RELATED APPLICATIONS

This application claims the benefit of 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.” Each of these is incorporated herein by reference in its entirety.

FIELD

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

BACKGROUND

Cultivated meat, or cell-based 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, or 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 for applications such as cultivated meat, and thus improvements are needed.

SUMMARY

The present disclosure generally relates to cultivated 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, to produce cultivated meat, myoblasts may be caused to proliferate and/or fuse, and in some cases, form a higher order construct, myotubes. Certain embodiments, for instance, are directed to strategies for microcarriers that may allow proliferation and/or differentiation of myoblasts to myotubes, e.g., in suspension bioreactors and/or in other reactors including those described herein.

In some embodiments, a product such as a cultivated meat product may include non human animal cells, for example, muscle cells. For instance, in one embodiment, myoblasts cultured on microcarriers may form a muscle replica. In some embodiments, the muscle replica may be a cultivated meat product. In addition, in certain embodiments, a cultivated meat product may comprise other materials, in addition to or instead of non-human animal cells.

As a non-limiting example, in some cases, a fat replica may be present in the product. In one embodiment, the fat replica comprises a fat emulsion. The fat replica may also comprise a non-human blood plasma. In some embodiments, a component of the non-human blood plasma, such as fibrin, may be crosslinked. In some cases, the non-human blood plasma may be clotted. In some embodiments, the fat replica comprises fat or adipose cells present on microcarriers.

In some embodiments, heme may be present in the product. In certain embodiments, heme may be obtained from hemoglobin. In some embodiments, hemoglobin may be obtained from a lysate of non-human red blood cells. In certain embodiments, hemoglobin may be obtained from plants. Hemoglobin may also be obtained from a genetically modified organism in some cases. In still other embodiments, the hemoglobin may be chemically synthesized.

Certain embodiments are directed toward the use of a non-human plasma (or serum) as a source of fibrinogen for preparing fibrin.

In some embodiments, the cultivated meat may be grown in a bioreactor comprising a hat is, at least partially, comprised of blood products. The blood products may be harvested from a human or non-human and may comprise components such as platelet rich plasma (PRP), platelet poor plasma (referred to as plasma), a platelet concentrate, a lysate of red blood cells, a platelet lysate (PL), growth factors, proteins, cytokines, or the like. In another embodiment, the non-human blood plasma may be used as a nutrient source in a bioreactor. In certain embodiments, the serum is fetal bovine serum. In some cases, the blood may be obtained from commercial vendors. However, in some embodiments, the non-human blood plasma may be obtained from living animal donors.

One aspect is generally drawn to a cultivated meat product. According to one set of embodiments, the cultivated meat product comprises a tissue mass of at least 10 g. In some embodiments, the cultivated meat product may also comprise a fat replica and a muscle replica. In some cases, the fat replica and the muscle replica are present in a mass ratio of between 1:99 to 50:50. In certain embodiments, the fat replica comprises a fat emulsion and a hydrogel comprising crosslinked non-human blood plasma. In some instances, the muscle replica comprises microcarriers comprising fibrin and non-human muscle cells.

In another set of embodiments, the cultivated meat product comprises a ground beef replica, having a mass of at least 10 g. In some embodiments, the cultivated meat product comprises a fat replica and a muscle replica. In certain cases, the fat replica comprises a fat emulsion and a hydrogel comprising crosslinked non-human blood plasma. In some instances, the muscle replica comprises microcarriers comprising fibrin and non-human muscle cells.

The cultivated meat product, in another set of embodiments, comprises a tissue mass of at least 10 g, comprising a fat replica, a muscle replica, non-human blood plasma and a non-human red blood cell lysate. In some embodiments, the fat replica comprises a fat emulsion and a hydrogel comprising crosslinked non-human blood plasma. In certain cases, the muscle replica comprises microcarriers comprising fibrin and non-human muscle cells.

The cultivated meat product, in still another set of embodiments, comprises a tissue mass of at least 10 g, comprising microcarriers comprising fibrin, non-human cells, a fat emulsion, non-human blood plasma, and a lysate of non-human red blood cells.

In yet another set of embodiments, the cultivated meat product comprises a tissue mass of at least 10 g. In some cases, the cultivated meat product comprises a mixture of microcarriers comprising fibrin, non-human muscle cells, a fat replica comprising a fat emulsion and non-human blood plasma, and a lysate of non-human red blood cells. In still another set of embodiments, the cultivated meat product comprises a tissue mass of at least 10 g, comprising between 0.1 to 10% wt% non-human red blood cell lysate, and an emulsion of fat and non-human blood plasma.

The cultivated meat product, according to another embodiment, comprises microcarriers comprising fibrin, and a fat replica comprising a fat emulsion and non-human blood plasma.

Another aspect is generally directed to a method. In one set of embodiments, the method comprises mixing a fat replica and a muscle replica in a mass ratio of between 1:99 and 50:50 to form a tissue mass of at least 10 g. In some embodiments, the fat replica comprises a fat emulsion and a hydrogel comprising crosslinked non-human blood plasma.

In certain cases, the muscle replica comprises microcarriers comprising fibrin and non-human muscle cells.

In another set of embodiments, the method comprises culturing non-human muscle cells in a bioreactor, and mixing the cultured cells and an emulsion of fat and non-human blood plasma and/or a lysate of non-human red blood cells to produce a tissue mass of at least 25 g.

The method, in still another set of embodiments, comprises culturing non-human cells on microcarriers comprising fibrin to produce cultured cells, and mixing the cultured cells and a fat replica comprising a fat emulsion and non-human blood plasma and/or a lysate of non-human red blood cells to produce a tissue mass of at least 10 g.

In yet another set of embodiments, the method comprises withdrawing a blood sample from a non-human animal, processing the blood sample to produce a non-human blood serum and non-human red blood cells, and using the non-human blood serum and non-human blood cells to produce a cultivated meat product.

In another aspect, the present disclosure encompasses methods of making one or more of the embodiments described herein, for example, cultivated meat. In still another aspect, the present disclosure encompasses methods of using one or more of the embodiments described herein, for example, cultivated 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 nee 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 illustrates a process of milling fibrin hydrogels to form fibrin microcarriers, in accordance with one embodiment;

Fig. 2A illustrates optical images of the fibrin microcarriers following milling for 40 seconds, 80 seconds, or 110 seconds, in other embodiments;

Fig. 2B illustrates the average diameter of fibrin microcarriers following milling for 40 seconds, 80 seconds, or 110 seconds, according to yet other embodiments;

Fig. 3A illustrates the cell viability of myoblasts encapsulated in fibrin microcarriers, post milling, following 3 days, 5 days, and 7 days of cell culture, using calcein-AM as a fluorescent biomarker for cell viability, in accordance with yet another embodiment;

Fig. 3B illustrates the quantification of the fluorescent signal from Fig. 3A using standard imaging techniques and normalized to the two dimensional projection of the microcarrier surfaces , in another embodiment;

Fig. 4 illustrates the concentration of myoblast cells as a function of time for cells cultured on the surface of milled fibrin microcarriers, in still another embodiment;

Fig. 5 illustrates the concentration of myoblast cells as a function of time for cells cultured on fibrin microcarriers fabricated under different conditions, using non-human blood plasma prepared at 1.1 mg/mL, 4.4 mg/mL, or 8.8 mg/mL, in still another embodiment;

Fig. 6 illustrates a scanning electron micrograph of fibrin microfibers fabricated by electro spinning solutions of non-human blood plasma, in yet another embodiment;

Figs. 7A-7B illustrate the cell viability of primary bovine satellite cells seeded on the surface of fibrin microfibers fabricated by electro spinning, according to still another embodiment;

Fig. 8 illustrates a process of forming a fat replica using fibrin hydrogels in accordance with one embodiment;

Fig. 9 illustrates an optical image of a fat replica following staining with Oil-O’ Red to highlight the fat component of a tissue construct, in another embodiment;

Fig. 10 illustrates a chylomicron, in accordance with yet another embodiment;

Fig. 11 Aillustrates a photomicrograph of a fat replica comprising alginate and ardance with one embodiment; Fig. 1 IB illustrates a photomicrograph of a fat replica comprising alginate, croconate oil, and sorbitan 80 in accordance with one embodiment; and

Fig. 11C illustrates a photomicrograph of a fat replica comprising alginate, sunflower oil, and sorbitan 80 in accordance with one embodiment..

Fig. 12 illustrates a process of separating whole blood to obtain packed non-human red blood cells and blood plasma, in accordance with one embodiment;

Fig. 13 illustrates the use of osmotic imbalance to lyse red blood cells to prepare a solution of lysate of non-human red blood cells, in accordance with another embodiment;

Fig. 14 illustrates the use of a sonicator (or other acoustic energy) to lyse red blood cells to prepare a solution of lysate of non-human red blood cells, in accordance with some embodiments;

Fig. 15 illustrates the use of freeze-thawing to lyse red blood cells to prepare a solution of lysate of non-human red blood cells, according to still another embodiment;

Fig. 16 illustrates the stiff constant of samples of cultivated meat as compared to native meat, in one embodiment;

Figs. 17A-17B illustrate color profiles and samples of various cultivated meat and native meat products, in yet another embodiment;

Fig 18 illustrates the stiffness constant of several commercially available ground meat products, in some embodiments;

Figs. 19A-19B illustrate the change in stiffness in cell-based meats compared to native ground beef meat products before and after cooking, in some embodiments;

Figs. 20A-20B illustrate the change in shrinkage of cell-based meats compared to native ground beef patties before and after cooking, in some embodiments;

Figs. 21A-21B illustrate the weight of cell-based meat products compared to native ground beef patties before and after cooking; in accordance with some embodiments;

Figs. 22A-22B illustrate the changes in color and volume of native meat products relative to cell-based meat products, in some embodiments;

Figs. 23A-23B illustrate the color profile for cell-based meat products and ground beef meat products before cooking, in some embodiments; and

Figs. 24A-24B illustrate the color profile for cell-based meat products and ground beef meat products after cooking, in some embodiments.

DETAILED DESCRIPTION

The present disclosure generally relates, in certain aspects, to cultivated meat and nal-derived products. In some embodiments, a cultivated meat product comprises a fat replica, muscle replica, and a cell lysate, e.g., forming a tissue mass. The muscle replica may comprise microcarriers and non-human muscle cells. The fat replica may comprise a fat emulsion and non-human blood plasma. The lysate may comprise a lysate of non-human red blood cells. In some cases, these may form a cultivated meat product, for example, a ground beef replica. Other embodiments are generally directed to methods of making or using such fat replicas, the microcarriers, or the cultivated meat products, kits involving these, or the like.

Cultivated meat is often described using terms such as cultivated meat, cultured meat, tissue mass, cellular (or cell-based) meat, slaughter-free meat, and synthetic meat, among other related terms. Cultivated 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.

In some embodiments, a cultivated meat product may comprise a muscle replica, a fat replica, and a cell lysate. Some or all of these may contain cells, e.g., from the same or different types of animals. For instance, the cells may all arise from cow, pig, chicken, etc., and/or the cells may arise from more than one type of animal. These may be formed into a tissue mass of, for example, at least 10 g, as a cultivated meat product. For example, the cultivated meat product may be a ground beef replica. The ratios of the muscle replica, the fat replica, and the cell lysate may be controlled, for example, to produce a cultivated meat product with a desirable appearance and/or taste. For instance, these ratios may be controlled such that the cultivated meat product resembles ground beef, or any of a number of other meats, including those described herein.

Certain embodiments are generally directed to a muscle replica. The muscle replica may comprise, for example, non-human muscle cells, which may in some case be present on microcarriers or scaffolds. Other non-limiting examples of muscle replicas may be found in a patent application entitled “Constructs Comprising Fibrin or Other Blood Products for Meat Cultivation and Other Applications,” filed on November 15, 2021, US Pat. Apl. Ser. No. 63/279,617, incorporated herein by reference in its entirety. In addition, in some cases, the microcarriers or scaffolds may have structures, such as grooves, that may allow the cells such as myoblasts to become aligned in a specific direction, although this is not a requirement. Such structures are described in U.S. Ser. No. 63/159,403, filed March 10, 2021, entitled “Constructs for Meat Cultivation and Other Applications,” by Khademhosseini, el ah, incorporated herein by reference in its entirety. In some embodiments, the microcarriers or scaffolds may comprise any material that forms an edible hydrogel, such as fibrin. For example, in one embodiment, a microcarrier may be formed from a non-human blood plasma which contains plasma-rich fibrinogen that can be crosslinked or otherwise processed to form a fibrin hydrogel. Such crosslinking can be achieved by exposure to thrombin, calcium, or other conditions such as those described herein. In some embodiments, fibrin hydrogels are formed using non-human blood plasma containing fibrinogen, e.g., at least 10 wt%, or more in some cases.

Certain embodiments are also generally directed to a fat replica. In some cases, the fat replica may comprise fat and a hydrogel. The fat may arise from any suitable source, or more than one source in some instances. For example, the fat may arise from animal cells and/or plants. Examples of plant-based fats include vegetable oil, corn oil, and other oils such as those described herein. Animal-based fats may, in some embodiments, be formed from fat (or adipose) cells that are cultivated, for example, on microcarriers, or other suitable scaffolds. The microcarriers or other scaffolds may comprise fibrin and/or other suitable materials such as those discussed below. In some cases, the fat cells may be grown in a reactor, such as those described herein. As yet another example, the fat may include fat that was synthetically prepared. Other non-limiting examples of fat replicas may be found in a patent application entitled “Systems and Methods of Producing Fat Tissue for Cell-Based Meat Products,” filed on November 15, 2021, US Pat. Apl. Ser. No. 63/279,642, incorporated herein by reference in its entirety.

In one set of embodiments, the fat in the fat replica may be present in an emulsion.

An emulsion of fat may be prepared, for example, by emulsifying fat with non-human blood plasma. In some cases, the fat may be caused to form a fat emulsion by mixing the fat with non-human blood plasma. Without wishing to be bound by any theory, it is believed that the plasma has components that can emulsify fat to form fat particles such as chylomicrons. For instance, the plasma may include proteins or surfactants that can from such fat particles.

The non-human blood plasma may be treated in some embodiments to form a fat replica. For example, fibrin within the plasma may be caused to clot and/or by causing the fibrin to crosslink, e.g., by exposing it to thrombin, calcium, or other clotting agents such as those described herein. In addition, in some embodiments, a fat replica may comprise a fat emulsion contained within a hydrogel. The hydrogel may be formed from non-human blood plasma, e.g., as discussed, and/or another component. Non-limiting examples of such hydrogels include alginate, gelatin, or others such as those described herein. Certain embodiments are also generally directed to a cell lysate. In some cases, the cell lysate may contain heme, which may be used as a colorant in the cultivated meat product. In one embodiment, the heme may be obtained from non-human red blood cells. For example, red blood cells may be lysed, e.g., by exposing the cells to hypoosmotic or distilled water to form a lysate of non-human red blood cells. Red blood cells contain hemoglobin, a structurally similar protein to myoglobin. Hemoglobin also contains a heme moiety. Lysing the red blood cells may release hemoglobin, e.g., into solution. Furthermore, in addition to these, other methods of lysing red blood cells can be used, including those discussed in more detail herein. Other non-limiting examples of lysates of non-human red blood cells may be found in a patent application entitled “Production of Heme for Cell-Based Meat Products,” filed on November 15, 2021, US Pat. Apl. Ser. No. 63/279,644, incorporated herein by reference in its entirety

Some embodiments are also directed toward repeated blood collection from non human animals, for example, to obtain blood, blood serum, blood plasma, red blood cells, or other blood products, etc. For instance, in some embodiments, blood may be withdrawn from the animal at spaced intervals, so as to allow the animal time to recover and produce new blood. For instance, blood may be withdrawn from the animal every 2 weeks, every 4 weeks, every 6 weeks, every 2 months, or the like. The blood draws may each be processed, for example, as discussed herein. For example, the blood may be used to form a cultivated meat product. In this way, blood can be obtained in certain embodiments in a sustainable and cost- effective manner, without killing the animal. This usage may result, in certain embodiments, in the reduction in carbon emissions, water use, land use, etc.

Certain structures and methods described herein can be useful, for example, by providing meat and other animal derived products for human consumption. Certain embodiments of structures and methods described herein may offer certain advantages as compared to existing agriculture-based methods of meat production, for example, by significantly reducing the number of animals bred for slaughter, thus decreasing the number of foodborne illnesses, diet related diseases, and the incidence of antibiotic resistance and infectious disease (e.g., zoonotic diseases such as Nipah vims and influenza A). In some cases, reducing the number of livestock worldwide may also have an effect on the environmental risks associated with agricultural farming due to, for example, ammonia emissions which contribute significantly to acid rain and acidification of ecosystems. In addition, in some instances, livestock, such as pigs and cows are a major agricultural source ; worldwide. In some embodiments, the structures and methods described herein may allow meat and other animal-derived products to be produced or cultivated in vitro, e.g., using blood and tissue donations obtained from living livestock donors (e.g., not intended for slaughter for human consumption). As a non-limiting example, certain embodiments as described herein are generally directed to a product comprising a muscle replica, a fat replica, and a lysate of red blood cell.

The above discussion is a non-limiting example of one aspect generally directed to a cultivated meat product. However, other embodiments are also possible besides those discussed above. Accordingly, more generally, various aspects are directed to various systems and methods for producing cultivated meat and other cultivated animal-derived products, as discussed herein.

In one aspect, a cultivated meat product may comprise a muscle replica, a fat replica, and a cell lysate. These may comprise non-human animal cells, and may independently arise from the same or different types of animals. For example, a beef substitute may comprise a muscle replica comprising cells originating from cow, a fat replica comprising cells originating from cow, and a cell lysate comprising cells originating from cow. Other non limiting examples include chicken, pig, sheep, goat, deer, fish, duck, turkey, shrimp, or other animals that are commonly recognized for widespread human consumption. However, it should be understood that in other embodiments, more than one animal type may be present in the cultivated meat product.

The muscle replica, a fat replica, and a cell lysate may be present in any suitable amounts within the cultivated meat product. For example, the muscle replica may be present at at least 10 wt%, at least 20 wt%, at least 30 wt%, at least 40 wt%, at least 50 wt%, at least 60 wt%, at least 70 wt%, at least 80 wt%, at least 90 wt%, etc., and/or no more than 95 wt%, no more than 90 wt%, no more than 80 wt%, no more than 70 wt%, no more than 60 wt%, no more than 50 wt%, no more than 40 wt%, no more than 30 wt%, no more than 20 wt%, no more than 10 wt%, etc. Similarly, the fat replica may be present at at least 10 wt%, at least 20 wt%, at least 30 wt%, at least 40 wt%, at least 50 wt%, at least 60 wt%, at least 70 wt%, at least 80 wt%, at least 90 wt%, etc., and/or no more than 95 wt%, no more than 90 wt%, no more than 80 wt%, no more than 70 wt%, no more than 60 wt%, no more than 50 wt%, no more than 40 wt%, no more than 30 wt%, no more than 20 wt%, no more than 10 wt%, etc.

The cell lysate may also be present at at least 10 wt%, at least 20 wt%, at least 30 wt%, at least 40 wt%, at least 50 wt%, at least 60 wt%, at least 70 wt%, at least 80 wt%, at least 90 wt%, etc., and/or no more than 95 wt%, no more than 90 wt%, no more than 80 wt%, no more than 70 wt%, no more than 60 wt%, no more than 50 wt%, no more than 40 wt%, no more than 30 wt%, no more than 20 wt%, no more than 10 wt%, etc.

In some cases, the muscle replica, a fat replica, and a cell lysate may be present at certain concentrations that allow the cultivated meat product to mimic an actual meat. For example, cultivated ground beef products may contain anywhere from 5% to 50% fat and from 50% to 95% lean muscle.

In some cases, the mimicry may be such that, if compared in “blind” taste tests, an average person would find it difficult to distinguish a cultivated meat product from the naturally occurring product. For example, if a sample of ground beef is compared to a cultivated meat ground beef by being given to a panel of randomly- selected people, at least 50% of the people being tested would be unable to correctly identify which is which, after tasting each.

Some embodiments, as described herein, are generally directed to microcarriers or scaffolds comprising fibrin. Fibrin is an edible fibrous protein involved in the clotting of blood. It can be formed, for example, by the action of the protease inhibitor thrombin on fibrinogen, which causes it to polymerize and form a clot. Fibrin can be used as a passive scaffolding material in some embodiments. However, in some embodiments, fibrin can specifically bind certain growth factors in the cell culture media that promote cell adhesion, proliferation, and migration. Non-limiting examples include fibronectin, hyaluronic acid, von Willebrand factor, or the like.

In certain embodiments, microcarriers or scaffolds such as those discussed herein may be treated to facilitate binding of cells, such as myoblasts. For example, the microcarriers or scaffolds may be exposed to non-human serum, which may include growth factors that bind to the microcarriers or scaffolds. The growth factors may, for example, promote cell adhesion, proliferation, and/or migration of cells into the microcarriers or scaffolds. In addition, in some cases, the microcarriers or scaffolds may have structures, such as grooves, that may allow the cells such as myoblasts to become aligned in a specific direction, although this is not a requirement. Such structures are described in U.S. Ser. No. 63/159,403, filed March 10, 2021, entitled “Constructs for Meat Cultivation and Other Applications,” by Khademhosseini, el ah, incorporated herein by reference in its entirety.

In some embodiments, the microcarriers or scaffolds may comprise any material that forms an edible hydrogel, such as fibrin. For example, in one embodiment, a microcarrier may be formed from a non-human blood plasma which contains plasma-rich fibrinogen that >r otherwise processed to form a fibrin hydrogel. Such crosslinking can be achieved by exposure to thrombin, calcium, or other conditions such as those described herein. In some embodiments, fibrin hydrogels are formed using non-human blood plasma containing fibrinogen, e.g., at least 10 wt%, or more in some cases.

In certain embodiments, non-human 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, myoblasts may be grown on microcarriers and, in some embodiments, allowed to differentiate or fuse to form aligned myotubes, e.g., within a bioreactor or other system.

For example, certain aspects are generally directed to cultivated animal-derived products, such as cultivated meat, or other products. These may be produced, for example, 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 their 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 “cultivated 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 cultivated meat may contain animal-derived cells (e.g., derived from a chicken, a cow, a pig, a sheep, a goat, a deer, a fish, a duck, a turkey, a shrimp, or other animals that are commonly recognized for widespread human consumption), such as muscle cells, fat cells, or the like. The cells may be wild-type or naturally-occurring cells (e.g., harvested from an animal), although in some embodiments, the cells may include genetically engineered cells, e.g., engineered in a way to increase proliferation. In addition, in some embodiments, the cultivated meat product may contain other edible materials, such as plant- originated materials. Non-limiting examples of edible materials include proteins, carbohydrates, sugars, saccharides, plant-based fats, etc., as well as polymers formed from these (for example, polylactic acid, polyglycolic acid, cellulose, etc.). In some cases, the y 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 cannot be digested, and/or 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 addition, it should be understood that the invention is not limited to only cultivated 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 embodiments, the cultivated meat products may be grown on microcarriers or other types of scaffolds, which may comprise fibrin in some embodiments. 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 meat product (or other cultivated animal-derived product). In some cases, the product thus formed can be used without additional processing. As a non-limiting example, a cultivated meat product may be grown by seeding myoblasts on microcarriers or scaffolds, then grown within a bioreactor to form a muscle replica or a cultivated meat product, etc. The cultivated meat product may not require subsequent separation or processing steps to convert the cultured cells into a product ready to be cooked or otherwise be used, e.g., as meat. However, it should be understood that in other embodiments, additional steps may be used to convert the muscle replica grown within the bioreactor into a cultivated 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 n 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 cultivated 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 meat 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 one or 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. Combinations of any of these dimensions are also possible in some embodiments.

The scaffold may comprise any suitable material. For example, in one set of embodiments, the scaffold may comprise fibrin, or another edible material. This may be

^' ins such as cultivated meat, where the cultivated animal-derived product will be eaten, e.g., by humans or other animals. In some embodiments, the microcarriers may comprise a hydrogel, e.g., a fibrin hydrogel, or other hydrogels such as those described herein.

In some cases, at least 50 wt%, at least 60 wt%, at least 70 wt%, at least 80 wt%, at least 90 wt%, or substantially all of a scaffold is formed from fibrin, and/or another edible material. The fibrin may arise from any suitable source. For example, the fibrin may arise from a non-human animal, such as a non-human mammal. Non-limiting examples include cows, pigs, sheep, goats, or the like. In some cases, the fibrin may arise from the blood of such an animal. For instance, in some embodiments, the fibrin may be prepared by acquiring blood or blood plasma from an animal, and processing it to produce fibrin. For example, in one set of embodiments, the blood is exposed to a protease inhibitor such as thrombin, which may cause fibrinogen to clot to form fibrin. The fibrin may be harvested, and used as discussed herein, e.g., to produce scaffolds such as microcarriers. In addition, in some cases, fibrin may be obtained from fibrinogen, which may be bought commercially, obtained from blood plasma, or the like.

In some cases, the blood may be acquired from the animal without killing the animal. For instance, blood may be withdrawn from the animal at spaced intervals, so as to allow the animal time to recover and produce new blood. For instance, blood may be withdrawn from the animal every 4 weeks, every 6 weeks, every 2 months, or the like, e.g., as discussed herein.

The fibrin may be processed to form a scaffold. 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 (for example, triangular) 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 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, ;ss 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, triangular, 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.

Fibrin itself may be edible. The microcarrier or scaffold may comprise, in addition to or instead of fibrin, other edible materials in certain embodiments. In addition, it should be understood that the scaffold is not limited to only edible or degradable materials. In other embodiments, the scaffold may comprise 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. lude 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.

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, chitin, collagen, soy protein, mycelium, gelatin, alginate, etc.

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. Additional examples of such materials include those described in U.S. Ser. No. 63/159,403, filed March 10, 2021, entitled “Constructs for Meat Cultivation and Other Applications,” by Khademhosseini, el ah, incorporated herein by reference in its entirety.

The microcarrier, or other scaffold may also 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 one aspect, at least some of the microcarriers or other types of scaffolds may have one or more grooves defined therein. (However, it should be understood that such grooves are not always required in other embodiments.) Examples of such grooves include those discussed in U.S. Ser. No. 63/159,403, filed March 10, 2021, entitled “Constructs for Meat ^~ er Applications,” by Khademhosseini, et ah, incorporated herein by reference in its entirety. 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. The grooves may also have any profile, e.g., square or rectangular, and any aspect ratio (i.e., width to height).

The microcarriers, or other scaffolds such as described herein, may be formed using any suitable technique, according to certain aspects. Non-limiting examples include extrusion, electro spinning, 3D-printing, molding, injection molding, or the like, e.g., of a precursor solution or a hydrogel block, etc. For example, a microcarrier or other scaffold maybe formed by milling, chopping, or otherwise processing hydrogel blocks. As still another non-limiting example, fibrin hydrogel blocks can be formed into millimeter-sized high speed homogenizers, or the like. In yet another other non-limiting example, 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.

In one set of embodiments, materials that will be used to form a microcarrier (e.g., comprising fibrin, 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., fibrin), 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., fibrin), 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. These precents are percent by weight.

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 gth 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 scaffolds) 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 cultivated 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.

In some embodiments, the microcarriers may be formed into a cultivated meat product, or other cultivated animal-derived product. For example, cells such as non-human animal cells may be grown on microcarriers or scaffolds, e.g., as discussed herein. Non limiting examples include muscle cells (e.g., myoblasts), adipose (fat) cells, or the like, and the cells may arise from the same or different species. In some cases, relatively large quantities of product may be prepared, e.g., by growing the cells in a bioreactor or other in vitro cell culture system, until at least a certain size or mass is reached. For example, the cells may be grown until they form a product that is, for example, at least 10 g, at least 25 g, at least 50 g, at least 100 g, at least 300 g, at least 1 kg, etc. Those of ordinary skill in the art will be aware of bioreactors and other cell culture systems.

In addition, in some cases, the cultivated meat product may contain a fat replica. One example of a fat replica is an emulsion of fat and non-human blood plasma, such as discussed in a patent application entitled “Systems and Methods of Producing Fat Tissue for Cell-Based Meat Products,” filed on November 15, 2021, US Pat. Apl. Ser. No. 63/279,642, incorporated herein by reference in its entirety.

Fat is typically an integral part of naturally-occurring meat, and exists in muscle tissue in varying percentages. Its presence can be useful in causing a cultivated meat product to taste more like naturally-occurring meat. Thus, as discussed herein, various fat replicas are provided in certain embodiments, which may be added to a cultivated meat product, or other cultivated animal-derived products such as those disclosed herein.

In one set of embodiments, a fat replica may comprise non-human fat cells, such as se progenitor cells, etc., and/or other types of fat cells. The cells may come from any suitable animal, such as a chicken, a cow, a pig, a sheep, a goat, a deer, a fish, a duck, a turkey, a shrimp, or the like. In some embodiments, the cells may be seeded onto microcarriers, or other scaffolding material. A variety of microcarriers can be used, for example microcarriers comprising fibrin, such as those discussed herein. Microcarriers are also discussed in U.S. Ser. No. 63/159,403, filed March 10, 2021, entitled “Constructs for Meat Cultivation and Other Applications,” by Khademhosseini, el al and a patent application entitled “Constructs Comprising Fibrin or Other Blood Products for Meat Cultivation and Other Applications,” filed on November 15, 2021, US Pat. Apl. Ser. No. 63/279,617, each incorporated herein by reference in its entirety. In certain cases, the cells may be cultured in a bioreactor or other in vitro cell culture system, such as described herein, to form a fat replica. In certain embodiments, for example, the cells are grown in serum, and induced to differentiate.

In some embodiments, the fat may be present within the fat replica as an emulsion.

For example, the fat may be contained within fat particles in the fat replica. In some cases, the fat may be dispersed (for example, homogenously) within the emulsion. In one set of embodiments, for example, fat particles may be formed by mixing fat with non-human blood plasma, which are suspended as an emulsion. The non-human blood plasma may come from any suitable source. As non-limiting examples, the plasma may arise from the blood of an animal, such as a chicken, a cow, a pig, a sheep, a goat, a deer, a fish, a duck, a turkey, a shrimp, etc. The plasma may arise from the same, or a different type of animal than the fat or fat cells that may be present. As previously discussed, and without wishing to be bound by any theory, it is believed that the plasma has components that can emulsify fat to form fat particles. Fat particles that may be formed include chylomicrons, very low density lipoproteins, low density lipoproteins, high density lipoproteins, and the like. These may be formed from various proteins, surfactants, etc. present with in the plasma. For example, particles such as chylomicrons may comprise apolipoproteins such as Al, A2, A3, A4, A5, B, B48, B 100, Cl, C2, C3, C4, D, E, F, H, L, M, or the like. Apolipoproteins such as these may be present within the plasma, e.g., naturally-occurring within the plasma. In some embodiments, however, various proteins, surfactants, or the like may be added.

In addition, in certain embodiments, a surfactant may be used to disperse the fat and/or stabilize the emulsion. Non-limiting examples of surfactants include phospholipids, monoglyercols, diglycerols, propylene glycol monoesters, lactylate esters, polyglycerol esters, sorbitan esters, ethoxylated esters, succinate esters, fruit acid esters, acetylated tylated diglycerols, phosphate monoglycerols, phosphate diglycerols, sucrose esters, etc. For example, a surfactant may be mixed with animal cells and/or non human blood plasma to form an emulsion.

Non-human blood plasma may be edible. In some cases, the non-human blood plasma may contain fibrinogen, which can be used to form fibrin hydrogels as discussed herein, for example, by the addition of thrombin or calcium. In some embodiments, the non human blood plasma may be concentrated or diluted, for example, to increase or decrease the crosslinking density of the hydrogel. Accordingly, in another set of embodiments, the fat replica may comprise a hydrogel. Fat may be present within the hydrogel, e.g., suspended within a fluid contained within the hydrogel, e.g., as an emulsion and/or present as fat particles, dissolved or suspended within the fluid, etc. Such a system may form a fat replica that can be used within a cultivated meat product, or other cultivated animal-derived product such as those described herein.

In one set of embodiments, the hydrogel may be formed, at least in part, by fibrin. Fibrin is a fibrous protein involved in the clotting of blood. It can be formed, for example, by exposing a non-human blood plasma to a clotting agent. For instance, the protease inhibitor thrombin is able to act on fibrinogen, causing the fibrinogen to form fibrin and thereby form a clot. The clot may be edible. Other techniques can also be used to crosslink fibrin in certain cases, e.g., artificially, rather than causing the clotting process to occur. For example, in one set of embodiments, calcium may be added to cause fibrin to crosslink. Thus, in certain embodiments, a fat replica may comprise a hydrogel comprising fibrin, e.g., that has been clotted to form the hydrogel. In other embodiments, the fibrin may not be clotted, but may form a hydrogel, e.g., by inducing crosslinking of the fibrin, for example, chemically.

However, it should be understood that the fat replica is not limited to only fibrin hydrogels. The hydrogel may be formed from other components, in addition to or instead of fibrin. Non-limiting examples of hydrogels include proteins (for example, collagen, gelatin, etc.), polymers (for example, polylactic acid, polyglycolic acid, etc.), and carbohydrates (for example, alginate, hyaluronan, chitosan, cellulose, hydroxymethyl cellulose etc.). In some cases, a hydrogel may be formed by causing components such as these to crosslink, e.g., in the presence of fat, to form a fat replica.

The hydrogels can be non-covalently and/or covalently crosslinked. Non-covalent hydrogels may be stabilized in some embodiments by hydrogen bonding, van der Waals interactions (e.g., hydrophobic interactions), etc. Covalent hydrogels may be formed, for example, by adding a crosslinking agent, bearing a first coupling group, to a crosslinkable ccond coupling group. The coupling groups can be any functional groups known to those of skill in the art that together form a covalent bond, for example, under mild reaction conditions or physiological conditions. Examples of couple groups include, but are not limited to, maleimides, N-hydroxysuccinimide (NHS) esters, carbodiimides, hydrazide, pentafluorophenyl (PFP) esters, phosphines, hydroxymethyl phosphines, psoralen, imidoesters, pyridyl disulfide, isocyanates, vinyl sulfones, alpha-haloacetyls, aryl azides, acyl azides, alkyl azides, diazirines, benzophenone, epoxides, carbonates, anhydrides, sulfonyl chlorides, cyclooctynes, aldehydes, and sulfhydryl groups, etc. In some embodiments, coupling groups may include free amines (-Nth), free sulfhydryl groups (-SH), free hydroxide groups (-OH), carboxylates, hydrazides, alkoxyamines, etc. In some embodiments, a coupling group can be a functional group that is reactive toward sulfhydryl groups, such as maleimide, pyridyl disulfide, or a haloacetyl.

In addition, it will be understood that the characteristics of the fat replica can be controlled, to some extent, by controlling the crosslinking density of the hydrogel, or other techniques such as those described herein. For example, characteristics such as texture, mechanical properties, rheological properties (e.g., elastic modulus, loss modulus, etc.), or the like may be controlled by controlling the crosslinking or hydrogel formation conditions and/or the components of the hydrogel. In addition, in some cases, the characteristics of the hydrogel may be controlled based on the type of non-human blood plasma used.

For example, in some embodiments, the crosslinking density may vary, e.g., by altering the concentration of the fibrinogen in the non-human blood plasma. In some embodiments, the crosslinking density may be determined, for example, using measurements such as rheological measurements, dynamical mechanical analysis measurements, or the like. Rheological measurements, e.g., the storage modulus, the loss modulus, etc., may be used to determine the characteristics of the fat replica and/or cultivated meat product (or other product). Examples of such characteristics include, but are not limited to textual proprieties, microstmctural proprieties, rheological proprieties, or the like. These may be determined, for example, using rheometers or other techniques known to those of ordinary skill in the art. In one set of embodiments, the stiffness of the cultivated meat product before cooking may be at least 0.5 (N/m), at least 1.0 (N/m), at least 1.5 (N/m), or at least 10 N/m (see, e.g., Figs 16,

18, 19A-B). In another set of embodiments, the stiffness of the cultivated meat product after cooking may increase to at least 50 N/m, at least 75 N/m, at least 100 N/m, or at least 150 N/m (see, e.g., Figs. 19A-B). In some embodiments, cooking the product may cause the cultivated meat to shrink up to 5%, up to 10%, up to 20%, or up to 30% (see, e.g., Figs. 20A- ^~ - -- - - - ther embodiments, cooking the cultivated meat product may result in weight loss of at least 10%, or at least 20%, of at least 30%, or of at least 40% (see, e.g., Figs. 21A-B).

In one set of embodiments, the fat replica and the muscle replica may be present in a mass ratio of at least 5:95, at least 10:90, at least 15:85, at least 20:80, at least 25:75, at least 30:70, at least 35:65, at least 40:60, at least 45:55, at least 50:50, at least 55:45, at least 60:40, at least 65:35, at least 70:30, at least 75:25, at least 80:20, at least 85:15, at least 90:10, etc.

In addition, in some embodiments, the mass ratio may be no more than 90:10, no more than 85:15, no more than 80:20, no more than 75:25, no more than 70:30, no more than 65:35, no more than 60:40, no more than 55:45, no more than 50:50, no more than 45:55, no more than 40:60, no more than 35:65, no more than 30:70, no more than 25:75, no more than 20:80, no more than 15:85, no more than 10:90, or no more than 5:95. Combinations of any these are also possible in certain embodiments. For example, the fat replica and the muscle replica are present in a mass ratio of between 5:95 and 35:65, between 10:90 and 40:60, between 20:80 and 30:70, etc.

Hemes are another non-limiting example of materials that can be added to a cultivated meat product. Hemes are further described in a patent application entitled “Production of Heme for Cell-Based Meat Products,” filed on November 15, 2021, US Pat. Apl. Ser. No. 63/279,644, incorporated herein by reference in its entirety.

Thus, one aspect of the present disclosure is generally directed toward providing a cultivated meat product (or other cultivated animal-derived product) having a relatively natural meat color. Cultivated meat products produced in vitro often do not contain sufficient myoglobin, the heme-containing protein that gives natural meat its color (usually a red color). For instance, natural meat appears red when myoglobin binds oxygen and changes the oxidation state of the iron group within the heme moiety (from Fe ²⁺ to Fe ³⁺ ).

As such, one set of embodiments is directed toward introducing heme into a product, such as a cultivated meat product. In one embodiment, the heme may be obtained from non human red blood cells. For example, red blood cells may be lysed, e.g., by exposing the cells to hypoosmotic or distilled water to form a lysate of non-human red blood cells. Red blood cells contain hemoglobin, a structurally similar protein to myoglobin. Hemoglobin also contains a heme moiety. Lysing the red blood cells may release hemoglobin, e.g., into solution. Furthermore, in addition to these, other methods of lysing red blood cells can be used, including those discussed in more detail herein.

One set of embodiments is generally directed to the production and use of heme,

1 to a product, such as a cultivated meat product, or other cultivated animal-derived products such as those discussed herein. The heme may arise from any suitable source. For instance, in certain embodiments, heme may be obtained from a lysate of non-human red blood cells. Such a lysate may be produced, for example, by lysing red blood cells, using any of a variety of lysing techniques. For example, the non-human red blood may arise from a chicken, a cow, a pig, a sheep, a goat, a deer, a fish, a duck, a turkey, a shrimp, or other animals.

For example, in one set of embodiments, cells may be lysed by exposing them to hypoosmotic water, such as distilled water. Cells typically have an osmolarity of about 300 mOsm; hypoosmotic solutions with osmolarities less than this (including distilled water, having an osmolarity of approximately zero) may cause water to enter the cells, ultimately causing them to burst or lyse. Accordingly, in certain embodiments, red blood cells are exposed to a hypoosmotic solution to cause the cells to lyse. In some embodiments the cells may be exposed to a volume of hypoosmotic water that is at least sufficient to lyse the red blood cells. For example, the volume may be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% of the volume of the solution containing the cells. These precents are percent by volume.

In addition, other methods of lysing red blood cells can be used in other embodiments. As one non-limiting example, acoustic energy may be used to lyse the cells. For example, the cells may be exposed to a sonicator or an ultrasonic bath to cause the cells to lyse. As yet another non-limiting example, the cells may be exposed to one or more freeze-thaw cycles, e.g., where the temperature is lowered to below the freezing point of the solution, thereby causing ice to form, killing at least some of the cells.

In certain embodiments, the cells may be lysed such that at least 50% of the cells have been lysed or ruptured, and in some embodiments, such that at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells have been lysed or ruptured. One or more than one lysing technique may be used in various embodiments.

In addition, it should be understood that red blood cells are not the only source of heme. In another embodiment, for example, the heme may be obtained from plant globins, for example, by extracting heme proteins from the root nodules of various plants. In some embodiments the plant globin is leghemoglobin. Non-limiting examples of plants containing globins include beans, soybeans, peas, chickpeas, peanuts, lentils, lupins, mesquites, carob, tamarind, alfalfa, clover, etc. In another embodiment, heme iron may be obtained from ysters, clams, mussels, chicken livers, organ meats, sardines, segmented worms, etc. The heme, in accordance with another embodiment, may also be produced using a genetically modified organism (GMO). In some embodiments the GMO is a yeast.

For example, in one embodiment, non-human blood plasma may be used as a source of fibrinogen to form fibrin hydrogels, which may be used to form fibrin microcarriers, for example by milling or other techniques. In some embodiments, fibrin microcarriers may be seeded with non-human cells, e.g., myoblasts cells, adipose cells, etc. In some embodiments, a fat replica may be formed using the non-human blood plasma to create a clot or a fibrin hydrogel, e.g., by exposure to thrombin, calcium, etc.

As another example, in some embodiments, non-human blood plasma may be used for the cultivation of cells in bioreactors. Cells typically require a variety of compounds such as soluble proteins, growth factors, cytokines, etc. for various activities, such as adhesion, proliferation, differentiation into different cell types, etc. In some embodiments, for example, non-human blood plasma may be applied to cells, e.g., in a bioreactor or other reactors such as those described herein.

As mentioned, some embodiments are directed toward repeated blood collection from non-human animals, for example, to obtain blood, blood serum, blood plasma, red blood cells, or other blood products, etc. For example, repeated blood collection from non-human animals may be used to obtain non-human blood plasma, lysate of non-human red blood cells, or the like.

In some cases, blood may be withdrawn from a donor animal at spaced intervals, which may be regular or irregular. Between blood draws, the animal can recover and produce new blood. Any suitable interval may be used. For example, the blood may be withdrawn from the animal every 2 weeks, every 4 weeks, every 6 weeks, every 2 months, or the like. As an example, in some embodiments, a blood draw may be withdrawn from an animal, and after a suitable interval, an additional blood draw may be withdrawn from the animal. This cycle can also be repeated any suitable number of times. The blood draws may each be processed, for example, as discussed herein. For example, the blood may be used to form a cultivated meat product.

For example, in one set of embodiments, one or more blood draws may be withdrawn or taken from a non-human animal. Exemplary embodiments of non-human animals include chicken, cow, pig, mutton, goat, deer, fish, duck, turkey, shrimp, or other animals that are commonly recognized for widespread human consumption. The blood may be processed in some cases to isolate the non-human red blood cells, e.g., using centrifugation, apheresis, or iques. In another set of embodiments, the non-human red blood cells may be lysed, e.g., as discussed herein. For example, the lysate may be made into a first cell- based meat product (e.g., using the lysate of non-human red blood as colorant). In some embodiments, the non-human red blood cells are lysed within 24 hours of withdrawal from a non-human living donor to produce the lysate. In another set of embodiments, non-human blood plasma may be used to form microcarriers, e.g., microcarriers comprising fibrin. In yet another set of embodiments, the non-human blood plasma may be used as a cell culture additive, e.g., to culture cells in a bioreactor.

In addition, the animal may also be subjected to second, third, etc. blood draws, e.g., at spaced intervals such as discussed herein. The blood withdrawn in each draw may be treated in the same way, or in different ways, depending on the application. Notably, however, the animal remains alive between blood draws, and can recover and produce new blood. In this way, although blood production from the animal occurs, the animal is not slaughtered in order for blood production from the animal to continue.

In some embodiments, it is believed that withdrawing blood from living animal donors, as opposed to farming them for slaughter, may translate into substantial environmental improvements, such as reductions in carbon emissions, water, land, etc.

As mentioned, in some aspects, a product such as a cultivated meat 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 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, one or 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 cases, some separation and/or processing of the cells may be used. As a non-limiting example, myotubes may be grown within a bioreactor or other cell culture system such as those described herein to produce a muscle replica. In some embodiments, s may be processed, e.g., by adding a fat replica to produce a cultivated meat 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. One non-limiting example of a fat replica is an emulsion of fat and non-human blood plasma, e.g., as discussed in a patent application entitled “Systems and Methods of Producing Fat Tissue for Cell-Based Meat Products,” filed on November 15, 2021, US Pat. Apl. Ser. No. 63/279,642, incorporated herein by reference in its entirety.

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, during cultivation, the cells may have a shear stress applied to them of at least 0.05 newton/meter squared, at least 0.1 newton/meter square, at least 0.2 newton/meter squared, at least 0.3 newton/meter squared, at least 0.4 newton/meter squared, at least 0.5 newton/meter squared, at least 0.6 newton/meter squared, at least 0.7 newton/meter squared, at least 0.8 newton/meter squared, 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. Those of ordinary skill in the art will be aware of methods and systems to induce differentiation in cells.

In one embodiment, the cell-based meat product may be grown in a bioreactor, or other in vitro cell culture system, comprising a cell growth medium. In one embodiment, the cell growth medium comprises an animal derived product, for example, platelet rich plasma (PRP), platelet poor plasma, platelet lysate (PL), platelet concentrate, a lysate of red blood cells, optionally comprising other nutrients, or the like. The cell growth medium may be used for the production of cell -based meat and/or to enhance the proliferation of primary cells, stem cells such as myoblasts, fibroblasts, adipocyte, vascular, osteoblasts, tenocyte, neural cells, etc. These cells may be isolated from human or non-human animals, grown in vitro, etc. These may include but are not limited to humans, cows, sheep, swine, horses, goats, camels, whales, fishes, crabs, shrimp and the like. In some embodiments, the blood products may be obtained from the blood of animals destined to slaughtered for food. In one embodiment, the platelet rich plasma (PRP) may be derived from whole blood from which red blood cells and white blood cells have been removed, such as by centrifugation, filtration, or other techniques known to those of ordinary skill in the art. Platelet rich plasma (PRP) may be generally categorized based on its leukocyte and fibrin content as (1) leukocyte- rich PRP (L-PRP), (2) leukocyte reduced PRP (P-PRP); (3) leukocyte reduced/pure PRP, or (4) leukocyte platelet-rich fibrin/pure platelet-rich fibrin (L- PRF). The platelet-rich plasma may be a blood derived composition having an increased concentration of platelets, compared to normal blood. For example, the PRP may have at least double, at least five times, or at least ten times or more the normal concentration of platelets in blood. In addition, in accordance with another embodiment, the platelet rich plasma may contain a variety of endogenous growth factors, such as transforming growth factor beta, fibroblast growth factor, insulin-like growth factor 1, insulin-like growth factor 2, vascular endothelial growth factor, epidermal growth factor, Interleukin 8, keratinocyte growth factor, connective tissue growth factor, etc.

In one embodiment, the platelet concentrate (PC) may be derived from the platelet rich plasma (PRP), for example, by centrifugation. In some embodiments, the concentration may be at least 10 ³ platelets/mL, at least 10 ⁴ platelets/mL, at least 10 ⁵ platelets/mL, at least 10 ⁶ platelets/mL, at least 10 ⁷ platelets/mL, at least 10 ⁸ platelets/mL, at least 10 ⁹ platelets/mL, at least 10 ¹⁰ platelets/mL, etc. In some embodiments, donated platelet concentrates may be stored at 4 °C prior to use for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days after donation. In another embodiment, expired human platelet concentrate may be obtained from blood banks, hospitals, and other institutions that routinely collect and store platelet rich plasma, and used as an additive in the cell growth medium.

In one embodiment, the platelet concentrate may impart the cell growth medium with antimicrobial properties. Platelets have certain properties similar to immune cells, and can in some cases induce potent anti-inflammatory responses when exposed to a number of chemical and biological triggers, for example, lipo saccharide protein (LPS). In some embodiments, the platelet concentrate may be added to the cell culture medium and stimulated by treatment with platelet activating reagents, for example, calcium, thrombin, citrate, EDTA, plasminogen, and other platelet activating reagents known to those skilled in the art, e.g., to release antimicrobial molecules that may neutralize common bacterial, fungal, or viral food pathogens. In some embodiments, an acellular antimicrobial cell growth pared by first culturing the platelet concentrate in the cell culture medium, stimulating them to release their antimicrobial payload, and then separating the antimicrobial cell growth medium from the platelet concentrate.

In one embodiment, the platelet concentrate may be lysed, for example by freeze- thawing or physical shearing (e.g. sonication or homogenization, etc.), to yield a platelet lysate (PL) comprising a plurality of cytokines and growth factors (e.g. transforming growth factor beta, fibroblast growth factor, insulin-like growth factor 1, insulin-like growth factor 2, vascular endothelial growth factor, epidermal growth factor, Interleukin 8, keratinocyte growth factor, connective tissue growth factor, etc.) that in some embodiments may enhance cell proliferation, for example, of myoblasts and adipocytes. In some embodiments, the platelet lysate comprises human platelets, and/or non-human platelets. For example, in one embodiment, the platelet rich plasma may include bovine platelet rich plasma.

In one embodiment, the cell growth medium comprises a combination of a platelet lysate (PL) and a platelet rich plasma (PRP). In one embodiment, the PL/PRP comprise at least 2 to 20% w/v, at least 5-15% w/v, or at least 10% w/v of the cell culture growth medium. In another embodiment, the total platelet component in the cell growth medium is at least 2 to 5 mg/mL, at least 2 to 10 mg/mL, at least 2 to 20 mg/mL, or at least 9 to 11 mg/mL.

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 the scaffold or microcarrier together may form a cultivated meat product (for example, if the scaffold or microcarrier is edible and/or degradable), or other cultivated animal-derived product. In some embodiments, the non-human cell-to-microcarrier ratio in the product is at least 95:5, at least 85:15, at least 75:25, at least 25:75, at least 15:85, or at least 5:95. 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 cultivated meat product or other cultivated animal-derived product, the scaffold or microcarrier may not necessarily be edible and/or degradable.

In some embodiments, a cultivated meat product may be formed by mixing a muscle (e.g., comprising an emulsion of fat and non-human blood plasma), and a lysate of non-human red blood cells. In some embodiments, the non-human cell to fibrin microcarrier ratio is at least 95:5, at least 85:15, at least 75:25, at least 25:75, at least 15:85, at least 5:95, etc. In certain other embodiments, the percent by weight of muscle replica to fat replica is at least 95:5, at least 90:10, at least 85:15, at least 80:20, at least 75:25, at least 70:30, at least 50:50, etc. In some embodiments, the color of the cultivated meat product may resemble that of native meat products before and after cooking (see, e.g., Fig. 22A-B, Fig. 23A-B, and Fig. 24A-B).

In some instances, a product such as a cultivated meat product further comprises binding agents that hold the various components together. Exemplary embodiments include transglutaminase, non-human plasma, fibrinogen, soy isolate, a soy concentrate, a soy milk, an egg, a soy flour, a wheat gluten isolate, or a pea isolate.

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 one method for producing fibrin microcarriers by milling preformed fibrin hydrogels (Fig. 1). In one embodiment, fibrin hydrogels are formed using non-human blood plasma isolated from freshly procured whole blood (such as from a chicken, goat, cow, turkey, etc.). Fresh whole blood can be obtained, for example, by placing a catheter into the animal’s vein, engaging the syringe tip with the catheter and retracting the plunger to remove blood from the animal, disengaging the syringe from the catheter, engaging the syringe tip with a vacutainer and depressing the syringe plunger to transfer the blood into the vacutainer, which contains an anticoagulant. Non-human blood plasma can be obtained from whole blood by centrifuging the whole blood at suitable speeds, such as 1000 g, at 2000 g, at 3000 g, at 4000 g, at 5000 g, etc. After centrifugation, the plasma fraction may be decanted, separating it from the pelleted cell fraction. Plasma rich fibrinogen (PRF), with varying concentrations of fibrinogen, may then be prepared by diluting the non-human blood plasma with cell culture media. In some cases, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 80%, at least 90% (vol/vol), or substantially all of a PRF is used to form the fibrin microcarriers.

In one embodiment, the PRFs were diluted with basal media to a final concentration of 10%, 20%, and 30% (vol/vol), respectively. The solutions were poured into a series of molds at 37 °C for 30 min to allow the fibrinogen time to gel. Here a mold may be a vessel, tank, or any other hollow shape that can hold the solution until gelled (e.g., sheets or rectangular blocks with or without grooves, etc.). Once gelled, the fibrin scaffolds were placed into a blender and homogenized between 100 to 1000 rpm for 40 s, 80 s, or 110 s to yield fibrin microcarriers with flake-like morphologies. In some embodiments, the shape of the microcarrier will depend on the blade geometry and the size of chamber used to homogenize the microcarriers. Fibrin microcarriers were than taken and imaged using optical microscopy and the average diameter of fibrin microcarriers determined using ImageJ by measuring the average diameter (Fig. 2A). The results showed that longer mill times corresponded to smaller microcarriers (Fig. 2B).

EXAMPLE 2

In this example, fibrin microcarriers with a PRF concentration of 20% vol/vol (with balance DMEM) were prepared as described in Example 1, and milled in a blender for either 20 s, 40 s, 90 s, or 110 s. The fibrin microcarriers (8.8 mg/mL) were placed in bioreactor containing myoblasts (100,000 cells/mL), resuspended in 100 mL of culture media, and ~¾e bioreactor was a stir-tank bioreactor, but other reactors including fluidized bed, packed bed, and aerated reactors, etc. may also be used. An aliquot of media was removed once a day and the number of cells per mL was determined. These results indicated that fibrin microcarriers with longer milling times had higher cell densities. It is believed that smaller particles have greater surface to volume ratios, which increases the available surface area for cells to grow and proliferation (Fig. 3).

EXAMPLE 3

This example tested the ability of fibrin microcarriers to promote growth and proliferation of myoblasts in a bioreactor following encapsulation within a carrier. Fibrin microcarriers were prepared as described in Example 1, except that bovine plasma was suspended in basal media containing bovine myoblasts (100,000 cells/mL) to a final PRF concentration of 10% vol/vol. The mixture was poured into a series of molds and kept 37 °C for 30 min to allow the fibrinogen time to gel. Once gelled, the fibrin scaffolds were placed into a blender and milled for 110 s at 5000 rpm to yield fibrin microcarriers with flake-like morphologies. The fibrin microcarriers were resuspended in growth media comprised of DMEM and 10% platelet rich plasma (PRP), loaded into a bioreactor (total volume, 200 mL), and allowed to cultivate for seven days. On days 3, 5, and 7, sample fibrin microcarriers were removed from the bioreactor, stained with calcein AM, imaged using fluorescence microscopy (see Fig. 4A), and the cell density quantified (see Fig. 4B). These results demonstrate that the cell density increased as a function of culture time, and that cells readily migrated throughout and proliferated within the fibrin microcarriers.

EXAMPLE 4

This experiment was designed to determine the effect of temperature and fibrinogen concentration on proliferation kinetics. In yet another embodiment, fibrin microcarriers containing varying concentrations of PRF were prepared by first diluting non-human blood plasma to 1.1 mg/mL, 4.4 mg/L or 8.8 mg/mL with basal media. The various PRF solutions were poured into molds and kept at either 37 °C or 4 °C (referred to as condensed fibrin carriers) for 30 min to allow the fibrinogen time to clot. Once gelled, the fibrin scaffolds were placed in a blender for 110 seconds for 5000 rpm and homogenized to yield fibrin microcarriers with flake-like morphologies. The fibrin microcarriers were subsequently placed into a 100 mL bioreactor vessel filled with growth media comprised of DMEM and 10% bovine platelet rich plasma and bovine myoblast cells (final concentration was 100,000 cells/mL) and allowed to culture for 7 days. On days 2, 3, 4, 5, 6, and 7 representative fibrin microcarriers were removed from culture and the cell density quantified by first dissolving .. a trypsin solution (0.25% in EDTA) followed by cell quantification using a hemocytometer to count the cells under microscopy These results showed that condensed fibrin microcarriers containing 8.8 mg/mL of PRF exhibited exponential cell growth, compared to uncondensed fibrin microcarriers, which exhibited near- linear proliferation kinetics (Fig. 5).

EXAMPLE 5

As mentioned above, in some embodiments, individual microcarriers can be fabricated by techniques such as extrusion, electro spinning, 3D printing, molding a fibrin solution, etc. In this non-limiting example, fibrin microcarriers were formed into fibers using two alternative methods. In one embodiment, solid fibrous fibrin microcarriers were formed by flowing a PRF solution containing a crosslinking agent inside a tubular mold (glass, plastic, or stainless steel), allowing it to gel, and then pushing it out to form solid microtubes.

In yet another embodiment, fibrous fibrin microcarriers were prepared using electro spinning. Electro spinning is generally a fiber production method which uses electric force to draw charged threads of polymer solutions or polymer melts to form fibers. These can have fiber diameters, for example, on the order of hundreds of nanometers. In this example, to produce fibrin fibers via electro spinning, non-human blood plasma (100 vol/vol%) was freeze-dried for 24 h to create a dry non-human blood plasma, which was finely ground using a mortar and pestle before use. The dried non-human blood plasma powder was dissolved in l,l,l,3,3,3-hexafluoro-2-propanol at a concentration of 200 mg/ml. Once in solution, the non-human blood plasma was loaded into a 3 mL syringe and placed in a syringe pump and the rate set to 2.8 ml/ h. A blunt metallic 18-gauge needle was placed on the syringe tip, and the positive voltage lead of a power supply was attached to the needle and set to 25 kV. A grounded piece of aluminum foil was used as a collection plate and was placed 20 cm away from the needle tip. All of these electro spinning processes were performed at 27 °C and 60% humidity.

The fiber morphology of the electrospun samples produced in this example was studied via scanning electron microscopy (SEM) operating at 14 kV. Electrospun samples were coated with Au-Pd at a thickness of 100 Angstroms (A) to reduce charging and produce a conductive surface (see Fig. 6; scale bar is 30 micrometers). The average fiber diameter of the electrospun fibrin fibers was determined from the SEM images using UTHSCSA ImageTool 3.0 software. Fiber diameter averages and standard deviations were calculated by taking the average of 60 random measurements per micrograph and was determined to be 0.82 +/- 0.14 micrometers. EXAMPLE 6

This example tested the ability of fibrous fibrin microcarriers generated using electro spinning to promote growth and proliferation of myoblasts in a bioreactor.

Electrospun fibrin fibers were created as described in Example 5. 10 mm diameter discs were punched from the electrospun fibrin mats, disinfected (30-minute soak in ethanol, followed by three 10-minute rinses in PBS), and placed in a 48-well plate. The electrospun fibrous fibrin microcarriers were subsequently seeded with 100,000 primary satellite cells (isolated from a cow) in 500 microliters of complete growth media (DMEM low glucose, supplemented with 10% FBS and 1% penicillin/streptomycin, Invitrogen, Carlsbad, CA, USA) and were cultured for up to 10 days in a humidified atmosphere (5% CO2) at 37 °C.

The media was changed every other day.

To evaluate cell viability, the electrospun fibrous fibrin microcarriers were stained using a solution containing calcein AM (2 mM) at 37 °C for 30 min, then imaged with fluorescent microscopy. Example fluorescence micrographs are shown in Figure 7. The majority of cells seeded on fibrin electrospun scaffolds were alive, as measured by live/dead staining (>98% cell viability). Likewise, the high number of live cells at day 10 indicated that the PRF electrospun scaffold was mechanically stable and appeared to actively promote adhesion, proliferation, and migration of the cells, as shown in Figs. 7A and 7B.

In particular, these figures illustrate the cell viability of primary bovine satellite cells seeded on the surface of fibrin microfibers fabricated by electro spinning. The cells were cultured for 10 days in standard cell culture conditions. Calcein- AM was used as a fluorescent biomarker for cell viability using fluorescent microscopy.

EXAMPLE 7

This example demonstrates the ability of fat emulsions stabilized within fibrin hydrogels to mimic the consistency and texture of fat content found in native meat products. Fat emulsions are made by adding a fat source to a solution of non-human blood plasma. In some embodiments, the fat source is an animal, a plant, or both. In certain embodiments, the fat source comprises a saturated fat, an unsaturated fat, or both. Non-human blood plasma can be derived from slaughtered animals or from living donors using apheresis by those skilled in the art and used to stabilize the fat. For example, blood plasma contains various globular proteins and macromolecules that can stabilize fat droplets following vigorous mixing to yield a homogenously dispersion which can be subsequently crosslinked to form a fat replica. To demonstrate these principals, fresh bovine blood was obtained and immediately processed to separate the plasma from the cells. In one embodiment, sunflower oil was mixed with the bovine blood plasma at a volumetric ratio of 80:20 (fahplasma), homogenized at 3000 rpm for 1 min minutes, and allowed to sit for 30 minutes at room temperature until gelation occurred. In another embodiment, thrombin or calcium maybe added to the solution after homogenization to accelerate the gelation process.

In another embodiment, the fat to plasma ratio of the above system was varied from 90:10 to 10:90. It was observed that ratios with higher fat contents produced softer tissue constructs, for instance, when using and unsaturated fat. When saturated fats, such as coconut oil, were used; however, an increase in the fat ratio, particularly at lower temperature, increased the brittleness of fat tissue. In other embodiments, fat oils also tested include canola oil, olive oil, coconut oil and milk fat.

EXAMPLE 8

In this example, a cultivated meat product was produced by mixing bovine muscle cells with a lysate of non-human red blood cells. A muscle replica comprising bovine muscle cells was prepared as discussed in a patent application entitled, “Constructs Comprising Fibrin or Other Blood Products for Meat Cultivation and Other Applications,” filed on November 15, 2021. Fresh whole blood was obtained from a living cow and the red blood cells were separated from the whole blood by centrifugation at 150 g for 25 minutes at 4 °C. The isolated red blood cells were then lysed using distilled water to a final concentration of 20% v/v. Although non-lysed red blood cells could also be used as a colorant in other embodiments, in these examples, it was observed that higher concentrations of unlysed red blood cells may be needed to obtain the same color profile as lysed red blood cells.

In some embodiments, a red colorant was obtained by mixing a 3% (vol/vol) lysate of non-human red blood cells with 77% bovine muscle replica and 20% fat replica. These were mixed together to form a cultivated meat product resembling ground beef. In some embodiments, the cultivated meat product may be mixed with a binding agent and a crosslinking agent. The binding agent may be non-human plasma (20% vol/vol) and the crosslinking agent may be calcium chloride (0.1% w/v) or thrombin. In some embodiments, the cultivated meat product may be cut and pressed to remove excess water.

EXAMPLE 9

In this example, red blood cell lysate was added to adjust the consistency and color of a ground beef cultivated meat product. The physical and mechanical properties of the ere measured using a combination of techniques including compressive modulus, weight loss, and volume loss in comparison to a selected native ground meat products. These determinations can be done before and/or after cooking, e.g., to ensure a better consumer response. For instance, the stiffness constant may be extrapolated using Hooke’s law from the linear regime of a stress versus strain plot. In one embodiment, the stiffness constant of the cultivated meat product increased from 5 +/- 5 N/m (i.e., uncooked) to 100 +/- 25 N/m (see, e.g., Fig. 16, Fig. 18, Fig. 19A-B) after cooking at 200 °C (380 °F) for 10 minutes. In another embodiment, the stiffness constant of the native meat product increased from 5 +/- 5 N/m (i.e., uncooked) to 200 +/- 50 N/m (see, e.g., Fig. 16, Fig. 18, Fig. 19A-B) after cooking at 200 °C (380 °F) for 10 min.

The weight loss upon cooking may be determined by placing the product on a food scale before and after cooking. The average weight of a cultivated meat product decreased from 10 +/- 2 grams (i.e., uncooked) to 7 +/- 1 grams after cooking at 200 °C (380 °F) for 10 minutes (see, e.g., Fig. 21A). The average weight of a native meat product decreased from 10 +/- 2 grams (i.e., uncooked) to 7 +/- 2 grams after cooking at 200 °C (380 °F) for 10 minutes (see, e.g., Fig. 21B).

The volume loss, or shrinkage, upon cooking may be determined by measuring the diameter of a meat patty made using a standard hamburger patty maker. In one embodiment, the average diameter of the cultivated meat product decreased from 35 +/- 1 mm (i.e., uncooked) to 30 +/- 2.5 mm after cooking at 200 °C (380 °F) for 10 minutes (see, e.g., Fig.

20 A). In another embodiment, the average diameter of the native meat product decreased from 35 grams (i.e., uncooked) to 20 +/- 2.5 grams after cooking at 200 °C (380 °F) for 10 minutes (see, e.g., Fig. 20B).

EXAMPLE 10

To quantify the redness and color profile of cell-based (cultivated meat) ground beef relative to native ground beef, and images of the uncooked and cooked samples were acquired and analyzed. 3 samples of cell-based ground beef and 3 samples of ground beef (purchased from a local supermarket) were molded into hamburger patties using a standard hamburger patty maker (i.e., they had about same thickness and diameters). Samples were placed on a piece of aluminon foil and photographs taken using a standard smart phone.

These samples were then cooked in a conventional oven at 200 °C (380 °F) for 10 minutes and imaged again. All images were then analyzed and the relative red, green, and blue intensities were determined (using IMAGEJ software, see Fig. 23A-B and Fig 24 A-B). The relative ratios of red:blue, red:green, and blue:green between groups was similar before and EXAMPLE 11

In this example, the cultivated meat product was used by a professional chef to prepare two dishes: a san choy bao and a meatball dish. The cultivated meat product and the final dishes were evaluated by a professional chef on a number of parameters before, during, and after cooking. Prior to cooking the cultivated meat product, the chef noted the following: (1) that the consistency of the cultivated meat product was paste like, but very smooth and held its form well when molded into various shapes, such as a meatball; (2) that the color of the cultivated meat product was light pink and appeared similar to pork mince, with a very even and smooth contrast of muscle and fat; (3) that the smell of the cultivated meat product was benign and while it did smell like a meat product it was extremely subtle; and (4) that the texture of the cultivated meat product was smooth and continuous as opposed to a granular appearance. During cooking, the chef noted the following: (1) that the consistency of the cultivated meat product changed as the product cooked, becoming firmer with time; (2) that the texture changed dramatically and appeared realistic with a granular texture reminiscent of native ground beef; (3) that the change in water content occurred as expected; (4) that the meat had almost no smell at all during preparation of two meals; (5) that the color change was immediate upon contact with heat and appeared identical to native meat products, and (6) that the while the cultivated meat product appeared to shrink, the product did not loose a significant amount of volume. The chef noted that the cultivated meat product lost about 25% of its initial mass following cooking. After cooking, the chef made the following remarks: (1) that the final meat color was grey /brown and appeared similar to native meat products; (2) the san choy bao dish mimicked outside minced beef products and that the meatball dish held its form, without the need for binders, and was extremely moist and tender; (3) that the san choy bao dish had no smell associated with it, whereas the meatball dish exhibited a subtle smell when cooking the meatballs; and (4) that the cultivated meat products did not have much flavor outside of the flavors added to the dish.

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, nfigurations 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 . >r” 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.

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