Method, device and system for producing cultured tissue

Abstract

Methods, devices, and systems for producing cultured tissue are provided. The method comprises the steps of: (a) providing a hydrogel composition and coagulating the hydrogel composition to form a hydrogel portion, wherein the hydrogel portion comprises a plurality of first channels in a predetermined first array; (b) providing at least one first cell composition to the single first channel to form at least one first cell culture therein, such that the tissue construct is formed. These methods, devices, and systems produce cultured meat having a controlled distribution of tissue components, and thus the texture of the cultured tissue is similar to that of natural tissue.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application Serial No. 63 / 386,495, filed on December 7, 2022. The entire contents of the foregoing application are hereby incorporated herein by reference in their entirety for all purposes. Technical Field

[0002] The present invention relates to a method, device and system for producing cultured cells. In particular, the present invention relates to a method, device and system for producing cultured tissues (such as cultured meat). Background Art

[0003] Meat's primary components are muscle fibers and intramuscular and intermuscular fat. Attempts to produce cultured meat that mimics meat's structure have been made, but challenges have been encountered. For example, creating a meat-like texture is difficult due to the low resolution of 3D printers or, in the case of cell sheet technology, the limited control over tissue architecture in three dimensions. Producing thick cultured meat structures while maintaining tissue viability is also challenging due to the lack of a vascular system. There is a significant need to produce cultured meat with a meat-like texture similar to natural meat and to provide a vascular system. Summary of the Invention

[0004] Disclosed herein are devices, systems, and methods that can be used to produce cultured tissue.

[0005] In some embodiments, a method for producing cultured tissue is provided, the method comprising the steps of: (a) providing a hydrogel composition and solidifying the hydrogel composition to form a hydrogel portion, wherein the hydrogel portion comprises a plurality of first channels in a predetermined first array; (b) providing at least one first cell composition to a single first channel to form at least one first cell culture therein, such that a tissue construct is formed.

[0006] In some embodiments, a device for producing cultured tissue is provided, the device comprising: a plurality of first channel-forming units, a single first channel-forming unit having a first diameter; and a holder assembly, the holder assembly comprising a holder unit; and at least one support unit, each support unit comprising a plurality of first channel-receiving portions, wherein the size and shape of the single first channel-receiving portion are determined to receive at least a portion of the single first channel-forming unit, wherein the plurality of first channel-receiving portions are constructed and arranged into a first pattern so that the plurality of first channel-forming units can be assembled with the holder assembly to form a predetermined first array and remaining space in the holder assembly; and wherein at least a portion of the remaining space is configured to receive a hydrogel composition to form a solidified hydrogel portion, so that a plurality of first channels in a predetermined first array are formed by removing the plurality of first channel-forming units from the hydrogel portion, and the single first channel is configured to receive at least one first cell composition therein to form at least one first cell culture, thereby producing a tissue construct.

[0007] In some embodiments, a system for producing cultured tissue is provided, comprising: an apparatus as described in any one of the embodiments herein to produce a hydrogel construct having a plurality of third channels; a hydrogel construct connector configured to connect the hydrogel construct to at least one tubing; a mixer for oxygenating the culture medium; at least one pump system for circulating the culture medium from the mixer through the plurality of third channels to the hydrogel construct; and optionally a culture medium recycler for removing any waste.

[0008] In some embodiments, provided is a cultured tissue prepared by any of the methods described in any of the embodiments herein, wherein the cultured tissue is derived from an animal selected from the group consisting of mammals, birds, fish, invertebrates, reptiles, and amphibians.

[0009] Advantages of this disclosure

[0010] The present invention provides numerous advantages. In certain embodiments, the provided devices, systems, and methods can produce cultured meat with aligned and controlled thin diameter (e.g., <100 μm) muscle fibers. In certain embodiments, the provided devices, systems, and methods can produce cultured meat with controlled inter-fiber distance (e.g., less than 20 μm).

[0011] In certain embodiments, the provided devices, systems, and methods can produce cultured meat in a three-dimensional manner with fully controllable tissue architecture. The distribution of muscle and fat areas can also be controlled, thereby improving the texture of the cultured meat. When all dimensions are optimized (channel diameter, distance between two adjacent channels, and specific cells inoculated into a specific channel), the cultured meat produced can be structurally similar to natural meat that cannot be achieved by other methods. And when tempered, the texture of the cultured meat can even be closer to that of natural meat.

[0012] In certain embodiments, cultured meat may contain fat to impart marbling and flavor, muscle as the primary portion of the meat, fibroblasts as structural cells, and / or endothelial cells for blood vessels.

[0013] In certain embodiments, the provided devices, systems, and methods can produce cultured meat having a controlled distribution of tissue components (e.g., muscle fibers, intramuscular fat, etc.), thereby improving the texture of cultured tissue (e.g., cultured meat) to be similar to natural tissue (e.g., natural meat, such as beef or pork).

[0014] In some embodiments, the tissue construct is configured to have final dimensions similar to those desired in meat (eg, adipose fiber diameter, adipose fiber length, fiber-fiber distance, etc.).

[0015] In some embodiments, the distance between two adjacent third channels is configured to be small enough to allow cell viability in the final meat construct to be at least a certain percentage, for example at least 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5%.

[0016] In some embodiments, the provided apparatus, systems, and methods for creating tissue constructs, such as meat, are highly customizable and flexible. By simply increasing the number of needles, needle length, and needle spacing, more tissue constructs, such as meat, can be produced. In some embodiments, by utilizing high-resolution laser cutting, metal plates (support units) with a variety of hole (channel-receiving portion) arrangements can be created to produce meat of different designs and textures. The present invention is highly beneficial in facilitating the creation of textures that mimic natural meat, or even creating new textures that do not exist in natural meat.

[0017] In some embodiments, the hydrogel portion holds the channels in place within the tissue construct.

[0018] In some embodiments, the hydrogel portion acts as an extracellular matrix mimic. Different meat muscle fibers can have different extracellular matrices (e.g., endomysial distance, which is the area between muscle fibers within a bundle; and perimysial distance, which is the sheath covering the thickness of the bundle). In some embodiments, the provided hydrogel portion effectively mimics these distances, provided that the plate (support unit) layout is well designed and appropriately contracted across the entire construct.

[0019] In some embodiments, provided apparatus, systems, and methods for producing tissue constructs promote consistent microchannel formation within hydrogels. In some embodiments, provided apparatus, systems, and methods for producing tissue constructs allow for the creation of a variety of plate designs, and therefore, designs of microchannel arrangements.

[0020] In some embodiments, provided devices, systems, and methods for producing tissue constructs support the formation of muscle fibers, because when at least one cell composition is injected into densely packed microchannels, densely packed muscle fibers can thereby be formed. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a schematic cross-sectional view of an example channel array simulating the dimensions of a slice of in vivo bovine tissue, according to an embodiment.

[0022] Figure 2 is a schematic cross-sectional view of another example channel array according to an embodiment.

[0023] Figure 3A is a schematic diagram showing an example support plate as described in Example 3.

[0024] Figures 3B to 3C is a schematic diagram and an enlarged view of an example channel component according to Example 3.

[0025] Figure 4A is a schematic diagram illustrating an example channel assembly filled with a hydrogel composition to form a hydrogel portion according to Example 4.

[0026] Figure 4B is a diagram showing an example channel assembly having a hydrogel portion according to Example 4, wherein a plurality of first channel-forming units are removed from the channel assembly.

[0027] Figure 4C and Figure 4D Schematic diagram and cross-sectional views of an example tissue construct according to Example 4.

[0028] Figures 5A to 5C Schematic diagrams and cross-sectional views of an example tissue construct extruded with a plurality of third channels according to Example 5.

[0029] Figure 6A and Figure 6B is a schematic diagram of example tissue construct linkers and tissue construct components according to Example 6.

[0030] Figure 6C is a schematic diagram illustrating an example perfusion assembly system according to Example 6.

[0031] 7A to 7C are schematic and cross-sectional views of an example tissue construct according to Example 7 after undergoing a shrinking step.

[0032] Figure 8A is a flow chart of an example method for producing cultured tissue.

[0033] Figure 8B is a flow chart of another example method for producing cultured tissue.

[0034] Figure 9 is a schematic cross-sectional view of another example channel array simulating the dimensions of a slice of in vivo Wagyu or Angus cattle tissue according to Example 10.

[0035] Figure 10 is a schematic cross-sectional view of another example channel array according to Example 10.

[0036] Figure 11A is a schematic diagram showing an example support plate as described in Example 11.

[0037] Figure 11B is a schematic diagram illustrating an example channel assembly according to Example 11.

[0038] Figure 11C is a schematic enlarged view of an example channel assembly according to Example 11.

[0039] Figure 12A is a schematic diagram illustrating an example channel assembly filled with a hydrogel composition to form a hydrogel portion according to Example 12.

[0040] Figure 12B is a diagram showing an example channel assembly having a hydrogel portion according to Example 12, wherein a plurality of first channel-forming units are removed from the channel assembly.

[0041] Figure 12C and Figure 12D Schematic diagram and cross-sectional views of an example tissue construct according to Example 12.

[0042] 13A to 13B Schematic diagrams and cross-sectional views of an example tissue construct extruded with a plurality of third channels according to Example 13.

[0043] Figure 14A and Figure 14B is a schematic diagram of example tissue construct linkers and tissue construct components according to Example 14.

[0044] Figures 14C to 14E is a schematic diagram showing an example tissue construct linker as described in Example 14 from different perspectives.

[0045] Figure 14F is a schematic diagram illustrating an example perfusion assembly system according to Example 14.

[0046] Figures 15A to 15C are schematic and cross-sectional views of an example tissue construct according to Example 7 after undergoing a shrinking step.

[0047] 16A to 16C is a schematic diagram showing another example channel assembly according to Example 16.

[0048] 17A to 17B is a schematic diagram showing another example channel assembly according to Example 17.

[0049] Figure 17C is a microscopic image showing a microchannel seeded with myoblasts in the presence of a fibrinogen thrombin hydrogel.

[0050] Figure 17D is a microscopic image showing aligned muscle fibers parallel to the microchannel.

[0051] Figure 17E is a microscopic image showing aligned muscle fibers parallel to the microchannel.

[0052] Figure 17F is a microscopic image showing a microchannel seeded with myoblasts in the absence of a hydrogel.

[0053] 18A to 18C is a schematic diagram showing another example channel according to Example 18.

[0054] Figure 18D is a photograph showing an example trimmed tissue construct prepared using Formulation #2 as the hydrogel portion.

[0055] Figure 18E is a photograph showing an example untrimmed tissue construct prepared using Formulation #3 as the hydrogel portion.

[0056] Figure 18F is a photograph showing an example untrimmed tissue construct prepared using Formulation #1 as the hydrogel portion.

[0057] 19A to 19Cis a schematic diagram showing another example channel assembly according to Example 19.

[0058] Figures 19D to 19F are photographs showing isometric, front cross-sectional, and side views of an example tissue construct prepared according to Example 19 using Formulation #1 as the hydrogel portion.

[0059] Figures 19G to 19H are photographs showing isometric and side views of an example tissue construct prepared according to Example 19 using Formulation #3 as the hydrogel portion.

[0060] Figures 19I to 19K are photographs showing isometric, front, and side views of another example tissue construct prepared according to Example 19 using Formulation #2 as the hydrogel portion.

[0061] Figure 19L is a microscopic image showing porcine myoblasts in Formulation #6 incorporated into microchannels according to Example 19.

[0062] 20A to 20C is a schematic front view illustration of a process for using an example device according to Example 20.

[0063] FIG. 20D to FIG. 20F is a schematic isometric view of an example apparatus according to Example 20.

[0064] Figures 21A to 21B Schematic illustrations of front and isometric views, respectively, of an example tissue construct according to Example 21.

[0065] Figures 21C to 21D Schematic illustrations of front and isometric views, respectively, of an example tissue construct according to Example 21 after the hydrogel has partially dissolved. DETAILED DESCRIPTION

[0066] Although the description refers to specific embodiments, this disclosure should not be construed as being limited to the embodiments set forth therein.

[0067] As used herein and in the claims, the terms "comprising" (or any related forms, such as "comprise" and "comprises"), "including" (or any related forms, such as "include" or "includes"), "containing" (or any related forms, such as "contain" or "contains"), are intended to mean the inclusion of the following elements but not the exclusion of other elements. It should be understood that for each embodiment where the terms "comprising" (or any related forms, such as "comprise" and "comprises"), "including" (or any related forms, such as "include" or "includes"), or "containing" (or any related forms, such as "contain" or "contains") are used, this disclosure / application also includes alternative embodiments where the terms "comprising," "including," or "containing" are replaced with "consisting essentially of" or "consisting of." These alternative embodiments using "consisting of" or "consisting essentially of are to be understood as narrower embodiments of the "comprising," "including," or "containing" embodiments.

[0068] For clarity, “comprise,” “include,” “contain,” and “have,” and any related forms, are open-ended terms that allow for additional elements or features other than the specified essential elements, whereas “consisting of,” is a closed term that is limited to the elements recited in a claim and excludes any element, step, or ingredient not specified in a claim.

[0069] “Consisting essentially of” limits the scope of a claim to specified materials, components, or steps (“essential elements”) that do not materially affect one or more essential features of the claimed invention. In some embodiments, the essential features are one or more basic and novel features of the claimed invention.

[0070] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. When a range is mentioned in the specification, the range should be understood to include every discrete point within the range. For example, 1 to 7 means 1, 2, 3, 4, 5, 6, and 7.

[0071] As used herein, the term "about" should be understood to mean within the normal tolerance range in the art and not exceeding ±20% of the recited value. By way of example only, about 50 means from 40 to 60, including all values ​​therebetween. As used herein, the phrase "about" a particular value also includes the particular value, for example, about 50 includes 50.

[0072] As used herein and in the claims, the terms "generally" or "generally" or "substantially" or "substantially" mean that the recited features, angles, shapes, states, structures or values ​​need not be achieved precisely, but rather deviations or variations (including, for example, tolerances, measurement errors, measurement accuracy limitations and other factors known to those skilled in the art) may occur in amounts that do not preclude the effects that the features are intended to provide.

[0073] As used herein, the terms "cultured meat," "cultivated meat," "in vitro meat," "cell-based meat," "clean meat," "synthetic meat," and "lab-grown meat" are used interchangeably to refer to meat products that contain tissue derived from cell culture. In some examples, cultured meat is produced by culturing animal cells or tissue in vitro without growing the entire animal (also known as "cellular agriculture"). For example, the process involves isolating meat-specific cells from an animal, an expansion step (where the cells are grown in a nutrient-rich medium to allow the cells to proliferate), and then ultimately harvesting the cells to form the final meat product.

[0074] As used herein, the term "proliferation" or "expansion" refers to the process of increasing the number or population of cells by culturing target cells.

[0075] As used herein, the term "culture medium" refers to a composition or formulation that supports the growth and / or other cellular activities of target cells or cell tissues.

[0076] As used herein, the term "basal medium" or "basal medium" refers to a growth medium that provides essential nutrients for the survival and cultivation of desired cells. Basal medium can serve as a starting point or basis, and specific additives can be added to meet the specific requirements of the desired cell culture. Examples include, but are not limited to, Dulbecco's modified Eagle's medium (DMEM), Ham's F12 medium (F12), Dulbecco's modified Eagle's medium F12 (DMEM:F12), Ham's F10 medium (F10), Roswell Park Memorial Institute 1640 medium (RPMI 1640), minimal essential medium (MEM), serum-free medium (SFM), or a combination thereof.

[0077] As used herein, the term "final concentration" refers to the concentration of a substance or component in a solution or mixture (such as a hydrogel composition or a cell composition) after all necessary dilutions or reactions have occurred. In some examples, the total concentration of the hydrogel composition or cell composition is calculated as weight to volume (100% (w / v)). In some other examples, the total concentration of the hydrogel composition or cell composition is calculated as volume to volume (100% (v / v)).

[0078] As used herein, the term "animal" refers to eukaryotic species, such as mammals (e.g., porcine, bovine, ovine, equine, canine, feline, rodent), birds or avians, reptiles, fish, amphibians, crustaceans, molluscs, cephalopods, etc.

[0079] As used herein, the term "perfusion" refers to the controlled delivery of at least one fluid to support oxygenation, nutrient replenishment, and / or waste removal. As an example, a perfusion assembly system can be used to perfuse oxygenated culture medium through a hydrogel construct to support cell growth and cell differentiation.

[0080] As used herein, the terms "device" or "channel assembly" are used interchangeably when referring to equipment that can be used to produce an end product or intermediate of cultured tissue or tissue constructs, such as cultured meat.

[0081] As used herein, the term "support unit" refers to a component of a device that has multiple channel-receiving portions to support at least a portion of a single channel-forming unit and define their relative position. In some embodiments, the support unit is or includes one or more plates having a plurality of holes as channel-receiving portions in a specific pattern. In some embodiments, the support unit is in the form of a plate. In some other embodiments, the support unit is in the form of a block having a greater thickness to support a larger portion of the channel.

[0082] As used herein, the terms "pattern" and "channel array" refer to the arrangement of channel-receiving portions on a support unit. In some embodiments, the pattern is regular (uniform) or irregular.

[0083] As used herein, the terms "array" and "needle array" refer to a regular or irregular spatial arrangement of channel-forming units positioned in desired relative positions (e.g., positions predetermined or defined by a pattern on a support unit). In some embodiments, the array (first array, second array, and / or third array) is predetermined in different corresponding patterns (first pattern, second pattern, and / or third pattern) on the support unit. In some embodiments, the array is regular or irregular.

[0084] As used herein, the term "holder unit" refers to a component of the device that is sized and shaped to receive and hold certain other components (e.g., at least one support unit, at least a portion of a channel-forming unit) and provide at least some remaining space for the hydrogel portion. The holder unit and the support unit together form a holder assembly.

[0085] As used herein, the terms "channel" and "microchannel" are used interchangeably to refer to a space or cavity formed within a hydrogel portion. Such a space or cavity can be introduced with certain elements, compositions, cells and / or compounds, etc.

[0086] As used herein, the term "channel forming unit" refers to a component of a device having a defined space to form a channel. In some embodiments, the channel forming unit is generally an elongated shaft. In some embodiments, the channel forming unit is flexible or extensible and can be manipulated into different shapes and / or orientations. In some embodiments, the channel forming unit is in the form of a needle or a wire.

[0087] As used herein, term " hydrogel " refers to the phase change material containing cross-linked polymer chains and the fluid (such as water or PBS) that can form gel-like structure. In certain embodiments, under certain conditions, hydrogel is prepared by hydrogel composition, and it is generally in the form of solid or semisolid. Example hydrogel includes but is not limited to protein or gelling agent, and alternatively has cross-linking agent such as enzyme. Under certain conditions (such as heating), the hydrogel in solid or semisolid form can be removed, decomposed, dissolved or melted. The example of hydrogel includes but is not limited to collagen, alginate, gelatin, gellan gum, fibrinogen, xanthan gum, cellulose, vegetable protein (such as soy protein hydrogel, pea protein or other vegetable protein), chitosan, carrageenan, starch hydrogel, agarose, pectin, guar gum, konjac glucomannan, lignin-based hydrogel and combination thereof.

[0088] As used herein, the term "sacrificial layer" refers to a layer made of a phase change material (liquid to solid or semisolid) such as ice and hydrogels, which is formed and can be sacrificed (e.g., removed, decomposed, melted, or dissolved) in a later step.

[0089] As used herein, the term "hydrogel construct" refers to an engineered product formed from a hydrogel portion having a plurality of channels in a predetermined array and optionally (i.e., with or without) one or more cell compositions introduced into the channels within the hydrogel portion. In some examples, the cells in the hydrogel construct are grown to form a tissue construct.

[0090] As used herein, the term "tissue construct" refers to an engineered product that mimics natural cell tissue, and such tissue is formed from at least one cell composition. In some embodiments, the tissue construct is cultured meat.

[0091] As used herein, the term "cell culture" refers to a population of cells grown under controlled conditions. In some examples, a cell culture is or includes a tissue culture in which cells are grown and / or differentiated in a channel.

[0092] It should be understood that terms such as "top," "bottom," "middle," "side," "length," "inside," "outside," "interior," "exterior," "lateral," "vertical," "horizontal," "proximal," "distal," etc., as may be used herein, merely describe reference points and do not limit the present invention to any particular orientation or configuration. Further, terms such as "first," "second," "third," "fourth," etc., merely designate one of the multiple parts, components, and / or reference points disclosed herein, and likewise do not limit the present invention to any particular configuration or orientation. The first, second, third, fourth, etc. parts, components, and / or reference points may be collectively referred to as the specific parts, components, and / or reference points. For example, a first cell, a second cell, a third cell, and / or a fourth cell may be collectively referred to as a "cell."

[0093] As used herein and in the claims, the term "fluid communication" refers to the flow of at least one fluid (such as a liquid), or at least one gas, or a combination thereof, as the case may be, from one component to another.

[0094] As used herein and in the claims, "connected" or "coupled" means directly or indirectly physically joined to other elements. Embodiments of the present invention Example 1

[0095] In certain embodiments, a method for producing a cultured tissue is provided, comprising the steps of: (a) providing a hydrogel composition and solidifying the hydrogel composition to form a hydrogel portion comprising a plurality of first channels and a plurality of second channels, and (b) providing at least one first cell composition to the plurality of first channels to form at least one first cell culture, and providing at least one second cell composition to the second channels to form at least one second cell culture, such that a tissue construct is formed.

[0096] In certain embodiments, the cultured tissue is cultured meat, the first cell composition comprises a plurality of first cells that are muscle-derived cells, muscle satellite cells, and / or myoblast-derived cells, and the second cell composition comprises a plurality of second cells that are adipose-derived cells.

[0097] In certain embodiments, the hydrogel composition comprises one or more of alginate, gelatin, gellan gum, and combinations thereof.

[0098] In certain embodiments, the hydrogel composition comprises about 2% alginate and about 10% gelatin.

[0099] In certain embodiments, the hydrogel composition further comprises adipose-derived cells and fibroblasts, and combinations thereof.

[0100] In certain embodiments, the first cell composition comprises muscle-derived cells, muscle satellite cells, and / or myoblast-derived cells, and optionally one or more of collagen, gellan gum, alginate, gelatin, and combinations thereof.

[0101] In certain embodiments, the second cell composition comprises adipose-derived cells, and optionally one or more of collagen, gellan gum, alginate, gelatin, and combinations thereof.

[0102] In certain embodiments, the method further comprises the step of: (c) providing a plurality of third channels, and optionally providing at least one third cell composition in the plurality of third channels.

[0103] In certain embodiments, the third cells comprise fibroblasts, endothelial cells, and combinations thereof.

[0104] In certain embodiments, the at least one or more first cells, second cells, and / or third cells are derived from fibroblasts, endothelial cells, myoblasts, muscle cells, adipocytes, skin cells, tendons, liver, brain, bone, heart, kidney, and combinations thereof.

[0105] In certain embodiments, the plurality of first channels, second channels, and / or third channels are formed by removing a plurality of first, second, and / or third channel-forming units from the solidified hydrogel composition.

[0106] In some embodiments, the plurality of first channels, second channels and / or third channels are formed by 3D printing or laser ablation.

[0107] In certain embodiments, the method further comprises the step of: (d) growing the first cell and the second cell until a desired tissue mass is obtained, thereby obtaining a cultured tissue product.

[0108] In certain embodiments, step (d) is performed by perfusing oxygenated culture medium through the tissue construct via the plurality of third channels.

[0109] In certain embodiments, the method further comprises the step of: (e) growing the third cells until a desired tissue mass is achieved.

[0110] In certain embodiments, step (c) is performed after step (b), and wherein the plurality of third channels are formed by extruding a plurality of perfusion channels from the plurality of third channel forming units.

[0111] In certain embodiments, the plurality of third channel forming units are biopsy punch tools.

[0112] In certain embodiments, the method further comprises the step of treating the tissue construct with a hydrogel shrinking agent.

[0113] In certain embodiments, the method further comprises the step of electrically and / or mechanically exercising the tissue construct.

[0114] In certain embodiments, before step (b), the method includes the step of coating the plurality of first channel-forming units and / or the plurality of second channel-forming units with a lubricant (such as oil).

[0115] In certain embodiments, the hydrogel contracting agent comprises low molecular weight (eg, 15 kDa) chitosan.

[0116] In certain embodiments, the method further comprises the step of cross-linking the hydrogel composition with a 0.3 M calcium chloride solution.

[0117] In certain embodiments, the step of solidifying the hydrogel composition is performed by incubating the hydrogel composition at a low temperature (eg, about 4°C).

[0118] In certain embodiments, the adipose-derived cells are derived from adipose-derived stem cells from bovine (eg, Wagyu rib-eye).

[0119] In certain embodiments, the muscle-derived cells are derived from myoblast-derived stem cells from bovine (eg, Wagyu rib-eye).

[0120] In certain embodiments, a device for producing cultured tissue is provided. The device includes a plurality of first channel-forming units, each having a first diameter, a plurality of second channel-forming units, each having a second diameter, and at least one support plate. The at least one support plate includes a plurality of first channel-receiving portions and a plurality of second channel-receiving portions, each first channel-receiving portion being sized and shaped to receive at least a portion of a first channel-forming unit and to confine a first needle at a first position, and each second channel-receiving portion being sized and shaped to receive at least a portion of a second channel-forming unit and to confine a second needle at a second position. The plurality of first channel-receiving portions and the plurality of second channel-receiving portions are constructed and arranged in an array such that the plurality of first channel-forming units and the plurality of second channel-forming units can be assembled together by the at least one support plate to form a channel array and a plurality of spaces therebetween. The plurality of spaces are configured to receive a hydrogel composition to form a solidified hydrogel portion, such that a plurality of first channels and a plurality of second channels are formed by removing the plurality of first channel-forming units and the plurality of second channel-forming units, respectively, from the hydrogel portion. Each first channel is configured to receive therein at least one first cell composition to form at least one first cell culture, and each second channel is configured to receive therein at least one second cell composition to form at least one second cell culture, thereby producing a tissue construct.

[0121] In certain embodiments, the device further comprises a plurality of third channel forming units to form a plurality of third channels for receiving at least one third cell therein.

[0122] In certain embodiments, the first diameter and the second diameter are larger (eg, about 1-20 times larger) than the diameters of the first cell culture and the second cell culture of interest, respectively.

[0123] In certain embodiments, the first cell composition comprises at least one first cell and the second cell composition comprises at least one second cell.

[0124] In certain embodiments, the cultured tissue is cultured meat, the first cells are muscle-derived cells, muscle satellite cells, and / or myoblast-derived cells, and the second cells are adipose-derived cells.

[0125] In certain embodiments, the first diameter is about 0.1-10,000 μm, such as about 20-500 μm.

[0126] In some embodiments, the first diameter is less than 200 μm, such as 20-90 μm.

[0127] In certain embodiments, the distance between two adjacent first channel-forming units is about 0.1-5,000 μm, for example, 0.1-500 μm.

[0128] In some embodiments, the distance between two adjacent first channel forming units is less than 100 μm, for example, 0.1-30 μm.

[0129] In certain embodiments, the second diameter is about 0.1-10,000 μm, such as about 20-500 μm.

[0130] In some embodiments, the second diameter is less than 500 μm, such as 100-300 μm.

[0131] In certain embodiments, the distance between two adjacent second channel-forming units is about 0.1-5,000 μm, for example, 0.1-500 μm, for example, about 1 μm.

[0132] In some embodiments, the distance between two adjacent second channel forming units is less than 100 μm, for example, 0.1-30 μm.

[0133] In certain embodiments, the third cells comprise at least one or more of fibroblasts, endothelial cells, myoblasts, muscle-derived cells, adipose-derived cells, and combinations thereof.

[0134] In certain embodiments, each of the plurality of first and / or second channel-forming units has a cylindrical tubular structure.

[0135] In certain embodiments, each of the plurality of first and / or second channel forming units has an open groove structure with a U-shaped cross-section.

[0136] In certain embodiments, the tissue construct comprises a plurality of fat regions and a plurality of muscle regions.

[0137] In certain embodiments, a system for producing cultured tissue is provided, the system comprising an apparatus for producing a tissue construct as described in any of the preceding embodiments, the apparatus having a plurality of third channels, a tissue construct connector configured to connect to the tissue construct, a bioreactor for oxygenating the culture medium, at least one pump system for circulating the culture medium from the bioreactor to the tissue construct through the plurality of third channels, and optionally a culture medium recycler for removing waste.

[0138] In certain embodiments, the cultured tissue is cultured meat, the first cells are muscle-derived cells, muscle satellite cells, and / or myoblast-derived cells, and the second cells are adipose-derived cells.

[0139] In certain embodiments, the cultured tissue is derived from an animal selected from the group consisting of a mammal, a bird, a fish, an invertebrate, a reptile, and an amphibian.

[0140] In certain embodiments, the animal is a human.

[0141] In certain embodiments, the animal is a non-human.

[0142] In certain embodiments, the cultured tissue is cultured meat.

[0143] In some embodiments, provided devices, systems, and methods have one or more of the following features: 1. Form microchannels (ie, a plurality of first and / or second channels) in the edible hydrogel in any desired manner (eg, using a channel-forming unit such as a needle, a biopsy punch, a wire, 3D printing, molding, laser ablation). 2. Incorporating multiple cell types (muscle cells, adipocytes, endothelial cells, fibroblasts, and / or other structural cells) into the microchannels. Cultured tissue need not be derived from skeletal muscle; cells can also be derived from skin, tendon, liver, brain, and so on. In other words, other types of cultured tissue can be produced for various purposes, such as, but not limited to, cultured meat, tissue engineering, and organ transplantation. 3. It becomes possible to exercise the vascular system (which can help enhance cell growth and texture). 4. Incorporation of a perfusion system (oxygenated medium pumped through the channels) to simulate a vascularized system. 5. The microchannels perfused with oxygenated medium had a channel-channel distance ranging from 0.5 μm to 5 mm. 6. Shrinking the microchannel to make the size smaller (using a shrinking agent). 7. Cell constructs formed by seeding cells (e.g., fat fibers, muscle fibers) in microchannels, as well as the diameter of the microchannels and the distance between any two adjacent microchannels, are fully controlled. For example, the diameter and distance are within the interquartile range of the desired meat tissue (e.g., within the interquartile range of muscle fibers found in Wagyu beef).

[0144] In some embodiments, an array of 200 μm microchannels has been created using gelatin / alginate hydrogels, where the distance between two adjacent microchannels is 100 μm. Muscle-derived cells suspended in gelatin / transglutaminase hydrogels have been injected into the channels for growth.

[0145] In some embodiments, the space between two adjacent individual channels is less than 150 μm. In some embodiments, the space between two adjacent individual channels is less than 20 μm.

[0146] In some embodiments, the hydrogel composition comprises an edible hydrogel.

[0147] In some embodiments, the hydrogel composition comprises a natural hydrogel and / or a non-natural hydrogel. In some embodiments, the hydrogel composition is plant-based and / or non-plant-based hydrogel. In some embodiments, the hydrogel composition is naturally derived. In other embodiments, the hydrogel composition is synthetically prepared.

[0148] In some embodiments, the cell (e.g., the first cell, the second cell, and / or the third cell) is derived from a mammalian species. In some embodiments, the cell is derived from a non-mammalian species.

[0149] In some embodiments, the cell is derived from a eukaryotic cell. In some embodiments, the cell is derived from a prokaryotic cell. In some embodiments, the cell is derived from a microorganism, such as a bacterium, fungus, or virus.

[0150] In some embodiments, the integrity of the plurality of third channels is maintained during incubation and perfusion.

[0151] In certain embodiments, the cultured tissue is derived from an animal selected from the group consisting of a mammal, a bird, a fish, an invertebrate, a reptile, and an amphibian.

[0152] In certain embodiments, the animal is a non-human.

[0153] In some embodiments, the tissue construct is formed by seeding and incubating at least one first cell culture and at least one second cell culture.

[0154] In certain embodiments, the first diameter and the second diameter are respectively about 1-20 times larger than the diameter of the target first cell culture and the target second cell culture. In certain embodiments, the first diameter and the second diameter are respectively about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 times (or even larger) larger than the diameter of the target first cell culture and the target second cell culture. In certain embodiments, the first diameter and the second diameter are respectively about 3 times larger than the diameter of the target first cell culture and the target second cell culture. In certain embodiments, the first diameter and the second diameter are respectively about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 times (or even larger) larger than the diameter of the target first tissue and the target second tissue. In certain embodiments, the first diameter and the second diameter are respectively about 3 times larger than the diameter of the target first tissue and the target second tissue.

[0155] For clarity, the at least one (first or second) cell comprises one or more cells of one or more cell types or cell lines.

[0156] In certain embodiments, the first diameter is less than about 90 μm, such as about 50 μm.

[0157] In certain embodiments, the second diameter is less than about 500 μm, such as about 135 μm.

[0158] In some embodiments, a distance between two adjacent first channel-forming units is less than about 15 μm, for example, about 5 μm.

[0159] In some embodiments, the distance between two adjacent second channel-forming units is less than about 15 μm, for example, about 5 μm.

[0160] In some other embodiments, the first cell and / or the second cell is derived from a fibroblast, an endothelial cell, a myoblast, a muscle cell, an adipocyte, a skin cell, tendon, liver, brain, bone, heart, kidney, and combinations thereof.

[0161] In some embodiments, the hydrogel composition comprises a natural hydrogel and / or a non-natural hydrogel. In some embodiments, the hydrogel composition is plant-based and / or non-plant-based hydrogel. In some embodiments, the hydrogel composition is naturally derived. In other embodiments, the hydrogel composition is synthetically prepared.

[0162] In some other embodiments, the hydrogel composition further comprises cells derived from fibroblasts, endothelial cells, myoblasts, muscle cells, adipocytes, skin cells, tendon, liver, brain, bone, heart, kidney, and combinations thereof.

[0163] In certain embodiments, the method further comprises the step of differentiating the first cells and / or second cells into mature cell tissues.

[0164] In certain other embodiments, step (e) is performed together with step (c), and wherein the plurality of third channels are formed by removing the plurality of third channel-forming units from the hydrogel portion. Examples

[0165] Provided herein are examples that describe certain embodiments of the present disclosure in more detail. The examples provided herein are for illustrative purposes only and are not intended to limit the scope of the present invention in any way. All references given below and elsewhere in this application are hereby incorporated herein by reference. Example 1: Designing desired muscle fiber / fat fiber dimensions based on selected slices of native Wagyu beef tissue Now refer to Figure 1, a cross-sectional view of an example design of a channel array (or microchannel array) having desired / target muscle fiber / fat fiber dimensions (all figures are shown in mm) that simulate the dimensions of a slice of bovine tissue in vivo. The microchannel array has an overall dimension of approximately 5×5 mm. The microchannel array includes a plurality of muscle regions formed by a plurality of first channels and a fat region formed by a plurality of second channels. In this example, each of the plurality of first channels has a smaller first diameter of approximately 60 μm, and the distance between two adjacent channels is approximately 30 μm. Each of the plurality of second channels has a larger second diameter of approximately 135 μm, and the distance between two adjacent second channels is approximately 30 μm.

[0166] Devices and systems for producing cultured tissue Example 2: Channel Array Now refer to Figure 2 , a cross-sectional view of another example design of a channel array. In this example, based on the design described in Example 1, the channel array has a size that is approximately three times larger (i.e., approximately 15×15 mm) than the example design in Example 1. Each of the plurality of first channels has a first diameter of 180 μm, and the distance between two adjacent first channels is approximately 90 μm. Each of the plurality of second channels has a second diameter of approximately 405 μm, and the distance between two adjacent second channels is approximately 90 μm. The overall size of the channel array is larger (e.g., approximately 3 times) than the desired final size of the channel array to allow for the production of larger sized cultured tissues and shrinkage in subsequent steps. Example 3: Channel Component

[0167] Now refer to Figures 3A to 3C , showing an example channel assembly (i.e., two support plates together with a first channel forming unit and a second channel forming unit (in this example, a first needle and a second needle are used as examples of the first channel forming unit and the second channel forming unit, respectively) to form a needle array and a plurality of spaces therebetween). Figure 3A A metal support plate is shown, comprising a plurality of first needle receiving portions (or holes) and a plurality of second needle receiving portions (or holes), the needle receiving portions being constructed and arranged based on the channel array as described in Example 2. The size and shape of each needle receiving portion are determined to receive at least a portion of a needle and will need to be defined at its location. The diameter of each needle receiving portion is slightly larger than the channel diameter to receive at least a portion of a needle. Each first needle has a first diameter of the first channel (approximately 180 μm in this example), and each second needle has a second diameter of the second channel (approximately 405 μm in this example).

[0168] Figure 3BAn example channel assembly is shown, comprising two support plates, with a plurality of first and second needles mounted through respective first and second needle receiving portions. The two support plates are shown in dark gray. The first needle for myogenesis is shown in green and has a first needle length of approximately 180 mm. The second needle for lipogenesis is shown in light gray and has a second needle length of approximately 90 mm.

[0169] Figure 3C is an enlarged view of the channel assembly showing a first needle (green) in the first needle receiving portion for later muscle formation, and a second needle (grey) inserted into the second needle receiving portion of the support plate for later fat formation. Example 4: Tissue Constructs

[0170] Now refer to Figures 4A to 4C , showing the formation of a tissue construct (including a hydrogel portion and a channel portion). Figure 4A 3 is an example channel assembly (as described in Example 3) filled with a hydrogel composition (mixture A) to form a hydrogel portion. The hydrogel composition is poured onto multiple spaces between the needle arrays of the channel assembly to form a hydrogel portion shown in red. In this example, the mixture A used contains about 2% sodium alginate and about 10% gelatin. In other examples, mixture A contains about 2% alginate and 20% gelatin. In other examples, 0.1%-10% (such as 0.5%) alginate can be used instead. Mixture A is allowed to solidify at low temperature (e.g., 4°C). Mixture A is cross-linked with 0.3M calcium chloride solution to form a hydrogel portion. In other examples, cross-linking is performed using a calcium chloride solution of less than 1%. In other examples, cross-linking is performed using a calcium carbonate solution of less than 1%.

[0171] Figure 4B An example channel assembly having a hydrogel portion is shown, wherein a plurality of first channel forming units (in this example, the first channel forming units are first needles, such as Figure 4A The green needle shown) is removed from the channel assembly. The first plurality of needles are removed from the hydrogel portion, thereby forming a plurality of first channels into which muscle cells can be seeded. In this example, a suspension of collagen type 1 ( Wagyu rib-eye-derived myoblasts (1 x 10 7 The first cell composition (mixture B) of 10 cells / ml was inoculated into the first channel. The first cell composition was first inoculated, and then the first needle was removed from the hydrogel portion to form the first channel together with the first cell composition therein. The neutralization solution (10 ml, Type 2 collagen solution, 6 mg / ml (bovine, Advanced BioMatrix) was added to mixture B (approximately 1 / 10 of the final volume was used) for cross-linking at approximately 37°C.

[0172] The plurality of second channel forming units (in this example, the second channel forming units are second needles) are removed from the hydrogel portion in no particular order, that is, before or after the step of removing the first needle, separately, or simultaneously. In this example, after the step of obtaining the cultured first cell culture, the plurality of second needles are removed from the hydrogel portion, thereby forming a plurality of second channels, into which adipose-derived cells can be seeded. Adipose-derived cells (1 x 10 7 A second cell composition (Mixture C) (100 cells / ml) was seeded into the remaining second channel. In this example, fat-derived stem cells from Wagyu rib-eye were used. Neutralization solution was also added to Mixture C (approximately 1 / 10 of the final volume) for cross-linking to occur at approximately 37°C.

[0173] Figure 4C and Figure 4D An example tissue construct comprising a hydrogel portion and a channel portion is shown after removal of the needles and support plate and seeding with adipose-derived cells and muscle-derived cells, wherein the tissue construct is seeded with both a first cell composition (comprising adipose-derived cells) and a second cell composition (comprising muscle-derived cells). Example 5: Tissue Constructs with Perfusion Channels

[0174] Now refer to Figures 5A to 5C . Figure 5A and Figure 5B Shown in Figure 4C Example tissue construct formed in the biopsy but subsequently extruded with multiple perfusion channels (third channel) through a biopsy punch tool. Figure 5B is a cross-sectional view of an example tissue construct perfused with multiple third channels. In this example, the perfusion channels form a 4×4 perfusion array. A biopsy punch tool is used to extrude the perfusion channels. In some embodiments, endothelial cells can be seeded in the third channels to form endothelial tubes along the perfusion channels.

[0175] In other examples, a perfusion channel can be first created by providing a plurality of third channel-forming units (e.g., needles) in a channel assembly (e.g., as described in Example 2), pouring a third cell composition (mixture D, comprising endothelial cells) and allowing the third cells to grow. In some embodiments, the third cells comprise endothelial cells, which will grow and differentiate along the surface of the third channel. The third channel is configured to be in fluid communication with the perfusion assembly system.

[0176] Figure 5C Shown Figure 5A and Figure 5B Dimensions of an example tissue construct. In this example, the diameter of each third channel is about 225 μm, and the distance between two adjacent third channels is about 3 μm. Example 6: Tissue Construct Connector and Perfusion Components

[0177] Figure 6A and Figure 6B Shown is a tissue construct sandwiched between tissue construct connectors.

[0178] Figure 6C A perfusion assembly system is shown, comprising a pump, a bioreactor, an oxygenated culture medium, a tissue construct (meat) and a tissue construct connector. The tissue construct connector (or pipe connector) is attached to the meat tissue construct (such as described in Example 5) to form a tissue construct assembly, and the pipeline is connected to the tissue construct assembly and the oxygenated culture medium source. In this example, the pump is a first micropump and a second micropump located upstream and downstream of the tissue construct assembly, respectively. The bioreactor is a rotary bioreactor with an agitator to oxygenate the culture medium, and the tissue construct assembly has a plurality of microchannels (a third channel for perfusion) connected to the perfusion assembly by the pipeline. In this example, the oxygenated culture medium source comes from a 100 mL bioreactor, which is provided with a PTFE filter to allow sterile air to enter the culture medium and contact the culture medium. When the culture medium is mixed, air (and oxygen) is dissolved to oxygenate the culture medium. One or more pumps are used to pump the oxygenated culture medium into the tissue construct, and deoxygenated culture medium and waste are pumped away from the tissue construct. Thus, the tissue construct (by the perfusion step) is perfused with culture medium. Optionally, a media recoverer is connected downstream of the tissue construct to remove any waste.The tissue construct is then allowed to mature. Example 7: Final product of tissue construct

[0179] Now refer to 7A to 7C . Figure 7A and Figure 7B The final target product of the meat tissue construct from Example 6 following a shrinkage step is shown. Figure 7BA cross-sectional view of the final meat tissue product simulating Wagyu beef tissue is shown. The tissue construct is treated with a shrinking agent to shrink the tissue. In this example, a low molecular weight chitosan (e.g., about 15kDa) solution is used, so the tissue construct is reduced by about 3 times. In this way, the tissue can be exercised to induce a larger matrix rearrangement and thus induce texture. In other examples, the tissue construct can be immersed in a solution that can shrink the fiber-fiber distance to, for example, about 20 μm, such as acetic acid (i.e., vinegar). In other examples, the force exerted by the muscle fibers will shrink the diameter (e.g., about 1-20 times, for example, about 3 times) (which can vary depending on the hydrogel used) to be more similar to the physiologically relevant diameter (50-66.7 μm muscle fiber diameter found in beef). In other examples, the shrinking step can be performed before the perfusion step as described in Example 6. Example 8: Method for producing cultured tissue

[0180] In this embodiment, cultured meat is used as an example of cultured tissue, and an example method of producing cultured meat includes the steps of preparing a channel component and then preparing a tissue construct.

[0181] In some embodiments, the channel assembly is prepared by the following steps: (1) providing an apparatus or system as described in the previous example; coating the one or more support plates and the channel forming units (such as needles) with oil, and providing the support plates with at least a plurality of first needles and a plurality of second needles to form a channel assembly; (2) rinsing the support plates; rinsing the plurality of first needles and the plurality of second needles in water twice, wherein each washing cycle is 15 minutes, and sterilizing the support plates and the plurality of first needles and the plurality of second needles (e.g., under UV light); (3) Assembling the support plates, the plurality of first needles, and the plurality of second needles into a channel assembly. In one example, the diameter of each of the plurality of first needles is approximately 180 μm, and the distance between two adjacent first needles is 50-60 μm, and the diameter of each of the plurality of second needles is approximately 405 μm, and the distance between two adjacent second needles is 50-60 μm. In other examples, other diameters and distances as described in the previous examples may be used. (4) Pouring mixture A onto the channel assembly, wherein mixture A comprises cells (e.g., one or more of muscle-derived cells, adipose-derived cells, fibroblasts, and combinations thereof), sodium alginate, gelatin, gellan gum, and combinations thereof. (5) Mixture A is allowed to solidify at a low temperature (eg, 4° C.) to form a hydrogel portion.

[0182] In some embodiments, a plurality of third needles are also provided in the initial step (1) and assembled with the channel assembly, so that a channel assembly including a plurality of first needles, second needles and third needles is formed.

[0183] In some embodiments, the first tissue of the tissue construct is prepared by: (1) pouring mixture B into a 6-well plate, wherein mixture B comprises first cells (in this example, muscle cells or myoblasts), collagen, sodium alginate, gelatin, gellan gum, a cross-linking agent (e.g., calcium chloride and / or calcium carbonate solution), a contracting agent, microbial transglutaminase, a culture medium (e.g., DMEM, FBS), a neutralizing solution, and a combination thereof, or as described in the previous examples; (2) removing the plurality of first needles from the channel assembly until the first needles are almost close to the end of the channel assembly, thereby forming a plurality of first channels. In some embodiments, the needles should still slightly extend out of the first channel section; (3) Slowly inoculating Mixture B as described in the previous example into multiple smaller channels without shearing the cells or introducing air bubbles; (4) ensuring that the first cells do not flow out of the first channel and allowing the mixture B to solidify; and (5) Cross-linking the mixture B in the first channel at a temperature below 37° C. (eg, about 4° C.).

[0184] In some embodiments, the method further comprises the step of growing the first cells until a desired tissue mass is obtained, thereby obtaining a cultured first tissue (ie, muscle tissue). In some embodiments, the growing step is performed as described in Example 6.

[0185] In some embodiments, before, simultaneously with, or after seeding the first cells and removing the first needle, a second cell culture of the tissue construct is prepared by: (1) pouring mixture C into a 6-well plate, wherein mixture C comprises second cells (in this example, adipose-derived cells), collagen, sodium alginate, gelatin, gellan gum, a cross-linking agent (e.g., calcium chloride and / or calcium carbonate solution), a contracting agent, microbial transglutaminase, a culture medium (e.g., DMEM, FBS), a neutralizing solution, and a combination thereof, or as described in the previous examples; (2) removing the plurality of second needles from the channel assembly until the second needles are almost close to the end of the channel assembly, thereby forming a plurality of second channels. In some embodiments, the second needles should still slightly extend out of the second channel section; (3) slowly inoculating the mixture C described in the previous example into the plurality of second channels without shearing the cells or introducing air bubbles; (4) ensuring that the second cells do not flow out of the second channel and allowing them to solidify; and (5) Cross-linking the mixture C in the second channel at a temperature below 37° C. (eg, about 4° C.).

[0186] In some embodiments, the method further comprises the step of growing the second cells until a desired tissue mass is obtained, thereby obtaining a cultured second tissue (ie, adipose tissue). In some embodiments, the growing step is performed as described in Example 6.

[0187] In some embodiments, the third tissue (if any) of the tissue construct is prepared by the following steps before, simultaneously with, or after the step of obtaining the cultured first and / or second tissue: (1) Pour mixture D into a 6-well plate, wherein mixture D comprises third cells (in this example, endothelial cells). Optionally, mixture D further comprises collagen, sodium alginate, gelatin, gellan gum, a cross-linking agent (e.g., calcium chloride and / or calcium carbonate solution), a contracting agent, microbial transglutaminase, a culture medium (e.g., DMEM, FBS), a neutralizing solution, and combinations thereof, or as described in the previous examples; (2) seeding the third cells and removing the plurality of third needles from the channel assembly until the third needles are nearly adjacent to the end of the assembly, thereby forming a plurality of third channels. In some embodiments, the third needles should still slightly extend beyond the third channel segment. In some other embodiments, the third channels are formed by extruding a plurality of perfusion channels, as described in the previous embodiments; (3) slowly inoculating the mixture D as described in the previous example into the plurality of third channels without shearing the cells or introducing air bubbles; and (4) optionally ensuring that the third cells do not flow out of the third channel, and allowing the third cells to attach to the third channel so as to form endothelial tissue along the surface of the third channel, and (5) Optionally, crosslink the mixture D in the third channel, for example, at a low temperature (e.g., below 37° C., such as about 4° C.).

[0188] In some other embodiments, after the tissue construct (or solidified hydrogel portion) is formed, the plurality of third needles are formed by perfusing the third channel.

[0189] In some embodiments, the method further comprises the step of growing the third cells until a desired tissue mass is obtained, thereby obtaining a cultured third tissue (ie, endothelial tissue along the third channel). In some embodiments, the growing step is performed as described in Example 6.

[0190] In some embodiments, the method further comprises treating the tissue construct with a shrinking agent to shrink the tissue construct. In some instances, the tissue construct shrinks to approximately 3 times its original size. In some embodiments, the shrinking agent is or comprises a low molecular weight (e.g., 15 kDa) chitosan solution. In some embodiments, the shrinking step is performed as described in Example 7. Example 9a: Example Method for Producing Cultured Tissue

[0191] Now refer to Figures 8A to 8B , a flow chart showing an example method for producing cultured tissue. Figure 8A As shown, the method 10 for producing cultured tissue includes the following steps.

[0192] In step 11, a hydrogel composition is provided and solidified to form a hydrogel portion, wherein the hydrogel portion includes a plurality of first channels in a predetermined first array. In one embodiment, the plurality of first channels is formed by providing a plurality of first channel-forming units in the hydrogel composition before solidification.

[0193] In one embodiment, in step 11, the hydrogel portion further comprises a plurality of second channels in a predetermined second array. In another embodiment, the plurality of second channels are formed by providing a plurality of second channel-forming units in the hydrogel composition before the hydrogel composition solidifies. In another embodiment, the plurality of first channels and / or second channels are formed after solidification. In one example, the first and second channel-forming units (and channels) are provided simultaneously or sequentially in any order.

[0194] In one embodiment, in step 11, the hydrogel portion further includes a plurality of third channels in a predetermined third array. In another embodiment, the plurality of third channels are formed by providing a plurality of third channel forming units in the hydrogel composition before the hydrogel composition solidifies. In one example, the first, second and third channel forming units (and channels) are provided simultaneously or sequentially in any order. In another embodiment, the plurality of third channels are formed by providing a plurality of third channel forming units in the hydrogel composition after the hydrogel composition solidifies (e.g., after step 12). For example, the plurality of third channel forming units are biopsy punching tools, and the plurality of third channels are formed after forming the hydrogel portion having the first channel and / or the second channel.

[0195] In step 12, at least one first cell composition is provided to form at least one first cell culture therein to single first passage, so that tissue construct is formed. For example, one or more different first cell compositions are provided in single or all first passages. The first cell composition can be introduced into successively simultaneously or in any order. In one embodiment, the first cell composition comprises a plurality of first cells (the first cell is a cell in muscle-derived cell, muscle satellite cell and / or myoblast-derived cell), and optionally further comprises one or more and combinations thereof in collagen, alginate, gelatin, gellan gum, fibrinogen. In one embodiment, the second cell composition optionally further comprises hydrogel, culture medium and other components. Example first cell composition will be further described in other examples.

[0196] In one embodiment, step 12 further comprises the following steps: provide at least one second cell composition to form at least one second cell culture therein to single second passage (if present). For example, one or more different second cell compositions are provided in single or all second passages. The second cell composition can be introduced into sequentially simultaneously or in any order. In one embodiment, the second cell composition comprises a plurality of second cells (the second cell is adipose-derived cells) and one or more and combinations thereof in collagen, alginate, gelatin, gellan gum, fibrinogen alternatively. In one embodiment, the second cell composition further comprises hydrogel, culture medium and other components alternatively. Example second cell composition will be further described in other examples.

[0197] In one embodiment, step 12 further comprises the following steps: provide at least one 3rd cell composition to form at least one 3rd cell culture therein to single 3rd passage (if present).For example, one or more different 3rd cell compositions are provided in single or all 3rd passages.The 3rd cell composition can be introduced into successively simultaneously or in any order.In one embodiment, the 3rd cell composition comprises one or more of a plurality of 3rd cells (the 3rd cell is fibroblast, endothelial cell and combination thereof) and alternatively collagen, alginate, gelatin, gellan gum, fibrinogen and combination thereof.In one embodiment, the 3rd cell composition further comprises hydrogel, culture medium and other components alternatively.Example 3rd cell composition will be further described in other examples.

[0198] In one embodiment, step 12 further comprises the step of growing the plurality of first cells, the plurality of second cells, and / or the plurality of third cells (if present) until a desired tissue mass is obtained. The growing step is performed by incubating or culturing the first cells, the second cells, and / or the third cells under desired conditions. In some embodiments, the growing step allows the cells to differentiate and / or grow.

[0199] In some embodiments, the tissue construct is prepared using a device as described in any of the examples described herein. Method 20 further comprises one or more of the following steps:

[0200] In step 21, an apparatus for producing cultured tissue is provided, the apparatus comprising a plurality of first channel-forming units and / or second channel-forming units, and a holder assembly, the holder assembly comprising: a holder unit; and at least one support unit, each support unit comprising a plurality of first channel-receiving portions, wherein the size and shape of a single first channel-receiving portion are determined to receive at least a portion of a single first channel-forming unit, and wherein the plurality of first channel-receiving portions are constructed and arranged into a first pattern such that the plurality of first channel-forming units can be assembled with the holder assembly to form a predetermined first array, and there is residual space in the holder assembly.

[0201] In step 22 , a hydrogel composition is provided to at least a portion of the remaining space and the hydrogel composition is solidified to form a hydrogel portion.

[0202] In step 23, individual first channel-forming units and / or second channel-forming units are removed from the hydrogel portion. In some embodiments, the first channel-forming units and / or second channel-forming units are removed after the hydrogel composition solidifies.

[0203] Optionally, the assembled device further comprises a plurality of third channel forming units, wherein the individual third channel forming units are arranged in a predetermined third array. The third channel forming units are provided before or after forming the hydrogel portion.

[0204] For the sake of clarity, the above-described method steps may be performed sequentially, simultaneously, or in any other order, where applicable. Example 9b: Preparation of Example Hydrogel Composition and Example Cell Composition

[0205] The hydrogel compositions and cell compositions of the examples are listed in Table 1 and Table 2, respectively. In general, the hydrogel compositions contain one or more proteins or gelling agents (e.g., gelatin, fibrinogen, alginate, and / or gellan gum, etc.) and optionally one or more corresponding cross-linking agents (enzymes, etc.). Unless otherwise specified, the hydrogel compositions and cell compositions were dissolved in phosphate-buffered saline (PBS). These examples are for illustrative purposes only and are not intended to be an exhaustive list of all possible embodiments of the present invention. Table 1. Example hydrogel compositions Note: rt = room temperature; U = enzyme unit; U / g = enzyme unit / gram of gelling agent

[0206] Methods for forming or preparing hydrogel portions using example hydrogel compositions are described below.

[0207] Preparation of Formulation #1:

[0208] To prepare hydrogel composition Formulation #1 as described in Table 1, the following steps were performed: (1) Prepare a 15% (w / v) gelatin (Sigma) solution by dissolving 15 g of gelatin in 100 mL of PBS. Heat the 15% gelatin solution to 50°C and autoclave. (2) Prepare a 10% (w / v) transglutaminase TI solution in PBS by dissolving 10 g of transglutaminase powder (yielding approximately 100 to 120 enzyme units (U) of transglutaminase TI / gram of powder) in 100 mL of PBS (10% Tg = 0.1 g / mL = 10 U / mL to 12 U / mL, where U represents enzyme units). Sterile filter the 10% transglutaminase solution through a 0.22 μm filter. (3) A hydrogel composition of Formulation #1 (approximately 13.5% gelatin + approximately 1% transglutaminase (1 U / mL)) was prepared by mixing 9 parts of a 15% gelatin solution with 1 part of a 10% transglutaminase solution (from (1) and (2)). In some embodiments, 1 part of the 10% transglutaminase was first added to a centrifuge tube, followed by the addition of 9 parts of the 15% gelatin solution and mixing for 30 seconds immediately before use. In this example, the final concentration of 1% transglutaminase contained approximately 7.4 to 8.88 U of transglutaminase per gram of gelatin.

[0209] To prepare a hydrogel portion using hydrogel composition Formulation #1, transfer the hydrogel composition to a desired device or container and allow crosslinking at room temperature for 48 hours. In some embodiments, Formulation #1 is further incubated at 37°C for 2 hours.

[0210] Preparation of Formulation #4:

[0211] Hydrogel compositions with varying concentrations of gelatin (or other gelling agents) were prepared using a similar process to that used for Formulation #1 above, with the transglutaminase TI concentration controlled within a range of 0.1-100 U transglutaminase per gram of gelatin (or other suitable gelling agent). In this example, to prepare Formulation #4, which contained 5% gelatin and 10 U / g transglutaminase TI (10 U transglutaminase per gram of gelatin), 10 U / g * 0.05 g = 0.5 U transglutaminase TI was required. If the 10% transglutaminase stock solution described above were used, the required volume would be 0.5 U / (10 U / mL) = 0.05 mL or 0.5 U / (12 U / mL) = 0.0416 mL. Formulation #4 was crosslinked at 37°C for 2-3 hours. In some embodiments, when Formulation #4 is used in a cell composition, the mixture is allowed to crosslink for 2-3 hours at 37°C with occasional mixing by pipetting up and down.

[0212] Preparation of Formulation #2:

[0213] Formulation #2 was obtained by preparing 20% ​​(w / v) gelatin (Sigma) + 2% (w / v) gellan gum by using PBS as a solvent, followed by autoclaving.

[0214] To prepare the hydrogel portion using hydrogel composition formulation #2, the hydrogel composition is first melted in boiling water. To prevent air bubbles from being trapped, the solution is occasionally transferred to a biosafety cabinet and the lid is opened to release pressure and air bubbles. If bubbles persist, the solution is allowed to solidify at 4°C and then the melt is repeated. Once the hydrogel composition is fully melted, it is transferred to the desired device or container. For crosslinking, hydrogel composition formulation #2 is immersed in 1% calcium chloride. In some embodiments, other crosslinking agents are, for example, other calcium salts (such as calcium chloride), zinc salts (such as zinc chloride), magnesium salts, barium salts, sodium tripolyphosphate, or DMEM + 10% FBS.

[0215] Preparation of Formulation #3:

[0216] First, a 2% sodium alginate solution was prepared using PBS as a solvent and heated to 50° C. While hot, the 2% sodium alginate solution was sterile filtered through a 0.45 μm filter. Sterile gelatin powder (8 g) was added to 40 mL of the 2% sodium alginate solution, and the mixture was then autoclaved to obtain a hydrogel composition formulation #3 with a final concentration of 20% (w / v) gelatin (Sigma) + 2% (w / v) sodium alginate.

[0217] To prepare the hydrogel portion using hydrogel composition formulation #3, first melt the hydrogel composition in a 37°C bath. Once the hydrogel composition is sufficiently melted, transfer it to the desired device or container. For crosslinking, hydrogel composition formulation #3 is immersed in 1% calcium chloride. In some embodiments, other crosslinking agents are, for example, other calcium salts (such as calcium chloride), zinc salts (such as zinc chloride), magnesium salts, or barium salts.

[0218] Preparation of Formulation #6:

[0219] Prepare stock solutions of 20 mg / mL fibrinogen (in PBS) and 20 U / mL thrombin (in water containing 0.1% (w / v) bovine serum albumin). Prepare hydrogel composition formulation #6 with a final concentration of 1 mg / mL fibrinogen + 1 U / mL thrombin by mixing 5 uL of 20 mg / mL fibrinogen, 5 uL of 20 U / mL thrombin, and 90 uL of PBS. Use the hydrogel composition immediately after mixing, as cross-linking occurs within approximately 1 minute.

[0220] Other example hydrogel compositions were prepared using methods similar to those described above with appropriate adjustments.

[0221] Example cell compositions are shown in Table 2. These examples are for illustrative purposes only and are not intended to be an exhaustive list of all possible embodiments of the invention. Table 2. Example cell compositions

[0222] Generally, the 2 The cells were rinsed twice with sterile PBS (passage 6 or less) and harvested from one or more passages to prepare the cell composition. Cells from passage 5 were used. The volume of trypsin and trypsin neutralizer solution used was 0.053 mL / cm 2. For example, 4 mL of 0.25% trypsin / EDTA is added to cells in a T75 flask (0.053 mL / cm2*75 cm2=about 4 mL) and incubated at about 37°C for about 5 minutes. After incubation for 5 minutes (occasionally tapping to promote cell separation), the trypsin solution is neutralized with an equal volume (4 mL) of trypsin neutralizer solution. Then, after trypsin separation and neutralization, the cells are centrifuged twice at 200 g for 5 minutes. After the first centrifugation, the cells are resuspended in 1 mL of PBS. After the second centrifugation, the cells are resuspended in an appropriate volume of PBS, or a solution of a hydrogel composition such as hydrogel formulation #6 (1% fibrinogen + 1% thrombin) or formulation #4 (5% gelatin (Sigma) and 10 U / g transglutaminase TI dissolved in PBS). In some embodiments, a range of about 1 x 10 2 –1x 10 11 In some embodiments, the cell composition further comprises a hydrogel composition, such as those selected from Table 1. Example 10: Pattern on the support unit

[0223] Now refer to Figure 9 , a cross-sectional view of an example design of a desired tissue construct having a pattern 100 having desired / target muscle fiber / fat fiber dimensions that simulate the dimensions of a slice of Wagyu or Angus cattle tissue in vivo. In this example, the pattern 100 has an overall size of approximately 5×5 mm. The pattern 100 includes a plurality of first cell (muscle) regions arranged in a first pattern formed by a plurality of first channels 110 and a plurality of second cell (fat) regions arranged in a second pattern formed by a plurality of second channels 120. These patterns are arranged irregularly, simulating the tissue arrangement of a slice of Wagyu or Angus cattle tissue in vivo. In this example, each of the plurality of first channels 110 has a smaller first diameter of approximately 60 μm, and the distance between two adjacent channels is approximately 30 μm. Each of the plurality of second channels 120 has a larger second diameter of approximately 135 μm, and the distance between two adjacent second channels is approximately 30 μm.

[0224] Now refer to Figure 10, a cross-sectional view of pattern 200 based on the design of pattern 100. In the present example, pattern 200 has a size that is approximately three times larger (i.e., approximately 15×15 mm) than the example design pattern 100. Each of the plurality of first channels 210 has a first diameter of 180 μm, and the distance between two adjacent first channels is approximately 90 μm. Each of the plurality of second channels 220 has a second diameter of approximately 405 μm, and the distance between two adjacent second channels is approximately 90 μm. The overall size of pattern 200 is larger (e.g., approximately 3 times) than the desired final size of pattern 100 to allow for the production of cultured tissues of larger sizes and to shrink in subsequent steps so that the final product can achieve the original size of the desired tissue construct after the shrinking step. Example 11: Example device for producing cultured tissue

[0225] Now refer to Figure 11A , an example support unit 1000 (also referred to as a "support plate" or "plate" in other examples) is shown, including a plurality of first channel receiving portions 1110 (also referred to as "holes" in other examples) and a plurality of second channel receiving portions 1120, the channel receiving portions 1110 and 1120 being constructed and arranged in a first pattern and a second pattern, respectively, similar to the pattern 100 described in Example 10. Each first channel receiving portion is sized and shaped to receive at least a portion of a first channel-forming unit, and each second channel receiving portion is sized and shaped to receive at least a portion of a second channel-forming unit, so that a plurality of channel-forming units can be assembled and defined by the corresponding channel receiving portions. In some embodiments, the diameter of each channel receiving portion is slightly larger than the diameter of the channel-forming unit so as to receive at least a portion of the channel-forming unit (or needle).

[0226] Now refer to Figures 11B to 11C , shows an example device 10000 (also referred to as a "channel assembly" in some examples), the device having a support unit 11000 configured to connect to the proximal side of a holder assembly, a support unit 11000' at the distal side of the holder assembly, and a plurality of first channel-forming units 12110 and second first channel-forming units 12120 (also referred to as "first needles" and "second needles," respectively) received in corresponding channel-receiving portions arranged based on first and second patterns, respectively. In this example, each of the first channel-forming units 12110 has a first diameter of the first channel (approximately 180 μm in this example), and each of the second first channel-forming units 12120 has a second diameter of the second channel (approximately 405 μm in this example). In this example, each of the first channel-forming units 12110 has a first length 12010 of approximately 180 mm, and each of the second channel-forming units 12120 has a second length 12020 of approximately 90 mm.

[0227] Figure 11C 1 is an enlarged view of example device 10000, showing a plurality of first channel receiving portions 11110 and second channel receiving portions 11120 on support units 11000 and 11000'. The plurality of first channel forming units 12110 and second first channel forming units 12120 are arranged based on pattern 11100 and are installed through the respective first channel receiving portions 11110 and second channel receiving portions 11120, so that the plurality of first channel forming units 12110 and second channel forming units 12120 can be assembled to form predetermined first and second arrays, respectively.

[0228] In some embodiments, the plurality of first channel forming units 12110 and the second first channel forming units 12120 are used for muscle fiber formation and fat fiber formation, respectively. Example 12: Example Devices and Hydrogel Constructs

[0229] Now refer to 12A to 12C , showing the process of forming an example device (including a hydrogel portion and a channel portion). Figure 12A Example device 10000 (as described in Example 11) is shown, comprising a plurality of first channel-forming units 12110 and a plurality of second channel-forming units 12120 assembled to form a predetermined first array and a predetermined second array, respectively, and a residual space therebetween, the residual space being defined by support units 11000 and 11000', and a hydrogel portion 13000 formed in the residual space. Hydrogel portion 13000 is formed by pouring a hydrogel composition (such as that described in Table 1) into the residual space and allowing it to solidify. In this example, a hydrogel composition containing approximately 2% sodium alginate and approximately 10% gelatin (such as mixture A1 described in Table 1) is used to form hydrogel portion 13000.

[0230] Figure 12B An example device 10000 (also referred to as a "channel assembly") is shown with a hydrogel portion 13000, wherein a plurality of first channel-forming units 12110 are removed and a plurality of second channel-forming units 12120 are present. In this example, the plurality of first channel-forming units 12110 are removed from the hydrogel portion 13000, thereby forming a plurality of first channels 13110 in a predetermined first array in the hydrogel portion 13000, wherein a single first channel 13110 is configured to receive at least one first cell composition. In this example, a cell composition comprising cells suspended in type 1 collagen ( Angus bovine myoblasts (approximately 1 x 10 7Cells / ml) of the first cell composition (cell composition B according to Table 2) were inoculated into the first channel 13110 to form a first cell culture. A neutralization solution (10 ml, A type 1 collagen solution, 6 mg / ml (bovine, Advanced BioMatrix) was added to the cell composition B (approximately 1 / 10 of the final volume was used) for cross-linking at approximately 37°C.

[0231] In some embodiments, the plurality of second channel forming units 12120 are removed from the hydrogel portion 13000 in no particular order, i.e., before or after the step of removing the first needle, separately, or simultaneously. In this example, after the step of obtaining the first cell culture, the plurality of second channel forming units 12120 are removed from the hydrogel portion 13000, so that a plurality of second channels (not shown) in a predetermined second array are formed, wherein a single second channel is configured to receive at least one second cell composition. In this example, the cells comprising cells suspended in type 1 collagen ( Adipose-derived stem cells from Angus cattle (1 x 10 7 A second cell composition (cell composition C according to Table 2) containing 10 cells / ml was seeded into the second channel. Neutralization solution was also added to cell composition C (approximately 1 / 10 of the final volume was used) for cross-linking at approximately 37°C.

[0232] Figure 12C and Figure 12D An example hydrogel construct 14000 is shown comprising a hydrogel portion 13000, a first channel 13110 seeded with a first cell composition (comprising muscle-derived cells), and a second channel 13120 seeded with a second cell composition (comprising adipose-derived cells) after removal of the second first channel-forming unit 12120 and the two support units 11000 and 11000'. Example 13: Hydrogel constructs with perfusion channels

[0233] Now refer to 13A to 13B . Figure 13A and Figure 13BAn example hydrogel construct 14000 prepared as described in Example 12 but subsequently extruded with a plurality of third channels 14130 (perfusion channels) by a biopsy punch tool is shown. In this example, the third channels 14130 form a 4×4 perfusion third array. In this example, each of the third channels 14130 has a diameter of approximately 225 μm, and the distance between two adjacent third channels is approximately 3 mm. A cutting tool (such as a biopsy punch tool) is used to extrude the third channels 14130. In some embodiments, endothelial cells can be seeded in the third channels 14130 to form endothelial tubes along the surface of the third channels 14130 serving as perfusion channels.

[0234] In another example, a third channel can be created in a hydrogel portion (e.g., hydrogel portion 13000) by providing a plurality of third channel-forming units in the device described in Example 11 along with the first and / or second channel-forming units, providing a third cell composition (comprising endothelial cells according to cell composition D in Table 2) in the third channel, and allowing the third cells to grow. In some embodiments, the third cells comprise endothelial cells, which will grow and differentiate along the surface of the third channel. The third channel is configured to be in fluid communication with a perfusion assembly system, which will be described in more detail later.

[0235] In yet another example, endothelial cells are seeded into a second (or other) channel (such as second channel 13120), and the second (or other) channel can be used for perfusion by connecting to a perfusion assembly system. Example 14: Example Hydrogel Construct Connector and Perfusion Component System

[0236] Figure 14A and Figure 14B An example hydrogel construct, such as the hydrogel construct 14000 described in any of the examples herein (such as Example 13), is shown sandwiched between two example hydrogel construct connectors 15000 to form an example hydrogel construct assembly 16800 .

[0237] Now refer to Figures 14C to 14E, shows an example hydrogel construct connector 15000 from different perspectives. Example hydrogel construct connector 15000 includes a hydrogel construct receiving portion 15100, a cavity 15200, and a tubing connection portion 15900. Hydrogel construct receiving portion 15100 is configured to receive at least one end of any of the hydrogel constructs described in the previous examples, such that a perfusion channel (such as the third channel 14130 described in Example 13) is in fluid communication with cavity 15200. Cavity 15200 extends through tubing connection portion 15900, which can be connected to the rest of the perfusion assembly system and allow fluid communication therebetween. In one example, the size and shape of the hydrogel construct receiving portion 15100 is determined to be larger than the hydrogel construct so that the end of the hydrogel construct can be partially fitted into the hydrogel construct receiving portion 15100, while leaving space between the end of the hydrogel portion and the hydrogel construct receiving portion 15100 so that the entrance of the perfusion channel (such as the third channel 14130 described in Example 13) is not blocked and allows fluid communication. In some examples, a sealing step as described in Example 18 can be included to prevent fluid leakage from the connector-hydrogel interface.

[0238] Figure 14F An example perfusion assembly system 16000 is shown, which generally includes a first pump 16100, a second pump 16100', a hydrogel construct assembly 16800, and a mixer 16200 containing a culture medium 16300, an agitator 16500, a culture medium recycler 16600, and a vent 16700. These components are connected in fluid communication with each other. Figure 14AThe hydrogel construct connector 15000 (or tubing connector) in the embodiment is attached to an example meat hydrogel construct (such as those described in Example 12) to form a hydrogel construct assembly 16800, and a conduit 16900 is connected to the hydrogel construct assembly 16800 and a source of oxygenated culture medium in fluid communication. In this example, a first pump 16100 and a second pump 16100' are located upstream and downstream of the hydrogel construct assembly 16800, respectively. The mixer 16200 is a rotary bioreactor with an agitator 16500 for oxygenating the culture medium 16300 and an exhaust port 16700. The hydrogel construct assembly 16800 has a plurality of perfusion channels (similar to or identical to the third channel 14130 discussed in Example 12) connected to the perfusion assembly via conduits 16900. In this example, the mixer 16200 is a 100 mL bioreactor with a PTFE filter to allow sterile air to enter and contact the culture medium 16300. As the culture medium 16300 is mixed, air (and oxygen) is dissolved to oxygenate the culture medium. One or more pumps are used to pump oxygenated culture medium into the hydrogel construct in the hydrogel construct assembly 16800, and deoxygenated culture medium and waste are pumped away from the hydrogel construct. Thus, the hydrogel construct is perfused with culture medium 16300 (via the perfusion step). Optionally, a culture medium recycler 16600 is connected downstream of the hydrogel construct to remove any waste. Optionally, the hydrogel construct is then allowed to mature to produce a tissue construct as the final cultured meat product. Example 15: Final Product of Example Tissue Construct

[0239] Now refer to FIG. 15A to FIG. 15B . Figure 15A and Figure 15B The final target product of the example tissue construct 17000 formed after perfusion as described in Example 14 is shown, wherein the plurality of third channels 14130 as described in Example 13 are still present. Figure 15BA cross-sectional view of a tissue product 17000 simulating Angus beef tissue is shown. In this example, tissue construct 17000 is then treated with a contraction agent to shrink the tissue. In this example, a low molecular weight chitosan solution (e.g., approximately 15 kDa) is used, resulting in tissue construct 17000 shrinking approximately three-fold. This allows the tissue construct to be exercised to induce greater matrix rearrangement and, therefore, texture. In other examples, the hydrogel construct or tissue construct can be immersed in a solution, such as acetic acid (i.e., vinegar), that shrinks the fiber-fiber distance to, for example, approximately 20 μm. In other examples, the force exerted by the muscle fibers will shrink the diameter (e.g., approximately 1-20 times, for example, approximately 3 times) (which can vary depending on the hydrogel used) to more closely resemble the physiologically relevant diameter (50-66.7 μm muscle fiber diameter found in beef). In other examples, a contraction step can be performed on a hydrogel construct (e.g., hydrogel construct 14000) prior to the perfusion step described in Example 14. Example 16: Example device with holder unit

[0240] Now refer to 16A to 16B , another example device 20000 is shown, comprising a support unit 21000 at the proximal end of a holder assembly, a support unit 21000′ at the distal end of the holder assembly (each having a predefined pattern 21100 and pattern 21100′, respectively), a plurality of first channel-forming units 22110, a plurality of second channel-forming units 22120, and a holder unit 27000. The connection of the two support units 21000, 21000′, the plurality of first channel-forming units 22110, and the plurality of second channel-forming units 22120 is similar to the connection discussed in Example 11, except that a holder unit 27000 is further included, which is sized and shaped to receive the two support units 21000, 21000′ and form the holder assembly. The corresponding channel receiving portions are constructed and arranged in a pattern 21100 so that the plurality of first channel forming units 22110 and second channel forming units 22120 can be assembled with the holder assembly to form a predetermined first array, a predetermined second array, and a remaining space in the holder assembly. In this example, the holder unit 27000 is a U-shaped holder. In this example, the remaining space is a hydrogel receiving portion 23100 where the hydrogel portion of the hydrogel construct can be formed, such as Figure 16B shown.

[0241] Figure 16C2 is an enlarged view of the channel assembly 20000, showing that the plurality of first channel forming units 22110 and the plurality of second channel forming units 22120 partially extend through the plurality of first channel receiving portions and the plurality of second channel receiving portions (not labeled) on the support plate 21000. The plurality of first channel forming units 22110 and the plurality of second channel forming units 22120 are arranged based on the pattern 21100 defined on the support units 21000 and 21000'. Example 17: Example Device Demonstrating Muscle Fiber Formation

[0242] Now refer to 17A to 17B , shows another example device 30000, which includes a support unit 31000, a plurality of channel forming units 32110, and a holder unit 37000. The support unit 31000 generally includes two support plates having channel receiving portions 31110 generally parallel to each other. The two support plates are connected by two additional connecting plates and integrated into one piece, forming a rectangular frame. The support unit 31000 includes a pattern 31100 and a corresponding pattern 31100' on opposite sides formed by the plurality of channel receiving portions 31110. In this example, the plurality of channel receiving portions form a 1x3 matrix as the pattern 31100. The support unit 31000 and the holder unit 37000 are assembled to form a holder assembly.

[0243] Similar to the previous examples, each channel receiving portion 31110 is sized and shaped to receive at least a portion of a channel forming unit 32110 (e.g., a needle) and to confine the channel forming unit 32110 to its relative position. The diameter of each channel receiving portion 31110 is slightly larger than the channel diameter to receive at least a portion of a single channel forming unit 32110. When the plurality of channel forming units 32110 are arranged based on the pattern 31100 and installed through the corresponding channel receiving portions 31110, the plurality of channel forming units 32110 can be assembled with the holder assembly to form a predetermined first array and a remaining space in the holder assembly. In this example, the remaining space is a hydrogel receiving portion 33100 that can receive a hydrogel composition to form the hydrogel portion of the hydrogel construct.

[0244] In this example, the holder unit 37000 is a silicone pad that is sized and shaped to fit over the support unit 31000 .

[0245] In this example, the support unit 31000 of the device 30000 is manufactured by 3D printing, with three holes on each side, using polylactic acid as the filament for printing. In this example, the diameter of each hole is about 1 mm, with a channel-to-channel distance of about 3 mm.

[0246] Experiments demonstrating muscle fiber formation

[0247] In this example, cell growth and alignment within a channel was demonstrated using device 30000. The hydrogel portion was formed according to the method described in Example 9a and the details are described herein.

[0248] The components of the device 30000, such as the support unit 31000 and the holder unit 37000 (also referred to as a "pad"), are sterilized and cleaned with detergent, then rinsed in water, wiped dry, and sprayed with ethanol to sterilize. These components are placed in a biosafety cabinet and allowed to dry under UV light. After drying, the support unit 31000 is placed on the holder unit 37000 and pressed firmly so that there is a slight adhesion between them to prevent subsequent leakage of the hydrogel.

[0249] A plurality of channel forming units 32110 are sterilized and inserted into the channel receiving portion 31110 of the support unit 31000. In this example, the plurality of channel forming units 32110 are 24G needles (with an outer diameter of about 0.566 mm) slightly smaller than the diameter of the channel receiving portion 31110.

[0250] Hydrogel composition Formulation #1, as described in Table 1, was prepared according to the procedure described in Example 9b, with a final concentration of approximately 13.5% gelatin and approximately 1% transglutaminase TI. Hydrogel composition Formulation #1 was quickly transferred to the device 30000 with a mixing time of approximately one minute and allowed to solidify at room temperature. The device 30000 containing hydrogel composition Formulation #1 was then transferred to a sterile container containing basal medium (DMEM:F12) containing approximately 100 ug / mL Primocin, and the hydrogel composition was allowed to crosslink at room temperature for 48 hours. After 48 hours, a hydrogel portion formed in the hydrogel receiving portion 33100, and the entire hydrogel construct was incubated in culture medium at approximately 37°C for approximately 2 hours.

[0251] The culture medium is then removed as much as possible and the entire construct is then incubated at about 75°C for about 5-10 minutes to inactivate transglutaminase TI.

[0252] The plurality of channel forming units 32110 are removed in a sterile environment, so that a plurality of channels (also referred to as "microchannels") in a predetermined array are formed in the hydrogel portion. The hydrogel construct can now be placed in a 6-well plate.

[0253] In some embodiments, the hydrogel construct is optionally sterilized again by ethanol and / or UV.

[0254] A cell composition (similar or identical to one of the examples described in Table 2) was prepared according to the following steps:

[0255] Prepare porcine primary myoblasts (passage 6 or younger) by rinsing the myoblasts with sterile PBS. In this example, cells from passage 5 were used, followed by the addition of 4 mL of 0.25% trypsin / EDTA to a T75 flask and incubation at approximately 37°C for approximately 5 minutes. Occasionally tap the T75 flask on the sides and bottom. After incubation for 5 minutes, neutralize the trypsin solution with an equal volume (4 mL) of trypsin neutralizer solution.

[0256] The cell mixture was then transferred to a centrifuge tube and centrifuged at 200 g for approximately 5 minutes. The supernatant was then discarded and the cells were resuspended in PBS. Another centrifugation was performed at approximately 200 g for 5 minutes. The supernatant was again discarded and the cells were resuspended in 100 uL of Formulation #6 prepared according to Example 9b. In this example, the final cell density was approximately 1 x 10 5 cells / mL.

[0257] Immediately inject or introduce approximately 20 uL of the cell composition into each channel. Leave the entire hydrogel construct in a 37°C incubator for approximately 30 minutes to complete crosslinking. After sufficient crosslinking, add approximately 5 mL of culture medium (DMEM + 10% FBS) to the well plate and allow the entire construct to incubate at approximately 37°C.

[0258] The above preparation process was repeated similarly to prepare a control, in which cells without hydrogel composition formulation #6 were injected into the channel. After centrifugation, another cell composition was prepared by resuspending the cells in approximately 100 uL of culture medium instead, and the final cell density was approximately 1 x 10 5 About 20 μL of the cell composition in culture medium (without hydrogel) was then injected into each channel. The cells were then allowed to attach overnight in an incubator, with the channel assembly occasionally flipped to allow attachment to the entire microchannel surface.

[0259] result

[0260] By injecting skeletal muscle cells into microchannels (both with and without hydrogels), the cells tend to align parallel to their microchannel templates. Figures 17C to 17E , Microscope images show microchannels seeded with porcine myoblasts within a fibrinogen + thrombin gel (i.e., hydrogel composition Formulation #6) and the resulting muscle fiber formation (scale bar = 200 μm). For cells in Formulation #6, cells were evenly distributed throughout the microchannel after seeding and crosslinking of Formulation #6, as shown in FIG. Figure 17C Within 1 day of seeding, cells rapidly formed aligned muscle fibers parallel to the microchannels, as shown in Figures 17D to 17EAs shown, a tissue construct was formed, in which some shrinkage of the hydrogel was observed. Unexpectedly, shrinkage occurred in Formulation #6 after one day, and the muscle fiber diameter became much smaller than the diameter of the microchannel. This can be exploited when smaller muscle fibers are desired to achieve a larger meat texture. In other instances, manual shrinkage was required via the addition of a shrinkage agent.

[0261] Now refer to Figure 17F , shows myoblasts seeded into a microchannel in the absence of a hydrogel, such as hydrogel composition formulation # 6. The cells are seen attached to the walls of the microchannel and tending to align parallel to the microchannel, with slight confluence, likely still proliferating and migrating to cover empty spaces.

[0262] These results indicate that the microchannels provided by the example devices can effectively support the formation of cell tissues (e.g., muscle fibers), and that the presence of a hydrogel in the cell composition can help promote tissue formation (e.g., regarding cell alignment, cell confluence and proliferation in the channels, and contraction of cell tissues (e.g., muscle fibers). Other example cell compositions with other additives will also improve tissue formation. Example 18: Example Device and Example Tissue Construct

[0263] Now refer to 18A to 18C, shows another example device 40000, which generally includes a support unit 41000 at the proximal end of the holder assembly, a support unit 41000' at the distal end of the holder assembly, a plurality of first channel forming units 42110, a plurality of second channel forming units 42120, and a holder unit 47000. In this example, these support units are in the form of plates. The two support units 41000 and 41000' are sized and shaped to match the proximal and distal sides of the holder unit 47000, respectively, thereby forming the holder assembly and defining a space therein. Support units 41000 and 41000' are generally rectangular in shape, in the form of a block having a certain thickness, and include a first channel receiving portion 41110 and a second channel receiving portion 41120 in a central region, as well as an outer region 41130. The connections of the two support units 41000, the first channel forming unit 42110, the second channel forming unit 42120, and the holder unit 47000 are the same or similar to those discussed in Example 16 and will not be reproduced here for the sake of clarity. In the assembled state, the corresponding channel receiving portions 41110 and 41120 on the support units 41000 and 41000' are constructed and arranged into a pattern 41100 and a corresponding pattern 41100' so that the proximal and distal ends of each of the first channel forming units 42110 can be inserted and received in the corresponding first channel receiving portion at the proximal and distal sides, respectively, to form a predetermined first array, and the proximal and distal ends of each of the second channel forming units 42110 can be inserted and received in the corresponding second channel receiving portion at the proximal and distal sides, respectively, to form a predetermined second array, with a remaining space therebetween. The remaining space is a hydrogel receiving portion 43100, which is configured to receive a hydrogel composition to form a solidified hydrogel portion, so that a plurality of first channels in a predetermined first array are formed by removing the plurality of first channel forming units 42110 from the hydrogel portion, and a single first channel is configured to receive at least one first cell composition therein to form at least one first cell culture.

[0264] like Figure 18CAs shown, pattern 41100 is formed by a uniform arrangement of a plurality of first channel receiving portions 41110 and a plurality of second channel receiving portions 41120. In this example, pattern 41100 is formed by overlaying a 24×24 matrix of first channel receiving portions 41110 with a 5×5 matrix of second needle receiving portions 41120. Each of first channel receiving portions 41110 and second channel receiving portions 41120 is sized and shaped to receive a first channel forming unit 42110 and a second channel forming unit 42120, respectively. In this example, each of the first channel receiving portions 41110 has a diameter of approximately 250 μm, and the distance between two adjacent first channel receiving portions 41110 is approximately 250 μm (center-to-center distance is approximately 500 μm), and each of the second channel receiving portions 41120 has a diameter of approximately 550 μm, and the distance between two adjacent second channel receiving portions 41120 is approximately 1450 μm (center-to-center distance is approximately 2000 μm). Each first channel-forming unit 42110 has a diameter of the first channel receiving portion 41110 (approximately 250 μm in this example), and each second channel-forming unit 42120 has a diameter of the second channel receiving portion 41120 (approximately 550 μm in this example).

[0265] In this example, experiments were conducted using the example apparatus 40000 to verify that the fabrication process was consistent and applicable to various hydrogel types.

[0266] The hydrogel compositions Formulation #2, Formulation #3, and Formulation #1 as described in Table 1 were prepared according to the procedure described in Example 9b.

[0267] Similar to the previous example, the components of the device 40000 (eg, the proximal and distal support units 41000 and 41000 ′, the first and second channel forming units 42110 and 42120 ) are sterilized and transferred into a biosafety cabinet.

[0268] The support unit 41000 and the support unit 41000 ′ are assembled to the holder unit 47000 such that the hole arrays (eg, of pattern 41100 ) at the proximal and distal sides are aligned.

[0269] In this example, the first channel forming units 42110 are elongated metal wires each having a diameter of approximately 0.2 mm, and the second channel forming units 42120 are elongated metal wires each having a diameter of approximately 0.6 mm. The plurality of first channel forming units 42110 and second channel forming units 42120 are individually inserted into the support unit 41000 and the support unit 41000′, starting with the first channel forming units 42110 and followed by the second channel forming units 42120.

[0270] Support unit 41000 and support unit 41000 ′ are positioned (pulled away from each other) to the desired plate-to-plate distance.

[0271] The plurality of first channel forming units 42110, second channel forming units 42120, and support plate 41000 are assembled to a holder unit 47000 (also referred to as a "U-shaped holder" in this example) to form an example device 40000, as shown in FIG. Figure 18B The entire device 40000 was sprayed with ethanol and allowed to dry under UV in a biosafety cabinet to sterilize.

[0272] seal: In this example, gelatin sealing mixture (GSM) was prepared by melting a mixture of approximately 20% (w / v) gelatin (Aladdin-e) at 37°C. Once the exemplary device 40000 was dry, the GSM was transferred to a biosafety cabinet. A small amount (approximately 100 μL per side) of GSM was added to the interface between the U-shaped holder unit 47000 and the support unit 41000 to create a seal without contacting the well array in the plate or the wire. While adding the GSM, the entire device 40000 was tilted so that the GSM rested horizontally on each side of the interface while solidifying. The GSM seal was then allowed to cool and solidify at room temperature. After the GSM solidified, another small amount (approximately 100 μL) of GSM was added to the outer surface of each support unit 41000, creating a thin layer on the support unit 41000 to prevent subsequent leakage of the hydrogel composition from the remaining space by flowing into the wells. In this example, GSM forms a thin (<1 mm) gelatin layer on the outer surface of the support unit 41000. Slowly add a few drops of hot sterile water (about 50°C) to the wire to melt the residual GSM in the wire that has leaked into the channel assembly. Use a sterile tissue to wipe away the residual water.

[0273] In some embodiments, the U-shaped holder is closed and the device is rotated so that GSM can be added to the interface on each side of the support unit (ie, left, right, top, and bottom) to achieve a proper seal.

[0274] In some embodiments, the above sealing steps are repeated on the outward-facing surface of support unit 41000 .

[0275] In some embodiments, the aforementioned sealing step is also applied to the interface between the U-shaped holder unit 47000 and the support unit 41000 ′.

[0276] After sealing, the device 40000 was sprayed with ethanol and allowed to dry under UV for 1 hour.

[0277] In case of using different hydrogel compositions according to Table 1, the following steps were performed.

[0278] Formulation #2 as the hydrogel portion:

[0279] Melt hydrogel composition formulation #2 in boiling water. To prevent air bubbles from becoming trapped, occasionally transfer the solution to a biosafety cabinet and open the lid to release pressure and air bubbles. If air bubbles persist, allow the solution to solidify at 4°C and then repeat the melt.

[0280] The entire apparatus 40000 is placed on ice. The ice promotes rapid cooling of the mixture to prevent the gelatin seal (GSM) created previously from melting.

[0281] Once the hydrogel composition is fully thawed, a sufficient volume is added to the hydrogel receiving portion 43100. The hydrogel composition formulation #2 is allowed to solidify at 4°C for 30 minutes and then immersed in 1% calcium chloride for 4 hours for cross-linking to solidify into a hydrogel portion.

[0282] Formulation #3 as the hydrogel portion:

[0283] The hydrogel composition Formulation #3 was thawed at 37°C.

[0284] The entire apparatus is placed on ice at 40000. The ice promotes rapid cooling of the mixture to prevent the gelatin seal created earlier from melting.

[0285] Once the hydrogel composition is fully thawed, a sufficient volume is added to the hydrogel receiving portion 43100. The hydrogel composition formulation #3 is allowed to solidify at 4°C for 30 minutes and then immersed in 1% calcium chloride for approximately 4 hours for cross-linking to solidify into a hydrogel portion.

[0286] Formulation #1 as the hydrogel portion:

[0287] The hydrogel composition was prepared as described in Example 9b and used immediately. The mixture was immediately mixed for approximately 30 seconds and then added to the hydrogel receiving portion 43100 of the device 40000. Hydrogel composition Formulation #1 was allowed to crosslink for approximately 48 hours at room temperature and then for approximately 2 hours at 37°C. In some embodiments, mixing and adding hydrogel composition Formulation #1 to the hydrogel receiving portion 43100 should occur within one minute.

[0288] By using a sterile container, the channel assembly filled with one of the above hydrogel compositions was kept at 4° C. for 30 minutes for low-temperature incubation. This step allowed the formed hydrogel portion to further harden to obtain greater temporary mechanical stability.

[0289] To prepare the cell composition, cells of interest were harvested from passages in a manner similar to that described in Example 17. After trypsinization and neutralization, the harvested cells were centrifuged twice. After the second centrifugation, the cells were resuspended in a solution of Formulation #6. In other examples, the cells were resuspended in PBS, sterile water, or any of the hydrogel compositions described in Table 1.

[0290] In this example, approximately 1x 10 6 In other examples, the final density of the cells is at least 1 x 10 6 cells / mL.

[0291] A small amount (about 100 uL) of cell composition is added to the interface between the channel forming units 42110, 42120 (i.e., metal wires) and the support units 41000, 41000' so that it completely covers all first channel receiving portions 41110 and second channel receiving portions 41120 of the support units 41000, 41000'. In some embodiments, the device 40000 is rotated vertically rather than horizontally so that the 100 uL volume does not fall toward one side. When the first channel forming unit 42110 is removed or pulled out, the cell composition flows into the first channel (also referred to as a "microchannel"). More cell composition is added as needed.

[0292] In some embodiments, the first channel-forming unit 42110 and the second channel-forming unit 42120 are removed one by one from the example apparatus 40000 , and a cell composition is introduced.

[0293] In some embodiments, the cell composition is injected into the microchannel.

[0294] In some embodiments, the cell composition (containing the hydrogel composition) is allowed to crosslink at room temperature for about 30 minutes.

[0295] In some embodiments, after the cell composition flows into the first channel in the hydrogel portion formed by the first channel-forming unit 42110 and solidified, the second channel-forming unit 42120 is removed.

[0296] In some embodiments, the first channel-forming unit 4211 and the second channel-forming unit 4212 (i.e., metal wires) are selectively removed, and different cell compositions containing different cell types are introduced into the selected channels and allowed to crosslink. In other words, more than one type of first cell composition and second cell composition can be sequentially and selectively introduced.

[0297] The final construct includes a central region containing a cell composition and a hydrogel portion therebetween, and an outer region (or hydrogel exterior) containing essentially only the hydrogel portion. The outer region is trimmed, and the central region containing the cell composition is placed in a culture dish with an appropriate amount of culture medium (in this example, DMEM + 10% FBS) to completely immerse the hydrogel construct in the culture dish to allow cell growth, thereby forming a tissue construct. Fresh culture medium (DMEM + 10% FBS) is provided to the tissue construct every two days.

[0298] In some embodiments, cells are resuspended in a cell composition using other suitable culture medium to grow tissue constructs.

[0299] result

[0300] Now refer to Figures 18D to 18F , showing various perspective views of a hydrogel portion having a microchannel prior to introduction of a cell composition and a trimmed or untrimmed hydrogel exterior. Figure 18D : is a side view image of a trimmed hydrogel construct prepared using Formulation #2 as the hydrogel portion. When the hydrogel exterior is further trimmed so that only the central microchannel segment remains, the hydrogel portion with microchannels resembles the texture seen in natural meat (such as pork). Figure 18E is a side view image of an untrimmed hydrogel section prepared using Formulation #3 as the hydrogel section. Figure 18F The figure shows a side view image of an untrimmed hydrogel portion prepared using Formulation #1 as the hydrogel portion. The results demonstrate that various hydrogel compositions can be successfully formed into hydrogel portions having microchannels using various example devices with various patterns or array arrangements and various microchannel diameters in the support unit, and that the microchannels can support the formation of densely packed muscle fibers. After introducing a cell composition into the microchannels and incubating the hydrogel construct under appropriate conditions to allow for cell proliferation, differentiation, and maturation, the microchannels in the hydrogel portion can support the formation of cellular tissues (e.g., muscle fibers), resulting in densely packed muscle fibers, thereby producing a desired tissue construct that resembles natural tissue constructs, such as meat. Example 19: Example device with fastener and example tissue construct

[0301] 19A to 19CAnother example device 50000 is shown, which generally includes a support unit 51000 configured to be connected to the proximal side of the holder assembly, a support unit 51000' configured to be connected to the distal side of the holder assembly, a plurality of support unit fasteners 51500, a plurality of channel forming units 52110, and a holder unit 57000. The connection of the support unit 51000, the support unit 51000', the channel forming unit 52110, and the holder unit 57000 is similar to the connection discussed in Examples 16 and 18, except that: the device in this example further includes a plurality of support unit fasteners 51500; the support unit 51000 and the support unit 51000' both include corresponding fastener receiving portions 51510; and the support unit 51000 and the support unit 51000' are further connected and fixed to each other by installing the plurality of support unit fasteners 51500 through the fastener receiving portions 51510. The support unit fasteners provide additional support and maintain the relative position of the support unit to the device.

[0302] like Figure 19C As shown, pattern 51100 is formed by a uniform arrangement of multiple channel receiving portions 51110. Multiple channel receiving portions 51110 are constructed and arranged in pattern 51100 so that the multiple channel forming units 52110 can be assembled to form a predetermined array and a remaining space. The remaining space is a hydrogel receiving portion 53100, which is configured to receive a hydrogel composition to form a solidified hydrogel portion, so that by removing the multiple channel forming units 52110 from the hydrogel portion, a plurality of channels in a predetermined array are formed, and a single channel is configured to receive at least one cell composition therein to form at least one cell culture. Support unit 51000 includes multiple fastener receiving portions 51510 around the perimeter of pattern 51100. Support unit 51000' also includes multiple fastener receiving portions around the perimeter of pattern 51100'. Each of the channel receiving portion 51110 and the fastener receiving portion 51510 is sized and shaped to respectively receive the corresponding channel forming unit 52110 and the supporting unit fastener 51500. In this example, each channel receiving portion 51110 has a diameter of approximately 280 μm, and the distance between two adjacent channel receiving portions 51110 is approximately 120 μm, and each fastener receiving portion 51510 has a diameter of approximately 600 μm, each channel forming unit 52110 has a diameter of the channel receiving portion 51110 (approximately 280 μm in this example), and each supporting unit fastener 51500 has a diameter of the fastener receiving portion 51510 (approximately 600 μm in this example).

[0303] Now refer to Figures 19D to 19L, shows a diagram of an example hydrogel portion prepared using apparatus 50000 before and after introduction of a cell composition. In this example, 1x10 6 Porcine myoblasts at a concentration of 10 cells / mL were incorporated into a 0.2 mm microchannel. Figures 19D to 19F are isometric, front cross-sectional, and side view images of an example hydrogel portion prepared using Formulation #1 as the hydrogel portion. Figures 19G to 19H are isometric and side view images of an example hydrogel portion prepared using Formulation #3 as the hydrogel portion. Figures 19I to 19K are isometric, front, and side view images showing another hydrogel portion prepared using Formulation #2 as the hydrogel portion. Figure 19L : is a microscopic image showing porcine myoblasts in Formulation #6 introduced into a 0.2 mm microchannel of an example hydrogel construct with Formulation #2 as the hydrogel portion (scale bar = 100 μm). The results show that various hydrogel compositions can be successfully formed into hydrogel portions having microchannels using various example devices having various patterns or array arrangements and various microchannel diameters in the support unit, and that the microchannels can support the formation of densely packed muscle fibers. After introducing the cell composition into the microchannel and incubating the hydrogel construct under appropriate conditions to allow cell proliferation, differentiation, and maturation, the microchannels in the hydrogel portion can support the formation of cell tissue (such as muscle fibers), which can form densely packed muscle fibers, thereby producing a desired tissue construct that resembles a natural tissue construct, such as meat. Example 20: Reusable Example Apparatus and Method for Forming a Sacrificial Portion

[0304] In this example, another example device similar to the device described in Example 16 is used to illustrate that when the formed cell construct is removed from the device, the device can be efficiently reused without completely disassembling the components (including the first channel forming unit or the second channel forming unit) from the holder assembly. 20A to 20F, shows another example device 60000, which generally includes a support unit 61000 at the proximal end of a holder assembly, a support unit 61000' at the distal end of the holder assembly, a plurality of first channel-forming units 62110, a plurality of second channel-forming units 62120, and a holder unit 67000. In this example, a 0.2 mm (diameter) wire and a 0.4 mm wire are used as the first channel-forming unit 62110 and the second channel-forming unit 62120, respectively. In this example, one or more sacrificial portions are provided to serve as temporary support units. In some embodiments, the first sacrificial portion 63100 and the second sacrificial portion 63200 are formed in the device 60000 using one or more materials, compounds, or compositions that can solidify into a solid or semi-solid state and can be removed (e.g., melted) under certain conditions, such as water (which can be in the form of ice at low temperatures) or a hydrogel composition (e.g., any of the hydrogel compositions described in Table 1).

[0305] A method of forming a hydrogel construct (with a sacrificial portion) using the reusable example device 60000 includes the following steps.

[0306] Similar to the previous example, two support units 61000 and 61000', a plurality of first channel forming units 62110, a plurality of second channel forming units 62120, and a holder unit 67000 are assembled to form a device 60000. The device 60000 is sprayed with ethanol and allowed to dry under UV in a biosafety cabinet for sterilization.

[0307] When the entire structure is dry, a drop of gelatin sealing mixture (GSM) containing 10% (w / v) gelatin (Aladdin-e) is added to the plate-holder interface (for both support units 61000 and 61000') and allowed to solidify. In some embodiments, the 10% (w / v) gelatin is warmed to about 37°C.

[0308] Place the device 60000 vertically in a sterile glass bottle (or any other container). Figure 20DAs shown, the support unit 61000 is set in a first position, which defines a first distance 63210 away from the proximal end of the holder assembly. In this example, the first distance 63210 is about 4 cm. Cold water is poured into the bottom of the glass bottle until the water level is about 2 cm from the support unit 61000'. The glass bottle is closed with a sterile cap and placed in a -20°C freezer to allow the water to freeze and form a first sacrificial portion 63100 (in the form of ice). The glass bottle containing the device 60000 is transferred back to the biosafety cabinet. While the support unit 61000' is maintained in the first position, a layer of GSM of about 2 cm is applied to the first sacrificial portion 63100 so that the GSM is in direct contact with the support unit 61000. In some embodiments, less GSM can be applied so that it does not come into direct contact with the plate (support unit). The GSM is allowed to solidify to form a second sacrificial portion 63200 on the first sacrificial portion. As shown Figure 20A and Figure 20D As shown, an ice layer (first sacrificial portion 63100) and a GSM layer (second sacrificial portion 63200) are then formed at the bottom (proximal side) of the example device 60000. After the GSM solidifies, the support unit 61000 is separated from the second sacrificial portion 63200.

[0309] The support unit 61000 is then moved and positioned in a second position defining a second distance 63410 away from the proximal side of the holder assembly. The above steps are repeated such that another layer of ice (third sacrificial portion 63300) and GSM (fourth sacrificial portion 63400) is subsequently formed on the first and second sacrificial portions at the bottom (proximal side) of the device 60000, as shown in FIG. Figure 20B and Figure 20E shown.

[0310] Now refer to Figure 20C and Figure 20F , the support unit 61000 is then moved and positioned in a third position, which defines a third distance 63510 away from the sacrificial portion. The device 60000 is then left at room temperature until the ice in the first sacrificial portion 63100 and the third sacrificial portion 63300 has melted, thereby forming a hydrogel receiving portion 63500 having a defined space defined by the second sacrificial portion 63200 and the fourth sacrificial portion 63400. The device 60000 is then removed from the glass bottle and placed horizontally, sterilized using ethanol, and allowed to dry under UV light for 1 hour.

[0311] Similar to the previous example, a hydrogel composition was added to the hydrogel receiving portion 63500. In this example, Formulation #3, as described in Table 1, was used. The device 60000 was immersed in 1% calcium chloride for 4 hours and maintained at 4°C for 30 minutes for low-temperature incubation. The hydrogel portion of the hydrogel construct was formed in the hydrogel receiving portion 63500 between the second sacrificial portion 63200 and the fourth sacrificial portion 63400.

[0312] To extract the hydrogel portion 63000 from the device 60000, the first channel forming unit 62110 and the second channel forming unit 62120 are pulled through the second sacrificial portion 63200, the hydrogel portion 63000 and the second sacrificial portion 63200', while at least a portion of the first channel forming unit 62110 and the second channel forming unit 62120 remain assembled with the support units 61000 and 61000'. The sacrificial portion can then be removed. In this way, when the formed construct is removed from the device and reassembled again, the device can be efficiently reused without completely disassembling the components (including the channel forming unit) from the holder assembly. In some embodiments, the cell composition or another hydrogel composition (with or without cells) can be loaded into the channel within the hydrogel portion 63000 via any method, such as injection, gravity flow, capillary action, and suction from the wire (channel forming unit) pull.

[0313] In some embodiments, the device 60000 is cleaned by immersing it in a solution of 0.2% type II collagenase (for degrading gelatin), 4% sodium citrate (for dissolving alginate hydrogel), and / or a boiling water solution, so that the device 60000 can be reused without having to reassemble the channel-forming units 62110 and 62120 and the plates 61000 and 61000'. In some other embodiments, the device 60000 is cleaned by autoclaving.

[0314] In some embodiments, it is sufficient for the device 60000 to include one supporting unit (ie, the supporting unit 61000 ′) because the sacrificial portion is prepared as a temporary supporting unit. Example 21: Post-processing of example tissue constructs

[0315] Described here are the post-processing of example tissue constructs prepared by the methods described in the previous examples. Figures 21A to 21B7000 is shown as an example tissue construct prepared by a similar process as described in the previous example. In this example, the example tissue construct 77000 includes a plurality of first cell cultures 73110 grown in a first channel, a plurality of second cell cultures 73120 grown in a second channel (as first cell tissue and second cell tissue), and a hydrogel portion 73000 disposed between the channel and an external area. In this example, the hydrogel portion 73000 was formed using hydrogel composition formulation #3 as described in Table 1. In this example, the hydrogel portion 73000 was formed using a hydrogel composition formulation #3 comprising approximately 1 x 10 7 The first cell culture 73110 was inoculated with porcine myoblasts containing approximately 1 x 10 cells / mL and the first cell composition of Formulation #6 to form a first tissue culture in the form of muscle fibers. 5 The second cell culture 73120 was inoculated with porcine mature adipocytes having a concentration of 10 cells / mL and a second cell composition of Formulation #6 to form a second tissue culture in the form of adipose fibers.

[0316] After forming the tissue construct 77000, the tissue construct 77000 is placed on the template 78000, as shown in FIG. Figures 21A to 21B In some embodiments, template 78000 is a diagonally rotated V-shaped or L-shaped holder.

[0317] The template 78000 containing the tissue construct 77000 was immersed in 4% sodium citrate and transferred to a 37°C incubator for approximately 30 minutes. As the hydrogel portion 73000 was dissolved or melted to be removed from the template 78000, the muscle fibers in the first cell culture 73110 and the fat fibers in the second cell culture 73120 began to settle to the bottom of the template 78000 to form a stacked fat and muscle fiber construct, as shown. Figures 21C to 21D shown.

[0318] After the hydrogel portion 73000 is melted or dissolved (or removed), the template 78000 containing the remaining tissue construct 77000 is gently aspirated and rinsed with sterile PBS without disturbing the stacked fat and muscle fiber constructs.

[0319] In some embodiments, the muscle fibers in the first cell culture 73110 and the fat fibers in the second cell culture 73120 are then bound together, such as by soaking the first cell culture 73110 and the second cell culture 73120 in about a 1% transglutaminase TI solution for about 10 minutes. In other embodiments, a small amount of another binding agent, such as the hydrogel composition described in Example 9b, is added to hold the muscle fibers in the first cell culture 73110 and the fat fibers in the second cell culture 73120 in place.

[0320] In some embodiments, template 78000 is populated with one or more tissue constructs, such as example tissue construct 77000, such that after hydrogel portion 73000 is dissolved or melted, multiple tissue constructs are stacked on top of each other to form a larger sized modular tissue construct.

[0321] In some embodiments, tissue constructs such as meat can undergo further processing to build muscle tissue (such as by electrical or mechanical means).

[0322] The exemplary embodiments of the present invention have been fully described. Although the description has been made with reference to specific embodiments, it will be clear to those skilled in the art that the present invention may be implemented with variations in these specific details. Therefore, the present invention should not be construed as being limited to the embodiments set forth herein.

[0323] For example, in the above examples, edible hydrogels have been used, but non-edible hydrogels may also be used. In some embodiments, the non-edible hydrogel may be biodegradable, such that the final product contains little or no trace of the non-edible hydrogel. In other examples, animal-derived or non-animal-derived hydrogels may be used.

[0324] For example, the provided devices, systems, and methods produce cultured tissues for consumption (e.g., cultured meat), but they can also be used for other applications, such as tissue engineering (e.g., for organ or tissue transplantation or replacement). Using similar vascularization concepts, the provided devices, systems, and methods can create thick, living tissue products, a truly elusive task currently in the tissue engineering industry.

[0325] For example, parameters such as dimensions (e.g., channel diameter, channel-channel distance, length), the number of needles (and therefore channels), the distribution of needle arrays (e.g., a first array, a second array, etc.), the size and shape of the needles and support plates, the type of hydrogel used, the type of cells used (e.g., adipose-derived cells in the hydrogel or channel (e.g., ADSC, DDFAT), the type of muscle-derived cells in the hydrogel or channel (e.g., myoblasts, satellite cells), the type of fibroblasts used (e.g., skeletal myofibroblasts, or from other tissue sources), the type of vascular cells used (e.g., endothelial cells, pericytes, vascular cells), the type of nerve cells used, the type of tendon cells used (e.g., tenocytes), the type of stem cells used) can be adjusted according to actual needs. Other types of cells (e.g., fibroblasts, endothelial cells, vascular cells, myoblasts, muscle cells, adipocytes, skin cells, neural cells, stem cells, tendon, liver, brain, bone, heart, kidney, and combinations thereof) for applications other than cultured meat (e.g., for bone tissue engineering, liver tissue engineering, etc.), the mixture of cells and hydrogel injected into the channel, the design of the channel (changing the channel size, cutting different channels for intermuscular fat, intramuscular fat, blood vessels, or muscle tissue bundles), the concentration of the hydrogel, the contractile agent, and the method of perfusing the culture medium through the channel or through the resulting blood vessels (e.g., the strength, duration, frequency, etc. of the pump) can be adjusted according to actual needs.

[0326] For example, in certain embodiments described herein, the cultured tissue is cultured meat, the first cells are muscle-derived cells, and the second cells are fat-derived cells, but in other embodiments, other cultured tissues can be produced, such as organ tissue, blood vessels, tendon cells, etc., and other cell types can be used for the first and second cells, depending on actual needs. For example, in certain embodiments, the cultured tissue is produced using first, second, and third cells, but a greater number of cells (or cell types) (such as a fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more cells) can be used in addition or alternatively.

[0327] For example, in certain embodiments as described herein, a first channel, a second channel, and / or a third channel are used to produce cultured tissue, but a greater number of channels (or corresponding channel-forming units and channel-receiving portions) (such as a fourth, fifth, sixth, seventh, eighth, ninth, tenth channel, or more) may additionally or alternatively be used.

[0328] For example, in some embodiments, a channel is created using a needle as an example of a channel forming unit, but the channel may be formed by other means, such as a wire, 3D printing, molding, and / or laser ablation.

[0329] For example, the cell composition can be provided by injection, aspiration, perfusion, or other available means to introduce, seed, or deposit the cell composition into the channel.

[0330] In some embodiments, the hydrogel portion holds the channels in place within the tissue construct.

[0331] In some embodiments, the hydrogel portion further comprises cells, such as fibroblasts, that will secrete extracellular matrix materials to form the network. For example, when the hydrogel portion is melted / dissolved, the network can remain, thereby maintaining the positioning of different tissues within the tissue construct.

[0332] For example, in some embodiments, these methods are performed using any of the devices described in any of the examples herein. In other embodiments, other devices having other types, structures, and configurations that can provide first channels, second channels, and / or third channels in the hydrogel portion to form a tissue construct can also be used.

[0333] For example, a tissue construct comprising first cells, second cells, and / or third cells is formed, but in other examples, different cell numbers and cell types, as well as different numbers, sizes, shapes, and patterns of channel-forming units and channels can be used.

[0334] For example, in some embodiments, the channel forming unit is an elongated straight wire or needle, but in other examples, other sizes, diameters, shapes, and forms of hydrogel forming units can be used. In other examples, the channel forming unit is flexible and can be deformed (such as twisted or compressed) at certain positions when forming the hydrogel construct. For example, when forming the hydrogel portion, a bundle of wires can be provided and compressed to form a capillary array for seeding endothelial cells, so that the final tissue construct simulates natural capillary tissue.

[0335] For example, in some embodiments, channel-forming units of uniform size and shape are used for specific cell types, but in other examples, channel-forming units of different sizes, shapes, and types may be used for each type of cell according to actual needs.

[0336] For example, in the above examples, edible hydrogels have been used, but non-edible hydrogels may also be used. In some embodiments, the non-edible hydrogel may be biodegradable, such that the final product contains little or no trace of the non-edible hydrogel.

[0337] For example, the provided devices, systems, and methods produce cell cultures for cultured meat, but they can also be used in tissue engineering, such as for organ replacement. Using the same vascularization concept, the provided devices, systems, and methods can create thick, living cell cultures, a truly elusive task currently in the tissue engineering industry.

[0338] For example, parameters such as dimensions (e.g., channel diameter, channel-channel distance, length), number of needles (and therefore channels), distribution of the channel array, size and shape of the needles and support plates, type of hydrogel used, type of fat cells in the hydrogel or channel (e.g., ADSC, DDFAT), type of muscle cells in the hydrogel or channel (e.g., myoblasts, satellite cells), type of fibroblasts used (e.g., skeletal myofibroblasts, or from other tissue sources), type of vascular cells used (e.g., endothelial cells, pericytes), type of neural cells used, type of tendon cells used (e.g., tenocytes), type of stem cells used, and other types of cells for applications other than cultured meat (e.g., for bone tissue engineering, liver tissue engineering, etc.), mixtures of cells and hydrogels injected into the channels, design of the channels (changing channel size, cutting different channels for intermuscular fat, intramuscular fat, blood vessels, or muscle tissue bundles), concentration of hydrogel, contractile agent, and the way in which culture medium is perfused through the channels or through the resulting blood vessels (e.g., pump strength, duration, frequency, etc.) can be adjusted according to actual needs.

[0339] For example, in certain embodiments described herein, the cultured tissue is cultured meat, the first cells are fat-derived cells, and the second cells are muscle-derived cells. However, in other embodiments, other cultured tissues can be produced, such as organ tissues, blood vessels, tendon cells, etc., and other cell types can be used for the first and second cells as needed. For example, the at least one or more first cells, second cells, and / or third cells can be derived from fibroblasts, endothelial cells, vascular cells, myoblasts, muscle cells, adipocytes, skin cells, neural cells, stem cells, tendon, liver, brain, bone, heart, kidney, and combinations thereof.

[0340] For example, in some embodiments, needles are used as an example of a channel forming unit to create or provide a channel in a hydrogel, but the channel can be formed by other means, such as wires, 3D printing, molding, and / or laser ablation.

[0341] For example, the support unit is in the form of a block having a certain thickness to support at least a portion of the channel forming unit, but the support unit may be in the form of a plate or a stack of plates, or in other forms or structures that can support the channel forming unit.

[0342] For example, in some embodiments, a first cell tissue (such as muscle fibers) and a second cell tissue (such as fat fibers) are produced simultaneously by these methods, systems and devices, but in other instances, each type of cell tissue can be produced separately, and the individual cell tissues can be combined into a desired array in a post-processing step.

[0343] For example, in some embodiments, ice or hydrogel is used as the sacrificial layer, but other phase change materials may be used instead.In some instances, the phase change material is non-toxic and safe to consume.

[0344] For example, the pattern (or plate design) used includes an array of holes of multiple diameters. For example, different array arrangements of microchannels can be created simply by changing the metal plate. For example, an array of uniformly and densely packed 0.2 mm microchannels can be used.

[0345] For example, the outer regions of the hydrogel construct are further trimmed so that only the microchannel segments remain.In some embodiments, the fiber template resembles the texture seen in traditional meats such as pork. Numbered Examples Numbered Example 1

[0346] Example 1. A method for producing cultured tissue, the method comprising the following steps: (a) providing a hydrogel composition and solidifying the hydrogel composition to form a hydrogel portion comprising a plurality of first channels and a plurality of second channels; (b) providing at least one first cell composition to the plurality of first channels to form at least one first cell culture, and providing at least one second cell composition to the second channels to form at least one second cell culture, so as to form a tissue construct.

[0347] Example 2. A method as described in Example 1, wherein the cultured tissue is cultured meat, the first cell composition comprises a plurality of first cells, the first cells are muscle-derived cells, muscle satellite cells and / or myoblast-derived cells, and the second cell composition comprises a plurality of second cells, the second cells are fat-derived cells.

[0348] Embodiment 3. The method according to any one of embodiments 1 to 2, wherein the hydrogel composition comprises one or more of alginate, gelatin, gellan gum, and combinations thereof.

[0349] Embodiment 4. The method of any one of embodiments 1 to 3, wherein the hydrogel composition comprises about 2% alginate and about 10% gelatin.

[0350] Example 5. The method according to any one of Examples 1 to 4, wherein the hydrogel composition further comprises adipose-derived cells and fibroblasts, and a combination thereof.

[0351] Example 6. A method as described in any one of Examples 1 to 5, wherein the first cell composition comprises muscle-derived cells, muscle satellite cells and / or myoblast-derived cells, and optionally one or more of collagen, gellan gum, alginate, gelatin and combinations thereof.

[0352] Example 7. The method of any one of Examples 1 to 6, wherein the second cell composition comprises adipose-derived cells, and optionally one or more of collagen, gellan gum, alginate, gelatin, and combinations thereof.

[0353] Example 8. The method of any one of Examples 1 to 7, further comprising the step of: (c) providing a plurality of third channels, and optionally providing at least one third cell composition in the plurality of third channels.

[0354] Example 9. The method of Example 8, wherein the third cells comprise fibroblasts, endothelial cells, and a combination thereof.

[0355] Example 10. The method of any one of Examples 8 to 9, wherein the at least one or more first cells, second cells and / or third cells are derived from fibroblasts, endothelial cells, myoblasts, muscle cells, adipocytes, skin cells, tendons, liver, brain, bones, heart, kidneys and combinations thereof.

[0356] Embodiment 11. The method of any one of embodiments 1 or 8, wherein the plurality of first channels, second channels, and / or third channels are formed by removing the plurality of first, second, and / or third channel-forming units from the solidified hydrogel composition.

[0357] Embodiment 12. The method of any one of Embodiments 1 or 8, wherein the plurality of first channels, second channels, and / or third channels are formed by 3D printing or laser ablation.

[0358] Example 13. The method of Example 8, further comprising the step of: (d) growing the first cells and the second cells until a desired tissue mass is obtained, thereby obtaining a cultured tissue product.

[0359] Example 14. The method of Example 13, wherein step (d) is performed by perfusing oxygenated culture medium through the tissue construct via the plurality of third channels.

[0360] Example 15. The method of Example 8, further comprising the step of: (e) growing the third cells until a desired tissue mass is obtained.

[0361] Embodiment 16. The method of any one of embodiments 8 to 14, wherein step (c) is performed after step (b), and wherein the plurality of third channels are formed by extruding a plurality of perfusion channels from the plurality of third channel forming units.

[0362] Example 17. The method of Example 16, wherein the plurality of third channel forming units are biopsy punch tools.

[0363] Embodiment 18. The method of any one of embodiments 1 to 17, further comprising the step of treating the tissue construct with a hydrogel shrinking agent.

[0364] Example 19. The method of any one of Examples 1 to 18, further comprising the step of electrically and / or mechanically exercising the tissue construct.

[0365] Embodiment 20. The method of any one of embodiments 1 to 19, wherein, before step (b), the plurality of first channel-forming units and / or the plurality of second channel-forming units are coated with a lubricant such as oil.

[0366] Example 21. The method of Example 18, wherein the hydrogel contracting agent comprises low molecular weight (eg, 15 kDa) chitosan.

[0367] Embodiment 22. The method of any one of embodiments 1 to 21, further comprising the step of cross-linking the hydrogel composition (eg, with a 0.3 M calcium chloride solution).

[0368] Embodiment 23. The method of any one of embodiments 1 to 22, wherein the step of solidifying the hydrogel composition is performed by incubating the hydrogel composition at a low temperature (eg, about 4°C).

[0369] Embodiment 24. The method of any one of embodiments 1 to 23, wherein the adipose-derived cells are derived from adipose-derived stem cells from cattle (such as Wagyu rib-eye).

[0370] Embodiment 25. The method of any one of embodiments 1 to 24, wherein the muscle-derived cells are derived from myoblast-derived stem cells from bovine (eg, Wagyu rib-eye).

[0371] Example 26. The method of any one of Examples 1 to 25, further comprising the step of differentiating the first cell and / or the second cell into mature cell tissue.

[0372] Example 27. A device for producing cultured tissue, comprising: a plurality of first channel-forming units, each of which has a first diameter; a plurality of second channel-forming units, each of which has a second diameter; and at least one support plate, the at least one support plate comprising: a plurality of first channel-receiving portions, each of which is sized and shaped to receive at least a portion of the first channel-forming unit and to confine a first needle at a first position; and a plurality of second channel-receiving portions, each of which is sized and shaped to receive at least a portion of the second channel-forming unit and to confine a second needle at a second position, wherein the plurality of first channel-receiving portions and the plurality of second channel-receiving portions are configured The tissue construct is constructed and arranged in an array so that the plurality of first channel-forming units and the plurality of second channel-forming units can be assembled together by the at least one support plate to form a channel array and a plurality of spaces therebetween, wherein the plurality of spaces are configured to receive a hydrogel composition to form a solidified hydrogel portion, so that a plurality of first channels and a plurality of second channels are formed by removing the plurality of first channel-forming units and the plurality of second channel-forming units from the hydrogel portion, respectively, each first channel is configured to receive therein at least one first cell composition to form at least one first cell culture, and each second channel is configured to receive therein at least one second cell composition to form at least one second cell culture, thereby producing a tissue construct.

[0373] Example 28. The device of Example 27, further comprising a plurality of third channel forming units to form a plurality of third channels for receiving at least one third cell therein.

[0374] Example 29. The device of Example 27, wherein the first diameter and the second diameter are larger (e.g., about 1-20 times larger) than the diameters of the target first cell culture and the target second cell culture, respectively.

[0375] Example 30. The device of Example 27, wherein the first cell composition comprises at least one first cell, and the second cell composition comprises at least one second cell.

[0376] Example 31. The device of any one of Examples 27 to 30, wherein the cultured tissue is cultured meat, the first cells are muscle-derived cells, muscle satellite cells, and / or myoblast-derived cells, and the second cells are adipose-derived cells.

[0377] Embodiment 32. The device of any one of Embodiments 27 to 31, wherein the first diameter is about 0.1-10,000 μm, such as about 20-500 μm.

[0378] Embodiment 33. The device of any one of Embodiments 27 to 31, wherein the first diameter is less than 200 μm, such as 20-90 μm.

[0379] Embodiment 34. The device of any one of embodiments 27 to 31, wherein a distance between two adjacent first channel forming units is about 0.1-5,000 μm, such as 0.1-500 μm.

[0380] Embodiment 35. The device of any one of embodiments 27 to 31, wherein the distance between two adjacent first channel forming units is less than 100 μm, such as 0.1-30 μm.

[0381] Embodiment 36. The device of any one of Embodiments 27 to 31, wherein the second diameter is about 0.1-10,000 μm, such as about 20-500 μm.

[0382] Embodiment 37. The device of any one of Embodiments 27 to 31, wherein the second diameter is less than 500 μm, such as 100-300 μm.

[0383] Embodiment 38. The device of any one of Embodiments 27 to 31, wherein the distance between two adjacent second channel forming units is about 0.1-5,000 μm, such as 0.1-500 μm, such as about 1 μm.

[0384] Embodiment 39. The device of embodiment 38, wherein the distance between two adjacent second channel forming units is less than 100 μm, for example, 0.1-30 μm.

[0385] Example 40. The device of any one of Examples 28 to 39, wherein the third cells comprise at least one or more of fibroblasts, endothelial cells, myoblasts, muscle-derived cells, adipose-derived cells, and combinations thereof.

[0386] Embodiment 41. The device of any one of Embodiments 27 to 40, wherein each of the plurality of first and / or second channel-forming units has a cylindrical tubular structure.

[0387] Embodiment 42. The device of any one of Embodiments 27 to 41, wherein each of the plurality of first and / or second channel forming units has an open groove structure with a U-shaped cross-section.

[0388] Example 43. The device of any one of Examples 27 to 42, wherein the tissue construct comprises a plurality of fat regions and a plurality of muscle regions.

[0389] Embodiment 44. A system for producing cultured tissue, the system comprising: an apparatus for producing a tissue construct having a plurality of third channels as described in any of the preceding embodiments; a tissue construct connector configured to connect to the tissue construct; a bioreactor for oxygenating the culture medium; at least one pump system for circulating the culture medium from the bioreactor through the plurality of third channels to the tissue construct; and optionally a culture medium recycler for removing waste.

[0390] Example 45. The system of Example 44, wherein the cultured tissue is cultured meat, the first cells are muscle-derived cells, muscle satellite cells, and / or myoblast-derived cells, and the second cells are adipose-derived cells.

[0391] Embodiment 46. A cultured tissue prepared by any of the methods described in any of embodiments 1 to 26, wherein the cultured tissue is derived from an animal selected from the group consisting of mammals, birds, fish, invertebrates, reptiles, and amphibians.

[0392] Example 47. The cultured tissue of Example 46, wherein the animal is non-human.

[0393] Example 48. The cultured tissue of Example 46, wherein the cultured tissue is cultured meat.

[0394] Embodiment 49. The cultured tissue of embodiment 46, wherein the animal is a human. Numbered Example 2

[0395] Example 1. A method for producing cultured tissue, the method comprising the following steps: (a) providing a hydrogel composition and solidifying the hydrogel composition to form a hydrogel portion, wherein the hydrogel portion comprises a plurality of first channels in a predetermined first array; (b) providing at least one first cell composition to a single first channel to form at least one first cell culture therein, thereby forming a tissue construct.

[0396] Example 2. A method as described in Example 1, wherein the hydrogel portion further includes a plurality of second channels in a predetermined second array, and the method further includes the following steps: (c) providing at least one second cell composition to a single second channel to form at least one second cell culture therein.

[0397] Example 3. A method as described in any of the preceding examples, wherein the cultured tissue is cultured meat, the first cell composition comprises a plurality of first cells, which are muscle-derived cells, muscle satellite cells and / or myoblast-derived cells; and the second cell composition, if present, comprises a plurality of second cells, which are fat-derived cells.

[0398] Embodiment 4. The method of any of the preceding embodiments, wherein the hydrogel composition comprises a first compound selected from the group consisting of collagen, alginate, gelatin, gellan gum, fibrinogen, xanthan gum, cellulose, plant protein, chitosan, carrageenan, starch hydrogel, agarose, pectin, guar gum, konjac glucomannan, lignin-based hydrogels, and combinations thereof, and optionally a second compound selected from the group consisting of transglutaminase, thrombin, tyrosinase, and combinations thereof.

[0399] Embodiment 5. A method as described in any of the preceding embodiments, wherein the hydrogel composition comprises any one of the following formulations: about 0.1-20 mg / ml type 1 collagen solution; about 0.5%-50% (w / v) gelatin and about 0.01%-5% (w / v) gellan gum; about 0.5%-50% (w / v) gelatin and about 0.1%-6% (w / v) sodium alginate; about 0.5%-50% (w / v) gelatin and about 0.1-100 U / gram of gelatin transglutaminase; and / or about 0.01-50 mg / mL fibrinogen and about 0.01-100 U / mL thrombin.

[0400] Embodiment 6. The method of any one of the preceding embodiments, wherein the hydrogel composition further comprises adipose-derived cells and fibroblasts, and combinations thereof.

[0401] Example 7. A method as described in any of the preceding embodiments, wherein the at least one first cell composition comprises muscle-derived cells, muscle satellite cells and / or myoblast-derived cells, and optionally one or more of collagen, alginate, gelatin, gellan gum, fibrinogen, xanthan gum, cellulose, plant protein, chitosan, carrageenan, starch hydrogel, agarose, pectin, guar gum, konjac glucomannan, lignin-based hydrogels, and combinations thereof.

[0402] Example 8. A method as described in any of the preceding embodiments, wherein the at least one second cell composition comprises adipose-derived cells, and optionally one or more of collagen, alginate, gelatin, gellan gum, fibrinogen, xanthan gum, cellulose, plant protein, chitosan, carrageenan, starch hydrogel, agarose, pectin, guar gum, konjac glucomannan, lignin-based hydrogels, and combinations thereof.

[0403] Example 9. A method as described in any of the preceding embodiments, wherein the hydrogel portion further includes multiple third channels as perfusion channels, and the method optionally further includes the following steps: (d) providing at least one third cell composition to a single third channel to form at least one third cell culture therein.

[0404] Example 10. The method of Example 9, wherein the third cell composition comprises fibroblasts, endothelial cells, smooth muscle cells, pericytes, and combinations thereof.

[0405] Embodiment 11. The method of any of the preceding embodiments, wherein the plurality of first cells, second cells, and / or third cells, if present, are derived from fibroblasts, endothelial cells, vascular cells, myoblasts, muscle cells, adipocytes, skin cells, neural cells, stem cells, tendon cells, liver cells, brain cells, bone cells, heart cells, kidney cells, neural cells, and combinations thereof.

[0406] Example 12. A method as described in any of the preceding embodiments, wherein the at least one first cell composition, the at least one second cell composition and / or the at least one third cell composition, if present, further comprises any of the following formulations: about 0.1-20 mg / ml type 1 collagen solution; about 0.5%-50% (w / v) gelatin and about 0.01%-5% (w / v) gellan gum; about 0.5%-50% (w / v) gelatin and about 0.1%-6% (w / v) sodium alginate; about 0.5%-50% (w / v) gelatin and about 0.1-100 U / gram of gelatin transglutaminase; and / or about 0.01-50 mg / mL fibrinogen and about 0.01-100 U / mL thrombin.

[0407] Example 13. A method as described in any of Examples 5 or 12, wherein the hydrogel composition, or the at least one first cell composition, the at least one second cell composition and / or the at least one third cell composition, if present, comprises any of the following formulations: about 6 mg / ml of type 1 collagen solution; about 20% (w / v) gelatin and about 2% (w / v) gellan gum; about 20% (w / v) gelatin and about 2% (w / v) sodium alginate; about 13.5% (w / v) gelatin and about 1 U / gram of gelatin transglutaminase; and / or about 1 mg / mL of fibrinogen and about 1 U / mL of thrombin.

[0408] Embodiment 14. A method as described in any of the preceding embodiments, wherein the single first channel and / or the single second channel, if present, is formed by the following steps: (1) providing an apparatus for producing cultured tissue, the apparatus comprising a plurality of first channel forming units and / or second channel forming units, and a holder assembly, the holder assembly comprising: a holder unit; and at least one support unit, each support unit comprising a plurality of first channel receiving portions, wherein the size and shape of the individual first channel receiving portions are determined to receive at least a portion of the individual first channel forming units, and wherein the plurality of first channel receiving portions are constructed and arranged into a first pattern so that the plurality of first channel forming units can be assembled with the holder assembly to form a predetermined first array, and there is residual space in the holder assembly; (2) providing a hydrogel composition to at least a portion of the residual space and solidifying the hydrogel composition to form a hydrogel portion; and (3) removing the individual first channel forming units and / or second channel forming units from the hydrogel portion.

[0409] Example 15. The method as described in Example 14, before step (2), further comprising the following steps: (i) providing a second sacrificial layer in the holder assembly; (ii) providing a third sacrificial layer on the second sacrificial layer; (iii) providing a fourth sacrificial layer on the third sacrificial layer; (iv) removing the third sacrificial layer so that the second sacrificial layer and the fourth sacrificial layer define a remaining space for the hydrogel portion within the holder assembly.

[0410] Embodiment 16. The method of embodiment 15, before step (i), further comprising the step of providing a first sacrificial layer in the holder assembly such that the second sacrificial layer is disposed on the first sacrificial layer.

[0411] Example 17. A method as described in Example 15 or 16, wherein step (i) further includes the following steps: setting the support unit at a first position close to the second sacrificial layer; and wherein step (iii) further includes the following steps: setting the support unit at a second position close to the fourth sacrificial layer.

[0412] Embodiment 18. The method of embodiment 15, wherein the first sacrificial layer, the second sacrificial layer, the third sacrificial layer, and / or the fourth sacrificial layer comprise ice and / or hydrogel.

[0413] Embodiment 19. The method of Embodiment 17, wherein the first sacrificial layer, if present, comprises ice, the second sacrificial layer comprises hydrogel, the third sacrificial layer comprises ice, and / or the fourth sacrificial layer comprises hydrogel.

[0414] Embodiment 20. The method of any one of the preceding embodiments, wherein the single first channel, second channel, and / or third channel, if present, is formed by 3D printing and / or laser ablation.

[0415] Embodiment 21. The method of any one of the preceding embodiments, further comprising the step of: (e) growing the plurality of first cells, the plurality of second cells, and / or the plurality of third cells, if present, until a desired tissue mass is obtained.

[0416] Example 22. The method of Example 21, wherein step (e) is performed by perfusing oxygenated culture medium through the tissue construct via the plurality of third channels.

[0417] Example 23. A method as described in any one of Examples 21 to 22, wherein step (e) is performed after step (be) or step (c), and wherein the plurality of third channels are formed by extruding at least a portion of the hydrogel portion as perfusion channels by a plurality of third channel forming units.

[0418] Example 24. The method of Example 23, wherein the single third channel forming unit is a cutting tool (such as a biopsy punch tool).

[0419] Embodiment 25. The method of any one of the preceding embodiments, further comprising the step of: (f) treating the tissue construct with a hydrogel shrinking agent.

[0420] Embodiment 26. The method of any one of the preceding embodiments, further comprising the step of: (g) electrically and / or mechanically exercising the tissue construct.

[0421] Embodiment 27. A method as described in any of the preceding embodiments, wherein, before step (2), the individual first channel forming units, second channel forming units and / or third channel forming units, if present, are coated with a lubricant (such as oil).

[0422] Example 28. The method of Example 25, wherein the hydrogel contracting agent comprises low molecular weight (eg, 15 kDa) chitosan.

[0423] Example 29. A method as described in any of the preceding embodiments, wherein the step of solidifying the hydrogel composition is performed by cross-linking the hydrogel composition with a cross-linking agent (e.g., about 0.03%-1% calcium chloride solution); and / or incubating the hydrogel composition at a low temperature (e.g., about 4°C).

[0424] Embodiment 30. The method of any one of the preceding embodiments, wherein the adipose-derived cells are adipose-derived stem cells derived from cattle (such as Angus cattle).

[0425] Embodiment 31. The method of any one of the preceding embodiments, wherein the muscle-derived cells are myoblast-derived stem cells or muscle satellite cells derived from cattle (such as Angus cattle).

[0426] Embodiment 32. The method of any one of the preceding embodiments, further comprising the step of allowing the first cell, the second cell, and / or the third cell, if present, to differentiate into mature cell tissue.

[0427] Embodiment 33. The method of any one of the preceding embodiments, wherein the first cell composition comprises porcine myoblasts.

[0428] Example 34. A device for producing cultured tissue, comprising: a plurality of first channel forming units, a single first channel forming unit having a first diameter; and a holder assembly, the holder assembly including a holder unit; and at least one support unit, each support unit including a plurality of first channel receiving portions, wherein the size and shape of the single first channel receiving portion are determined to receive at least a portion of the single first channel forming unit, and wherein the plurality of first channel receiving portions are constructed and arranged into a first pattern so that the plurality of first channel forming units can be assembled with the holder assembly to form a predetermined first array and remaining space in the holder assembly; and wherein at least a portion of the remaining space is configured to receive a hydrogel composition to form a solidified hydrogel portion, so that a plurality of first channels in the predetermined first array are formed by removing the plurality of first channel forming units from the hydrogel portion, and the single first channel is configured to receive at least one first cell composition therein to form at least one first cell culture, thereby producing a tissue construct.

[0429] Example 35. A device as described in Example 34, further comprising a plurality of second channel forming units, a single second channel forming unit having a second diameter, wherein the at least one support unit further comprises a plurality of second channel receiving portions, a single second channel receiving portion being sized and shaped to receive at least a portion of a single second channel forming unit, wherein the plurality of second channel receiving portions are constructed and arranged into a second pattern so that the plurality of second channel forming units can be assembled with the holder assembly to form a predetermined second array and the remaining space, and wherein the remaining space is configured to receive a hydrogel composition to form a solidified hydrogel portion, so that a plurality of second channels in the predetermined second array are formed by removing the plurality of second channel forming units from the hydrogel portion, and a single second channel is configured to receive at least one second cell composition therein to form at least one second cell culture.

[0430] Embodiment 36. The device of embodiment 35, further comprising a plurality of third channel forming units to form a plurality of third channels for perfusion and optionally for receiving at least one third cell therein.

[0431] Embodiment 37. The device of any one of Embodiments 35 to 36, wherein the first diameter and the second diameter are larger than the diameters of the target first cell culture and the target second cell culture, respectively.

[0432] Example 38. An apparatus as described in any one of Examples 35 to 37, wherein the cultured tissue is cultured meat, the first composition comprises a plurality of first cells, the first cells are muscle-derived cells, muscle satellite cells and / or myoblast-derived cells; and the second composition, if present, comprises a plurality of second cells, the second cells are adipose-derived cells.

[0433] Embodiment 39. The device of any one of Embodiments 34 to 38, wherein the first diameter is about 0.1-10,000 μm, such as about 20-500 μm.

[0434] Embodiment 40. The device of any one of Embodiments 34 to 39, wherein the first diameter is less than 200 μm, such as 20-90 μm.

[0435] Embodiment 41. The device of any one of Embodiments 34 to 40, wherein a distance between two adjacent first channel-forming units is about 0.1-5,000 μm, such as 0.1-500 μm.

[0436] Embodiment 42. The device of embodiment 41, wherein the distance between two adjacent first channel forming units is less than 100 μm, for example, 0.1-30 μm.

[0437] Embodiment 43. The device of any one of Embodiments 35 to 42, wherein the second diameter is about 0.1-10,000 μm, such as about 20-500 μm.

[0438] Embodiment 44. The device of any one of Embodiments 35 to 43, wherein the second diameter is less than 500 μm, such as 100-300 μm.

[0439] Embodiment 45. The device of any one of embodiments 35 to 44, wherein the distance between two adjacent second channel forming units is about 0.1-5,000 μm, such as 0.1-500 μm, such as about 1 μm.

[0440] Embodiment 46. The device of embodiment 45, wherein the distance between two adjacent second channel forming units is less than 100 μm, for example, 0.1-30 μm.

[0441] Example 47. The device of any one of Examples 36 to 46, wherein the third cells comprise at least one or more of fibroblasts, endothelial cells, myoblasts, muscle-derived cells, adipose-derived cells, and combinations thereof.

[0442] Embodiment 48. The device of any one of Embodiments 36 to 47, wherein a single first channel-forming unit, a second channel-forming unit, and / or a third channel-forming unit, if present, has a generally cylindrical elongated structure.

[0443] Embodiment 49. The device of any one of Embodiments 36 to 48, wherein a single first channel-forming unit, a second channel-forming unit, and / or a third channel-forming unit, if present, has an open trough-like structure with a generally U-shaped cross-section.

[0444] Example 50. The device of any one of Examples 34 to 49, wherein the tissue construct comprises a plurality of fat regions and a plurality of muscle regions.

[0445] Example 51. A system for producing cultured tissue, the system comprising: an apparatus for producing a hydrogel construct having a plurality of third channels as described in any one of Examples 34 to 50; a mixer for oxygenating a culture medium; a hydrogel construct connector configured to connect the hydrogel construct to the mixer; at least one pump system for circulating the culture medium from the mixer through the plurality of third channels to the hydrogel construct; and optionally a culture medium recycler for removing any waste.

[0446] Example 52. A system as described in Example 51, wherein the cultured tissue is cultured meat, the first cell composition comprises a plurality of first cells, which are muscle-derived cells, muscle satellite cells and / or myoblast-derived cells; and the second cell composition, if present, comprises a plurality of second cells, which are fat-derived cells.

[0447] Embodiment 53. A cultured tissue prepared by any one of the methods described in embodiments 1 to 33, wherein the cultured tissue is derived from an animal selected from the group consisting of mammals, birds, fish, invertebrates, reptiles, and amphibians.

[0448] Example 54. The cultured tissue of Example 53, wherein the animal is non-human, such as a pig, sheep, cow, or chicken.

[0449] Example 55. The cultured tissue of Example 53, wherein the cultured tissue is cultured meat.

[0450] Embodiment 56. The cultured tissue of embodiment 53, wherein the animal is a human.

Claims

1. A method for producing cultured tissue, comprising the steps of: (a) providing a hydrogel composition and solidifying the hydrogel composition to form a hydrogel portion, wherein the hydrogel portion comprises a plurality of first channels in a predetermined first array; (b) providing at least one first cell composition to a single first channel to form at least one first cell culture therein, such that a tissue construct is formed.

2. The method according to claim 1, wherein The hydrogel portion further comprises a plurality of second channels in a predetermined second array, and the method further comprises the step of: (c) providing at least one second cell composition to an individual second channel to form at least one second cell culture therein.

3. A method according to any one of the preceding claims, wherein The cultured tissue is cultured meat, the first cell composition comprises a plurality of first cells, which are muscle-derived cells, muscle satellite cells and / or myoblast-derived cells; and the second cell composition, if present, comprises a plurality of second cells, which are fat-derived cells.

4. A method according to any one of the preceding claims, wherein The hydrogel composition comprises a first compound selected from the group consisting of collagen, alginate, gelatin, gellan gum, fibrinogen, xanthan gum, cellulose, plant protein, chitosan, carrageenan, starch hydrogel, agarose, pectin, guar gum, konjac glucomannan, lignin-based hydrogels, and combinations thereof, and optionally a second compound selected from the group consisting of transglutaminase, thrombin, tyrosinase, and combinations thereof.

5. A method according to any one of the preceding claims, wherein The hydrogel composition comprises any one of the following formulations: About 0.1-20 mg / ml type 1 collagen solution; about 0.5%-50% (w / v) gelatin and about 0.01%-5% (w / v) gellan gum; about 0.5%-50% (w / v) gelatin and about 0.1%-6% (w / v) sodium alginate; About 0.5%-50% (w / v) gelatin and about 0.1-100 U / gram of gelatin of transglutaminase; and / or About 0.01-50 mg / mL of fibrinogen and about 0.01-100 U / mL of thrombin.

6. A method according to any one of the preceding claims, wherein The hydrogel composition further comprises adipose-derived cells and fibroblasts, and combinations thereof.

7. A method according to any one of the preceding claims, wherein The at least one first cell composition comprises muscle-derived cells, muscle satellite cells and / or myoblast-derived cells, and optionally one or more of collagen, alginate, gelatin, gellan gum, fibrinogen, xanthan gum, cellulose, plant protein, chitosan, carrageenan, starch hydrogel, agarose, pectin, guar gum, konjac glucomannan, lignin-based hydrogels, and combinations thereof.

8. A method according to any one of the preceding claims, wherein The at least one second cell composition comprises adipose-derived cells, and optionally one or more of collagen, alginate, gelatin, gellan gum, fibrinogen, xanthan gum, cellulose, plant protein, chitosan, carrageenan, starch hydrogel, agarose, pectin, guar gum, konjac glucomannan, lignin-based hydrogel, and combinations thereof.

9. A method according to any one of the preceding claims, wherein The hydrogel portion further comprises a plurality of third channels as perfusion channels, and the method optionally further comprises the step of: (d) providing at least one third cell composition to a single third channel to form at least one third cell culture therein.

10. The method according to claim 9, wherein: The third cell composition comprises fibroblasts, endothelial cells, smooth muscle cells, pericytes, and combinations thereof.

11. A method according to any one of the preceding claims, wherein The plurality of first cells, second cells and / or third cells, if present, are derived from fibroblasts, endothelial cells, vascular cells, myoblasts, muscle cells, adipocytes, skin cells, tendon cells, liver cells, brain cells, bone cells, heart cells, kidney cells, neural cells, stem cells, and combinations thereof.

12. A method according to any one of the preceding claims, wherein The at least one first cell composition, the at least one second cell composition and / or the at least one third cell composition, if present, further comprises any of the following formulations: About 0.1-20 mg / ml type 1 collagen solution; about 0.5%-50% (w / v) gelatin and about 0.01%-5% (w / v) gellan gum; about 0.5%-50% (w / v) gelatin and about 0.1%-6% (w / v) sodium alginate; About 0.5%-50% (w / v) gelatin and about 0.1-100 U / gram of gelatin of transglutaminase; and / or About 0.01-50 mg / mL of fibrinogen and about 0.01-100 U / mL of thrombin.

13. The method according to any one of claims 5 or 12, wherein: The hydrogel composition, or the at least one first cell composition, the at least one second cell composition and / or the at least one third cell composition, if present, comprises any of the following formulations: About 6mg / ml type 1 collagen solution; about 20% (w / v) gelatin and about 2% (w / v) gellan gum; about 20% (w / v) gelatin and about 2% (w / v) sodium alginate; About 13.5% (w / v) gelatin and about 1 U / gram of gelatin of transglutaminase; and / or About 1 mg / mL of fibrinogen and about 1 U / mL of thrombin.

14. A method according to any one of the preceding claims, wherein The single first channel and / or the single second channel, if present, are formed by the following steps: (1) A device for producing cultured tissue is provided, the device comprising a plurality of first channel forming units and / or second channel forming units, and a holder assembly, the holder assembly comprising: a holder unit; and at least one support unit, each support unit comprising a plurality of first passage receiving portions, wherein an individual first passage receiving portion is sized and shaped to receive at least a portion of an individual first passage forming unit, and wherein the plurality of first channel receiving portions are constructed and arranged in a first pattern such that the plurality of first channel forming units can be assembled with the holder assembly to form a predetermined first array and a remaining space in the holder assembly; (2) providing a hydrogel composition to at least a portion of the remaining space and solidifying the hydrogel composition to form a hydrogel portion; and (3) Removing individual first channel-forming units and / or second channel-forming units from the hydrogel portion.

15. The method according to claim 14, before step (2), further comprising the following steps: (i) providing a second sacrificial layer in the holder assembly; (ii) providing a third sacrificial layer on the second sacrificial layer; (iii) providing a fourth sacrificial layer on the third sacrificial layer; (iv) removing the third sacrificial layer such that the second sacrificial layer and the fourth sacrificial layer define a remaining space for the hydrogel portion within the holder assembly.

16. The method according to claim 15, further comprising the following steps before step (i): A first sacrificial layer is provided in the holder assembly such that the second sacrificial layer is disposed on the first sacrificial layer.

17. The method according to claim 15 or 16, wherein Step (i) further includes the steps of: disposing the support unit at a first position close to the second sacrificial layer; and Wherein, step (iii) further includes the following steps: arranging the supporting unit at a second position close to the fourth sacrificial layer.

18. The method according to claim 15, wherein The first sacrificial layer, the second sacrificial layer, the third sacrificial layer and / or the fourth sacrificial layer include ice and / or hydrogel.

19. The method according to claim 17, wherein The first sacrificial layer, if present, comprises ice, the second sacrificial layer comprises hydrogel, the third sacrificial layer comprises ice, and / or the fourth sacrificial layer comprises hydrogel.

20. A method according to any one of the preceding claims, wherein The single first channel, second channel and / or third channel, if present, is formed by 3D printing and / or laser ablation.

21. The method according to any one of the preceding claims, further comprising the steps of: (e) growing the plurality of first cells, the plurality of second cells, and / or the plurality of third cells, if present, until a desired tissue mass is obtained.

22. The method according to claim 21, wherein Step (e) is performed by perfusing oxygenated culture medium through the tissue construct via the plurality of third channels.

23. The method according to any one of claims 21 to 22, wherein Step (e) is performed after step (be) or step (c), and wherein the plurality of third channels are formed by extruding at least a portion of the hydrogel portion as perfusion channels by a plurality of third channel forming units.

24. The method according to claim 23, wherein The single third channel forming unit is a cutting tool (such as a biopsy punch tool).

25. The method according to any one of the preceding claims, further comprising the steps of: (f) treating the tissue construct with a hydrogel shrinking agent.

26. The method according to any one of the preceding claims, further comprising the steps of: (g) electrically and / or mechanically exercising the tissue construct.

27. A method according to any one of the preceding claims, wherein Prior to step (2), the individual first passage-forming units, the second passage-forming units, and / or the third passage-forming units, if present, are coated with a lubricant such as oil.

28. The method according to claim 25, wherein The hydrogel shrinking agent comprises low molecular weight (eg, 15 kDa) chitosan.

29. A method according to any one of the preceding claims, wherein The step of solidifying the hydrogel composition is performed as follows: cross-linking the hydrogel composition with a cross-linking agent (e.g., about 0.03%-1% calcium chloride solution); and / or The hydrogel composition is incubated at low temperature (eg, about 4°C).

30. A method according to any one of the preceding claims, wherein The adipose-derived cells are stem cells derived from adipose tissue derived from cattle (such as Angus cattle).

31. A method according to any one of the preceding claims, wherein The muscle-derived cells are myoblast-derived stem cells or muscle satellite cells derived from cattle (such as Angus cattle).

32. The method according to any one of the preceding claims, further comprising the steps of: The first cell, the second cell and / or the third cell, if present, is allowed to differentiate into mature cell tissue.

33. A method according to any one of the preceding claims, wherein The first cell composition comprises porcine myoblasts.

34. A device for producing cultured tissue, comprising: a plurality of first channel forming units, each of the first channel forming units having a first diameter; as well as Retainer assembly, The holder assembly includes a holder unit; as well as at least one support unit, each support unit comprising a plurality of first channel receiving portions, wherein the size and shape of the single first passage receiving portion are determined to receive at least a portion of the single first passage forming unit, and wherein the plurality of first channel receiving portions are constructed and arranged in a first pattern such that the plurality of first channel forming units can be assembled with the holder assembly to form a predetermined first array and a remaining space in the holder assembly; and At least a portion of the remaining space is configured to receive a hydrogel composition to form a solidified hydrogel portion, such that a plurality of first channels in a predetermined first array are formed by removing the plurality of first channel-forming units from the hydrogel portion, and a single first channel is configured to receive at least one first cell composition therein to form at least one first cell culture, thereby producing a tissue construct.

35. The device according to claim 34, further comprising a plurality of second channel forming units, each of the second channel forming units having a second diameter, wherein The at least one support unit further includes a plurality of second passage receiving portions, each second passage receiving portion being sized and shaped to receive at least a portion of a single second passage forming unit, wherein the plurality of second channel receiving portions are constructed and arranged in a second pattern so that the plurality of second channel forming units can be assembled with the holder assembly to form a predetermined second array and the remaining space, and The remaining space is configured to receive a hydrogel composition to form a solidified hydrogel portion, so that a plurality of second channels in the predetermined second array are formed by removing the plurality of second channel-forming units from the hydrogel portion, and a single second channel is configured to receive at least one second cell composition therein to form at least one second cell culture.

36. The device according to claim 35, further comprising a plurality of third channel forming units to form a plurality of third channels for perfusion and optionally for receiving at least one third cell therein.

37. The device according to any one of claims 35 to 36, wherein The first diameter and the second diameter are larger than the diameters of a first cell culture of interest and a second cell culture of interest, respectively.

38. The device according to any one of claims 35 to 37, wherein The cultured tissue is cultured meat, the first composition comprises a plurality of first cells, which are muscle-derived cells, muscle satellite cells and / or myoblast-derived cells; and the second composition, if present, comprises a plurality of second cells, which are fat-derived cells.

39. The device according to any one of claims 34 to 38, wherein The first diameter is about 0.1-10,000 μm, for example about 20-500 μm.

40. The device according to any one of claims 34 to 39, wherein The first diameter is less than 200 μm, for example, 20-90 μm.

41. The device according to any one of claims 34 to 40, wherein The distance between two adjacent first channel forming units is about 0.1-5,000 μm, for example, 0.1-500 μm.

42. The apparatus according to claim 41, wherein The distance between two adjacent first channel forming units is less than 100 μm, for example, 0.1-30 μm.

43. The apparatus according to any one of claims 35 to 42, wherein The second diameter is about 0.1-10,000 μm, for example about 20-500 μm.

44. The device according to any one of claims 35 to 43, wherein The second diameter is less than 500 μm, for example, 100-300 μm.

45. The apparatus according to any one of claims 35 to 44, wherein The distance between two adjacent second channel forming units is about 0.1-5,000 μm, for example, 0.1-500 μm, for example, about 1 μm.

46. ​​The apparatus of claim 45, wherein The distance between two adjacent second channel forming units is less than 100 μm, for example, 0.1-30 μm.

47. The apparatus according to any one of claims 36 to 46, wherein The third cells include at least one or more of fibroblasts, endothelial cells, myoblasts, muscle-derived cells, adipose-derived cells, and combinations thereof.

48. The apparatus according to any one of claims 36 to 47, wherein A single first channel-forming unit, a second channel-forming unit, and / or a third channel-forming unit, if present, has a generally cylindrical elongated structure.

49. The apparatus according to any one of claims 36 to 48, wherein A single first channel-forming unit, a second channel-forming unit, and / or a third channel-forming unit, if present, has an open groove-like structure with a substantially U-shaped cross-section.

50. The apparatus according to any one of claims 34 to 49, wherein The tissue construct includes a plurality of fat regions and a plurality of muscle regions.

51. A system for producing cultured tissue, the system comprising: The device for producing a hydrogel construct having a plurality of third channels according to any one of claims 34 to 50; a mixer for oxygenating the culture medium; a hydrogel construct connector configured to connect the hydrogel construct to the mixer; at least one pump system for circulating the culture medium from the mixer through the plurality of third channels to the hydrogel construct; as well as Optional media recoverer to remove any waste.

52. The system of claim 51, wherein: The cultured tissue is cultured meat, the first cell composition comprises a plurality of first cells, which are muscle-derived cells, muscle satellite cells and / or myoblast-derived cells; and the second cell composition, if present, comprises a plurality of second cells, which are fat-derived cells.

53. A cultured tissue prepared by any one of the methods of claims 1 to 33, wherein The cultured tissue is derived from an animal selected from the group consisting of mammals, birds, fish, invertebrates, reptiles, and amphibians.

54. The cultured tissue according to claim 53, wherein The animal is a non-human, such as a pig, sheep, cow, or chicken.

55. The cultured tissue according to claim 53, wherein The cultured tissue is cultured meat.

56. The cultured tissue according to claim 53, wherein The animal is a human.

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