RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial No. 63/159,403, filed March 10, 2021, entitled “Constructs for Meat Cultivation and Other Applications”; US Provisional Patent Application No. 63/279,617, filed November 15, 2021, entitled “Constructs Comprising Fibrin or Other Blood Products for Meat Cultivation and Other Applications”; US Provisional Patent Application No. 63/279,631, filed November 15, 2021, entitled, “Methods and Systems of Preparing Cultivated Meat from Blood or Cellular Biomass”; US Provisional Patent Application No. 63/279,642, filed November 15, 2021, entitled, “Systems and Methods of Producing Fat Tissue for Cell-Based Meat Products”; US Provisional Patent Application No. 63/279,644, filed November 15, 2021, entitled “Production of Heme for Cell-Based Meat Products”; US Provisional Patent Application Serial No. US 63/300,577, filed January 18, 2022, entitled “Animal-Derived Antimicrobial Systems and Methods”; US Provisional Patent Application No. 63/164,397, filed March 22,

2021, entitled “Growth Factor for Laboratory Grown Meat”; US Provisional Patent Application No. 63/164,387, filed March 22, 2021, entitled, “Methods of Producing Animal Derived Products”; US Provisional Patent Application No. 63/314,171, filed February 25,

2022, entitled “Growth Factors for Laboratory Grown Meat and Other Applications”; and US Provisional Patent Application No. 63/314,191, filed February 25, 2022, entitled “Methods and Systems of Producing Products Such as Animal Derived Products.” Each of these is incorporated herein by reference in its entirety.

FIELD

The present disclosure generally relates to clean meat and other cultivated animal- derived products.

BACKGROUND

Clean meat, or cultivated meat, is meat that is produced using in vitro cell culture or bioreactors, instead of being harvested from live animals. In many cases, the meat that is produced may include muscle cells and fat cells. Such meat may include, for example, chicken, beef, pork, and fish. Such technologies have the potential to revolutionize agriculture, for example, by decreasing the amount of land necessary to produce meat, avoiding unethical farming of animals, or increasing the available food supply. However, it is still difficult and expensive to culture cells, and thus improvements are needed. SUMMARY

The present disclosure generally relates to clean meat and other applications. The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems and/or articles.

In accordance with certain embodiments, in order to produce cell-based cultivated meat, myoblasts should be caused to proliferate, fuse, and form a higher order construct, myotubes. Myoblasts alignment may be important for efficient fusion myoblasts to myotubes. Certain embodiments are directed to design and fabrication strategies of microcarriers that allow both proliferation and differentiation of myoblasts to myotubes, e.g., in suspension bioreactors, based on edible plant proteins. For example, in one set of embodiments, zein-based constructs are produced using molding or extrusion techniques to produce certain shapes such as microfibers, micro-whiskers, micro-flake, etc. In some cases, these may contain micro grooves, for example, as a secondary hierarchical architecture, which may be used to promote alignment during the growth of the myoblasts on the microcarrier. Such microcarriers may allow cell proliferation and/or spontaneous differentiation of myoblasts to myotubes, e.g., without additional steps, in bioreactors in some cases. In certain embodiments, such techniques may be used to produce the final product, e.g., an edible plant- based protein cell-laden microcarrier may be directly used as the final product, e.g., with minimum intermediate processing.

Certain aspects of the present disclosure are generally directed to articles. In one set of embodiments, the article comprises a cultivated animal-derived product. In some cases, the product comprises aligned myotubes on microcarriers comprising a plurality of grooves.

The article, in accordance with another set of embodiments, comprises a cultivated animal-derived product, which comprises aligned myotubes on microcarriers formed from a plant-originated material.

In yet another set of embodiments, the article comprises a cultivated animal-derived product, comprising an anisotropic scaffold.

In addition, some aspects of the present disclosure are generally directed to methods. In accordance with one set of embodiments, for example, the method comprises seeding myoblasts on microcarriers, and causing the myoblasts to fuse to form aligned myotubes on the microcarriers. The method, in another set of embodiments, comprises seeding myoblasts on microcarriers, growing the myoblasts in a bioreactor to produce aligned myotubes on the microcarriers, and producing a cultivated animal-derived product comprising the aligned myotubes within the bioreactor.

In still another set of embodiments, the method comprises fabricating microcarriers comprising a plurality of grooves, and seeding myoblasts on the microcarriers.

According to yet another set of embodiments, the method comprises seeding myoblasts on microcarriers comprising a plurality of grooves, and growing the myoblasts to produce myotubes aligned with the plurality of grooves of the microcarriers.

In another set of embodiments, the method comprises seeding myoblasts on microcarriers formed from a plant-originated material, and growing the myoblasts to produce a plurality of aligned myotubes on the microcarriers.

Additionally, in still another set of embodiments, the method comprises seeding myoblasts on an anisotropic scaffold formed from a plant-originated material, and growing the myoblasts to produce a tissue mass comprising the anisotropic scaffold.

In addition, in one set of embodiments, the method comprises acts of extruding a polymer to produce a microcarrier comprising a plurality of grooves, and seeding myoblasts on the microcarrier.

The method, in yet another set of embodiments, comprises 3D-printing a polymer as a microcarrier comprising a plurality of grooves, and seeding myoblasts on the microcarrier.

In another aspect, the present disclosure encompasses methods of making one or more of the embodiments described herein, for example, clean meat. In still another aspect, the present disclosure encompasses methods of using one or more of the embodiments described herein, for example, clean meat.

Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure when considered in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present disclosure 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 disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure. In the figures:

Fig. 1 is schematic illustrating the formation of myotubes on a grooved microcarrier, in accordance with one embodiment;

Figs. 2A-2C illustrate zein microfibers produced in accordance with certain embodiments; and

Figs. 3A-3B illustrate myotubes grown on microgrooved flakes and 3D-printed substrates, in other embodiments.

DETAILED DESCRIPTION

The present disclosure generally relates, in certain aspects, to clean meat and other cultivated animal-derived products. In some embodiments, muscle or fat cells can be grown on microcarriers or other scaffolds, for example, in a bioreactor or other in vitro cell culture system. These may contain grooves or other structures in some instances. The grooves may be useful, for example, for helping myoblasts to form aligned myotubes. In addition, in some cases, the microcarriers or other scaffolds can be formed from materials such as plant- originated materials, e.g., which may be edible. These may also be present within the final product in certain embodiments. Other embodiments are generally directed to methods of making or using microcarriers or cultivated animal-derived products, kits involving these, or the like.

One aspect of the present disclosure is generally directed to a clean meat product that includes muscle cells that are cultivated, e.g., on microcarriers or other suitable scaffolds. As mentioned, clean meat products are typically produced using in vitro cell culture or bioreactors, as opposed to “regular” meat that is grown and harvested from live animals. Myoblasts can be seeded on the scaffolds and allowed to grow, e.g., in a cell culture system. The myoblasts can fuse together to form myotubes, which are the foundation of muscle fibers and meat in general. However, in cell culture, the myoblasts may not have much existing structure, and the resulting myoblasts are not structured and may instead be randomly oriented. Such random myoblast structures do not adequately form muscle fibers, and are often a poor meat substitute.

However, in certain embodiments as discussed herein, the microcarriers or other scaffolds may have a plurality of grooves or channels, in which cells such as myoblasts are able to orient in and thereby become aligned, e.g., when cultured. See, e.g., Fig. 1. In this way, myotubes generally aligned with the grooves may be formed as the myoblasts fuse together to form the myotubes. It should be understood that the alignment of the myotubes relative to the grooves is not necessarily perfect, but the myotubes may still exhibit a strong preference to the direction of the grooves.

In some cases, for example, for applications such as clean meat, which are intended to be consumed as food, the microcarriers or other scaffolds may be formed from materials that are edible, for example, plant-originated materials such as plant-originated proteins. The microcarriers or other scaffolds may, in some cases, contain grooves such as those described herein, although this is not a requirement. Non-limiting examples of plant-originated proteins that may be used as microcarriers or scaffolds include gliadin, secalin, zein, kafirin, avenin, or others such as those described herein. In addition, in some cases, other materials can be used, e.g., instead of and/or in addition to plant-originated materials, for example, polymers, carbohydrates, sugars, saccharides, etc. However, it should be understood that in other embodiments, other applications for cultivated animal-derived products are possible, including applications that are not intended for food consumption. Thus, in certain embodiments, the scaffolds or microcarriers need not be formed from materials that are edible and/or degradable.

In certain embodiments, cells such as myoblasts may be seeded on the microcarriers or other scaffolds, and grown in a bioreactor or other in vitro cell culture system. For instance, the myoblasts may be grown on the microcarriers and allowed to differentiate or fuse to form aligned myotubes, e.g., while contained within the bioreactor or other system. In contrast, in certain other systems where the myoblasts are not aligned, the cells are often then removed from the microcarrier or scaffold, then reprocessed in order to form clean meat or other cultivated products.

The above discussion is a non-limiting example of one aspect generally directed to microcarriers or other scaffolds containing grooves. These can be used for growing cells such as myoblasts, e.g., to produce aligned myotubes in a clean meat product, or another cultivated animal-derived product. However, other embodiments are also possible besides those discussed above. Accordingly, more generally, various aspects of the invention are directed to various systems and methods for producing clean meat and other cultivated animal-derived products, as discussed herein.

For example, certain aspects are generally directed to cultivated animal-derived products, such as clean meat, or other products. These are produced using cells taken from an animal, but then the cells are cultured in vitro, e.g., using bioreactors, flasks, petri dishes, microwell plates, or other cell culture systems. Many cell culture systems will be known to those of ordinary skill in the art. This is in stark contrast to traditional techniques of sacrificing animals and harvesting meat or other organs (e.g., skin, internal organs, etc.) for food or other uses. Although the original cells seeded to form the product may have originated or otherwise have originally been derived from a living animal, the bulk of the cells forming the actual product were grown or cultured in an in vitro setting, rather than naturally as part a living animal.

A variety of products may be formed from cells cultured in vitro. For instance, in certain embodiments, the products may form “clean meat,” or meat that is intended to be eaten, for example, by humans. It will be appreciated that, because it is to be eaten, such products will often be formed of edible or digestible materials, e.g., materials that can be digested, or degraded to form generally nontoxic materials within the digestive system. For instance, the clean meat may contain animal-derived cells (e.g., derived from chicken, beef, pork, mutton, goat, venison, fish, duck, turkey, shrimp, or other animals that are commonly recognized for widespread human consumption), such as muscle cells, fat cells, or the like.

In addition, in some embodiments, the clean meat product may contain other edible materials, such as plant-originated materials. Non-limiting examples of edible materials include proteins, carbohydrates, sugars, saccharides, etc., as well as polymers formed from these (for example, polylactic acid, polyglycolic acid, cellulose, etc.). In some cases, the edible materials may be digested to form nutrients, e.g., such as amino acids, sugars, etc. that have nutritional value, for example, when taken up into the body. However, in some cases, the edible materials can be digested to form non-nutrients that cannot be absorbed as nutrients, but can be passed through the digestive system without detrimental effects.

In certain embodiments, the plant-originated materials may have relatively high amounts of glutamine and/or proline. For instance, the plant-originated materials may have at least 10 wt%, at least 15 wt%, at least 20 wt%, at least 25 wt%, at least 30 wt%, at least 35 wt%, at least 40 wt%, at least 45 wt%, or at least 50 wt% glutamine, and/or at least 10 wt%, at least 15 wt%, at least 20 wt%, at least 25 wt%, at least 30 wt%, at least 35 wt%, at least 40 wt%, at least 45 wt%, or at least 50 wt% proline. In some cases, the presence of glutamine and/or proline may facilitate the attachment of cells, e.g., to a scaffold. In addition, in some cases, the plant-originated materials may be poorly soluble in water.

In addition, it should be understood that the invention is not limited to only clean meat products. In some cases, products such as those described herein may be cultivated from animal-derived cells, but the product is not necessarily one that is intended to be eaten. For instance, cells from an animal may be cultured to form various organs that can be harvested, such as skin, hair, fur, or the like. Thus, as a non-limiting example, leather, cultivated fur, etc. can be formed by growing cells in culture, for example as discussed herein, without the traditional method of sacrificing animals to harvest their skin or other organs.

In certain aspects, the cultivated animal-derived products may be grown on microcarriers or other types of scaffolds. For example, cells derived from an animal may be seeded onto microcarriers or scaffolds, and grown in vitro , e.g., in a bioreactor or other cell culture systems such as are described herein, to produce a cultivated product. In some cases, the product thus formed can be used without additional processing. As a non-limiting example, a clean meat product may be grown by seeding myoblasts on microcarriers or scaffolds, then grown within a bioreactor to form the clean meat product. The clean meat product may not require subsequent separation or processing steps to convert the cultured cells into a product ready to be cooked or otherwise use as meat. However, it should be understood that in other embodiments, additional steps may be used to convert the cells grown within the bioreactor into a clean meat product, or other cultivated animal-derived product.

As mentioned, the cells seeded on the microcarriers or other types of scaffolds may arise or be derived from any suitable animal. Non-limiting examples of animals typically consumed as food include chicken, cow, pig, sheep, goat, deer, fish, duck, turkey, shrimp, or any other suitable animals. In addition, in some cases, the cells may be cells that are not from an animal intended to be consumed by humans as food. For instance, as discussed herein, in some cases, the cells may be cultured to grow leather or cultivated fur, and may be derived from an appropriate animal type, e.g., mink or racoon. In yet another embodiment, the cells may be cultured to grow a product that is to be implanted in a subject. For instance, the cells may be derived from a human, and the product may be a muscle or other organ to be implanted in a human. In one set of embodiments, the cells are derived from the subject (e.g., a human subject) that will receive the implant; this may be useful, for example, to avoid an immunological reaction with the implanted product. As another non-limiting example, organs, tissues, etc. of endangered animals can be grown in accordance with certain embodiments, for example, tiger liver, rhinoceros hom, etc.

In addition, in some embodiments, other types of cells may be seeded on a microcarrier or other type of scaffolds, e.g., in addition (or instead of) myoblasts. As a non limiting example, adipose cells can be added to produce fat within the final clean meat product. Cells such as these may be derived from the same animal species as the myoblasts, and/or the cells may be derived from different animal species. Accordingly, one or more than one type of cell may be seeded, e.g., sequentially and/or simultaneously, etc. Accordingly, in one set of embodiments, cells are seeded onto scaffolds. A scaffold may define a substrate that the cells are able to divide and proliferate on, e.g., forming tissue that forms the basis of the cultivated animal-derived product. A variety of cell scaffold structures can be used, including scaffolds known by those of ordinary skill in the art. The scaffold may thus have any suitable size or shape. In some cases, the scaffold may be anisotropic, i.e., not exhibiting radial or spherical symmetry. In addition, in certain embodiments, the scaffold may be relatively solid, or have holes or pores. Thus, for example, the scaffold may have any suitable degree of porosity. One or more than one scaffold may be present. For instance, in certain cases, the scaffold comprises a plurality of microcarriers, e.g., as described herein. If more than one scaffold is present, the scaffolds may be independently the same or different. In addition, in certain embodiments, the scaffold may have one or more grooves, e.g., as discussed herein.

In one set of embodiments, a scaffold may have a largest or maximum internal dimension of less than 100 mm, less than 80 mm, less than 70 mm, less than 60 mm, less than 50 mm, less than 40 mm, less than 30 mm, less than 20 mm, less than 10 mm, less than 5 mm, less than 3 mm, less than 2 mm, or less than 1 mm. In addition, in some cases, the microcarriers may have a maximum internal dimension that is at least 1 mm, at least 2 mm, at least 3 mm, at least 5 mm, at least 10 mm, at least 20 mm, at least 30 mm, at least 40 mm, at least 50 mm, at least 60 mm, at least 70 mm, at least 80 mm, at least 90 mm, at least 100 mm, etc.

The scaffold may comprise any suitable material. For example, in one set of embodiments, the scaffold may comprise a material that cells are able to bind. The material may be biocompatible in some instances. In addition, in certain embodiments, the scaffold may comprise a polymer, e.g., one that is biodegradable. For example, in some cases, the scaffold may be one that begins to spontaneously degrade (for example, via hydrolysis reactions, dissolution, etc.) when maintained in contact with water, e.g., for at least 12 hours.

In addition, according to one set of embodiments, the scaffold may comprise an edible material. This may be useful for applications such as clean meat, where the cultivated animal-derived product will be eaten, e.g., by humans or other animals. While almost anything can physically be eaten, materials that are edible include those that are found naturally occurring in foods that are commonly eaten by significant percentages of the general population. Examples of edible materials include, but are not limited to proteins or peptides, polysaccharides, carbohydrates, or the like. In some cases, such materials may be broken down by the digestive system to produce nutrients such as amino acids, monosaccharides, simple sugars, etc. However, in some cases, the edible materials need not be digestible into such nutrients. Specific non-limiting examples of edible materials include cellulose, fibrin, chitin, collagen, soy protein, mycelium, gelatin, alginate, etc. In some cases, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or substantially all of a scaffold is formed from an edible material.

Additionally, in some but not all embodiments, the scaffold may comprise a plant- originated material, such as a plant-originated protein. Such plant-originated materials may be harvested directly from a plant, be grown in vitro (e.g., in cell culture from a culture initially originating in a plant), be synthetically produced (e.g., without using a plant, e.g., chemically produced), etc. Examples of protein-originated material include, but are not limited to, cellulose or certain proteins, such as prolamin, zein, fibrin, gliadin, hordein, secalin, kafirin, avenin, gliadine, 2S albumin, globulin, glutelin, etc. The plant that material originates from may be any plant, including but not limited to food crop plants. Non-limiting examples of plants include, but are not limited to, wheat, barley, rye, com, sorghum, oats, quinoa, hemp, potato, soy, etc.

In one set of embodiments, the scaffold may take the form of one or more microcarriers. The microcarriers may have any shape or size. In some cases, more than one type of microcarrier may be present, e.g., some of which may have various materials, shapes, sizes, etc., such as are described herein. For example, in some embodiments, at least some of the microcarriers may be substantially spherical or exhibit spherical symmetry, although in other embodiments, at least some of the microcarriers may be non-spherically symmetric or may be anisotropic. In addition, in certain cases, at least some of the microcarriers may have a plurality of grooves, e.g., as discussed herein.

In addition, it should be understood that the scaffold is not limited to only edible or degradable materials. In other embodiments, the scaffold may be formed from materials, such as polymers, that are not necessarily edible and/or degradable. Non-limiting examples of such materials include natural polymers such as proteins (e.g., silk, collagen, gelatin, fibrinogen, elastin, keratin, actin, myosin, etc.), polysaccharides (e.g., cellulose, amylose, dextran, chitin, glycosaminoglycans), or the like. Other examples include polymers such as polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid), polyhydroxyalkanoates, polycaprolactones, etc., bioactive ceramics such as hydroxyapatite, tricalcium phosphate, silicates, phosphate glasses, glass-ceramic composites (such as apatite-wollastonite), etc., or the like. In certain embodiments, the microcarriers may have a largest or maximum internal dimension of less than 100 mm, less than 80 mm, less than 70 mm, less than 60 mm, less than 50 mm, less than 40 mm, less than 30 mm, less than 20 mm, less than 10 mm, less than 5 mm, less than 3 mm, less than 2 mm, less than 1 mm, less than 0.5 mm, less than 0.3 mm, less than 0.2 mm, less than 0.1 mm, less than 0.05 mm, less than 0.03 mm, less than 0.02 mm, or less than 0.01 mm. In addition, in some cases, the microcarriers may have a maximum internal dimension that is at least 0.01 mm, at least 0.02 mm, at least 0.03 mm, at least 0.05 mm, at least 0.1 mm, at least 0.2 mm, at least 0.3 mm, at least 0.5 mm, at least 1 mm, at least 2 mm, at least 3 mm, at least 5 mm, at least 10 mm, at least 20 mm, at least 30 mm, at least 40 mm, at least 50 mm, at least 60 mm, at least 70 mm, at least 80 mm, at least 90 mm, at least 100 mm, etc. In addition, in certain cases, combinations of any of these dimensions are also possible. As non-limiting examples, the microcarriers may have a maximum internal dimension of between 10 mm and 30 mm, between 5 mm and 20 mm, between 3 mm and 10 mm, between 50 mm and 70 mm, between 1 mm and 3 mm, etc. In some cases, the maximum internal dimension is the length of longest straight line that can be contained entirely within the microcarrier and/or the interior of the microcarrier (e.g., if the microcarrier defines a hollow sphere).

In some cases, however, some or all of the microcarriers may not necessarily be spherical. For example, at least some of the microcarriers may have shapes such as cubical, rectangular solid, tetrahedral, octahedral, irregular, etc. In some cases, at least some of the microcarriers have a shape that is substantially planar. For instance, the microcarrier may have a generally rectangular shape where the smallest dimension of the rectangular solid is substantially smaller than either of the other two dimensions, for example, by a factor of at least 3, at least 5, or at least 10, etc.

In addition, in certain cases, at least some of the microcarriers have a relatively large surface to volume ratio. This may be important, for example, in embodiments where the microcarriers contain a plurality of grooves, e.g., as discussed herein. In contrast, a perfect sphere would have the smallest possible surface to volume ratio for a given volume of material. As a nonlimiting example, the surface to volume ratio may be at least 100, at least 200, at least 300, etc., e.g., for a sheet thickness of 0.01 mm surface and an area of 1 mm x 10 mm.

In one aspect, at least some of the microcarriers or other types of scaffolds may have one or more grooves defined therein. Without wishing to be bound by any theory, it is believed that grooves may promote cellular alignment during growth on the microcarriers or other types of scaffolds. Thus, for example, myoblasts seeded within grooves on microcarriers may be induced to grow together to form substantially aligned myotubes, e.g., that are substantially parallel to each other. This can result in muscle fibers can be grown on the microcarriers. As this is a biological system, those of ordinary skill in the art would understand that the myotubes will not necessarily grow to be perfectly parallel to a high degree of mathematical precision. Nonetheless, the myotubes may still be readily identified as having substantially parallel myotubes within the cultivated animal-derived product, for example, as opposed to myotubes grown on spherical particles not containing grooves, where the myotubes are formed randomly from the myoblasts. For instance, the myotubes may exhibit a strong preference to the direction of the grooves, e.g., having an average directionality that varies by less than 20°, less than 15°, less than 10°, or less than 5° relative to the direction of the grooves.

Thus, in some embodiments, the grooves may be positioned or sized within the microcarriers or other scaffolds to allow the myoblasts to be directional or aligned, e.g., to allow them to fuse together to become myotubes. One or more grooves may be present. For instance, a microcarrier or other scaffold may have at least 2, at least 3, at least 4, at least 5, at least 7, at least 10, at least 12, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 70, or at least 100 or more grooves defined therein. If more than one type of microcarrier or scaffold is present, they may independently have the same or different numbers of grooves. In some cases, the average number of grooves present within the microcarriers may have the ranges described here. The grooves may be positioned in any orientation on the microcarriers. For instance, the grooves may be substantially parallel to each other, e.g., to promote the formation of substantially aligned myotubes.

In some cases, the grooves may have an average cross-sectional width of less than 500 micrometers, less than 400 micrometers, less than 300 micrometers, less than 200 micrometers, less than 100 micrometers, less than 50 micrometers, less than 30 micrometers, less than 20 micrometers, less than 10 micrometers, less than 5 micrometers, less than 3 micrometers, less than 2 micrometers, less than 1 micrometers, less than 500 nm, less than 300 nm, less than 200 nm, less than 100 nm, less than 50 nm, less than 30 nm, less than 20 nm, less than 10 nm, less than 5 nm, less than 3 nm, less than 2 nm, etc. In addition, in some cases, the grooves may have an average cross-sectional width of at least 1 nm, at least 2 nm, at least 3 nm, at least 5 nm, at least 10 nm, at least 20 nm, at least 30 nm, at least 50 nm, at least 100 nm, at least 200 nm, at least 300 nm, at least 500 nm, at least 1 micrometer, at least 2 micrometers, at least 3 micrometers, at least 5 micrometers, at least 10 micrometers, at least 20 micrometers, at least 30 micrometers, at least 50 micrometers, at least 100 micrometers, at least 200 micrometers, at least 300 micrometers, etc. In addition, combinations of any of these are possible. For instance, in some cases, the groove may have an average cross- sectional width of between 50 and 300 micrometers, between 300 and 500 micrometers, between 500 nm and 2 micrometers, etc.

The grooves may have a substantially square or rectangular profile. Other shapes are also possible in certain cases, e.g., triangular, semicircular, etc. The grooves may also have any aspect ratio (i.e., width to height). For example, the grooves may have an average aspect ratio of at least 1:10, at least 1:8, at least 1:6, at least 1:5, at least 1:4, at least 1:3, at least 1:2, at least 1:1, at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 8:1, at least 10:1, and/or an average aspect ratio of no more than 10:1, no more than 8:1, no more than 6: 1, no more than 5: 1, no more than 4: 1, no more than 3: 1, no more than 2: 1, no more than 1:1, no more than 1:2, no more than 1:3, no more than 1:4, no more than 1:5, no more than 1:6, no more than 1:8, or no more than 1:10.

The microcarriers, or other scaffolds such as described herein, may be formed using any suitable technique. Non-limiting examples include extrusion, electro spinning, 3D- printing, molding, injection molding, or the like. As still other non-limiting examples, cells may be confined on an engineered surface or material having a micro-nanotopography as contact guidance, or by applying mechanical forces generated either by the contractile activity of the cells or by an external strain, e.g., as discussed in U.S. Pat. Apl. Pub. No. 2020/0054793.

In one set of embodiments, materials that will from the scaffold (e.g., plant-originated materials such as plant-originated proteins, polymers, etc.) may be formed into a paste or other mixture that is extruded, e.g., at low temperatures (e.g., temperatures below 20 °C, 15 °C, 10 °C, or 5 °C, etc.) and/or into a water bath to solidify and/or coagulate the materials into microcarriers. The mixture may have, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% material (e.g., a plant-originated material), and/or no more than 80%, no more than 70%, no more than 60%, no more than 50%, no more than 40%, or no more than 30% material, by weight. In some cases, combinations of any of these ranges are also possible, e.g., the mixture may have between 40% and 60% material (e.g., a plant-originated material), between 20% and 80% material, between 30% and 50% material, etc. In some cases, the materials may be dissolved and/or suspended in a suitable liquid, e.g., water, a strong alcohol (e.g., 70% to 80% aqueous solution by volume), an acid solution, an alkaline solution, or the like.

In some embodiments, the microcarriers (or other scaffolds) may be formed to have any of a wide variety of shapes, such as flakes, plates, fibers, whiskers, or the like, e.g., having dimensions such as any of those described herein. In addition, as previously noted, in certain embodiments, some of these shapes may contain grooves.

As a non-limiting example, the microcarriers may have the form of fibers, e.g., having an average length of at least 1 micrometer, at least 2 micrometers, at least 3 micrometers, at least 4 micrometers, at least 5 micrometers, at least 10 micrometers, at least 20 micrometers, at least 30 micrometers, at least 40 micrometers, at least 50 micrometers, at least 100 micrometers, at least 200 micrometers, at least 300 micrometers, at least 400 micrometers, at least 500 micrometers, at least 1 mm, at least 2 mm, at least 3 mm, at least 4 mm, at least 5 mm, at least 1 cm, at least 2 cm, at least 5 cm, at least 10 cm, at least 20 cm, at least 30 cm, at least 50 cm, etc. In addition, in certain embodiments, the fibers may have an average length of no more than 100 cm, no more than 50 cm, no more than 30 cm, no more than 20 cm, no more than 10 cm, no more than 5 cm, no more than 4 cm, no more than 3 cm, no more than 2 cm, no more than 1 cm, no more than 5 mm, no more than 4 mm, no more than 3 mm, no more than 2 mm, no more than 1 mm, no more than 500 micrometers, no more than 400 micrometers, no more than 300 micrometers, no more than 200 micrometers, no more than 100 micrometers, etc. Combinations of any of these are also possible, e.g., the fibers may have an average length of between 200 micrometers and 500 micrometers, between 500 micrometers and 5 mm, between 300 micrometers and 1 mm, between 10 micrometers and 400 micrometers, etc.

In some cases, the microcarriers (or other scaffold) may be purified, e.g., by extracting impurities prior to use, e.g., prior to seeding with cells. For example, impurities such as citric acid or ethanol may interfere with cell culture, and/or may interfere with the taste of the clean meat product. Thus, in some cases, such microcarriers or other scaffolds may be exposed to water, e.g., washed, to remove potential contaminants.

In addition, in certain embodiments, the microcarriers or other scaffolds may be sterilized before use, e.g., prior to seeding with cells. A variety of techniques for sterilizing the microcarriers can be used, including but not limited to, applying ultraviolet light or high temperatures (e.g., a temperature of at least 100 °C) to the microcarriers. Those of ordinary skill in the art will be aware of various sterilization techniques that may be used.

As mentioned, in some aspects, the cultivated animal-derived product may be produced within a bioreactor or other cell culture system. A wide variety of bioreactors can be used in various embodiments including, but not limited to, suspension bioreactors, continuous stirred-tank bioreactors, rocker bioreactors, airlift bioreactors, fixed bed bioreactors, bubble column bioreactors, fluidized bed bioreactors, packed bed bioreactors, or the like.

In some cases, cells may be seeded on microcarriers or other or other scaffolds, then introduced into the bioreactor or other cell culture system. Those of ordinary skill in the art will be familiar with techniques for seeding cells on a scaffold. For instance, the scaffold may be exposed to a suspension containing animal-derived cells, which are allowed to settle from the suspension onto the scaffold. In some cases, more than one type of cell may be present in suspension and allowed to settle.

In some cases, a product can be formed within the bioreactor without additional processing, for example, without separating the cells or tissues grown within the bioreactor. However, in other case, some separation and/or processing of the cells may still be used. As a non-limiting example, myotubes may be grown within a bioreactor or other cell culture system such as those described herein, and such myotubes may be processed, e.g., by adding adipose cells (fat) to produce a cultivated animal-derived product having any desired ratio of muscle to fat in it. For instance, the ratio of muscle to fat may be at least 95: 1, at least 90:1, at least 70:1, at least 50:1, at least 30:1, at least 20:1, at least 10:1, at least 5:1, at least 1:1, etc. by weight.

A variety of techniques may be used to grow cells within the bioreactor or other cell culture system. For instance, the cells may be grown at body temperature (e.g., about 38.5 °C for cow cells, about 41 °C for chicken cells, about 39-40 °C for pig cells, about 40-42 °C for duck cells, etc.).

In some embodiments, cells within the bioreactor or other cell culture system may be induced to differentiate, e.g., by adding suitable factors and/or altering the cell culture conditions therein. As a non-limiting example, myoblasts may be grown in serum, while removing or reducing the serum from the myoblasts may cause the myoblasts to differentiate to from myotubes. For instance, in one set of embodiments, the serum may be reduced from 10% to 2% to induce differentiation of myoblasts.

The cells may be grown within the bioreactor or other cell culture system for any suitable length of time, e.g., to produce a cultivated product. For example, the cells may be grown for at least 3 days, at least 5 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, etc.

In some cases, the scaffold or microcarrier containing cells may be usable with the scaffold or microcarrier in place. For example, the cells and scaffold or microcarrier together may form a clean meat product (for example, if the scaffold or microcarrier is edible and/or degradable), or other cultivated animal-derived product. However, in some embodiments, the cells may be separated from the scaffold or microcarrier. For example, the scaffold or microcarrier may be removed and reused or discarded, while the cells may be used without the scaffold present. Thus, for example, if the cells are used in a clean meat product or other cultivated animal-derived product, the scaffold or microcarrier may not necessarily be edible and/or degradable.

The following are each incorporated herein by reference in their entireties: US Provisional Patent Application Serial No. 63/159,403, filed March 10, 2021, entitled “Constructs for Meat Cultivation and Other Applications”; US Provisional Patent Application Serial No. 63/279,617, filed November 15, 2021, entitled “Constructs Comprising Fibrin or Other Blood Products for Meat Cultivation and Other Applications”; US Provisional Patent Application Serial No. 63/279,631, filed November 15, 2021, entitled, “Methods and Systems of Preparing Cultivated Meat from Blood or Cellular Biomass”; US Provisional Patent Application Serial No. 63/279,642, filed November 15, 2021, entitled, “Systems and Methods of Producing Fat Tissue for Cell-Based Meat Products”; US Provisional Patent Application Serial No. 63/279,644, filed November 15, 2021, entitled “Production of Heme for Cell- Based Meat Products”; US Provisional Patent Application Serial No. US 63/300,577, filed January 18, 2022, entitled “Animal-Derived Antimicrobial Systems and Methods”; US Provisional Patent Application Serial No. 63/164,397, filed March 22, 2021, entitled “Growth Factor for Laboratory Grown Meat”; US Provisional Patent Application Serial No. 63/164,387, filed March 22, 2021, entitled, “Methods of Producing Animal Derived Products”; US Provisional Patent Application Serial No. 63/314,171, filed February 25, 2022, entitled “Growth Factors for Laboratory Grown Meat and Other Applications”; and US Provisional Patent Application Serial No. 63/314,191, filed February 25, 2022, entitled “Methods and Systems of Producing Products Such as Animal Derived Products.”

The following examples are intended to illustrate certain embodiments of the present disclosure, but do not exemplify the full scope of the disclosure.

EXAMPLE 1

This example illustrates a cultivation technique for primary myoblasts from farm animals such as cows, lamb, chickens, fish, etc., in accordance with one embodiment. In some cases, edible microcarriers having various shapes such as fibers, flakes, whiskers, etc., may be seeded with myoblasts, which are allowed to proliferate and differentiate to produce aligned myotubes. This process may be scalable, e.g., using suspension bioreactors. The microcarriers may be formed, for example, using various non water-soluble plant-originated proteins.

Examples of such plant-based proteins include, but are not limited to, beans and peas such as soy bean, grains and seed proteins like as quinoa, hemp protein, potato protein, wheat protein (e.g., gliadine, albumin, globulin, etc.) or the like. Other examples include prolamin- rich proteins found in plants, e.g., in the seeds of cereal grains such as wheat (gliadin), barley (hordein), rye (secalin), corn (zein), sorghum (kafirin), oats (avenin), etc. In some cases, such proteins may have a high glutamine and proline content, and/or have poor solubility in water. In addition, in some cases, such proteins may be solubilized in strong alcohol (e.g., 70-80%), light acid, and/or alkaline solutions.

This example illustrates the application of such proteins in the development of cell culture microcarriers for mass cultivation of cells and induction of alignment to myoblasts for the production of high quality myotubes. Zein protein was used as an example microcarrier material to promote myoblast adhesion, alignment and differentiation to myotubes. Cell culture in suspension may ensure scalability of this approach, which can be important for the mass production of cell-based meat. In addition, microcarriers having shapes such as microfibers and/or microwhiskers can be used in some cases for the direct production highly aligned fibrillar structure of meat, for example, using extrusion techniques that may allow the emulation of meat fibrillar structure and aligned myotubes.

In one embodiment, zein (obtained as a powder) at concentration of 50% w/v (or a range of between 20-80% w/v) is dissolved mixture of in citric acid (8% w/v, or a range of 2- 10% w/v) in ethanol (e.g., 70% v/v in water) at pH 5. After dispersion and dissolution, the mixture was allowed to rest for 48 hours (or a range of between 36-60 hours) at ambient temperatures (e.g., 18-23 °C). During this time, the viscosity of the mixture was observed to increase to form a paste-like viscous material. This paste was easily processed with different techniques to create desire shapes and forms for the microcarriers. For example, the zein paste could be extruded into a water bath, e.g., preferably at low temperatures (for example, 2-8 °C), to solidify and coagulate the protein into microfibers (or other microcarriers). The head of extruder could also be easily tuned to produce a variety of desired diameters or other characteristics of the microfibers. Thus, one can induce the formation of microwhiskers having microgrooved surfaces by application of suitable extrusion mold as illustrated in Fig.

1. This figure shows an example process for inducing cell seeding proliferation and differentiation in a suspension bioreactor on edible plant-based protein microcarriers such as microgrooved micro- whiskers.

The final product in this example was a long fiber that could be broken to small pieces, e.g., by harsh agitation or other suitable techniques. The zein microfibers were observed to be more dense than water, and precipitated upon stopping the stirring collected from the water container by removing the supernatant or filtration (Fig. 2A). To make the zein microcarrier suitable for cell culturing, several washing steps were used to remove water-soluble additives such as excessive citric acid, ethanol, and/or other impurities in the zein. Such impurities, if allowed to remain, may alter the taste, and/or may be toxic to cells, for example, at relatively high concentrations. In some cases, the microcarrier was also sterilized using ultraviolet light or by autoclave to avoid or reduce contamination of the cell culturing media.

To culture myoblasts on zein microfiber, the fiber was initially be treated with a culture medium overnight. Then, the fiber was transferred to non-adhesive containers to avoid unwanted cell attachment to the undesired surfaces. After adding cell suspension (e.g., primary lamb myoblasts), after half an hour, the cells began to attach to the fibers. In some cases, the fibers could be mixed and/or addition cells added and allowed to settle, e.g., for another half an hour. 4 hours after cell seeding process, the zein microcarriers were moved to the bioreactor for cell proliferation phase. It was observed that the microfibers remained suspended with slight shaking in the bioreactor vessels. Minimal shaking or agitation may be important for cultivation of certain types of cells that are sensitive to shear stress in certain cases, such as myoblasts or stem cells.

After 7 days of culture, the microfibers were collected and the presence of cells checked using microscopy. In addition, the presence of cells on microfibers was confirmed by a metabolic activity assay (an MTT assay) as compared with zein without cells.

Fig. 2 illustrates the results of these experiments. Fig. 2A shows photomicrographs of zein microfibers that were collected at the bottom of the reactor. Fig. 2B shows zein microcarriers after 7 days of cell seeing, showing the formation of cell layers on top of the fibers. Fig. 2C shows that the presence of cells on microfibers was confirmed by several samples that were taken from the bioreactor, when compared to a sample without initial cell seeding.

EXAMPLE 2

This example tested zein paste for 3D printing applications as well as molding of microgrooved flakes or sheet of zein. Microgrooved sheets could be used to obtain microflakes, which may be useful in suspension bioreactors. 3D printing is a versatile method to produce a variety of geometries, including scaffolding or microcarriers.

In some experiments, extrusion heads as small as 100 micrometers were used.

Patterns of zein were 3D-printed, then moved to a water bath for coagulation and stabilization of the pattern. Some structures were printed under water. This caused the immediate coagulation of the extruded pattern, which may be suitable for some applications.

In another experiment, a microgrooved zein sheet was made by compression molding of zein paste. After molding, the zein paste was allowed to dry and demolded. A microgrooved sheet could also be ground to produce microflakes. The results of these experiments are shown in Fig. 3A (microgrooved flakes) and Fig. 3B (3D-printed).

While several embodiments of the present disclosure have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present disclosure. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present disclosure is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the disclosure may be practiced otherwise than as specifically described and claimed. The present disclosure is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present disclosure.

In cases where the present specification and a document incorporated by reference include conflicting and/or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and/or inconsistent disclosure with respect to each other, then the document having the later effective date shall control. All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”

As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one,

B (and optionally including other elements); etc.

When the word “about” is used herein in reference to a number, it should be understood that still another embodiment of the disclosure includes that number not modified by the presence of the word “about.”

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. In the claims, as well as in the specification above, all transitional phrases such as

“comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.