Patent Application
20240400979
As the world's population continues to grow, cell-based or cultured food products (e.g., cell-based meat products) for consumption have emerged as an attractive alternative (or supplement) to conventional meat from animals. For instance, cell-based, cultivated, or cultured meat represents a technology that could address the specific dietary needs of humans. Cell-based food products can be prepared from a combination of cultivated adherent and suspension cells derived from a non-human animal. Because the cells for cell-based meat are made in a food cultivation facility, cell masses are often formed and shaped to mimic familiar forms of conventional meat.
In addition to addressing dietary needs, cell-based food products help alleviate several drawbacks linked to conventional food products for humans, livestock, and the environment. For instance, conventional meat production involves controversial practices associated with animal husbandry, slaughter, and harvesting. Other drawbacks associated with harvested or slaughtered meat production include low conversion of caloric input to edible nutrients, microbial contamination of the product, emergence and propagation of veterinary and zoonotic diseases, relative natural resource requirements, and resultant industrial pollutants, such as greenhouse gas emissions and nitrogen waste streams.
Despite advances in creating cell-based food products, existing methods or systems for cultivating and processing cell-based food products face several shortcomings, such as challenges or failures to mimic the textures and flavors of slaughtered or harvested meat. In particular, some existing methods or systems often produce cell-based food products with undesirable textures. For instance, certain systems often grow cells in a pure single cell suspension. While the cells grow in the pure single cell suspension, they are typically unable to form muscle fibers or other multicellular structures. This inability to form multicellular structures can cause cell-based food products comprised of the grown cells to be too soft or suffer from other textural drawbacks.
In addition to poor texture, existing methods or systems of forming cell-based or cultured meat often utilize inefficient scaffolds to improve the textures of cell-based food products. For example, some existing scaffolds are large, unitary structures that require surfaces with specific geometric properties or surface coatings to adhere to cells. In certain cases, scaffolds must be removed from the adhered cells prior to forming the cell-based food product. Such removal requires further processing and can decrease the collection of cell deposited protein and/or disrupt the multicellular structures or cell morphologies that improve cell-based food product texture. In some existing systems, scaffolds must use specialized vessels that anchor the scaffolds while the non-human cells grow and differentiate.
Moreover, several existing methods or systems of forming cell-based or cultured meat utilize inflexible or undesirable cell culture media compositions. For example, many existing systems must use animal serum (e.g., Fetal Bovine Serum) in cell culture media to promote the growth and differentiation of non-human cells. The cost and sustainability of utilizing animal serum make these approaches suboptimal. Thus, many existing systems are limited to and by certain cell media compositions.
These, along with additional problems and issues exist in existing methods for cultivating cell-based food products.
This disclosure generally describes methods for growing cells on chickpea microcarriers as part of a process of creating cell-based food products (e.g., cell-based meat products). For example, the disclosed method can include adding chickpea microcarriers comprising ground and textured chickpea protein to cell culture media in a vessel for cell suspension, such as a bioreactor or cultivator. The chickpea microcarriers can further be mixed with non-human cells in the cell culture media. In some cases, for instance, the chickpea microcarriers and non-human cells are mixed within the cell culture media at different rates during different time periods. During mixing, the non-human cells can adhere to (and grow on) the surface of the chickpea microcarriers. After a mixing period, a growth period, a maturation period, a differentiation period, or some combination thereof, the textured cell tissue, made up of the chickpea microcarriers and adhered cells, can be harvested from the vessel and formed into a comestible food product.
Additional features and advantages of one or more embodiments of the present disclosure will be set forth in the description that follows.
Various embodiments will be described and explained with additional specificity and detail through the use of the accompanying drawings, which are summarized below.
This disclosure describes one or more implementations of a method for growing adhering, and/or maturing non-human cells on a chickpea microcarrier as part of a process of creating cell-based food products. In one or more embodiments, the disclosed method comprises adding chickpea microcarriers comprising textured chickpea protein to a cell culture media. The disclosed method further comprises mixing the chickpea microcarriers with non-human cells in the cell culture media. In some cases, the chickpea microcarriers, non-human cells, and cell culture media are mixed at different rates, such as by increasing a mixing rate over a time period or by using different mixing rates at different time periods. The disclosed method further comprises harvesting textured cell tissue comprising the chickpea microcarriers and non-human cells adhering to a surface of the chickpea microcarriers. Such harvested textured cell tissue can comprise grown, matured, and/or differentiated non-human cells. In some embodiments, the disclosed method forms the harvested textured cell tissue into a comestible food product (e.g., a comestible meat product).
As indicated above, the disclosed method includes forming chickpea microcarriers. In one or more embodiments, the disclosed method (i) extracts chickpea protein from chickpeas and (ii) adds chickpea flour to the extracted chickpea proteins to create a textured chickpea protein. Either before or after adding chickpea flour, in certain embodiments, the disclosed method micronizes or otherwise reduces the textured chickpea protein into chickpea microcarriers by grinding the textured chickpea protein to a particular granularity or size. In addition to size-reducing, in certain embodiments, the disclosed method ensures that the chickpea microcarrier is the particular size by filtering the chickpea microcarrier through a sieve or other filter. Once the chickpea microcarrier reaches the particular size, in one or more embodiments, the disclosed method sterilizes the chickpea microcarrier.
As mentioned, the disclosed method comprises adding chickpea microcarriers to cell culture media within a vessel. In some embodiments, the cell culture media is free of animal serums (e.g., Fetal Bovine Serum “FBS”). In one or more embodiments, the cell culture media contains non-human cells before adding the chickpea microcarriers. As depicted and described further below, in other embodiments, the disclosed methods comprise adding both non-human cells and chickpea microcarriers to the cell culture media at the same time.
The disclosed method further includes mixing the non-human cells with the chickpea microcarriers within the cell culture media. In particular, the disclosed method can utilize various agitation methods to enable adherence of the non-human cells to the surface of the chickpea microcarriers. For instance, in some cases, an agitation method is required for non-human cells and the chickpea microcarriers to stay in suspension, but gentle enough to allow the non-human cells to attach and stay attached to the chickpea microcarrier. In general, agitation is antithetical to adherence between chickpea microcarriers and non-human cells, so a proper balance must be achieved such that agitation is strong enough to maintain the coated microcarriers in suspension, but weak enough to allow the non-human cells to remain adhered to the chickpea microcarriers. The combination of proper agitation and cell adherence to the chickpea microcarriers in cell culture media facilitates or creates an environment for cell differentiation over time. To illustrate, in some cases, the disclosed method sets an initial mixing speed (e.g., rate) of 60 rotations per minute (“RPM”) (or other mixing speed suitable for a scale of the equipment) to promote an environment in which the cells can adhere to the textured chickpea microcarriers. After a period of time, the disclosed method increases the initial mixing speed to 100 RPMs (or other increased mixing speed suitable for the scale of the equipment), which further fosters an environment in which the non-human cells grow, differentiate, exhibit a certain phenotype or some combination thereof. In some embodiments, the disclosed method maintains the increased rate until the non-human cells are ready to harvest.
As mentioned above, by adhering to the chickpea microcarriers, the non-human cells and the chickpea microcarriers together form a textured cell tissue. In some cases, the textured cell tissue is harvested by removing the textured cell tissue from the cell culture media. For instance, the disclosed method concentrates the textured cell tissue by draining the cell culture media through a sieve. In some cases, the disclosed method reduces a moisture content of the textured cell tissue by additionally or alternatively pressing the textured cell tissue. In other cases, the disclosed method reduces the moisture content by centrifuging the textured cell tissue.
After harvesting, the disclosed method can form the harvested textured cell tissue into a comestible food product. For instance, the textured cell tissue may be arranged within a mold having a shape of a target cut of meat to mimic the muscular architecture/shape of the target cut of meat. In particular, the mold may comprise grooves that cause the textured cell tissue to have a surface texture that mimics the surface texture of the target cut of meat.
The disclosed method provides several benefits relative to existing methods for growing cell-based meats. In particular, the disclosed method provides an improved adherent surface for non-human cells and, in some cases, facilitates the production of additional extracellular matrix (“ECM”). By facilitating the production of the ECM, the disclosed method provides a substrate and environment within which non-human cells can differentiate to a cell type and/or produce a protein that, when embedded with the chickpea microcarriers in a product, exhibits a desirable texture. As part of improving texture relative to existing cell-based food products—because the adherence of the non-human cells to chickpea microcarriers—the disclosed method produces, and thus, collects more ECM than existing methods and provides a structure that forms a more robust three-dimensional (3D) tissue. Accordingly, the disclosed method forms cell-based food products (e.g., cell-based meat products) having improved texture, structure, and shape relative to existing methods.
In addition to increasing the growth of non-human cells exhibiting an improved texture, the disclosed method improves the efficiency of growing cells and harvesting textured cell tissue. In particular, because chickpea microcarriers are edible, stable, textured, and hypoallergenic, they likewise expedite the process of growing differentiated cells and forming a cell-based food product that includes not only the cells but also suitable microcarriers that do not require removal. For instance, it is hypothesized that due to their texture, the chickpea microcarriers do not require lengthy processing to ensure adherence between them and the non-human cells. Moreover, the disclosed method reduces the degree of processing for harvested cell tissue because the chickpea microcarriers remain in the harvested cell tissue.
Beyond efficient cell growth, the disclosed method expands the type of cell culture media that can be used to effectively grow non-human cells for cell-based food products. For example, the disclosed method creates an environment in which non-human cells adhere to the chickpea microcarriers in animal-serum-free media. By eliminating animal serum, the disclosed method can utilize various cell culture media formulations without the limits of existing, animal-serum-based methods.
As described further below, in contrast to chickpea microcarriers, soy and pea textured microcarriers lack the media range-media range referring to, for example, amenability to different media types and formulation in which the microcarriers retain of function—and adherence of chickpea microcarriers. For example, after mixing the non-human cells and the soy and pea textured microcarriers in the cell culture media, the non-human cells showed minimal attachment to soy and pea textured microcarriers in cell culture media without FBS. The soy and pea textured microcarriers lack media range relative to chickpea microcarriers because the soy and pea textured microcarriers require cell culture media containing animal serum (e.g., FBS) to provide an environment where non-human cells can adhere to a surface of the soy and pea textured microcarriers in a suspension culture environment. Additionally, soy and pea textured microcarriers lack the adherence capabilities of chickpea microcarriers. For example, non-human cells either do not attach to or have minimal attachment to soy and pea textured microcarriers in serum free cell culture media. Conversely, non-human cells attach to chickpea microcarriers in serum free cell culture media, which allows the non-human cells to grow, differentiate, and exhibit a certain phenotype, or some combination thereof on the chickpea microcarrier. The ability of cells to attach to chickpeas but not to soy and pea under serum free conditions was surprising to the inventors.
As illustrated by the foregoing discussion, the present disclosure utilizes a variety of terms to describe features and advantages of the disclosed method. Additional detail is now provided regarding the meaning of such terms. As used herein, the term “cells” (or “non-human cells”) refers to cells that form food products (e.g., meat products). Generally, non-human cells may comprise at least one of muscle cells, muscle progenitor cells, or muscle support cells. In particular, non-human cells may comprise different cell types, such as one or more of myoblasts, mesoangioblasts, myofibroblasts, mesenchymal stem cells, hepatocytes, fibroblasts, pericytes, adipocytes, epithelial cells, chondrocytes, osteoblasts, osteoclasts, pluripotent cells, somatic stem cells, endothelial cells, embryonic stem cells, induced pluripotent stem cells, or other similar cell types. Furthermore, cells may comprise different types of progenitor cells, including myogenic progeny and progenitors, adipogenic progeny or progenitors, mesenchymal progeny or progenitors, or other types of progenitor cells. When seeding, in some embodiments, the disclosed method includes seeding telomerase reverse transcriptase (TERT) immortalized chicken fibroblasts or other immortalized cells, spontaneously immortalized or otherwise.
As used herein, the term “textured cell tissue” refers to a tissue or mass comprising non-human cells and chickpea microcarriers. For instance, the textured cell tissue can include cells of cultivated meat (e.g., non-human cells) adhered to chickpea microcarriers which are gathered into a collective mass. Such a textured cell tissue may nevertheless be raw or uncooked. In some embodiments, the textured cell tissue is comestible. Additionally, a textured cell tissue may include grown non-human cells that have been nourished by a growth medium (e.g., cell culture media) to grow during a formation period within a cultivator. In some embodiments, a textured cell tissue may include matured or differentiated non-human cells that may have been exposed to differentiation media and/or conditions favoring differentiation to form structures such as, for example, myotubes. In some examples, textured cell tissue is grown from mixing non-human cells and chickpea microcarriers floating and/or suspended in liquid or gel in a suspension reactor/cultivator.
As used herein, the term “microcarrier” refers to a spherical or irregular-shaped and distinct support matrix that adheres or attaches to cells within a cultivator. A microcarrier can be a discrete, free-floating granule (e.g., unit) in a suspension. During use, a microcarrier is placed within a cell cultivator and suspended within a cell culture media but the microcarrier does not otherwise maintain a fixed position or orientation relative to a cell cultivator. Microcarriers may comprise different materials. In some instances, microcarriers can be edible or inedible. For example, microcarriers can comprise edible materials, such as chickpea protein, soybean protein, pea protein, polysaccharides, polypeptides, lipids, pectin, gelatin, dextran, or cellulose. In other cases, microcarriers can comprise inedible materials, such as, glass, plastic, dextran, or polystyrene. While this disclosure describes chickpea-protein-based microcarriers that are edible, in some embodiments, such microcarriers may include other edible components in addition to chickpea protein.
As used herein, the term “differentiation” refers to a process by which a cell changes from an initial cell type to a different cell type with a more specialized form and/or role. For instance, differentiated cells have specific structures and functions. Differentiation can be used so that cells mature to exhibit a certain phenotype characteristic of cells, such as, myoblasts, mesoangioblasts, myofibroblasts, mesenchymal stem cells, hepatocytes, fibroblasts, pericytes, adipocytes, epithelial cells, chondrocytes, osteoblasts, osteoclasts, pluripotent cells, somatic stem cells, endothelial cells, embryonic stem cells, induced pluripotent stem cells, or myogenic stem cells.
As used herein, the term “extracellular matrix” (or “ECM”) refers to a three-dimensional network that supports cell growth, adhesion, and differentiation. For instance, an ECM is an interwoven mesh comprising of glycosaminoglycans (e.g., hyaluronan), glycoproteins (e.g., fibronectin, laminin, etc.), and/or fibrous proteins (e.g., collagen, elastin, etc.). The ECM is secreted by cells and forms a network surrounding the cells that helps the cells communicate with one another. For example, the ECM can refer to the structure adhering cells to each other. In some embodiments, the ECM adheres to a microcarrier. Additionally, the ECM helps regulate cell differentiation. For example, based on the rigidity of the ECM, the non-human cells may develop into different cell types.
As used herein, the term “cell culture media” refers to a media (e.g., liquid, gel, etc.) that provides nutrients to cells and supports cell growth, cell differentiation, or both. In some cases, cell culture media assists in regulating environmental conditions. Cell culture media can be natural (e.g., extracted from animal tissue or animal body fluids) or synthetic (e.g., combinations of organic or inorganic compounds). For instance, cell culture media can comprise any combination of amino acids, vitamins, carbohydrates, inorganic salts, minerals, supplements, glucose, serums, or hormones. In certain cases, cell culture media can be animal component free (“ACF”). For instance, ACF cell culture media does not contain FBS.
As used herein, the term “harvest” or “harvesting” refers to a process of removing cells from a controlled environment. For example, harvesting refers to the process of removing textured cell tissue from a controlled environment by draining a portion of the cell culture media. In one or more implementations, harvesting comprises reducing the moisture content of the textured cell tissue by pressing the textured cell tissue.
Additional detail will now be provided regarding disclosed methods in relation to illustrative figures portraying example embodiments and implementations of the disclosed methods and apparatuses.
In some implementations, the act 102 of adding chickpea microcarriers to cell culture media includes adding the chickpea microcarriers at the same time as adding the non-human cells. By adding the chickpea microcarriers and the non-human cells at the same time, the disclosed method allows the non-human cells to grow, mature, differentiate, or some combination thereof while attached to the surface of the chickpea microcarrier.
By contrast, in one or more embodiments, the act 102 of adding chickpea microcarriers to cell culture media includes adding the chickpea microcarriers to cell culture media already containing non-human cells. For instance, non-human cells may be in the cell culture media as single cell suspension to grow the non-human cells. In some cases, the non-human cells grow to high density prior to adding the chickpea microcarriers. In some embodiments, the non-human cells are grown to high density, subsequently chickpea microcarriers are added, and subsequently the non-human cells are further grown, matured, differentiated, or some combination thereof.
In some cases, the act 102 includes adding dry chickpea microcarriers to the cell culture media. For instance, the disclosed method can transfer dry chickpea microcarriers from a container vessel to a bioreactor that mixes together the chickpea microcarriers, the non-human cells, and the cell culture media. In addition or in the alternative to drying chickpea microcarriers prior to adding them to cell culture media, in some embodiments, the disclosed method hydrates the chickpea microcarriers prior to adding them to the cell culture media. For instance, the disclosed method hydrates the chickpea microcarriers by adding cell culture media to dry chickpeas in an aseptic container. In some cases, the disclosed method transfers the hydrated chickpea microcarriers to a larger bioreactor that mixes the chickpea microcarriers, non-human cells, and cell culture media. As explained further below, in some embodiments, the chickpea microcarriers are coated with an adhesive material or other coating prior to adding to the cell culture media.
In one example, the act 104 comprises mixing the chickpea microcarriers, non-human cells, and cell culture media at different rates and/or intensities. For example, the act 104 includes an initial mixing speed during an initial time period where the non-human cells adhere to the chickpea microcarrier. In some embodiments, after the initial time period that is customized for cell attachment to the chickpea microcarriers, the disclosed method increases the initial mixing speed to allow increased nutrient mixing and growth relative to the initial time period until the disclosed method harvests the non-human cells adhered to the chickpea microcarriers.
In some embodiments, the act 104 facilitates and helps create an environment for cell differentiation. In particular, during mixing, the non-human cells differentiate to exhibit a certain phenotype. For instance, while the cells adhere to the chickpea microcarrier, the cells contact each other and exchange biochemical signals. In some cases, the cell-to-cell contact allows the cells to fuse together to form myoblasts and myotubes on a surface of the chickpea microcarrier.
As further illustrated in
In addition to harvesting, in certain embodiments, the disclosed method further comprises washing the textured cell tissue. For instance, the disclosed method includes rinsing the textured cell tissue with a wash buffer. In some implementations, the disclosed method rinses the textured cell tissue over a filter. In certain embodiments, the disclosed method further comprises drying the textured cell tissue. For example, the disclosed method comprises reducing the moisture content of the textured cell tissue by pressing the textured cell tissue on a cell press.
As further illustrated in
As previously mentioned, in some implementations, the disclosed method comprises forming chickpea microcarriers.
As shown in
In some embodiments, the disclosed method utilizes a burr grinder, a mill, or a biopsy punch that crushes or otherwise reduces the size of the textured chickpea protein granules by force between two surfaces. In other embodiments, the disclosed method utilizes a blade grinder to cut the textured chickpea protein granules into smaller units. In some embodiments, the disclosed method grinds the textured chickpea protein multiple times. For instance, the disclosed method may grind the textured chickpea protein three times before filtering the discrete granules of the textured chickpea protein. In additional embodiments, the disclosed method grinds the textured chickpea proteins for a length of time until they reach a specific granularity or size.
As further illustrated in
For example, in some embodiments, the filter, strainer, or sifter includes filtering holes of 200 μm or less in size. For example, in one or more embodiments, the disclosed method utilizes a filter comprising filtering holes that do not exceed 80 μm. Thus, in certain cases, the disclosed method filters the micronized, discrete granules of the textured chickpea protein to generate chickpea microcarriers 212 that do not exceed a certain size. For instance, the chickpea microcarriers do not exceed 150 μm or the size of chickpea microcarriers is 80 μm or less.
In some implementations, the disclosed method further comprises sanitizing the chickpea microcarriers. To illustrate, the disclosed method may sterilize the chickpea microcarriers with sterile solutions. For instance, the disclosed method sterilizes the chickpea microcarriers with a phosphate buffered saline (PBS) wash, such as antibiotic-antimycotic (“anti-anti”) PBS wash. In other embodiments, the disclosed method sterilizes the chickpea microcarriers with heat. For instance, the disclosed method places the chickpea microcarriers into an autoclave and heats them under pressure with steam for a period of time. For example, the disclosed method may place the microcarriers in the autoclave a temperature of 110-130° C. for at least 20-40 minutes under 10-20 pounds per square inch (e.g., a temperature of 121° C. for at least 30 minutes under 15 pounds per square inch). In some embodiments, the disclosed method may store the sanitized chickpea microcarriers in sanitized containers prior to adding them to the cell culture media.
As discussed above, the disclosed method adds the chickpea microcarriers to a cell culture media and mixes the chickpea microcarriers, non-human cells, and cell culture media.
Generally, the type of cell culture media affects how the non-human cells adhere to a surface. For instance, cell culture media comprising animal sourced serum enables cells to attach to several surfaces. However, animal sourced serum is expensive, and extraction of animal sourced serum raises ethical issues. In the alternative to animal sourced serum, the disclosed method can use serum-free cell culture media. For example, in some cases, the cell culture media does not contain any animal sourced serum (e.g., FBS, newborn calf serum, horse serum, etc.). In some embodiments, the media comprises suspension yellow lion (“SusYL”), a proprietary animal-component-free (ACF) media. In addition to an underlying media, in one or more cases, the cell culture media contains other reagents that feed the non-human cells.
In the alternative to serum-free media, the cell culture media can contain animal sourced serum. For example, in certain embodiments, the cell culture media contains a portion of animal sourced serum. Such cell culture media can comprise a mixture of SusYL and 10% FBS or other type of animal serum.
As further shown in
In some implementations, the disclosed method directly adds dry chickpea microcarriers 310 to the cell culture media 318. For instance, the disclosed method transfers the dry chickpea microcarriers 310 from a container to the bioreactor 316 that mixes the chickpea microcarriers 310, non-human cells 312, and cell culture media 318. To illustrate, the disclosed method can deposit the dry chickpea microcarriers 310 from an aseptic container to an enclosed bioreactor through a sterile tube connecting the enclosed container and the enclosed bioreactor.
Alternatively, in some implementations, chickpea microcarriers 310 are hydrated prior to adding them to the cell culture media 318 in the bioreactor 316. For example, the disclosed method may include adding a portion of the cell culture media 318 to the chickpea microcarriers 310 in an aseptic container. Once hydrated, the disclosed method can pump the hydrated chickpea microcarriers 310 from the aseptic container to an enclosed bioreactor containing a remaining portion of the cell culture media 318 and the non-human cells 312.
In some implementations, the chickpea microcarriers are coated with an adhesive material prior to adding the chickpea microcarriers 310 to the cell culture media 318. For instance, the disclosed method may coat the chickpea microcarriers 310 with transglutaminase and/or other bonding materials prior to adding the chickpea microcarriers 310 to the cell culture media 318. In some cases, coating the chickpea microcarriers 310 with the adhesive materials further enhances adherence, differentiation, ECM production, and allows for higher agitation rates.
As shown in
In some embodiments, prior to adding the non-human cells 312, the disclosed method prepares the non-human cells 312 for seeding. For instance, the disclosed method thaws the non-human cells 312 and, in some instances, scales up the cells to the desired density. To illustrate, the disclosed method thaws a cryovial containing frozen non-human cells 312 (e.g., chicken fibroblast, bovine fibroblast, etc.). After thawing, the disclosed method can grow the thawed non-human cells 312 in the cell culture media 318 or another growing reagent until they reach a target density (e.g., 250,000 to 30 million non-human cells/ml).
As further shown in
As indicated above, the disclosed method can mix the non-human cells chickpea microcarriers, and cell culture media at various rates for different lengths of time. For instance, as shown in
At very small scales, e.g., 30 ml, the RPM may range from 40-75 RPM. However, as someone skilled in the art would understand, when scaling up to larger volumes, RPM will need to be changed and is typically determined empirically at each scale. For example, at larger scales, impellers are often bigger and can rotate more slowly to provide the same level of mixing. In some embodiments, RPM is as slow as possible to ensure adherence of non-human cells to the chickpea microcarriers while avoiding dead zones of no stirring and settling of the chickpea microcarriers while ensuring accurate sampling/testing results. In one or more cases, once the non-human cells attach to the chickpea microcarriers, the disclosed method can raise the RPM to ensure adequate oxygenation and optimal growth without causing excess or detrimental shear.
As further shown in
In alternative implementations, the disclosed method may utilize various means of mixing (e.g., agitating) the chickpea microcarriers 310, the non-human cells 312, and the cell culture media 318. To illustrate, the disclosed method may mix the chickpea microcarriers 310, the non-human cells 312, and the cell culture media 318 through shaking, rolling, stirring, blending, rocking, or some combination thereof. Moreover, in some implementations, based on the scale and/or type of the bioreactor 316, the disclosed method can modify and/or scale the mixing rate (e.g., degree of agitation) in order to enable adhesion while avoiding excessive shear according to known methods. For example, the velocity of the cell culture media 318 within the bioreactor 316 should not shear the majority of the non-human cells 312 from the chickpea microcarriers 310.
As further shown in
As further illustrated in
In some embodiments, the disclosed method maintains a subsequent mixing speed of 90-110 RPMs (e.g., 100 RPM) from the subsequent time period 306 through a final time period 308. At the final time period 308, the disclosed method harvests textured cell tissue 314 comprising a proliferated population of the non-human cells 312 and the chickpea microcarriers 310. As shown in
As discussed above, the disclosed method may add the chickpea microcarriers 310 to the cell culture media 318 after growing the non-human cells 312 to high density.
As indicated above, in some embodiments, the non-human cells 332 are seeded and grown in ACF cell culture media (e.g., SusYL). In other embodiments, the non-human cells 332 are seeded and grown in a growth media with reagents necessary to feed the non-human cells. Alternatively, the cell culture media 334 contains sera derived from animals (e.g., SusYL and 10% FBS).
During the seeding phase 322 depicted in the example embodiment of
As further shown in the example embodiment of
As further illustrated by the example embodiment of
In some embodiments, mixing the non-human cells 332, the chickpea microcarriers 338, and the cell culture media 334 during the initial time period 326 causes the non-human cells 332 to attach to a surface of the chickpea microcarriers 338. In some implementations, the disclosed method adheres the non-human cells 332 to the chickpea microcarriers 338 by stopping a mixing motion, reducing a mixing rate, adding adherent factors or some combination thereof. For instance, the disclosed method may decrease the initial mixing speed to 30-49 RPMs (e.g., 45 RPMs) during the initial time period 326. Alternatively, the disclosed method may utilize adherent culture to adhere the non-human cells 332 to the chickpea microcarriers 338 and/or to create cell-to-cell adhesions.
As further illustrated in the example embodiment of
As discussed above, the disclosed method forms a textured cell tissue comprising the chickpea microcarriers and the non-human cells.
As shown in
In certain cases, the non-human cells 406 form more than one layer over a surface of the chickpea microcarrier 402. For example, in one or more implementations, the non-human cells 406 form a first layer over the surface of the chickpea microcarrier 402. In some embodiments, the non-human cells 406 form a second layer over a surface of the chickpea microcarrier 402. As illustrated in
As mentioned above, the non-human cells 406 form the ECM 404 through secretion. In some embodiments, the ECM 404 helps the non-human cells 406 communicate and differentiate (e.g., turn into more specialized cells with specialized functions). In particular, the non-human cells 406 differentiate to exhibit a certain phenotype characteristic of cells, such as, myoblasts, mesoangioblasts, myofibroblasts, mesenchymal stem cells, hepatocytes, fibroblasts, pericytes, adipocytes, epithelial cells, chondrocytes, osteoblasts, osteoclasts, pluripotent cells, somatic stem cells, endothelial cells, or myogenic stem cells.
As shown in
Additionally, or alternatively, separating the textured cell tissue refers to removing the textured cell tissue from the bioreactor. In some implementations, the act 514 further comprises removing the cells from the bioreactor by passing the cell culture media and textured cell tissue through a filter. For example, transferring the textured cell tissue to a harvest container, such as a jacketed bag filter. The textured cell tissue may be kept in the bioreactor or transferred to a harvest container for additional processing.
As further illustrated in
In some embodiments, the disclosed method aligns portions of the textured cell tissue to exhibit textural variation. For instance, the disclosed method stacks the roughly spherical particles (or other portions) of the textured cell tissue so that the comestible food product has an internal texture and an external texture mimicking the textures of a target meat. In some implementations, the disclosed method forms the comestible food product with textured cell tissue that includes extracellular matrix proteins.
As indicated above, chickpea microcarriers outperform other alternative materials as edible microcarriers. To illustrate,
As a skilled artisan would understand, however, when scaling up to larger volumes, the mixing rate or speed (e.g., RPM) will change and is typically determined empirically at each scale for a larger orbital diameter. For example, at larger scales, impellers are often bigger and can rotate more slowly to provide the same level of mixing. Accordingly, in some cases, each well plate has an initial mixing speed of 25-40 RPMs with relatively larger orbital diameter (e.g., 0.20 m orbital diameter) during the initial time period of 16 hours. After 16 hours, the initial mixing speed increased to 60-80 RPM during the subsequent time period.
As just mentioned, the scale of the orbital diameter correlates to the mixing rate (e.g., RPM). In certain implementations, the mixing rate (e.g., RPM) changes based on the orbital diameter according to the following equation:
where d1 represents the orbital diameter for the first shaker, d2 represents the diameter for a second (e.g., different) shaker, r1 represents the RPM for the first shaker and r2 represents the RPM for the second (e.g., different) shaker, as described by Mary Kay Bates, Orbital Shaker Benchmarks: Best Practices for Use and Maintenance, ThermoFisher Scientific Application Note (2017), available at https://assets.thermofisher.com/TFS-Assets/LED/Application-Notes/Orbital-Shaker-Benchmarks-Best-Practices-App-Note-ANMAXQBEST.pdf, which is hereby incorporated by reference in its entirety. For instance, the RPM for scaling up from a system utilizing 25 mm orbital diameter at 100 RPM to a system utilizing a 50 mm orbital diameter is 65-72 RPM.
In certain implementations, when scaling up to larger volumes, the mixing rate may be calculated based on reaching a certain suspension state (e.g., just suspended, nearly homogenous suspension, or homogenous suspension) for various chickpea microcarrier concentrations. For example, in some cases, a Zwietering correlation can help predict the just suspended state, where the mixing rate suspends particles (e.g., chickpea microcarriers) in the cell culture media such that the chickpea microcarriers stay in motion and do not settle on the bottom of the suspension bioreactor for more than 1 to 2 seconds. In some embodiments, the just suspended state can be calculated according to the following equation:
where Njs represents the just suspended speed, S represents the Zwietering Njs constant, v represents the kinematic viscosity
g represents the gravitation constant
ρs represents the solid density
ρ1 represents liquid density (kg/m3), X represents solids loading
dp represents particle diameter (m), and D represents impeller diameter (m). In some cases, a change in the just suspended state Njs can be determined based on a change in the impeller diameter (D), as described in Determining Agitation Requirements for Microcarrier Processes: Method Development Using the Mobius® 50 L Single-Use Bioreactor, Millipore Sigma Application Note (2018), available at https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/marketing/global/documents/359/769/mobius-cellready-50l-appnote-an4462en-ms.pdf, which is hereby incorporated by reference in its entirety. In one or more implementations, the mixing rate may be based on a nearly homogenous suspension, where a small population of microcarriers settled on the bottom of the suspension bioreactor nears zero. In certain embodiments, the mixing rate may be based on maintaining a completely homogenous suspension.
As shown in
As further illustrated in
Regarding the data in
As described above, the disclosed methods can utilize various seeding densities for seeding non-human cells on chickpea microcarriers.
As further shown in
In some cases, too high of a seeding density can inhibit the growth (e.g., proliferative capacity) of the non-human cells by forming the non-human cells into thick clumps or tissue that can induce necrosis. To prevent inducing necrosis, in some embodiments, the disclosed methods can utilize a seeding density that supports proliferation of the non-human cells without inhibiting the growth of the non-human cells. For example, in one or more embodiments, the seeding density of the non-human cells can be between 1M/mL and 4 M/mL. In some embodiments, based on the scale of production, mixing speeds, length of the initial time period, and/or length of the subsequent time period, the disclosed methods can increase or decrease the seeding density of the non-human cells as to enable the proliferation of the non-human cells.
As described above with respect to
In
As just mentioned, the initial mixing speed of 40 RPM resulted in the highest number of the non-human cells 812 attaching to the chickpea microcarriers 814. As shown in the images 804a-c, 806, 808, and 810 in
As indicated by
As
A skilled artisan would, of course, understand that the relative effectiveness of a given initial mixing speed on adherence of non-human cells to chickpea microcarriers depends on seeding density, volume of cell culture media, size of bioreactor, method of agitation, and other conditions. Thus, in some embodiments, the disclosed method can utilize an initial mixing speed from a range of initial mixing speeds during the initial time period that allows the non-human cells 812 to adhere to the chickpea microcarriers 814. In some cases, the range of initial mixing speeds can comprise between 40 RPM and 80 RPM.
As further shown in
Indeed, in some cases, the disclosed methods can utilize an initial mixing speed that generates clumps of the non-human cells 812 and the chickpea microcarriers 814 within a clump size range. In some embodiments, for instance, the disclosed method can utilize an initial mixing speed that generates clumps of the non-human cells 812 adhered to the chickpea microcarriers 814 with a clump size ranging between 75 micrometers and 125 micrometers. Relatedly, the disclosed method can utilize the initial mixing speed so that the diameter, width, and/or length of the clumps of the non-human cells 812 adhered to the chickpea microcarriers 814 falls between 75 micrometers and 125 micrometers.
As just discussed,
As shown in
As further indicated by
Similarly, as further shown in
Finally, as depicted in
As discussed above, the initial mixing speed and subsequent mixing speed can affect the adherence or attachment of non-human cells to chickpea microcarriers and to other non-human cells.
In particular,
As previously described in
As previously discussed, non-human cells can attach to chickpea microcarriers and form textured cell tissue comprising the non-human cells and the chickpea microcarriers.
As shown in
As indicated in
As just mentioned, the disclosed method tested the effects of combining the media additive 1008 with the non-human cells and the chickpea microcarriers. During testing, the non-human cells and the chickpea microcarriers combined with the magnesium chloride 1012 had less clumping and fewer single non-human cells than another media additive 1008. Thus, indicating that magnesium chloride can aid in the adhesion of the non-human cells to the chickpea microcarriers while diminishing the clumping of the non-human cells to each other.
As illustrated in
The series of acts 1100 illustrated in
As further illustrated in
In some embodiments, the series of acts 1100 further comprises an act where the cell culture media comprises animal serum-free media. In one or more embodiments, the series of acts also includes an act where the non-human cells differentiate to exhibit a phenotype characteristic of cells comprising myoblasts, mesoangioblasts, myofibroblasts, mesenchymal stem cells, hepatocytes, fibroblasts, pericytes, adipocytes, epithelial cells, chondrocytes, osteoblasts, osteoclasts, pluripotent cells, somatic stem cells, endothelial cells, or myogenic stem cells prior to harvesting.
In certain embodiments, the series of acts 1100 includes an act where the textured cell tissue comprises the non-human cells forming at least one layer over a surface of a chickpea microcarrier of the chickpea microcarriers. In some embodiments, the series of acts 1100 includes an act where the textured cell tissue comprises the non-human cells forming at least one myotube a chickpea microcarrier of the chickpea microcarriers.
In particular embodiments, the series of acts 1100 further comprises mixing the cell culture media, the non-human cells, and the chickpea microcarriers for a time period of no more than 15 days. In one or more embodiments, the series of acts 1100 includes an act where the chickpea microcarriers comprise discrete granules. In some embodiments, the series of acts 1100 further comprises an act where an individual chickpea microcarrier of the chickpea microcarriers does not exceed 150 microns in diameter.
In one or more embodiments, the series of acts 1100 further comprises an act where the chickpea microcarriers suspend in the cell culture media. In some embodiments, the series of acts 1100 further comprises an act where the chickpea microcarriers are hydrated in a separate vessel with media or dried by freeze-drying prior to mixing.
In one or more cases, the series of acts 1100 includes an act where the range of initial mixing speeds comprises 40 RPM to 80 RPM. In one or more implementations the series of acts 1100 includes an act of adding the non-human cells to the cell culture media with a seeding density of 1 million non-human cells per milliliter of the cell culture media to 4 million non-human cells per milliliter of the cell culture media. In particular embodiments, the series of acts 1100 further comprises mixing the non-human cells with the chickpea microcarriers at an initial mixing speed within a range of initial mixing speeds and increasing the initial mixing speed after a time period in which the non-human cells adhere to the chickpea microcarriers. In one or more cases, the series of acts 1100 includes an act where the range of initial mixing speeds comprises 40 rotations per minute (RPM) to 80 RPM.
In certain cases, the series of acts 1100 includes an act where the initial mixing speed within the range of initial mixing speeds supports proliferation of the non-human cells. In one or more instances, the series of acts 1100 includes an act where the initial mixing speed generates clumps of one or more non-human cells and one or more chickpea microcarriers within a threshold range of clump sizes. In some embodiments, the series of acts 1100 includes an act where the threshold range of clump sizes of one or more non-human cells and one or more chickpea microcarriers comprises between 75 micrometers and 125 micrometers. In some cases, the series of acts 1100 includes an act where the initial mixing speed within the range of initial mixing speeds causes the non-human cells to adhere to the chickpea microcarriers while limiting formation of aggregates of the non-human cells.
In certain embodiments, the series of acts 1100 further comprises an act where mixing the non-human cells with the cell culture media comprises: causing the non-human cells to adhere to the chickpea microcarriers by stopping a mixing motion, reducing a mix rate, or adding adherent factors.
In one or more embodiments, the series of acts 1100 further comprises an act where the initial mixing speed does not generate dead zones and suspends the chickpea microcarriers during an initial time period. In some embodiments, the series of acts 1100 includes an act where the initial mixing speed of the initial time period increases to a subsequent mixing speed within a range of subsequent mixing speeds that sufficiently mixes the non-human cells, the chickpea microcarriers, and cell culture media during a subsequent time period. In one or more embodiments, the series of acts 1100 includes an act where the range of subsequent mixing speeds comprises 60 rotations per minute (RPM) to 125 RPM.
In particular embodiments, the series of acts 1100 comprises an act where the textured cell tissue comprises the chickpea microcarriers, extracellular matrix proteins, and the non-human cells. In certain embodiments, the series of acts 1100 further comprises concurrently adding the non-human cells and the chickpea microcarriers to the cell culture media.
In one or more embodiments, the series of acts 1100 also comprises adding the non-human cells to the cell culture media, growing the non-human cells in the cell culture media, and adding the chickpea microcarriers to the cell culture media comprising a plurality of grown non-human cells.
In certain embodiments, the series of acts 1100 includes an act of adding the non-human cells to the cell culture media with a seeding density of 250,000 to 20,000,000 non-human cells per milliliter. In some embodiments, the series of acts 1100 includes an act in which harvesting textured cell tissue comprising the non-human cells adhered to the chickpea microcarriers further comprises removing at least a portion of the cell culture media, and reducing moisture content by pressing the textured cell tissue comprising the non-human cells adhered to the chickpea microcarriers. In one or more implementations, the series of acts 1100 includes an act of adding magnesium chloride to non-human cells and chickpea microcarriers within the cell culture media. In some cases, the series of acts 1100 includes an act where protein of the non-human cells adhering to the chickpea microcarriers ranges between 900 micrograms per milliliter and 1500 micrograms per milliliter.
In some embodiments, the series of acts 1200 includes an act in which filtering the discrete granules further comprises utilizing a filter not exceeding 150 microns. In certain embodiments, the series of acts 1200 includes an act where the chickpea protein is dry prior to grinding. In one or more implementations, the series of acts 1200 comprises mixing, within the cell culture media, non-human cells with the chickpea microcarriers, harvesting textured cell tissue comprising the non-human cells adhered to the chickpea microcarriers, and forming the textured cell tissue into a comestible food product.
In some embodiments, the series of acts 1200 includes an act where the discrete granules are hydrated in a separate vessel with media or dried by freeze-drying prior to providing the discrete granules of chickpea protein as the chickpea microcarriers.
As described, this disclosure describes various steps to create a comestible food product and describes various embodiments of a comestible food product. In some embodiments, the comestible food product comprises non-human cells grown from cell culture media; and chickpea microcarriers comprising textured chickpea protein and adhering to the non-human cells. Additionally, in some implementations, the textured cell tissue further comprises a first layer of the non-human cells over a surface of the chickpea microcarriers.
In some embodiments, the textured cell tissue further comprises a second layer of the non-human cells over a surface of the chickpea microcarriers. In one or more embodiments, the non-human cells form a myotube over a surface of the chickpea microcarriers. In certain implementations, the textured cell tissue further comprises extracellular matrix proteins.
The paragraphs above describe methods for forming a textured cell tissue into a cell-based food product.
As illustrated by step 1302 in
Cells may be extracted from the tissue 1304 that was removed from the animal. More specifically, the tissue 1304 is broken down by enzymatic and/or mechanical means. To illustrate,
Cells in the digested tissue 1306 may be proliferated under appropriate conditions to begin a primary culture. As illustrated in
In some examples, cells are stored and frozen (i.e., banked) at different steps along the cell culture process. Cryopreservation generally comprises freezing cells for preservation and long-term storage. In some implementations, tissue and/or cells are removed from a surface or substrate, centrifuged to remove moisture content, and treated with a protective agent for cryopreservation. For example, as part of cryopreservation, tissues and cells are stored at temperatures at or below −80 C. The protective agent may comprise dimethyl sulfoxide (DMSO) or glycerol.
Cells stored through cryopreservation may be used to replenish working cell stock. For instance, while a portion of the digested tissue 1306 is used as the cells 1308 spread on a surface or substrate, the remaining or excess digested tissue 1306 is transferred to cryovials 1310 for storage. Furthermore, the cells 1312 may be banked once reaching confluence and stored in cryovials 1313.
Once the cells 1312 have reached confluence, or just before the cells 1312 have reached confluence (e.g., occupation of about 80% of the substrate), the disclosed process comprises a series of cell passage steps. During cell passage, the cells 1312 are divided into one or more new culture vessels for continued proliferation. To illustrate, the cells 1312 may be diluted or spread on one or more surfaces or substrates to form the cells 1318. The cells 1318 are then grown 1316 to confluence, or just before confluence.
The cycle of dividing the cells 1312 into the cells 1318 for continued proliferation in new culture vessels may be repeated for a determined number of cycles. Typically, cell lines derived from primary cultures have a finite life span. Passaging the cells allows cells with the highest growth capacity to predominate. In one example, cells are passaged for five cycles to meet a desired genotypic and phenotypic uniformity in the cell population.
In some implementations, the disclosed method comprises immortalizing cells that have been grown and passaged for the determined number of cycles. For instance, the cells 1318 may be immortalized. As shown in
Cells that have reached immortality or a target growth capacity by living through a target passage level may be adapted to suspension culture. In one example, a suspension culture media and agitation of cells in this suspension environment help cells to adapt and start proliferating in the new growth environment. The cells adapted to suspension 1326 may be stored in cryovials 1328 for cryopreservation and banking. Cells in suspension 1326 will begin to proliferate and the process begins a series of dilute and expand steps.
During dilution and expansion, cells are moved from growth vessels into newer, and progressively larger, growth vessels. For example, cells in suspension 1326 may begin in a single tube. The cells will proliferate and increase in cellular density. Once the cells have reached a target cell number (i.e., viable cell density (VCD) at desired volume), they are diluted and moved to a larger growth vessel. Optionally, the cells are banked in cryovials throughout expansion. For example, once cells in suspension reach a maximum VCD, the cells may begin to leave exponential growth due to overcrowding. After reaching a target density, the suspension cells may be transferred to a larger vessel 1330 and diluted with additional media. The dilute-and-expand steps are repeated using progressively larger vessels (e.g., the vessel 1331 and the vessel 1332) and/or progressive dilution until the cells reach a production-ready volume. For example, cells may be production ready at about a 1,000-100,000-liter scale at 5 million cells per mL. The cells may be banked in cryovials at any of the dilution and expansion cycles.
As part of preparing cells to form a comestible food product, the disclosed process comprises growing the cells on microcarriers in a suspension. The cells grown in suspension may remain in the vessel 1332 or may be transferred to a different bioreactor.
In some implementations, and as illustrated in
Prior to optionally finishing the cells of the present disclosure in the adherent bioreactors 1348, the cells are grown in suspension conditions to grow into cell tissue adhering to the microcarriers. Once they have grown to a target density, either according to a learned timing or according to a measured fluctuation in cell metabolism of components such as glucose and oxygen, then the cell tissue is ready for removal or further processing. The removal process of the disclosed method uses filters to separate the cell tissue from the media. The wash buffer 1356 and cell tissue are flowed through a filter 1352 where the cell tissue is collected into one or more cell tissue masses 1354.
As described above and depicted in
In some implementations, the non-human cells are prepared by flowing the non-human cells and chickpea microcarriers suspended in cell culture media across substrates in the plurality of adherent bioreactors. More particularly, the non-human cells and chickpea microcarriers from the suspension bioreactor vessel may contact or land on the substrates in the plurality of adherent bioreactors. The non-human cells, chickpea microcarriers, and cell culture media that flowed through the adherent bioreactors are cycled back to the suspension bioreactor vessel. The cell culture media, non-human cells and chickpea microcarriers can be cycled through the adherent bioreactors until a target adherent cell volume is reached. For instance, in some implementations, the disclosed method comprises measuring a cell density of outflow from the adherent bioreactors to infer an adherent cell volume.
The non-human cells grow into adherent textured cell tissue within the adherent bioreactors. Once they have grown to a target volume or quality, either according to a learned timing or according to a measured fluctuation in cell metabolism of components such as glucose and oxygen, then the adherent textured cell tissue is ready for removal. The removal process of the disclosed method uses a high-pressure flow to shear the adherent textured cell tissue comprising the non-human cells and chickpea microcarriers off the substrate surfaces. In one example, wash buffer from a wash tank is flowed across the substrates in the adherent bioreactors. The wash buffer and textured cell tissue mixture are flowed through a filter where the textured cell tissue is collected into one or more cell tissue masses.
The cell tissue masses 1354 may be further processed to adjust moisture content.
In accordance with common practice, the various features illustrated in the drawings may not be drawn to scale. The illustrations presented in the present disclosure are not meant to be actual views of any particular apparatus (e.g., device, system, etc.) or method, but are merely idealized representations that are employed to describe various embodiments of the disclosure. Accordingly, the dimensions of the various features may be arbitrarily expanded or reduced for clarity. In addition, some of the drawings may be simplified for clarity. Thus, the drawings may not depict all of the components of a given apparatus (e.g., device) or all operations of a particular method.
Terms used herein and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including, but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes, but is not limited to,” etc.).
Additionally, if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations.
In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” or “one or more of A, B, and C, etc.” is used, in general such a construction is intended to include A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B, and C together, etc. For example, the use of the term “and/or” is intended to be construed in this manner.
Further, any disjunctive word or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” should be understood to include the possibilities of “A” or “B” or “A and B.”
However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations.
Additionally, the use of the terms “first,” “second,” “third,” etc., are not necessarily used herein to connote a specific order or number of elements. Generally, the terms “first,” “second,” “third,” etc., are used to distinguish between different elements as generic identifiers. Absent a showing that the terms “first,” “second,” “third,” etc., connote a specific order, these terms should not be understood to connote a specific order. Furthermore, absent a showing that the terms “first,” “second,” “third,” etc., connote a specific number of elements, these terms should not be understood to connote a specific number of elements. For example, a first widget may be described as having a first side and a second widget may be described as having a second side. The use of the term “second side” with respect to the second widget may be to distinguish such side of the second widget from the “first side” of the first widget and not to connote that the second widget has two sides.
All examples and conditional language recited herein are intended for pedagogical objects to aid the reader in understanding the invention and the concepts contributed by the inventor to furthering the art and are to be construed as being without limitation to such specifically recited examples and conditions. Although embodiments of the present disclosure have been described in detail, it should be understood that the various changes, substitutions, and alterations could be made hereto without departing from the spirit and scope of the present disclosure.
The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Indeed, the described embodiments are to be considered in all respects only as illustrative and not restrictive. For example, the methods described herein may be performed with less or more steps/acts or the steps/acts may be performed in differing orders. Additionally, the steps/acts described herein may be repeated or performed in parallel to one another or in parallel to different instances of the same or similar steps/acts. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes that come within the meaning and range of equivalency of the claims are to be embraced within their scope.
This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63/505,989, titled, “CHICKPEA MICROCARRIERS,” filed on Jun. 2, 2023, which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63505989 | Jun 2023 | US |
