Compositions and methods for production of edible cell-infused scaffolds

Abstract

Edible scaffolds prepared from plant protein and / or fungal protein and methods for their production and use are described. The edible scaffolds can be used for various methods, such as use as supports for animal cell cultures and for culturing animal cells on the edible scaffolds. The edible scaffolds can also be used for the production of laboratory grown and / or cultured meat and fish.

Description

COMPOSITIONS AND METHODS FOR PRODUCTION OF EDIBLE CELL-INFUSED SCAFFOLDSCross-Reference to Related Application

[0001] This application claims priority to Singapore Provisional Application No. 10202303501Y filed December 14, 2023, entitled “Compositions and Methods for Production of Edible Cell-Infused Scaffolds”, the contents of which are incorporated in their entirety by reference.Incorporation of Sequence Listing

[0002] The material in the accompanying sequence listing is hereby incorporated by reference into the application. The accompanying sequence listing .xml file, named Sequence_Listing.xml, was created on December 13, 2024 and is 6,384 bytes in size.

[0003] The present disclosure relates to methods for production of edible scaffolds used as supports for animal cell cultures. In addition, the present disclosure provides methods for culturing animal cells on the edible scaffolds and the resultant three-dimensional cell compositions, also known as laboratory grown meat and / or cultured meat.Background

[0004] The world population continues to increase while the amount of arable agricultural land decreases due to urban sprawl and changing weather conditions. The United Nations estimated in 2015 that 11% of greenhouse gases result from livestock; however, this value has been questioned by several authors who contend that the contribution is significantly higher (Twine (2021) Sustainability 13:6276; Xu et al. (2021) Nature Food 2:724-732). Furthermore, in 2020, the Food and Agriculture Organization of the United Nations indicated that global agriculture land area is approximately 5 billion hectares, or 38% of the global land surface. About one-third of that is used as cropland, while the remaining two-thirds consist of meadows and pastures for grazing livestock.

[0005] Not only does agriculture demand a large area of land, but since 1940 an estimated 50% of zoonotic disease emergence has been associated with agriculture, in particular from intensive management which confines animals and their wastes (Hayek (2022) Sci Adv 8 eadd6681). Hence the rise of laboratory grown / cultivated / cultured / clean meat as a potential solution to reducing the impact of agriculture, especially agriculture directed towards production of animal products.

[0006] Yet while laboratory methods for individual cell reproduction / production, such as stem cells, have long existed, the ability to generate meat tissues that appear identical or equal in visual appearance and / or mouth-feel to naturally occurring meat tissues (for example, muscle, fat, tendon, bone, and so on) has lagged. This has been due, in part, to the difficulty associated with generating a microenvironment that simulates that which is required for stem cell attachment, differentiation, and growth.

[0007] In nature, cells exist within the extracellular matrix (ECM), which is a complex matrix of secreted proteins and proteoglycans which has an inherent stiffness. The ECM provides cues to the cells to develop in particular ways (see, for example Wang et al. (2015) Biochem Insights 8(Suppl 2):15-21; Engler et al. (2006) Cell 126(4):677-689; Calve et al. (2010) Dev Biol 344(1):259-271; and Guneta et al. (2016) J Biomed Mater Res Part A 104A:1090-1101). A significant challenge for laboratory grown meat has been generating appropriate microenvironments and / or scaffolds upon which various different types of cells can grow in a manner that resembles natural muscle tissue. While using ECM proteins can provide an environment for cell adhesion and proliferation, because they are animal-derived and / or are expensive to produce recombinantly, they are not favored for mass production of cultured tissues.

[0008] Currently, scaffolds used in tissue engineering can be found in fiber form (1-dimensional or 1-D) and film form (2-dimensional or 2-D), as well as in microcarrier form, sponge form, hydrogel form or other types of three dimensional (3-D) forms. While synthetic polymer materials are available, these typically do not have FDA Generally Recognized as Safe (GRAS) status, in part due to the crosslinking chemicals required, and so cannot be used for edible food products. Instead, naturally occurring polysaccharides and / or proteins are preferred, such as plant-based polysaccharides and proteins.

[0009] Plant polymers are often used for scaffolds upon which cells can be grown for cultured meat (see, for example, Harris et al. (2021) Int J Mol Sci 22:12347). Many plant polymers are inexpensive, easily acquired, and are naturally occurring plant polymers, such as cellulose which is a naturally occurring polymer of sugar molecules (a polysaccharide). Other types of polymers are also used, especially for electrospinning, such as polylactic acid (PLA) (Singh et al. (2023) Cell and Tissue Research 391:235-247).

[0010] The recent attempts to generate scaffolds from plant proteins have met with limited success. Scaffolds comprised of plant components typically decay more quickly than synthetic and composite scaffolds. The presents a challenge when the scaffold must be sustained in cell culture media while the initial cell population reproduces and differentiates. In addition, as the scaffold deteriorates, the byproducts must remain harmless and not alter the flavor or the texture of the cultured meat (Ramachandraiah (2021) Sustainability 13:938).

[0011] Consequently, challenges remain in developing scaffolds for laboratory grown meat that are cost-effective for mass production, meet GRAS requirements, provide robust environments for cell adhesion, proliferation, and differentiation, and which result in an eating experience that is pleasurable, tasty, and similar to that experienced with naturally occurring meat tissues / products.

[0012] Despite considerable advances made to date, there still exists a need for improved methods of preparing three-dimensional cell compositions and the compositions themselves.Summary

[0013] Protein scaffolds

[0014] The present disclosure provides edible, plant- and / or fungal based protein scaffolds formed without application of external chemical crosslinking agents for use as substrates in culturing animal cells for laboratory grown meat. The disclosed scaffolds are stable in culture media and provide the texture and physical properties associated with naturally occurring meat and fish products.

[0015] In some aspects, the plant-based protein is at least one gluten-containing protein. Gluten containing proteins include, but are not limited to, wheat, barley, rye, triticale, malt, semolina, spelt, farina, farro, graham, einkorn, durum, emmer, khosan wheat, gliadin, glutenin, and combinations thereof.

[0016] In other aspects, the plant-based protein is at least one non-gluten containing protein. Non-gluten containing proteins include, but are not limited to, soy, pea, rice, oat, hemp, quinoa, buckwheat, sorghum, teff, lentil, chickpea, potato, chia, and combinations thereof.

[0017] In yet other aspects the fungal-based protein is a mycelium protein(s), a fungal collagen, or combinations thereof.

[0018] In some aspects, the edible, plant- and / or fungal-based protein scaffolds have a hardness in the range of about 235 kPa to about 400 kPa. In other aspects, the edible plant- and / or fungal-based protein scaffolds have a springiness of about 0.8 to about 0.95. In some additional aspects, the edible plant- and / or fungal-based protein scaffolds have a cohesiveness of about 0.65 to about 0.86. In still other aspects, the edible plant- and / or fungal-based protein scaffolds have a resilience of about 0.22 to about 3.1.

[0019] In some aspects, the edible plant- and / or fungal-based protein scaffolds have a porosity of about 30% to about 50% (2-D). In other aspects, the edible plant- and / or fungal-based protein scaffolds have a mean pore size of about 2 µm to about 20 µm.

[0020] Methods of preparing protein scaffolds

[0021] The present disclosure also provides methods for generating the edible plant- and / or fungal-based protein scaffolds formed without application of external chemical crosslinking agents by exposing a solution of plant- and / or fungal-based protein to sonication or ultrasonic pressure waves to form a colloid prior to further processing.

[0022] In some aspects, a plant- and / or fungal-based protein solution is formed with an acid. In some aspects, the acid is an organic acid. Suitable organic acids include, but are not limited to, acetic acid, ascorbic acid, citric acid, formic acid, lactic acid, malic acid, oxalic acid, tartaric acid, uric acid, and combinations thereof.

[0023] In other aspects, a plant- and / or fungal-based solution is formed with a mineral acid. Suitable mineral acids include, but are not limited to, phosphoric acid, hydrochloric acid, and combinations thereof.

[0024] In some aspects, the further processing comprises directional freezing. In other aspects, the further processing comprises freeze drying. In yet other aspects, the further processing involves steam sterilization / crosslinking. In still other aspects, the further processing comprises removing residual acid.

[0025] The present disclosure further provides methods for generating laboratory grown meat and / or tissue products by introducing to the plant- and / or fungal-based protein scaffolds living cells and culturing and / or differentiating the cells on the plant- and / or fungal-based protein scaffolds.

[0026] In some aspects, the cells are animal cells. Suitable animal cells include, but are not limited to those obtained from bovine, porcine, ovine, caprine, cervid, fish, crustacean, bivalves, poultry, and combinations thereof.

[0027] In some aspects, the cells introduced to the scaffolds are all of the same cell type. In other aspects, the cells introduced to the scaffolds are of different cell types. Suitable cell types include, but are not limited to mesenchymal stem cells, muscle stem cells, adipose stem cells, embryonic stem cells, hematopoietic stem cells, neural stem cells, epithelial stem cells, totipotent stem cells, pluripotent stem cells, multipotent stem cells, oligopotent stem cells, unipotent stem cells, satellite cells, myofibers, adipocytes, fibroblasts, chondrocytes, endothelial cells, recombinant cells, and combinations thereof.Brief Description of the Drawings

[0028] shows an example of a mold for casting plant protein solutions and directional freezing. Protein solution is placed into the mold space of the left side of a copper sheet and liquid nitrogen is introduced on the right side of the copper sheet.

[0029] shows a freeze-dried soy protein scaffold after directional freezing.

[0030] shows freeze-dried scaffolds after washing.

[0031] presents SEM images of the disclosed scaffolds, including porosity(2D) and mean pore size.

[0032] shows the texture analyzer used to generate the texture profile analysis.

[0033] presents a graph of force over time for the texture analyzer. Hardness (N) = F1 and is the highest peak force measured during the first compression; Cohesiveness = (d+e) / ((a+b) and is the area underneath the second compression curve divided by the area underneath the firs compression curve; Springiness (%) = (Distance 2) / (Distance 1)* 100 or (Time 2) / (Time 1)* 100 and is a ratio or percentage of a product’s recovery to its original height; Resilience = b / a and is the area under the curve after peak force is reached divided by the area under the curve before the peak force is reached.

[0034] presents hardness data obtained from the texture analyzer and the texture profile generated for extensively washed scaffolds (“Exten Washed Scaffold”), using pan-fried strip-loin as a control.

[0035] presents springiness, cohesiveness, and resilience data obtained from the texture analyzer and the texture profile generated for extensively washed scaffolds (“Exten Washed Scaffold”), using pan-fried strip-loin as a control.

[0036] presents texture profile data obtained from the texture analyzer and the texture profile generated for extensively washed scaffolds (“Exten Washed Scaffold”), using pan-fried strip-loin as a control.

[0037] presents the results from PCR amplification of cDNA generated from RNA isolated from (a) immortalized porcine cells (as control cells; columns 1, 5, and 9), (b) immortalized bovine fibroblasts (iBF; columns 2, 6, and 10), (c) blank scaffold (SC; columns 3, 7, and 11) and (d) iBF-loaded scaffold (iBF-SC; columns 4, 8, and 12) using primers for porcine species-specific genes (columns 1-4), primers for bovine species-specific genes (columns 5-8), or primers for an internal, scaffold control (columns 9-12). Bovine specific genes were identified in column 8, indicating successful iBF loading into the scaffold.

[0038] shows the appearance of freeze-dried immortalized bovine fibroblasts. As shown in Figures 9A and 9B, no difference is shown between the appearance of the freeze-dried cell-loaded and blank scaffolds.

[0039] shows the appearance of freeze-dried blank scaffolds. As shown in Figures 9A and 9B, no difference is shown between the appearance of the freeze-dried cell-loaded and blank scaffolds.

[0040] shows the appearance of steam-sterilized immortalized bovine fibroblasts. As shown in Figures 10A, 10B, 10C, and 10D, no difference is shown between the appearance of the steam-sterilized and hydrated cell-loaded and blank scaffolds.

[0041] shows the appearance of hydrated immortalized bovine fibroblasts. As shown in Figures 10A, 10B, 10C, and 10D, no difference is shown between the appearance of the steam-sterilized and hydrated cell-loaded and blank scaffolds.

[0042] shows the appearance of steam-sterilized blank scaffolds. As shown in Figures 10A, 10B, 10C, and 10D, no difference is shown between the appearance of the steam-sterilized and hydrated cell-loaded and blank scaffolds.

[0043] shows the appearance of hydrated blank scaffolds. As shown in Figures 10A, 10B, 10C, and 10D, no difference is shown between the appearance of the steam-sterilized and hydrated cell-loaded and blank scaffolds.

[0044] is a bar graph depicting hardness results for blank scaffolds (SC), scaffolds containing bovine fibroblasts (BMF-SC), and pan-fried striploin. As shown in Figures 11A and 11B, there is no statistically significant difference between hardness, springiness, and cohesiveness for SC, BMF-SC, and pan-fried striploin.

[0045] is a bar graph depicting springiness, cohesiveness, and resilience results for blank scaffolds (SC), scaffolds containing bovine fibroblasts (BMF-SC), and pan-fried striploin. As can be seen in Figures 11A and 11B, there is no statistically significant difference between hardness, springiness, and cohesiveness for SC, BMF-SC, and pan-fried striploin, while a slightly higher value of resilience was found for scaffolds containing bovine fibroblasts (BMF-SC; 0.31) vs. blank scaffolds (SC; 0.26).

[0046] shows a comparison of condensation over time for the disclosed scaffold and two similar prior art products. Both CN ‘0 3and CN ‘014 show condensation over time with no apparent change in viscosity. After sonication, the disclosed scaffold shows no condensation and remains homogeneously distributed as a colloid with a visual increase in viscosity.

[0047] shows the scaffold of CN ‘013 after freeze drying. The scaffold of CN ‘013 appears brittle and weak, being easily crushed and difficult to pick up with tweezers as well as appearing powdery or flakey, suggesting no cohesive bonding between the protein units.

[0048] shows the scaffold of CN ‘014 after freeze drying. The scaffold of CN ‘014 appears brittle and weak, being easily crushed and difficult to pick up with tweezers as well as appearing powdery or flakey, suggesting no cohesive bonding between the protein units.

[0049] shows the disclosed scaffold after freeze drying. The disclosed scaffold is strong and stable, and is easily picked up and handled using tweezers, suggesting bonding / connectivity between the protein units.

[0050] shows the scaffold of CN ‘013 after steam-sterilization. The scaffold of CN ‘013 is brittle and still slightly powdery, although mechanically more stable after steam-sterilization, but with significant shrinking.

[0051] shows the scaffold of CN ‘014 after steam-sterilization. The scaffold of CN ‘014 is brittle and still slightly powdery, although mechanically more stable after steam-sterilization, but with significant shrinking.

[0052] shows the disclosed scaffold after steam-sterilization. The disclosed scaffold remains strong and stable when handled using tweezers with no apparent diminishment of cohesion between the protein units.

[0053] compares a mixture of the scaffolds of CN ‘013 and CN ‘014 after hydration with the disclosed scaffolds. The scaffolds of CN ‘013 and CN ‘014 show visible degradation in PBS buffer whereas the PBS buffer associated with the disclosed scaffolds remains clear, suggesting no or little degradation.

[0054] shows the results of shear forces on the scaffold of CN ‘013. The scaffold of CN ‘013 has almost zero shear strength, is easily crumbled under shear, and has a powdery appearance.

[0055] shows the results of shear forces on the scaffold of CN ‘014. The scaffold of CN ‘014 has almost zero shear strength, is easily crumbled under shear, and has a powdery appearance.

[0056] shows the results of shear forces on the disclosed scaffold. The disclosed scaffold has meat-like shear strength and meat-like morphology.

[0057] presents the scaffold Texture Profile Analysis (TPA) depicting hardness results for the CN ‘013 scaffold, the CN ‘014 scaffold, the disclosed scaffold, and pan-fried striploin.

[0058] presents the scaffold Texture Profile Analysis (TPA) depicting springiness, cohesiveness, and resilience results for the CN ‘013 scaffold, the CN ‘014 scaffold, the disclosed scaffold, and pan-fried striploin.

[0059] presents the scaffold Texture Profile Analysis (TPA) depicting Texture Profile Examples for the CN ‘013 scaffold, the CN ‘014 scaffold, the disclosed scaffold (n=5), and pan-fried striploin (n=9).

[0060] provides the actual values obtained for the data presented in Figures 11A and 11B as well as Figures 7A and 7B.Description of the Sequence Listing

[0061] SEQ ID NO:1 shows a porcine specific forward primer.

[0062] SEQ ID NO:2 shows porcine specific reverse primer.

[0063] SEQ ID NO:3 shows bovine specific forward primer.

[0064] SEQ ID NO:4 shows bovine specific reverse primer.

[0065] SEQ ID NO:5 shows an internal control forward primer.

[0066] SEQ ID NO:6 shows an internal control reverse primer.Definitions

[0067] As used herein, “colloid,” “colloidal solution,” “colloidal suspension,” and “colloidal dispersion” refer to a mixture that has particles ranging between 1 and 1,000 nanometers in diameter that remain evenly dispersed throughout a medium or solution. The particles always remain dispersed and do not settle to the bottom of the medium or solution. The small particles needed for colloids can be generated by breaking apart the product to be dispersed into the medium or solution by chemical reaction or physical forces (for example, agitation, stirring, pounding, shaking, grinding, milling, spraying, and so on). Pressure forces in the form of pressure waves, high pressure, shear, cavitation, impact, and sonication can also be used.

[0068] As used herein, the terms “laboratory grown meat,” “lab-grown meat,” “clean meat,” “cultured meat,” “cell-based meat,” and “cultivated meat” are used interchangeably and refer to cell populations grown in tissue culture media, oftentimes on scaffolds.

[0069] As used herein, “scaffold” refers to an artificial structure that structurally and biologically supports cell growth and differentiation. Cell supporting scaffolds aim to resemble not only the naturally occurring structural elements found in organs and tissues, but also the biochemical properties of the extracellular matrix found in naturally occurring organs and tissues.

[0070] As used herein, “sonicate” and “sonication” refers to a process by which a sample is exposed to ultrasonic pressure waves and is typically, but not exclusively, conducted with a sonicator device that produces ultrasonic pressure ways.Detailed Description

[0071] This disclosure is not limited to the particular systems, devices and methods described, as these may vary. The terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope.

[0072] Protein scaffolds

[0073] The present disclosure provides an edible macroporous three-dimensional structure (“scaffold”) for animal cell cultivation. The scaffolds disclosed are biocompatible with muscle, fat / adipose, and / or stem cells from agricultural animals and permit the growth, development, and / or differentiation of the cells. In addition, the scaffolds preferably are robust enough to maintain their shape and consistency during food preparation; that is, preferably are capable of manipulation without tearing, shearing, and / or flaking. Furthermore, they preferably have the “mouth feel” associated with that of cooked natural meat. The disclosed scaffolds meet all of these desired properties.

[0074] The scaffold disclosed is composed primarily of a protein source comprising at least one plant or fungal protein. The scaffold can comprise, consist essentially of, or consist of the at least one plant or fungal protein. Suitable plants from which the plant protein can be obtained are, without limitation, members of the Poaceae family (for example, rice, wheat, barley, rye, teff, millet, corn, sorghum, oats, and so on), Fabaceae family (for example, peas, soybeans, beans, lentils, chickpeas, peanut, carob, alfalfa, kudzu, and so on), the Brassicaceae family (for example, broccoli, cauliflower, kale, mustard, cabbage, kohlrabi, turnip, and so on), and the Sargassaceae family. Suitable fungi from which the fungal protein can be obtained are, without limitation, edible members of the Agaricaceae family (for example, Agaricus species, Pleurotus species, Morchella species, Lentinula species, Auricularia species, Volvariella species, Flammulina species, Tremella species, Hypsizygus species, Stropharia species, Cyclcybe species, Hericium species, Phallus species, Boletus species, Calbovista species, Craterellus species, Tuber species, and so on). The protein can be one protein, or a combination of two proteins, three proteins, four proteins, five proteins, six proteins, and so on. In some examples, the plant protein is a wheat gluten protein, a soy protein, a pea protein, or combinations thereof. In some examples, the plant protein is a wheat gluten protein. In other examples, the plant protein is a soy protein. In other examples, the plant protein is a pea protein.

[0075] As an example, without limitation, the type of plant or fungal protein can be a protein isolate, an alginate, a prolamin, a glutenin, a gliadin, a zein, a fungal collagen, a mycelium protein, a laminin, a protein hydrolysate, or combinations thereof.

[0076] Methods

[0077] Directional freezing of plant and / or fungal proteins is known to cause freezing (generation of ice crystals) to take place throughout the thickness of the mass to produce generally parallel fibers that are aligned generally perpendicularly to the cooling surfaces (Zhang et al. (2005) Nature Materials 4:787-793; CA 1038224). Directional freezing typically involves placing the substance to be frozen into a partitioned mold which has minimum thermal conductivity (for example, polytetrafluoroethylene (PTFE), polydimethylsiloxane (PDMS), and so on). Partitioning is typically provided by a non-insulating material, such as a metal. Suitable metals are copper, steel, aluminum, aluminum nitride, silver, tungsten, and others to name a few. Suitable synthetic crystalline minerals can also be used, such as silicon carbide and others.

[0078] Directional freezing is then typically accomplished by placing the substance to be frozen on one side of the partition and a supercooled gas on the other side of the partition. Suitable supercooled gases are, for example, nitrogen (liquid nitrogen), hydrogen (liquid hydrogen), and carbon dioxide (liquid carbon dioxide), combinations thereof, and others, to name but a few. The directionally frozen substance is optionally stabilized prior to removal of the ice crystals. Stabilization can be accomplished by incubation, for example, at -80 °C for 30 minutes or for longer periods of time at less extreme temperatures, such as for 24 hours at -20 °C.

[0079] Removal of the ice crystals via freeze drying produces scaffolds with distinct, generally parallel aligned zones and a porous structure (Guan et al. (2010) J Applied Polymer Science 118(3): 1658-1665; Xiang et al. (2022) Biomaterials 11:1-11). Freeze drying can be performed at temperatures of about -1 °C to about -80 °C under a vacuum level of about 5 Pa to about 20 Pa for about 12 hours to about 48 hours, such as at a temperature of about -1 °C at a vacuum level of about 9 Pa to about 12 Pa for about 48 hours, or at a temperature of about -5 °C at a vacuum level of about 9 Pa to about 12 Pa for about 24 hours. Freeze-dried scaffolds can then optionally be further dried for about 15 minutes to about 1 hour at about 45 °C to about 100 °C, such as for about 30 minutes at about 60 °C.

[0080] Ultimately, scaffolds useful for generation of laboratory grown meat preferably have low concentrations of or are free of toxic substances. Typically, protein-type scaffolds have depended upon crosslinking with chemical (for example, formaldehyde or glutaraldehyde) or naturally occurring (for example, gallic acid or tannins) to bind the protein molecules together, improve tensile strength, and increase aqueous stability (Jahangirian et al. (2019) Biomolecules 9(10):619). The literature has reported a physical crosslinking method to generate protein self-crosslinking by exposure to high-pressure and high temperature, such as autoclaving (see CN114438013 and CN114438014). However, while the method produces self-crosslinked proteins, the scaffolds produced from that process do not withstand manipulation well, making the scaffolds inadequate for laboratory grown meat preparations.

[0081] Applicant has surprisingly found an important aspect of using directional freezing followed by freeze drying and autoclaving is to first generate a colloidal suspension of the plant and / or fungal protein. This can be accomplished by breaking a plant and / or fungal protein into particles ranging from about 1 nanometer and about 1000 nanometers in diameter or by using a plant and / or fungal protein that already has particles in that size range. An alternative range is about 0.001 um to about 300 um. When breaking is required, that can be accomplished using chemicals or physical force such as, without limitation, agitation, stirring, pounding, shaking, grinding, milling, and / or spraying, Alternatively, pressure forces can be used such as, without limitation, pressure waves, high pressure, shear, cavitation, pressure impact, and sonication with or without a pulse-rest interval.

[0082] After directional freezing and freeze drying, scaffolds produced with a colloidal suspension of the plant and / or fungal protein can be physically crosslinked by steam sterilization / autoclaving. Suitable autoclaving temperatures, times, and pressures are about 50 °C to about 150 °C for about 10 minutes to about 60 minutes, and at about 15 psi (103 kPa) to about 30 psi (207 kPa). A specific example can be about 121 °C for 20 minutes at about 18 psi (124 kPa).

[0083] Crosslinked scaffolds can be washed. For example, crosslinked scaffolds are washed three times, each for about 30 minutes to about 1 hour, to remove any residual acid, for example washing in PBS buffer, in 0.37% sodium bicarbonate solution, or in another suitable solution (for example, 0.9% sodium chloride) with or without shaking. Washes can be carried out at about 4 °C to about 25 °C, such as at room temperature. Washed scaffolds optionally can then be freeze dried again as noted above for storage purposes.

[0084] In some examples, the described methods do not include contacting with one or more chemical crosslinking agents. For example, the protein source, frozen protein source, or protein scaffold are not contacted with one or more chemical crosslinking agents.

[0085] Scaffolds have, in their final form, an aligned / layered type of morphology and are porous in nature. Porosity ranges from about 30% to about 50%, such as, for example about 42%, with a mean pore size in the range of about 2 µm to about 20 µm.

[0086] Scaffolds exhibited a hardness of about 235 kPa to about 400 kPa, a springiness of about 0.8 to about 0.95, cohesiveness of about 0.65 to about 0.86, and resilience of about 0.22 to about 0.31 via Texture Profile Analysis (TPA; Figures 17A and 17B), having an extremely similar texture profile to pan-fried striploin (). Scaffolds exhibited significant bonding / connectivity between protein units, significant shear resistance, little shrinking during the steam-sterilization / crosslinking process, and little to no degradation / high stability in wash solution(s).

[0087] If desired, one or more additional cell biomolecules can be used to coat the scaffolds to further encourage cell adhesion and differentiation. Examples of such biomolecules include, without limitation, RGD peptide(s), vitronectin, fibronectin, fibrin, and so on.

[0088] Cells can be incorporated into scaffolds after or immediately after generation of a plant and / or fungal protein colloid. Here, cells can be mixed in the colloid suspension at a rate (cells: colloid) of about 1:5, about 1:10, about 1:15, or about 1:20 (w / w) prior to directional freezing, freeze drying, crosslinking, and washing. Cells can be contacted with the protein scaffold to produce a populated protein scaffold.

[0089] Alternatively or additionally, cells can be introduced to the scaffold post-scaffold production via direct seeding. In this case, scaffolds are placed in appropriate cell culture media that has been inoculated with a desired cell type. The cell suspension can be contacted with the protein scaffold to produce a populated protein scaffold. In some cases, the cell suspension is forced through the scaffold via either internal or external vacuum pressure. As cells travel through the scaffold, they are lodged in the pores and thus seed the scaffold.

[0090] The cells can generally be any type of cell, such as animal cells, plant cells, seafood cells, fish cells, or combinations thereof. Fish cells can include fish, shrimp, prawn, crab, crayfish, lobster cells, or combinations thereof. In other examples, the cells can be teleost fish cells, crustacea cells, cephalopod cells, or combinations thereof. Animal cells can include cow cells, pig cells, chicken cells, deer cells, bear cells, sheep cells, or combinations thereof.Examples

[0091] Example 1: Generation of Aligned Fiber Scaffold

[0092] The following solutions were prepared: (a) a 10% (v / v) aqueous acetic acid solution by mixing glacial acetic acid with deionized water at room temperature, (b) a 0.9% (w / v) sodium chloride solution by dissolving sodium chloride in deionized water at room temperature, (c) a 0.37% sodium bicarbonate solution by dissolving sodium bicarbonate in 0.9% sodium chloride solution, pH adjusted to 7.4 with glacial acetic acid, (d) a 10% soy protein solution by mixing soy protein powder in 10% acetic acid at room temperature, and (e) a PBS solution (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4).

[0093] A soy protein colloid was generated by sonicating the 10% soy protein solution using 80% power under a pulse-rest interval of 0.7s-0.3s for 20 minutes at room temperature Directional freezing was performed by inserting a T2 copper sheet in the middle of a PTFE mold to divide the mold space into two. The soy protein colloid was cast into one side of the copper sheet and liquid nitrogen added to the other side ().

[0094] The frozen colloid was stabilized at -80 °C for 30 minutes. Freeze drying was then performed under a vacuum level of 9-12 Pa at -1 °C for 48 hours or -5 °C for 48 hours and produced a freeze-dried scaffold with the morphology of that shown in. Freeze-dried scaffolds were further dried for 30 minutes at 60 °C.

[0095] The freeze-dried scaffolds were physically crosslinked via steam sterilization (autoclaving) at 121 °C for 20 minutes at 18 psi.

[0096] Residual acid was removed by standard washing with PBS (two changes with 1 hour duration per wash with 80 rpm shaking) prior to freeze drying. Washed scaffolds were then freeze-dried again under a vacuum level of 9-12 Pa at -1 °C for 48 hours prior to cellular experiments.

[0097] Alternatively, scaffolds were extensively washed. Scaffolds were first washed in the 0.37% (sodium bicarbonate solution three times for 1 hour each at room temperature with 80 rpm shaking. A fourth was conducted overnight at 4 °C without shaking. Scaffolds were then submitted to two further washes in 0.9% sodium chloride solution for one hour / wash at room temperature with 80 rpm shaking followed by a final was in deionized water for 30 minutes without shaking. Washed scaffolds were then freeze-dried again under a vacuum level of 9-12 Pa at -1 °C for 48 hours prior to cellular experiments. As discussed below, the extensive washing procedure was not used due to its degradative effects.

[0098] After washing, the scaffolds had the morphology shown in. These attractive results display a similar morphology to finely flaked fish that can be formed into a fish fillet.

[0099] Example 2: Scanning Electron Microscope (SEM) Analysis

[0100] The structure and pore size of the scaffolds produced with standard washing was analyzed via SEM (). The porosity (2D) was determined to be 42.0% via image analysis on a single layer plane of the scaffold. Pore size measurement was determined by the shortest diagonal distance between the solid edges for each pore. Mean pore size was determined to be 8.85 ± 5.75 µm (n = 100).

[0101] Example 3: Scaffold Texture Profile Analysis (TPA)

[0102] To analyze the texture of the scaffolds produced, TPA was performed using a TA.XT2i texture analyzer (Stable Micro Systems, Surrey, United Kingdom;), which imitates the action of the jaw on bite-sized pieces. This procedure correlates well with sensory evaluation.

[0103] Two samples were tested and values generated for hardness, springiness, cohesiveness, and resilience. Referring to the graph in, hardness (N) is equal to F1 and is the highest peak force measured during the first compression.

[0104] Cohesiveness is equal to (d+e) / (a+b); that is the area under the second compression curve divided by the area under the first compression curve ().

[0105] Springiness (%) is equal to (Distance 2) / (Distance 1)*100 or (Time 2 / Time 1)*100 and provides a ratio or percentage of a product’s recovery to its original height ().

[0106] Resilience is equal to b / a and is the area under the curve after peak force is reached divided by the area under the curve before peak force is reached ().

[0107] Force data (N) were normalized by the scaffold dimensions to generate stress data (kPa) so data from samples of different sizes could be compared. Pan-fried striploin was used as a control. Results were similar to the control sample.

[0108] Example 4: Effect of Extensive Washing on Scaffolds

[0109] Figures 7A and 7B present the hardness, springiness, cohesiveness, and resilience values obtained for extensively washed scaffolds, andpresents their texture profile. Figures 17A and 17B presents hardness, springiness, cohesiveness, and resilience values obtained for standardly washed scaffolds. As can be seen from a comparison of the values for the disclosed scaffolds having extensive washing (Figures 7A and 7B) and standard washing (Figures 17A and 17B), also summarized in, the extensive / long duration washing (24 hours) and vigorous shaking (about 100 rpm) of the extensively washed scaffolds resulted in a loss of hardness (about 66%) as compared to standard washed scaffolds (see Figures 17A and 17B). There was, however, no significant loss of cohesiveness, springiness, and resilience.

[0110] A comparison of the texture profile of the extensively washed scaffolds () and the standardly washed scaffolds (), indicated that standardly washed scaffolds had a texture profile which essentially mirrored that of pan-fried striploin, suggesting that the mouth-feel of the standardly washed scaffold would be fundamentally equivalent to that of pan-fried striploin. In contrast, the extensively washed scaffolds showed a texture profile that was noticeably different from that of pan-fried striploin, suggesting that the mouth-feel would be noticeably different from that of pan-fried striploin.

[0111] As a consequence, extensive washing of the scaffolds was not required.

[0112] Example 5: Incorporation of Cells into Scaffolds

[0113] The efficacy for cell incorporation into the scaffolds was determined. Immediately after generation of a soy protein colloid (see above), immortalized bovine fibroblasts (iBFs) were directly mixed into the colloid at a ratio of 1 iBF : 10 Colloid (w / w). Directional freezing using liquid nitrogen, a PTFE mold, and a copper plate was conducted as described above. Cell-infused scaffolds were freeze dried at -1 °C for 48 hours.

[0114] RNA was isolated from (a) iBFs, (b) immortalized porcine cells (as negative control cells), (c) iBF-loaded scaffold (iBF-SC), and (d) blank scaffold (SC). cDNA was synthesized from the isolated RNA and PCR performed using IPROOF™ High-Fidelity DNA Polymerase (BIO-RAD, Hercules, CA) to amply species-specific genes via bovine or porcine-specific primers (see Table 1).

[0115] [Table 1] Primer sequences for DNA amplificationSize (bp)Primer SequenceNote460Pi F: 5’-CTACTATCCCTGCCAGTT-3’ (SEQ ID NO:1)Pi R: 5’-GAATAGGAAGATGAAGCC-3’ (SEQ ID NO:2)Porcine Specific Primer102Cw F: 5’-GCTATTCCAACCGGGGTAAAAGTC-3’ (SEQ ID NO:3)Cw R: 5’-GAAAATAAAGCCTAGGGCTCAC-3’ (SEQ ID NO:4)Bovine Specific Primer70F: 5’-CGGGGAATYAGGGTTCGATTC-3’ (SEQ ID NO:5)Ic R: 5’-GCCTGCTGCCTTCCTTKGATG-3’ (SEQ ID NO:6)Internal Control

[0116] PCR products were electrophoresed on a 1% agarose gel stained with SYBR Safe (Bio Basic Asia Pacific, Singapore). Results are shown in, indicating that bovine specific genes were detected from the iBF-incorporated scaffolds (iBF-SC) and successful iBF loading into the scaffold.

[0117] No differences were observed between the cell-loaded and blank scaffolds which were strong and stable, easily handled using tweezers and exhibiting cohesiveness; that is, bonding / connectivity between the protein units (Figures 9A-9B). In addition, no difference was observed between the cell-loaded and blank scaffolds which had the same fiber morphology and similar shear strength as tested manually (Figures 10A-10D). Furthermore, no statistical difference was found in (a) hardness, (b) springiness, and (c) cohesiveness, although a slightly higher value of resilience was found in iBF-SC (0.31) compared to SC (0.26) (see Figures 11A-11B). These results were as expected, as cells were mixed with scaffold. If cells were allowed to grow and differentiate within the scaffold, the texture may change due to extracellular matrix secretion, just like with meat.

[0118] Example 6: Comparison of parameters and traits to demonstrate commercial feasibility and increased probability of consumer acceptance

[0119] Chinese patent application CN114438013A (“CN ‘013”) was published May 6, 2022, and is entitled “Method for Preparing Cell Culture Meat Biological Scaffold Through Physical Crosslinking.” Its companion Chinese patent publication, CN114438014A (“CN ‘014”) was published May 8, 2022, and is entitled “Edible Chitosan Glutenin Biomimetic Orientation Cell Culture Meat Biological Scaffold.” While there are some general similarities between the two CN ‘013 and CN ‘014 publications and the method disclosed above, there are some critical aspects of the disclosed method that are absent from the CN ‘013 and CN ‘014 publications. Consequently, the following comparative experiment was conducted.

[0120] Both the CN ‘013 and CN ‘014 publications use soy protein powder as the protein basis for the scaffolds and state that water is used as the dispersing agent or solvent, although the two methods divulge somewhat from each other at that point. Table 2 below shows the ingredients / reagents used for the comparative experiment.

[0121] CN ‘013 publicationCN ‘013 usedCN ‘014 publicationCN ‘014 usedDisclosed methodProtein sourceSoy Protein Isolate powder (SPI), etc.Soy Protein Isolate powder (SPI)Soy Protein Isolate powder (SPI), etc.Soy Protein Isolate powder (SPI)Soy Protein Isolate powder (SPI)SPI solventWaterWaterWaterWater10% acetic acid solutionSPI concentration1-200 mg / ml10% (w / v)100 mg / ml1-200 mg / ml10 % (w / v) 100 mg / ml10% (w / v)SPI protein solution pH1-5 or 7-123.03 (adjusted with food-grade acetic acid (10% v / v)1-5 or 7-126.262.96Further treatmentNoneNoneNoneNoneSonicationDirectional FreezingLiquid Nitrogen, PDMS mold, copper plateLiquid Nitrogen, PTFE mold, copper plateLiquid Nitrogen, PDMS mold, copper plateLiquid Nitrogen, PTFE mold, copper plateLiquid Nitrogen, PTFE mold, copperFreeze drying temperature-40 to-100 °C-40 °C-40 to-100 °C-40 °C-1 °CFreeze drying duration24-72 hours72 hours24-72 hours72 hours48 hoursSteam SterilizationAutoclaveAutoclaveAutoclaveAutoclaveAutoclaveAutoclave temperature50-200oC121oC50-200oC121oC121oCAutoclave duration1-60 minutes20 minutes1-60 minutes20 minutes20 minutesAutoclave pressure1-100 psi18 psi1-100 psi18 psi18 psiRehydrate scaffold / washingPBSPBSPBSPBSPBSRehydrate or washing duration1 h x 3 times (2 changes of PBS): total 3 h1 h x 3 times (2 changes of PBS): total 3 h1 h x 3 times (2 changes of PBS): total 3 hRehydrate or washing temperatureRoom temperature (20 °C)Room temperature (20 °C)Room temperature (20 °C)

[0122] shows the results after preparation and treatment of the protein solution prior to freeze drying. There was a visual increase in viscosity of the sonicated protein solution which was absent from CN’013 and CN ‘014, which retained a water-like appearance. While CN’013 and CN ‘014 showed condensation over time, the sonicated solution remained a homogeneously distributed colloid.

[0123] Figures 13A, 13B, and 13C show the results after directional freezing and freeze-drying. Both the CN ’013 and CN ‘014 scaffolds were brittle and weak, easily crushed, and difficult to pick up with tweezers. Their powdery or flakey appearance indicated little cohesive bonding between the protein units. The disclosed scaffold, however, was strong and stable, easily picked up and handled with tweezers, with good cohesive bonding / connectivity between the protein units.

[0124] Steam sterilization also showed differences between the scaffolds (Figures 14A, 14B, and 14C). Here, the CN ’013 and CN ‘014 scaffolds remained brittle and still slightly powdery, although being more stable mechanically after steam sterilization. Both did, however, exhibit significant shrinkage. In contrast, the disclosed scaffolds remained strong and stable with no apparent change in size (that is, no visible shrinkage) or the ease of manipulation with tweezers. No apparent change in cohesiveness was noted.

[0125] Changes were also seen after washing / hydration of the scaffolds. As seen in, there was visible degradation in CN ‘013 and CN ‘014 after washing, causing the PBS wash solution to become cloudy, while no visible degradation occurred for the disclosed scaffolds. Importantly, the strength of the CN ‘013 and CN ‘014 scaffolds was poor with almost zero shear strength, the scaffolds being easily crumbled with finger strength shear force, and the scaffolds having a powdery appearance (Figures 16A, 16B, and 16C). In contrast, the disclosed scaffolds exhibited meat-like shear strength, continued to have cohesiveness, be easily manipulated using tweezers, and having a meat-like fiber morphology.

[0126] TPA analysis was performed on the CN ‘013, CN ‘014, and the disclosed scaffolds (as described above), using pan-fried striploin as a control. CN ‘013 had approximately twice the hardness value as CN ‘014, while the disclosed scaffolds exhibited no statistical difference for hardness versus pan-fried striploin (2-sample t test;). With respect to springiness, the CN ‘013, CN ‘014, and disclosed scaffolds all had higher values than the pan-fried striploin, while the cohesiveness values were slightly higher for the CN ‘013, CN ‘014, and disclosed scaffolds compared to the pan-fried striploin (2-sample t test) (). While the CN ‘013 and CN ‘014 scaffolds had greater resilience values than pan-fried striploin, the resilience of the disclosed scaffolds had no statistical difference from pan-fried striploin (2-sample t test;).

[0127] Texture profiles were generated for the disclosed scaffolds (n=9) and pan-fried striploin (n=5). As can be seen in, the texture profiles are very similar.

[0128] presents the TPA analysis data for CN ‘013, CN ‘014, and the disclosed scaffolds with standard and extensive washing. Considering this, as well as the step-wise variances noted above, while the differences between the methods of CN ‘013 and CN ‘014 and the disclosed method are arguably small, those changes had an unexpectedly significant effect on each step of the production of the scaffolds and resulted in surprisingly substantial differences in the scaffold product produced.

[0129] Example 7: Incorporation of live or non-living animal cells on scaffolds

[0130] Scaffolds made from plant- or fungal-based protein are commonly used as scaffolds for growing animal or human cells. In most of the cases, coating of plant-based scaffold with biomolecule such as RGD peptide, serum, gelatin, collagen, fibronectin, vitronectin, laminin, or anything extracellular matrix protein is required. By coating the scaffolds with biomolecules, animal cells could be incorporated in or on the scaffolds, which can further proliferate and / or differentiate into mature cells such as muscle and fat cells.

[0131] For example, the sterile scaffolds could be produced by autoclaving or through gamma irradiation. The sterile scaffolds can then be coated with 2% gelatin overnight by submerging scaffolds in gelatin and placed in a sterile vacuum chamber. The vacuum pressure will force out the air within scaffolds and replace it with gelatin solution. This process can also be further simplified by autoclaving the scaffolds together with 2% gelatin solution in an autoclave. Once coated, the scaffolds can be placed in shake flasks together with immortalized bovine / porcine myoblasts and / or pre-adipocytes. Cells will attach to the scaffolds overtime and migrate into the scaffolds, as the surface of the flasks are cell-repellent. By adjusting the concentration of plant / fungal protein colloid, the pore size of the scaffolds could be increased or decreased accordingly to fit various cell line. Once the cells has proliferated to significant amount, the cell culture media could be switch from proliferation media to differentiation media to convert stem cells into mature muscle and fat cells.

[0132] The texture of the scaffolds is predicted to improve towards actual meat as cells are secreting extracellular matrix and attaching firmly to the scaffolds. The sarcomere within the mature muscle cells will also contribute to the texture, while fatty acids from the fat cells will contribute to the flavor, taste and aroma of the final product. In general, the nutritional value and composition of the final product will also be improved due to the presence of animal protein and fats. If fish cells were used, it can further enhance the nutritional value by providing healthy fats such as omega 3 and micronutrients such as selenium.

[0133] By means of example, cells (including fibroblasts, stem-, fat- and muscle- cells) can also be incorporated into the scaffolds without culturing them further. This could be done in the forms of microneedles arrays injections, with diameter of the microneedles ranging between 0.1-1 mm. This will enhance the nutritional value and taste, flavor and aroma of the finished product.

[0134] Example 8: Testing the integration of flavor compounds in edible scaffolds

[0135] Incorporation of flavor-enhancing compounds into an edible protein scaffold on the flavor profile and consumer sensory experience of cultured meat products will be evaluated.

[0136] A soy protein solution (10% w / v) is prepared by dissolving soy protein isolate in a 10% acetic acid solution. The solution is sonicated at 80% power using a pulse-rest interval of 0.7s-0.3s for 20 minutes to create a homogeneous colloid. The sonicated solution is subjected to directional freezing using a PTFE mold and liquid nitrogen, followed by freeze-drying at -1°C for 48 hours under a vacuum level of 9–12 Pa to produce a porous scaffold.

[0137] The following flavor-enhancing compounds are prepared in a sterile solution: Glutamic acid (0.5% w / v) for umami flavor; Oleic Acid (0.2% w / v) for fatty mouthfeel; Heme Compound (0.1% w / v) derived from plant sources for iron-rich meat-like notes; and Maillard precursors: A mixture of glucose (0.3% w / v) and lysine (0.2% w / v) for browning reaction flavors.

[0138] The flavor solution is infused into the scaffold by immersion for 1 hour at 25°C under gentle agitation (80 rpm). Excess solution is removed, and the scaffold is air-dried under sterile conditions.

[0139] Immortalized bovine muscle cells and adipocytes are seeded onto the flavored scaffold at a ratio of 2:1 (muscle:fat) and cultured in a standard growth medium. Cell-laden scaffolds are incubated at 37°C with 5% CO₂ for 14 days.

[0140] After incubation, scaffolds are pan-fried at 180°C for 2 minutes per side to simulate a typical consumer cooking process. Controls include scaffolds without flavor compounds and scaffolds with plain cell culture medium infusion.

[0141] The expected results are as follows.

[0142] Sensory Evaluation: Flavored scaffolds are expected to score higher on umami, fattiness, and overall meat-likeness compared to controls.

[0143] Chemical Analysis: Enhanced production of meat-like volatiles, including sulfur compounds and aldehydes, is anticipated in the flavored scaffolds.

[0144] TPA: No significant difference in mechanical properties (e.g., hardness, springiness) is expected between flavored and control scaffolds.

[0145] Example 9: Modifying scaffold texture to mimic different meat types

[0146] The ability to modify the texture of an edible protein scaffold to match the mechanical properties and mouthfeel of different types of meat products such as steak, chicken, and fish will be determined.

[0147] A protein colloid is prepared using soy protein isolate (10% w / v) dissolved in a 10% acetic acid solution. The solution is sonicated at 80% power with a pulse-rest interval of 0.7s-0.3s for 20 minutes to ensure a homogeneous suspension. Directional freezing is performed using a PTFE mold and liquid nitrogen, followed by freeze-drying at -1°C for 48 hours under vacuum (9–12 Pa), producing the base scaffold.

[0148] Pore Size Adjustments:

[0149] The freezing rate is varied to control pore size:

[0150] Rapid Freezing: Using liquid nitrogen directly to create small pore sizes (5–10 µm) for denser textures (for example, steak).

[0151] Slow Freezing: Using a -20°C freezer to create larger pore sizes (20–50 µm) for softer, flakier textures (for example, fish).

[0152] Protein Composition:

[0153] Different protein blends are tested to adjust scaffold hardness and elasticity:

[0154] For Steak: Soy protein isolate and wheat gluten (2:1 ratio) for a firm, springy texture.

[0155] For Chicken: Soy protein isolate and pea protein (1:1 ratio) for moderate firmness and cohesiveness.

[0156] For Fish: Soy protein isolate and alginate (3:1 ratio) for a softer, flaky texture.

[0157] Crosslinking Intensity:

[0158] Autoclaving is performed at different temperatures and durations to modulate mechanical properties:

[0159] High Crosslinking: 121 °C for 20 minutes to enhance scaffold hardness.

[0160] Low Crosslinking: 100 °C for 10 minutes to maintain flexibility.

[0161] Mechanical Testing:

[0162] Texture Profile Analysis (TPA):

[0163] Hardness, springiness, cohesiveness, and resilience are measured using a texture analyzer.

[0164] Target benchmarks:

[0165] Steak: Hardness 300–400 kPa, springiness 0.85–0.95.

[0166] Chicken: Hardness 200–300 kPa, springiness 0.75–0.85.

[0167] Fish: Hardness 100–200 kPa, springiness 0.65–0.75.

[0168] Scanning Electron Microscopy (SEM):

[0169] Pore structure and alignment are analyzed to confirm modifications.

[0170] Sensory Testing:

[0171] Scaffolds are seeded with bovine muscle cells for steak, chicken muscle cells for chicken, and fish muscle cells for fish, then cultured for 14 days.

[0172] Cooked samples (pan-fried at 180°C for 2 minutes per side) are evaluated by a sensory panel for texture and mouthfeel.

[0173] Expected Results:

[0174] Mechanical Testing:

[0175] Rapid freezing produces dense scaffolds with smaller pores and higher hardness values, mimicking steak.

[0176] Slow freezing produces flaky scaffolds with larger pores and lower hardness values, suitable for fish textures.

[0177] Protein blends and crosslinking variations effectively modulate springiness and cohesiveness.

[0178] Sensory Evaluation:

[0179] Steak-like scaffolds are firm and chewy, chicken-like scaffolds are moderately firm, and fish-like scaffolds are soft and flaky, closely replicating the textures of their natural counterparts.

[0180] Microscopy Analysis:

[0181] Aligned fiber structures are evident in steak-like scaffolds, random porous structures in fish-like scaffolds, and intermediate structures in chicken-like scaffolds.

[0182] In the above detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be used, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

[0183] The present disclosure is not to be limited in terms of the particular embodiments described in this application, which are intended as illustrations of various aspects. Many modifications and variations can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this disclosure is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0184] As used in this document, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Nothing in this disclosure is to be construed as an admission that the embodiments described in this disclosure are not entitled to antedate such disclosure by virtue of prior invention. As used in this document, the term “comprising” means “including, but not limited to.”

[0185] While various compositions, methods, and devices are described in terms of "comprising" various components or steps (interpreted as meaning "including, but not limited to"), the compositions, methods, and devices can also "consist essentially of" or "consist of" the various components and steps, and such terminology should be interpreted as defining essentially closed-member groups.

[0186] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity.

[0187] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (for example, bodies of the appended claims) are generally intended as “open” terms (for example, the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (for example, “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (for example, the bare recitation of "two recitations," without other modifiers, means at least two recitations, or two or more recitations). 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 one having skill in the art would understand the convention (for example, “ 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, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (for example, “ a system having at least one of A, B, or 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, etc.). 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, claims, or drawings, 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.”

[0188] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0189] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

[0190] Various of the above-disclosed and other features and functions, or alternatives thereof, may be combined into many other different systems or applications. Various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art, each of which is also intended to be encompassed by the disclosed embodiments.

Claims

A method of preparing a protein scaffold, the method comprising:providing at least one protein source, comprising at least one plant protein, at least one fungal protein, or at least one plant protein and at least one fungal protein;directional freezing the protein source to form ice crystals in a frozen protein source;removing the ice crystals from the frozen protein source; andcrosslinking the frozen protein source after the removing the ice crystals step to prepare the protein scaffold.The method of claim 1, wherein the protein source is at least one plant protein.The method of claim 1, wherein the protein source is at least one fungal protein.The method of claim 1, wherein the protein source is one plant protein.The method of claim 1, wherein the protein source is one fungal protein.The method of claim 1, wherein the protein source is wheat gluten protein, a soy protein, a pea protein, or combinations thereof.The method of claim 1, wherein the plant protein is from the Poaceae family, rice, wheat, barley, rye, teff, millet, corn, sorghum, oats, or combinations thereof.The method of claim 1, wherein the plant protein is from the Fabaceae family, peas, soybeans, beans, lentils, chickpeas, peanut, carob, alfalfa, kudzu, or combinations thereof.The method of claim 1, wherein the plant protein is from the Brassicaceae family, broccoli, cauliflower, kale, mustard, cabbage, kohlrabi, turnip, or combinations thereof.The method of claim 1, wherein the plant protein or fungal protein is a protein isolate, an alginate, a prolamin, a glutenin, a gliadin, a zein, a fungal collagen, a mycelium protein, a laminin, a protein hydrolysate, or combinations thereof.The method of claim 1, wherein the directional freezing is performed using at least one supercooled gas.The method of claim 1, wherein the directional freezing is performed with liquid nitrogen, liquid hydrogen, liquid carbon dioxide, or combinations thereof.The method of claim 1, further comprising stabilizing the frozen protein source after the directional freezing step and before the removing the ice crystals step.The method of claim 1, further comprising stabilizing the frozen protein source by incubation after the directional freezing step and before the removing the ice crystals step.The method of claim 1, wherein the removing the ice crystals step is performed by freeze drying.The method of claim 1, further comprising crosslinking the protein scaffold using an autoclave after the removing the ice crystals step.The method of claim 1, further comprising contacting cells and the protein scaffold to produce a populated protein scaffold.The method of claim 1, further comprising contacting a cell suspension and the protein scaffold to produce a populated protein scaffold.The method of claim 1, wherein the method does not comprise contacting the protein source, frozen protein source, or protein scaffold with one or more chemical crosslinking agents.A protein scaffold prepared by a method, the method comprising:providing at least one protein source, comprising at least one plant protein, at least one fungal protein, or at least one plant protein and at least one fungal protein;directional freezing the protein source to form ice crystals in a frozen protein source;removing the ice crystals; andcrosslinking the protein scaffold after the removing the ice crystals step to prepare the protein scaffold.The protein scaffold of claim 20, wherein the method further comprises contacting a cell suspension and the protein scaffold to produce a populated protein scaffold.

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