WO2023023309A1

WO2023023309A1 - Systems and methods for fish tissue preservation

Info

Publication number: WO2023023309A1
Application number: PCT/US2022/040860
Authority: WO
Inventors: I-Hsiung Brandon CHEN; Julia MARSCHALLINGER; Brian WYRWAS
Original assignee: Finless Foods Inc.
Priority date: 2021-08-20
Filing date: 2022-08-19
Publication date: 2023-02-23

Abstract

The present disclosure describes systems, devices, compositions, and methods for tissue storage, transportation, and/or preservation.

Description

SYSTEMS AND METHODS FOR FISH TISSUE PRESERVATION

Cross Reference to Related Application

[0001] This application claims the benefit of U.S. Application No. 63/235,214, filed on August 20, 2021, the disclosure of which is hereby incorporated by reference in its entirety.

Background

[0002] Preserving and/or storage of fish tissue on a commercial scale is difficult. Unfortunately, for example, transportation and/or storage of fish typically damages or kills fish tissue, which leads to inferior products compared to fresh tissue products. Although fish can be frozen and/or transported in tanks full of water sparged with oxygen, tissue viability prior to consumption remains marginal at best.

Summary

[0003] The present disclosure recognizes a need for improved systems, compositions, and methods for fish tissue preservation. In some embodiments, fish tissue is or comprises post-mortem fish tissue. In some embodiments, fish tissue is or comprises live fish tissue. In some embodiments, fish tissue is or comprises engineered fish tissue.

[0004] Among other things, the present disclosure recognizes that preserving tissue such as fish tissue during transport and/or storage improves the quality of the tissue. For example, in some embodiments, the present disclosure recognizes that contacting and/or storing fish cells, tissues, organs in a fluid improves the quality of the fish tissue while minimizing costs.

[0005] Among other things, the present disclosure recognizes that freezing fish tissue on a commercial scale substantially kills all live cells and/or damages the tissues, which leads to an inferior product after thawing as compared to fresh tissue. As another example, the present disclosure recognizes that even without freezing, live cells during transportation die at an accelerated rate due to lack of factors such as oxygen and nutrients. For instance, in some embodiments, the present disclosure recognizes that depriving tissue of factors such as oxygen and nutrients triggers tissue breakdown, thereby leading to inferior products (e.g., to the point of inedibility). Among other things, the present disclosure recognizes that maintaining fish cells, tissues, or organs in water alone reduced the viability of fish cells, tissues, or organs. [0006] Among other things, the present disclosure recognizes that transporting fish in full tanks of water sparged with oxygen can physically damage the fish over travel durations longer than thirty minutes. Such restrictions require heavy transport weights that increase costs and often limit the number of fish that can be transported in such a fashion.

[0007] Among other things, in some embodiments, the present disclosure provides systems, compositions, devices, and methods for fish tissue preservation and/or storage during transportation and/or preservation. Among other things, in some embodiments, the present disclosure provides systems, compositions, devices, or methods that extend fish tissue integrity. Among other things, in some embodiments, the present disclosure provides systems, compositions, devices, or methods that extend the shelf life of fish. In some embodiments, a system or composition for extending fish tissue integrity and/or shell life comprises food-grade storage media. In some embodiments, a system or composition for extending fish tissue integrity and/or shell life is food-grade storage media. For example, the present disclosure provides particular systems, compositions, devices, and methods of transporting, storing, extending the viability of, and/or preserving fish tissue and/or preventing tissue damage to fish tissue by contacting fish tissue with a fluid and/or storing fish tissue in a fluid. In some embodiments, a fluid suitable for fish tissue preservation and/or storage comprises at least one or more antimicrobial agents, one or more antioxidant agents, or any combination thereof. For example, in some embodiments, a fluid suitable for fish tissue preservation and/or storage reduces contamination of fish tissue. In some embodiments, contamination can change properties (such as pH) of fish tissue.

[0008] In another example, the present disclosure provides method comprising contacting and/or perfusing fish tissues, cells, or organs with a fluid via a device. In some embodiments, device comprises a needle. In some embodiments, a needle is placed into an organ of a circulatory system of one or more fish, including, for instance, with reference to systems, methods, and devices specifically exemplified herein. In some embodiments, the present disclosure provides methods, systems and devices as described by International Application No. PCT/US2021/019210 filed on February 23, 2021, entitled “SYSTEMS AND METHODS FOR LIVE FISH TISSUE PRESERVATION”, the disclosure of which is hereby incorporated by reference in its entirety.

[0009] In some embodiments, a needle is placed into an organ of a circulatory system for receiving a fluid for perfusing a circulatory system. [0010] In some embodiments, a fluid suitable for contacting and/or perfusing and/or storing fish cells, tissues, or organs in accordance with the systems, compositions, methods, and/or devices is exemplified herein. In some embodiments, a fish is live fish. In some embodiments, a fish is post-mortem. In some embodiments, a fish comprises engineered cells, engineered tissue, and/or engineered organs.

[0011] Among other things, the present disclosure further provides for systems, methods, compositions, and devices in preserving cell, tissue and organs for fish in research and/or commercial settings where viable cell, organ and other culture techniques are needed for basic and applied biomedical research and/or diagnostic procedures and/or food-related industries requiring preserving tissue viability in vitro.

[0012] Elements of embodiments involving one aspect of the disclosure (e.g., methods) can be applied in embodiments involving other aspects of the disclosure, and vice versa.

Brief Description of Drawing

[0013] FIGS. 1A-1B show images of cells at 0 hours (FIG. 1A) and 24 hours (FIG. IB) post mortem. FIG. 1 A shows an image of cells immediately after fish death. FIG. IB shows an image of cells after storing fish tissue in Hank’s Balanced Salt Solution (HBSS) for 24 hours.

[0014] FIGS. 2A-2B show images of fish tissue cells after storing in HBSS and HypoThermo® FRS. FIG. 2 A shows an image of cells after storing fish tissue in HBSS. FIG 2B shows an image of cells after storing fish tissue in HypoThermo® FRS.

[0015] FIGS. 3A-3F show images of microbial colonies after incubating fish tissue in storage fluids comprising one or more antimicrobial agents and images of microbial colonies under control conditions. FIG. 3 A shows an image of colonies under negative control as described in Example 4. FIG. 3B shows an image of colonies under positive control as described in Example 4. FIG. 3C shows an image of colonies resulting from a NNC (nisin + natamycin + chitosan) condition as described in Example 4. Black arrow indicates an observed microbial colony. FIG. 3D shows an image of colonies resulting from a chitosan condition as described in Example 4. FIG. 3E shows an image of colonies resulting from a nisin condition as described in Example 4. FIG. 3F shows an image of colonies resulting from a natamycin condition as described in Example 4. Definitions

[0016] “About”: The term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0017] “Amino acid”: in its broadest sense, as used herein, refers to any agent, compound and/or substance that can be incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally- occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and/or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and/or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and/or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term “amino acid” may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.

[0018] “Buffer”: The term “buffer,” as used herein, refers to aqueous solutions or compositions that resist changes in pH when acids or bases are added to the solution. Solutions that exhibit buffering activity are often referred to in the art as “buffers” or “buffer solutions.” Buffers typically are able to maintain the pH of the solution within defined ranges, often for example between pH 5.5 and pH 7.5. Buffer solutions that are typically able to maintain a pH of approximately 7, are often referred to a “physiological buffers.” Exemplary biological buffers include, but are not limited to, Lactated Ringer's solution, physiological saline solution, N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES); N-2-acetamido-2- iminodiacetic acid (ADA); N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES); N,N-bis(2-hydroxyethyl)glycine (BICINE); 2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)- 1,3 -propanediol (BIS-TRIS); 3 -(cyclohexylamino)- 1 -propanesulfonic acid (CAPS); 3- (cyclohexylamino)-2-hydroxy-l -propanesulfonic acid (CAPSO); 2- (cyclohexylamino)ethanesulfonic acid (CHES); (N,N-bis[2-hydroxyethyl]amino)-2- hydroxypropanesulfonic acid (DIPSO); 4-(2 -Hydroxyethyl)- 1 -piperazinepropanesulfonic acid (EPPS); 4-(2-hydroxyethyl)piperazine-l -ethanesulfonic acid (HEPES); 4-(2- hydroxyethyl)piperazine-l-(2-hydroxypropane sulfonic acid) (HEPPSO); 2-(N- morphilino)ethanesulfonic acid (MES); 3-(N-morpholino)propanesulfonic acid (MOPS); 3- morpholino-2-hydroxypropanesulfonic acid (MOP SO); piperazine- l,4-bis(2-ethanesulfonic acid) (PIPES); piperazine-N,N'-bis(2-hydroxypropane sulfonic acid) (POPSO); [(2-hydroxy-

1.1-bis(hydroxy methyl)ethyl)amino]-l -propanesulfonic acid (TAPS); 3-(N- tris[hydroxymethyl]methylamino)-2-hydroxypropanesulfonic acid (TAPSO); 2-[(2-hydroxy-

1.1-bis(hydroxylmethyl)ethyl)amino]ethanesulfonic acid (TES); N- [tris(hydroxymethyl)methyl]glycine (TRICINE); and tris(hydroxymethyl)aminomethane) (TRIS); mixtures or derivatives thereof, as well as other biological buffers including those developed by Good et al. (1966).

[0019] “Comparable” . As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0020] “Comprising” : A composition or method described herein as "comprising" one or more named elements or steps is open-ended, meaning that the named elements or steps are essential, but other elements or steps may be added within the scope of the composition or method. To avoid prolixity, it is also understood that any composition or method described as "comprising" (or which "comprises") one or more named elements or steps also describes the corresponding, more limited composition or method "consisting essentially of' (or which "consists essentially of') the same named elements or steps, meaning that the composition or method includes the named essential elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristic(s) of the composition or method. It is also understood that any composition or method described herein as "comprising" or "consisting essentially of' one or more named elements or steps also describes the corresponding, more limited, and closed-ended composition or method "consisting of' (or "consists of') the named elements or steps to the exclusion of any other unnamed element or step. In any composition or method disclosed herein, known or disclosed equivalents of any named essential element or step may be substituted for that element or step.

[0021] “Engineered” : The term “engineered”, as used herein, refers to an aspect of having been manipulated and altered by the hand of man. For example, the term “engineered fish cell” refers to a fish cell that has been subjected to a manipulation, so that its genetic, epigenetic, and/or phenotypic identity is altered relative to an appropriate reference fish cell such as otherwise identical fish cell that has not been so manipulated. In some embodiments, the manipulation is or comprises a genetic manipulation. In some embodiments, an engineered fish cell is one that has been manipulated so that it contains and/or expresses a particular agent of interest (e.g., a protein, a nucleic acid, and/or a particular form thereof) in an altered amount and/or according to altered timing relative to such an appropriate reference fish cell. In some embodiments, an engineered fish tissue can be produced by incorporating either engineered fish cells or non-engineered fish cells, or both to create a 3-dimentional structure via manipulation by the hand of man. In some embodiments, an engineered fish tissue contains scaffolding that is not present in a non-manipulated fish tissue obtained from a wild-caught fish. In some embodiments, an engineered fish tissue is created via an engineering method (e.g., 3D bioprinting, stereolithography (SLA), electrospinning, etc.).

[0022] “Fluid”: The term “fluid” as are used herein in a broad sense is intended to encompass a variety of solutions, buffers, formulations, agents, and/or compounds, in which a specific biological organism, cell, tissue, organ, or other type of biological samples or materials may reside for any period of time that is conducive to the preservation of viability of the biological material placed within such buffers, solutions, formulations, agents, and/or compounds.

[0023] “Improve ” “increase" , “inhibit” or “reduce": As used herein, the terms “improve”, “increase”, “inhibit’, “reduce”, or grammatical equivalents thereof, indicate values that are relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions absent presence of (e.g., prior to and/or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement may be or comprise a measurement in comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment.

[0024] “Perfusion” and/or “Perfusing”: As used herein, the terms “perfusion” and/or “perfusing” refers to the passage (or passaging) of blood, a blood substitute, or other fluid through the blood vessels or other natural channels in an organ or tissue.

[0025] “Substantially” : As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0026] “Salt”: As used herein, the term “salt” may be used to refer to a salt form of such compounds; in some embodiments, compound(s) described herein may be provided and/or utilized in an appropriate salt form (e.g., in systems, methods, devices, and/or fluids exemplified and/or otherwise described herein). Those skilled in the art, reading the present disclosure, will appreciate salt form(s) which are, within the scope of sound judgment, suitable for use in contact with the tissues of humans and/or lower animals (e.g., fish and/or fish tissues or cells as described herein) without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Various salts (e.g., that may be utilized to prepare and/or may be included in salt form(s) of compound(s) as described and/or utilized herein) are known in the art. For example, S. M. Berge, et al. describes various pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). In some embodiments, salts may include, but are not limited to, nontoxic acid addition salts, which are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. In some embodiments, salts may include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. In some embodiments, a provided compound comprises one or more acidic groups, and a salt form of such compound is an alkali, alkaline earth metal, or ammonium (e.g., an ammonium salt of N(R)s, wherein each R is independently defined and described in the present disclosure) salt. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, a salt is a sodium salt. In some embodiments, a salt is a potassium salt. In some embodiments, a salt is a calcium salt. In some embodiments, salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate. In some embodiments, a provided compound comprises two or more acid groups. In some embodiments, a salt form, or generally a salt, of such a compound comprises two or more cations, which can be the same or different. In some embodiments, in a salt form, all ionizable hydrogen (e.g., in an aqueous solution with a pKa no more than about 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2; in some embodiments, no more than about 7; in some embodiments, no more than about 6; in some embodiments, no more than about 5; in some embodiments, no more than about 4; in some embodiments, no more than about 3) in one or more (or, in some embodiments, all) acidic groups are replaced with cations.

[0027] “Tissue” and/or “Organ”: As used herein, the term, “tissue” and/or “organ” refers to viable cellular materials in an aggregate form, e.g., small portions of an organ, as well as dispersed cells, e.g., cells dispersed, isolated and/or grown from muscle, heart muscle, liver or kidney, including bone marrow cells and progeny cells, blood born stem cells and progeny, and the various other blood elements, unless otherwise specified. In some embodiments, the tissue and/or organ refers to kidney, heart liver, stomach, spleen, pancreas, lung, brain, eye, intestines, bladder, muscle, skin or dermal tissue, blood vessel, veins, arteries, heart valves, sperm, and oocyte(s). As used herein, the term “organ” encompasses both solid organs, e.g., kidney, heart, liver, lung, as well as functional parts of organs, e.g., segments of skin, sections of artery, veins, transplantable lobes of a liver, kidney, lung, and the like.

Detailed Description of Certain Embodiments

[0028] The present disclosure provides methods, compositions, systems, and devices for transporting, preserving, storing, and/or extending the viability of fish cells, tissues, or organs, and/or preventing tissue damage to fish tissue. Among other things, in some embodiments, the present disclosure provides systems, compositions, devices, or methods that extend post-mortem fish tissue integrity. Among other things, in some embodiments, the present disclosure provides systems, compositions, devices, or methods that extend the shelf life of fish. In some embodiments, a system or composition for extending post-mortem fish tissue integrity and/or shell life comprises food-grade storage media. In some embodiments, a system or composition for extending fish tissue integrity and/or shell life is food-grade storage media.

[0029] Among other things, in some embodiments, the present disclosure improves the ability to transport fish tissue while the brain of the fish is no longer functioning. Among other things, in some embodiments, one advantage of the present disclosure includes decreasing weight of the transported unit as compared to traditional methods of live fish transport. Another example of an advantage of the present disclosure is that the fish can be transported for an increased amount of time. For example, transportation can be increased from a couple of days to a couple of weeks or months or more. Among other things, the present disclosure allows for temperature of the transported unit to not be at extremely low levels, thereby reducing costs on cooling. Among other things, the present disclosure increases the perceived freshness to the consumer due to reduce tissue breakdown via necrosis.

Fish

[0030] The present disclosure provides for a variety of fish that can be used in accordance with the systems, methods, devices, and fluids exemplified herein. As exemplified herein, the present disclosure provides systems, compositions, and methods to transport all types of fish and is not limited to transporting fish that can only survive traditional transportation methods. As described herein, the term “fish” can be used to indicate fish tissues, fish cells, fish organs, or combinations thereof. In some embodiments, fish include halibut, teleosts, primitive bichirs, sturgeons, paddlefish, freshwater garfishes, or bowfins. In some embodiments, fish include fish listed in “Appendix: List of Fish” as described by https://en.wikti0nary.0rg/wiki/Appendix:List_0f_f1sh, the contents of which is hereby incorporated by reference in its entirety.

[0031] The present disclosure provides engineered fish tissues, cells, or organs that can be used in accordance with the systems, methods, devices, and fluids exemplified herein. In some embodiments, engineered fish tissues, cells, or organs are plant based (e.g., having been derived in part or in whole from plants).

Reducing stress experienced by fish

[0032] Among other things, the present disclosure provides for a variety of systems, methods, and devices for reducing and/or mitigating stress one or more fish experiences using any of the systems, methods, or devices exemplified herein. In some embodiments, stress experienced by fish can promote tissue damage and/or necrosis which can lead to poor product quality. In some embodiments, methods and compositions for improving fish product quality is described by U.S. Patent No. 6,001,396A, the contents of which is hereby incorporated by reference in its entirety. In some embodiments, methods and compositions for improving fish product quality is described by U.S. Patent No. 9,468, 197B2, the contents of which is hereby incorporated by reference in its entirety.

Temperature

[0033] In some embodiments, fish freeze at or below 0°C. In some embodiments, fish freeze at or below -4°C in salt water. [0034] In some embodiments, one or more fish can be cooled to a temperature to reduce and/or eliminate stress perceived by the fish. In some embodiments, one or more fish has a temperature that is in a range from about -4°C to about 20°C. In some embodiments, one or more fish are cooled to have a temperature of about 20°C. In some embodiments, one or more fish are cooled to have a temperature of about 19°C. In some embodiments, one or more fish are cooled to have a temperature of about 18°C. In some embodiments, one or more fish are cooled to have a temperature of about 17°C. In some embodiments, one or more fish are cooled to have a temperature of about 16°C. In some embodiments, one or more fish are cooled to have a temperature of about 15°C. In some embodiments, one or more fish are cooled to have a temperature of about 14°C. In some embodiments, one or more fish are cooled to have a temperature of about 13°C. In some embodiments, one or more fish are cooled to have a temperature of about 12°C. In some embodiments, one or more fish are cooled to have a temperature of about 11°C. In some embodiments, one or more fish are cooled to have a temperature of about 10°C. In some embodiments, one or more fish are cooled to have a temperature of about 9°C. In some embodiments, one or more fish are cooled to have a temperature of about 8°C. In some embodiments, one or more fish are cooled to have a temperature of about 7°C. In some embodiments, one or more fish are cooled to have a temperature of about 6°C. In some embodiments, one or more fish are cooled to have a temperature of about 5°C. In some embodiments, one or more fish are cooled to have a temperature of about 4°C. In some embodiments, one or more fish are cooled to have a temperature of about 3°C. In some embodiments, one or more fish are cooled to have a temperature of about 2°C. In some embodiments, one or more fish are cooled to have a temperature of about 1°C. In some embodiments, one or more fish are cooled to have a temperature of about 0°C. In some embodiments, one or more fish are cooled to have a temperature of about -1°C In some embodiments, one or more fish are cooled to have a temperature of about -2°C In some embodiments, one or more fish are cooled to have a temperature of about -3 °C In some embodiments, one or more fish are cooled to have a temperature of about -4°C

[0035] Among other things, in some embodiments, the present disclosure describes systems, methods, compositions, and devices for reducing stress of one or more fish. In some embodiments, one or more fish can be placed in an ice bath to reduce stress. In some embodiments, one or more fish can be placed in a cold water bath to reduce stress. In some embodiments, one or more fish can be placed in a refrigerator to reduce stress. In some embodiments, one or more fish can be placed in an ice-water slurry bath to reduce stress. In some embodiments, an ice-water slurry bath comprises about 0.1% to about 99% ice by weight. In some embodiments, ice can be crushed. In some embodiments, ice can be cubed. In some embodiments, ice can be shaved. In some embodiments, ice can be flaked-shaped. In some embodiments, ice can be pear-shaped. In some embodiments, ice can be crescentshaped. In some embodiments, a cooling process that be used as an embodiment of the present disclosure is described in U.S. Patent No. 9,468, 197B2, the disclosure of which is hereby incorporated by reference in its entirety.

Antioxidants

[0036] Among other things, the present disclosure describes methods, systems, compositions, and/or devices that comprise exposing a fish to an antioxidant to reduce stress to the fish and/or enhance the quality of the fish and/or preserve the cells, tissues, and/or organs of the fish. In some embodiments, a fluid comprises an antioxidant such as a tocopherol. In some embodiments, a fluid comprises an antioxidant such as an ethoxyquin. In some embodiments, a fluid comprises an antioxidant such as butylated hydroxytoluen (BHT). In some embodiments, a fluid comprises an antioxidant such as butylated hydroxyanisole (BHA). In some embodiments, a fluid comprises an antioxidant such as a tert-Butylhydroquinone (TBHG). In some embodiments, a fluid comprises an antioxidant such as a carotenoid. In some embodiments, a fluid comprises an antioxidant such as a retinoid.

[0037] Among other things, in some embodiments, the present disclosure describes methods, systems, compositions and/or devices for reducing potential oxygen damage to fish tissue. Examples of methods, systems, compositions and/or devices are described by U.S. Patent No. 8,268, 547B2, the contents of which is hereby incorporated by reference in its entirety.

Incapacitating fish

[0038] In some embodiments, the present disclosure does not require for one or more fish to be incapacitated.

[0039] Among other things, the present disclosure provides for a variety of systems, methods, and devices for incapacitating one or more fish as exemplified herein. In some embodiments, one or more fish are incapacitated via asphyxiation. In some embodiments, one or more fish are incapacitated via an ice bath. In some embodiments, one or more fish are incapacitated via decapitation. For example, in some embodiments, decapitation is performed using a knife, using a hydraulic press attached to a sharp edge, or using a saw (e.g., a circular saw, e.g., a reciprocating saw). In some embodiments, one or more fish are incapacitated via one or more blows to the head.

[0040] In some embodiments, one or more fish are incapacitated using a hammer, axe, knife, scissors, or jig saw as described herein. In some embodiments, one or more fish are incapacitated using electrical power, human strength, steam power or gas power.

[0041] In some embodiments, one or more fish are incapacitated via one or more blows to the head using a hammer. Types of hammers include a ball peen hammer, a claw hammer, a club hammer, a dead blow hammer, a framing hammer, a sledge hammer, a tack hammer, a brick hammer, an electrician’s hammer, an engineering hammer, a rock hammer, a scutch hammer, a shingle hammer, a spike maul hammer, a soft-faced hammer, a toolmaker’ s hammer, a welding hammer, or a power hammer. In some embodiments, a power hammer can include a steam-powered hammer, an electrical powered hammer, or an air powered hammer. In some embodiments, a hammer can be a rubber hammer, a metal hammer, a plastic hammer, a wooden hammer, a brass hammer, or a copper hammer.

[0042] In some embodiments, one or more fish are incapacitated via one or more blows to the head using an axe. Types of axes include a felling axe, a hatchet, a throwing tomahawk, a splitting axe, a double bit axe, a Viking or dane axe, a tactical axe, a battle axe, a hewing axe, a carpenter’s axe, a fireman’s axe, a crash axe or a throwing axe. In some embodiments, an axe can be a rubber axe, a metal axe, a plastic axe, a brass axe, or a copper axe.

[0043] In some embodiments, one or more fish are incapacitated via pithing as exemplified herein. In some embodiments, one or more fish are pithed with a metal material. In some embodiments, one or more fish are pithed with an alloy material. In some embodiments, one or more fish are pithed with a rubber material. In some embodiments, one or more fish are pithed with a plastic material. In some embodiments, one or more fish are pithed with a wooden material. In some embodiments, one or more fish are pithed with a brass material. In some embodiments, one or more fish are pithed with a copper material. In some embodiments, one or more fish are pithed with a carbon fiber material. In some embodiments, one or more fish are pithed using a material having a diameter from about 1 mm to about 15 cm. [0044] In some embodiments, one or more fish are pithed in the brain (e.g., in the olfactory lobe, e.g., in the telencephalon, e.g., in the optic lobe, e.g., in the cerebellum, e.g., in the myelencephalon, e.g., in the spinal cord). In some embodiments, one or more fish are pithed in the spinal cord (e.g., such that the nervous system is destroyed, e.g., such that nerves are destroyed). In some embodiments, the tail of one or more fish are cut to gain access to the spinal cord (e.g., through any cut/opening on the posterior side of the fish) (e.g., through any cut/opening on the anterior side of the fish). In some embodiments, access to the spinal cord is obtained through an opening to the brain. In some embodiments, one or more fish are pithed in the heart. In some embodiments, one or more fish are pithed in the gills (e.g., at the base of the gills, e.g., at the top of the gills, e.g., at the top of the gills near the front of the one or more fish, e.g., at the middle of the gills).

[0045] Among other things, in some embodiments, one or more fish are incapacitated using ikejime as described herein. Ikejime or Ikijime is a humane method of killing fish to maintain the quality of its meat. In some embodiments, ikejime involves the insertion of a spike quickly into the hindbrain or other organ of a fish (e.g., wherein the hindbrain and/or other organ is located slightly behind and above the eye, thereby causing immediate brain death). In some embodiments, ikejime involves insertion of a spike directly into the hindbrain or other organ of a fish (e.g., wherein the hindbrain and/or other organ is located slightly behind and above the eye, thereby causing immediate brain death). In some embodiments, when spiked correctly, fish fins flare. In some embodiments, when spiked correctly, fish relax. In some embodiments, when spiked correctly, fish immediately ceasing all motion. Among other things, in some embodiments, destroying brain and spinal cord of fish may prevent reflex action from happening. In some embodiments, such muscle movements may otherwise consume adenosine triphosphate in the muscle, which may produce lactic acid and ammonia (e.g., making fish sour, soggy and/or less tasteful). Furthermore, in some embodiments, the present disclosure describes that blood contained in fish flesh retracts to the gut cavity, which, for example, may produce a better colored and flavored fillet. In some embodiments, the present disclosure describes that blood contained in fish flesh retracts to the gut cavity, which, for example, may prolong shelf life. As described herein, ikejime may be considered to be a fastest and humane method of killing fish. For example, ikejime-killed fish is sought-after by restaurants as it also allows the fish to develop more umami when aged. Among other things, the present disclosure describes using ikejime as exemplified herein. In some embodiments, ikejime is used to pith the nervous system (e.g., to stop neurons from firing).

[0046] Among other things, in some embodiments, one or more fish are incapacitated by poisoning. In some embodiments, poisoning is performed by saturating water with carbon dioxide (e.g., wherein carbon dioxide is introduced in a range greater than 5 mg/L, e.g., greater than 10 mg/L). In some embodiments, poisoning is performed by introducing tricaine mesylate (MS222) as described by https://en.wikipedia.org/wiki/Tricaine_mesylate, the contents of which is hereby incorporated by reference in its entirety. It is noted that MS222 would not be used in embodiments where the one or more fish are to be consumed by humans.

[0047] Among other things, in some embodiments, one or more fish are incapacitated by electricity.

Introducing and/or removing fluid into and/or from the circulatory system

[0048] The present disclosure provides methods, systems, and devices for live fish transporting and/or preserving fish tissue and/or preventing tissue damage to fish tissue. Among other things, the present disclosure describes introducing fluid into the circulatory system of one or more fish using any of the methods, systems, and/or devices exemplified herein.

Needles

[0049] Among other things, in some embodiments, the present disclosure provides methods, systems, and devices for live fish transporting and/or preserving fish tissue and/or preventing tissue damage to fish tissue using one or more needles. In some embodiments, a needle has a NeedlePoint style such as 2, 3, 3T, 4 (at 1 degree through 90 degrees), 5, or AS. In some embodiments, a needle has a needle gauge of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33. In some embodiments, a needle has a material comprising medical grade stainless steel, advanced alloys, exotic metals, MRI compatible materials, or polymers.

[0050] In some embodiments, one or more needles are introduced into the heart of the one or more fish as exemplified herein. In some embodiments, a needle head is introduced into the heart of the one or more fish as exemplified herein. [0051] In some embodiments, one needle is introduced into an organ of the circulatory system for pumping fluid into the circulatory system as exemplified herein. In some embodiments, one needle is introduced into an organ of the circulatory system for pumping fluid into and out of an organ of the circulatory system as exemplified herein. In some embodiments, a first needle is introduced into an organ of the circulatory system for pumping fluid into an organ of the circulatory system and a second needle is introduced into the circulatory system for pumping fluid out of the circulatory system as exemplified herein. In some embodiments, one needle is introduced into an organ of the circulatory system for pumping fluid simultaneously into and out of an organ of the circulatory system as exemplified herein.

[0052] In some embodiments, a needle is introduced into the heart. In some embodiments, a needle is introduced into the hepatic vein. In some embodiments, a needle is introduced into the portal vein. In some embodiments, a needle is introduced into the caudal vein. In some embodiments, a needle is introduced into the caudal artery. In some embodiments, a needle is introduced into the dorsal aorta. In some embodiments, a needle is introduced into the mesenteric artery. In some embodiments, a needle is introduced into the hepatic artery. In some embodiments, a needle is introduced into the efferent gill arteries. In some embodiments, a needle is introduced into the hepatic artery. In some embodiments, a needle is introduced into the afferent gill arteries.

Tubes and pumps

[0053] Among other things, in some embodiments, the present disclosure provides methods, systems, and devices for live fish transporting and/or preserving fish tissue and/or preventing tissue damage to fish tissue using a system of one or more tubes as exemplified herein. Type of tubes include aluminum, copper, steel, stainless steel, nylon, Polyethylene (PE), Polypropylene, Polyurethane, PVC, Vinyl, rubber tubing, Fiberglass and composites. In some embodiments, a back end of a needle is attached to one tube and that tube is connected to a pump for pumping fluid through the circulatory system (e.g., to perfuse the circulatory system) of one or more fish. Types of pumps include peristaltic pumps, lobe pumps, diaphragm pumps, or piston pumps.

[0054] In some embodiments, a fluid is pumped through the circulatory system of one or more fish. For example, in some embodiments, a system comprises one fish. As another example, a system comprises one or more fish. Closed System

[0055] Among other things, in some embodiments, movement of fluid is within a closed system (for example, as described by International Application No.

PCT/US2021/019210 filed on February 23, 2021, entitled “SYSTEMS AND METHODS FOR LIVE FISH TISSUE PRESERVATION”, the disclosure of which is hereby incorporated by reference in its entirety). In some embodiments, the present disclosure provides for systems, methods, and/or devices comprising one or more tanks for storing and/or moving fluid as exemplified herein. For example, in some embodiments, fluid is depleted from a first tank, perfused through the circulatory of one or more fish, and accumulated into a second tank.

[0056] For example, in some embodiments, fluid comprises minimal negative factors that harm tissue viability. In some embodiments, fluid comprises one or more positive factors that keep and/or replenish positive factors that maintain and/or promote and/or preserve tissue viability. Types of negative factors include ammonia, acids, or toxic cell waste. Types of positive factors include water, elements to balance pH, elements to maintain osmolarity and/or osmolality, growth factors, oxygen, oxygen products, organic agents, thickening agents, vitamins, ions, ATP, or cofactors.

Fluid

[0057] Among other things, the present disclosure provides particular systems, compositions, devices, and methods of transporting, storing, extending the viability of, and/or preserving fish tissue and/or preventing tissue damage to fish tissue by contacting fish tissue with a fluid. For example, among other things, the present disclosure describes a fluid comprising a variety of factors that reduces contamination of fish tissue.

[0058] In some embodiments, a fluid is described herein for transporting fish tissue. In some embodiments, a fluid is described herein for storing fish tissue. In some embodiments, a fluid is described herein for extending the viability of fish tissue. In some embodiments, a fluid is described herein for preventing damage to fish tissue. In some embodiments, a fluid is described herein for extending shelf life of fish tissue.

[0059] In some embodiments, a fluid comprises water. In some embodiments, a fluid is water. In some embodiments, a fluid comprises ice. In some embodiments, a fluid comprises ice slurry water. In some embodiments, a fluid is ice slurry water. [0060] Among other things, the present disclosure describes a variety of factors that can be used in a fluid for transporting, storing, extending the viability of, and/or preserving fish tissue and/or preventing tissue damage to fish tissue.

[0061] Among other things, the present disclosure describes a variety of factors that can be used in a fluid for perfusing a circulatory system of a fish. In some embodiments, a fluid comprises spent fish blood devoid of toxic cell wastes, acids, or ammonium.

[0062] In some embodiments, a fluid comprises a factor selected from: elements to balance pH, elements to maintain osmolality, growth factors, gasses, organic agents, salts, antimicrobial agents, antioxidants, vitamins, ions, ATP, cofactors, buffers, and combinations thereof. In some embodiments, a fluid comprises chemically-defined pH and/or osmolality- balanced media comprising nutrients (e.g., carbon, nitrogen, or phosphate sources). In some embodiments, a fluid comprises sugars. In some embodiments, a fluid comprises elements to balance pH. In some embodiments, a fluid comprises elements to balance osmolality. In some embodiments, a fluid comprises gasses. In some embodiments, a fluid comprises organic agents. In some embodiments, a fluid comprises salts. In some embodiments, a fluid comprises antimicrobial agents. In some embodiments, a fluid comprises antioxidants. In some embodiments, a fluid comprises antimicrobial agents, antioxidants, or gasses, or combinations thereof. In some embodiments, a fluid comprises thickening agents. In some embodiments, a fluid comprises vitamins. In some embodiments, a fluid comprises growth factors. In some embodiments, a fluid comprises ions. In some embodiments, a fluid comprises ATP. In some embodiments, a fluid comprises cofactors. In some embodiments, a fluid comprises NADH/NADPH. In some embodiments, a fluid comprises buffers. In some embodiments, a fluid comprises enzymes. In some embodiments, a fluid comprises combinations of factors as exemplified herein.

[0063] In some embodiments, a fluid comprises at least one factor comprising one or more antimicrobial agents, one or more antioxidant agents, or any combination thereof.

[0064] Among other things, in some embodiments, as exemplified herein, a fluid comprises isotonic saline solutions, that may contain, in various proportions, salts, sugars, osmotic agents, local anesthetic, buffers, and other such agents, as described, simply by way of example, by Berdyaev et al., U.S. Pat. No. 5,432,053; Belzer et al., and the product ViaSpan®, described by U.S. Pat. Nos. 4,798,824, 4,879,283; and 4,873,230; Taylor, U.S. Pat. No. 5,405,742; Dohi et al., U.S. Pat. No. 5,565,317; Stern et al., U.S. Pat. Nos. 5,370,989 and 5,552,267, the disclosures of which are incorporated by reference in their entireties herein. The ViaSpan® product data sheet describes the product as a sterile, non-pyrogenic solution for hypothermic flushing and storage of organs. The solution has an approximate calculated osmolarity of 320 mOsM, a sodium concentration of 29 mEq/L, a potassium concentration of 125 mEq/L, and a pH of 7.4.

[0065] Among other things, in some embodiments, a fluid comprises pyruvate or inorganic salts supporting cell membrane potential such as described in U.S. Patent No. 5,066,578, the disclosure of which is hereby incorporated by reference in its entirety. As another example, in some embodiments, a fluid comprises one or more phosphatidic acids or sugars, and lysophosphotidic acids or sugars, together with enhancers such as albumen, optionally delivered in liposomal compositions such as described by U.S. Pat. Nos. 6,495,532 and 6,004,579, the disclosures of which is hereby incorporated by reference in its entirety.

[0066] Among other things, in some embodiments, a fluid comprises compositions described by International Patent Publication No. W01999015011 Al, U.S. Patent No. US7960098B2, International Patent Publication No. W02008100636A2, and U.S.

Publication No. US20050037330A1, the contents of which is hereby incorporated by reference in their entireties.

[0067] Among other things, in some embodiments, a fluid comprises compositions as described by U.S. Patent No. 6,001,396A, the contents of which is hereby incorporated by reference in its entirety.

[0068] Among other things, in some embodiments, a fluid comprises compositions as described by U.S. Patent No. 8,268, 547B2, the contents of which is hereby incorporated by reference in its entirety.

[0069] Among other things, in some embodiments, a fluid comprises compositions as described by U.S. Patent No. 6,824,389B1, the contents of which is hereby incorporated by reference in its entirety.

[0070] Among other things, in some embodiments, a fluid comprises one or more of the following factors: one or more salts, one or more sugars, one or more antimicrobial agents, one or more antioxidants, or a combination thereof. For example, in some embodiments, a fluid comprises one or more of the following factor described herein. For example, in some embodiments, a fluid comprises one or more of the following: Calcium Chloride (CaCh) (e.g., within a range of from about 0.1-10 rnM), Potassium Chloride (KC1) (e.g., within a range of from about 0.1-10 mM), Potassium Phosphate Monobasic (KH2PO4) (e.g., within a range of from about 0.1-10 mM), Magnesium Chloride (MgCh 6H2O) (e.g., within a range of from about 0.1-10 mM), Magnesium Sulfate (MgSC 7H2O) (e.g., within a range of from about 0.1-10 mM), Sodium Chloride (NaCl) (1-1000 mM), Sodium Bicarbonate (NaHCCh) (e.g., within a range of from about 0.1-10 mM), Sodium Phosphate Dibasic (Na2HPO4) (e.g., within a range of from about 0.1-10 mM), Glucose (e.g., within a range of from about 0.1-100 mM), chitosan (e.g., within a range of from about 1-10,000 kDa), natamycin (e.g., within a range of from about 0.1-100 mg/kg), Butylated hydroxyanisole (e.g., within a range of from about 1 -10,000 mg/kg), Butylated hydroxytoluene (e.g., within a range of from about 1-10,000 mg/kg), a-, B-, 5-, y- tocopherols (e.g., within a range of from about 1-10,000 mg/kg), or any combination thereof.

Water

[0071] Among other things, the present disclosure describes methods, systems, and/or devices comprising a fluid that comprises water as exemplified herein. In some embodiments, water is or comprises tap water. In some embodiments, water is or comprises mineral water. In some embodiments, water is or comprises spring water. In some embodiments, water is or comprises glacier water. In some embodiments, water is or comprises sparkling water. In some embodiments, water is or comprises distilled water. In some embodiments, water is or comprises purified water. In some embodiments, water is or comprises non-alkaline water. In some embodiments, water is or comprises well water. In some embodiments, water is or comprises ocean water. In some embodiments, water is or comprises sea water. In some embodiments, water is or comprises lake water. In some embodiments, water is or comprises sterile water. In some embodiments, water is ozonized. In some embodiments, water is ozonized at a concentration of ozone from about 0 to about 100 ppm. In some embodiments, water is ozonized at a concentration of ozone from about 0 to about 50 ppm. In some embodiments, water is ozonized at a concentration of ozone from about 0 to about 25 ppm. In some embodiments, water is ozonized at a concentration of ozone from about 0 to about 15 ppm. In some embodiments, water is ozonized at a concentration of ozone from about 0 to about 14 ppm. In some embodiments, water is ozonized at a concentration of ozone from about 0 to about 13 ppm. In some embodiments, water is ozonized at a concentration of ozone from about 0 to about 12 ppm. In some embodiments, water is ozonized at a concentration of ozone from about 0 to about 11 ppm. In some embodiments, water is ozonized at a concentration of ozone from about 0 to about 10 ppm. In some embodiments, water is ozonized at a concentration of ozone from about 0 to about 9 ppm. In some embodiments, water is ozonized at a concentration of ozone from about 0 to about 8 ppm. In some embodiments, water is ozonized at a concentration of ozone from about 0 to about 7 ppm. In some embodiments, water is ozonized at a concentration of ozone from about 0 to about 6 ppm. In some embodiments, water is ozonized at a concentration of ozone from about 0 to about 5 ppm. In some embodiments, water is ozonized at a concentration of ozone from about 0 to about 4 ppm. In some embodiments, water is ozonized at a concentration of ozone from about 0 to about 3 ppm. In some embodiments, water is ozonized at a concentration of ozone from about 0 to about 2 ppm. In some embodiments, water is ozonized at a concentration of ozone from about 0 to about 1 ppm. In some embodiments, water is ozonized at a concentration of ozone from about 1 to about 10 ppm. In some embodiments, water is ozonized at a concentration of ozone from about 2 to about 8 ppm. In some embodiments, water is ozonized at a concentration of ozone from about 3 to about 6 ppm. In some embodiments, water is ozonized at a concentration of ozone about 0.1 ppm. In some embodiments, water is ozonized at a concentration of ozone about 0.2 ppm. In some embodiments, water is ozonized at a concentration of ozone about 0.3 ppm. In some embodiments, water is ozonized at a concentration of ozone about 0.4 ppm. In some embodiments, water is ozonized at a concentration of ozone about 0.5 ppm. In some embodiments, water is ozonized at a concentration of ozone about 0.6 ppm. In some embodiments, water is ozonized at a concentration of ozone about 0.7 ppm. In some embodiments, water is ozonized at a concentration of ozone about 0.8 ppm. In some embodiments, water is ozonized at a concentration of ozone about 0.9 ppm. In some embodiments, water is ozonized at a concentration of ozone about 1 ppm. In some embodiments, water is ozonized at a concentration of ozone about 2 ppm. In some embodiments, water is ozonized at a concentration of ozone about 3 ppm. In some embodiments, water is ozonized at a concentration of ozone about 4 ppm. In some embodiments, water is ozonized at a concentration of ozone about 4.5 ppm. In some embodiments, water is ozonized at a concentration of ozone about 5 ppm. In some embodiments, water is ozonized at a concentration of ozone about 6 ppm. In some embodiments, water is ozonized at a concentration of ozone about 7 ppm. In some embodiments, water is ozonized at a concentration of ozone about 8 ppm. In some embodiments, water is ozonized at a concentration of ozone about 9 ppm. In some embodiments, water is ozonized at a concentration of ozone about 10 ppm. [0072] In some embodiments, water has been sterilized using an autoclave, a filter (e.g., a tangential flow filer, e.g., a PES filter, e.g., a PVDF filer, e.g., a nylon filter). In some embodiments, water is or comprises non-sterile water.

Sugars

[0073] Among other things, the present disclosure describes methods, systems, and/or devices comprising a fluid that comprises a sugar as exemplified herein. In some embodiments, sugar is or comprises glucose. In some embodiments, sugar is or comprises fructose. In some embodiments, sugar is or comprises sucrose. In some embodiments, a fluid comprises a plurality of sugars. In some embodiments, a fluid comprises sugar in a range from about 100 mg/L to about 2000 mg/L. In some embodiments, a fluid comprises sugar in a range from about 500 mg/L to about 1500 mg/L. In some embodiments, a fluid comprises sugar in a range from about 1000 mg/L to about 1300 mg/L. In some embodiments, a fluid comprises sugar (e.g., glucose) at a concentration of about 1250 mg/L.

Elements to balance pH

[0074] Among other things, the present disclosure describes methods, systems, and/or devices that comprise a fluid comprising an element to balance pH. In some embodiments, a fluid comprises an element such as sodium bicarbonate. In some embodiments, a fluid comprises an element such as (4-(2-hydroxy ethyl)- 1 -piperazineethanesulfonic acid) (HEPES). In some embodiments, a fluid comprises phosphate-buffered saline. An example of phosphate-buffered saline is described by https://en.wikipedia.org/wiki/Phosphate- buffered saline, the contents of which is hereby incorporated by reference in its entirety. In some embodiments, a fluid comprises a plurality of elements to balance pH.

[0075] In some embodiments, a fluid comprises an element to balance pH within a range from about a pH of about 4.0 to a pH of about 9.0. In some embodiments, a fluid comprises an element to balance pH within a range from about a pH of about 5.0 to a pH of about 8.0. In some embodiments, a fluid comprises an element to balance pH within a range from about a pH of about 6.0 to a pH of about 8.0. In some embodiments, a fluid comprises an element to balance pH within a range from about a pH of about 7.2 to a pH of about 7.2. In some embodiments, the pH is as described by https://www.intechopen.com/books/biomedical-tissue-culture/culture-conditions-and-types- of-growth-media-for-mammalian-cells, the contents of which is hereby incorporated by reference in its entirety. Elements to maintain osmolality

[0076] Among other things, the present disclosure describes methods, systems, and/or devices that comprise a fluid comprising an element to maintain osmolality (or osmotic pressure). In some embodiments, a fluid comprises an element such as sodium chloride. In some embodiments, a fluid comprises an element such as magnesium chloride. In some embodiments, a fluid comprises a plurality of elements to maintain osmolality.

[0077] In some embodiments, a fluid comprises an element to maintain osmolality in a range from about 0.1 mOSM/kg to about 10,000 mOSM/kg. In some embodiments, a fluid comprises an element to maintain osmolality in a range from about 100 mOSM/kg to about 5,000 mOSM/kg. In some embodiments, a fluid comprises an element to maintain osmolality in a range from about 500 mOSM/kg to about 2,000 mOSM/kg. In some embodiments, a fluid comprises an element to maintain osmolality in a range from about 1000 mOSM/kg. In some embodiments, a fluid comprises an element to maintain osmolality in a range from about 100 mOSM/kg to about 500 mOSM/kg. In some embodiments, a fluid comprises an element to maintain osmolality in a range from about 200 mOSM/kg to about 400 mOSM/kg. In some embodiments, a fluid comprises an element to maintain osmolality in a range from about 260 mOSM/kg to about 320 mOSM/kg. In some embodiments, the osmolality (or osmotic pressure) is in a range as described by https://www.intechopen.com/books/biomedical-tissue-culture/culture-conditions-and-types- of-growth-media-for-mammalian-cells, the contents of which is hereby incorporated by reference in its entirety.

Growth Factors

[0078] Among other things, the present disclosure describes methods, systems, and/or devices that comprise a fluid comprising a growth factor. In some embodiments, a fluid comprises a growth factor such as FGF. In some embodiments, a fluid comprises a growth factor such as TGF-b. In some embodiments, a fluid comprises a plurality of growth factors. In some embodiments, a fluid comprises growth factors such as epithelial and endothelial growth factors, vascular endothelial growth factors, platelet derived endothelial growth factors, epithelial growth factors, hepatocyte growth factors, and mixtures thereof.

[0079] In some embodiments, a fluid comprises a growth factor in an amount from about 1 fg/mL to about 10 g/mL. In some embodiments, the growth factor is in an amount as described by https://www.intechopen.com/books/biomedical-tissue-culture/culture- conditions-and-types-of-growth-media-for-mammalian-cells, the contents of which is hereby incorporated by reference in its entirety.

Gasses

[0080] Among other things, the present disclosure describes methods, systems, and/or devices that comprise a fluid comprising a gas. In some embodiments, a fluid comprises a gas such as oxygen. In some embodiments, a fluid comprises a gas such as ozone. In some embodiments, a fluid comprises a gas such as nitrogen. In some embodiments, a fluid comprises a gas such as hydrogen. In some embodiments, a fluid comprises a gas such as air. In some embodiments, a fluid comprises a plurality of gasses.

[0081] In some embodiments, a fluid comprises a gas in an amount from about 0.001 mg of gas/L to about 100 mg of gas/L. In some embodiments, a fluid comprises oxygen in an amount from about 0.001 mg of O2/L to about 100 mg of O2/L.

[0082] In some embodiments, a fluid lacks excess carbon dioxide. In some embodiments, a fluid has been degassed to remove excess carbon dioxide. In some embodiments, the systems and methods comprise vigorously aerating a fluid (e.g., with a pump). In some embodiments, degassing techniques are described by https://www.fdacs.gov/Consumer-Resources/Recreation-and-Leisure/Aquarium- Fish/Aquarium-Water-Quality-Carbon-Dioxide, the contents of which is hereby incorporated by reference in its entirety.

[0083] In some embodiments, a gas is sparged into a fluid prior to perfusion of the one of more fish. In some embodiments, a gas is sparged into a fluid during perfusion of the one of more fish.

Organic agents

[0084] Among other things, the present disclosure describes methods, systems, and/or devices that comprise a fluid comprising an organic agent. In some embodiments, a fluid comprises an organic agent such as amino acids. In some embodiments, a fluid comprises insulin, L- Alanine, L- Arginine, L- Asparagine, L- Aspartic Acid, L-Cysteine, L-Cysteine, L- Glutamic Acid, L-Glutamine, L-Histidine, L-Isoleucine, L-Leucine, L-Lysine, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, or salt thereof, or any combination thereof. [0085] In some embodiments, a fluid comprises an organic agent such as fatty acids. In some embodiments, fatty acids include oleic acid, linoleic acid, palmitic, stearic acid, myristic acid, lauric acid, eicosapentaenoic acid, docosahexaenoic acid, and salt thereof, and combinations thereof. In some embodiments, a fluid comprises an organic agent such as a carbohydrate. In some embodiment, a fluid comprises an organic agent such as a carbohydrate for cellular metabolism. In some embodiments, a fluid comprises a plurality of organic agents or salts thereof.

[0086] In some embodiments, a fluid comprises an organic agent in an amount from about 1 fg/mL to about 10 g/mL. In some embodiments, a fluid comprises an organic agent in an amount from about 1 pg/mL to about 10 mg/mL. In some embodiments, a fluid comprises an organic agent in an amount from about 1 pg/mL to about 1 g/rnL.

Salts

[0087] Among other things, the present disclosure describes methods, systems, and/or devices that comprise a fluid comprising a salt. In some embodiments, a fluid comprises a salt such as sodium chloride. In some embodiments, a fluid comprises a salt such as potassium chloride. In some embodiments, a fluid comprises a salt such as calcium chloride. In some embodiments, a fluid comprises a salt such as calcium sulfate. In some embodiments, a fluid comprises a salt such as sodium bicarbonate. In some embodiments, a fluid comprises a salt such as potassium phosphate monobasic. In some embodiments, a fluid comprises a salt such as magnesium chloride. In some embodiments, a fluid comprises a salt such as magnesium sulfate. In some embodiments, a fluid comprises a salt such as sodium phosphate dibasic. In some embodiments, a fluid comprises a plurality of salts.

[0088] In some embodiments, a fluid comprises a salt in an amount from about 0.001 g/L to about 1000 g/L. In some embodiments, a fluid comprises a salt in an about from about 0.01 g/L to about 100 g/L. In some embodiments, a fluid comprises a salt in an about from about 0.1 g/L to about 100 g/L. In some embodiments, a fluid comprises a salt in an about from about 1 g/L to about 100 g/L. In some embodiments, a fluid comprises a salt in an about from about 1 g/L to about 50 g/L. In some embodiments, a fluid comprises a salt in an about from about 1 g/L to about 25 g/L. In some embodiments, a fluid comprises a salt in an about from about 5 g/L to about 10 g/L. In some embodiments, a fluid comprises a salt in an about from about 1 g/L to about 9 g/L. In some embodiments, a fluid comprises a salt in an about from about 1 g/L to about 10 g/L. In some embodiments, a fluid comprises a salt in an about from about 0.1 g/L to about 0.5 g/L. In some embodiments, a fluid comprises a salt in an about from about 0.05 g/L to about 0.25 g/L. In some embodiments, a fluid comprises a salt in an about from about 0.1 g/L to about 0.3 g/L.

[0089] In some embodiments, a fluid comprises sodium chloride in an amount from about 5 g/L to about 10 g/L. In some embodiments, a fluid comprises sodium chloride in an amount of about 8 g/L.

[0090] In some embodiments, a fluid comprises calcium sulfate in an amount of about 125 mg/L to about 250 g/L

[0091] In some embodiments, a fluid comprises sodium bicarbonate in an amount of about 100 mg/L to about 200 mg/L.

[0092] In some embodiments, a fluid comprises a salt such as described by https://freshwater-aquaculture.extension.org/wp-content/uploads/2019/08/UsingSalt.pdf, the contents of which is hereby incorporated by reference in its entirety. In some embodiments, a fluid comprises a salt in an amount as described by https://freshwater- aquaculture.extension.org/wp-content/uploads/2019/08/UsingSalt.pdf, the contents of which is hereby incorporated by reference in its entirety.

Antimicrobial agents

[0093] Among other things, the present disclosure describes methods, systems, and/or devices that comprise a fluid comprising one or more antimicrobial agents. In some embodiments, a fluid comprising one or more antimicrobial agents is suitable for contacting and/or storing and/or extending viability of fish cells, tissues, or organs (e.g., post-mortem fish cells, post-mortem fish tissue, or post-mortem fish organs). In some embodiments, an antimicrobial agent is used to increase the shelf life of fish tissues, cells, or organs. In some embodiments, an antimicrobial agent is selected from chitosan, nisin, or natamycin, or salt thereof, or combinations thereof. In some embodiments, an antimicrobial agent comprises nisin (nisin has an “E-number” of E234 as a food additive). In some embodiments, an antimicrobial agent is nisin. In some embodiments, nisin is added to a fluid so that the concentration of nisin is in a range from about 5.5 to about 7 mg per kilogram of fish. In some embodiments, nisin is in a range from about 1 mg to about 5.5 mg per kilogram of fish. In some embodiments, nisin is in a range from about 1 mg to about 3 mg per kilogram of fish. In some embodiments, nisin is in a range from about 1 mg to about 2 mg per kilogram of fish. In some embodiments, nisin is about 1 mg per kilogram of fish. In some embodiments, nisin is about 2 mg per kilogram of fish. In some embodiments, nisin is about 3 mg per kilogram of fish. In some embodiments, nisin is about 4 mg per kilogram of fish. In some embodiments, nisin is about 5 mg per kilogram of fish. In some embodiments, nisin is added to a fluid so that the concentration of nisin is in a range from about 100 to about 10,000 international units (IU) per gram fish.

[0094] In some embodiments, an antimicrobial agent comprises natamycin (natamycin has an “E-number” of E235 as a food additive). In some embodiments, an antimicrobial agent is natamycin. In some embodiments, natamycin is added to a fluid so that the concentration of natamycin is in a range from about 3 to about 20 mg per kilogram of fish. In some embodiments, natamycin is in a range from about 3 to about 15 mg per kilogram of fish. In some embodiments, natamycin is in a range from about 10 to about 20 mg per kilogram of fish. In some embodiments, natamycin is in a range from about 5 to about 15 mg per kilogram of fish. In some embodiments, natamycin is in a range from about 8 to about 12 mg per kilogram of fish. In some embodiments, natamycin is in a range from about 9 to about 11 mg per kilogram of fish. In some embodiments, natamycin is about 5 mg per kilogram of fish. In some embodiments, natamycin is about 6 mg per kilogram of fish. In some embodiments, natamycin is about 7 mg per kilogram of fish. In some embodiments, natamycin is about 8 mg per kilogram of fish. In some embodiments, natamycin is about 9 mg per kilogram of fish. In some embodiments, natamycin is about 10 mg per kilogram of fish. In some embodiments, natamycin is about 11 mg per kilogram of fish. In some embodiments, natamycin is about 12 mg per kilogram of fish. In some embodiments, natamycin is about 13 mg per kilogram of fish. In some embodiments, natamycin is about 14 mg per kilogram of fish. In some embodiments, natamycin is about 15 mg per kilogram of fish.

[0095] In some embodiments, an antimicrobial agent comprises chitosan. In some embodiments, an antimicrobial agent is chitosan. In some embodiments, a fluid comprises chitosan in an amount from about 0.001 mg/L to about 1000 mg/L. In some embodiments, a fluid comprises chitosan in an amount from about 1 pg/L to about 100 mg/L. In some embodiments, a fluid comprises chitosan in an amount from about 10 pg/L to about 50 mg/L. In some embodiments, a fluid comprises chitosan in an amount from about 50 pg/L to about 100 pg/L. In some embodiments, a fluid comprises chitosan in an amount from about 50 pg/L to about 10 mg/L. In some embodiments, a fluid comprises chitosan in an amount of about 90 pg/L. In some embodiments, a fluid comprises chitosan in an amount of about 1 mg/L. In some embodiments, a fluid comprises chitosan in an amount of about 1 fM to about 10M. In some embodiments, chitosan is added to a fluid so that the concentration of chitosan is in a range from about 10 mg to about 100 mg per kilogram of fish. In some embodiments, chitosan is in a range from about 20 mg to about 90 mg per kilogram of fish. In some embodiments, chitosan is in a range from about 30 mg to about 70 mg per kilogram of fish. In some embodiments, chitosan is in a range from about 40 mg to about 60 mg per kilogram of fish. In some embodiments, chitosan is in a range from about 50 mg to about 100 mg per kilogram of fish. In some embodiments, chitosan is in a range from about 50 mg to about 70 mg per kilogram of fish. In some embodiments, chitosan is in a range from about 55 mg to about 65 mg per kilogram of fish. In some embodiments, chitosan is in a range from about 70 mg to about 90 mg per kilogram of fish. In some embodiments, chitosan is in a range from about 30 mg to about 50 mg per kilogram of fish. In some embodiments, chitosan is in a range from about 10 mg to about 30 mg per kilogram of fish. In some embodiments, chitosan is about 10 mg per kilogram of fish. In some embodiments, chitosan is about 20 mg per kilogram of fish. In some embodiments, chitosan is about 30 mg per kilogram of fish. In some embodiments, chitosan is about 40 mg per kilogram of fish. In some embodiments, chitosan is about 50 mg per kilogram of fish. In some embodiments, chitosan is about 60 mg per kilogram of fish. In some embodiments, chitosan is about 70 mg per kilogram of fish. In some embodiments, chitosan is about 80 mg per kilogram of fish. In some embodiments, chitosan is about 90 mg per kilogram of fish. In some embodiments, chitosan is about 100 mg per kilogram of fish.

[0096] In some embodiments, antimicrobial agent comprises ozone. In some embodiments, an antimicrobial agent is ozone. In some embodiments, a fluid comprises ozone at a concentration of about 0.1 ppm. In some embodiments, a fluid comprises ozone at a concentration of about 0.2 ppm. In some embodiments, a fluid comprises ozone at a concentration of about 0.3 ppm. In some embodiments, a fluid comprises ozone at a concentration of about 0.4 ppm. In some embodiments, a fluid comprises ozone at a concentration of about 0.5 ppm. In some embodiments, a fluid comprises ozone at a concentration of about 0.6 ppm. In some embodiments, a fluid comprises ozone at a concentration of about 0.7 ppm. In some embodiments, a fluid comprises ozone at a concentration of about 0.8 ppm. In some embodiments, a fluid comprises ozone at a concentration of about 0.9 ppm. In some embodiments, a fluid comprises ozone at a concentration of about 1 ppm. In some embodiments, a fluid comprises ozone at a concentration of about 1.5 ppm. In some embodiments, a fluid comprises ozone at a concentration of about 2 ppm. In some embodiments, a fluid comprises ozone at a concentration of about 2 ppm. In some embodiments, a fluid comprises ozone at a concentration of about 2.5 ppm. In some embodiments, a fluid comprises ozone at a concentration of about 3 ppm. In some embodiments, a fluid comprises ozone at a concentration of about 3.5 ppm. In some embodiments, a fluid comprises ozone at a concentration of about 4 ppm. In some embodiments, a fluid comprises ozone at a concentration of about 4.5 ppm. In some embodiments, a fluid comprises ozone at a concentration of about 5 ppm. In some embodiments, a fluid comprises ozone at a concentration of about 10 ppm. In some embodiments, a fluid comprises ozonized slurry ice comprising 0.2 mg ozone/L. In some embodiments, a fish is in contact with ozonized slurry ice and the fish: ice ratio is 1 : 1.

[0097] In some embodiments, a fluid comprises a plurality of antimicrobial agents. In some embodiments, a plurality of antimicrobial agents comprise combinations of antimicrobial agents as described herein.

[0098] In some embodiments, antimicrobial agent is as described in Juneja, Vijay K. et al. "Novel natural food antimicrobials." Annual review of food science and technology 3 (2012): 381-403, the disclosure of which is hereby incorporated by reference in its entirety.

[0099] Among other things, the present disclosure describes methods, systems, and/or devices that comprise contacting fish with a fluid comprising an antimicrobial agent. In some embodiments, an antimicrobial agent comprises ozone. In some embodiments, an antimicrobial agent is ozone. Among other things, the present disclosure recognizes that contacting a fish with a fluid described herein for a certain amount of time may influence preservation and/or shelf-life of fish cells, tissues, and/or organs.

[0100] In some embodiments, a fish is in contact with a fluid comprising an antimicrobial agent for a period of time within a range of about 2 seconds to about 5 days. In some embodiments, a period of time is within a range of about 2 seconds to about 3 days. In some embodiments, a period of time is within a range of about 2 seconds to about 2 days. In some embodiments, a period of time is within a range of about 2 seconds to about 24 hours. In some embodiments, a period of time is within a range of about 2 seconds to about 10 hours. In some embodiments, a period of time is within a range of about 2 seconds to about 5 hours. In some embodiments, a period of time is within a range of about 2 seconds to about 2 hours. In some embodiments, a period of time is within a range of about 2 seconds to about 1 hour. In some embodiments, a period of time is within a range of about 2 seconds to about 30 min. In some embodiments, a period of time is within a range of about 2 seconds to about 10 min. In some embodiments, a period of time is within a range of about 2 seconds to about 5 min. In some embodiments, a period of time is within a range of about 2 seconds to about 2 min. In some embodiments, a period of time is within a range of about 2 seconds to about 1 min. In some embodiments, a period of time is within a range of about 2 seconds to about 30 seconds. In some embodiments, a period of time is within a range of about 2 seconds to about 20 seconds. In some embodiments, a period of time is within a range of about 2 seconds to about 10 seconds. In some embodiments, a period of time is within a range of about 2 seconds to about 5 seconds. In some embodiments, a period of time is within a range of about 5 seconds to about 10 seconds. In some embodiments, a period of time is within a range of about 10 seconds to about 30 seconds. In some embodiments, a period of time is within a range of about 30 seconds to about 1 min. In some embodiments, a period of time is within a range of about 1 min to about 2 min. In some embodiments, a period of time is within a range of about 2 min to about 5 min. In some embodiments, a period of time is within a range of about 5 min to about 10 min. In some embodiments, a period of time is within a range of about 10 seconds to about 15 min. In some embodiments, a period of time is within a range of about 5 min to about 15 min. In some embodiments, a period of time is within a range of about 10 min to about 30 min. In some embodiments, a period of time is within a range of about 30 min to about 1 hour. In some embodiments, a period of time is within a range of about 1 hour to about 2 hours. In some embodiments, a period of time is within a range of about 2 hours to about 5 hours. In some embodiments, a period of time is within a range of about 5 hours to about 10 hours. In some embodiments, a period of time is within a range of about 10 hours to about 24 hours. In some embodiments, a period of time is within a range of about 24 hours to about 2 days. In some embodiments, a period of time is within a range of about 2 days to about 3 days. In some embodiments, a period of time is within a range of about 3 days to about 4 days. In some embodiments, a period of time is within a range of about 4 days to about 5 days.

[0101] In some embodiments, a fish is in contact with a fluid comprising an antimicrobial agent for about 2 seconds. In some embodiments, a fish is in contact with a fluid comprising an antimicrobial agent for about 3 seconds. In some embodiments, a fish is in contact with a fluid comprising an antimicrobial agent for about 4 seconds. In some embodiments, a fish is in contact with a fluid comprising an antimicrobial agent for about 5 seconds. In some embodiments a fish is in contact with a fluid comprising an antimicrobial agent for about 6 seconds. In some embodiments, a fish is in contact with a fluid comprising an antimicrobial agent for about 7 seconds. In some embodiments, a fish is in contact with a fluid comprising an antimicrobial agent for about 8 seconds. In some embodiments, a fish is in contact with a fluid comprising an antimicrobial agent for about 9 seconds. In some embodiments, a fish is in contact with a fluid comprising an antimicrobial agent for about 10 seconds. In some embodiments, a fish is in contact with a fluid comprising an antimicrobial agent for about 11 seconds. In some embodiments, a fish is in contact with a fluid comprising an antimicrobial agent for about 12 seconds. In some embodiments, a fish is in contact with a fluid comprising an antimicrobial agent for about 13 seconds. In some embodiments, a fish is in contact with a fluid comprising an antimicrobial agent for about 14 seconds. In some embodiments, a fish is in contact with a fluid comprising an antimicrobial agent about 15 seconds. In some embodiments, a fish is in contact with a fluid comprising an antimicrobial agent for about 16 seconds. In some embodiments, a fish is in contact with a fluid comprising an antimicrobial agent for about 17 seconds. In some embodiments, a fish is in contact with a fluid comprising an antimicrobial agent for about 18 seconds. In some embodiments a fish is in contact with a fluid comprising an antimicrobial agent for about 19 seconds. In some embodiments, a fish is in contact with a fluid comprising an antimicrobial agent for about 20 seconds. In some embodiments, a fish is in contact with a fluid comprising an antimicrobial agent for about 30 seconds. In some embodiments, a fish is in contact with a fluid comprising an antimicrobial agent for about 1 min. In some embodiments a fish is in contact with a fluid comprising an antimicrobial agent for about 2 min. In some embodiments, a fish is in contact with a fluid comprising an antimicrobial agent for about 5 min. In some embodiments, a fish is in contact with a fluid comprising an antimicrobial agent for about 10 min. In some embodiments a fish is in contact with a fluid comprising an antimicrobial agent for about 30 min. In some embodiments, a fish is in contact with a fluid comprising an antimicrobial agent for about 1 hour. In some embodiments, a fish is in contact with a fluid comprising an antimicrobial agent for about 2 hours. In some embodiments a fish is in contact with a fluid comprising an antimicrobial agent for about 5 hours. In some embodiments, a fish is in contact with a fluid comprising an antimicrobial agent for about 10 hours. In some embodiments a fish is in contact with a fluid comprising an antimicrobial agent for about 24 hours. In some embodiments a fish is in contact with a fluid comprising an antimicrobial agent for about 2 days. In some embodiments, a fish is in contact with a fluid comprising an antimicrobial agent for about 3 days. In some embodiments a fish is in contact with a fluid comprising an antimicrobial agent for about 5 days.

Antioxidants

[0102] Among other things, the present disclosure describes methods, systems, and/or devices that comprise a fluid comprising an antioxidant. In some embodiments, a fluid comprising an antioxidant is suitable for contacting and/or storing and/or extending viability of fish cells, tissues, or organs (e.g., post-mortem fish cells, post-mortem fish tissue, or postmortem fish organs). In some embodiments, antioxidant is used to increase the shelf life of fish tissues, cells, or organs. In some embodiments, an antioxidant is selected from butylated hydroxyanisole (BHA; “E-number” E320), butylated hydroxytoluene (BHT; “E-number” E321), Tocopherols (Vitamin E; “E-numbers” E307-E309), ascorbic acid (e.g., Vitamin C; E300), rosemary extract, green tea extract, or salt thereof, or combinations thereof. In some embodiments, an antioxidant comprises butylated hydroxyanisole (BHA). In some embodiments, an antioxidant is butylated hydroxyanisole (BHA). In some embodiments, butylated hydroxyanisole (BHA) is added to a fluid so that the concentration of butylated hydroxyanisole (BHA) is in a range from about 50 to about 1000 mg per kilogram of fish. In some embodiments, butylated hydroxyanisole (BHA) is added to a fluid so that the concentration of butylated hydroxyanisole (BHA) is in a range from about 50 to about 500 mg per kilogram of fish. In some embodiments, butylated hydroxyanisole (BHA) is added to a fluid so that the concentration of butylated hydroxyanisole (BHA) is in a range from about 50 to about 200 mg per kilogram of fish. In some embodiments, butylated hydroxyanisole (BHA) is added to a fluid so that the concentration of butylated hydroxyanisole (BHA) is in a range from about 50 to about 100 mg per kilogram of fish. In some embodiments, butylated hydroxyanisole (BHA) is added to a fluid so that the concentration of butylated hydroxyanisole (BHA) is in a range from about 50 to about 75 mg per kilogram of fish.

[0103] In some embodiments, an antioxidant comprises butylated hydroxytoluene (BHT). In some embodiments, an antioxidant is hydroxytoluene (BHT). In some embodiments, hydroxytoluene (BHT) is added to a fluid so that the concentration of hydroxytoluene (BHT) is in a range from about 50 to about 200 mg per kilogram of fish. In some embodiments, hydroxytoluene (BHT) is added to a fluid so that the concentration of hydroxytoluene (BHT) is in a range from about 50 to about 150 mg per kilogram of fish. In some embodiments, hydroxytoluene (BHT) is added to a fluid so that the concentration of hydroxytoluene (BHT) is in a range from about 50 to about 100 mg per kilogram of fish. In some embodiments, hydroxytoluene (BHT) is added to a fluid so that the concentration of hydroxytoluene (BHT) is in a range from about 50 to about 75 mg per kilogram of fish.

[0104] In some embodiments, an antioxidant comprises Tocopherols (Vitamin E; “E- numbers” E307-E309). In some embodiments, an antioxidant is Tocopherols (Vitamin E; “E- numbers” E307-E309). In some embodiments, an antioxidant is a-Tocopherol (E-307). In some embodiments, an antioxidant is y-Tocopherol (E-308). In some embodiments, an antioxidant is 6-Tocopherol (E-309). In some embodiments, Tocopherols are added to a fluid so that the concentration of Tocopherols are in a range from about 100 to about 600 mg per kilogram of fish. In some embodiments, Tocopherols are added to a fluid so that the concentration of Tocopherols are in a range from about 100 to about 500 mg per kilogram of fish. In some embodiments, Tocopherols are added to a fluid so that the concentration of Tocopherols are in a range from about 100 to about 400 mg per kilogram of fish. In some embodiments, Tocopherols are added to a fluid so that the concentration of Tocopherols are in a range from about 100 to about 300 mg per kilogram of fish. In some embodiments, Tocopherols are added to a fluid so that the concentration of Tocopherols are in a range from about 100 to about 200 mg per kilogram of fish. In some embodiments, Tocopherols are added to a fluid so that the concentration of Tocopherols are in a range from about 100 to about 150 mg per kilogram of fish.

[0105] In some embodiments, an antioxidant comprises ascorbic acid (e.g., Vitamin C). In some embodiments, an antioxidant is ascorbic acid (e.g., Vitamin C). In some embodiments, a fluid comprises ascorbic acid in an amount from about 0.001 mg/L to about 1000 mg/L. In some embodiments, a fluid comprises ascorbic acid in an amount from about 1 pg/L to about 100 mg/L. In some embodiments, a fluid comprises ascorbic acid in an amount from about 10 pg/L to about 50 mg/L. In some embodiments, a fluid comprises ascorbic acid in an amount from about 50 pg/L to about 100 pg/L. In some embodiments, a fluid comprises ascorbic acid in an amount from about 50 pg/L to about 10 mg/L. In some embodiments, a fluid comprises ascorbic acid in an amount of about 90 pg/L. In some embodiments, a fluid comprises ascorbic acid in an amount of about 1 mg/L. In some embodiments, a fluid comprises ascorbic acid in an amount of about 1 fM to about 10M.

[0106] In some embodiments, an antioxidant comprises rosemary extract. For example, among other things, certain preparation of antioxidants from rosemary and composition of rosemary extract are as described in Nieto, Gema et al. “Antioxidant and Antimicrobial Properties of Rosemary (Rosmarinus officinalis, L.): A Review.” Medicines (Basel, Switzerland) vol. 5,3 98. 4 Sep. 2018, and the disclosure of which is incorporated by reference in its entirety herein.

[0107] In some embodiments, an antioxidant comprises green tea extract. For example, among other things, certain antioxidant effects of green tea are as described in Forester, Sarah C, and Joshua D Lambert “The role of antioxidant versus pro-oxidant effects of green tea polyphenols in cancer prevention.” Molecular nutrition & food research vol.

55,6 (2011): 844-54, and the disclosure of which is incorporated by reference in its entirety herein.

[0108] In some embodiments, a fluid comprises a plurality of antioxidants. In some embodiments, a plurality of antioxidants comprise combinations of antioxidants as described herein.

[0109] In some embodiments, antioxidants are as described in WO1997039632A1, US 2011/0195171 Al, and W02017085099A1, the disclosures of which are incorporated by reference in their entireties herein.

Thickening Agents

[0110] Among other things, the present disclosure describes methods, systems, and/or devices that comprise a fluid comprising a thickening agent. In some embodiments, a fluid comprises a thickening agent such as polyvinyl alcohol (PVA). In some embodiments, a fluid comprises a thickening agent such as polyvinylpyrrolidone (PVP). In some embodiments, a fluid comprises a plurality of thickening agents. In some embodiments, a fluid comprises a thickening agent such as polyethylene glycol (PEG). In some embodiments, a fluid comprises a thickening agent such as methyl cellulose.

[OHl] In some embodiments, a fluid comprises a thickening agent in an amount from about 0.001% to about 99%. In some embodiments, a fluid comprises a thickening agent in an amount from about 0.001% to about 50%. In some embodiments, a fluid comprises a thickening agent in an amount from about 0% to about 20%. In some embodiments, a fluid comprises a thickening agent in an amount from about 0% to about 10%. In some embodiments, a fluid comprises a thickening agent in an amount from about 0% to about 5%.

[0112] In some embodiments, a fluid comprises PVP in an amount from about 0% to about 10%. In some embodiments, a fluid comprises PVP in an amount of about 0%, 2%, 4%, 6%, 8%, 9%, or 10%. In some embodiments, a fluid comprises PVP in an amount as described by Steven Denniss and James Rush “Polyvinylpyrrolidone can be Used to Cost- Effectively Increase the Viscosity of Culture Media” (published online 1 Apr 2015 in the FASEB Journal) (https://www.fasebj.org/doi/abs/10.1096/fasebj.29.1 supplement.1029.19), the contents of which is hereby incorporated by reference in its entirety.

[0113] In some embodiments, a fluid comprises methyl cellulose in an amount from about 0% to about 10%. In some embodiments, a fluid comprises methyl cellulose in an amount from about 0% to about 5%. In some embodiments, a fluid comprises methyl cellulose in an amount from about 1% to about 2%. In some embodiments, a fluid comprises methyl cellulose in an amount from about 0% to about 2%. In some embodiments, a fluid comprises methyl cellulose in an amount from about 0.5% to about 1%. In some embodiments, a fluid comprises methyl cellulose in an amount as described by https://www.sigmaaldrich.com/content/dam/sigma- aldrich/docs/Sigma/Product_Information_Sheet/2/m0512pis.pdf, the contents of which is hereby incorporated by reference in its entirety. In some embodiments, a fluid comprises methyl cellulose in an amount as described by https://www.sigmaaldrich.com/content/dam/sigma- aldrich/docs/Sigma/Product_Information_Sheet/2/m0512pis.pdf, the contents of which is hereby incorporated by reference in its entirety.

Vitamins

[0114] Among other things, the present disclosure describes methods, systems, and/or devices that comprise a fluid comprising a vitamin. In some embodiments, a fluid comprises a vitamin such as Vitamin A. In some embodiments, a fluid comprises a vitamin such as Vitamin C. In some embodiments, a fluid comprises a vitamin such as Vitamin B12. In some embodiments, a fluid comprises a vitamin such as partial polyoxometalates calcium. In some embodiments, a fluid comprises a vitamin such as choline chloride. In some embodiments, a fluid comprises a vitamin such as folic acid. In some embodiments, a fluid comprises a vitamin such as inositol. In some embodiments, a fluid comprises a vitamin such as pyridoxine. In some embodiments, a fluid comprises a vitamin such as riboflavin. In some embodiments, a fluid comprises a vitamin such as thiamine. In some embodiments, a fluid comprises a plurality of vitamins.

[0115] In some embodiments, a fluid comprises a vitamin in an amount from about 0.001 mg/L to about 1000 mg/L. In some embodiments, a fluid comprises a vitamin in an amount from about 1 pg/L to about 100 mg/L. In some embodiments, a fluid comprises a vitamin in an amount from about 10 pg/L to about 50 mg/L. In some embodiments, a fluid comprises a vitamin in an amount from about 50 pg/L to about 100 pg/L. In some embodiments, a fluid comprises a vitamin in an amount from about 50 pg/L to about 10 mg/L. In some embodiments, a fluid comprises a vitamin in an amount of about 90 pg/L. In some embodiments, a fluid comprises a vitamin in an amount of about 1 mg/L. In some embodiments, a fluid comprises a vitamin in an amount of about 1 fM to about 10M.

[0116] In some embodiments, a fluid comprises Vitamin A in an amount of about 90 pg/L. In some embodiments, a fluid comprises Vitamin E in an amount of about 1 mg/L.

[0117] In some embodiments, a fluid comprises a vitamin in an amount as described by https://www.intechopen.com/books/biomedical-tissue-culture/culture-conditions-and- types-of-growth-media-for-mammalian-cells, the contents of which is hereby incorporated by reference in its entirety.

Ions

[0118] Among other things, the present disclosure describes methods, systems, and/or devices that comprise a fluid comprising an ion. In some embodiments, a fluid comprises an ion such as Na⁺. In some embodiments, a fluid comprises an ion such as Cl'. In some embodiments, a fluid comprises an ion such as H⁺. In some embodiments, a fluid comprises an ion such as K⁺. In some embodiments, a fluid comprises an ion such as Mg²⁺. In some embodiments, a fluid comprises an ion such as Ca²⁺. In some embodiments, a fluid comprises a plurality of ions.

[0119] In some embodiments, a fluid comprises an ion in an amount from about 0.1 mM to about 1000 mM. In some embodiments, a fluid comprises an ion in an amount as described by http://book.bionumbers.org/what-are-the-concentrations-of-different-ions-in- cells/, the contents of which is hereby incorporated by reference in its entirety.

[0120] In some embodiments, a fluid comprises an ion in an amount from about 0.1 mM to about 500 mM. In some embodiments, a fluid comprises an ion in an amount from about 1 mM to about 500 mM. In some embodiments, a fluid comprises an ion in an amount from about 10 mM to about 300 mM. In some embodiments, a fluid comprises an ion in an amount from about 10 mM to about 100 mM.

ATP [0121] Among other things, in some embodiments, the present disclosure provides for restoring of organ viability that may be accomplished by restoring high energy nucleotide (<?.g, adenosine triphosphate (ATP)) levels and enzyme levels in the organ which were reduced by warm ischemia time and/or hypoxia.

[0122] In some embodiments, ATP is produced using a substrate such as phosphocreatine, creatine ethyl ester, dicreatine malate, creatine gluconate, fructose, sucrose, ribose, hexose, pentose, creatine orotate, creatine monohydrate, adenosine, dextro se/glucose, Dichloroacetate, malate, fumarate or pyruvate.

[0123] Among other things, in some embodiments, a fluid comprises energy substrates to replenish the intracellular ATP energy pool. For example, the present disclosure provides for aerobic metabolism during the perfusion and preservation process; antioxidants and/or xanthine oxidase inhibitors to mitigate reperfusion injury due to the free oxygen radicals.

[0124] In some embodiments, a fluid comprises ATP in an amount from about 1 fM to about 10 M. In some embodiments, a fluid comprises ATP in an amount from about 0.1 mM to about 100 mM. In some embodiments, a fluid comprises ATP in an amount from about 0.1 mM to about 50 mM. In some embodiments, a fluid comprises ATP in an amount from about 0.5 mM to about 10 mM.

Cofactors

[0125] Among other things, the present disclosure describes methods, systems, and/or devices that comprise a fluid comprising a cofactor. In some embodiments, a fluid comprises a cofactor such as NADH. In some embodiments, a fluid comprises a cofactor such as NADPH. In some embodiments, a fluid comprises a cofactor such as thiamine HC1. In some embodiments, a fluid comprises a cofactor such as biotin. In some embodiments, a fluid comprises a cofactor such as a vitamin as described herein. In some embodiments, a fluid comprises a plurality of cofactors.

[0126] In some embodiments, a fluid comprises a cofactor in an amount from about 0.001 mg/L to about 1000 mg/L. In some embodiments, a fluid comprises a cofactor in an amount from about 1 pg/L to about 100 mg/L. In some embodiments, a fluid comprises a cofactor in an amount from about 10 pg/L to about 50 mg/L. In some embodiments, a fluid comprises a cofactor in an amount from about 50 pg/L to about 100 pg/L. In some embodiments, a fluid comprises a cofactor in an amount from about 50 pg/L to about 10 mg/L. In some embodiments, a fluid comprises a cofactor in an amount of about 90 pg/L. In some embodiments, a fluid comprises a cofactor in an amount of about 1 mg/L. In some embodiments, a fluid comprises a cofactor in an amount of about 1 fM to about 10M.

[0127] In some embodiments, a fluid comprises a cofactor in an amount as described by https://www.intechopen.com/books/biomedical-tissue-culture/culture-conditions-and- types-of-growth-media-for-mammalian-cells, the contents of which is hereby incorporated by reference in its entirety.

Buffers

[0128] Among other things, the present disclosure describes methods, systems, and/or devices that comprise a fluid comprising a buffer or a combination of buffers as provided herein. In some embodiments, the buffer comprises phosphate buffered saline (PBS). In some embodiments, the buffer comprises from about 0.1 % to about 99% (e.g, 0.1% of HEPES [v/v with water]). In some embodiments, the buffer comprises 2% of HEPES, 96% of Sodium Bicarbonate, and 2% water. In some embodiments, the buffer comprises a buffer such as described by https://www.intechopen.com/books/biomedical-tissue- culture/culture-conditions-and-types-of-growth-media-for-mammalian-cells, the contents of which is hereby incorporated by reference in its entirety. In some embodiments, the buffer comprises a buffer such as described by http://vvwvv.endmemo.com/chem/massmolarconcentration.php, the contents of which is hereby incorporated by reference in its entirety.

Enzymes

[0129] Among other things, the present disclosure describes methods, systems, and/or devices that comprise a fluid comprising an enzyme. In some embodiments, a fluid comprises an enzyme such as phosphokinase. In some embodiments, a fluid comprises a substrate such as phosphocreatine. In some embodiments, a fluid comprises an enzyme such as NAD kinase. In some embodiments, a fluid comprises an enzyme such as Polyphosphate- AMP phosphotransferase (PAP). In some embodiments, a fluid comprises an enzyme such as polyphosphate kinase (PPK). In some embodiments, a fluid comprises an enzyme such as Phosphoenolpyruvate (PEP). In some embodiments, a fluid comprises a substrate such as pyruvate. In some embodiments, a fluid comprises an enzyme such as pyruvate kinase. In some embodiments, a fluid comprises an enzyme for ATP regeneration. In some embodiments, a fluid comprises an enzyme for NADPH regeneration. In some embodiments, a fluid comprises an enzyme for generation of a cofactor described herein. In some embodiments, a fluid comprises a combination of enzymes. In some embodiments, a fluid comprises an enzyme such as creatine kinase.

[0130] In some embodiments, a fluid comprises an enzyme in an amount from about 1 fM to about IM. In some embodiments, a fluid comprises an enzyme in an amount as described by Love et al., “NAD kinase controls animal NADP biosynthesis and is modulated via evolutionarily divergent calmodulin-dependent mechanisms” first published on January 20, 2015 in the Proceedings of the National Academy of Science of the United States of America, the contents of which is hereby incorporated by reference in its entirety. In some embodiments, a fluid comprises an enzyme in an amount as described by https://en.wikipedia.org/wiki/Creatine_kinase, the contents of which is hereby incorporated by reference in its entirety. In some embodiments, a fluid comprises an enzyme in an amount as described by Kameda et al., “A novel ATP regeneration system using polyphosphate- AMP phosphotransferase and polyphosphate kinase,” Journal of Bioscience and Bioengineering, Volume 91, Issue 6, 2001, pages 557-563, the contents of which is hereby incorporated by reference in its entirety.

Use

[0131] In some embodiments, a fluid as described herein preserves and/or prevents damage and/or extends viability to fish cells, tissues, and organs for an extended period of time, e.g., for 5 minutes, e.g., 30 minutes, e.g., 1 day, e.g., 1 week, e.g., 1 month, e.g., 6 months, e.g, for 1 year, e.g, greater than a year.

[0132] In some embodiments, a fluid as described herein preserves and/or prevents damage and/or extends viability to fish cells, tissues, and organs for an extended period of time (e.g., from about 5 minutes to about 1 year, e.g., from about 30 minutes to about 9 months, e.g., from about 7 days to about 6 months, e.g., from about 1 month to about 6 months, e.g., from about 2 months to about 6 months, e.g., from about 2 months to about 12 months, e.g., from about 6 days to about 15 days, e.g., from about 1 day to about 3 days, e.g., from about 1 day to about 15 days, from about 1 day to about 21 days, e.g., from about 1 day to about 30 days, e.g., from about 1 day to 3 days, e.g., from about 1 day to about 5 days, e.g., from about 1 day to 7 days, e.g., from about 3 days to about 5 days, e.g., from about 5 days to about 7 days, e.g., from about 7 days to about 15 days) as compared to the maintenance and/or storage of the fish tissue, cells, or organs maintained in water (e.g., iced water) alone. [0133] In some embodiments, an extended shelf life of fish tissue can be used in a variety of industries and purposes. For example, in some embodiments, the present disclosure provides that increasing shelf life of material derived from a fish allows a wider window of time for supply chain logistics. In some embodiments, the present disclosure provides that increasing shelf life of material derived from a fish allows transportation and/or preservation of fish tissue.

[0134] In some embodiments, the present disclosure provides a method of transporting and/or preserving fish tissue and/or preventing tissue damage to fish tissue, the method comprising: contacting fish tissue (e.g., post-mortem fish tissue) with a fluid as described herein. In some embodiments, the present disclosure provides a method of transporting and/or preserving fish tissue and/or preventing tissue damage to fish tissue, the method comprising: storing fish tissue (e.g., post-mortem fish tissue) in a fluid as described herein. In some embodiments, a fluid comprises at least one factor comprising one or more antimicrobial agents, one or more antioxidant agents, or any combination thereof.

[0135] In some embodiments, the present disclosure provides fish tissue (e.g., postmortem fish tissue) for use in transportation and/or preservation, wherein the fish tissue is stored in a fluid as described herein.

[0136] Among other things, the present disclosure provides for systems, methods, devices, and fluids that can be used in cellular agriculture. For example, in some embodiments, a fish can be acquired from its natural habitat and materials derived from the fish (e.g., fish cells, tissues, and/or organs) can be used in a laboratory setting for cellular agricultural purposes. In some embodiments, for example, a fish can be acquired from its natural habitat and materials derived from the fish can be exposed to (or contacted with or stored in) a fluid to allow the derived materials to stay alive for an extended period of time. In some embodiments, for example, a fish can be acquired from its natural habitat and materials derived from the fish can be exposed to (or contacted with or stored in) a fluid to allow the derived materials to stay alive for an extended period of time for a purpose described herein (e.g., for later use in a laboratory setting for cellular agricultural purposes). Among other things, in some embodiments, materials derived from a fish are stored in a container (c.g, a sealed container) for preserving cell viability during transport.

[0137] Among other things, in some embodiments, live cells from fish tissues could be isolated by researchers that are studying fish cell biology. [0138] Among other things, the present disclosure provides that the systems, methods, devices, and fluids can be used in fish transporting companies.

[0139] Among other things, the present disclosure provides that the systems, methods, devices, and fluids can be used in food industry. Examples of food industries include fast food industries, fine dining, mom and pop stores, restaurants, cafeterias, prison, airline industries, schools, and the like.

[0140] Among other things, the present disclosure provides that systems, methods, devices, and fluids can be used in seafood distribution industries. Among other things, the present disclosure provides that systems, methods, devices, and fluids can be used in wholesaler industries.

Types of sterilization

[0141] In some embodiments, the present disclosure provides that the systems, methods, devices, and fluids can be sterilized or substantially sterilized. Examples of sterilization techniques include autoclaving, filtering (e.g, using tangential flow, polyethersulfone (PES), polyvinylidene fluoride (PVDF), or nylon), using ultraviolet light, antibiotics, or antimycotics.

Exemplary Enumerated Embodiments

[0142] Among other things, the present disclosure provides the following enumerated embodiments.

[0143] 1. A fluid suitable for contacting and/or storing and/or extending viability of fish cells, tissues, or organs (e.g., post-mortem fish cells, post-mortem fish tissue, postmortem fish organs, e.g, live fish cells, live fish tissue, live fish organs, e.g, engineered fish cells, engineered fish tissue, engineered fish organs).

[0144] 2 The fluid of embodiment 1, wherein the viability of the fish cells, tissue, or organs maintained substantially in the fluid is greater than 5% (e.g, 10%, 15%, 20%, 25%, 30%) of the viability of the fish cells, tissues, or organs stored compared to a reference fluid.

[0145] 3. The fluid of embodiment 1 or 2, comprising at least one factor comprising one or more antimicrobial agents, one or more antioxidant agents, or any combination thereof. [0146] 4. The fluid of embodiment 3, wherein each factor of the at least one factor is present in the fluid in an amount effective to prolong the viability of the fish cells, tissues, or organs when maintained substantially in the fluid, as compared to the maintenance of the fish cells, tissues, or organs stored in the fluid alone.

[0147] 5. The fluid of embodiment 4, wherein the viability of the fish cells, tissue, or organs maintained substantially in the fluid is greater than 5% (e.g., 10%, 15%, 20%, 25%, 30%) of the viability of the fish cells, tissues, or organs stored in the fluid alone.

[0148] 6. The fluid of any one of embodiments 3-5, wherein the one or more antimicrobial agents comprises ozoned slurry ice, ozonated water, natural food antimicrobrials (e.g., chitosan, nisin, or natamycin), or any combination thereof.

[0149] 7. The fluid of embodiment 6, wherein the natural food antimicrobials comprises or is chitosan, nisin, or natamycin, or any combination thereof.

[0150] 8. The fluid of any one of embodiments 3-7, wherein the one or more antioxidant agents comprises butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ascorbic acid (e.g., Vitamin C), a tocopherol (e.g., Vitamin E) , rosemary extract, green tea extract, or any combination thereof.

[0151] 9. The fluid of embodiment 8, wherein the ascorbic acid is Vitamin C.

[0152] 10. The fluid of any one of embodiments 3-8, wherein the one or more antioxidant agents comprises a tocopherol.

[0153] 11. The fluid of embodiment 10, wherein the tocopherol is Vitamin E.

[0154] 12. The fluid of any one of embodiments 1-11, wherein the fluid has a pH within a range from about 5 to about 8, e.g., about 6.

[0155] 13. The fluid of any one of embodiments 1-12, wherein the fluid comprises one or more of the following factors: one or more salts, one or more sugars, one or more antimicrobial agents, one or more antioxidants, or a combination thereof.

[0156] 14. The fluid of any one of embodiments 1-13, wherein the fluid comprises one or more of

(i) Calcium Chloride (CaCE) (e.g, within a range of from about 0.1-10 mM),

(ii) Potassium Chloride (KC1) (e.g, within a range of from about 0.1-10 mM), (iii) Potassium Phosphate Monobasic (KH2PO4) e.g., within a range of from about (0.1-10 mM),

(iv) Magnesium Chloride (MgCh 6H2O) (e.g., within a range of from about 0.1-10 mM),

(v) Magnesium Sulfate (MgSC 7H2O) (e.g., within a range of from about 0.1-10 mM),

(vi) Sodium Chloride (NaCl) (e.g., within a range of from about 1-1000 mM),

(vii) Sodium Bicarbonate (NaHCCh) (e.g., within a range of from about 0.1-10 mM),

(viii) Sodium Phosphate Dibasic (Na2HPO4) (e.g., within a range of from about 0.1- 10 mM),

(ix) Glucose (e.g., within a range of from about 0.1-100 mM),

(x) chitosan (e.g., within a range of from about 1-10,000 kDa, e.g., within a range from about 0.01%- 10% w/w),

(xi) natamycin (e.g., within a range of from about 0.1-100 mg/kg),

(xii) Butylated hydroxyanisole (e.g., within a range of from about 1 -10,000 mg/kg),

(xiii) Butylated hydroxytoluene (e.g., within a range of from about 1-10,000 mg/kg),

(xiv) a-, B-, 8-, y- tocopherols (e.g., within a range of from about 1-10,000 mg/kg), or any combination thereof.

[0157] 15. The fluid of any one of embodiments 1-12, wherein the fluid comprises

HBSS and one or more of natural food antimicrobials.

[0158] 16. The fluid of embodiment 15, wherein the one or more natural food anitmicrobials comprises or is nisin.

[0159] 17. The fluid of embodiment 15, wherein the one or more natural food anitmicrobials comprises or is natamycin.

[0160] 18. The fluid of embodiment 15, wherein the one or more natural food anitmicrobials comprises or is chitosan. [0161] 19. The fluid of embodiment 15, wherein the one or more natural food anitmicrobials comprises nisin, natamycin, chitosan, or any combination thereof.

[0162] 20. The fluid of any one of embodiments 14-19, wherein the Calcium

Chloride (CaCh) is within a range of from about 0.1-10 rnM.

[0163] 21. The fluid of any one of embodiments 14-20, wherein the Potassium

Chloride (KC1) is within a range of from about 0.1-10 rnM.

[0164] 22. The fluid of any one of embodiments 14-21, wherein the Potassium

Phosphate Monobasic (KH2PO4) is within a range of from about 0.1-10 mM.

[0165] 23. The fluid of any one of embodiments 14-22, wherein the Magnesium

Chloride (MgCh 6H2O) is within a range of from about 0.1-10 mM.

[0166] 24. The fluid of any one of embodiments 14-23, wherein the Magnesium

Sulfate (MgSCU 7H2O) is within a range of from about 0.1-10 mM.

[0167] 25. The fluid of any one of embodiments 14-24, wherein the Sodium

Chloride (NaCl) is within a range of from about 1-1000 mM.

[0168] 26. The fluid of any one of embodiments 14-25, wherein the Sodium

Bicarbonate (NaHCCfi) is within a range of from about 0.1-10 mM.

[0169] 27. The fluid of any one of embodiments 14-26, wherein the Glucose is within a range of from about 0.1-100 mM.

[0170] 28. The fluid of any one of embodiments 14-27, wherein the chitosan is within a range of from about 1-10,000 kDa, and is within a range from about 0.01%-10% w/w.

[0171] 29. The fluid of embodiment 28, wherein the chitosan is at a concentration of about 60 mg per kilogram of fish.

[0172] 30. The fluid of any one of embodiments 14-29, wherein the natamycin is within a range of from about 0.1-100 mg/kg.

[0173] 31. The fluid of embodiment 30, wherein the natamycin is at a concentration of about 10 mg per kilogram of fish.

[0174] 32. The fluid of any one of embodiments 14-31, wherein the nisin is at a concentration of about 2 mg per kilogram fish. [0175] 33. The fluid of any one of embodiments 14-32, wherein the Butylated hydroxyanisole is within a range of from about 1 -10,000 mg/kg.

[0176] 34. The fluid of any one of embodiments 14-33, wherein the Butylated hydroxytoluene is within a range of from about 1-10,000 mg/kg.

[0177] 35. The fluid of any one of embodiments 14-34, wherein the a-, B-, 8-, y- tocopherols are within a range of from about 1-10,000 mg/kg.

[0178] 36. The fluid of any one of embodiments 1-35, wherein the fluid preserves and/or prevents damage and/or extends viability to the fish cells, tissues, and organs for an extended period of time (e.g., from about 5 minutes to about 1 year, e.g., from about 30 minutes to about 9 months, e.g., from about 7 days to about 6 months, e.g., from about 1 month to about 6 months, e.g., from about 2 months to about 6 months, e.g., from about 2 months to about 12 months, e.g., from about 6 days to about 15 days, e.g., from about 1 day to about 3 days, e.g., from about 1 day to about 15 days, from about 1 day to about 21 days, e.g., from about 1 day to about 30 days) as compared to the maintenance and/or storage of the fish tissue, cells, or organs maintained in water (e.g., iced water) alone.

[0179] 37. The fluid of embodiment 36, wherein the fluid preserves and/or prevents damage and/or extends viability to the fish cells, tissues, and organs for an extended period of time from about 1 day to about 5 days as compared to the maintenance and/or storage of the fish tissue, cells, or organs maintained in water alone.

[0180] 38. Fish tissue (e.g., post-mortem fish tissue) for use in transportation and/or preservation, wherein the fish tissue is stored in a fluid.

[0181] 39. A method of transporting and/or preserving fish tissue and/or preventing tissue damage to fish tissue, the method comprising: contacting fish tissue (e.g., post-mortem fish tissue) with a fluid comprising at least one factor comprising one or more antimicrobial agents, one or more antioxidant agents, or any combination thereof.

[0182] 40. The method of embodiment 39, comprising storing the fish tissue at a temperature range less than 10 degrees Celsius, e.g., less than 4 degrees Celsius.

[0183] 41. The method of embodiment 40, wherein the temperature range is less than 4 degrees Celsius. [0184] 42. The method of any one of embodiments 39-41, wherein the one or more antimicrobial agents comprises ozoned slurry ice, ozonated water, natural food antimicrobrials (e.g., chitosan, nisin, or natamycin), or any combination thereof.

[0185] 43. The method of embodiment 42, wherein the natural food antimicrobial comprises or is chitosan, nisin, or natamycin, or any combination thereof.

[0186] 44. The method of any one of embodiments 39-43, wherein the one or more antioxidant agents comprises butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ascorbic acid (e.g., Vitamin C), a tocopherol (e.g., Vitamin E), rosemary extract, green tea extract, or any combination thereof.

[0187] 45. The method of embodiment 44, wherein the ascorbic acid is Vitamin

[0188] 46. The method of embodiment 44, wherein the tocopherol is Vitamin E.

[0189] 47. The method of embodiments 39-46, comprising keeping the fish tissue in contact with the fluid for at least 1 day (e.g., at least 7 days, e.g., at least 14 days, e.g., at least 21 days, e.g, at least 28 days).

[0190] 48. The method of embodiment 47, where the fish tissue is in contact with the fluid for at least 5 days.

[0191] 49. The method of any one of embodiments 39-48, wherein the fluid preserves and/or prevents damage to the fish cells, tissues, and organs for an extended period of time (e.g, from about 5 minutes to about 1 year, e.g, from about 30 minutes to about 9 months, e.g., from about 7 days to about 6 months, e.g., from about 1 month to about 6 months, e.g., from about 2 months to about 6 months, e.g., from about 2 months to about 12 months, e.g., from about 6 days to about 15 days, e.g., from about 1 day to about 3 days, e.g., from about 1 day to about 15 days, from about 1 day to about 21 days, e.g., from about 1 day to about 30 days) as compared to the maintenance and/or storage of the fish tissue, cells, or organs maintained in water (e.g, iced water) alone.

[0192] 50. The method of embodiment 49, wherein the fluid preserves and/or prevents damage to the fish cells, tissues, and organs for an extended period of time from about 1 day to about 5 days, as compared to the maintenance and/or storage of the fish tissue, cells, or organs maintained in water alone. [0193] 51. The method of any one of embodiments 39-50, wherein the method comprises: contacting fish tissue with the fluid via a device.

[0194] 52. The method embodiment 51, wherein the device comprises a needle adapted for contacting and/or perfusing and/or storing fish cells, tissues, or organs (e.g., of one or more incapacitated fish).

[0195] 53. The method of embodiment 52, wherein the needle is placed into a first location (e.g., a chamber) of an organ of the circulatory system (e.g., wherein the first chamber is or comprises a member selected from the group consisting of a bulbus arteriosus, a ventricle, an Atrium, and a sinus venosus) of one or more fish.

[0196] 54. The method of embodiment 53, wherein the organ of the circulatory system is or comprises a member selected from the group consisting of a bulbus arteriosus, a ventricle, an Atrium, and a sinus venosus.

[0197] 55. The method of any one of embodiments 52-54, wherein the needle is simultaneously placed into both a first location of the organ and a second location of the organ.

[0198] 56. The method embodiment 55, wherein the first needle comprises a head, and wherein the head is placed into a second chamber of the heart.

[0199] 57. The method of any one of embodiments 52-56, wherein the first needle simultaneously is adapted to flow the fluid into and out of the organ.

[0200] 58. A method of characterizing a fluid of any one of embodiments 1-37.

[0201] 59. A method of characterizing fish cells, tissue, or organs stored in a fluid of any one of embodiments 1-37.

Exemplification

Example 1 :

[0202] The present Example demonstrates systems, methods, compositions, and/or devices for transporting and/or preserving fish tissue and/or preventing tissue damage.

Exemplary protocol [0203] The present Example provides exemplary systems, compositions, methods, and/or devices for transporting and/or preserving fish tissue and/or preventing tissue damage. It is noted that the term “fish” can refer to a single or multiple (or “array of’) fish.

[0204] For example, after a fish has been incapacitated via damage to its brain or by other means described herein, a needle is introduced into the heart and/or circulatory system of the fish. Afterwards, a fluid mixture is introduced into the circulatory system of the fish using a system of pumps. A fluid mixture supplies every tissue of the fish with nutrients and gasses in order to keep the fish alive while the brain is no longer functioning. The spent fluid is then pulled out of the body of the fish via another needle that is introduced into the atrium and threaded through the sinus venosus. The present Example further provides an exemplary protocol of making and using the described systems, methods, and/or devices for transporting and/or preserving fish tissue and/or preventing tissue damage.

1. _ Obtain One or More Live Fish.

[0205] Among other things, the present Example provides for obtaining one or more (e.g., five to ten) live fish. First, one or more live fish as described by International Application No. PCT/US2021/019210 filed on February 23, 2021, entitled “SYSTEMS AND METHODS FOR LIVE FISH TISSUE PRESERVATION”, the disclosure of which is hereby incorporated by reference in its entirety, is obtained. Live fish can include halibut, teleosts, primitive bichirs, sturgeons, paddlefish, freshwater garfishes, bowfins, or other fish known in the art. For example, a person having ordinary skill in the art would understand that other types of fish can be stored and/or preserved using the methods and systems described herein.

2. _ Euthanize Live Fish.

[0206] Among other things, the present Example provides methods for euthanizing live fish. For example, fish can be euthanized by cooling to a temperature from about -4 to about 20 degrees Celsius. In some embodiments, fish can be cooled by placing fish in an ice bath (e.g., having a temperature from about -4 to about 20 degrees Celsius). Fish can also be cooled by placing fish in an ice-water slurry bath (e.g., having a temperature from about -4 to about 20 degrees Celsius). In some embodiments, the ice-water bath has a percentage of ice by weight from about 0.1% to about 99.9%. Types of ice can include crushed, cubed ice, shaved ice, flake(s) of ice, pear ice, or crescent ice. Fish can also be cooled by placing fish in a cool water bath (e.g., having a temperature from about -4 to about 20 degrees Celsius).

Fish can also be cooled by placing fish in a refrigerator (e.g., having a temperature from about -4 to about 20 degrees Celsius). A person of ordinary skill in the art would understand that other systems and methods can be used to cool fish to a temperature from about -4 to about 20 degrees Celsius.

3. Cool Fish to a Specific Temperature.

[0207] Among other things, the present Example provides for cooling one or more fish to a specific temperature to preserve fish tissue. For example, fish are then cooled to a specific temperature, e.g., any temperature from about -4 to about 20 degrees Celsius, or any temperature in between.

4. Optionally Incapacitate Fish.

[0208] Among other things, the present Example provides for optionally incapacitating one or more fish to preserve live fish tissue. In some embodiments, after cooling fish to a specific temperature as described herein, fish are optionally incapacitated, for example, as described by International Application No. PCT/US2021/019210 filed on February 23, 2021, entitled “SYSTEMS AND METHODS FOR LIVE FISH TISSUE PRESERVATION”, the disclosure of which is hereby incorporated by reference in its entirety. Exemplary methods of incapacitation include asphyxiation, an ice bath, decapitation, one or more blows to the head, pithing, ikejime, poisoning, or electricity. A person having ordinary skill in the art would understand that other methods of incapacitation can be used with the systems and methods described herein.

[0209] Exemplary methods of decapitation include using a knife, using a hydraulic press attached to a sharp edge, or a saw (e.g., a circular saw or a reciprocating saw). A person having ordinary skill in the art would understand that other methods of decapitation can be used with the systems and methods described herein.

[0210] Blows to the head can be performed using a hammer or axe. Exemplary hammers include ball peen hammer, claw hammer, club hammer, dead blow hammer, framing hammer, sledge hammer, tack hammer, brick hammer, electricians hammer, engineering hammer, rock hammer, scotch hammer, shingle hammer, spike maul hammer, soft-faced hammer, toolmakers hammer, welding hammer, or power hammer (e.g., steam- powered hammer, electrical power hammer, air powered hammer). Exemplary axes include felling axes, hatchets, throwing tomahawks, splitting axes, double bit axes, Viking/dane axes, tactical axes, battle axes, hewing axes, adze axes, carpenters axes, fireman’s axes, crash axes, or throwing axes. Exemplary hammers and/or axes can comprise a material such as rubber, metal, plastic, wooden, brass or copper. A person having ordinary skill in the art would understand that other methods of blows to the head can be used with the systems and methods described herein.

[0211] Pithing is a technique that can be used to immobilize or kill an animal (such as fish) by inserting a needle or rod into an area of the animal. Pithing can be performed using a material such as metal, rubber, plastic, wood, brass, copper, or carbon fiber. In some embodiments, the pithing material is from about 1 mm in diameter to about 15 cm in diameter. The area that is pithed can include the brain (e.g., olfactory lobe, telencephalon, optic lobe, cerebellum, myelencephalon), spinal cord, heart or gills of the fish.

[0212] Ikejime is another method by which fish can be incapacitated. First, fish are pithed as described herein. Second, gills of the fish are cut. Gills can be cut at the base of the gills, the top of the gills near the front of the fish, or the middle of the gills. Gills can be cut using a knife, scissors, an axe, or a jigsaw using a material as described herein and/or using power such as electricity, human strength, steam power, or gas power. Third, a tail of the fish are cut. A tail can be cut at the base of the caudal fin, 20 cm out from the Caudal fin, or anywhere in between. A tail can be cut using a knife, scissors, axe, or jigsaw using a material as described herein and/or using power such as electricity, human strength, steam power, or gas power. Fourth, the spinal cord of the fish is destroyed. The spinal cord of the fish can be destroyed at the port of entry (e.g., any cut/opening on the posterior side of the fish, any cut/opening on the posterior side of the fish, or any cut/opening on the anterior side of the fish). The spinal cord can be destroyed using a utensil having a material described herein. The utensil can be powered by electricity, human strength, steam power or gas power. Fifth, the fish is bled. Fish can be bled at room temperature or at a temperature from about -4 to about 20 degrees Celsius. In some embodiments, fish are placed in an ice bath, ice-water slurry bath, cold water bath, refrigerator, or in open air. In some embodiments, the ice bath, ice-water slurry bath, cold water bath, refrigerator, or in open air has a temperature from about -4 to about 20 degrees Celsius. In some embodiments, ice-water slurry bath has a percentage of ice by weight of about 0.1% to about 99.9%. Types of ice in the ice-water slurry bath can include crushed ice, cubed ice, shaved ice, flake(s) of ice, pear ice, or crescent ice. Sixth, fish are optionally cauterized at any opening that may expel fluid. For example, fish can be poisoned by being immersed in water saturated with carbon dioxide (e.g., where carbon dioxide levels in water are greater than 5 mg/L as described by https://www.fdacs.gov/Consumer-Resources/Recreation-and-Leisure/Aquarium- Fish/Aquarium-Water-Quality-Carbon-Dioxide, the contents of which are hereby incorporated by reference herein in its entirety). As another example, fish can be poisoned by being exposed to MS22 as described by https://en.wikipedia.org/wiki/Tricaine_mesylate, the contents of which are hereby incorporated by reference herein in its entirety. Fish can also be cauterized by electricity. A person having ordinary skill in the art would understand that different types of cauterization techniques could be employed.

5. Introduce Fluid into Circulatory System of Fish.

[0213] Among other things, the present Example also describes that a fluid is optionally introduced into the circulatory system of the fish through one of the methods to preserve fish tissue, for example, as described by International Application No. PCT/US2021/019210 filed on February 23, 2021, entitled “SYSTEMS AND METHODS FOR LIVE FISH TISSUE PRESERVATION”, the disclosure of which is hereby incorporated by reference in its entirety. A fluid can be flowed at a rate from about 1 nL per minute to about 1 L per minute, or at any rate in between. A fluid can be at a temperature from about -1 to about 20 degrees Celsius, or any temperature in between.

[0214] The present Example provides for a variety of needles that can be used according to the methods described herein. In some embodiments, the needle has a NeedlePoint style such as 2, 3, 3T, 4 (at 1 degree through 90 degrees), 5, AS. In some embodiments, the needle has a needle gauge of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33. The needle can be comprised of medical grade stainless steel, Advanced alloys, exotic metals, MRI compatible materials, or Polymers. A person having ordinary skill in the art would understand that other types of needles could be used in accordance with the systems and methods described herein.

[0215] The present Example also provides methods for introducing fluid into the circulatory system by placing one or more needles into the heart of the fish. While the present Example describes placement into the heart, a person having ordinary skill in the art would understand that needles can also be placed into other organs of the circulatory system. Exemplary organs include hepatic vein, portal veins, caudal vein, caudal artery, dorsal aorta, mesenteric artery, hepatic artery, efferent gill arties, or afferent gill arteries.

A. One needle that is introduced into the heart pumping fluid into the circulatory system. [0216] Among other things, the present Example also describes that a needle is introduced into the heart and is used to pump fluid into the circulatory system, for example, as described by International Application No. PCT/US2021/019210 filed on February 23, 2021, entitled “SYSTEMS AND METHODS FOR LIVE FISH TISSUE PRESERVATION”, the disclosure of which is hereby incorporated by reference in its entirety.

[0217] The needle can be placed into a variety of positions within the chambers of the heart, and can be positioned such that it is in only one chamber of the heart. For example, in some embodiments, a needle is introduced into the Bulbus Arteriosus only and is used to pump fluid into the circulatory system. In some embodiments, a needle is introduced into the Ventricle only and is used to pump fluid into the circulatory system. In some embodiments, a needle is introduced into the Atrium only and is used to pump fluid into the circulatory system. In some embodiments, a needle is introduced into the Sinus Venosus only and is used to pump fluid into the circulatory system. In some embodiments, a needle is introduced into the into the Sinus Venosus only and is used to pump fluid into the circulatory system.

[0218] As another example, in some embodiments, a needle can be placed into a variety of positions within the chambers of the heart, and can be positioned such that it is in more than one chamber of the heart simultaneously, for example, as described by International Application No. PCT/US2021/019210 filed on February 23, 2021, entitled “SYSTEMS AND METHODS FOR LIVE FISH TISSUE PRESERVATION”, the disclosure of which is hereby incorporated by reference in its entirety. For instance, in some embodiments, a needle is introduced Into the Bulbus Arteriosus and the head of the needle is introduced into the Ventricle. In some embodiments, a needle is introduced Into the Bulbus Arteriosus and the head of the needle is introduced into the Ventricle. In some embodiments, a needle is introduced into the Ventricle and the head of the needle is introduced into the Bulbus Arteriosus. In some embodiments, a needle is introduced into the Ventricle and the heard of the needle is introduced into the Atrium. In some embodiments, a needle is introduced into the Atrium and the head of the needle is introduced into the Ventricle. In some embodiments, a needle is introduced into the Atrium and the needle head is introduced into the Sinus Venosus. In some embodiments, a needle is introduced into the Sinus Venosus and the head of the needle is introduced into the Atrium.

B. One needle that is introduced into the heart pumping fluid into the circulatory system and one needle that is introduced into the heart pumping fluid out o f the circulatory system. [0219] Among other things, the present Example also describes that a first needle is introduced into the heart for pumping fluid into the circulatory system, and a second needle is introduced into the heart for pumping out of the circulatory system. A needle can be shaped and sized for placement into the circulatory system as described herein.

[0220] The needles can be placed into a variety of positions within the chambers of the heart, and can be positioned such that both needles are in only one chamber of the heart. For example, in some embodiments, both needles are introduced into the Bulbus Arteriosus only. For example, in some embodiments, both needles are introduced into the Ventricle only. For example, in some embodiments, both needles are introduced into the Atrium only. For example, in some embodiments, both needles are introduced into the Sinus Venosus only.

[0221] Alternatively, in some embodiments, the needles can be placed into a variety of positions within the chambers of the heart, and can be positioned such that the first needle is positioned into a first chamber of the heart, and the second needle is positioned into a second chamber of the heart. For example, the first needle is placed into the Bulbus Arteriosus and the second needle is placed in the Ventricle. For example, the first needle is placed into the Ventricle and the second needle is placed in the Bulbus Arteriosus. For example, the first needle is placed into the Ventricle and the second needle is placed in the Atrium. For example, the first needle is placed into the Atrium and the second needle is placed in the Ventricle. For example, the first needle is placed into the Atrium and the second needle is placed in the Sinus Venosus. For example, the first needle is placed into the Sinus Venosus and the second needle is placed in Atrium.

C. One specially designed needle that is introduced into the heart that allows fluids to be simultaneously pumped in and out o f the heart.

[0222] Among other things, the present Example also describes that a specially designed needle that is introduced into the heart that allows fluids to be simultaneously pumped in and out of the heart.

[0223] In such embodiments, the needle can be placed into a variety of positions within the chambers of the heart, and can be positioned such that it is in only one chamber of the heart. For example, in some embodiments, a needle is introduced into the Bulbus Arteriosus only and is used to pump fluid into the circulatory system. In some embodiments, a needle is introduced into the Ventricle only and is used to pump fluid into the circulatory system. In some embodiments, a needle is introduced into the Atrium only and is used to pump fluid into the circulatory system. In some embodiments, a needle is introduced into the Sinus Venosus only and is used to pump fluid into the circulatory system. In some embodiments, a needle is introduced into the Sinus Venosus only and is used to pump fluid into the circulatory system.

[0224] As another example, in some embodiments, a needle can be placed into a variety of positions within the chambers of the heart, and can be positioned such that it is in more than one chamber of the heart simultaneously. For instance, in some embodiments, a needle is introduced into the Bulbus Arteriosus and the head of the needle is introduced into the Ventricle. In some embodiments, a needle is introduced Into the Bulbus Arteriosus and the head of the needle is introduced into the Ventricle. In some embodiments, a needle is introduced into the Ventricle and the head of the needle is introduced into the Bulbus Arteriosus. In some embodiments, a needle is introduced into the Ventricle and the heard of the needle is introduced into the Atrium. In some embodiments, a needle is introduced into the Atrium and the head of the needle is introduced into the Ventricle. In some embodiments, a needle is introduced into the Atrium and the needle head is introduced into the Sinus Venosus. In some embodiments, a needle is introduced into the Sinus Venosus and the head of the needle is introduced into the Atrium.

D. Fluid is pumped through the circulatory system through a system o f tubes.

[0225] Among other things, the present Example describes that fluid is pumped through the circulatory system of the fish through a system of tubes for the purpose of preserving fish tissue. In some embodiments, for example, the back end of the needle attaches to a tube, and the tube is connected to a pump. Exemplary pumps include peristaltic pumps, lobe pumps, diaphragm pumps, or piston pumps. Exemplary tubes include: aluminum tubes, copper tubes, steel tubes, stainless steels tubes, nylon tubes, polyethylene tubes, polypropylene tubes, polyurethane tubes, PVC tubes, vinyl tubes, rubber tubing, or fiberglass or composite tubes. A person having ordinary skill in the art would understand that other types of pumps and/or tubes can be used with the systems and methods described herein.

6. Fluid is Pumped through the Circulatory System of One or More Fish.

[0226] The present Example also describes that fluid is pumped through the circulatory system of one or more fish in order to preserve live fish tissue. For instance, the present Example describes a system that hooks up to one fish as described by International Application No. PCT/US2021/019210 filed on February 23, 2021, entitled “SYSTEMS AND METHODS FOR LIVE FISH TISSUE PRESERVATION”, the disclosure of which is hereby incorporated by reference in its entirety. The present Example also describes a system that hooks up to many fish as described by International Application No. PCT/US2021/019210 filed on February 23, 2021, entitled “SYSTEMS AND METHODS FOR LIVE FISH TISSUE PRESERVATION”, the disclosure of which is hereby incorporated by reference in its entirety.

7. Fluid Comprises Many Factors to Preserve Fish Tissue.

[0227] Among other things, the present Example are describes a fluid that comprises a variety of factors to preserve fish tissue. For instance, the fluid described herein allows fish to stay alive for a longer period of time. Exemplary factors include water, sugars, elements to balance pH, elements to maintain osmolality, growth factors, organic agents, salts, antimicrobial agents, antioxidants, thickening agents, vitamin, ions, ATP, cofactors, buffers, and enzymes. Examples of types of such factors are provided herein. A person having ordinary skill in the art would understand that other factors may also be used to preserve live fish tissue in accordance with the systems and methods described herein.

8. Movement of Fluid Stays Within Closed System.

[0228] Among other things, the present Example described that fluid moves within a closed system as described by International Application No. PCT/US2021/019210 filed on February 23, 2021, entitled “SYSTEMS AND METHODS FOR LIVE FISH TISSUE PRESERVATION”, the disclosure of which is hereby incorporated by reference in its entirety. For example, in some embodiments, fluid is depleted from a first tank (Tank 1), perfused through the body of a fish (or multiple fish) and then accumulates into a second tank (Tank 2).

Example 2:

[0229] The present Example demonstrates that a fluid comprising a variety of factors extends fish tissue integrity and/or shell life of fish tissue. For example, among other things, the present disclosure describes a fluid comprising a variety of factors that reduces contamination of fish tissue (as compared to water alone). In some embodiments, contamination itself can alter properties of fish tissue (such as pH of fish tissue) that affect fish tissue integrity and/or shelf life of fish tissue. 1. Fish Fillet Extraction.

[0230] Among other things, the present Example describes that more than one 3x3 inch of fish fillet is extracted from fish as described herein. In some embodiments, more than one or more 3x3 inch of fish fillet are extracted from the same fish. In some embodiments, more than one 3x3 inch of fish fillet are extracted from the same part of different fish of the same kind. In some embodiments, more than one 3x3 inch of fish fillet are extracted from the same part of different fish of different kind. In some embodiments, more than one 3x3 inch of fish fillet are randomly extracted from the different part of different fish of different kind.

2. Preparing Various Fluids Comprising Factors to Preserve and/or Store Fish Tissue.

[0231] Among other things, the present Example describes preparing a fluid that comprises a variety of factors to preserve fish tissue. For instance, a fluid described herein allows for storing and/or extending viability of fish cells, tissues, or organs (e.g., post-mortem fish cells, post-mortem fish tissue, or post-mortem fish organs, e.g., live fish cells, live fish tissue, live fish organs, e.g., engineered fish cells, engineered fish tissue, engineered fish organs) for a longer period of time. Exemplary factors include water, sugars, elements to balance pH, elements to maintain osmolality, growth factors, organic agents, salts, antimicrobial agents, antioxidants, thickening agents, vitamin, ions, ATP, cofactors, buffers, and enzymes. Examples of types of such factors are provided herein. A person having ordinary skill in the art would understand that other factors may also be used to preserve fish tissue in accordance with the systems and methods described herein.

3. _ Transferring Fish Fillet to Various Fluids.

[0232] Among other things, the present Example describes that 3x3 inch of fish fillet are each placed into various fluids with a fish: fluid ratio of 1 : 1 by weight and stored in a sealed container. For example, a sealed container can be a sealed plastic bag, a plastic bin with lid, etc. The present Example also describes that the sealed container is stored at a specified temperature. For example, the sealed container is stored at 4 °C.

[0233] Among other things, the present Example describes that six 3x3 fillet are stored in the following fluids comprising various factors at 4 °C for 28 days (see Table 1 and Table 2).

Table 1

^aThe concentration of various factors is calculated based on the weight of fish. For example, for 1 kg of fish, 6 mg of nisin was added to water. In some embodiments, for 1 kg of fish, 6 mg of nisin was added to 1 L water. In some embodiments, for 1 kg of fish, 6 mg of nisin was added to 500 mL water. In some embodiments, for 1 kg of fish, 6 mg of nisin was added to 200 mL water. In some embodiments, for 1 kg of fish, 6 mg of nisin was added to 2 L water.

Table 2

^aThe concentration of various factors is calculated based on the weight of fish. For example, for 1 kg of fish, 6 mg of nisin was added to HBSS. In some embodiments, for 1 kg of fish, 6 mg of nisin was added to 1 L HBSS. In some embodiments, for 1 kg of fish, 6 mg of nisin was added to 500 mL HBSS. In some embodiments, for 1 kg of fish, 6 mg of nisin was added to 200 mL HBSS. In some embodiments, for 1 kg of fish, 6 mg of nisin was added to 2 L HBSS. 4. Measurements of Data.

A. pH

[0234] Among other things, the present Example describes measurements of pH of various fluids as described herein. In some embodiments, the pH of a fluid is adjusted to about 6 using the buffer as described herein. In some embodiments, the pH of a fluid is adjusted to about 7 using the buffer as described herein. In some embodiments, the pH of a fluid is adjusted to about 8 using the buffer as described herein. Among other things, the present Example describes measuring pH values of the fluid set forth in Conditions 1-6 at days 0, 1, 7, 14, 21, 28.

B. Sensory Test

[0235] Among other things, the present Example describes performing sensory test on the fish fillet under Conditions 1-6 on days 1, 7, 14, 21, and 28. For example, sensory test provides information regarding appearance, texture, taste, and smell of fish fillet. These sensory components are indicative the freshness and viability of fish tissues under Conditions 1-6.

C. Optional Microbial Test

[0236] Among other things, the present Example describes optionally performing microbial tests on fish fillet under Conditions 1-6 on days 1, 7, 14, 21, and 28. For example, microbial test is performed on LB agar plates. Specific protocols of culturing E. coli on LB agar plates is well understood in the art and as described in https://www.sigmaaldrich.com/US/en/technical-documents/protocol/cell-culture-and-cell- culture-analysis/microbial-cell-culture/microbial-growth, the contents of which is hereby incorporated by reference in its entirety.

5. Fluid Allows Fish Tissue and/or Cells to Stay Alive for An Extended Period of Time.

[0237] Among other things, the present Example also describes that contacting fluid with fish tissue and/or fish cells allows for fish tissue and/or cells to stay alive for an extended period of time. For instance, in some embodiments, contacting or storing fish tissue and/or fish tissue in a fluid which can extend the life time of the fish tissue and/or cells for a time period from about 5 minutes to about one year, or any time in between. 6. Fluid Extends Shelf Life of Fish Tissue to Help Multiple Industries.

[0238] Among other things, the present Example describes a fluid that extends shelf life of fish tissue. Extending shelf life of live tissue is important to a variety of industries such as cellular agriculture, fish transporting companies, and the food industry. In the cellular agriculture industry, for example, it is important that the fish tissue and/or cells stay fresh from material acquisition from where the natural habitat of the fish is to transportation of the fish to a laboratory of a company. Increasing the shelf life of the material allows scientists more time to process the tissue. In addition, for fish transporting companies or the food industry, increasing the shelf life of fish material allows a wider window of time for companies supply chain logistics. Exemplary food industries include fast food industries, fine dining, mom and pop store, restaurants, cafeteria, prison, airline, schools, food distributors, and wholesalers.

7. The Described Systems are Sterile or Close to Sterile.

[0239] Among other things, the present Example also provides for a closed system that is sterile or close to sterile that preserves fish tissue. Types of sterilization are described herein. A person of ordinary skill in the art would understand that other methods for sterilization can be used.

Example 3 :

[0240] Among other things, the present Example demonstrates that HBSS is capable of preserving viability of fish cells post mortem. For example, among other things, the present Example describes that viability of fish muscle tissue was preserved when such tissue was stored in HBSS for 24 hours post mortem. In particular, in some embodiments, the present Example demonstrates the surprising finding that there was no significant change in the number of viable fish cells derived from fish tissue at 24 hours post mortem as compared to the number of viable fish cells derived from fish tissue immediately after death. 1. Materials and Methods.

[0241] Fresh sea bass was euthanized and 10 g tissue pieces were dissected from axial muscle tissue. Tissue pieces were stored in HBSS at 4 °C. Subsequently, cells were isolated according to a standard cell isolation procedure at different time points post mortem, e.g., 0 hours, 24 hours after death. In the instance where cells were isolated 0 hours after death, cells were isolated immediately after tissues were taken from fish, without storage in HBSS. Isolated cells were seeded into T25 flasks and kept in culture for 4 days and images were then taken and the amount of live cells in culture was analyzed.

[0242] Standard cell isolation procedures. Tissues were removed from storage fluid and minced. Tissues were then dissociated with TrypLE™ at room temperature in 100 RPM shaker and the resulting mixture was filtered with 100 pm nylon filter. The resulting solution was transferred to a new 50 mL collection tube and quenched with L15/FS + 5% FBS + IxAA and the mixture was centrifuged at 400G for 4 min. Subsequently, the solution was resuspended in 30 mL of PBS prior to cell count.

2. Results.

[0243] The present Example describes that fish muscle tissue that was stored in HBSS for 24 hours post mortem maintained a high number of viable cells. Images of fish tissue cells immediately after death (0 hour post mortem) and fish tissue cells 24 hours post mortem are shown in FIGS. 1 A-1B. Among other things, the present Example demonstrates that HBSS can be used a storage buffer to preserve fish tissue.

Example 4:

[0244] Among other things, the present Example demonstrates that HBSS can preserve fish tissue and cell viability better than commercially available storage buffer. For example, among other things, the present Example describes that the number of viable fish cells stored in HBSS for 5 days was greater than the number of viable fish cells stored in HypoThermosol® FRS, a commercially available storage buffer.

1. Materials and Methods.

[0245] Fresh carp was euthanized and 10 g tissue pieces were dissected from axial muscle tissue. Tissue pieces were stored in HBSS or HypoThermosol® FRS at 4 °C. Subsequently, cells were isolated according to the standard cell isolation procedures described in Example 3 at different time points post mortem, e.g., 5 days after death. Isolated cells were seeded into T25 flasks and maintained in culture for 4 days and images were then taken and the amount of live cells in culture was analyzed.

2. Results.

[0246] The present Example describes that viable cells were surprisingly isolated from fish tissue muscle 5 days post mortem. For example, in some embodiments, more viable cells were observed in tissues stored in HBSS than in tissues stored in HypoThermosol® FRS under otherwise same conditions (see also Example 3). Images of fish tissue cells from HBSS and HypoThermosol® FRS are shown in FIGS. 2A-2B. Among other things, in some embodiments, the present Example describes that HBSS performed better than HypoThermosol® FRS in storing fish tissues.

[0247] Accordingly, the present Example demonstrates that HBSS can be used as a fluid suitable for storing fish tissue and for extending viability of fish cells, tissues, and/or organs.

Example 5:

[0248] Among other things, the present Example demonstrates that HBSS comprising a variety of factors inhibited growth of microbes and reduced contamination of fish tissues. The present Example also demonstrates that inhibition of microbial growth and reduced contamination of fish tissue extended fish tissue integrity and/or shell life of fish tissue. In particular, the present Example describes that inhibition of microbial growth and reduced contamination of fish tissue were observed by measuring microbial colonies incubated from samples taken from various fish storage fluids as described in Example 2.

[0249] The present Example also describes that fish tissue stored in HBSS comprising nisin showed no microbial colonies when incubated for 24 hours in culture. The present Example also describes that fish tissue stored in HBSS comprising natamycin showed no microbial colonies when incubated for 24 hours in culture. The present Example also describes that fish tissue stored in HBSS comprising chitosan showed no microbial colonies when incubated for 24 hours in culture.

1. Materials and Methods.

[0250] Tuna whole fish (dead) was stored on ice for 4 days and 5 g tissue pieces were obtained 4 days post mortem. [0251] HBSS fluids comprising one or more antimicrobial agents were prepared according to Table 3. Fish tissues were stored in various fluids according to Table 3 for 5 days. 1 mL sample were taken from each of the various storage fluids and were added into 4 mL PBS. Sterile cotton tips were dipped into such PBS solution and then samples were spread over LB petri dish. The LB petri dish was incubated at 37 °C for 24 hours.

Table 3

2. Results.

[0252] It was found that that when fish tissue was stored in HBSS comprising one or more antimicrobial agents, no colonies of microbial were observed after incubating the storage fluid in culture for 24 hours. After incubation for 24 hours, samples taken from the fish tissue storage fluid comprising nisin (Table 3, entry 1) showed no colonies, samples taken from the fish tissue storage fluid comprising natamycin (Table 3, entry 2) showed no colonies, samples taken from the fish tissue fluid comprising chitosan (Table 3, entry 3) showed no colonies, and samples taken from the fish tissue storage fluid comprising nisin, natamycin, and chitosan (NNC, Table 3, entry 4) showed one colony. In comparison, samples seeded with staphylococcus epidermidis (Positive Control) showed more than 100 colonies after incubation in culture for 24 hours under otherwise identical conditions. In comparison, sterile PBS (Negative Control) showed no colonies after incubation in culture for 24 hours under otherwise identical conditions. The pictures of LB petri dish after incubation for 24 hours are shown in FIGS. 3A-3F.

[0253] Accordingly, the present Example demonstrates that storing fish cells, tissues, or organs in a fluid comprising one or more antimicrobial agents resulted in reduced contamination. Therefore, fluids comprising antimicrobial agents can extend viability of fish cells, tissues, and/or organs.

Example 6:

[0254] Among other things, the present Example describes that fish cells derived from fish tissue demonstrated significantly improved cell viability when fish tissues were stored in fluids comprising one or more antimicrobial agents, compared to a reference.

[0255] In this Example, fish muscle tissue was stored for 5 days in HBSS with nisin, natamycin, or chitosan to determine conditions to increase cell viability. Fish cells were successfully isolated in all conditions, and cell number and cell viability measurements were obtained as described herein. Surprisingly, when maintained under each of these conditions, cell viability was significantly improved to be greater than 30% (data not shown).

[0256] Accordingly, the present Example demonstrates that storing fish cells, tissues, or organs in a fluid comprising one or more antimicrobial agents resulted in reduced contamination. Moreover, the present Example demonstrates that fluids comprising one or more antimicrobial agents can extend viability of fish cells, tissues, and/or organs.

Equivalents

[0257] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. The scope of the present disclosure is not intended to be limited to the above Description, but rather is as set forth in the following claims.

Claims

Claims We claim:

1. A fluid suitable for contacting and/or storing and/or extending viability of fish cells, tissues, or organs.

2. The fluid of claim 1, wherein the viability of the fish cells, tissue, or organs maintained substantially in the fluid is greater than 5% of the viability of the fish cells, tissues, or organs stored compared to a reference fluid.

3. The fluid of claim 1 or 2, comprising at least one factor comprising one or more antimicrobial agents, one or more antioxidant agents, or any combination thereof.

4. The fluid of claim 3, wherein each factor of the at least one factor is present in the fluid in an amount effective to prolong the viability of the fish cells, tissues, or organs when maintained substantially in the fluid, as compared to the maintenance of the fish cells, tissues, or organs stored in the fluid alone.

5. The fluid of claim 4, wherein the viability of the fish cells, tissue, or organs maintained substantially in the fluid is greater than 5% of the viability of the fish cells, tissues, or organs stored in the fluid alone.

6. The fluid of any one of claims 3-5, wherein the one or more antimicrobial agents comprises ozoned slurry ice, ozonated water, natural food antimicrobrials, or any combination thereof.

7. The fluid of claim 6, wherein the natural food antimicrobials comprises or is chitosan, nisin, or natamycin, or any combination thereof.

8. The fluid of any one of claims 3-7, wherein the one or more antioxidant agents comprises butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ascorbic acid, a tocopherol , rosemary extract, green tea extract, or any combination thereof.

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9. The fluid of claim 8, wherein the ascorbic acid is Vitamin C.

10. The fluid of any one of claims 3-8, wherein the one or more antioxidant agents comprises a tocopherol.

11. The fluid of claim 10, wherein the tocopherol is Vitamin E.

12. The fluid of any one of claims 1-11, wherein the fluid has a pH within a range from about 5 to about 8.

13. The fluid of any one of claims 1-12, wherein the fluid comprises one or more of the following factors: one or more salts, one or more sugars, one or more antimicrobial agents, one or more antioxidants, or a combination thereof.

14. The fluid of any one of claims 1-13, wherein the fluid comprises one or more of

(i) Calcium Chloride (CaCh),

(ii) Potassium Chloride (KC1),

(iii) Potassium Phosphate Monobasic (KH2PO4),

(iv) Magnesium Chloride (MgCh 6H2O),

(v) Magnesium Sulfate (MgSCU 7H2O),

(vi) Sodium Chloride (NaCl),

(vii) Sodium Bicarbonate (NaHCCh),

(viii) Sodium Phosphate Dibasic (Na2HPO4),

(ix) Glucose,

(x) chitosan,

(xi) natamycin,

(xii) Butylated hydroxyanisole,

(xiii) Butylated hydroxytoluene,

(xiv) a-, B-, 5-, y- tocopherols, or any combination thereof.

15. The fluid of any one of claims 1-12, wherein the fluid comprises HBSS and one or more of natural food antimicrobials.

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16. The fluid of claim 15, wherein the one or more natural food anitmicrobials comprises or is nisin.

17. The fluid of claim 15, wherein the one or more natural food anitmicrobials comprises or is natamycin.

18. The fluid of claim 15, wherein the one or more natural food anitmicrobials comprises or is chitosan.

19. The fluid of claim 15, wherein the one or more natural food anitmicrobials comprises nisin, natamycin, chitosan, or any combination thereof.

20. The fluid of any one of claims 14-19, wherein the Calcium Chloride (CaCh) is within a range of from about 0.1-10 mM.

21. The fluid of any one of claims 14-20, wherein the Potassium Chloride (KC1) is within a range of from about 0.1-10 mM.

22. The fluid of any one of claims 14-21, wherein the Potassium Phosphate Monobasic (KH2PO4) is within a range of from about 0.1-10 mM.

23. The fluid of any one of claims 14-22, wherein the Magnesium Chloride (MgCh 6H2O) is within a range of from about 0.1-10 mM.

24. The fluid of any one of claims 14-23, wherein the Magnesium Sulfate (MgSC 7H2O) is within a range of from about 0.1-10 mM.

25. The fluid of any one of claims 14-24, wherein the Sodium Chloride (NaCl) is within a range of from about 1-1000 mM.

26. The fluid of any one of claims 14-25, wherein the Sodium Bicarbonate (NaHCOs) is within a range of from about 0.1-10 mM.

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27. The fluid of any one of claims 14-26, wherein the Glucose is within a range of from about 0.1-100 mM.

28. The fluid of any one of claims 14-27, wherein the chitosan is within a range of from about 1-10,000 kDa, and is within a range from about 0.01%-10% w/w.

29. The fluid of claim 28, wherein the chitosan is at a concentration of about 60 mg per kilogram of fish.

30. The fluid of any one of claims 14-29, wherein the natamycin is within a range of from about 0.1-100 mg/kg.

31. The fluid of claim 30, wherein the natamycin is at a concentration of about 10 mg per kilogram of fish.

32. The fluid of any one of claims 14-31, wherein the nisin is at a concentration of about 2 mg per kilogram fish.

33. The fluid of any one of claims 14-32, wherein the Butylated hydroxyanisole is within a range of from about 1 -10,000 mg/kg.

34. The fluid of any one of claims 14-33, wherein the Butylated hydroxytoluene is within a range of from about 1-10,000 mg/kg.

35. The fluid of any one of claims 14-34, wherein the a-, B-, 5-, y- tocopherols are within a range of from about 1-10,000 mg/kg.

36. The fluid of any one of claims 1-35, wherein the fluid preserves and/or prevents damage and/or extends viability to the fish cells, tissues, and organs for an extended period of time as compared to the maintenance and/or storage of the fish tissue, cells, or organs maintained in water alone.

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37. The fluid of claim 36, wherein the fluid preserves and/or prevents damage and/or extends viability to the fish cells, tissues, and organs for an extended period of time from about 1 day to about 5 days as compared to the maintenance and/or storage of the fish tissue, cells, or organs maintained in water alone.

38. Fish tissue for use in transportation and/or preservation, wherein the fish tissue is stored in a fluid.

39. A method of transporting and/or preserving fish tissue and/or preventing tissue damage to fish tissue, the method comprising: contacting fish tissue with a fluid comprising at least one factor comprising one or more antimicrobial agents, one or more antioxidant agents, or any combination thereof.

40. The method of claim 39, comprising storing the fish tissue at a temperature range less than 10 degrees Celsius

41. The method of claim 40, wherein the temperature range is less than 4 degrees Celsius.

42. The method of any one of claims 39-41, wherein the one or more antimicrobial agents comprises ozoned slurry ice, ozonated water, natural food antimicrobrials, or any combination thereof.

43. The method of claim 42, wherein the natural food antimicrobial comprises or is chitosan, nisin, or natamycin, or any combination thereof.

44. The method of any one of claims 39-43, wherein the one or more antioxidant agents comprises butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ascorbic acid, a tocopherol, rosemary extract, green tea extract, or any combination thereof.

45. The method of claim 44, wherein the ascorbic acid is Vitamin C.

46. The method of claim 44, wherein the tocopherol is Vitamin E.

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47. The method of claims 39-46, comprising keeping the fish tissue in contact with the fluid for at least 1 day.

48. The method of claim 46, where the fish tissue is in contact with the fluid for at least 5 days.

49. The method of any one of claims 39-48, wherein the fluid preserves and/or prevents damage to the fish cells, tissues, and organs for an extended period of time as compared to the maintenance and/or storage of the fish tissue, cells, or organs maintained in water alone.

50. The method of claim 49, wherein the fluid preserves and/or prevents damage to the fish cells, tissues, and organs for an extended period of time from about 1 day to about 5 days, as compared to the maintenance and/or storage of the fish tissue, cells, or organs maintained in water alone.

51. The method of any one of claims 39-50, wherein the method comprises: contacting fish tissue with the fluid via a device.

52. The method claim 51, wherein the device comprises a needle adapted for contacting and/or perfusing and/or storing fish cells, tissues, or organs.

53. The method of claim 52, wherein the needle is placed into a first location of an organ of the circulatory system of one or more fish.

54. The method of claim 53, wherein the organ of the circulatory system is or comprises a member selected from the group consisting of a bulbus arteriosus, a ventricle, an Atrium, and a sinus venosus.

55. The method of any one of claims 52-54, wherein the needle is simultaneously placed into both a first location of the organ and a second location of the organ.

56. The method claim 55, wherein the first needle comprises a head, and wherein the head is placed into a second chamber of the heart.

57. The method of any one of claims 52-56, wherein the first needle simultaneously is adapted to flow the fluid into and out of the organ.

58. A method of characterizing a fluid of any one of claims 1-37.

59. A method of characterizing fish cells, tissue, or organs stored in a fluid of any one of claims 1-37.