US20230287317A1 - Cultured tissue and bioreactor systems and methods for production thereof - Google Patents

Cultured tissue and bioreactor systems and methods for production thereof Download PDF

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US20230287317A1
US20230287317A1 US18/041,371 US202118041371A US2023287317A1 US 20230287317 A1 US20230287317 A1 US 20230287317A1 US 202118041371 A US202118041371 A US 202118041371A US 2023287317 A1 US2023287317 A1 US 2023287317A1
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cell
bioreactor
laden
cultured tissue
cells
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David L. Kaplan
John Yuen
Natalie R. Rubio
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Tufts University
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Tufts University
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/58—Reaction vessels connected in series or in parallel
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/16—Hollow fibers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2513/00—3D culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2537/00—Supports and/or coatings for cell culture characterised by physical or chemical treatment

Definitions

  • the present disclosure generally relates to cultured tissue and to methods for producing cultured tissue.
  • the present disclosure further relates to bioreactor systems for manufacturing the cultured tissue.
  • the cultured tissue may be cultured meat that resembles whole muscle meat.
  • Cultured meat (also called in vitro, cultivated, lab grown meat) prepared using tissue and bioengineering techniques in vitro is another alternative to traditional animal agriculture.
  • meat muscle and fat tissue
  • the time frame to generate cultured meat tissues in vitro is also thought to be faster compared to traditional animal agriculture, and may only require weeks as opposed to months or years for pork and beef, for example.
  • tight control over cell biology during tissue cultivation, as well as the production process allows for the fine tuning of nutritional parameters by engineering muscle or fat cells to produce vital nutrients that would otherwise not be found (or found only at low concentrations) in conventional meat.
  • cultured meat production systems may offer healthier, more efficient, and more environmentally friendly alternatives to animal-derived meats.
  • tissue engineering for the production non-animal derived foods
  • a particular challenge is not only cell and tissue density, but also the alignment of the cells and matrices (scaffolds, extracellular matrix) to emulate the native structure and function of tissues and food.
  • achieving mechanical requirements as well as mastication and organoleptic features are important goals.
  • animal skeletal muscles are striated and packed into dense arrangements of fiber bundles.
  • these features provide the specific texture and mouthfeel obtained when biting into a whole muscle cut of meat (e.g., steak).
  • the system may include a first bioreactor.
  • the first bioreactor may include an internal chamber containing culture medium, a fiber inlet for feeding a fiber scaffold into the internal chamber, and a cell inlet for feeding precursor cells into the internal chamber.
  • the precursor cells may proliferate and differentiate on a surface of the fiber scaffold in the culture medium to provide a cell-laden fiber composed of cells attached to the fiber scaffold.
  • the first bioreactor may further include an outlet through which the cell-laden fiber emerges from the first bioreactor.
  • the cell-laden fiber may be used in the production of the cultured tissue.
  • the method may include feeding a fiber scaffold into a chamber containing culture medium, seeding the chamber with precursor cells, and allowing the precursor cells to proliferate and differentiate on a surface of the fiber scaffold to provide a cell-laden fiber composed of cells adhered to the fiber scaffold.
  • the method may further include twisting a plurality of cell-laden fibers to provide a cell-laden yarn, and weaving or knitting the cell-laden yarn into a three-dimensional (3D) structure to provide the cultured tissue.
  • cultured tissue including a plurality of cell-laden fibers each comprised of cells attached to a fiber scaffold.
  • the plurality of cell-laden fibers may be twisted into a cell-laden yarn, and the cell-laden yarn may be further woven or knitted into a three-dimensional (3D) shape.
  • the cultured tissue may exhibit a structural organization that mimics skeletal muscle tissue.
  • FIG. 1 is a schematic representation of a system for the production of cultured tissue, according to an embodiment of the present disclosure.
  • FIG. 2 is a flow chart of steps that may be involved in producing the cultured tissue, according to an embodiment of the present disclosure.
  • FIG. 3 is a schematic representation of a first bioreactor of the system of FIG. 1 , according to an embodiment of the present disclosure.
  • FIG. 4 is a side, transparent view of the first bioreactor, in accordance with the present disclosure.
  • FIG. 5 is a perspective, transparent view of the first bioreactor, according to an embodiment of the present disclosure.
  • FIG. 6 is a top, transparent view of the first bioreactor, according to an embodiment of the present disclosure.
  • FIG. 7 is a schematic representation of an operation at a second bioreactor of the system of FIG. 1 , according to an embodiment of the present disclosure.
  • FIG. 8 is a schematic representation of operations performed at a third bioreactor of the system of FIG. 1 , according to an embodiment of the present disclosure.
  • FIG. 9 shows scanning electron microscopy (SEM) images of a silk fiber cord manufactured with a twisting machine, and human mesenchymal stem cells (hMSCs) that attached, spread, and formed confluent cells sheets on the sill fiber cord (scale bar: 100 micrometers ( ⁇ m), in accordance with the present disclosure.
  • SEM scanning electron microscopy
  • FIG. 10 shows results of live/dead fluorescence staining of C2C12 cells on commercially available fibers and tissue culture polystyrene (TSP) (control) over 8 days of culture, in accordance with the present disclosure.
  • TSP tissue culture polystyrene
  • the cultured tissue 12 may be cultured whole muscle meat suitable for consumption and having a structural organization and hierarchy that mimics natural whole muscle meat.
  • the system 10 employs principles from textile engineering to generate the cultured tissue 12 , whereby fibers of muscle and fat are first cultured in vitro and then twisted into yarns and knitted/weaved and folded or stacked into large, macroscale two-dimensional (2D) or three-dimensional (3D) tissue constructs. This process imparts strength in the resulting cultured tissue 12 and provides a structural organization and hierarchy reminiscent of that in skeletal muscle tissue.
  • the cultured tissue 12 may have a stiffness that approaches, matches, or surpasses that of native bovine muscle (about 12 kilopascals (kPa)).
  • the cultured tissue 12 may further exhibit marbling of fat tissue that resembles fat marbling in whole muscle meat.
  • the cultured tissue 12 has a rectangular prism structure in FIG. 1 for simplicity, it will be understood that the cultured tissue 12 may have many other 2D or 3D shapes in practice.
  • the system 10 may include one or more bioreactors 14 or bioreactor stations which operate to produce the cultured tissue 12 .
  • the system 10 may be run in separate unit operations, or as a continuous, robotically-controlled, and automated process. For the continuous, automatic process, the output from each stage/bioreactor 14 may be fed directly into the next, allowing for minimal human intervention, sterility, and reduced risk of cell contamination.
  • One or more computer controllers 16 may be in communication with the bioreactors 14 for automating and controlling the operations thereof.
  • the system 10 may include a first bioreactor 18 , a second bioreactor 20 downstream of the first bioreactor 18 , and a third bioreactor 22 downstream of the second bioreactor 20 .
  • precursor cells 24 may proliferate and differentiate on a fiber scaffold 26 in culture media to provide a cell-laden fiber 28 composed of mature cells 29 (mature muscle or fat cells) attached to the fiber scaffold 26 (also see FIG. 3 ).
  • the fiber scaffold 26 may be composed of a fiber.
  • a “fiber” is a basic building block of a fabric that is significantly longer than it is wide.
  • a plurality of the cell-laden fibers 28 emerging from one or more of the first bioreactors 18 may be combined and twisted to form a cell-laden yarn 30 at the second bioreactor 20 (also see FIG. 7 ).
  • a “yarn” is a continuous strand of fibers that are spun or twisted together.
  • the cell-laden yarn 30 emerging from the second bioreactor 20 may be knitted or woven and folded, rolled, and/or stacked into various 2D and 3D constructs at the third bioreactor 22 (also see FIG. 8 ).
  • Inputs into the first bioreactor 18 may include the fiber scaffold 26 , the precursor cells 24 , and culture medium, and the output of the first bioreactor 18 may be the cell-laden fiber 28 (also see FIG. 3 ).
  • the input and the output of the second bioreactor 20 may be cell-laden fibers 28 and the cell-laden yarn 30 , respectively (also see FIG. 7 ).
  • the input and the output of the third bioreactor 22 may include cell-laden fiber yarns 30 and the cultured tissue 12 , respectively.
  • more or fewer bioreactors 14 may be used for the production of the cultured tissue 12 , with the above operations of the bioreactors 14 delegated in various different ways.
  • the fiber scaffold 26 may be fed into an internal chamber 34 of the first bioreactor 18 containing a culture medium (also see FIG. 3 ).
  • a block 36 may involve seeding the internal chamber 34 containing the culture medium with precursor cells 24 (also see FIG. 3 ).
  • the precursor cells 24 may be seeded onto the fiber scaffold 26 using a sol-gel dispensing system.
  • the blocks 32 and 36 may be carried out in different orders or simultaneously in practice.
  • the precursor cells 24 may be permitted to proliferate and differentiate on a surface of the fiber scaffold 26 in the culture medium to provide the cell-laden fiber 28 .
  • the transit time and culture medium in the first bioreactor 18 may be tuned to provide a desired degree of coverage of differentiated cells on the fiber scaffold 26 .
  • confluence (or a desired degree of coverage of differentiated cells on the surface of the fiber scaffold 26 ) may be identified when at least 70-80% of a surface area of the fiber scaffold 26 is coated with the mature cells.
  • the cells 24 may be cultured to a confluence of at least 75% surface area coverage in the first bioreactor 18 .
  • Cell differentiation may be indicated by the expression of myosin heavy chain (WIC) in muscle cells, and by the accumulation of lipid in fat cells.
  • WIC myosin heavy chain
  • a plurality of cell-laden fibers 28 emerging from first bioreactors 18 may be combined and twisted to impart densification into the fiber-cell matrices and provide the cell-laden yarn 30 .
  • the cell-laden yarn 30 emerging from the second bioreactor 20 may be knitted or woven and folded or stacked into various 2D or 3D structures to provide the culture tissue 12 .
  • the block 40 may be performed at the second bioreactor 20
  • the block 42 may be performed at the third bioreactor 22 .
  • the first bioreactor 18 may include a body 44 having the internal chamber 34 containing the culture medium, one or more fiber inlets 46 for feeding the fiber scaffold 26 into the internal chamber 34 , and one or more cell inlets 48 for feeding the precursor cells 24 into the internal chamber 34 .
  • the fiber scaffold 26 and the precursor cells 24 may be fed into the first bioreactor 18 via the same inlet.
  • the first bioreactor 18 may further include one or more outlets 50 through which the cell-laden fiber 28 emerges from the first bioreactor 18 .
  • the first bioreactor 18 may include one or more translucent sections 52 to allow observation of the internal chamber 34 and monitoring of the cell seeding/differentiation processes.
  • the precursor cells 24 may attach to and proliferate on the surface of the fiber scaffold 26 , and differentiate into mature cells 29 on the surface of the fiber scaffold 26 .
  • Different bioreactors fiber scaffold lines
  • the fiber scaffold lines with different cell types may be combined in later stages of the process with continuous cultivation for expansion and differentiation with sufficient residence time in the system to optimize tissue outcomes (e.g., myotubes for muscle, fat droplets for fat, extracellular matrix depositions representative of those found in meat).
  • a time for the cells 24 to attach to and reach confluence on the fiber scaffold 26 may range from 12 to 48 hours, and a time for cell differentiation into the mature cells 29 may range from 7 to 21 days.
  • cell growth may be continued until a surface of the fiber scaffold 26 is at least 70% or at least 80% covered by differentiated cells.
  • cell differentiation of at least 90% may be achieved in the first bioreactor 18 .
  • Cell growth may continue once the cell-laden yarns 30 are woven into their desired forms, and may be halted by freezing during storage/transport. It may not be necessary for the cells to be alive once the fibers are formed, as the cultured tissue may be cooked prior to consumption.
  • Factors such as, but not limited to, the rate of translation of the fiber scaffold 26 through the first bioreactor 18 and the composition of the culture medium may be tuned/adjusted to provide a desired level of cell coverage or confluence on the fiber scaffold 26 and/or to control cell differentiation.
  • cell proliferation to differentiation may be driven by a shift in media composition.
  • satellite cells may be proliferated in a growth factor-rich proliferation media, and triggered for differentiation in a growth factor-poor differentiation media, with the concentration of the growth factor decreasing along the length of the internal chamber 34 from a proximal end 54 to a distal end 56 .
  • Performing cell proliferation and differentiation initially on the fiber scaffold 26 at the first bioreactor 18 addresses mass transport issues of tissue engineering, as tissue densification (and its associated nutrient/O 2 diffusion constraints) is decoupled/delayed until after maturation of individual cell-laden fibers 28 . Further, the use of cell-laden fibers 28 as the cultured meat building blocks fosters cell and extracellular matrix alignment along the fiber axis, thereby enhancing mechanics and texture.
  • the first bioreactor 18 may include a first spool 58 run by a motor which may provide a continuous feed of the fiber scaffold 26 into the fiber inlet 46 and into the internal chamber 34 .
  • the first spool 58 may be mounted on an outside of the first bioreactor 18 at or near a proximal end 60 of the first bioreactor 18 .
  • a cover 62 may be placed over the first spool 58 to maintain sterility of the spooled fiber scaffold.
  • a second spool 64 run by a motor may re-spool the cell-laden fiber 28 that emerges from the outlet 50 of the first bioreactor 18 .
  • the second spool 64 may be mounted outside of the first bioreactor 18 at or near a distal end 66 of the first bioreactor 18 .
  • the second spool 64 having the re-spooled cell-laden fiber 28 may be removed, shipped, and/or stored if desired.
  • one or more rods 68 may extend into the culture medium for keeping the fiber scaffold 26 submerged inside of the culture medium and preserving cell viability. As the rods 68 may keep the fiber scaffold 26 submerged in the culture media, the internal chamber 34 may not need to be completely filled with culture media and smaller volumes of culture media may be used. In one embodiment, the rods 28 may be posts having rotating sponges on their ends.
  • the internal chamber 34 may also include a media divider 70 to keep proliferation and differentiation media separated in two compartments 72 of the internal chamber 34 . A media divider may not be included in some embodiments which use a single, combination proliferation/differentiation culture medium (see below). Culture media may be replenished via one or more inlet ports 74 and outlet ports 76 .
  • Sterility of the developing cell-laden fiber 28 may be maintained by the closed system of the internal chamber 34 .
  • the first bioreactor 18 may have a slide-on lid 78 (see FIGS. 5 - 6 ).
  • all or a portion of the body of the first bioreactor 18 may be composed of a translucent material, such as polycarbonate.
  • FIG. 7 A schematic representation of the second bioreactor 20 is shown in FIG. 7 .
  • the cell-laden fibers 28 emerging from one or more first bioreactors 18 may be transferred to the second bioreactor 20 for densification.
  • Versatility in the resulting cell-laden yarn 30 may be provided by varying which process lines are combined at the second bioreactor 20 .
  • cell-laden yarns 30 composed of muscle cells, fat cells, and combinations thereof may be generated at the second bioreactor 20 by combining/twisting various combinations of muscle cell-laden fibers and fat cell-laden fibers. Twisting and/or braiding of the cell-laden fibers 28 using programmable strain rates and extents at the second bioreactor 20 may impart controlled densification into the resulting cell-laden yarn 30 .
  • the second bioreactor 20 may include wheels 80 driven by motors which attach to each end of the cell-laden fibers 28 .
  • the wheels 80 may rotate at a rotation rate to provide fiber bundles or yarns.
  • the degree of twisting, yarn diameter, and yarn density may be determined based on combinations of mechanical targets (e.g., food-like for meats) and desired cell outcomes (e.g., survival, function, retention).
  • the second bioreactor 20 may produce muscle cell-laden yarns with controlled densification that mimic the density of mammalian skeletal tissue (about 1.06 kilograms (kg)/liter (L), and fat cell-laden yarns with a density similar to adipose tissue (about 0.92 kg/L).
  • the resulting yarns may have varying diameters.
  • the densified cell-laden yarns 30 emerging from the second bioreactor 20 may have diameters that range from about 50 micrometers ( ⁇ m) to about 100 ⁇ m.
  • One or more of the densified cell-laden yarns 30 from the second bioreactor 20 may proceed to the weaving and knitting phase at the third bioreactor 22 which may build up 2D or 3D structures from the previously constructed muscle and adipose yarns.
  • the inputs into the third bioreactor may include one or more muscle cell-laden yarns, one or more fat cell-laden yarns, or combinations of muscle cell-laden yarns and fat cell-laden yarns.
  • the ratio of the muscle cell-laden yarns to the fat cell-laden yarns may be selected/controlled to provide various muscle and fat contents in the resulting cultured tissue 12 , as well as to mimic marbling in whole muscle meat. As shown in FIG.
  • the cell-laden yarns 30 may be woven, braided, or knitted into 2D sheets 82 , and one or more of the 2D sheets 82 may be folded, twisted, rolled 84 , and/or stacked 86 to provide the 3D construct of the cultured tissue 12 .
  • the resulting cultured tissue 12 may emulate steak or meat rolls, for example.
  • the cell-laden yarns 30 may be directly knitted or weaved into a 3D structure.
  • the fiber scaffold 26 may be made of an edible biomaterial that supports cell and tissue growth and is compatible for continuous culture in a flow through device.
  • the fiber scaffold 26 may be composed of edible fibers from natural sources such as collagen, silk, and chitosan which have used in textile-based engineering.
  • other edible and economic biomaterials such as wheat gluten, cellulose, zein, starch, and soy may also be used. Fabrication of these materials into fibers may be achieved by electrospinning (see, for example, Woerdeman, D. L.; Ye, P.; Shenoy, S.; Parnas, R. S.; Wnek, G.
  • Additional fiber materials for the fiber scaffold include, but are not limited to, fungal mycelia, mung bean fibers, or combinations of any of the foregoing materials. Fibrous materials from natural sources may be used to foster cell expansion and tissue alignment, and to support cell differentiation on the fibers.
  • the fiber materials may be commercially available as large-scale agricultural products and byproducts.
  • Custom options for fiber materials include, but are not limited to, cellulose fibers suspended within a bioreactor by magnetic beads, vacuum-based suspension, or gel fibers with a sacrificial outer gel and an inner core fiber composed of cells that secrete their own extracellular matrix over several weeks.
  • the fiber scaffold 26 may support cell viability at greater than 80%, and cell adhesion at greater than 70% after 48 hours of culture with a differentiation efficiency within 20% of control (i.e., tissue culture plastic) conditions. In some aspects, the fiber scaffold 26 may support more than 90% cell coverage within 48 hours of culture when using a high cell seeding density. Furthermore, the fiber scaffold 26 may be strong enough to be handled and loaded between bioreactor components in the bioreactors without breaking or deforming. The fiber scaffold 26 may also be windable during operations. In some embodiments, the fiber scaffold 26 may have an ultimate tensile strength that ranges from 3 kilopascals (kPa) to 40 kPa.
  • kPa kilopascals
  • the fiber scaffold 26 may conform to the mechanical properties of meats with Warner Bratzler Shear force values of 2 to 8 kg, thus capturing the required strength for textile engineering as well as consumer expectations in terms of bite and chew. These properties may be attributes of the fiber scaffold 26 alone or with one or more coatings.
  • the fiber scaffold 26 may include one or more coatings to provide desirable properties such as those mentioned above, and/or to improve cell attachment to the fiber scaffold 26 .
  • Various cost-effective biopolymers or complex extracts from natural sources may be used as coating materials.
  • extracellular matrix proteins and/or chemical/synthetic coatings may be used as coatings to improve cell attachment to the natural fibers and mimic in vivo cell behavior.
  • coating materials may include commercially available products such as, but not limited to, fibronectin, laminin, vitronectin, collagen, cadherin, elastin, hyaluronic acid, poly-D-lysine, poly-L-lysine, poly-L-ornithine, concanavalin A, soy, and other adhesive, non-toxic chemicals.
  • Conconavalin A, laminin, and hyaluronic acid may be obtained from animal-free origins, and have been shown to enhance muscle cell attachment to various biomaterials.
  • the fiber scaffold 26 may have a gel coating.
  • the coating component may be integrated into the continuous bioreactor system to produce the coated fiber scaffold on demand.
  • the cells 29 may be edible cells including muscle cells, fat cells, and combinations thereof.
  • the precursor cells 24 may be muscle precursor cells or adipoctye precursor cells. Examples of suitable cell types include, but are not limited to, satellite cells, fat cells (i.e., adipocytes), fibroblasts, myoblasts, muscle cells, precursors thereof, and combinations thereof.
  • the cells may be from animal source including, without limitation, from bovine, avian (e.g., chicken, quail), porcine, or murine sources.
  • the cells may also be derived from seafood such as fish (e.g., salmon, tuna, etc.), shellfish (e.g., clams, mussels, and oysters); crustaceans (e.g., lobsters, shrimp, prawns, and crayfish), and echinoderms (e.g., sea urchins and sea cucumbers).
  • the cells 29 may be engineered to produce vital nutrients such as proteins and essential fatty acids.
  • transgenic cells may be used to decrease the time needed for cell differentiation.
  • media formulations may include transgenic components to drive cell differentiation.
  • tetracycline-responsive promoters inserted into transgenic cells may be activated by including tetracycline in the culture medium, resulting in forced expression of myogenic or adipogenic genes in edible cell lines (e.g., chicken fibroblasts, bovine satellite cells, etc.).
  • edible cell lines e.g., chicken fibroblasts, bovine satellite cells, etc.
  • a single combination proliferation/differentiation culture medium may be used for both cell proliferation and differentiation in the first bioreactor 18 .
  • a simultaneous proliferation/differentiation culture medium may be beneficial for rapid cell attachment and differentiation, while also minimizing the amount of medium needed within the chamber 34 .
  • An exemplary combination proliferation-differentiation culture medium for muscle cells may be Dulbecco's Modified Eagle Medium (DMEM) with fetal bovine serum (FBS), insulin-like growth factor 1 (IGF-1), and insulin.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • IGF-1 insulin-like growth factor 1
  • a suitable proliferation-differentiation medium which achieves both high cell density and efficient myogenesis of the cell-laden fibers 28 may be DMEM, 10% FBS, IGF-1 (1 nanogram (ng)/milliliter (mL), 10 ng/mL, or 100 ng/mL) and 10 micrograms ( ⁇ g)/mL insulin.
  • the combination proliferation-differentiation culture medium may be DMEM, 10% FBS, 100 ng/mL IGF-1, and 10 ⁇ g/mL insulin.
  • two different media formulations for cell proliferation and cell differentiation may be used within two different compartments 72 of the first bioreactor 18 .
  • bovine satellite cells may be continuously seeded onto the fiber scaffold (with or without coatings).
  • Bovine satellite cells may be cultured in growth media with growth factors (e.g., DMEM with Glutamax, 20% FBS, and 1% antiobiotic-antimycotic, and 1 ng/mL human fibroblast growth factor 2 (FGF-2)).
  • growth factors e.g., DMEM with Glutamax, 20% FBS, and 1% antiobiotic-antimycotic, and 1 ng/mL human fibroblast growth factor 2 (FGF-2)
  • FGF-2 human fibroblast growth factor 2
  • Bovine fat cells may also be coated onto the fiber scaffold 26 and cultured in growth media (e.g., DMEM with Glutamax, 20% FBS, 1% antibiotic-antimycotic).
  • growth media e.g., DMEM with Glutamax, 20% FBS, 1% antibiotic-antimycotic.
  • DMEM fetal bovine serum
  • FBS fetal bovine serum
  • free fatty acid solution may be 50 millimolar (mM) free fatty acid solutions containing elaidic acid, erucic acid, myristoleic acid, oleic acid, palmitoleic acid, phytanic acid, and pristanic acid.
  • ORO Oil Red O
  • Various parameters of the system 10 may be controlled/programmed via the computer controller 16 (or controlled manually) to optimize features such as cell proliferation/differentiation, cell attachment to the fiber scaffold 26 , and the composition, density, bite, and texture of the cultured tissue 12 .
  • a time frame for proliferation and differentiation of the precursor cells 24 in the first bioreactor 18 may be controlled to reach target percentages for differentiation and degree of cell attachment on the fiber scaffold 26 .
  • Other controlled parameters may include the degree of twisting of the cell-laden fibers 28 at the second bioreactor 20 , the diameter of the yarns 30 , the rotation rate of the wheels 80 of the second bioreactor 20 , the size and shape of the cultured tissue 12 , the packing density of the cultured tissue 12 , and the composition of the cultured tissue 12 including the cell types, fiber scaffold composition, and the ratio of muscle-cell laden fibers and fat-cell laden fibers in the cultured tissue product.
  • the structural hierarchy and marbling of the cultured tissue construct may be tunable by changing the ratio of muscle cell fibers and fat cell fibers. Warner-Bratzler shear force test may be used to assess the texture and tenderness of the cultured tissue product.
  • the present disclosure provides a bioreactor system based on textile engineering principles for cultured meat production.
  • Cultured muscle and adipose cells on edible fibers are integrated into tissue assemblies via twisting, weaving or knitting and rolling, stacking, and/or folding to provide versatile outputs that meet target metrics pertaining to properties such as texture, thermal response upon cooking, composition, nutrition, density, alignment, composition, and marbling.
  • This textile engineering-based system is cost-efficient, scalable, and generates cultured meats that mimic whole muscle through the recapitulation of structural hierarchy present in in vivo skeletal muscle.
  • the technique facilitates fabrication of constructs with controlled microstructure, mechanical properties, and cellular distribution which plays an important role in the engineering of structured hierarchical tissues.
  • fibril scaffolds enables an effective mass (nutrition)/oxygen transfer in the cell culture system as cell-laden fibers are fully surrounded by culture media, avoiding complications inherent to perfusion systems.
  • the technology disclosed herein may enable economic mass production of cultured whole muscle meat.
  • FIG. 9 B shows initially attached hMSCs on the silk cord shown in FIG. 9 A .
  • FIG. 9 C shows initial spreading of the hMSCs on the silk cord 1 hour after seeding.
  • FIG. 9 D shows hMSCs and extracellular matrix coating the silk cord 7 days post-seeding.
  • FIG. 9 E shows thick encapsulation of the silk cord by hMSCs and extracellular matrix 14 days following seeding.
  • a bioreactor (first bioreactor) was machined from polycarbonate (McMaster Carr, Lancaster, Pa.) and assembled using interlocking components. The prototype is translucent to allow observation of the cell seeding and differentiation processes.
  • a fiber scaffold is fed through a spool run by a motor (Honeywell, Columbus, Ohio). Spool sterility is maintained by a cover over the spool. Culture medium is replenished through inlet ports and sterility is maintained by the closed system of the chamber. Small amounts of culture medium ( ⁇ 100 mL) may be used. Rods in the chamber keep the fiber scaffold submerged in the culture medium so that the chamber does not need to be completely filled with medium (per cost considerations).
  • the cell-laden fiber may proceed directly to the second bioreactor or be re-spooled at another spool run by a motor at a distal end of the chamber.
  • the re-spooled fiber may be removed, shipped, or stored as needed, or used directly to enter the second bioreactor (see FIG. 7 ).
  • the fibers may be spooled at the second bioreactor in some arrangements.
  • Fibers were screened and selected based on availability in fiber format, edibility, cost, mechanical properties, and ability to support cell culture (e.g., viability, adhesion, differentiation).
  • Selected fibers included cellulose, chitosan, soy protein, starch, wheat gluten, other plant-based materials, and silk.
  • the fibers were purchased from Amazon. As described above, many other biomaterials may be used for the textile systems.
  • Fibers were mechanically tested after soaking for 24 hours in DMEM at 37° C. Testing was conducted at 37° C. in a saline bath to mimic conditions during spooling at a rate of 1% strain per second. Stress, strain at maximum stress, and elastic modulus were determined from the stress-strain curve. Elastic modulus was calculated as the slope of the linear region of the curve.
  • All fibers had a circular cross-section, and stress was calculated by inputting the cross-section diameter into the Instron software.
  • the cross-section was measured 3 times using electronic calipers (Carerra, New York, N.Y.) and averaged.
  • the linear region was defined by fitting a linear equation to the portion of the stress-strain curve that resulted in a R 2 value of >95% for the fit.
  • Mechanical testing was conducted for coated and uncoated fibers to evaluate if coating the materials impacted the mechanics.
  • C2C12 cells are mouse skeletal muscle myoblasts that form multinucleated myotubes, and are an established cell line for evaluating myogenesis in response to various stimuli. Viability and attachment were assessed via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay and Live/Dead staining, respectively (Invitrogen, Carlsbad, Calif.).
  • MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
  • viable and metabolically active cells metabolize purple formazan into yellow salts, and the change in color can be used to generate a viability curve with a standard plate reader (Thermo Fisher, Pittsburgh, Pa.).
  • Live/Dead staining results in a fluorescent green stain for viable cells, and a fluorescent red stain for dead cells, allowing for a rapid visual assessment of cell viability following attachment onto a substrate.
  • Live/Dead staining was imaged using a Keyence BZX fluorescent microscope (Keyence Corporation, Pittsburgh, Pa.).
  • FIG. 10 shows results of Live/Dead staining of C2C12 cells on commercially available fibers and tissue culture polystyrene (TCP) (control) over 8 days culture. Soy protein fibers supported roughly 40% cell attachment and viability. Results from mung bean noodle ( ⁇ 1% attachment), spaghetti (30% attachment), cotton fiber (15% attachment), and TCP (>90% attachment) are also shown.
  • a combination muscle cell proliferation/differentiation culture medium was designed to achieve both high cell density and efficient myogenesis of the cell-laden fibers.
  • a combination proliferation-differentiation culture medium for muscle cells is desirable as it will enable the use of a single medium with the first bioreactor.
  • C2C12 cells were grown in baseline culture medium (DMEM, 10% FBS, 1% antibiotic-antimycotic), low-serum medium (DMEM, 1% FBS, 1% antibiotic-antimycotic), serum-free medium (DMEM, 1% insulin-transferrin-selenium, 1% N 2 ), and proliferation-differentiation medium (DMEM, 10% FBS, 1, 10, or 100 ng/mL IGF-1, 10 ⁇ g/mL insulin).
  • the C2C12 cells were grown on 2D culture wells and coverslips and in 3D sponges in different media combinations for up to 28 days. The cells were then evaluated for myogenic differentiation and proliferation by Western blot, and for morphology via staining and imaging. Cells were harvested for Western blot analysis via radioimmunoprecipitation (RIPA) cell lysis buffer and HaltTM protease inhibitor (Invitrogen). Sodium dodecyl sulfate (SDS) was added to the collection buffer at a 1:1 ratio and samples were sonicated, heated to 100° C. for 5 minutes, and loaded into Novex Wedgewell 4-20% Tris-Glycine Gels (Invitrogen). Protein content was normalized across timepoints by varying gel loading volume.
  • RIPA radioimmunoprecipitation
  • HaltTM protease inhibitor Invitrogen
  • SDS Sodium dodecyl sulfate
  • Blots were transferred to nitrocellulose membranes (Invitrogen), blocked in 5% milk in tris-buffered saline (Boston Bioproducts, Ashland, Mass.) with 0.1% Tween20 (TBST) (Acros Organics, Morris Plains, N.J.), and incubated at 4° C. overnight on an orbital shaker in 5% bovine serum albumin (BSA) in TBST with primary antibodies for MyoD, myosin heavy chain (WIC), and ⁇ -actin (Abcam, Cambridge, Mass.), that were diluted to 1:2000, 1:2000, or 1:10000, respectively.
  • BSA bovine serum albumin
  • HRP horseradish peroxidase
  • ECL electrogenerated chemiluminescence
  • DAPI 4,6-diamidino-2-phenylindole
  • actin cytoskeleton was visualized using fluorescein isothyanate (FITC)-phalloidin (Life Tech., Waltham, Mass.).
  • FITC fluorescein isothyanate
  • Cells were imaged using a Keyence BZX fluorescent microscope (Keyence Corporation).
  • the combination proliferation-differentiation medium having DMEM, 10% FBS, 100 ng/mL IGF-1, and 10 ⁇ g/mL insulin resulted in the highest cell growth and formed multinucleated myotubes at 14 days.

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