. No. 166118.01557 precursor cells, smooth muscle cells, keratinocytes, melanocytes, macrophages, t-cells, b-cells, dendritic cells, neuronal cells, endothelial cells, epithelial cells, chondrocytes, osteoblasts, mammary epitheilial cells, parietal cells, podocytes, mesangial cells, renal tubule cells, pericytes, ovarian cells, pluripotent stem cells, mesenchymal stem cells, satellite cells, myoblast cells, myofibroblast cells, fibroblasts cells, endothelial cells, adipose cells, hepatocytes, cardiomyocytes, insect cells, algal cells, fungal cells, reptilian cells, amphibian cells, avian cells, fish cells, mollusc cells, lobster cells, crayfish cells, and mammalian cells. In some aspects, the plurality of cells are introduced for a period of at least one hour, and, in some aspects, the plurality of cells is introduced at a concentration of about 1 million cells / mL to about 200 million cells / mL. In some aspects, the plurality of cells adheres and / or spreads faster compared to untreated scaffolding.

[0344] In another aspect, the present disclosure provides a composition produced by any one of the methods disclosed herein.

[0345] In another aspect, the present disclosure provides a composition comprising a scaffold, wherein the scaffold is treated with a treatment and wetted with a wetting system. In some aspects, the treatment is selected from the group consisting of plasma, flame, corona discharge, ion beam, electron beam, ionizing radiation, UV, ozone, chemical etching, laser treatment, enzymatic modification, grafting polymerization, surface polymerization, peroxides, and supercritical fluids. In some aspects, the wetting system comprises a wetting fluid. In some aspects, the wetting system comprises a payload, and, in some aspects, the payload comprises a plurality of cells, and, in some aspects, the plurality of cells is selected from the group consisting of muscle cells, muscle precursor cells, smooth muscle cells, keratinocytes, melanocytes, macrophages, t-cells, b-cells, dendritic cells, neuronal cells, endothelial cells, epithelial cells, chondrocytes, osteoblasts, mammary epitheilial cells, parietal cells, podocytes, mesangial cells, renal tubule cells, pericytes, ovarian cells, pluripotent stem cells, mesenchymal stem cells, satellite cells, myoblast cells, myofibroblast cells, fibroblasts cells, endothelial cells, adipose cells, hepatocytes, cardiomyocytes, insect cells, algal cells, fungal cells, reptilian cells, amphibian cells, avian cells, fish cells, mollusc cells, lobster cells, crayfish cells, mammalian cells, and / or combinations thereof. In some aspects, the plurality of cells comprises at least two different cell types, and, in some aspects, the at least two cell types is selected from the group consisting of muscle cells, muscle precursor cells, smooth muscle cells, keratinocytes, melanocytes, macrophages, t-cells, b-cells, dendritic cells, neuronal cells, endothelial cells, epithelial cells, chondrocytes, osteoblasts, mammary epitheilial cells,Atty. Dkt. No. 166118.01557 parietal cells, podocytes, mesangial cells, renal tubule cells, pericytes, ovarian cells, pluripotent stem cells, mesenchymal stem cells, satellite cells, myoblast cells, myofibroblast cells, fibroblasts cells, endothelial cells, adipose cells, hepatocytes, cardiomyocytes, insect cells, algal cells, fungal cells, reptilian cells, amphibian cells, avian cells, fish cells, mollusc cells, lobster cells, crayfish cells, and mammalian cells.

[0346] In some aspects, the wetted scaffold has decreased surface tension, hydrophilicity, and / or capillary action relative to the scaffold before it is wetted but after it is treated. In some aspects, the scaffold has increased cell coverage and / or cell uniformity.

[0347] In some aspects, the scaffold is a thread, and in some aspects, the thread comprises gelatin, collagen, fibrin, elastin, animal protein, silk fibroin, seaweed gel, alginate, carrageenan, agarose, algal protein, fungal protein, chitosan, bacterial gel, gellan gum, plant protein, leaf protein, soy protein, wheat gluten, glutenin, gliadin, zein, mung bean protein, rice protein, potato protein, sweet potato protein, fava bean protein, chickpea protein, rapeseed protein, lentil protein, hemp protein, quinoa protein, oat protein, sunflower seed protein, pumpkin seed protein, heparin, heparin sulfate, pullulan, amylose, dextran, starch, cellulose, hemicellulose, lignin, glucomannan, chondroitin sulfate, chitin, polynucleotides, polysaccharide, a glycosaminoglycan, natural polyesters, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic) acid, polycaprolactone, polyhydroxyalkanoates, bioesters, or a polymer material made of a monomer selected from the group consisting of: polylactic acid, polyglycolic acid, poly(lactic-co-glycolic) acid, polyhydroxyalkanoates, monosaccharides, glucose, fructose, mannose, galactose or any derivatives or variants thereof, glycosamines, aminosugars, aminoacids and derivatives thereof, nucleotides and derivatives thereof, and fatty acids and derivatives thereof, cotton, flax, wool, hemp, jute, sisal, or combinations thereof.

[0348] In another aspect, the present disclosure provides a kit for seeding a scaffold, the kit comprising the components of a treatment and a wetting system. Components of a treatment will include the means necessary to provide a treatment which may be selected from at least one member from the group consisting of plasma, flame, corona discharge, ion beam, electron beam, ionizing radiation, UV, ozone, chemical etching, laser treatment, enzymatic modification, grafting polymerization, surface polymerization, peroxides, and supercritical fluids. In some aspects, the wetting system comprises a wetting fluid.Device for generating treated edible fiber scaffoldAtty. Dkt. No. 166118.01557

[0349] In an aspect of the current disclosure, devices for generating treated edible fiber scaffold are provided, (a) a movement mechanism to extend an edible fiber scaffold into the device, wherein the movement mechanism is controlled by at least one controller; (b) a first chamber housing a component for treating the edible scaffold; (c) a second chamber housing a wetting system. The at least one controller may be configured to drive the edible fiber scaffold into the device such that the edible fiber passes through the first chamber and into the second chamber. The component for treating the edible scaffold may comprise a component for treating the edible fiber scaffold with at least one of the following: cold plasma, flame, corona discharge, ion beam, electron beam, ionizing radiation, UV, ozone, chemical etching, laser treatment, enzymatic modification, grafting polymerization, surface polymerization, peroxides, or supercritical fluids. The component for treating the edible fiber scaffold may comprise a component that generates cold plasma.

[0350] The movement mechanism may comprise a series of rollers, conveyors, and / or belts, the rollers, conveyors, and / or belts individually serving to at least one of support, apply motive force to, and / or direct the scaffold (e.g., as in FIGs. 5, 12 and / or 55). The movement mechanism may comprise at least one roller or pinch rollers.

[0351] The second chamber housing the wetting system may comprise an inlet, a chamber well, and an outlet, and wherein the diameter of the outlet is narrower than the diameter of the chamber well (see FIG. 55). The device may be oriented vertically with (a), (b), and (c) being arranged from top to bottom (see FIGs. 14 and 55). The outlet of the second chamber may comprise an aqueous fluid and creates a meniscus to wet the edible fiber scaffold as it passes through the second chamber. The device may further comprise a movement mechanism to place the edible fiber scaffold leaving the second chamber into a bioreactor or vessel. The second chamber may comprise cell culture medium. The bioreactor or vessel may comprises cell culture medium. The second chamber may comprise a plurality of cells in suspension in the cell culture medium. The plurality of cells may comprise muscle and / or adipose cells. The plurality of cells may comprises porcine, galline, or bovine cells. The plurality of cells may comprise precursor cells, pluripotent stem cells, mesenchymal stem cells, satellite cells, myoblast cells, myofibroblast cells, fibroblasts cells, endothelial cells, adipose cells, hepatocytes, cardiomyocytes, or combinations thereof.Additional DefinitionsAtty. Dkt. No. 166118.01557

[0352] It is to be understood that the invention is not limited to the particular embodiments described. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. The scope of the present invention will be limited only by the claims. It should be apparent to those skilled in the art that many additional modifications beside those already described are possible without departing from the inventive concepts. In interpreting this disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise. For example, the term “a cell” or “an edible fiber” should be interpreted to mean “one or more cells” and “one or more edible fibers,” respectively, unless the context clearly dictates otherwise. As used herein, the term “plurality” means “two or more.”

[0353] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term, up to plus or minus 20% of the particular term.

[0354] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0355] The phrase “such as” should be interpreted as “for example, including.” Moreover the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0356] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinaryAtty. Dkt. No. 166118.01557 skill in the art would understand the convention (e g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0357] All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.

[0358] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can. ’’The following abbreviations are used herein: Analysis of Variance (ANOVA), Bovine Satellite Cells (BSCs), Dulbecco’s modified Eagle medium (DMEM), Feed Conversion Ratios (FCR), Fetal Bovine Serum (FBS), Fibroblast Growth Factor (FGF), Green Fluorescent Protein (GFP), Myosin Heavy Chain (MHC), Platinum / Palladium (Pt / Pd), Radioimmunoprecipitation Assay (RIP A), Sarcomeric Myosin Heavy Chain (MF-20), Scanning Electron Microscopy (SEM), sodium carbonate (Na2CO), Western Blot (WB).EXAMPLESExample 1: FIBER SCREENING AND SELECTION

[0359] Suitable biomaterial fibers were screened and selected based on: commercial availability in fiber format, edibility, cost, mechanical properties, purity, and ability to support cells in cultureAtty. Dkt. No. 166118.01557(e g., viability, adhesion, differentiation). Suitable candidates included cotton (cellulose), chitosan, soy protein, wheat gluten (from commercially available spaghetti), and silk (all Amazon Basics brand, Amazon, Seattle, WA). All fiber candidates were sterilized via autoclave (steam cycle) prior to use in cell culture experiments. In addition to autoclave treatment (steam and / or pressure) chemical treatment such as ethylene oxide (ETO), or physical treatment such as heat or radiation (for instance gamma radiation) may also be used to sterilize textiles and fibers [37,38], along with combinations of the foregoing. Autoclave sterilization was selected due to its availability, relatively low cost and ease of use, and applications in the food industry. ETO sterilization is effective without altering fiber characteristics, but can be costly and challenging due to the safety requirements

[0037] , Gamma irradiation can damage fibers but is preferred when fibers need to be treated for insect removal

[0039] , rather than for use in food applications. In one case, a “raw” fiber was not treated beyond autoclave sterilization, but in other cases combination treatments may be performed. A “degummed” silk fiber was boiled according to standard protocols to remove the sericin protein and preserve only the silk fibroin

[0040] , A “pretreated” silk and cotton fiber was degummed (in the case of silk) and subjected to the boiling and detergent wash described herein. Additionally, for the purpose of the present study we utilize the term ‘fiber’. However, the materials utilized are actually yams (e.g., multifiber bundles) when examining them as purchased materials.

[0360] C2C12 murine skeletal muscle myoblasts (ATCC, Manassas, VA) were cultured in tissue-culture treated flasks

[0041] , Briefly, cells were grown to -70% confluency in standard growth medium (Dulbecco’s Modified Eagle’s Medium (DMEM) + Glutamax, 10% fetal bovine serum (FBS), and 1% antibiotic / antimycotic) (all from Gibco, Waltham, MA) and used between passages 5 and 16. For screening experiments, cells were detached using 0.25% Trypsin-EDTA and seeded onto sterilized fibers at 20,000 cells / mL. Cells were cultured up to 48 h to assess initial interactions with the fibers. Initial attachment was quantified via fluorescence imaging of GFP cells, LIVE / DEAD staining, CyQuant cell counting, and Alamar Blue assays (expanded below). All experiments were repeated a minimum of 3 times for statistical interpretations.

[0361] PRETREATED SILK AND COTTON FIBERS FOR EXPERIMENTS

[0362] C2C12 cells were cultured as described above, detached using 0.25% Trypsin-EDTA, and seeded onto pretreated and sterilized fibers at 20,000 cells / mL, with and without coatings.Atty. Dkt. No. 166118.01557Cells were cultured for 12 h, 24 h, 48 h, 5 weeks, or 8 weeks to assess both short- and long-term interactions with the fibers.BOVINE SATELLITE CELL CULTURE ON PRETREATED SILK AND COTTON FIBERS

[0363] Testing with C2C12 cells demonstrates proof-of-concept showing cytocompatibility of the fibers, however, this murine cell line may be less desired commercially for human food consumption (though it could be used for food). To demonstrate feasibility with another cell line, bovine satellite cells (BSCs), an edible cell source from livestock

[0042] , were cultured on the fibers using the parameters established with the C2C12s. BSCs were isolated and cultured per established methods

[0043] , BSCs were cultured in DMEM supplemented with 20% FBS, 1% antibiotic / antimycotic and Ing / mL fibroblast growth factor (FGF)-basic (154 a.a., PeproTech, White Plains, NJ). Tissue culture flasks were coated with recombinant laminin-511 (iMatrix-611 , Fisher, USA) at a concentration of 1.5 pg / cm2. The BSCs were incubated at 37 °C with 5% CO2. All BSCs were used at passage 5.SEM IMAGING OF CELL-LADEN FIBERS

[0364] Cell-laden fiber samples were cross-linked with 1% glutaraldehyde solution (solvent: DPBS) for 1 hr. The cross-linked scaffolds were then dehydrated in EtOH under progressively higher concentrations (30%, 50%, 70%, 90%, and 100% EtOH). The scaffolds were soaked in each concentration of EtOH for 30 min. Samples were then dried in a fume hood overnight. A thin layer (10 nm) of platinum / palladium (Pt / Pd) was applied onto the dried scaffolds using a sputter coater (208HR, Cressington Scientific Instruments Inc., UK). The samples were subsequently imaged using a scanning electron microscope (Zeiss UltraPlus SEM, Carl Zeiss SMT Inc., USA) at 5KeV.FLUORESCENCE MICROSCOPY

[0365] For initial fiber studies, GFP-C2C12 cells (ATCC) were used and visualized without staining with a Keyence BZ-X700 series fluorescence microscope (Keyence Corp, Japan). For subsequent experiments, C2C12s without GFP were used to allow staining with dyes in the GFP emission range. To confirm cell viability and to observe changes in cell morphology with time on the fibers, whole fibers with seeded cells were stained using a LIVE / DEAD kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions and imaged on the Keyence microscope. Fibers and cells were also stained with Alexa Fluor 488 phalloidin (Invitrogen),Atty. Dkt. No. 166118.01557 sarcomeric myosin heavy chain (MF-20) (Developmental Studies Hybridoma Bank, Towa City, IA), and 4,6-Diamidino-2-phenylindole (DAPI) (Life Tech., Waltham, MA) to observe the actin cytoskeleton, MF-20 and cell nuclei, respectively. MF-20 was visualized by incubating whole fibers in Al exaFluor 594 goat anti-rabbit secondary antibody (Invitrogen) using the manufacturer’s recommended staining technique. The fibers were stained with antibodies and imaged using a Leica SP2 confocal microscope (Leica Microsystems, Germany).MECHANICAL ANALYSIS

[0366] Mechanical testing was performed on the cotton, soy protein, wheat gluten, silk fibers and bean noodles with pull to failure in tension using an Instron load frame (Instron, White Plains, NY) and 1 kN load cell. Fibers were soaked for 24 h in DMEM at 37°C and then mechanically tested at a strain rate of 1% per second until failure. Stress, strain at maximum stress, and elastic moduli were determined from the stress-strain curve, with elastic modulus calculated as the slope of the linear region of the curve. All fibers had a circular cross-section, and stress was calculated by inputting the cross-sectional diameter into the Instron software. The cross-section was measured 3 times using electronic calipers (Carerra, New York, NY) and averaged. The linear region was defined by fitting a linear equation to the portion of the stress-strain curve that resulted in a R2 value of >95% for the fit. Mechanical testing was conducted as part of the initial fiber sweep for coated and uncoated fibers and for untreated versus pretreated fibers to evaluate the impact of fiber processing on the mechanical properties.FIBER COATING

[0367] Prior to coating, fibers were cut into 4 cm length pieces, washed 3 times with phosphate buffered saline (PBS, Gibco, Grandview, NY) for 20 min each, and air dried inside a chemical hood. The fibers were then autoclaved for sterilization on a steam cycle, and sterile coating solutions were prepared. Fibers were submerged within each coating solution overnight (2% sodium alginate in 4% CaC12 crosslinking bath, fibronectin 5 ug / mL, 1% transglutaminase; all materials from Invitrogen, Carlsbad, CA) or plasma coated using a plasma cleaner (PDC-32G, Harrick Scientific, New York, USA). Specifically, the fibers were coated in the plasma chamber for 5 min on the “high voltage” setting using ambient air. Fibers were not dried prior to use. C2C12 and C2C12-GFP cells (ATCC) were then seeded on the fibers in 24-well ultra-low attachment plates (Coming, Corning, NY), at 20,000 cells / well. Cells were cultured for 12, 24, or 48 hours for cell attachment tests (Cy Quant cell counting and fluorescence microscopy imaging).Atty. Dkt. No. 166118.01557SILK AND COTTON FIBER PRETREATMENT

[0368] Mulberry silk yam was supplied by The Fibre Co. (Rochester, NY) and raw cotton yarn was purchased from Aurora Silk (Portland, OR). Silk and cotton materials, both 100% compositions, were pre-treated based on an established protocol

[0044] , For the treatment, 5 g of silk yarn was soaked in a solution containing 0.3% non-ionic detergent (TritonX-100, Sigma) and 2.12 g / L sodium carbonate (Na2CO; Sigma) at 90°C for 1 h. The silk was then rinsed in deionized water (DI H2O) 3 times for 20 min. Silk and raw cotton yarns were dried in a chemical hood for 12 h at room temperature (25°C). Silk was autoclaved prior to use. The same procedure was repeated for degummed silk boiled for 30 min and prepared based on established protocols

[0040] , For the cotton yarn, 5 g was treated by boiling for 4 h in a solution containing 0.5 g / L Na2COs and 3 g / L TritonX- 100. The cotton was then washed 3 times for 20 min in DI H2O and dried at 25 °C in the chemical hood for 12 h. All fibers were also sterilized in an autoclave.CELL VIABILITY

[0369] Alamar blue (ThermoFisher) was used to monitor the metabolic activity of C2C12s and bovine satellite cells. The samples were incubated in 10% Alamar blue solution (stock reagent mixed with culture media) for 2 hr at 37°C with 5% CO2. Then, 100 pL of solution was taken from the incubated samples and pipetted into 96-well plates. The fluorescence of the solutions was measured under 560 / 590 (Excitation / Emission) using a microplate reader (BioTek Instruments, Winooski, VT). All measurement were done in triplicate.WESTERN BLOT ANALYSIS

[0370] To confirm the formation of myosin heavy chain (MHC) and the viability of the cells, Western Blot analyses were carried out on cells grown on the pretreated cotton and silk fibers at various time points. Cell-laden fibers were collected for Western blot (WB) analysis in radioimmunoprecipitation assay (RIP A) cell lysis buffer and Halt protease inhibitor (Invitrogen). Cells and fibers were then homogenized using a standard handheld homogenizer probe (Fisher) and centrifuged at 2,000 RPM for 5 minutes to separate the fiber components from the cell lysate. The pellets (fiber components) were discarded, and the supernatant was kept for further analysis of the cellular components. Sodium dodecyl sulfate (SDS) was added at a 1: 1 ratio and samples were sonicated, heated to 95oC for 5 minutes, and loaded into Novex Wedgewell 4-20% Tris Glycine Mini Gels (Invitrogen). Lanes were loaded differentially to normalize total proteinAtty. Dkt. No. 166118.01557 content. Cell lysate collected from each fiber sample was run in its own lane. At least 3 wells of each condition were run per individual experiment. Following separation via electrophoresis, gels were transferred to nitrocellulose membranes (Invitrogen), blocked in 5% milk in Tris buffered saline (Boston Bioproducts, Ashland, MA) with 0.1% Tween20 (TBST) (Acros Organics, Morris Plains, NJ), and incubated overnight at 4o C on an orbital shaker with 1 :2000 MHC primary antibody raised in rabbit (Abeam, Cambridge, MA) in 5% bovine serum albumin (BSA) in TBST. P-actin (Abeam) was used as the loading and viability control. Blots were washed 3x for 5 min in TBST and incubated for 1 h at room temperature with goat anti-rabbit HRP-linked secondary antibody (Invitrogen). Blots were then washed in TBST, developed using enhanced chemiluminescence (ECL) reagents (Invitrogen), and imaged using a Genesis Pi6 imager (Syngene, Frederick, MD). No statistical analysis was carried out as the objective was to confirm the presence of MHC following extended culture on the fibers.STATISTICAL ANALYSIS

[0371] Cell attachment data was quantified using the Cy-Quant counting system and analyzed using 2-tailed unpaired T-tests with Welch’s correction, or using a one-way analysis of variance (ANOVA). Mechanical data was analyzed using either a two-tailed unpaired T-test with Welch’s correction or a one-way ANOVA in Prism 9 (GraphPad, LaJolla, CA) with Sidak’s multiple comparison test, depending on the specific set of fibers. Significance was set at p < 0.05. Results are reported as mean ± standard deviation.

[0372] RESULTSINITIAL FIBER ANALYSES REQUIRED ADDITIONAL PROCESSING FOR CELL ATTACHMENT

[0373] To identify a suitable fiber candidate, C2C12 cells were seeded onto a variety of edible and commercially available fibers as a proof-of-concept cell line prior to validation with the potentially edible BSCs, and initial attachment was evaluated. While some cell attachment was observed on all fiber types within the first 24 h (Figure 21), overall cell attachment was poor and insufficient for the required rapid fiber coating. Additionally, the mung bean noodle (Figure 21 D- F), wheat gluten (Figure 21 G-I), and soy protein (Figure 21 M-O) fibers dissolved in the culture medium within the first 2 weeks of the experiment, and thus were not subjected to long-term mechanical testing (Figure 22 shows mechanical testing of dry fibers). Commercially available cotton and silk fibers were stable in the medium, and thus were considered further in the study.Atty. Dkt. No. 166118.01557COATINGS DID NOT SIGNIFICANTLY IMPROVE CELL ATTACHMENT ON COMMERCIALSILK AND COTTON FIBERS

[0374] Though plasma coating of the fibers prior to use in cell culture significantly improved initial cell attachment (Figure 16 C-D), cell attachment remained insufficient for rapid cell coating and growth on the fibers (Figure 16 A-B). The Cyquant cell counting analysis showed that of the 20,000 cells / mL (>60,000 total) seeded into the fibers, an average of 12,000 per fiber were attached following 24 h in culture on the cotton fibers, corresponding to approximately 6,000 cells per cm of fiber length and about 20,000 cells per cm2of fiber area (assuming average fiber dimensions of 2 cm long and 1 mm in diameter, for a total surface area of 0.64 mm2). On the silk fibers, the average was less than 10,000 cells attached after 24 h in culture (Figure 16 C-D). The goal was for at least 80% of the cells, or roughly 16,000 cells per fiber, to still attach after 24 h in culture, as this level of cell coverage would best support myofiber proliferation and differentiation into myotubes (43), which give meat its structure and texture.

[0375] To improve initial cell attachment to the cotton and silk fibers, silk (Figure 23) and cotton (Figure 24) fibers were coated overnight in transglutaminase, alginate, fibronectin or plasma treated. Transglutaminase was tested as a potential way to form enzyme-loaded fibers that could generate peptide bonds between cell-secreted proteins to improve cell attachment to the fibers. Alginate was tested as a physical encapsulant to keep the cells physically close to the fibers to optimize cell-fiber interactions, as the cells historically would not adhere to the polysaccharide directly. LIVE / DEAD staining showed poor overall C2C12 attachment and spreading, suggesting that the fibers and coatings did not support sufficient cell interactions with the fiber surface (Figures 18, 19). In addition, autofluorescence from the fibers showed smooth surfaces that would likely be challenging for robust cell attachment, highlighting the need for alterative fiber pretreatment to support cell attachment.DETERGENT PRE-TREATMENT SIGNIFICANTLY IMPROVED CELL ATTACHMENT ONCOMMERCIAL SILK AND COTTON FIBERS

[0376] Following pre-treatment of the fibers with nonionic detergents and boiling, cell attachment significantly improved at 24 h on both silk (Figure 17 A-F) and cotton (Figure 17 G- L). Fluorescence imaging showed cells spread on the fibers, with an even coating and few dead cells. Cells covered the fibers sufficiently to reduce autofluorescence from the underlying polymerAtty. Dkt. No. 166118.01557 material (Figure 17). Cell coverage remained high throughout the experiment (Figure 18 A; Figures 25, 26, 27). Initial increases in fluorescence intensity were sustained over several weeks in culture and fiber coverage was maintained throughout the experiment, plateauing at its maximal value at week 8 (Figure 18).DETERGENT AND BOILING PRETREATMENT OF FIBERS SUPPORTS LONGER-TERM CELLGROWTH

[0377] Over 8 weeks in culture, SEM imaging showed cells completely coated both the silk and cotton fibers and secreted an extensive extracellular matrix that obscured the surface morphology of the fibers (Figures 18 C, D, Figures 19, 20). Compared to acellular control silk fibers (Figure 19 A, B, E, F) and acellular control cotton fibers (Figure 20 A-B), cellular fibers displayed extensive coating with cell-secreted ECM that smoothed the surface of the fibers and covered them in sheet-like structures (Figure 19 C, D, G, H-L; Figure 20 E-H). Alamar blue assays carried out over 10 weeks of cell culture on the fibers indicated significantly higher cell viability on all pretreated fibers (degummed and commercial silk and cotton) compared to the control samples (Figure 18A, Figure 25 C, D). Cell viability on the various pretreated fibers was comparable, with slightly higher viability in degummed silk fibers at week 8, and there were significant differences between the different types of fibers (Figure 25 C). Western Blot data showed that cells survived and produced MHC as early as 7 days following seeding on both cotton and silk fibers (Figure 25 A), and over the course of at least 35 days on the pretreated silk fibers (Figure 25 B), indicating cells were differentiating in addition to proliferating.FIBER COATINGS AND PRETREATMENT DID NOT IMPACT MECHANICAL PROPERTIES,WHILE THE CELLS DID

[0378] Mechanical testing of fibers during the initial assessments showed a range of mechanical properties that could support muscle tissue growth. The elastic modulus of muscle reported in the literature varies, but common values are in the range of 1 to 3 MPa (44-46). Fibers that are less than an order of magnitude outside of this range will likely be suitable for supporting muscle cell growth, as the mechanical properties of the environment will support cell differentiation (47). For the specific fibers tested in this study, elastic moduli ranged from less than 10 MPa for soy protein and mungbean fibers, to between 12 and 18 MPa for wheat gluten fibers, and over 1 GPa for cotton and silk fibers (Figure 18 B, C and Figure 22). Pretreatment of the cotton and silk fibers used inAtty. Dkt. No. 166118.01557 the further studies with the detergent process did not significantly alter the mechanical properties (Figure 18 B, C). The deformation of the pretreated fibers was fully reversible in the elongation range (<10%) that is appropriate for culturing cells. Following 8 weeks in culture, both the silk and cotton cell-laden fibers had significantly lower stress at failure compared to acellular fibers kept in growth media for 8 weeks (controls), suggesting the cells were remodeling the fibers or depositing new ECM that enhanced mechanics (Figure 18 C).PRETREATED COTTON AND SILK FIBERS SUPPORT BSC GROWTH AND DIFFERENTIATION

[0379] Following validation of the ability of the pretreated silk and cotton fibers to support C2C12 cell attachment, growth, and differentiation, the fibers were seeded with BSCs, which are potentially edible and would be used in cultured meat applications. BSCs attached to the cotton (Figure 27 A-C) and Silk (Figure 27 D-F) fibers and formed multi nucleated myotubes (Figure 27, white arrows and blue punctate staining) over 4 weeks in culture. Immunofluorescence staining showed that the BSCs produced MHC (Figure 27 red staining), indicating that the BSCs maintained myogenic differentiation potential.DISCUSSION

[0380] The application of textile engineering similar techniques for the production of cultivated meat at scale depends on the availability of an inexpensive, large quantity, edible and cellcompatible fiber-based scaffolds to support muscle cell growth and differentiation. Among other things, this disclosure describes screening, coating, evaluating, commercially available fiber candidates for this function and identified two preferred candidates: silk and cotton. Both fiber types supported robust cell attachment, growth, and differentiation, while requiring minimal pretreatment. To reach this outcome, a series of inexpensive and commercially available biomaterial fibers were initially screened and selected based on availability in fiber format, edibility, cost, mechanical properties, and the ability to support cell culture (e.g., cell viability, adhesion, differentiation). Initial candidates included cotton (cellulose), chitosan, soy protein, starch, wheat gluten, other plant-based materials, and silk protein. These materials were considered for cell-laden fiber designs. The unique demands of cell culture, including a liquid environment and 37°C resulted in a preference for silk and cotton over wheat gluten, soy protein and mung bean. Both silk and cotton withstood prolonged storage and the required culture conditions. Silk and cotton are not the only widely available, edible fibers, however, they show suitability of a protein-based and a polysaccharide-based fiber option that survived the initial screen.Atty. Dkt. No. 166118.01557Additionally, cotton and silk are widely used in the textile industry, and their mechanical properties and other attributes are well-characterized, providing a solid foundation for utility for the longer- term goals of this study. Cotton / cellulose has been used in other food applications, such as in the form of cellulose nanofibers. Cellulose nanofibers have been proposed as tools for increasing fiber density and decreasing the caloric value of food, as a way of delivering bioactive molecules with food, as food preservation agents, as coatings or packaging for food, and as fat mimetics (48-50). Many other biomaterials can also be considered as fiber candidates. For example, glutenin, zein or chitosan fibers can be considered for support of cell growth and also be edible (40,51-53).

[0381] Although coatings are used in tissue engineering to improve cytocompatibility on a variety of biomaterial substrates, under the solution-based conditions utilized here, results showed coatings did not significantly improve cell attachment. Commercially available cotton and silk fibers often have residues or chemicals left over from their harvesting and packaging, as well as proprietary additives, even if labeled “raw,” and these may inhibit initial cell attachment even with fibronectin, transglutaminase (54), or other previously validated coatings. The nonionic detergent and extensive boiling pretreatment some of these coatings, and, combined with autoclaving, supported the improved cell attachment onto and within the cotton fibers, even in the absence of ligands, a phenomenon reported previously (55). Based on the SEM images of the fibers before and after boiling (data not shown) and in the acellular versus cellular conditions (Figures 18, 19, 20), we hypothesize that the pretreatment i) removed waxy or cytotoxic coatings, ii) created spaces or “pores” within the fibers for cells to attach via increased surface area, and iii) resulted in enough chemical functionalization of the fiber surfaces to provide areas for cell attachment. With high enough initial cell attachment and subsequent secretion of ECM, these fibers were able to support prolonged cell growth. The same outcome was found for the degummed and commercial silk fibers, where processing denatured parts of the silk protein matrix exposed sufficient binding sites to support initial cell attachment (56,57). With enough initial cell attachment, cells appeared to secrete ECM and continued to grow and proliferate on and within the new matrix, as observed via SEM imaging for both the silk and the cotton fibers (Figures 19, 20). Cotton or silk, when modified to increase cell attachment, would likely support even better initial cell attachment, though such custom fibers are not currently commercially available at the scale needed for cultivated meat applications.Atty. Dkt. No. 166118.01557

[0382] Silk and cellulose, while edible, are not readily digestible in the human gut (58). This issue could be mitigated by enzymatically pre-digesting the fibers. Other approaches may involve the use of genetically modified cells that secrete enzymes to digest the fibers once the desired 3D structure has been achieved, assuming genetically modified cells would be acceptable towards food-related goals (59). Cooking (thermal treatment) may also impact the properties of the fibers, potentially improving digestibility. Interestingly, following 8 weeks in culture, the stress at failure of the cell-laden fibers had decreased significantly compared to the acellular controls, suggesting cellular activity had begun to weaken the fibers (Figure 18 C). The exact mechanisms by which C2C12 cells may be degrading cotton fibers are unclear, but since the cells do not secrete cellulase, this outcome may be more related to physical phenomena vs biochemical, such as cells infiltrating the spaces between the strands of the fibers and separating them, resulting in an overall weakening of the fibers under tensile strain. Our initial biological analysis via Alamar blue assays, fluorescence imaging, and WB immunoassays suggests the cells are displaying normal proliferative behavior, with evidence of differentiation. These data highlight the feasibility of the cotton and silk fiber platforms for translating scale-up of textile engineering to the cultivated meat. The fibers were still well within the mechanical range required for a textile engineering process, and utilizing cells to modulate fiber mechanical and digestibility properties is also possible.

[0383] For the BSCs, our experiments showed that they proliferate and adhere to both cotton and degummed silk yarns (Figure 27). The samples showed MHC expression, which indicates the cells maintain their phenotypes and can undergo myogenic differentiation (62). The cell coverage ratio of BSCs (Figure 27) appeared lower compared to the C2C12s (Figure 26). Coating the fibers with substances to specifically enhance BSC attachment (recombinant proteins or hydrogels) may accelerate BSC coverage and differentiation (60,61). The results demonstrate that the fibers support muscle cells and maintain mechanical integrity.

[0384] The results suggest that since MF20 and MHC were produced consistently during longterm cell culture on the fibers (Figures 25, 26, 27), the cells have the ability to differentiate. Additionally, non-livestock animal cells (such as insect cells or genetically modified C2C12 murine lines) may be suitable for cultivated meats as pet foods of the future.

[0385] The results showed that muscle cells attached and grew on commercially available silk and cotton fibers with minimal pre-treatment. These fibers have utility in cultivated meat technology, particularly for scaling production using textile engineering-based methods. Cell-Atty. Dkt. No. 166118.01557 laden fibers may be used as building blocks for food engineering, providing mechanically robust biomaterial substrates to enhance meat-like textures. These fibers have the potential contribute towards sustainable, healthy, and affordable cultivated meat alternatives in the future.ReferencesN. Alexandratos, J. Bruinsma, World Agriculture towards 2030 / 2050: the 2012 revision, Food and Agriculture Organization of the United Nations, 2012. http: / / www.fao. org / 3 / ap 106e / ap 106e.pdf.N.R.W. Geiker, H.C. Bertram, H. Mejborn, L.O. Dragsted, L. Kristensen, J.R. Carrascal, S. Biigel, A. 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Microdevices. 14 (2012) 1115-1127. https: / / doi.org / 10.1007 / sl0544-012-9677-0.A. Townsend-Nicholson, S.N. Jayasinghe, Cell Electrospinning: a Unique Biotechnique for Encapsulating Living Organisms for Generating Active Biological Microthreads / Scaffolds, Biomacromolecules. 7 (2006) 3364-3369. https: / / doi.org / 10.1021 / bm060649h.S. Ghorbanian, M.A. Qasaimeh, M. Akbari, A. Tamayol, D. Juncker, Microfluidic direct writer with integrated declogging mechanism for fabricating cell-laden hydrogel constructs, Biomed. Microdevices. 16 (2014) 387-395. https: / / doi.org / 10.1007 / sl0544-014-9842-8.K. Drabkova, J. Krejci, M. Skrdlantova, M. Durovic, B. Bacilkova, Influence of Disinfectants on Natural Textile Fibres, Restaur. Int. J. Preserv. Libr. Arch. Mater. 42 (2021) 67- 86. https: / / doi.org / 10.1515 / res-2021-0002.Front Matter, in: Antiseps. Disinfect. Steriliz., John Wiley & Sons, Ltd, 2017: pp. i-xix. https: / / doi.org / 10.1128 / 9781555819682.fmatter.Irradiation of Cellulosic Pulps: Understanding Its Impact on Cellulose Oxidation | Biomacromolecules, (n.d.). https: / / pubs.acs.org / doi / 10.1021 / bm3014457 (accessed September 6, 2022).D.N. Rockwood, R.C. Preda, T. Yucel, X. Wang, M.L. Lovett, D.L. Kaplan, Materials fabrication from Bombyx mori silk fibroin, Nat Protoc. 6 (2011).N. Xiang, J.S.K. Yuen, AJ. Stout, N.R. Rubio, Y. Chen, D.L. Kaplan, 3D porous scaffolds from wheat glutenin for cultured meat applications, Biomaterials. 285 (2022) 121543. https: / / doi.Org / 10.1016 / j.biomaterials.2022.121543.AJ. Stout, MJ. Arnett, K.M. Chai, T. Guo, L. Liao, A.B. Mirliani, M.L. Rittenberg, M. Shub, E.C. White, J.S.K. Yuen, X. Zhang, D.L. Kaplan, Immortalized bovine satellite cells for cultured meat applications, Cell Biology, 2022. https: / / doi.org / 10.1101 / 2022.12.02.518927.Atty. Dkt. No. 166118.01557R. Simsa, J. Yuen, A. Stout, N. Rubio, P. Fogelstrand, D.L. Kaplan, Extracellular heme proteins influence bovine myosatellite cell proliferation and the color of cell-based meat., Foods. 8 (2019) 521.R.L. Horan, K. Antle, A.L. Collette, Y. Wang, J. Huang, J.E. Moreau, V. Volloch, D.L. Kaplan, G.H. Altman, In vitro degradation of silk fibroin, Biomaterials. 26 (2005) 3385-3393. https: / / doi.Org / 10.1016 / j.biomaterials.2004.09.020.Example 2: Foundational Studies in Textural Engineering for Alternative Protein Products

[0386] This body of work is critical to the advancement of soy protein isolate (SPI) fiber technology for applications in meat analog development. Through systematic modulation of fiber composition and spinning parameters, the studies demonstrate the ability to precisely control key mechanical properties, including stiffness, tensile strength, and extensibility, all of which are essential for replicating the structural and textural attributes of conventional meat. Furthermore, the research examines the mechanical stability and microbial resistance of SPI fibers under varying storage conditions, ensuring their functional integrity and practical applicability. The successful assembly of fiber bundles and the demonstrated capacity for direct cell seeding onto fiber surfaces underscore the potential for the development of hybrid products that integrate cellular components. Collectively, these findings establish a comprehensive foundation for the creation of nextgeneration meat analogs, offering enhanced textural fidelity while addressing broader objectives related to consumer acceptance and environmental sustainability.

[0387] Project Results - The study successfully demonstrated the creation of soy protein isolate (SPI) fibers with tunable mechanical properties suitable for meat analog applications. By adjusting the concentrations of pectin and glycerol, key characteristics such as stiffness, tensile strength, and extensibility were modulated. The [25% / 0P / 2G] formulation achieved a stiffness comparable to pork tissue, highlighting the potential for fine-tuning plant-based fibers to match specific textural targets. Additionally, the mechanical stability and microbial resistance of SPI fibers were evaluated under different storage conditions, showing stable Young’s modulus and maximum strain over 28 days. The fiber bundle creation process yielded a prototype with increased tensile properties, providing a basis for further optimization and testing. These findings offer a foundational framework for developing next-generation meat analogs with authentic, meat-like texture and performance.

[0388] Furthermore, the study demonstrated the feasibility of seeding cells directly onto the surface of the fibers. Stromal Vascular Cells (SVCs) showed better attachment than PrimaryAtty. Dkt. No. 166118.01557Bovine Satellite Cells (PBSCs), and cells were successfully differentiated into adipocytes on the fiber surface. This data serves as proof of concept for developing hybrid products that include either cells or cell-derived components, advancing efforts to create next-generation meat analogs with authentic, meat-like texture and performance.

[0389] Introduction:

[0390] Meat — particularly chicken, beef, and pork — plays a central role in diets around the world, offering high- quality protein, essential nutrients, and a rich sensory experience that includes flavor, juiciness, and texture. Global demand for meat, especially chicken, has soared due to its relatively low cost and favorable health profile. Yet this rising demand places mounting pressure on environmental resources, including land, water, and feed, while contributing to greenhouse gas emissions. In response, researchers and companies are exploring sustainable meat alternatives through technologies like cellular agriculture and plant-based analogs. The success of these alternatives depends not only on their nutritional equivalence but also on their ability to convincingly replicate the structural and sensory properties of conventional meat. Meat is composed primarily of muscle fibers, which are highly organized bundles of multinucleated cells packed with fibrous proteins. These structures influence mechanical attributes such as hardness, chewiness, and elasticity — factors that directly affect consumer perception. Although new approaches, such as electrospinning and fat-cell engineering, show promise in mimicking meat’s form, a key challenge remains there is no unified dataset describing the mechanical properties of commonly consumed meats like chicken, beef, and pork. Without this data, it is difficult to engineer analogs that truly match the original. To address this gap, the present study systematically measures the fundamental mechanical properties of raw and cooked meat samples from these three species using standard analytical tools.

[0391] Methods:

[0392] Meat Sample Selection -One of the fundamental challenges in measuring the mechanical properties of meat is the variability introduced by several factors. These include the specific anatomical location of the muscle, the physiological function of that muscle in the animal, storage conditions and duration, and more subjective factors such as overall tissue “vigor”, a genetic or epigenetic trait unique toa particular farm. To minimize variability due to storage, beef loin samples were purchased from Stop & Shop on the day they were packaged. Similarly, pork loin and chicken breast were purchased to represent pork and chicken, respectively. After acquisition,Atty. Dkt. No. 166118.01557 all samples were stored at 5 °C until they were prepared for mechanical testing. Each sample was then processed using standardized protocols to ensure consistency across tests.

[0393] Sample Preparation - For each type of meat, two sample types were prepared. The first consisted of individual muscle fascicles. Muscle fascicles are bundles of muscle myotubes, which are the contractile elements responsible for the mechanical function of skeletal muscle. Although myotubes are the fundamental unit of muscle tissue, their small size makes them prohibitively difficult to isolate and mechanically test using conventional equipment. To address this, muscle fascicles were chosen as the smallest reliably measurable unit of muscle. These samples were used to evaluate the tensile properties of meat, as fascicles bear most of the tensile load during contraction.

[0394] Fascicles were isolated using blunt dissection to avoid damaging the tissue, as any structural compromise could affect mechanical measurements. The dissection process began by creating a small opening in the surface of the muscle and gently pushing aside visible fibers. This often revealed a seam where the perimysium — the connective tissue surrounding fascicles — was naturally separated. By following the path of the perimysium, individual fascicles could be carefully freed from the surrounding tissue. The samples were periodically rinsed with phosphate- buffered saline (PBS) to keep the tissue moist during preparation.

[0395] The second sample type consisted of bulk sections of meat, cut into rectangular segments measuring 2 cm x 1 cm x 4 cm. These segments were cut such that the longest dimension was perpendicular to the direction of muscle fibers. This orientation ensured that tensile loading would primarily stress the perimysium, the connective tissue that binds fascicles together. These bulk samples provided insight into the tearing mechanics of meat — specifically, the force required to pull muscle fibers apart, which is critical for replicating the texture of meat during consumption. Prepared samples were stored in PBS until mechanical testing.

[0396] Mechanical Testing - Tensile tests for individual muscle fascicles were conducted using the TA.XTPlus 100 Connect texture analyzer, equipped with a 5 Kg load cell and configured for high-resolution force detection (system gain: 10334.7). Each fascicle was assumed to have a cylindrical geometry, with a measured diameter of 3 mm and a strain height of 25 mm, resulting in a calculated stress area of 7.06 mm2. The strain rate was set to 1 mm / s and maintained until failure. Fascicles, isolated by blunt dissection, were mounted between cardboard leaflets to minimize slippage and avoid preloading. Phosphate-buffered saline (PBS) was applied duringAtty. Dkt. No. 166118.01557 mounting to preserve tissue hydration. Force calibration was performed immediately before testing, and the system was tared to ensure accurate baseline measurements.

[0397] To evaluate the mechanical properties of the perimysium, rectangular bulk tissue samples were prepared and oriented so that the tensile force was applied perpendicular to the direction of the muscle fibers. This setup ensured that the connective tissue network binding the fascicles — primarily the perimysium — bore most of the tensile load. The strain rate for these tests was also set to 1 mm / s and maintained until failure. As with the fascicle tests, force calibration and taring were performed prior to testing to ensure reliable and reproducible data.

[0398] Data Analysis - The output from the TA.XTPlus 100 Connect texture analyzer was exported as a spreadsheet containing the following variables: force, time, strain, stress (as reported by the analyzer), and distance. For consistency and accuracy, the stress values provided by the analyzer were not used. Instead, stress was recalculated at each time point by dividing the measured force by the known cross-sectional area of each sample.

[0399] The recalculated stress and corresponding strain values were used to generate stress-strain curves for each sample. Three key mechanical properties were extracted from these curves:

[0400] Young’s Modulus - Calculated as the slope of the linear region of the stress-strain curve between 0% and 10% strain.

[0401] Ultimate Tensile Strength (UTS) - Defined as the maximum stress recorded before failure.

[0402] Maximum Strain - Defined as the highest strain value achieved prior to sample failure.

[0403] All data are reported as the mean ± standard deviation. Statistical analysis and figure generation were performed using GraphPad Prism.

[0404] ResultsTable 1 : Mechanical Characterization of Skeletal Muscle Tissues Across Common Meat SpeciesMaatType Tissue Young’s Modulus (Kpa) OTS(Kpa Maximum StrainAtty. Dkt. No. 166118.01557

[0405] Fascicle testing revealed chicken fascicles had a significantly higher young’s modulus and UTS than the other common meat species with beef having the lowest. In terms of Maximum strain, beef was the highest followed by pork and chicken. Transverse tensile testing showed lower young’s modulus and UTS across all species when compared to the corresponding fascicles. Whereas perimysium maximum strain dwarfed fascicles across all species. In terms of Young’s modulus and UTS, chicken showed the highest average values followed by beef and pork. However, pork exhibited the highest maximum strain followed by beef and chicken.

[0406] Expanded Condition Testing for SPI Fibers

[0407] Introduction:

[0408] Tuning the mechanical properties of biomaterials is essential for developing structures that accurately replicate the texture and performance of conventional meat products. Modifying the composition of protein- based fibers offers a promising strategy for achieving targeted mechanical characteristics that align with specific meat analog applications. In this study, the effects of pectin and glycerol incorporation on the mechanical behavior of soy protein isolate (SPI) fibers were evaluated.

[0409] Using the most reliable SPI fiber formulation established in prior work as the baseline, increasing amounts of pectin and glycerol were introduced to the fibers. Pectin, a polysaccharide commonly used for its gel- forming capabilities, and glycerol, a known plasticizer, were selected for their potential to modify fiber strength, elasticity, and flexibility. Through this investigation, the goal is to develop a deeper understanding of how compositional adjustments can fine-tune SPI fiber properties to better match the mechanical profiles of different meat products

[0410] Methods:

[0411] SPI Dope Production-10412] 8M Urea - Solvent

[0413] 25% w / v Soy Protein Isolate (SPI)

[0414] 1.1% w / w(l.l% wt of SPI) Sodium Sulfite Additives

[0415] Weigh out all dry products in falcon tubes. Vortex and shake to mix homogenously. To a beaker, add appropriate volume of Urea. Using an overhead stirrer with a whisk attachment. Start spinning Urea at 300 RPM. Slowly add small increments of dry powder (SPI w / Sodium Sulfite + Additives), roughly 1 gram at a time. For each addition of powder, wait until the prior addition of powder has been incorporated into the Urea. Do not add all powder at once. Once all powder hasAtty. Dkt. No. 166118.01557 been incorporated into solution and dissolving, cover the beaker with 3 piece of parafilm in a triangle, allowing the whisk to spin while preventing evaporation. Let the polymer solution(dope) spin and dissolve for at least 8 hours. If there are additives that impact the dope solution, adjust spin time until fully dissolved and incorporated homogenously. Once dope is done stirring, stop overhead stirrer and remove. Pour dope into falcon tube for storage. Clean beaker.

[0416] Example / Current: 12.5 g SPI ; 82mg Sodium Sulfite ; 50mL 8M Urea ; Stir for 8-24 hours

[0417] SPI Fiber Production - To load a syringe with dope solution for the spinning process, two methods can be employed depending on the viscosity of the dope. In the first method, the plunger is removed from a plastic syringe, and a luer lock stopper is attached to the end of the syringe. The dope solution is poured into the back of the syringe, which is then placed into a 50 mL falcon tube for centrifugation to remove bubbles. Initial centrifugation at 400g is recommended, although higher speeds may be necessary for thicker solutions. After centrifuging, the syringe is removed from the falcon tube, and the plunger is carefully inserted into the back of the syringe using a thin needle to allow excess air to escape. Once the plunger is fully inserted, the needle is removed, and the syringe is ready for use.

[0418] In the second method, the tip of a plastic syringe is submerged into the dope solution to suck up the dope. The outside of the syringe is cleaned, and the plunger is removed from a second syringe. The two syringes are connected using a transfer luer lock, and the dope is pushed into the second syringe without the plunger. A cap luer lock is attached to the end of the second syringe, which is then placed into a 50 mL falcon tube for centrifugation at speeds ranging from 400g to 2000g to remove bubbles. After centrifugation, the plunger is inserted into the back of the syringe using a thin needle to allow air to escape. Once all air has escaped, the needle is removed, and the syringe is ready for use.

[0419] For the current process, 25% SPI dope with sodium sulfite in 8M urea is sucked into a 5 mL BD plastic syringe, and the outside of the syringe is cleaned. A transfer luer lock connector is attached, and the plunger is removed from a second 5 mL plastic syringe. The two syringes are connected, and the dope is transferred into the second syringe without the plunger. The connector and first syringe are removed, and a luer lock end cap is attached to the syringe with the dope. The syringe is placed into a 50 mL falcon tube for centrifugation at 400g for 3 minutes to remove bubbles. If bubbles remain, the speed is increased to 800g for 5 minutes. The plunger is then attached with a 2’ ’ 30g needle to allow air to escape, and the syringe is ready for use.Atty. Dkt. No. 166118.01557

[0420] To begin the wet spinning process, high-pressure tubing is attached to the end of the syringe with the dope solution, and a needle is attached to the end of the tube. The plunger is pressed by hand to fill the tube with dope solution until a droplet is extruded. The syringe is placed onto a syringe pump, and the diameter indicator on the pump is set to the inner diameter of the syringe being used. The speed / rate of extrusion for the wet spinning process is set, and the needle at the end of the tube is placed into the coagulation bath opposite the collection spool. The needle is secured such that the tip is submerged, and the syringe pump is turned on to begin extruding dope solution into the coagulation bath. A bulb of dope should begin collecting around the tip of the needle, which will start crosslinking immediately upon entering the bath. The bulb is removed, and the syringe pump is allowed to build up pressure within the syringe for 5-10 minutes to ensure consistent extrusion. The bulb of crosslinked dope is periodically cleared from the tip of the needle with tweezers.

[0421] During this time, the collection spool is wrapped in aluminum foil and turned on at a rotation velocity of 5.4 m / min (90 mm / s). Once the extrusion is up to speed, the fiber is grabbed with tweezers and pulled through the bath at roughly 90 mm / s. The fiber is guided around the guiding rod under the collector and pulled up out of the bath, then wrapped around the collection spool. The spool adheres the fiber and begins pulling it at a constant rate. The wet spinning process continues until 100 m of fiber is collected, at which point the syringe pump is turned off. Monitoring the spinning process is necessary to reattach the fiber to the collector if it breaks due to bubbles introduced into the fiber.

[0422] Mechanical Testing - At each designated timepoint (0, 1, 7, 14, and 28 days), Tensile tests for individual SPI fibers were conducted using the TA.XTPlus 100 Connect texture analyzer, equipped with a 5 Kg load cell and configured for high-resolution force detection (system gain: 10334.7). Each fiber was assumed to have a cylindrical geometry, with a measured diameter of 0.160 mm and a strain height of 25 mm, resulting in a calculated stress area of 0.020 mm2. The test speed was set to 1 mm / s and maintained until failure. Force calibration was performed immediately before testing, and the system was tared to ensure accurate baseline measurements.

[0423] Data Analysis - The output from the TA.XTPlus 100 Connect texture analyzer was exported as a spreadsheet containing the following variables: force, time, strain, stress (as reported by the analyzer), and distance. For consistency and accuracy, the stress values provided by theAtty. Dkt. No. 166118.01557 analyzer were not used. Instead, stress was recalculated at each time point by dividing the measured force by the known cross-sectional area of each sample.

[0424] The recalculated stress and corresponding strain values were used to generate stress-strain curves for each sample. Three key mechanical properties were extracted from these curves:

[0425] Young’s Modulus - Calculated as the slope of the linear region of the stress-strain curve between 0% and 10% strain.

[0426] Ultimate Tensile Strength (UTS) - Defined as the maximum stress recorded before failure.

[0427] Maximum Strain - Defined as the highest strain value achieved prior to sample failure.

[0428] All data are reported as the mean ± standard deviation. Statistical analysis and figure generation were performed using GraphPad Prism

[0429] Results:

[0430] Samples containing pectin concentrations higher than 2% could not be successfully formed into fibers. Concentrations between 2% and 4% could be dissolved to create the spinning dope; however, the resulting solutions were too viscous to be spun into fibers. At concentrations greater than 4%, the pectin could not be fully dissolved during mixing, resulting in a near-solid consistency that prevented dope formation altogether.

[0431] The mechanical properties of soy protein isolate (SPI) fibers were evaluated following the incorporation of varying concentrations of pectin and glycerol. Analysis focused on Young’s modulus, ultimate tensile strength (UTS), and maximum strain to assess how compositional adjustments influenced fiber performance. In terms of Young’s modulus, the inclusion of glycerol consistently reduced the stiffness of the fibers. This softening effect was more pronounced at higher concentrations of pectin, suggesting a synergistic interaction between glycerol and pectin in modulating mechanical flexibility. In the absence of glycerol, increasing pectin concentration resulted in a steady increase in fiber stiffness, consistent with the known gel-forming properties of pectin and its ability to reinforce the protein network. Regarding UTS, both glycerol and pectin contributed to increases in tensile strength. However, the strengthening effect of glycerol plateaued at concentrations above 2%, whereas pectin continued to enhance UTS across the tested concentration range from 0% to 2%. These trends highlight distinct roles for each additive: glycerol primarily influencing early-stage mechanical improvements and pectin providing continued reinforcement at higher concentrations. Comparison to native pork fascicle properties showed that most SPI fiber formulations exhibited mechanical characteristics that exceeded thoseAtty. Dkt. No. 166118.01557 of pork muscle tissue. Among the tested formulations, samples containing 0% pectin and 2% glycerol, demonstrated a Young’s modulus statistically comparable to that of pork fascicles, indicating a close match in stiffness. However, despite this similarity, significant differences remained in terms of maximum strain and UTS between the SPI fibers and native pork tissue.

[0432] SPI Fiber Storage Conditions

[0433] Introduction:

[0434] The stability and longevity of biomaterials are critical factors when considering their use in food products and cultivated meat applications. Over time, exposure to environmental conditions can lead to microbial contamination and degradation of mechanical properties, compromising material performance and safety. In this study, soy protein isolate (SPI) fibers were selected as a model system to investigate the effects of storage conditions on microbial growth and mechanical stability over time.

[0435] SPI fibers were stored at controlled timepoints — 0, 1, 7, 14, and 28 days — to assess changes in material integrity and the potential for bacterial or mold contamination. Two storage solutions were tested: filtered phosphate-buffered saline (PBS) and filtered 2% acetic acid. PBS served as a neutral aqueous environment, while the acidic conditions provided by 2% acetic acid were intended to explore the potential for enhanced microbial resistance. Through this analysis, the aim is to better understand how storage duration and solution composition impact the shelf-life and performance of protein-based fibrous materials.

[0436] Methods:

[0437] SPI Dope Production-10438] 8M Urea - Solvent

[0439] 25% w / v Soy Protein Isolate (SPI)

[0440] 1.1% w / w(l .1% wt of SPI) Sodium Sulfite Additives

[0441] Weigh out all dry products in falcon tubes. Vortex and shake to mix homogenously. To a beaker, add appropriate volume of Urea. Using an overhead stirrer with a whisk attachment. Start spinning Urea at 300 RPM. Slowly add small increments of dry powder(SPI w / Sodium Sulfite + Additives), roughly 1 gram at a time. For each addition of powder, wait until the prior addition of powder has been incorporated into the Urea. Do not add all powder at once. Once all powder has been incorporated into solution and dissolving, cover the beaker with 3 piece of parafilm in a triangle, allowing the whisk to spin while preventing evaporation. Let the polymer solution(dope)Atty. Dkt. No. 166118.01557 spin and dissolve for at least 8 hours. If there are additives that impact the dope solution, adjust spin time until fully dissolved and incorporated homogenously. Once dope is done stirring, stop overhead stirrer and remove. Pour dope into falcon tube for storage. Clean beaker.

[0442] Exampl e / Curr ent: 12.5 g SPI ; 82mg Sodium Sulfite ; 50mL 8M Urea ; Stir for 8-24 hours

[0443] SPI Fiber Production - To load a syringe with dope solution for the spinning process, two methods can be employed depending on the viscosity of the dope. In the first method, the plunger is removed from a plastic syringe, and a luer lock stopper is attached to the end of the syringe. The dope solution is poured into the back of the syringe, which is then placed into a 50 mL falcon tube for centrifugation to remove bubbles. Initial centrifugation at 400g is recommended, although higher speeds may be necessary for thicker solutions. After centrifuging, the syringe is removed from the falcon tube, and the plunger is carefully inserted into the back of the syringe using a thin needle to allow excess air to escape. Once the plunger is fully inserted, the needle is removed, and the syringe is ready for use.

[0444] In the second method, the tip of a plastic syringe is submerged into the dope solution to suck up the dope. The outside of the syringe is cleaned, and the plunger is removed from a second syringe. The two syringes are connected using a transfer luer lock, and the dope is pushed into the second syringe without the plunger. A cap luer lock is attached to the end of the second syringe, which is then placed into a 50 mL falcon tube for centrifugation at speeds ranging from 400g to 2000g to remove bubbles. After centrifugation, the plunger is inserted into the back of the syringe using a thin needle to allow air to escape. Once all air has escaped, the needle is removed, and the syringe is ready for use.

[0445] For the current process, 25% SPI dope with sodium sulfite in 8M urea is sucked into a 5 mL BD plastic syringe, and the outside of the syringe is cleaned. A transfer luer lock connector is attached, and the plunger is removed from a second 5 mL plastic syringe. The two syringes are connected, and the dope is transferred into the second syringe without the plunger. The connector and first syringe are removed, and a luer lock end cap is attached to the syringe with the dope. The syringe is placed into a 50 mL falcon tube for centrifugation at 400g for 3 minutes to remove bubbles. If bubbles remain, the speed is increased to 800g for 5 minutes. The plunger is then attached with a 2’ ’ 30g needle to allow air to escape, and the syringe is ready for use.

[0446] To begin the wet spinning process, high-pressure tubing is attached to the end of the syringe with the dope solution, and a needle is attached to the end of the tube. The plunger is pressed byAtty. Dkt. No. 166118.01557 hand to fill the tube with dope solution until a droplet is extruded. The syringe is placed onto a syringe pump, and the diameter indicator on the pump is set to the inner diameter of the syringe being used. The speed / rate of extrusion for the wet spinning process is set, and the needle at the end of the tube is placed into the coagulation bath opposite the collection spool. The needle is secured such that the tip is submerged, and the syringe pump is turned on to begin extruding dope solution into the coagulation bath. A bulb of dope should begin collecting around the tip of the needle, which will start crosslinking immediately upon entering the bath. The bulb is removed, and the syringe pump is allowed to build up pressure within the syringe for 5-10 minutes to ensure consistent extrusion. The bulb of crosslinked dope is periodically cleared from the tip of the needle with tweezers.

[0447] During this time, the collection spool is wrapped in aluminum foil and turned on at a rotation velocity of

[0448] 5.4 m / min (90 mm / s). Once the extrusion is up to speed, the fiber is grabbed with tweezers and pulled through the bath at roughly 90 mm / s. The fiber is guided around the guiding rod under the collector and pulled up out of the bath, then wrapped around the collection spool. The spool adheres the fiber and begins pulling it at a constant rate. The wet spinning process continues until 100 m of fiber is collected, at which point the syringe pump is turned off. Monitoring the spinning process is necessary to reattach the fiber to the collector if it breaks due to bubbles introduced into the fiber.

[0449] Mechanical Testing - At each designated timepoint (0, 1, 7, 14, and 28 days), Tensile tests for individual SPI fibers were conducted using the TA.XTPlus 100 Connect texture analyzer, equipped with a 5 Kg load cell and configured for high-resolution force detection (system gain: 10334.7). Each fiber was assumed to have a cylindrical geometry, with a measured diameter of 0.160 mm and a strain height of 25 mm, resulting in a calculated stress area of 0.020 mm2. The test speed was set to 1 mm / s and maintained until failure. Force calibration was performed immediately before testing, and the system was tared to ensure accurate baseline measurements.

[0450] DNA Analysis - At each designated timepoint (0, 1, 7, 14, and 28 days), the supernatant from each SPI

[0451] fiber sample was thoroughly mixed by pipetting up and down to ensure homogeneity. A 500 pL aliquot of the supernatant was collected from each sample and immediately stored at -80Atty. Dkt. No. 166118.01557°C until DNA analysis could be performed. All collected samples were analyzed simultaneously to minimize batch variability.

[0452] DNA content was quantified using the CyQUANT assay, a fluorescence-based method designed for sensitive detection of nucleic acids. Following the manufacturer’s instructions, samples were thawed on ice and prepared in 96-well plates alongside appropriate standards and controls. Fluorescence was measured using a microplate reader, and DNA concentrations were determined based on a standard curve generated during each assay run. The CyQUANT assay provided a comparative measure of microbial load in each sample over the course of the storage study.

[0453] Data Analysis - The output from the TA.XTPlus 100 Connect texture analyzer was exported as a spreadsheet containing the following variables: force, time, strain, stress (as reported by the analyzer), and distance. For consistency and accuracy, the stress values provided by the analyzer were not used. Instead, stress was recalculated at each time point by dividing the measured force by the known cross-sectional area of each sample.

[0454] The recalculated stress and corresponding strain values were used to generate stress-strain curves for each sample. Three key mechanical properties were extracted from these curves:

[0455] Young’s Modulus - Calculated as the slope of the linear region of the stress-strain curve between 0% and 10% strain.

[0456] Ultimate Tensile Strength (UTS) - Defined as the maximum stress recorded before failure.

[0457] Maximum Strain - Defined as the highest strain value achieved prior to sample failure.

[0458] All data are reported as the mean ± standard deviation. Statistical analysis and figure generation were performed using GraphPad Prism.

[0459] Results:

[0460] The mechanical properties of SPI fibers were monitored over a 28-day storage period under two different conditions: filtered PBS and filtered 2% acetic acid. Young’s modulus measurements revealed no statistically significant changes in stiffness over time in either storage condition, indicating that the elastic properties of the fibers remained stable throughout the experiment.

[0461] In contrast, ultimate tensile strength (UTS) increased noticeably over time for fibers stored under both conditions. This trend suggests that the fibers became more resistant to tensile failure as storage time progressed, although the underlying mechanisms remain to be explored. MaximumAtty. Dkt. No. 166118.01557 strain measurements showed no statistically significant changes over time in either storage group, suggesting that the extensibility of the fibers was maintained throughout the 28-day period.

[0462] DNA analysis of the supernatant revealed a steady increase in exogenous DNA content over time in the PBS-stored samples, indicative of microbial contamination or cellular debris accumulation. In contrast, no detectable DNA was found in the supernatants from the acetic acid- stored samples. However, it is suspected that the absence of detectable DNA in these samples may be due to interference of the acetic acid with the CyQUANT assay, potentially leading to false negative results.

[0463] Scaffold Bundling and Prototype development

[0464] Introduction:

[0465] Meat — particularly chicken, beef, and pork — plays a central role in diets around the world, offering high- quality protein, essential nutrients, and a rich sensory experience that includes flavor, juiciness, and texture. Global demand for meat, especially chicken, has soared due to its relatively low cost and favorable health profile. Yet this rising demand places mounting pressure on environmental resources, including land, water, and feed, while contributing to greenhouse gas emissions. In response, researchers and companies are exploring sustainable meat alternatives through technologies like cellular agriculture and plant-based analogs. The success of these alternatives depends not only on their nutritional equivalence but also on their ability to convincingly replicate the structural and sensory properties of conventional meat. Meat is composed primarily of muscle fibers, which are highly organized bundles of multinucleated cells packed with fibrous proteins. These structures influence mechanical attributes such as hardness, chewiness, and elasticity — factors that directly affect consumer perception. Although new approaches, such as electrospinning and fat-cell engineering, show promise in mimicking meat’s form, a key challenge remains: there is no unified dataset describing the mechanical properties of commonly consumed meats like chicken, beef, and pork. Without this data, it is difficult to engineer analogs that truly match the original. To address this gap, the present study systematically measures the fundamental mechanical properties of raw and cooked meat samples from these three species using standard analytical tools.

[0466] Methods:

[0467] SPI Dope Production-10468] 8M Urea - SolventAtty. Dkt. No. 166118.01557

[0469] 25% w / v Soy Protein Isolate (SPI)

[0470] 1.1% w / w(l.l% wt of SPI) Sodium Sulfite Additives

[0471] Weight out all dry products in falcon tubes. Vortex and shake to mix homogenously. To a beaker, add appropriate volume of Urea. Using an overhead stirrer with a whisk attachment. Start spinning Urea at 300 RPM. Slowly add small increments of dry powder(SPI w / Sodium Sulfite + Additives), roughly 1 gram at a time. For each addition of powder, wait until the prior addition of powder has been incorporated into the Urea. Do not add all powder at once. Once all powder has been incorporated into solution and dissolving, cover the beaker with 3 piece of parafilm in a triangle, allowing the whisk to spin while preventing evaporation. Let the polymer solution(dope) spin and dissolve for at least 8 hours. If there are additives that impact the dope solution, adjust spin time until fully dissolved and incorporated homogenously. Once dope is done stirring, stop overhead stirrer and remove. Pour dope into falcon tube for storage. Clean beaker.

[0472] Example / Current: 12.5 g SPI ; 82mg Sodium Sulfite ; 50mL 8M Urea ; Stir for 8-24 hours

[0473] SPI Fiber Production - To load a syringe with dope solution for the spinning process, two methods can be employed depending on the viscosity of the dope. In the first method, the plunger is removed from a plastic syringe, and a luer lock stopper is attached to the end of the syringe. The dope solution is poured into the back of the syringe, which is then placed into a 50 mL falcon tube for centrifugation to remove bubbles. Initial centrifugation at 400g is recommended, although higher speeds may be necessary for thicker solutions. After centrifuging, the syringe is removed from the falcon tube, and the plunger is carefully inserted into the back of the syringe using a thin needle to allow excess air to escape. Once the plunger is fully inserted, the needle is removed, and the syringe is ready for use.

[0474] In the second method, the tip of a plastic syringe is submerged into the dope solution to suck up the dope. The outside of the syringe is cleaned, and the plunger is removed from a second syringe. The two syringes are connected using a transfer luer lock, and the dope is pushed into the second syringe without the plunger. A cap luer lock is attached to the end of the second syringe, which is then placed into a 50 mL falcon tube for centrifugation at speeds ranging from 400 G to 2000 Gto remove bubbles. After centrifugation, the plunger is inserted into the back of the syringe using a thin needle to allow air to escape. Once all air has escaped, the needle is removed, and the syringe is ready for use.Atty. Dkt. No. 166118.01557

[0475] For the current process, 25% SPI dope with sodium sulfite in 8M urea is sucked into a 5 mL BD plastic syringe, and the outside of the syringe is cleaned. A transfer luer lock connector is attached, and the plunger is removed from a second 5 mL plastic syringe. The two syringes are connected, and the dope is transferred into the second syringe without the plunger. The connector and first syringe are removed, and a luer lock end cap is attached to the syringe with the dope. The syringe is placed into a 50 mL falcon tube for centrifugation at 400g for 3 minutes to remove bubbles. If bubbles remain, the speed is increased to 800g for 5 minutes. The plunger is then attached with a 2” 30g needle to allow air to escape, and the syringe is ready for use.

[0476] To begin the wet spinning process, high-pressure tubing is attached to the end of the syringe with the dope solution, and a needle is attached to the end of the tube. The plunger is pressed by hand to fill the tube with dope solution until a droplet is extruded. The syringe is placed onto a syringe pump, and the diameter indicator on the pump is set to the inner diameter of the syringe being used. The speed / rate of extrusion for the wet spinning process is set, and the needle at the end of the tube is placed into the coagulation bath opposite the collection spool. The needle is secured such that the tip is submerged, and the syringe pump is turned on to begin extruding dope solution into the coagulation bath. A bulb of dope should begin collecting around the tip of the needle, which will start crosslinking immediately upon entering the bath. The bulb is removed, and the syringe pump is allowed to build up pressure within the syringe for 5-10 minutes to ensure consistent extrusion. The bulb of crosslinked dope is periodically cleared from the tip of the needle with tweezers.

[0477] During this time, the collection spool is wrapped in aluminum foil and turned on at a rotation velocity of 5.4 m / min (90 mm / s). Once the extrusion is up to speed, the fiber is grabbed with tweezers and pulled through the bath at roughly 90 mm / s. The fiber is guided around the guiding rod under the collector and pulled up out of the bath, then wrapped around the collection spool. The spool adheres the fiber and begins pulling it at a constant rate. The wet spinning process continues until 100 m of fiber is collected, at which point the syringe pump is turned off. Monitoring the spinning process is necessary to reattach the fiber to the collector if it breaks due to bubbles introduced into the fiber.

[0478] Binder and Protype Production -

[0479] Producing Binding PrototypeAtty. Dkt. No. 166118.01557

[0480] During the spinning process, the collection spool can be coated with crosslinking solution. During this time, periodically spray the fibers with crosslinking solution to evenly coat them. Do so even 1 minute.

[0481] Example / Current: During the spinning process a 15% w / v solution of transglutaminase is used as a crosslinking agent. This solution is loaded into a spray bottle and periodically sprayed onto the rotating collector.

[0482] Results:

[0483] The fiber bundle was successfully created using the described method. The spool, with a diameter of 50 mm and a circumference of 157 mm, rotated and moved back and forth, forming a fiber sheet with a width of 30 mm and a length of 157 mm. The spool completed one back-and- forth cycle for every 100 rotations, resulting in a consistent 30 mm wide region where fibers were collected. The prototype had a thickness of approximately 3 mm. The final fiber sheet exhibited increased tensile properties upon hand-feel. Individual fibers did not bind together, indicating the need for an additional crosslinker in future tests to improve fiber cohesion. The overall process demonstrated the feasibility of creating a fiber bundle with the specified materials and methods, providing a basis for further optimization and testing.

[0484] Introduction:

[0485] The integration of cells and cell-derived products into biomaterial scaffolds is a critical step toward developing more advanced and biologically realistic meat analogs. Beyond mechanical properties alone, the incorporation of biological components can enhance the functional, nutritional, and sensory characteristics of engineered food products, making them closer to conventional meat. In this study, SPI fibers, previously optimized for mechanical performance, were used as abase material to evaluate the feasibility of integrating cells and cellular products. This investigation focuses on assessing how cells interact with the SPI fibers, including adhesion, viability, and differentiation. By understanding these interactions, we aim to inform strategies for constructing more complex, tissue-like structures that combine mechanical robustness with biological authenticity, advancing the next generation of cultivated and hybrid meat products.

[0486] Methods:

[0487] Isolation of PBSCs- Bovine satellite cells were isolated from muscle tissue using enzymatic digestion. Fresh tissue was placed into a sterile petri dish containing DPBSAtty. Dkt. No. 166118.01557 supplemented with 10% antibiotic- antimycotic (Anti / Anti) to maintain moisture. The tissue was minced thoroughly with a scalpel and tweezers into 1-3 mm3fragments and transferred into a 50 mL conical tube. DMEM supplemented with Glutamax was added, followed by 2% collagenase II solution to achieve a final collagenase concentration of 0.2%. The suspension was incubated at 37 °C for 1 hour with trituration (pipetting up and down) every 20 minutes to facilitate digestion. If necessary, an additional 30-minute incubation was performed.

[0488] After digestion, 20 mL of growth media was added to neutralize the collagenase activity. The cell

[0489] suspension was sequentially filtered through 70 pm and then 40 pm cell strainers into a new 50 mL tube. Cells were pelleted by centrifugation at 400 G for 10 minutes. The supernatant was carefully aspirated without disturbing the pellet, and the cells were resuspended in growth media. Approximately 50,000- 100,000 cells / cm2were plated onto uncoated tissue culture plates or flasks. The following day, the non- adherent cells were collected by transferring the supernatant to a new flask of equal size to enrich for satellite cells. Cultures were then incubated without disturbance for six days before initiating regular feeding every 2-3 days.

[0490] Isolation of SVCs - Bovine stromal vascular cells were isolated from adipose tissue through enzymatic digestion. Tissue was placed into a sterile petri dish containing DPBS supplemented with 10% antibiotic- antimycotic (Anti / Anti) to maintain hydration. Using a scalpel and tweezers, visible connective tissue and fascia were removed, and the fat was minced into small fragments (1-3 mm3) or a thick paste. Minced tissue was transferred into a 50 mL conical tube, and DMEM supplemented with Glutamax was added, followed by 2% collagenase II solution to achieve a final concentration of 0.2% collagenase.

[0491] The mixture was incubated at 37 °C for 1 hour, with inversion of the tube every 20 minutes to promote thorough digestion. If necessary, incubation was extended by an additional 30 minutes. The resulting cell suspension was sequentially filtered through 750 pm, 300 pm, and 70 pm strainers into fresh 50 mL tubes, avoiding use of 40 pm strainers to prevent loss of larger stromal vascular cells. Cells were pelleted by centrifugation at 400 G for 10 minutes. The supernatant was carefully aspirated, minimizing disturbance to the pellet and avoiding collection of buoyant mature adipocytes at the surface. The cell pellet was resuspended in 5 mL of growth media and counted. Approximately 15,000 cells / cm2were plated onto uncoated plates or flasks. Cells were maintained with regular feeding every 2-3 days using fresh growth media.Aty. Dkt. No. 166118.01557

[0492] Cell Tracking - To visualize and monitor cell distribution within the SPI fibers, cells were labeled using CellTracker Green dye prior to integration. Cells were first harvested and resuspended in serum-free DMEM. A working solution of CellTracker Green was prepared according to the manufacturer’s instructions, and added to the cell suspension at a final concentration of 5 pM. Cells were incubated with the dye at 37 °C for 30 minutes, protected from light to prevent photobleaching. Following incubation, cells were washed twice with fresh growth media to remove any excess dye. Labeled cells were then resuspended in growth media and immediately used for seeding onto SPI fibers. Fluorescence microscopy was used to track and confirm cell attachment and distribution post-seeding.

[0493] Cell Seeding - SPI fibers were prepared for cell seeding by separating them into small bundles approximately 1 cm in length. Fiber bundles were sterilized by immersion in 70% ethanol for 30 minutes, followed by thorough rinsing with sterile distilled water to remove residual ethanol. This rinsing step was critical to prevent precipitation of phosphates from subsequent exposure to PBS or media.

[0494] Polystyrene cloning cylinders were similarly sterilized by soaking in 70% ethanol for 30 minutes and rinsed thoroughly with sterile distilled water. For each sample, a sterile cylinder was briefly dipped into distilled water to wet its surface. Using sterile forceps, fiber bundles were carefully draped over the cylinder, with surface tension aiding in the adhesion of fibers to the cylinder’ s surface. Once properly mounted, the cylinders with attached fiber bundles were inverted and placed into a low-attachment 48-well plate.

[0495] The fibers were pre-incubated in growth media for 1 hour to equilibrate before cell seeding. After pre- incubation, the media was aspirated and replaced with either PBSCs or SVCs at a seeding density of 10,000 cells / cm2. The surface area for seeding calculations was estimated based on the thickness of the fiber bundles. Plates were returned to the incubator for subsequent culture and monitoring.

[0496] SVC Differentiation- For adipogenic induction, growth media was replaced with induction media (DMEM

[0497] + 10% FBS + 10 pM biotin, 5.67 pM pantothenate, 3 pg / mL insulin, 0.3 pM dexamethasone, 0.1 mM IBMX, and 10 pM rosiglitazone). After 2-3 days in induction media, wells were transitioned to accumulation media (DMEM, 3 pg / mL insulin, 10 pM biotin, 113 pMAtty. Dkt. No. 166118.01557 ascorbic acid, and 500 pg / mL Intralipid). Media changes were performed every 2-3 days. SVCs were maintained in accumulation media for 14 days.

[0498] Viability Assessment - Cell viability on SPI fibers was assessed using the PrestoBlue assay, which measures metabolic activity as an indicator of viable cells. Following seeding, cells were cultured on the fibers for 7 days in growth media, with media replacement every 48 hours to maintain optimal nutrient conditions.

[0499] At the end of the 7-day culture period, the media was aspirated and replaced with fresh growth media containing 10% PrestoBlue reagent, prepared according to the manufacturer’s instructions. Samples were incubated at 37 °C for 1 hour, protected from light. After incubation, 100 pL aliquots of the supernatant were transferred to a 96-well plate for fluorescence measurement. Fluorescence intensity was recorded using a plate reader, with excitation at 560 nm and emission at 590 nm. Fluorescence values were used as a proxy for metabolic activity and thus overall cell viability.

[0500] Lipid Staining - Lipid accumulation in differentiated stromal vascular cells (SVCs) was assessed using BODIPY staining. After the 14-day culture period on SPI fibers, samples were gently washed with PBS to remove residual media. Cells were then fixed with 4% paraformaldehyde for 15 minutes at room temperature, followed by three PBS washes to remove any residual fixative. A BODIPY staining solution was prepared by diluting BODIPY 493 / 503 dye to a final concentration of 5 pg / mL in PBS. The fixed samples were incubated with the BODIPY solution for 30 minutes at room temperature, protected from light. Following incubation, samples were washed three times with PBS to remove excess dye.

[0501] Lipid droplets were visualized using fluorescence microscopy with appropriate filter sets (excitation -493 nm, emission -503 nm). Representative images were captured to document lipid accumulation within the seeded cell populations.

[0502] Data Analysis - Viability data are reported as the mean ± standard deviation. Statistical analysis and figure generation were performed using GraphPad Prism.

[0503] Conclusions & Future Needs

[0504] Overall, the findings from this study provide important mechanical benchmarks that reflect the structural organization and functional roles of muscle and connective tissues in conventional meats. These benchmarks offer critical guidance for the design of meat analogs, emphasizing theAtty. Dkt. No. 166118.01557 need to replicate not only the stiffness and strength of muscle fibers but also the extensibility and toughness of connective tissues to achieve realistic textural profiles.

[0505] This study demonstrates the potential of compositional tuning to control the mechanical properties of soy protein isolate (SPI) fibers for meat analog applications. By systematically adjusting the concentrations of pectin and glycerol, it was possible to modulate key mechanical characteristics such as stiffness, tensile strength, and extensibility. Glycerol primarily served to reduce stiffness and increase flexibility, particularly in the presence of pectin, while pectin alone reinforced the fiber network, enhancing both Young’s modulus and ultimate tensile strength. Although most formulations exhibited mechanical properties exceeding those of native pork fascicles, the [25% / 0P / 2G] formulation achieved a stiffness statistically comparable to pork tissue, highlighting the feasibility of fine-tuning plant-based fibers to match specific textural targets. However, differences in maximum strain and tensile strength indicate that further optimization is necessary to fully replicate the complex mechanical behavior of animal muscle.

[0506] The study also evaluated the mechanical stability and microbial resistance of SPI fibers stored under two different conditions: filtered PBS and filtered 2% acetic acid. Over a 28-day period, Young’s modulus and maximum strain remained stable in both conditions, indicating that the fibers retained their basic elastic and extensibility properties during storage. An increase in ultimate tensile strength over time was observed in both groups, suggesting some degree of material strengthening with prolonged storage. DNA analysis revealed a progressive accumulation of exogenous DNA in the PBS-stored samples, consistent with microbial growth or contamination. In contrast, no DNA was detected in the acetic acid-stored samples; however, potential assay interference by the acetic acid suggests that this result may represent a false negative rather than true sterility.

[0507] The fiber bundle was successfully created using the described method, forming a fiber sheet with a width of 30 mm and a length of 157 mm. The prototype had a thickness of approximately 3 mm and exhibited increased tensile properties upon hand-feel. Individual fibers did not bind together, indicating the need for an additional crosslinker in future tests to improve fiber cohesion. The overall process demonstrated the feasibility of creating a fiber bundle with the specified materials and methods, providing a basis for further optimization and testing.

[0508] Future directions for this research include enhancing cell attachment to the fibers, as initial experiments demonstrated that SVCs attach better than PBSCs. Modifications to the fibers may beAtty. Dkt. No. 166118.01557 necessary to improve cell attachment. Additionally, the successful differentiation of cells into adipocytes on the surface of the fibers serves as proof of concept for a hybrid product that includes either cells or cell-derived products. Further studies will focus on optimizing the mechanical properties and biological functionality of SPI fibers to create next-generation meat analogs with authentic, meat-like texture and performance.

[0509] For the mechanical stability and microbial resistance experiments, future research should investigate alternative storage conditions and additives that could further enhance the stability and sterility of SPI fibers. Exploring different concentrations and combinations of coagulation bath components may also provide insights into improving fiber properties and reducing microbial contamination.

[0510] In the context of fiber bundle creation, future experiments should aim to refine the process parameters to minimize fiber breakage and improve the consistency of fiber collection. Investigating the use of different materials for the collection spool and optimizing the wrapping technique could enhance fiber drying and

[0511] overall quality. Additionally, incorporating advanced monitoring systems to detect and address fiber breakage in real-time could significantly improve the efficiency and reliability of the fiber spinning process.

[0512] To achieve the target thickness of 1 cm for fiber bundles, future research should explore modifications to the spinning process, such as adjusting the extrusion rate, increasing the number of fibers collected per cycle...

Claims

1. Atty. Dkt. No. 166118.01557CLAIMS1. An edible fiber scaffold comprising about 10% (weight) soy protein isolate (SPI) to about 30% (weight) SPI and at least one scaffold additive selected from pectin and glycerol.

2. The edible fiber scaffold of claim 1, wherein the edible fiber scaffold comprises about 20% (weight) SPI to about 30% (weight) SPI.

3. The edible fiber scaffold of claim 1 or 2, wherein the edible fiber scaffold further comprises sodium sulfite.

4. The edible fiber scaffold of claim 3, wherein the sodium sulfite is at a concentration of about 0.1% (weight / weight of SPI) to about 2.1% (weight / weight of SPI).

5. The edible fiber scaffold of claim 4, wherein the sodium sulfite is at a concentration of about 1.1% (weight / weight of SPI).

6. The edible fiber scaffold of any one of the preceding claims, wherein the at least one scaffold additive comprises pectin.

7. The edible fiber scaffold of claim 6, wherein the pectin is at a concentration of about 0.01% (weight) to about 2% (weight).

8. The edible fiber scaffold of any one of the preceding claims, wherein the at least one scaffold additive comprises glycerol.

9. The edible fiber scaffold of claim 8, wherein the glycerol is at a concentration of about 0.01% (weight) to about 20% (weight).

10. The edible fiber scaffold of claim 9, wherein the glycerol is at a concentration of about 1% (weight) to about 4% (weight).

11. The edible fiber scaffold of claim 10, wherein the glycerol is at a concentration of about 2% (weight).

12. A method of generating an edible fiber scaffold, the method comprising(a) preparing an edible fiber dope, wherein the edible fiber dope comprises about 10% (weight / volume) soy protein isolate (SPI) to about 30% (weight / volume) SPI and at least one scaffold additive;(b) extruding the edible fiber dope to form an extruded edible fiber dope; and(c) contacting the extruded edible fiber dope with a coagulation solution to generate the edible fiber scaffold.Atty. Dkt. No. 166118.0155713. The method of claim 12, wherein the at least one scaffold additive selected from pectin and glycerol.

14. The method of claim 12 or 13, wherein the edible fiber dope comprises about 20% SPI (weight / volume) to about 30% SPI (weight / volume).

15. The method of any one of claims 12-14, wherein the edible fiber dope further comprises sodium sulfite.

16. The method of claim 15, wherein the sodium sulfite is at a concentration of about 0.1% (weight / weight of SPI) to about 2.1% (weight / weight of SPI).

17. The method of claim 16, wherein the sodium sulfite is at a concentration of about 1.1% (weight / weight of SPI).

18. The method of any one of claims 12-17, wherein the at least one scaffold additive comprises pectin.

19. The method of claim 18, wherein the pectin is at a concentration of about 0.01% (weight / volume) to about 2% (weight / volume).

20. The method of any one of claims 12-19, wherein the at least one scaffold additive comprises glycerol.

21. The method of claim 20, wherein the glycerol is at a concentration of about 0.01% (weight / volume) to about 20% (weight / volume).

22. The method of claim 21, wherein the glycerol is at a concentration of about 1% (weight / volume) to about 4% (weight / volume).

23. The method of claim 22, wherein the glycerol is at a concentration of about 2% (weight / volume).

24. The method of any one of claims 12-23, the method further comprising treating the edible fiber scaffold, wherein the treating the edible fiber scaffold comprises one of the following treatments cold plasma, flame, corona discharge, ion beam, electron beam, ionizing radiation, UV, ozone, chemical etching, laser treatment, enzymatic modification, grafting polymerization, surface polymerization, peroxides, and supercritical fluids.

25. The method of claim 24, wherein treating the edible fiber scaffold comprises treating the edible fiber scaffold with cold plasma.

26. The method of claim 25, wherein the scaffold has increased surface energy, surface charge, hydrophilicity, and / or capillary action relative to the scaffold before it is treated.Atty. Dkt. No. 166118.0155727. The method of any one of claims 12-25, further comprising seeding a plurality of cells on the edible fiber scaffold.

28. The method of claim 26, wherein the plurality of cells comprise muscle and / or adipose cells.

29. The method of claim 26 or 27, wherein the plurality of cells comprises porcine, galline, or bovine cells.

30. The method of any one of claims 24-28, wherein the plurality of cells comprise precursor cells, pluripotent stem cells, mesenchymal stem cells, satellite cells, myoblast cells, myofibroblast cells, fibroblasts cells, endothelial cells, adipose cells, hepatocytes, cardiomyocytes, or combinations thereof.

31. The method of any one of claims 24-30, wherein seeding the plurality of cells comprises activating a movement mechanism to move an elongated edible fiber scaffold into contact with a culture medium, wherein the culture medium is optionally inside a bioreactor or vessel; seeding cells onto the scaffold in a seeding area; and growing the cells to increase the number and / or density of the cells on the edible fiber scaffold.

32. The method of claim 31, wherein the step of activating a movement mechanism comprises activating at least one of a roller, conveyor, and / or belt to apply a motive force on the scaffold thereby causing the scaffold to move from outside the bioreactor or vessel to inside the bioreactor or vessel.

33. The method of claim 32, the method further comprising extracting the scaffold seeded with the cells from the bioreactor or vessel.

34. The method of any one of claims 30-33, the method further comprising differentiating the cells to form a meat tissue comprising differentiated cells, optionally, wherein the meat tissue comprises muscle cells and fat cells and wherein the culture medium varies over the length of the bioreactor or vessel.

35. The method of any one of claims 12-34, further comprising arranging two or more edible fiber scaffolds to form an edible scaffold bundle.

36. The method of claim 35, wherein the two or more edible fiber scaffolds adhere to one another based on intrinsic adhesiveness of the two or more edible fiber scaffolds derived from the generating the two or more edible fiber scaffolds.Atty. Dkt. No. 166118.0155737. The method of claim 35 or 36, wherein the method further comprises applying at least one binder to the two or more edible fiber scaffolds.

38. The method of claim 37, wherein the at least one binder comprises transglutaminase (TG).

39. The method of any one of claims 12-38, further comprising generating two or more edible fiber scaffolds and winding the two or more edible fiber scaffolds to form an edible scaffold thread.

40. A composition comprising the edible fiber scaffold generated by the method of any one of claims 12-39.

41. A strand of cultured meat tissue comprising the edible fiber scaffold of any one of claims 1-11 and a plurality of cells grown on the edible fiber scaffold.

42. A food product comprising a plurality of the edible fiber scaffolds of any one of claims 1- 11 or a plurality of the strands of cultured meat tissue of claim 36.

43. A device for generating a modified edible fiber scaffold, the device comprising(a) a movement mechanism to extend an edible fiber scaffold into the device, wherein the movement mechanism is controlled by at least one controller;(b) a first chamber housing a component for treating the edible scaffold;(c) a second chamber housing a wetting system.

44. The device of claim 43, wherein the at least one controller is configured to drive the edible fiber scaffold into the device such that the edible fiber passes through the first chamber and into the second chamber.

45. The device of claim 43 or 44, wherein the component for treating the edible scaffold comprises a component for treating the edible fiber scaffold with at least one of the following: cold plasma, flame, corona discharge, ion beam, electron beam, ionizing radiation, UV, ozone, chemical etching, laser treatment, enzymatic modification, grafting polymerization, surface polymerization, peroxides, or supercritical fluids.

46. The device of claim 45, wherein the component for treating the edible fiber scaffold comprises a component that generates cold plasma.Atty. Dkt. No. 166118.0155747. The device of any one of claims 43-46, wherein the movement mechanism comprises a series of rollers, conveyors, and / or belts, the rollers, conveyors, and / or belts individually serving to at least one of support, apply motive force to, and / or direct the scaffold.

48. The device of claim 47, wherein the movement mechanism comprises at least one roller or pinch rollers.

49. The device of any one of claims 43-48, wherein the second chamber housing the wetting system comprises an inlet, a chamber well, and an outlet, and wherein the diameter of the outlet is narrower than the diameter of the chamber well.

50. The device of any one of claims 43-49, wherein the device is oriented vertically with (a), (b), and (c) being arranged from top to bottom.

51. The device of claim 50, wherein the outlet of the second chamber comprises an aqueous fluid and creates a meniscus to wet the edible fiber scaffold as it passes through the second chamber.

52. The device of any one of claims 43-41, wherein the device further comprises a movement mechanism to place the edible fiber scaffold leaving the second chamber into a bioreactor or vessel.

53. The device of any one of claims 43-52, wherein the second chamber comprises cell culture medium.

54. The device of claim 52 or 53, wherein the bioreactor or vessel comprises cell culture medium.

55. The device of claim 54, wherein the second chamber comprises a plurality of cells in suspension in the cell culture medium.

56. The device of claim 55, wherein the plurality of cells comprises muscle and / or adipose cells.

57. The device of claim 55 or 56, wherein the plurality of cells comprises porcine, galline, or bovine cells.

58. The method of any one of claims 55-57, wherein the plurality of cells comprise precursor cells, pluripotent stem cells, mesenchymal stem cells, satellite cells, myoblast cells, myofibroblast cells, fibroblasts cells, endothelial cells, adipose cells, hepatocytes, cardiomyocytes, or combinations thereof.Atty. Dkt. No. 166118.0155759. The device of any one of claims 43-58, wherein the edible fiber scaffold comprises the edible fiber scaffold of any one of claims 1-11.

60. A system for producing strands of cultured meat tissue, the system comprising:(a) an edible fiber scaffold;(b) a movement mechanism configured to move the scaffold into a culture medium contact area in a bioreactor or vessel to put the edible fiber scaffold in contact with a culture medium;(c) a seeding mechanism configured to seed cells onto the scaffold; and(d) a cell growth area inside the bioreactor or vessel configured to grow the cells seeded on the scaffold to increase the number and / or density of the cells on the scaffold to form a strand of meat tissue.

61. The system of claim 60, wherein the edible fiber scaffold comprises the edible fiber scaffold of any one of claims 1-11.

Images