How to preserve tissue for transplantation

Abstract

Provided herein are systems and methods for storing and transporting tissue for transplantation. Specifically, the tissue to be transplanted is encapsulated in an alginate gel and exposed to a culture medium to improve cell viability and suppress inflammatory factors during long-term storage and transport.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 960,298, filed January 13, 2020, the entire contents of which are incorporated by reference herein. [Technical field]

[0002] FIELD OF THE DISCLOSURE The present disclosure relates generally to the field of tissues, such as allografts, and more specifically to the long-term storage and preservation of tissues for transplantation. [Background technology]

[0003] Osteochondral allografts (OCA) can transplant functional viable tissue for the treatment of defective cartilage without the need for immunosuppression. Therefore, OCA is one of the most widely used methods applied for the repair of moderate to large articular cartilage defects (Mandelbaum, et al., Am J Sports Med. 1998; 853). Recent clinical results have shown that OCA is successful in 60% to 80% of cases at 10 years after surgery (Cavendish, et al., JBJS Rev. 2019; e7). These grafts must be obtained from organ donors and stored to allow viral and bacterial testing for safety before being transported to a surgical center for implantation in patients. Based on studies investigating the viability of cells in the grafts, it is recommended that the tissue be transplanted as soon as possible after harvesting to maximize the success rate.

[0004] One of the key factors for obtaining satisfactory clinical outcomes is to improve the quality of stored OCA (including maximizing chondrocyte viability and cartilage matrix content). Although various studies have been conducted previously to improve storage conditions, the quality of long-term stored grafts has not been satisfactorily maintained (Ball, et al., Clin Orthop Relat Res. 2004:246; Williams, et al., J Bone Joint Surg. Am. 2003:2111; and Pallante, et al., Am J Sports Med. 2009:24S). Improved storage conditions would improve the shelf life of OCA and the potential for improved clinical outcomes in its clinical use. Summary of the Invention

[0005] The present disclosure is based on the discovery that encapsulating the tissue to be transplanted in an alginate gel improves the quality and viability of the tissue after prolonged storage or transport, and after both storage and transport.

[0006] Thus, the present disclosure provides methods for preserving osteochondral tissue or other tissue(s) or organ(s) (such as articular cartilage without any subchondral bone, fibrocartilage tissue (such as meniscus, e.g., medial or lateral meniscus or both), tendons (such as patellar tendon or Achilles tendon), cornea, or cornea with vitreous body). In various embodiments, the osteochondral tissue comprises, consists essentially of, or consists of an osteochondral core trimmed to have a bone to cartilage thickness ratio of about 20:1 to about 1:10 and ranges or ratios therebetween (e.g., about 4:1). The method comprises (e.g., comprises, consists essentially of, or further consists of) encapsulating the osteochondral tissue or other suitable tissue(s) or organ(s) in an alginate gel and exposing the gel-encapsulated osteochondral tissue or other suitable tissue(s) or organ(s) to a culture medium. In some embodiments, the gel encapsulated tissue is placed in culture medium for about 24 hours to 8 weeks, or at least about 2 weeks (such as at least about 4 weeks, at least about 6 weeks, or longer, and ranges therebetween). In various embodiments, the gel encapsulated tissue in culture medium is then used for transplantation. In various embodiments, the method further comprises encapsulating the tissue(s) or organ(s) in a semipermeable membrane. In various embodiments, the semipermeable membrane is characterized by one or more of a pore size of at least about 250 pm, a pore size of about 0.22 μm to about 1000 μm, and ranges therebetween (such as about 0.22 μm to about 100 μm or about 0.22 μm to about 100 μm). Additionally or alternatively, the semipermeable membrane is characterized by a molecular weight cut off (MWCO) of about 50 kDa or about 200,000 kDa. Additionally or alternatively, the semipermeable membrane has a mesh openness of about 1% to about 50% and ranges or percentages therebetween (e.g., 2%). In various embodiments, the tissue is encapsulated in the gel before, during, or after encapsulation in the semipermeable membrane. In various embodiments, the alginate gel is a highly purified alginate (UPAL) gel. In various embodiments, an alginate such as UPAL is present in the gel at a concentration of about 0.5% to about 2.0% and ranges or percentages therebetween (e.g., about 1.2%).In various embodiments, the culture medium is a complete medium. In various embodiments, the culture medium is not replenished after exposure of the gel-encapsulated osteochondral tissue or other gel-encapsulated tissue(s) or organ(s) to the culture medium. In various embodiments, the method further comprises exposing the tissue(s) or organ(s) to less than about 10°C, for example, between about 0°C and about 4°C, or to about 4°C. In various embodiments, the encapsulated tissue(s) or organ(s) are not stored with one or more of artificially controlled CO2 levels, artificially controlled O2 levels, or artificially controlled humidity. In various embodiments, the method further comprises dissolving the alginate gel after exposure to the culture medium. In further embodiments, the alginate gel is dissolved by a method comprising, consisting essentially of, or consisting of exposing the encapsulated tissue(s) or organ(s) to a chelating solution. In still further embodiments, the chelating solution comprises, consists essentially of, or further consists of a sodium citrate solution or an ethylenediaminetetraacetic acid (EDTA) solution or a solution comprising or consisting essentially of sodium citrate and EDTA. In various embodiments, the method further comprises degrading the semipermeable membrane after exposure to culture medium, e.g., mechanically or by the chelating solution. In various embodiments, the encapsulated tissue(s) or organ(s) reduce the release of inflammatory factor(s), e.g., compared to tissue or organ stored without encapsulation. In further embodiments, the inflammatory factor(s) comprises, consists essentially of, or further consists of one or more of nitric oxide, cytokines, or both. In various embodiments, the encapsulated tissue(s) or organ(s) prior to implantation maintains a cell viability of at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85% or higher than the cell viability prior to the encapsulation step. In various embodiments, prior to exposure to culture medium, the encapsulated tissue(s) or organ(s) are washed with a buffer solution, such as saline.In various embodiments, the encapsulated tissue(s) or organ(s) (eg, cornea) is placed in culture medium for at least about two weeks.

[0007] In another aspect, the disclosure provides methods for storing osteochondral tissue or other tissue(s) or organ(s) (such as articular cartilage without any subchondral bone, fibrocartilage tissue (such as meniscus, e.g., medial or lateral meniscus or both), tendons (such as patellar tendon or Achilles tendon), cornea, or cornea with vitreous), e.g., prior to implantation. In various embodiments, the osteochondral tissue comprises, consists essentially of, or consists of an osteochondral core trimmed to have a bone to cartilage thickness ratio of about 20:1 to about 1:10 (e.g., about 4:1). The method includes (e.g., comprises, or consists essentially of, or further consists of) the steps of obtaining osteochondral tissue or other suitable tissue(s) or organ(s) to be transplanted as required, placing the obtained osteochondral tissue or other suitable tissue(s) or organ(s) in a semipermeable membrane chamber containing an alginate solution, placing the semipermeable membrane chamber in a culture chamber, adding a calcium solution or a barium solution or both to the culture chamber, whereby the osteochondral tissue or other suitable tissue(s) or organ(s) are encapsulated in the alginate gel, injecting a culture medium into the alginate gel or culture chamber, or injecting an alginate gel into the culture chamber, and placing the culture chamber in a storage container. In some embodiments, the encapsulated osteochondral tissue or other suitable tissue(s) or organ(s) remains viable, e.g., for transplantation. Additionally or alternatively, the encapsulated osteochondral tissue or other suitable tissue(s) or organ(s) remain viable, for example, for at least about 2 weeks (such as at least about 4 weeks or at least about 6 weeks) or up to 8 weeks after the obtaining step. In various embodiments, the calcium solution comprises, consists essentially of, or further consists of a calcium chloride solution. In various embodiments, the barium solution comprises, consists essentially of, or further consists of a barium chloride solution. In various embodiments, the reservoir further comprises a temperature sensor. In various embodiments, the tissue(s) or organ(s) are encapsulated in a semipermeable membrane chamber.In various embodiments, the semipermeable membrane has one or more characteristics of a pore size of at least about 250 pm; or about 0.22 μm to about 1000 μm; and ranges therebetween (such as about 0.22 μm to about 100 μm or about 100 μm to about 1000 μm). Additionally or alternatively, the semipermeable membrane has a molecular weight cut off (MWCO) of about 50 kDa or about 200,000 kDa. In various embodiments, the semipermeable membrane has a mesh opening percentage of about 1% to about 50% and ranges or percentages therebetween (e.g., about 2%). In various embodiments, the alginate solution is a high purity alginate solution. In various embodiments, the alginate solution comprises or consists essentially of about 0.5% to about 2.0% alginate and ranges or percentages therebetween, optionally about 1.2% alginate. In various embodiments, the culture medium is a complete medium. In various embodiments, the method further comprises transporting the storage container to a hospital after a period of storage. In various embodiments, the method further comprises maintaining a temperature in the semipermeable membrane, or in the culture chamber, or in the storage container below 10° C. (e.g., about 0° C. to about 4° C., or about 4° C.). In various embodiments, the CO2 level, or O2 level, or humidity, or any combination thereof, in the semipermeable membrane, or in the culture chamber, or in the storage container is not artificially controlled. In various embodiments, the method further comprises dissolving the alginate gel, e.g., by a method comprising, consisting essentially of, or consisting of exposing the encapsulated tissue(s) or organ(s) to a chelating solution, e.g., prior to implantation. In further embodiments, the chelating solution comprises, consists essentially of, or further consists of a sodium citrate solution or an ethylenediaminetetraacetic acid (EDTA) solution or a solution comprising or consisting essentially of sodium citrate and EDTA. In various embodiments, the method further comprises decomposing the semipermeable membrane, e.g., prior to implantation. In various embodiments, for example, the tissue(s) or organ(s) survived for transplantation will have reduced release of inflammatory factor(s) as compared to a tissue or organ stored without encapsulation, for example, in a gel, in a semipermeable membrane, or both in a gel and in a semipermeable membrane.In further embodiments, the inflammatory factor(s) comprises, consists essentially of, or further consists of one or more of nitric oxide, or cytokines, or both nitric oxide and one or more of cytokines. In various embodiments, the tissue(s) or organ(s) remain viable, for example for transplantation. Additionally or alternatively, the tissue(s) or organ(s) maintain a cell viability of at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85% or higher than the cell viability before the encapsulation step. In various embodiments, the method further comprises injecting a buffer solution (such as saline) into the alginate gel or the culture chamber or into the alginate gel in the culture chamber before injecting the culture medium. In further embodiments, the buffer solution (such as saline) reduces or removes soluble calcium or barium. In various embodiments, the encapsulated tissue(s) or organ(s) (eg, cornea) is, for example, in one aspect, stored for at least about two weeks prior to transplantation.

[0008] In another aspect, the present disclosure provides a tissue transport system. The system includes a tissue chamber formed in part or in whole from a semipermeable membrane and configured to contain the tissue and alginate solution to be transported; a culture chamber having an inlet and an optional outlet, the culture chamber configured to contain the tissue chamber, and upon addition of a calcium solution or a barium solution or both via the inlet, the alginate solution gels and encapsulates the tissue; a transport box or storage container configured to contain the culture chamber; and an optional temperature sensor disposed in the transport box or storage container and configured to measure the temperature of the culture chamber (e.g., includes, consists essentially of, or further consists of). In various embodiments, the tissue chamber is enclosed. In various embodiments, the culture chamber is enclosed. In various embodiments, the alginate solution is a high purity alginate (UPAL) solution. In various embodiments, the tissue chamber is an enclosed tissue chamber configured to encapsulate the tissue by the semipermeable membrane. In various embodiments, the semipermeable membrane has / includes a pore size of at least about 250 pm, for example, about 0.22 μm to about 1000 μm and ranges therebetween (e.g., 0.22 μm to about 100 μm, or about 100 μm to about 1000 μm). In various embodiments, the semipermeable membrane has a mesh openness of about 1% to about 50% and ranges or percentages therebetween (e.g., about 2%). Additionally or alternatively, the semipermeable membrane has a molecular weight cut off (MWCO) of about 50 kDa or about 200,000 kDa. In various embodiments, the alginate solution comprises, consists essentially of, or further consists of about 0.5% to about 2.0% alginate and ranges or percentages therebetween, optionally about 1.2% alginate. In various embodiments, the transport box or storage container or culture chamber further comprises an insulating component or a cooling component or both a thermal insulating component and a cooling component configured to maintain a temperature in the transport box or storage container or culture chamber below 10°C (e.g., about 0°C to about 4°C and ranges therebetween, or about 4°C).In various embodiments, the system does not include sensors or controllers for the CO2 levels, or O2 levels, or humidity, or any combination thereof, in the semi-permeable membrane or in the culture chamber or storage vessel. In various embodiments, the transport box or storage vessel is sealed against continuous flow of gas or liquid.

[0009] In some embodiments, the tissue culture device includes (e.g., includes, or consists essentially of, or further consists of) an outer chamber defining an inlet, the inlet constructed to allow the inflow of fluid through said inlet, and an outlet, the outlet constructed to allow the outflow of fluid through said outlet, the outer chamber being enclosed. The inner chamber is disposed at least in part within the outer chamber, the inner chamber having at least one wall formed from a semipermeable membrane, the inner chamber being enclosed and constructed to hold tissue within the inner chamber. The tissue culture device also optionally includes (e.g., further includes) an enclosed storage vessel, the outer chamber disposed within an interior volume defined by the storage vessel, and the resulting inner chamber. In various embodiments, the tissue culture device further includes a temperature sensor operably coupled to the storage vessel and configured to measure the temperature within the storage vessel. In various embodiments, the tissue is encapsulated by an alginate gel. In various embodiments, the fluid comprises, consists essentially of, or consists of at least one of an alginate solution, a calcium solution, a barium solution, a saline solution, a culture medium, or a storage medium. In various embodiments, the semipermeable membrane has a molecular weight cut-off (MWCO) of about 50 kDa or about 200,000 kDa. In various embodiments, the semipermeable membrane has a pore size of at least about 250 pm. In various embodiments, the semipermeable membrane has a pore size in the range of about 0.22 μm to about 100 μm and ranges therebetween (such as about 100 μm to about 1000 μm, or about 0.22 μm to about 1000 μm). In various embodiments, the semipermeable membrane has a mesh openness of about 1% to about 50% and ranges or percentages therebetween (e.g., about 2%). In various embodiments, the tissue culture device further comprises at least one of an insulating component or a cooling component disposed within the storage container, wherein the at least one of the insulating component or the cooling component is configured to maintain a temperature within the storage container and / or the outer chamber below 10°C.

[0010] In one aspect, a kit as disclosed herein is provided that comprises or consists essentially of a tissue culture device as disclosed herein and optional instructions for use. In various embodiments, the kit as disclosed herein further comprises one or more of the following: an optional solution of alginate or any other hydrogel monomer, a calcium solution, a barium solution, a calcium salt, a barium salt, a solvent, a washing solution, or a culture medium. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a pictorial diagram showing an exemplary storage condition scheme.

[0012] [Diagram 2] FIG. 2 provides a series of graphs showing the results of chondrocyte regional viability.

[0013] [Figure 3A] FIG. 3A provides a pictorial representation of representative tissue images (FIG. 3A) and a graphical representation of the histological score results (FIG. 3B). [Figure 3B] FIG. 3B provides a pictorial representation of representative tissue images (FIG. 3A) and a graphical representation of the histological score results (FIG. 3B).

[0014] [Figure 4A] FIG. 4A is a pictorial diagram illustrating an exemplary tissue storage and transport system.

[0015] [Figure 4B] FIG. 4B shows various projections of a tissue culture device according to an embodiment. [Figure 4C] 4A-4C are various projections of a tissue culture device according to an embodiment. [Figure 4D] 4A-4D are various projections of a tissue culture device according to an embodiment. [Figure 4F] FIG. 4F shows various projections of a tissue culture device according to an embodiment.

[0016] [Diagram 5] FIG. 5 is a pictorial representation showing exemplary storage conditions and UPAL gel coated samples.

[0017] [Figure 6A] FIG. 6A shows the normalized percentage of viable cells in pooled articular cartilage (FIG. 6A), meniscus (FIG. 6B), patellar tendon (FIG. 6C), and cornea (FIG. 6D). [Figure 6B] FIG. 6B shows the normalized percentage of viable cells in pooled articular cartilage (FIG. 6A), meniscus (FIG. 6B), patellar tendon (FIG. 6C), and cornea (FIG. 6D). [Figure 6C] FIG. 6C shows the normalized percentage of viable cells in pooled articular cartilage (FIG. 6A), meniscus (FIG. 6B), patellar tendon (FIG. 6C), and cornea (FIG. 6D). [Figure 6D] Figure 6D shows the normalized percentage of viable cells in pooled articular cartilage (Figure 6A), meniscus (Figure 6B), patellar tendon (Figure 6C), and cornea (Figure 6D). Mean ± SE, *=p<0.05, **=p<0.01.

[0018] [Figure 7] FIG. 7 provides examples of partial structures of alginates. Detailed Description of the Invention

[0019] The present disclosure is based on the discovery that encapsulating the tissue to be transplanted in an alginate gel improves the quality and viability of the tissue after prolonged storage or transportation, or after both storage and transportation.

[0020] (definition) It is understood that the section or subsection headings used herein are for organizational purposes only and are not to be construed as limiting or isolating, or both limiting and isolating, the subject matter of the described invention.

[0021] Before describing the compositions and methods of the present invention, it should be understood that this disclosure is not limited to the specific compositions, methods, and experimental conditions described, and as such, the compositions, methods, and conditions may vary. It should also be understood that the technology used herein is for the purpose of illustrating specific embodiments only, and is not intended to be limiting, since the scope of the present disclosure is limited only by the appended claims.

[0022] The practice of the present technology will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, within the skill of the art. See, e.g., Green and Sambrook eds. (2012) Molecular Cloning: A Laboratory Manual, 4 th edition;the series Ausubel et al.eds.(2015)Current Protocols in Molecular Biology;the series Methods in Enzymology(Academic Press,Inc.,NY);MacPherson et al.(2015)PCR 1:A Practical Approach(IRL Press at Oxford University Press);MacPherson et al.(1995)PCR 2:A Practical Approach;McPherson et al. al. (2006) PCR: The Basics (Garland Science); Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Greenfield ed. (2014) Antibodies, A Laboratory Manual; Freshney (2010) Culture of Animal Cells: A Manual of Basic Technique, 6 thedition;Gait ed.(1984)Oligonucleotide Synthesis;U.S.Pat.No.4,683,195;Hames and Higgins eds.(1984)Nucleic Acid Hybridization;Anderson(1999)Nucleic Acid Hybridization;Herdewijn ed.(2005)Oligonucleotide Synthesis:Methods and Applications;Hames and Higgins eds.(1984)Transcription and Translation;Buzdin and Lukyanov ed.(2007)Nucleic Acids Hybridization:Modern Applications;Immobilized Cells and Enzymes(IRL Press(1986));Grandi ed.(2007)In Vitro Transcription and Translation Protocols,2 nd edition;Guisan ed.(2006)Immobilization of Enzymes and Cells;Perbal(1988)A Practical Guide to Molecular Cloning,2 nd edition;Miller and Calos eds,(1987)Gene Transfer Vectors for Mammalian Cells(Cold Spring Harbor Laboratory);Makrides ed.(2003)Gene Transfer and Expression in Mammalian Cells;Mayer and Walker eds.(1987)Immunochemical Methods in Cell and Molecular Biology(Academic Press,London);Lundblad and Macdonald eds.(2010)Handbook of Biochemistry and Molecular Biology,4 thedition; and Herzenberg et al. eds. (1996) Weir's Handbook of Experimental Immunology, 5th edition. th edition; and more recent editions of each of the foregoing available at the time of filing.

[0023] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods, one or more steps, and both one or more methods and one or more steps of the type described herein, as would become apparent to one of skill in the art upon reading this disclosure and so forth.

[0024] The term "comprising" is used interchangeably with "including," "containing," or "characterized by" and is an inclusive or open-ended term that does not exclude additional, unrecited elements or method steps. The phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The phrase "consisting essentially of" limits the scope of the claim to those materials or steps specified and that do not materially affect the basic and novel characteristics of the claimed invention. The present disclosure contemplates composition and method embodiments that fall within the scope of each of these phrases. Thus, a composition or method that includes a recited element or step contemplates specific embodiments in which the composition or method consists essentially of or consists of the element or step.

[0025] "Optionally" or "as needed" means that the subsequently described circumstance may or may not occur, and thus the description includes instances where the circumstance occurs and instances where the circumstance does not occur.

[0026] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted as an alternative ("or").

[0027] All numerical designations, including ranges (e.g., pH, temperature, time, concentration, and molecular weight), are approximate and may vary (+) or (-) by increments of 1.0 or 0.1, as appropriate, or by + / - 15%, or 10%, or 5%, or 2%. It is to be understood, although not always expressly stated, that all numerical designations are preceded by the term "about." It is also to be understood, although not always expressly stated, that the reagents described herein are merely exemplary and that equivalents of such reagents are known in the art.

[0028] The term "subject" as used herein refers to any individual or patient on whom the subject method is performed. Generally, the subject is a human, but as will be appreciated by those skilled in the art, the subject may be an animal. Thus, other animals, including mammals such as rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, farm animals (including cows, horses, goats, sheep, pigs), sport animals, pets, etc., and primates (including monkeys, chimpanzees, orangutans, and gorillas), are included within the definition of a subject.

[0029] As used herein, the term "animal" refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term "mammal" includes both human and non-human mammals.

[0030] As used herein, the terms "sample" and "biological sample" refer to any sample suitable for the methods provided by the present disclosure. In one embodiment, the biological sample of the present disclosure is a tissue sample (e.g., a biopsy specimen, such as a sample derived from a needle biopsy (i.e., a biopsy sample)).

[0031] As used herein, the terms "reduce" and "inhibit" are used together, as it is recognized that in some cases, the decrease may be less than the detection level of a particular assay (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% of the basal level). As such, it may not always be clear whether an expression level or activity is "reduced" to less than the detection level of an assay or is "inhibited" completely. That said, it will be clearly determinable after treatment according to the present method.

[0032] Chondrocytes, as used herein, refer to cells in cartilage. Unaffected chondrocytes generate and maintain the cartilaginous matrix, which is mainly composed of collagen and proteoglycan (PG). Mesenchymal stem cells are the naturally occurring precursors of chondrocytes. Other stem cells may induce chondrocytes under suitable conditions, non-limiting examples of which are provided in the examples.

[0033] As used herein, the term "tissue" refers to a level of cellular organization between cells and complete organs. A tissue comprises, consists essentially of, or further consists of a set of cells (similar or dissimilar) of the same origin that together perform a specific function as needed, and their extracellular matrix (such as epithelial tissue, connective tissue, muscle tissue, nervous tissue, osteochondral tissue, or fibrocartilaginous tissue). In some embodiments, tissues as used herein are animal tissues, such as human tissues. In other embodiments, tissues are non-human animal tissues.

[0034] As used herein, the term "organ" refers to a group of tissues that structurally form a functional unit specialized to perform a particular function (such as the kidney, liver, heart, lung, bone, or eye). In some embodiments, an organ as used herein is an animal organ (such as a human organ). In other embodiments, the tissue is a non-human animal organ.

[0035] As used herein, osteochondral tissue comprises, consists essentially of, or further consists of articular cartilage and subchondral bone regions. In some embodiments, osteochondral tissue comprises, consists essentially of, or further consists of an osteochondral core. Additionally or alternatively, osteochondral tissue is obtained from an animal (human or non-human) and optionally trimmed. In further embodiments, osteochondral tissue has or comprises a bone to cartilage thickness ratio of about 20:1 to about 1:10. In yet further embodiments, for example, osteochondral tissue has or comprises a bone to cartilage thickness ratio of about 4:1.

[0036] As used herein, articular cartilage refers to the hyaline cartilage on the articular surfaces of bones, which lines the articular cavity of a synovial joint and is filled with synovial fluid produced by the synovial membrane lining the walls of the joint cavity.

[0037] As used herein, fibrocartilage tissue refers to a mixture of white fibrous tissue and cartilage tissue in various ratios. (For example, fibrocartilage syndesmosis (such as pubic symphysis, annulus fibrosis of intervertebral disc, or manubrium syndesmosis)); labrum of shoulder joint; acetabular labrum of hip joint; meniscus (including but not limited to medial and lateral meniscus of knee joint); or where tendons and ligaments attach to bone. In some embodiments, fibrocartilage tissue disclosed herein refers to meniscus (for example, medial or lateral meniscus or both).

[0038] As used herein, tendon refers to fibrous connective tissue that connects muscle to bone and can withstand tension. In some embodiments, the tendons disclosed herein refer to the patellar tendon. Additionally or alternatively, the tendons disclosed herein refer to the Achilles tendon. In some embodiments, the tendons used herein are human tendons. In other embodiments, the tendons used herein are tendons of non-human animals.

[0039] As used herein, the cornea refers to the transparent front part of the eye that covers the iris, pupil, and anterior chamber. In some embodiments, the cornea as used herein refers to the cornea with the vitreous body, a transparent gel that fills the space between the lens and the retina of the human and other vertebrate eye. In some embodiments, the cornea as used herein is a human cornea. In other embodiments, the cornea as used herein is a non-human animal cornea.

[0040] As used herein, the terms "encapsulated," "embedded," or grammatical variations thereof refer to an orientation or position of a structure or component such that it is at least partially (e.g., at least 99%, or at least 98%, or at least 97%, or at least 96%, or at least 95%, or at least 90%, or at least 85%, or at least 80%, or at least 75%, or at least 70%, or at least 65%, or at least 60%, or at least 55%, or at least 50%, or at least 45%, or at least 40%, or at least 35%, or at least 30%, or at least 25%, or at least 20%, or at least 15%, or at least 10%, or at least 5%) of the surface of the structure), and in some embodiments completely (e.g., about 100% of the surface of the structure), surrounded (directly or indirectly) by one or more other structures or one or more other components. In various embodiments, the term "encapsulation" or grammatical variants thereof refers to the orientation or position of a structure or component such that it is completely surrounded by one or more other structures or one or more other components. In some embodiments, the term "encapsulation" refers to being at least partially or completely disposed within. One non-limiting example is an alginate gel, or a tissue surrounded by an alginate gel and a semipermeable membrane, referred to herein as an encapsulated tissue.

[0041] The term "exposure" or grammatical variants thereof refers to contact (meaning direct or indirect interaction between two or more components). In various embodiments, the term "exposure" refers to envelopment or immersion. One non-limiting example as used herein is exposure of tissue (such as tissue encapsulated in alginate gel, or tissue encapsulated in alginate gel and semipermeable membrane, or tissue without any encapsulation) to culture medium via a method that includes, consists essentially of, or consists of contacting the tissue with culture medium (e.g., enveloping or immersing the tissue in culture medium). A further non-limiting example is exposure of tissue in a chamber (such as tissue encapsulated in alginate gel, or tissue encapsulated in alginate gel and a semipermeable membrane, or tissue without any encapsulation) to culture medium via a method that includes, consists essentially of, or consists of adding culture medium to the chamber containing the tissue (e.g., replacing at least a portion (e.g., about 50%, about 60%, about 70%, about 80%, about 90%, or more) or complete (i.e., about 100%) of the solution initially present in the chamber, if any).

[0042] As used herein, the term storage or grammatical variants thereof refers to maintaining the viability of the stored cells, tissues, organs, or other components (e.g., at about 100%, or at least 99%, or at least 98%, or less than the viability prior to storage) for a certain period of time (e.g., at least 48 hours, at least 3 days, at least 5 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks or longer). "Storage" refers to storing tissue or organs with a viability of at least 97%, or at least 96%, or at least 95%, or at least 90%, or at least 85%, or at least 80%, or at least 75%, or at least 70%, or at least 65%, or at least 60%, or at least 55%, or at least 50%, or at least 45%, or at least 40%, or at least 35%, or at least 30%, or at least 25%, or at least 20%, or at least 15%, or at least 10%, or at least 5%. Methods for assessing the viability of tissues or organs are available to those skilled in the art (see, for example, the methods disclosed in the Examples).

[0043] As used herein, culture medium refers to a liquid substance used in the culture of cells or tissues or organs, for example, to support cell growth, maintain cell viability, or maintain tissue viability. In some embodiments, the culture medium comprises, consists essentially of, or further consists of: one or more amino acids, or one or more carbohydrates as an energy source, or one or more trace elements, or one or more vitamins, or one or more salts, or one or more possible additional components (e.g., to affect cell growth and / or productivity and / or product quality), or any combination thereof. In some embodiments, the culture medium comprises, consists essentially of, or further consists of Dulbecco's Modified Essential Medium (DMEM), or Ham's F-12 Medium (F12), or Roswell Park Memorial Institute (RPMI) 1640 Medium, or any combination thereof. In a further embodiment, the culture medium comprises, consists essentially of, or further consists of DMEM / F12 (a 1:1 mixture of DMEM and F12).

[0044] In some embodiments, complete medium refers to a culture medium that further comprises one or more additional components that may contribute to cell growth (such as one or more growth factors, one or more hormones, one or more proteins, one or more serum, one or more serum substitutes, etc.). In further embodiments, complete medium refers to a culture medium that further comprises serum, such as fetal bovine serum (FBS). Additionally or alternatively, complete medium further comprises about 1% to about 20% serum (such as about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10%, or about 11%, or about 12%, or about 13%, or about 15%, or about 20%).

[0045] As used herein, semipermeable membranes, also referred to as dialysis membranes, refer to a type of biological or synthetic polymeric membrane that allows certain particles (such as molecules or ions) to pass through it by osmosis, or sometimes by more specialized processes that facilitate diffusion, passive transport, or active transport, thereby allowing the passage of particles by size or molecular weight.

[0046] For example, semipermeable membranes have pores of various sizes that allow particles smaller than the pore size to pass through. In some embodiments, the pore size of the semipermeable membrane is given by the diameter of the membrane pores. In further embodiments, the pore size of the semipermeable membrane is given by the average diameter of the membrane pores. In some embodiments, the semipermeable membranes disclosed herein have a diameter of at least about 250 picometers (referred to herein as "pm"), at least about 300 pm, at least about 500 pm, at least about 750 pm, at least about 1 nm, at least about 20 nm, at least about 50 nm, at least about 100 nm, at least about 150 nm, at least about 200 nm, at least about 300 nm, at least about 400 nm, at least about 500 nm, at least about 600 nm, at least about 700 nm, at least about 800 nm, at least about 900 nm, at least about 1 μm, at least about 20 μm, at least about 50 μm, at least about 100 μm, at least about 150 μm, at least about 200 μm, at least about 300 μm, at least about 400 μm, at least about 500 μm, at least about 600 μm, at least about 700 μm, at least about 800 μm, at least about 9 00 μm, at least about 1000 μm, at least about 1100 μm, at least about 1200 μm, at least about 1300 μm, at least about 1400 μm, at least about 1500 μm, at least about 1600 μm, at least about 1700 μm, at least about 1800 μm, at least about 1900 μm, at least about 2000 μm, at least about 2100 μm, at least about 2200 μm, at least about 2300 μm, at least about 2400 μm, μm, at least about 2500 μm, at least about 2600 μm, at least about 2700 μm, at least about 2800 μm, at least about 3900 μm, at least about 3000 μm, at least about 4000 μm, at least about 5000 μm, at least about 6000 μm, at least about 7000 μm, at least about 8000 μm, at least about 9000 μm, at least about 10 mm, or more, and ranges of pore sizes therebetween.Additionally or alternatively, the semipermeable membranes disclosed herein may be up to about 300 pm, up to about 500 pm, up to about 750 pm, up to about 1 nm, up to about 20 nm, up to about 50 nm, up to about 100 nm, up to about 150 nm, up to about 200 nm, up to about 300 nm, up to about 400 nm, up to about 500 nm, up to about 600 nm, up to about 700 nm, up to about 800 nm, up to about 900 nm, up to about 1 μm, up to about 20 μm, up to about 50 μm, up to about 100 μm, up to about 150 μm, up to about 200 μm, up to about 300 μm, up to about 400 μm, up to about 500 μm, up to about 600 μm, up to about 700 μm, up to about 800 μm, up to about 900 μm, up to about 1000 μm up to about 1100 μm, up to about 1200 μm, up to about 1300 μm, up to about 1400 μm, up to about 1500 μm, up to about 1600 μm, up to about 1700 μm, up to about 1800 μm, up to about 1900 μm, up to about 2000 μm, up to about 2100 μm, up to about 2200 μm, up to about 2300 μm, up to about 2400 μm, The pore size is up to about 2500 μm, up to about 2600 μm, up to about 2700 μm, up to about 2800 μm, up to about 3900 μm, up to about 3000 μm, up to about 4000 μm, up to about 5000 μm, up to about 6000 μm, up to about 7000 μm, up to about 8000 μm, up to about 9000 μm, up to about 10 mm, or more. In some embodiments, the semipermeable membranes disclosed herein have a pore size of about 0.22 μm to about 1000 μm, and ranges or sizes therebetween, such as about 0.22 μm to about 100 μm and ranges therebetween, or about 100 μm to about 1000 μm and ranges therebetween. In some embodiments, the semipermeable membranes disclosed herein have pore sizes of about 100 μm to about 1000 μm and ranges therebetween (e.g., 100 μm to about 200 μm, 100 μm to about 300 μm, 100 μm to about 400 μm, 100 μm to about 500 μm, 100 μm to about 600 μm, 100 μm to about 700 μm, 100 μm to about 800 μm, or about 100 μm to about 900 μm). In various embodiments, one of skill in the art will select a pore size for the semipermeable membranes disclosed herein such that one or more of the components of the culture medium (e.g., complete medium) can pass through the membrane, but not one or more of the cells, tissues (e.g., encapsulated tissues), or alginate gels.

[0047] In some embodiments, molecular weight cut-off (MWCO) is used to describe the permeability of a semipermeable membrane and refers to the molecular weight (such as Daltons (Da) or kilodaltons (kDa)) at which more than 90% of the particles with the lowest molecular weight are retained by the membrane. In various embodiments, the MWCO of the semipermeable membrane is selected or determined by selecting the pore size. In other embodiments, the pore size of the semipermeable membrane is selected or determined by selecting the MWCO. In various embodiments, the MWCO is selected based on the solution and its concentration used herein (such as a calcium solution, a barium solution, or a chelating solution). In various embodiments, the skilled artisan selects the MWCO of the semipermeable membrane disclosed herein such that one or more of the components of the culture medium (such as a complete medium) can pass through the membrane, but one or more of the cells, tissues (e.g., encapsulated tissues), or alginate gels cannot pass through. In some embodiments, the MWCO of the semipermeable membranes disclosed herein is from about 1 kDa to about 10,000,000 kDa and ranges therebetween (eg, from about 1 kDa to about 2,000,000 kDa).In some embodiments, the MWCO of the semipermeable membranes disclosed herein is at least about 1 kDa, at least about 10 kDa, at least about 50 kDa, at least about 100 kDa, at least about 200 kDa, at least about 300 kDa, at least about 400 kDa, at least about 500 kDa, at least about 600 kDa, at least about 700 kDa, at least about 800 kDa, at least about 900 kDa, at least about 1,000 kDa, At least about 2,000 kDa, at least about 3,000 kDa, at least about 4,000 kDa, at least about 5,000 kDa, at least about 6,000 kDa, at least about 7,000 kDa, at least about 8,000 kDa, at least about 9,000 kDa, at least about 10,000 kDa, at least about 20,000 kDa, at least about 30,000 kDa, at least about 40,000 kDa, at least about 50,000 kDa a, at least about 60,000 kDa, at least about 70,000 kDa, at least about 80,000 kDa, at least about 90,000 kDa, at least about 100,000 kDa, at least about 200,000 kDa, at least about 300,000 kDa, at least about 400,000 kDa, at least about 500,000 kDa, at least about 600,000 kDa, at least about 700,000 kDa, at least about 800,000 kDa kDa, at least about 900,000 kDa, at least about 1,000,000 kDa, at least about 2,000,000 kDa, at least about 3,000,000 kDa, at least about 4,000,000 kDa, at least about 5,000,000 kDa, at least about 6,000,000 kDa, at least about 7,000,000 kDa, at least about 8,000,000 kDa, or at least about 9,000,000 kDa.Additionally or alternatively, the MWCO of the semipermeable membranes disclosed herein can be up to about 50 kDa, up to about 100 kDa, up to about 200 kDa, up to about 300 kDa, up to about 400 kDa, up to about 500 kDa, up to about 600 kDa, up to about 700 kDa, up to about 800 kDa, up to about 900 kDa, up to about 1,000 kDa, up to about 2,000 kDa, up to about 3,000 kDa. up to about 4,000kDa, up to about 5,000kDa, up to about 6,000kDa, up to about 7,000kDa, up to about 8,000kDa, up to about 9,000kDa, up to about 10,000kDa, up to about 20,000kDa, up to about 30,000kDa, up to about 40,000kDa, up to about 50,000kDa, up to about 60,000kDa, up to about 70,000kDa Up to about 80,000kDa, up to about 90,000kDa, up to about 100,000kDa, up to about 200,000kDa, up to about 300,000kDa, up to about 400,000kDa, up to about 500,000kDa, up to about 600,000kDa, up to about 700,000kDa, up to about 800,000kDa, up to about 900,000kDa, up to about 1,000,000kDa up to about 2,000,000 kDa, up to about 3,000,000 kDa, up to about 4,000,000 kDa, up to about 5,000,000 kDa, up to about 6,000,000 kDa, up to about 7,000,000 kDa, up to about 8,000,000 kDa, up to about 9,000,000 kDa, up to about 10,000,000 kDa or more. In some embodiments, the MWCO of the semipermeable membranes disclosed herein is about 10 kDa, or about 20 kDa, or about 30 kDa, or about 40 kDa, or about 50 kDa, or about 60 kDa, or about 70 kDa, or about 80 kDa, or about 90 kDa, or about 100 kDa, or about 200,000 kDa.

[0048] In some embodiments, the semipermeable membrane may be a partially semipermeable membrane or a completely semipermeable membrane. For example, a partially semipermeable membrane may be able to selectively transport certain fluids through. In contrast, a completely semipermeable membrane may transport fluids through without restriction, but may prevent transport through alginate gel and tissue. Such a completely semipermeable membrane may comprise, consist essentially of, or consist of a mesh (e.g., medical grade mesh). Thus, in some embodiments, mesh opening size refers to the pore size of the mesh or its average, and may be used interchangeably with membrane pore size.

[0049] In some embodiments, the semipermeable membrane has an open area ratio (also referred to herein as mesh open area ratio) of about 1% to about 50% and ranges or percentages therebetween. In some embodiments, the semipermeable membrane has a mesh open area ratio of at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, or at least about 45%. Additionally or alternatively, the semipermeable membrane has an open area ratio of less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, less than about 75%, less than about 80%, less than about 85%, less than about 90%, or less than about 95%. In some embodiments, the semipermeable membrane has a mesh open area ratio of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%.

[0050] In some embodiments, the open area of ​​a membrane refers to the percentage or ratio of the open area to the total surface of the membrane.

[0051] As used herein, percentage (%) is used to indicate the concentration of a component in a mixture (solid or liquid). In some embodiments, percentage (%) refers to mass or weight ratio. In other embodiments, percentage (%) refers to volume ratio. In still other embodiments, percentage (%) refers to molar ratio. In some embodiments, x% of a component in a solution refers to x grams of a component in 100 mL of solution.

[0052] As used herein, chelating solution refers to a solution containing a chelating agent, such as a calcium chelating agent. Non-limiting examples of chelating agents include edetic acid, citric acid, edetate disodium anhydrous, edetate calcium disodium anhydrous, ethylenediaminetetraacetic acid (EDTA), or ethyleneglycol-bis(β-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). In some embodiments, the chelating agent is sodium citrate or ethylenediaminetetraacetic acid (EDTA). In some embodiments, the chelating solution is a sodium citrate solution or an EDTA solution, or a mixture thereof.

[0053] In some embodiments, the solution is an aqueous solution, which may include additional components (such as saline).

[0054] Inflammatory response, used interchangeably with inflammation herein, refers to the immune response that occurs when tissue is injured by bacteria, trauma, toxins, heat, or any other cause (such as ex vivo storage). Tissues release inflammatory factors (such as histamine, bradykinin, serotonin, cytokines, nitric oxide, etc.) during an inflammatory response. As used herein, cytokines (such as interleukin (IL)-1, IL-1β, IL-6, and tumor necrosis factor-α (TNFα)) are produced by immune cells (such as activated macrophages), tissues or organs, or chondrocytes.

[0055] As used herein, the term "allograft" refers to a tissue transplant from a donor that is the same species as the recipient but is not genetically identical. Allograft tissue can be removed from the donor by techniques known in the art. For example, general sterile surgical procedures or other physical interventions of allografts can include, but are not limited to, removal, excision, amputation, transplantation, microsurgery, general surgery, laser surgery, robotic surgery, or autopsy.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, the preferred methods and materials are described herein. EMBODIMENTS FOR CARRYING OUT THE DISCLOSURE

[0057] The source of the tissue or allograft can be cells, tissues, or organs from all organism types, including but not limited to human, porcine, ovine, bovine, canine, and equine. In one embodiment, the source of the tissue or allograft is human. Potential tissue or allograft sources can include, but are not limited to, eye, brain, heart, kidney, liver, intestine, bone, cartilage, skin, lung, thyroid, stomach, ligament, tendon tissue, or any other source of tissue or cells that may require transplantation. In various embodiments, the allograft or tissue can include bone or cartilage or meniscus tissue of the spine, scapula, humerus, radius, ulna, pelvis, femur, tibia, fibula, patella, talus, phalanges, or temporomandibular joint, or any combination thereof. In various embodiments, the allograft or tissue can be osteochondral tissue. Although the descriptions herein may refer to allograft tissue, those of skill in the art will recognize that other tissues may be used in the methods described herein.

[0058] In some embodiments, the tissue encapsulated by the gel, semipermeable membrane, or both the gel and semipermeable membrane has a dimension in any direction of the tissue that is greater than about 1 millimeter (mm) (e.g., at least about 2 mm, at least about 3 mm, at least about 4 mm, at least about 5 mm, at least about 6 mm, at least about 7 mm, at least about 8 mm, at least about 9 mm, at least about 1 centimeter (cm), or more). Additionally or alternatively, the tissue encapsulated by the gel, semipermeable membrane, or both the gel and semipermeable membrane has a dimension in any direction of the tissue that is less than about 20 cm (e.g., less than about 15 cm, less than about 10 cm, less than about 5 cm, less than about 4 cm, less than about 3 cm, less than about 2 cm, less than about 1 cm, or less).

[0059] Osteochondral allografts (OCA) are currently stored at 4°C and used within 28 days of donor harvest. The disease testing protocol requires 2 weeks to implement, limiting the window of opportunity to 14 days, thereby severely limiting availability to potential recipients. This study was undertaken to find improved storage conditions that would improve the shelf life of OCA and potentially improve clinical outcomes in its clinical use.

[0060] High-purity alginate (UPAL) is highly purified and has low endotoxin toxicity (less than 10,000 times lower than conventional alginate). In some embodiments, UPAL is purified from conventional alginate by a method comprising, consisting essentially of, or consisting of adding acid to conventional alginate to precipitate the alginate and isolating the insoluble alginate. See, e.g., Igarashi, et al., Cartilage 2012:70. As a scaffolding material, UPAL has been shown to enhance the repair of osteochondral defects in animal models (Igarashi, et al., Cartilage 2012:70; and Baba, et al., Am J Sports Med. 2018; 1970) and, as an injectable material into joints, to delay the progression of osteoarthritis (Tsukuda, et al., J Biomed Mater Res A. 2015:3441). Since alginate is similar to articular cartilage matrix and is preferred for three-dimensional culture as an embedding material in the form of alginate beads (Chiba, et al., Spine J. 2007:694), it is suggested that UPAL can be expected to act as an optimal storage medium to prevent OCA degradation. Therefore, it was hypothesized that modifying the current storage conditions using UPAL would improve the quality of OCA after long-term storage. The specific objective of this study was to evaluate the effect of UPAL on cell viability and cartilage matrix status after 4 weeks of storage of OCA obtained from porcine knees.

[0061] Alginic acid, also called algin, is a polysaccharide widely distributed in the cell walls of brown algae, which exhibits hydrophilicity and forms a viscous gum when hydrated. Its salts with metals such as sodium and calcium are known as alginates. The sugars in alginates consist of guluronate (G), mannuronate (M), or guluronate-mannuronate blocks, and the strength of the gel formed is determined by the difference in the ratio of sugars. In some embodiments, the M / G ratio of the alginic acid and / or its salts used is usually in a range selected from about 0.2 to about 4.0, in the range of about 0.4 to about 3.0, and in the range of about 0.5 to about 3.0, and in between. Polymeric substances derived from natural products are generally aggregates of molecules of various molecular weights, and are therefore measured as a molecular weight distribution having a certain range. The molecular weight of alginic acid can be measured by standard calculation methods (ASTM F2605-16 (2016), ASTM International Publication) by SEC-MALS (size exclusion chromatography with multi-angle light scattering detector). In some embodiments, the molecular weight of the alginic acid and / or its salt used is usually in the range of about 10,000 to about 2,000,000, about 15,000 to about 1,500,000, about 20,000 to about 1,000,000, or about 25,000 to about 500,000, and the ranges selected therebetween, as measured by the SEC-MALS method.

[0062] It is well known that high chondrocyte viability leads to better in vivo function (Pallante, et al., Am J Sports Med. 2012:1814). Encapsulation of OCA with UPAL gel significantly increased chondrocyte viability in the surface section compared to other groups. The viability and total thickness of the middle section in the UPAL gel group also tended to be higher than in other groups. These results are consistent with the results that the surface section was fragile and that the cell viability of the surface section is sensitive to its storage conditions (Pallante, et al., Am J Sports Med. 2009:24S). Encapsulation with UPAL gel did not show any structural deformation during the storage period. In addition, the maintenance of cell viability and matrix content in alginate gel, as well as polyglycan (PG) synthesis, is supported by the low total nitric oxide (NO) concentration in the storage medium. Embedding in alginate gel may also provide some protection to the tissue from mechanical damage during transportation to the clinical site. The use of UPAL gel encapsulation could be effective in improving the quality of OCA after 28 days of storage. Therefore, the use of UPAL gel during storage may improve clinical outcomes after osteochondral allograft transplantation and minimize cell death during the storage period.

[0063] As illustrated in the examples disclosed herein, embedding (i.e., encapsulation) of osteochondral allografts (OCAs) in UPAL gel improves cell viability and matrix composition after 28 days or 6 weeks of storage. Matrix synthesis is preserved compared to OCA stored in other control methods. Furthermore, cell viability was higher with alginate than without alginate (control culture). Cell viability was significantly higher when graft tissue was stored with UPAL gel than tissue stored in medium alone. The UPAL membrane group, with tissue embedded in UPAL gel and semipermeable membrane, had higher cell viability than all other test conditions. Without wishing to be bound by theory, the conditions in the UPAL membrane group provide better access to nutrients through the UPAL gel and membrane, and also provide physical protection during storage. Furthermore, embedding in a single alginate gel provides some protection to the tissue from mechanical damage during storage. At the end of the storage period, the tissue containing the alginate gel is removed from the device or system by replacing the medium with a chelating solution. Any remaining alginate gel around the tissue is dissolved by exposure to a chelating solution (such as EDTA or sodium citrate); dissolution may be performed in the operating room immediately prior to implantation. Additionally, embedding in alginate gel provides some protection to the tissue from mechanical damage during transport of the tissue to the clinical site.

[0064] Although there are several methods available for storing cartilage tissue, the methods, devices, or systems disclosed herein have advantage(s) over other methods, for example, during transportation. For example, such advantages over liquid storage systems may include, but are not limited to, improved tissue preservation conditions by embedding in alginate gel; protection of the tissue from shock, vibration, or shaking during transportation of the tissue to the hospital, thereby preventing tissue damage due to transportation; improved cell viability and suppression of inflammatory factors (such as cytokines and nitric oxide); and storage and transportation stages in one enclosed system. Thus, preserving allograft tissue by embedding in alginate gel and an optional semipermeable membrane improves the maintenance of cells in the tissue. In some embodiments, the semipermeable membrane may be a partial or complete semipermeable membrane. For example, a partial semipermeable membrane may be capable of selectively transporting certain fluids through it. In contrast, a complete semipermeable membrane may transport fluids through it without restriction, but may prevent transport through the alginate gel and the tissue. Such a complete semi-permeable membrane may comprise (may comprise, consist essentially of, or consist of) a mesh (e.g., a medical grade mesh). Such modification of storage conditions improves the quality of the tissues disclosed herein, such as osteochondral allografts.

[0065] Thus, in one aspect, the disclosure provides a method, system, or device for tissue preservation. The method includes encapsulating tissue in UPAL gel and storing the encapsulated tissue, for example, for about 24 hours to about 8 weeks prior to implantation. In various embodiments, the tissue is osteochondral tissue. In various embodiments, the tissue is a tissue disclosed herein other than osteochondral tissue.

[0066] An unexpected advantage of the procedures disclosed herein in any aspect or embodiment is that the tissue sample can remain viable and sterile for an extended period of time compared to prior art methods. For example, typically in the prior art, upon removal of the allograft from the donor, the tissue was stored on ice or at approximately 4°C. Tissue prepared according to this method tended to remain viable for approximately 21-28 days. However, the procedures described herein surprisingly and unexpectedly increase the viability of the allograft tissue. Tissue prepared and stored according to the procedures described herein remains viable for an extended period of time compared to storage at 4°C. By extended period of time, it is meant at least about 7-100 days, at least about 20-80 days, or at least about 29-70, 40-70, 50-70, or 60-70 days. In various embodiments, the encapsulated tissue is stored for about 1 day, 2 days, 7 days, 14 days, 28 days, 4 weeks, 6 weeks, or 8 weeks prior to use (such as implantation). In various embodiments, at least about 40% of the cells of the tissue remain viable after storage, compared to the viability of the cells of the tissue on day 0.

[0067] In another aspect, the present disclosure provides a method for storing the osteochondral tissue or other tissue(s) or organ(s) disclosed herein, for example, prior to transplantation. The method includes (e.g., comprises, consists essentially of, or consists of) the steps of obtaining the osteochondral tissue or other tissue(s) or organ(s) to be transplanted, placing the obtained osteochondral tissue or other tissue(s) or organ(s) in a semipermeable membrane chamber containing an alginate solution, placing the semipermeable membrane chamber in a culture chamber, and adding a calcium solution or a barium solution or both to the culture chamber, thereby encapsulating the osteochondral tissue or other tissue(s) or organ(s) in the alginate gel, injecting a culture medium into the culture chamber, and optionally placing the culture chamber in a storage container. In various embodiments, the encapsulated osteochondral tissue remains viable for transplantation up to 8 weeks after obtaining.

[0068] Provided herein are methods, materials, formulations, systems, or devices for preserving allograft tissue embedded in an alginate gel and an optional semi-permeable membrane that improves cell retention in the tissue. In some embodiments, the methods, materials, formulations, systems, or devices also provide some protection to the tissue from mechanical damage during transport to the clinical site.

[0069] In one aspect, the present disclosure provides a method for preserving osteochondral tissue. In another aspect, the present disclosure provides a method for preserving other tissue(s) or organ(s) disclosed herein. In some embodiments, the tissue(s) or organ(s) is selected from osteochondral tissue (such as a trimmed osteochondral core having a bone to cartilage thickness ratio of about 20:1 to about 1:10 and ranges therebetween, for example, about 4:1), articular cartilage without any subchondral bone, fibrocartilage tissue (such as a meniscus, for example, the medial or lateral meniscus or both), tendon (such as the patellar tendon or Achilles tendon), cornea, or cornea with vitreous body.

[0070] In some embodiments, the method comprises (e.g., comprises, or consists essentially of, or further consists of) encapsulating osteochondral tissue or other suitable tissue(s) or organ(s) in an alginate gel and exposing the gel-encapsulated osteochondral tissue or other suitable tissue(s) or organ(s) to culture medium. In some embodiments, the gel-encapsulated tissue is placed in culture medium for a period selected from about 24 hours to about 8 weeks, and ranges therebetween, about 28 days, at least about 6 weeks, for example, about 24 hours to 8 weeks, prior to implantation. Additionally or alternatively, the method comprises (e.g., comprises, or consists essentially of, or further consists of) encapsulating osteochondral tissue or other suitable tissue(s) or organ(s) in an alginate gel and exposing the encapsulated osteochondral tissue or other suitable tissue(s) or organ(s) to culture medium for at least about 2 weeks (such as at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, or longer). In some embodiments, the method includes encapsulating osteochondral tissue or other suitable tissue(s) or organ(s) in an alginate gel and incubating the encapsulated osteochondral tissue or other suitable tissue(s) or organ(s) in a culture medium for about 2 weeks to about 3 weeks, about 2 weeks to about 4 weeks, about 2 weeks to about 5 weeks, about 2 weeks to about 6 weeks, about 2 weeks to about 7 weeks, about 2 weeks to about 8 weeks, about 2 weeks to about 9 weeks, about 2 weeks to about 10 weeks, about 3 weeks to about 4 weeks, about 3 weeks to about 5 weeks, about 3 weeks to about 6 weeks, about 3 weeks to about 7 weeks, about 3 weeks to about 8 weeks, about 3 weeks to about 9 weeks, about 2 weeks to about 10 weeks, about 3 ... The method includes (e.g., includes, or consists essentially of, or further consists of) a step of exposing for a period of about 8 weeks, about 3 weeks to about 9 weeks, about 3 weeks to about 10 weeks, about 4 weeks to about 5 weeks, about 4 weeks to about 6 weeks, about 4 weeks to about 7 weeks, about 4 weeks to about 8 weeks, about 4 weeks to about 9 weeks, about 4 weeks to about 10 weeks, about 5 weeks to about 6 weeks, about 5 weeks to about 7 weeks, about 5 weeks to about 8 weeks, about 5 weeks to about 9 weeks, about 5 weeks to about 10 weeks, about 6 weeks to about 7 weeks, about 6 weeks to about 8 weeks, about 6 weeks to about 9 weeks, about 6 weeks to about 10 weeks, or a period of range between each of the aforementioned.

[0071] In various embodiments, the method further comprises encapsulating the tissue(s) or organ(s) in a semipermeable membrane. In various embodiments, the semipermeable membrane has a pore size of at least about 250 pm (such as about 0.22 μm to about 100 μm and ranges therebetween, about 0.22 μm to about 1000 μm and ranges therebetween, or about 100 μm to about 1000 μm and ranges therebetween). Additionally or alternatively, the semipermeable membrane has a molecular weight cut off (MWCO) of about 50 kDa or about 200,000 kDa. In some embodiments, the semipermeable membrane has a mesh openness of about 1% to about 50% and ranges or percentages therebetween (e.g., about 2%). In various embodiments, the tissue is encapsulated in the gel before, during, or after encapsulation in the semipermeable membrane.

[0072] In various embodiments, the alginate gel is a purified gel, or a highly purified alginate gel (UPAL). In various embodiments, the alginate gel comprises or consists essentially of about 0.5% to about 2.0% alginate and ranges or percentages therebetween (e.g., about 1.2% alginate). In various embodiments, UPAL is present in the gel at a concentration of about 0.5% to about 2.0% and ranges or percentages therebetween (e.g., about 1.2%).

[0073] Alginates are natural polysaccharides that contain 30-60% of brown algae (e.g. kelp). Alginates are heteropolymeric aggregates that contain, consist essentially of, or consist of D-mannuronic acid (M) and L-guluronic acid (G). The carboxyl groups composed of M and G have high ion exchange capacity and easily bind various cations. See examples of alginate moieties in Figure 7. The physical properties of alginates change in various ways depending on different types of cations. In particular, cross-linking of alginates by calcium ions results in the formation of water-insoluble hydrated gels. Alginates with an average molecular weight of 10 4 ~10 6Alginates are available in the Da range. The higher the molecular weight of the alginate, the higher the viscosity of the solution. Sodium alginate is a water-soluble, white, freeze-dried powder, while calcium alginate is a water-insoluble, white powder. The purification process of alginate from raw materials includes, consists essentially of, or consists of: drying and grinding; washing and swelling; extraction; filtration; solidification and precipitation; and drying of alginate (wet). It is widely applied in food (e.g., as a thickener, gelling agent, emulsifier, noodle quality improver, pet food, farmed fish feed, binder, and thickener, etc.), cosmetics, textile printing paste, etc., and pharmaceutical field (e.g., as a disintegrant for tablets, or as a gastroprotectant). Alginate is also the main component of impression materials used in industry. Alginate also has excellent cartilage formation ability.

[0074] Highly purified alginate (UPAL) has been developed. Conventional alginates have high endotoxin levels and cannot be used clinically except for oral administration or wound dressing. High endotoxin levels cause inflammatory responses in vivo. This adverse effect hinders the clinical application of alginate-based materials. Mochida has developed a highly purified alginate with very low endotoxin levels called UPAL. UPAL eliminates the inflammatory response in vivo caused by endotoxin, making clinical application of alginate-based materials possible. For example, UPAL gel enhances cell proliferation of bone marrow stromal cells (BMSCs). See, for example, Igarashi, Iwasaki, et al. JBMR A, 2010. Various tests have revealed that UPAL gel promotes the regeneration of hyaline-like cartilage. Clinical studies are being conducted with volunteers who wish to use sodium alginate in the field of regenerative medicine or cell therapy.

[0075] In various embodiments, alginates (ALGs) as used herein are of various molecular weights (10 4 ~10 6The molecular weight of the alginate is 0.01 to 0.01 Da, and values ​​therebetween. The higher the molecular weight of the alginate, the harder and more elastic the alginate gel will be formed. One of skill in the art will be prepared to select an alginate of appropriate molecular weight in the practice of the methods disclosed herein.

[0076] In various embodiments, the culture medium is a complete medium.

[0077] In various embodiments, the encapsulated osteochondral tissue or other suitable tissue(s) or organ(s) are stored for about 28 days (about 4 weeks) prior to implantation or for about 6 weeks, etc. prior to implantation. In some embodiments, the encapsulated tissue(s) or organ(s) are stored for at least about 2 weeks (such as at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, or longer) prior to implantation. In some embodiments, the encapsulated tissue(s) or organ(s) may be incubated for, for example, about 2 weeks to about 3 weeks, about 2 weeks to about 4 weeks, about 2 weeks to about 5 weeks, about 2 weeks to about 6 weeks, about 2 weeks to about 7 weeks, about 2 weeks to about 8 weeks, about 2 weeks to about 9 weeks, about 2 weeks to about 10 weeks, about 3 weeks to about 4 weeks, about 3 weeks to about 5 weeks, about 3 weeks to about 6 weeks, about 3 weeks to about 7 weeks, about 3 weeks to about 8 weeks, about 3 weeks to about 9 weeks, The cornea may be stored for a period of time ranging from about 3 weeks to about 10 weeks, about 4 weeks to about 5 weeks, about 4 weeks to about 6 weeks, about 4 weeks to about 7 weeks, about 4 weeks to about 8 weeks, about 4 weeks to about 9 weeks, about 4 weeks to about 10 weeks, about 5 weeks to about 6 weeks, about 5 weeks to about 7 weeks, about 5 weeks to about 8 weeks, about 5 weeks to about 9 weeks, about 5 weeks to about 10 weeks, about 6 weeks to about 7 weeks, about 6 weeks to about 8 weeks, about 6 weeks to about 9 weeks, about 6 weeks to about 10 weeks, or any range between the foregoing. In various embodiments, the encapsulated cornea is stored for at least about 2 weeks prior to transplantation.

[0078] In various embodiments, the culture medium is not replenished after the exposure of the gel-encapsulated tissue to the culture medium. In various embodiments, the culture medium is replenished no more than once, or no more than twice, or no more than three times, or no more than four times, or no more than five times after the exposure of the gel-encapsulated tissue to the culture medium.

[0079] In various embodiments, the encapsulated tissue(s) or organ(s) are stored below 10°C, for example, at about 0°C to about 4°C and in any range therebetween, or at about 4°C.

[0080] In various embodiments, the encapsulated tissue(s) or organ(s) are not stored with artificially controlled CO2 levels, or artificially controlled O2 levels, or artificially controlled humidity, or any combination thereof.

[0081] In various embodiments, the method further comprises dissolving the alginate gel after exposure to the culture medium and, optionally, prior to implantation, for example, by a method comprising, consisting essentially of, or consisting of exposing the encapsulated tissue(s) or organ(s) to a chelating solution. In further embodiments, the chelating solution comprises, consists essentially of, or further consists of a sodium citrate solution or an ethylenediaminetetraacetic acid (EDTA) solution or a solution comprising both sodium citrate and EDTA.

[0082] In various embodiments, the method further comprises degrading the semipermeable membrane after exposure to the culture medium and prior to implantation.

[0083] In various embodiments, the encapsulated tissue(s) or organ(s) reduce the release of inflammatory factor(s) compared to tissue or organs stored without encapsulation, for example, in an alginate gel, or in a semipermeable membrane, or both in an alginate gel and a semipermeable membrane. In further embodiments, the inflammatory factor(s) comprises, consists essentially of, or further consists of one or more of cytokines, or nitric oxide, or both nitric oxide and one or more of cytokines.

[0084] In various embodiments, the encapsulated tissue(s) or organ(s) prior to implantation maintains at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least 90%, or at least 95% or higher cell viability prior to the encapsulation step or after the obtaining step (i.e., a step comprising, consisting essentially of, or consisting of obtaining the tissue(s) or organ(s) from a subject, etc.).

[0085] In various embodiments, prior to exposure to culture medium, the encapsulated tissue(s) or organ(s) are washed with a buffer solution, such as saline or culture medium.

[0086] In one aspect, the present disclosure provides a method for storing osteochondral tissue. In another aspect, the present disclosure provides a method for storing the tissue(s) or organ(s) disclosed herein. In some embodiments, the tissue(s) or organ(s) is selected from osteochondral tissue (such as a trimmed osteochondral core having a bone to cartilage thickness ratio of about 20:1 to about 1:10, for example, about 4:1), articular cartilage without any subchondral bone, fibrocartilage tissue (such as meniscus, for example, medial or lateral meniscus, or both medial and lateral meniscus), tendon (such as patellar tendon or Achilles tendon), cornea, or cornea with vitreous body, prior to implantation.

[0087] In some embodiments, the method comprises (e.g., comprises, or consists essentially of, or further consists of) the steps of optionally obtaining osteochondral tissue or other suitable tissue(s) or organ(s) to be transplanted, placing the obtained osteochondral tissue or other suitable tissue(s) or organ(s) in a semi-permeable membrane chamber containing an alginate solution, placing the semi-permeable membrane chamber in a culture chamber, and adding a calcium solution or a barium solution or both to the culture chamber, whereby the osteochondral tissue or other suitable tissue(s) or organ(s) are encapsulated in the alginate gel, injecting culture medium into the alginate gel or into the culture chamber or injecting alginate gel into the culture chamber, and optionally placing the culture chamber in a storage container.

[0088] In some embodiments, the encapsulated osteochondral tissue or other suitable tissue(s) or organ(s) remain viable for implantation for at least about 2 weeks (such as at least about 4 weeks or at least about 6 weeks). In some embodiments, the encapsulated osteochondral tissue or other suitable tissue(s) or organ(s) remain viable for implantation for up to 8 weeks after the obtaining step. In some embodiments, the encapsulated osteochondral tissue or other suitable tissue(s) or organ(s) remain viable for implantation for at least about 2 weeks (such as at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, or more). In some embodiments, the encapsulated osteochondral tissue or other suitable tissue(s) or organ(s) may be cultured for about 2 weeks to about 3 weeks, about 2 weeks to about 4 weeks, about 2 weeks to about 5 weeks, about 2 weeks to about 6 weeks, about 2 weeks to about 7 weeks, about 2 weeks to about 8 weeks, about 2 weeks to about 9 weeks, about 2 weeks to about 10 weeks, about 3 weeks to about 4 weeks, about 3 weeks to about 5 weeks, about 3 weeks to about 6 weeks, about 3 weeks to about 7 weeks, about 3 weeks to about 8 weeks, about 3 weeks to about 8 weeks, about 3 weeks to about 9 weeks, about 3 weeks to about 10 weeks, about 3 weeks to about 4 weeks, about 3 weeks to about 5 weeks, about 3 weeks to about 6 weeks, about 3 weeks to about 7 weeks, about 3 weeks to about 8 weeks, about 3 weeks to about 9 weeks, about 3 weeks to about 10 ... The encapsulated cornea remains viable for transplantation for at least about 2 weeks to about 9 weeks, about 3 weeks to about 10 weeks, about 4 weeks to about 5 weeks, about 4 weeks to about 6 weeks, about 4 weeks to about 7 weeks, about 4 weeks to about 8 weeks, about 4 weeks to about 9 weeks, about 4 weeks to about 10 weeks, about 5 weeks to about 6 weeks, about 5 weeks to about 7 weeks, about 5 weeks to about 8 weeks, about 5 weeks to about 9 weeks, about 5 weeks to about 10 weeks, about 6 weeks to about 7 weeks, about 6 weeks to about 8 weeks, about 6 weeks to about 9 weeks, or about 6 weeks to about 10 weeks. In various embodiments, the encapsulated cornea remains viable for transplantation for at least about 2 weeks.

[0089] In various embodiments, the calcium solution comprises, consists essentially of, or further consists of a calcium chloride solution. In some embodiments, the calcium solution used herein may be replaced with a barium solution (e.g., a barium chloride solution). In further embodiments, the calcium solution used herein may be replaced with a solution comprising or consisting essentially of a calcium salt and a barium salt (such as calcium chloride and barium chloride).

[0090] In various embodiments, the reservoir further includes a temperature sensor.

[0091] In various embodiments, the tissue(s) or organ(s) are encapsulated in a semipermeable membrane chamber. In various embodiments, the semipermeable membrane has a pore size of at least about 250 pm (such as about 0.22 μm to about 100 μm and ranges therebetween, about 0.22 μm to about 1000 μm and ranges therebetween, or about 100 μm to about 1000 μm and ranges therebetween). Additionally or alternatively, the semipermeable membrane has a molecular weight cut off (MWCO) of about 50 kDa or about 200,000 kDa. In some embodiments, the semipermeable membrane has / includes a mesh opening ratio of about 1% to about 50% and ranges or percentages therebetween (e.g., about 2%).

[0092] In various embodiments, the alginate solution is a high purity alginate solution, hi various embodiments, the alginate solution comprises or consists essentially of about 0.5% to about 2.0% alginate and ranges or percentages therebetween, optionally about 1.2% alginate.

[0093] In various embodiments, the culture medium is a complete medium.

[0094] In various embodiments, the method further comprises transporting the storage container to a hospital after a period of storage, which period may be any period disclosed herein.

[0095] In various embodiments, the method further includes maintaining the temperature in the semi-permeable membrane, or in the culture chamber, or in the storage vessel at less than 10° C. (e.g., about 0° C. to about 4° C. and any range therebetween, or about 4° C.).

[0096] In various embodiments, the CO2 levels, O2 levels, humidity, or any combination thereof in the semi-permeable membrane, or culture chamber, or storage vessel is not artificially controlled.

[0097] In various embodiments, the method further comprises dissolving the alginate gel prior to implantation, for example, by a method comprising exposing the encapsulated tissue(s) or organ(s) to a chelating solution. In further embodiments, the chelating solution comprises, consists essentially of, or further consists of a sodium citrate solution or an ethylenediaminetetraacetic acid (EDTA) solution or a solution comprising both sodium citrate and EDTA. Additionally or alternatively, the alginate gel may be mechanically removed, such as by manual extraction.

[0098] In various embodiments, the method further comprises the step of degrading the semipermeable membrane, for example, prior to implantation.

[0099] In various embodiments, tissue(s) or organ(s) advanced using the methods disclosed herein (e.g., those that remain viable for transplantation at the time of donation) have reduced release of inflammatory factor(s) compared to tissue or organs stored without encapsulation, e.g., in an alginate gel, or in a semipermeable membrane, or both in an alginate gel and in a semipermeable membrane. In further embodiments, the inflammatory factor(s) comprises, consists essentially of, or further consists of nitric oxide, or one or more cytokines, or both nitric oxide and one or more cytokines.

[0100] In various embodiments, tissue(s) or organ(s) processed using the methods disclosed herein (e.g., those that remain viable for transplantation at the time of donation) maintain at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% or more cell viability prior to the encapsulation step or after the obtaining step (i.e., a step that includes, consists essentially of, or consists of, for example, obtaining the tissue(s) or organ(s) from a subject).

[0101] In various embodiments, the method further comprises injecting a buffer solution (such as saline) into the alginate gel or the culture chamber or into the alginate gel in the culture chamber prior to injecting the culture medium. In further embodiments, the buffer solution (such as saline) reduces or removes soluble calcium or barium.

[0102] In a non-limiting example of the method disclosed herein, the tissue is placed in a dialysis device (such as Tube-O-DIALYZER™, G-Bioscience, MO, USA) that serves as a semipermeable membrane chamber and filled with an alginate solution. In a further embodiment, the alginate solution comprises or consists essentially of 1.2% alginate. Additionally or alternatively, the dialysis device is equipped with a 50 kDa cut-off semipermeable membrane. In yet a further embodiment, the alginate solution is crosslinked / gelled with a calcium or barium solution or both, washed with normal saline as needed, and then stored in complete medium.

[0103] In another aspect, the present disclosure provides a tissue transport system. For example, FIG. 4A shows a tissue culture device that can form part of the tissue transport system. The tissue culture device includes an outer chamber (also referred to herein as a "culture chamber") that defines an inlet, the inlet being constructed to allow the inflow of fluid through said inlet, and an outlet, the outlet being constructed to allow the outflow of fluid through said outlet. In some embodiments, instead of having separate inlets and outlets, the outer chamber can have a single port that can be selectively used to communicate fluids in and out of the outer chamber. In some embodiments, the fluid can include at least one of an alginate solution, a calcium solution, a barium solution, a saline solution, a culture medium, or a storage medium. The outer chamber is enclosed (i.e., defines a closed internal volume).

[0104] The tissue culture device also includes an inner chamber (also referred to herein as a "tissue chamber") disposed within the outer chamber. The inner chamber has at least one wall (e.g., a portion of the wall, cap, or bottom formed from a semipermeable membrane). In various embodiments, the semipermeable membrane comprises, consists essentially of, or consists of a mesh. In some embodiments, the semipermeable membrane can be a bag-strainer structure or a cell-strainer-like structure. For example, the inner chamber can include a bag formed from a semipermeable membrane or mesh such that the tissue is placed into the bag, which is then placed within the outer chamber. In some embodiments, the inner chamber can include walls formed from a non-porous material (e.g., a plastic, polymer, or metal), and the cap of the inner chamber can be formed at least in part from the semipermeable membrane. The inner chamber is enclosed (i.e., defines a closed internal volume), is configured to hold or contain tissue within the inner chamber (e.g., for tissue storage or transport), and includes an alginate gel that surrounds or encapsulates the tissue or organ. In some embodiments, after placing the tissue to be stored / transported in the inner chamber, an alginate solution is added to the inner chamber. For example, the tissue may be immersed in the alginate solution and placed in the inner chamber, or the inner chamber containing the tissue may be filled with the alginate solution. Then, a calcium solution or a barium solution or both are communicated into the outer chamber via an inlet. In other embodiments, both the calcium solution or the barium solution are communicated into the inner chamber (e.g., by immersing the inner chamber in a container filled with calcium solution and / or barium solution) before the inner chamber is placed in the outer chamber, and the inner chamber is placed in the outer chamber at the time when an alginate or other polymer network that encapsulates the tissue is formed in the inner chamber. In some embodiments, the tissue in the inner chamber may be immersed in a solution such as one or more of an alginate solution, a calcium solution, a barium solution, or a culture medium. In further embodiments, the solution may be removed or replaced without releasing the tissue from the inner chamber.

[0105] Although alginate gels are generally described herein, it should be appreciated that tissues may be encapsulated in any other crosslinked gel (e.g., hydrogels or hydrophilic polymer networks such as agarose, methylcellulose, hyaluronan, elastin-like polypeptides, polyethylene glycol (PEG), poly(2-hydroxyethyl methacrylate) (PHEMA), 2-hydroxyethyl methacrylate (HEMA), methacrylic acid (MAA), PEG-PEGMA, carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), acrylamide / acrylic acid copolymers, linear cationic polyallyl ammonium chloride, poly(N-isopropylacrylamide) (PNIPAM), chitosan, acrylate-modified PEG and acrylate-modified hyaluronic acid, amine-terminated functionalized 4-arm star PEG, any other suitable hydrogel or polymer, or combinations thereof). In some embodiments, the gels used herein partially or completely depolymerize in vivo, for example, by contact with a solution (such as dissolving the alginate gel in a chelating solution). Additionally or alternatively, the gels used herein allow the flow of culture medium, calcium solution, barium solution, or chelate solution through the gel. The gel (e.g., alginate gel or any other gel or polymer described herein) that encapsulates the tissue has the advantage of protecting the tissue placed in the inner chamber until the tissue is used. Furthermore, the semipermeable membrane or mesh that forms at least a portion of the inner chamber prevents the alginate gel or any other gel or polymer that encapsulates the tissue from leaking out of the inner chamber while allowing fluid (e.g., calcium solution, barium solution, saline, culture medium, or storage medium) to flow through the semipermeable membrane or mesh to communicate with the inner volume of the inner chamber.

[0106] In various embodiments, the gels disclosed herein (such as alginate gels) may be dissolved by a method comprising, consisting essentially of, or consisting of exposing the gel to a chelating solution disclosed herein (such as an EDTA solution or a sodium citrate solution). Additionally or alternatively, the gels disclosed herein may be dissolved by a method comprising, consisting essentially of, or consisting of exposing the gel to an excess amount of saline.

[0107] In some embodiments, the semipermeable membrane may have a porosity (e.g., a pore size of at least about 250 μm, e.g., about 0.22 μm to about 1,000 μm, about 0.22 μm to about 100 μm, about 100 μm to about 1,000 μm, and any range therebetween) constructed to prevent leakage of the alginate solution or alginate gel from the inner chamber while allowing fluid communication into the inner chamber. In some embodiments, the inner chamber may include a dialysis cassette (e.g., a dialysis cassette under the trade name SLIDE-A-LYZER available from SIGMA ALDRICH®).

[0108] If not already present in the outer chamber, the inner chamber is then placed into an outer chamber having an inlet and an outlet. The alginate solution can then be gelled by adding a calcium or barium solution or both to the inlet of the culture chamber. After gelling, the calcium or barium solution or both are washed out by injecting saline or culture medium (e.g., by draining the calcium or barium solution or both from the outlet of the outlet chamber).

[0109] The tissue culture device or system also includes an enclosed storage container (also referred to herein as a "box") within which the outer chamber, and thus the inner chamber, are disposed within an interior volume defined by the storage container. For example, after washing the alginate gel from the outer chamber, the outer chamber is placed into the storage container. In some embodiments, a temperature sensor is operably coupled to the storage container and configured to measure the temperature within the storage container, for example, to ensure that the tissue is maintained at a desired temperature (e.g., less than 10 degrees Celsius) during transport. In some embodiments, the tissue culture device may also include at least one of an insulating component or a cooling component disposed within the storage container. The insulating component or the cooling component is configured to maintain a temperature within the storage container and / or the outer chamber below 10°C.

[0110] The storage containers may be stored for up to 4-8 weeks and transported to a hospital for implantation. In the operating room, the storage medium is replaced with a lysis buffer for a few minutes. The inner chamber is then removed and the tissue encapsulated in the alginate gel is transferred to an EDTA or sodium citrate solution to dissolve the alginate gel. Once the alginate gel has dissolved, the tissue is retrieved and washed with saline in preparation for implantation into the subject.

[0111] 4B-4F are various pictorial views of another embodiment of a tissue culture device 100 and various components of the tissue culture device 100. The tissue culture device 100 includes an outer chamber 110 or culture chamber and an inner chamber 120 or tissue chamber disposed at least partially inside the outer chamber 110.

[0112] As shown in FIG. 4D, the outer chamber 110 includes a fluid containment portion 111 and an outer chamber cap 112 coupled to an upper end of the fluid containment portion 111. The fluid containment portion 111 defines an interior volume configured to receive at least a portion of the inner chamber 120 and contain a fluid (e.g., culture medium, saline solution, calcium solution, washing solution, chelating solution, etc.). As shown in FIGS. 4B-4D, the fluid containment portion 111 may define a circular cross-section. In other embodiments, the fluid containment portion 111 may have a square, rectangular, elliptical, oval, polygonal, or any other suitable cross-sectional shape. In some embodiments, the fluid containment portion 111 may be formed from a transparent or translucent material (e.g., acrylic, plastic, glass, etc.).

[0113] The outer chamber cap 112 may be removably or fixedly coupled to an upper end of the fluid containment 111. In some embodiments, the outer chamber cap 112 may be removably coupled to the containment 111 by, for example, threads, a friction fit, a snap fit, an interference fit, or any other suitable coupling mechanism. The outer chamber cap 112 defines an opening 114 therethrough that is configured to receive at least a portion of the inner chamber 120 therethrough. An inlet 115 for communicating fluid into the fluid containment 111 and an outlet 117 for communicating fluid to an exterior of the fluid containment 111 are defined therethrough. In some embodiments, the inlet 115 and the outlet 117 may be defined oppositely between the opening 114 and an outer periphery of the outer chamber cap 112. As described herein, threads 116 may be defined on an inner wall of opening 114 and configured to mate with corresponding mating threads defined on interior chamber 120 .

[0114] As shown in FIGS. 4E-4F, the internal chamber 120 includes a tissue containment section 121 and an internal chamber cap 122 coupled to an upper end of the tissue containment section 121. The tissue containment section 121 defines an internal volume constructed to hold tissue therein (e.g., any of the tissues described herein). In some embodiments, at least a portion of the tissue containment section 121 is formed from a semipermeable membrane (e.g., any of the semipermeable membranes described herein or a mesh such as a medical grade mesh). For example, only the bottom of the tissue containment section 121 may be formed completely or at least partially (e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or more) from a semipermeable membrane, or only the walls of the tissue containment section 121 may be formed completely or at least partially (e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or more) from a semipermeable membrane. 0%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or more) or both the walls and bottom of the tissue containment portion 121 may be formed completely or at least partially (at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or more) of a semipermeable membrane. In some embodiments, the tissue disposed within the tissue containment portion 121 is encapsulated in an alginate gel.

[0115] The internal chamber cap 124 couples to the top end of the tissue store 121. For example, a first series of threads 123 can be defined on an inner surface of the top end of the tissue store 121. The internal chamber cap 124 can include a protrusion 126 that protrudes from a bottom of the internal chamber cap 124 toward the tissue store 121. A series of mating threads 125 can be defined radially on an outer surface of the protrusion 121 and can mate with the first series of threads 123 to couple the internal chamber cap 124 to the tissue store 121.

[0116] A second set of threads 127 may also be defined radially on the outer surface of the upper end of the tissue containment portion 121 and configured to mate with the threads 116 defined on the inner wall of the opening 114. The tissue containment portion 121 is configured to be inserted through the opening 114 in the outer chamber cap 112 and into the fluid containment portion 111 until the second set of threads 127 mate with the threads 116 to secure the inner chamber 120 to the outer chamber 110. In some embodiments, the cross-sectional width (e.g., diameter) of the inner chamber cap 124 is greater than the cross-sectional width (e.g., diameter) of the opening 114 such that when the tissue containment portion 121 is positioned inside the fluid containment portion 111, the cap 124 cannot be inserted through the opening 114 and remains positioned outside the outer chamber 110. In some embodiments, to prevent removal of the inner chamber cap 124 from the tissue containment portion 121 when the tissue containment portion 121 is unscrewed from the outer chamber cap 112, a first set of threads 123 coupled to the mating threads 125 of the inner chamber cap 124 may face in the opposite direction to a second set of threads 127 coupled to the threads 116 of the opening 114. In operation, a user may hold the inner chamber 120 by the inner chamber cap 124 to couple or uncouple the inner chamber 120 from the outer chamber 110. The tissue culture device 100 is placed in a storage container or box for transport as described herein.

[0117] In one aspect, a system is provided that comprises, consists essentially of, or further consists of an outer chamber (also referred to herein as a culture chamber) that includes an inner chamber with a semipermeable membrane (also referred to herein as a tissue chamber or semipermeable membrane chamber). In various embodiments of any aspect disclosed herein, the wall of the inner chamber can be formed completely or at least partially (such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or more) of the semipermeable membrane.

[0118] In some embodiments, the semipermeable membrane has a pore size of at least about 250 pm, for example, from about 0.22 μm to about 100 μm and ranges therebetween, from about 0.22 μm to about 1000 μm and ranges therebetween, or from about 100 μm to about 1000 μm and ranges therebetween.

[0119] In some embodiments, the semipermeable membrane has a mesh openness of about 1% to about 50% and any range or percentage therebetween (eg, about 2%).

[0120] In some embodiments, the MWCO of the semi-permeable membrane is about 50 kDa or about 200,000 kDa.

[0121] In various embodiments, the inner container (also referred to herein as the inner chamber) can be filled with the alginate solution, closed, and secured into the outer container (also referred to herein as the outer chamber). Additionally or alternatively, the inner container / inner chamber can be removed from the outer container / outer chamber for retrieving tissue in the operating room for transplantation.

[0122] In various embodiments, the outer container (also referred to herein as the outer chamber) includes an inlet and an outlet for injecting a crosslinking solution (i.e., a solution that gels the alginate solution, such as a calcium solution, a barium solution, or both (e.g., a calcium chloride solution or a barium chloride solution)) or a dissolving solution (i.e., a solution that dissolves the alginate gel, such as a chelating solution (e.g., an EDTA solution or a sodium citrate solution or a solution containing both EDTA and sodium citrate)). Thus, gelling of the alginate or dissolving of the alginate can be performed aseptically without opening the inner container (also referred to herein as the inner chamber).

[0123] In one embodiment, provided herein is a newly designed implantable system or device comprising an alginate gel and an optional semi-permeable membrane.

[0124] In some embodiments, the system or device provides some protection to the tissue from mechanical damage during transport of the tissue to the clinical site, hi some embodiments, the tissue is embedded in a device or system with alginate.

[0125] In some embodiments, the system or device provides some protection to the tissue from mechanical damage during transport of the tissue to the clinical site. In some embodiments, the tissue is embedded in the device or system with alginate, stored at 4° C., and transported from a first location (such as an organ bank) to a second location (such as a hospital).

[0126] In some embodiments, a single device (i.e., the system described herein) is used to embed, store, and transfer. In some embodiments, after the tissue is placed in the device or system, an alginate solution and a calcium solution are added to the device or system to surround or encapsulate the tissue. In some embodiments, the tissue is placed in a semi-permeable membrane chamber, and an alginate solution and a calcium solution (or a barium solution or a solution comprising or consisting essentially of a barium salt and a calcium salt) are added to the chamber. In some embodiments, the alginate is gelled by adding a calcium solution or a barium solution, or both, to the chamber. In some embodiments, after gelling, the alginate gel is washed by injecting saline or culture medium to remove excess calcium or barium (i.e., soluble calcium or barium).

[0127] In some embodiments, the semi-permeable membrane chamber (i.e., the inner chamber) is enclosed or fixed in the culture chamber (i.e., the outer chamber). In some embodiments, the two bilayer chambers are enclosed in an outer box (i.e., a storage vessel or box) equipped with a temperature sensor.

[0128] In some embodiments, the tissue is then stored for at least about 2 weeks (e.g., at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, or longer). Additionally or alternatively, the tissue is stored for up to 4 weeks, or up to 6 weeks, or up to 8 weeks, or up to 10 weeks. In some embodiments, the tissue is then stored for about 2 weeks to about 3 weeks, about 2 weeks to about 4 weeks, about 2 weeks to about 5 weeks, about 2 weeks to about 6 weeks, about 2 weeks to about 7 weeks, about 2 weeks to about 8 weeks, about 2 weeks to about 9 weeks, about 2 weeks to about 10 weeks, about 3 weeks to about 4 weeks, about 3 weeks to about 5 weeks, about 3 weeks to about 6 weeks, about 3 weeks to about 7 weeks, about 3 weeks to about 8 weeks, about 3 weeks to about 9 weeks, about 3 weeks to about 10 weeks, The tissue may be stored for about 4 weeks to about 5 weeks, about 4 weeks to about 6 weeks, about 4 weeks to about 7 weeks, about 4 weeks to about 8 weeks, about 4 weeks to about 9 weeks, about 4 weeks to about 10 weeks, about 5 weeks to about 6 weeks, about 5 weeks to about 7 weeks, about 5 weeks to about 8 weeks, about 5 weeks to about 9 weeks, about 5 weeks to about 10 weeks, about 6 weeks to about 7 weeks, about 6 weeks to about 8 weeks, about 6 weeks to about 9 weeks, about 6 weeks to about 10 weeks, or any range therebetween. In some embodiments, the tissue is stored for up to 4 to 8 weeks.

[0129] In some embodiments, the outer box with two chambers is transported to the hospital. In some embodiments, after arrival at the hospital, the alginate gel is dissolved in a device or system in the operating room using sodium citrate or EDTA. In some embodiments, in the operating room, the storage medium (e.g., culture medium) is replaced with a lysis buffer for a few minutes. In some embodiments, the semi-permeable membrane chamber is disassembled and the tissue with the alginate gel is transferred to an EDTA solution to dissolve the alginate gel. In some embodiments, the tissue is retrieved from the alginate gel for transplantation and washed with saline.

[0130] The tissue transport system offers several advantages over standard liquid-based tissue storage systems. Tissue preservation conditions are improved by embedding in alginate gel. The tissue is protected from shock, vibration, or shaking during transport of the tissue to the hospital. As such, tissue damage due to transport can be prevented. Furthermore, as shown herein, cell viability is improved and inflammatory factors (such as cytokines and nitric oxide) are suppressed. One enclosed system provides storage and transport steps.

[0131] In some embodiments, the tissue transport system and / or tissue culture device may be provided in a kit. For example, the kit may include (e.g., may include, may consist essentially of, or may consist of) a tissue culture device including an outer chamber (e.g., outer chamber 110 or inner chamber described in FIG. 4A) and an inner chamber (e.g., inner chamber 120 or inner chamber described in FIG. 4A). The kit may also include (e.g., may further include) a storage container or box (e.g., as described in FIG. 4A) that may have insulation and / or cooling components, as well as a temperature sensor. The kit may also include (e.g., may further include) a container or vial containing an alginate solution or any other hydrogel solution, a container or vial containing a calcium solution or a barium solution or both, a container or vial containing a calcium salt or a barium salt or both, a container or vial filled with a washing solution (e.g., saline or any other solution described herein) and / or a culture medium (e.g., any of the culture media described herein). The kits may also include written or downloadable instructions for using the tissue culture devices described herein. In various embodiments, the kits disclosed herein comprise or consist essentially of the tissue culture devices disclosed herein and optional instructions for use. In various embodiments, the kits disclosed herein further comprise one or more of the following: an optional solution of alginate or any other hydrogel monomer, a calcium solution, a barium solution, a calcium salt, a barium salt, a solvent, a washing solution, or a culture medium. EXAMPLES

[0132] The following examples are intended to illustrate, but not limit, the present disclosure. Example 1 In vitro evaluation of the effect of highly purified alginate on the performance of osteochondral allografts harvested from porcine knees

[0133] method Explant preparation and storage conditions - Osteochondral cores (n=25) were harvested from 10 knees of 5 mature sows obtained from an abattoir within 48 hours after slaughter. Under sterile conditions, osteochondral cores were harvested from either the medial or lateral femoral condyle by coring with a custom-made 4.0 mm bit drill. Prior to storage, core bones were trimmed to a bone to cartilage thickness ratio of 4:1. Subchondral bone was subjected to pulsed lavage with air to remove bone marrow components, followed by thorough rinsing with normal saline. Each osteochondral core was stored overnight in DMEM / F12 with 10% fetal bovine serum (FBS), then placed in an individual sterile 5 mL culture tube and stored at 4° C. according to the following conditions assigned to each core ( FIG. 1 ): (1) control group—stored in 1.5 mL complete medium (DMEM / F12+10% FBS); (2) UPAL solution (sol) group—stored in 1.5 ml UPAL solution (1.2%) dissolved in complete medium; (3) UPAL gel group—encapsulated with 1.2% UPAL gel dissolved by normal saline and stored in 1.5 ml complete medium. After 28 days of storage, each sample was warmed at 37° C. for 24 hours, and then the full-thickness cartilage was removed from the bone and evaluated as follows:

[0134] Chondrocyte viability - Chondrocyte viability (n=4) across all full thickness cartilage depths was analyzed using the LIVE / DEAD kit and imaged using confocal laser microscopy. For vertical profiles, viability was calculated for the entire cartilage thickness and for the following cartilage zones: superficial (top 15%), middle (35%), and deep (50%).

[0135] Proteoglycan (PG) content - PG content in each cartilage core (n=6) was measured using the dimethylmethylene blue (DMMB) assay (normalized by wet weight).

[0136] PG synthesis - radiolabeled 35 Incorporation of S-sulfate into sulfated PG (n=6) was measured by a rapid Alcian blue filtration assay (Masuda, et al., Anal Biochem. 1994:167) as an index of PG synthesis.

[0137] PG turnover - in culture medium and cartilage digest (n=6) 35 Measure the number / amount of S-PGs 35 S-PG synthesis in tissues relative to total synthesis 35 S-PG remaining amount was assessed as PG turnover (O'Driscoll, et al., J Bone Joint Surg Am. 1986:1017).

[0138] Nitric oxide (NO) and matrix metalloproteinase-3 (MMP-3) concentrations - Storage media collected on day 28 was assayed for total NO (n=6) and MMP-3 (n=4) concentrations using commercially available assay kits.

[0139] Histology - Vertical midsection sections (5 μm) of each OCA (n=7) were stained with either hematoxylin and eosin or safranin-O. An observer blinded to the study analyzed the histological sections and graded them using a modified O'Driscoll histological scoring system (Aota, et al., Spine 2005:722).

[0140] Statistical analysis - All data are expressed as mean ± standard deviation (SD). Data were statistically analyzed using two-way repeated measures analysis of variance (ANOVA) or one-way ANOVA for comparison. P value less than 0.05 was considered statistically different.

[0141] result Chondrocyte viability - The viability of chondrocytes in the surface area of ​​the UPAL gel group (45.4 ± 20.1%) was significantly higher than that of the control group (18.6 ± 10.7%, p < 0.05; Figure 2).

[0142] Proteoglycan (PG) content – ​​No significant differences were observed in PG content on day 28 (control: 276.0 ± 40.5 μg / mg, UPAL solution: 329.1 ± 54.0 μg / mg, UPAL gel: 314.2 ± 49.3 μg / mg).

[0143] PG synthesis – PG synthesis in the UPAL gel group (33192.8±15906.4 CPM / mg) was significantly higher than that in the control group (12748±2699 CPM / mg, p<0.05) on day 28. 35 S incorporation was significantly decreased in all groups compared to day 1 (2,073,811 ± 496,258 CPM / mg, p < 0.01).

[0144] PG turnover - in tissues on day 28 35 No significant difference was observed in S-PG survival.

[0145] Nitric oxide (NO) and matrix metalloproteinase-3 (MMP-3) concentrations in storage medium - Total NO production in the control group (3.8±2.4 μM / mg) was significantly higher than that in the UPAL solution group (1.0±0.8 μM / mg, p<0.01) and the UPAL gel group (1.5±0.8 μM / mg, p<0.01) on day 28. No significant differences were observed in MMP-3 levels among the three conditions in the storage medium on day 28.

[0146] Histology - The cartilage matrix in the control group showed poor Safranin-O staining and low cellularity, whereas the OCA in the UPAL gel group showed good matrix staining and near-normal cellularity. The total histological score in the UPAL gel group (13.9 ± 1.1) was significantly higher than that in the control group (12.0 ± 1.3, p < 0.01; Figure 3).

[0147] Example 2 Example 1 showed that the use of high-purity alginate (UPAL) gel encapsulation was effective in improving the quality of osteochondral grafts after 28 days of storage. To increase the availability of grafts in clinical practice, it is important to extend the storage period. First, Example 2 showed that the storage period of cartilage was successfully extended up to 6 weeks. Furthermore, the application was extended to other tissues (meniscus, tendon, and cornea). Second, to improve the storage conditions with UPAL, the effect of different gelling conditions of UPAL on cell viability during extended storage periods was studied. It should be noted that the endpoint of this experiment is 1.5 times (6 weeks) that of the current tissue preservation protocol. Importantly, the availability of graft tissues is higher when the viable tissue storage period is extended. The use of UPAL gel during tissue storage is favorable for cell viability and structural maintenance of tissues during storage, and also provides physical protection as a filling material during transportation.

[0148] method Graft preparation and storage conditions - Osteochondral cores, meniscus, patellar tendon, and cornea were harvested from juvenile minipigs (6 months old). Under sterile conditions, osteochondral cores were harvested from either the medial or lateral femoral condyle by coring with a custom-made 4.0 mm drill bit. The subchondral bone was subjected to pulsatile lavage with air to remove bone marrow components, followed by thorough rinsing with normal saline. Prior to storage, the core bones were trimmed to a bone to cartilage thickness ratio of 4:1. Tissues from the medial and lateral meniscus were punched out using skin pinches (4 mm), and the patellar tendon was aseptically dissected and minced into 5 x 10 mm pieces. Corneal tissue with vitreous was also dissected and 5 x 10 mm pieces of graft tissue were prepared. Each explant tissue was stored overnight in DMEM / F12 with 10% fetal bovine serum (FBS) and then placed in an individual sterile 5 mL culture tube and stored at 4° C. according to the following conditions assigned to each core ( FIG. 5 ): (1) control group—stored in 3 mL complete medium (DMEM / F12+10% FBS); (2) UPAL solution (sol) group—stored in 3 ml UPAL solution (1.2%) dissolved in complete medium; (3) UPAL gel-coated group—encapsulated with 1.2% UPAL gel (approximately 1 ml UPAL cross-linked by calcium solution and washed with normal saline) and stored in 3 ml complete medium; and (4) UPAL membrane group: tissue prepared as described in the UPAL gel-coated group was placed in a dialysis device (Tube-O-DIALYZER™, G-Bioscience, MO, USA) with a 50 kDa cut-off semipermeable membrane and filled with 1.2% alginate solution. Tubes containing one graft were immersed in 0.1 M CaCl2 solution for 30 min to promote gelation. The tubes were then washed with saline and placed in a 50 ml culture tube filled with 40 ml complete medium. Articular cartilage, meniscus, and Achilles tendon were stored for 6 weeks, whereas corneal tissue was stored for 2 weeks (all at 4°C).

[0149] After 6 weeks of storage, each sample was warmed at 37°C for 24 hours, and then the tissue was placed in 30 mM EDTA solution for 5 minutes to dissolve the alginate gel. Cell viability was analyzed using the LIVE / DEAD kit and imaged using confocal laser scanning microscopy. For vertical profiles, viability was calculated using Otsu's binarization method followed by the particle analysis function of the Image-J program. Viability was expressed as endpoint viability / original cell viability.

[0150] Statistical analysis - All data are expressed as mean ± standard error (SE). Data were statistically analyzed using one-way analysis of variance (ANOVA) or Kruskal-Wallis test adjusted by Bonferroni correction for multiple testing. P values ​​less than 0.05 were considered statistically different.

[0151] result Articular Cartilage - At 6 weeks, the viability of chondrocytes in the surface area of ​​the UPAL membrane group was significantly higher than that of the medium group (p<0.05; Fig. 6A). At 6 weeks, the viability of chondrocytes in the UPALsol and UPAL gel-coated groups showed a tendency to be higher, but statistical significance was not observed due to sample size.

[0152] The viability of meniscus-meniscus cells was significantly decreased in the medium group after 6 weeks (Figure 6B). The UPALsol group, UPAL gel coating group, and UPAL membrane group had significantly higher cell viability than the medium group.

[0153] As seen in the patellar tendon-meniscus, the cell viability in the patellar tendon was significantly decreased in the medium group after 6 weeks. The cell viability in the UPAL gel-coated group and the UPAL membrane group was significantly higher than that in the medium group (Figure 6C).

[0154] Cornea - After 2 weeks, the cell viability of the corneal tissue in the UPAL membrane group was significantly higher than that in the medium group (Figure 6D). The cell viability of the UPAL membrane group was not significantly different from that of the original tissue.

[0155] Equivalent Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this technology pertains.

[0156] The technology illustratively described herein may suitably be practiced in the absence of any component(s), limitation(s) not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," and the like, shall be interpreted broadly and without limitation. Furthermore, the terms and expressions used herein are used for purposes of description and not limitation, and the use of such terms and expressions is not intended to exclude any equivalents of the features shown and described or portions thereof, but is recognized as being capable of various modifications within the scope of the technology described in the claims.

[0157] Accordingly, it should be understood that the materials, methods, and examples provided herein are representative of preferred embodiments and examples, and are not intended to limit the scope of the technology.

[0158] The present technology is described broadly and generically herein. Each of the narrower species and subgeneric groups falling within the scope of the generic disclosure also form part of the present technology. This includes the generic description of the present technology, including any negative limitation that excludes any subject matter from the genus or genus, regardless of whether the excised material is specifically recited herein.

[0159] Furthermore, when features or aspects of the technology are described in terms of Markush groups, one of skill in the art will recognize that the technology is also described with respect to any individual member or subgroup of members of the Markush group.

[0160] All publications, patent applications, patents, and other documents mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each was individually incorporated by reference. In the case of conflict, the present specification, including definitions, will control.

[0161] Other aspects are within the scope of the following claims.

[0162] Although the disclosure has been described with reference to the above examples, it will be understood that modifications and variations are within the spirit and scope of the disclosure. Accordingly, the disclosure is limited only by the scope of the following claims.

Claims

1. 1. A tissue transport system comprising: a tissue chamber formed in part or in whole from a semipermeable membrane and configured to contain the tissue to be transported and a high purity alginate solution, said tissue chamber being enclosed; a culture chamber having an inlet and an outlet and configured to contain the tissue chamber, wherein upon addition of a calcium solution or a barium solution or both via the inlet, the alginate solution gels and encapsulates the tissue and the culture chamber; , a tissue transport system.

2. one or more storage vessels configured to contain the incubation chamber; The tissue transport system of claim 1 .

3. The alginic acid solution comprises 0.5% to 2.0% alginate.

3. The tissue transport system of claim 1 or 2.

4. The alginic acid solution contains 1.2% alginate. The tissue transport system of any one of claims 1 to 3.

5. a temperature sensor operably disposed in the storage container and configured to measure a temperature within the storage container; The tissue transport system of claim 2.

6. the semi-permeable membrane has one or more of the following characteristics: a pore size of at least 250 pm, a pore size of 100 μm to 1000 μm, a pore size of 0.22 μm to 1000 μm, or a pore size of 0.22 μm to 100 μm, a molecular weight cut off (MWCO) of 50 kDa or 200,000 kDa, or a mesh open area of ​​1% to 50%; The tissue transport system of any one of claims 1 to 5.

7. further comprising a thermal insulation component configured to maintain a temperature within one or more of said storage vessels or said incubation chambers below 10° C.; 3. The tissue transport system of claim 1 or 2.

8. The tissue transport system of claim 7, wherein the insulating component is configured to maintain a temperature within one or more of the storage containers or culture chambers between 0°C and 4°C.

9. The tissue transport system of claim 8, wherein the insulating component is configured to maintain a temperature within one or more of the storage containers or culture chambers at 4°C.

10. further comprising a cooling component configured to maintain a temperature within one or more of said storage containers or said incubation chambers below 10° C.; 3. The tissue transport system of claim 1 or 2.

11. The tissue transport system of claim 10, wherein the cooling component is configured to maintain a temperature within one or more of the storage containers or culture chambers between 0°C and 4°C.

12. The tissue transport system of claim 11, wherein the cooling component is configured to maintain a temperature within one or more of the storage containers or culture chambers at 4°C.

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