Methods of preserving tissues for transplantation

Abstract

To provide systems and methods for preserving and transporting tissues for transplantation.SOLUTION: Tissue to be implanted is encapsulated in an alginate gel, wherein the encapsulated tissue is exposed to a culture medium to improve cell viability and suppress inflammatory factors during long-term storage and transport.SELECTED DRAWING: Figure 4A

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a joint venture of U.S. Provisional Patent Application No. 119(e) filed on January 13, 2020. No. 62 / 960,298, the entire contents of which are incorporated herein by reference. ) priority is claimed. [Technical field]

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

[0003] Osteochondral allografts (OCA) are a promising treatment for defective cartilage without the need for immunosuppression. Therefore, OCA is a treatment for moderate to large joint soft tissue. It is one of the most widely used methods applied for the repair of bone defects (M andelbaum,et al.,Am J Sports Med.1998;85 3) Recent clinical results show that OCA is successful in 60% to 80% of cases 10 years after surgery. It has been shown (Cavendish, et al., JBJS Rev. 2019; e7). These grafts are obtained from organ donors and delivered to surgical centers for transplant into patients. They must be stored in a manner that allows for viral and bacterial testing for safety before being transported to the Based on studies investigating cell viability in grafts, Therefore, it is recommended that tissue be transplanted as soon as possible after harvesting.

[0004] One of the key factors for obtaining a satisfactory clinical outcome is improving the quality of the stored OCA. (including maximizing chondrocyte viability and cartilage matrix content) Although various studies have been conducted previously to improve storage conditions, long-term stored grafts The quality of the orthopedic implants has not been satisfactorily maintained (Ball, et al., Clin Orthop Relat Res.2004:246;Williams,et al.,J Bon e Joint Surg. Am. 2003:2111; and Pallante, et al., Am J Sports Med. 2009:24S). Storage conditions have improved. This could potentially improve the shelf life of OCA and its clinical outcomes in clinical applications. It will improve the sex. Summary of the Invention

[0005] The present disclosure provides a method for the long-term storage or transport of tissue by encapsulating the tissue to be transplanted in an alginate gel. Based on the discovery that the quality and viability of tissue after both storage and transportation is improved after delivery, Made.

[0006] Thus, the present disclosure provides a method for preserving osteochondral tissue or other tissue(s) or organ (e.g., (multiple possible) (articular cartilage without any subchondral bone, fibrocartilage tissue (meniscus, e.g., medial meniscus or lateral meniscus or both), tendons (such as the patellar tendon or Achilles tendon), cornea In various embodiments, the present invention provides a method for preserving a bone, a cartilage, or a cornea having a vitreous body. The tissue may have a bone to cartilage thickness ratio of about 20:1 to about 1:10 and any range or ratio therebetween. 1. The method of claim 1, further comprising: The method may comprise the step of: encapsulating organ(s) in alginate gel and forming gel-encapsulated osteochondral tissue or other suitable tissue(s) or organ(s) to the culture medium. In some embodiments, the compound may include (e.g., comprise, consist essentially of, or further consist of). The gel-encapsulated tissue can be stored for approximately 24 hours to 8 weeks, or for at least approximately 2 weeks (at least for at least about 4 weeks, at least about 6 weeks, or longer, and any range therebetween) In various embodiments, the gel capsule is then placed in culture medium. The transformed tissue is used for transplantation. In various embodiments, the method comprises the steps of: Further comprising the step of encapsulating the organ(s) in a semi-permeable membrane. The permeable membrane has a pore size of at least about 250 μm, a pore size of about 0.22 μm to about 1000 μm, and and the range therebetween (approximately 0.22 μm to approximately 100 μm or approximately 0.22 μm to approximately 100 μm, etc. Additionally or alternatively, the semipermeable membrane has one or more of the following characteristics: or characterized by a molecular weight cut-off (MWCO) of about 200,000 kDa. Alternatively, the semipermeable membrane has a mesh opening ratio of about 1% to about 50% and ranges therebetween. In various embodiments, the tissue has a percentage (such as 2%) prior to encapsulation in the semipermeable membrane. During or after encapsulation, the gel is encapsulated. In various embodiments, The alginate gel is a highly purified alginate (UPAL) gel. Alginates such as PAL range from about 0.5% to about 2.0% and any range or percentage therebetween. (e.g., about 1.2%). In various embodiments, the culture medium is In various embodiments, the culture medium is a gel-encapsulated osteochondral tissue or other The gel is replenished after exposure of the encapsulated tissue(s) or organ(s) to culture medium. In various embodiments, the method comprises heating the tissue(s) or organ(s) at about 10° C. 0°C to about 4°C, or about 4°C. In various embodiments, the encapsulated tissue(s) or organ(s) are artificially artificially controlled CO2 levels, artificially controlled O2 levels, or artificially controlled In various embodiments, the method further comprises storing the culture medium at one or more of the following temperatures: In a further embodiment, the method further comprises dissolving the alginate gel after exposure to the substrate. 2. Exposing the encapsulated tissue(s) or organ(s) to a chelating solution The dissolution is achieved by a process comprising, consisting essentially of, or consisting of the step of: In further embodiments, the chelating solution is a sodium citrate solution or an ethylenediamine Containing or consisting essentially of sodium tetraacetic acid (EDTA) solution or sodium citrate and EDTA In various embodiments, the method comprises the steps of: The method includes, for example, separating the semipermeable membrane after exposure to culture medium, either mechanically or by a chelating solution. In various embodiments, the encapsulated tissue(s) or organ (e.g., (s) may, for example, reduce inflammation compared to tissues or organs stored without encapsulation. In a further embodiment, the release of the proinflammatory factor(s) is / are reduced. , nitric oxide, cytokines, or both. In various embodiments, the encapsulated tissue(s) prior to implantation or further comprises The organ or organs should have at least about 60% of the cell viability prior to the encapsulation process, or at least about 60% of the cell viability prior to the encapsulation process. At least about 65%, or at least about 70%, or at least about 75%, or less The cell viability is preferably at least about 80%, or at least about 85% or higher. In various embodiments, the encapsulated tissue(s) or organ(s) are cultured prior to exposure to culture medium. The tissue (or tissue fragments) may be washed with a buffer, such as saline. In various embodiments, the encapsulated tissue may be The culture(s) or organ(s) (e.g., cornea) are cultured for at least about 2 weeks. It is placed in the medium.

[0007] In another aspect, the present disclosure relates to a method for the treatment of osteochondral or other tissue(s) or organ(s). ) (articular cartilage without any subchondral bone, fibrocartilage tissue (meniscus, e.g., medial meniscus) tendons (such as the patellar or Achilles tendons), cornea, or The present invention provides a method for storing a cornea with a vitreous body, for example, prior to transplantation. In terms of morphology, the osteochondral tissue has a bone to cartilage thickness ratio of about 20:1 to about 1:10 (e.g., or consisting essentially of an osteochondral core trimmed to have a diameter of about 4:1; The method comprises the steps of: providing osteochondral tissue or other suitable tissue(s) to be transplanted; Optionally, obtaining organ(s), and then extracting the resulting osteochondral tissue or other suitable tissue (e.g., placing the organ(s) or organ(s) in a semipermeable membrane chamber containing an alginate solution; placing the membrane chamber in a culture chamber; whereby osteochondral tissue or other suitable tissue(s) or or the organ(s) are encapsulated in an alginate gel; adding a culture medium; Injecting the alginate gel into the culture chamber or injecting the alginate gel into the culture chamber and placing the incubation chamber in a storage container (e.g., comprising, or consisting essentially of). In some embodiments, the encapsulated osteochondral tissue or Alternatively, other suitable tissue(s) or organ(s) may be used to obtain a viable tissue for, e.g., transplantation. Additionally or alternatively, encapsulated osteochondral tissue or other suitable tissue(s) may be used. The organ or organs may be, for example, preserved for at least about two weeks (or at least The cells remain viable for up to about 4 weeks or at least about 6 weeks or up to 8 weeks. In embodiments, the calcium solution comprises, consists essentially of, or comprises a calcium chloride solution. In various embodiments, the barium solution comprises or further comprises a barium chloride solution. In various embodiments, the storage vessel comprises a temperature In various embodiments, the tissue(s) or organ(s) further comprises a semi-transducer. In various embodiments, the semi-permeable membrane is at least about 250 pm or from about 0.22 μm to about 1000 μm; and ranges therebetween (from about 0.22 μm to about 1 The porous membrane has one or more characteristics of a pore size of about 100 μm or about 100 μm to about 1000 μm. Additionally or alternatively, the semipermeable membrane may have a molecular weight cut off (MWCO) of about 50 kDa or about In various embodiments, the semipermeable membrane has a mesh opening ratio of 200,000 kDa. From about 1% to about 50% and any range or percentage therebetween (e.g., about 2%). In embodiments, the alginate solution is a high purity alginate solution. The alginate solution may contain from about 0.5% to about 2.0% alginate and any range therebetween or 100%. fraction, optionally comprising or consisting essentially of about 1.2% alginate. In embodiments, the culture medium is a complete medium. In various embodiments, the method includes storing the culture medium for a period of time. The method further comprises transporting the storage container to a hospital after storage. Keep the temperature in the membrane, culture room, or storage container below 10°C (e.g., about 0°C to about 4°C). In various embodiments, the semi-permeable membrane or the medium may be maintained at a temperature of about 4° C. or about 4° C. CO2 levels, O2 levels, humidity, or both in the culture room or storage container Any combination of is not artificially controlled. In various embodiments, the method comprises the steps of: The acid gel may be applied to, for example, the encapsulated tissue(s) or organ(s) prior to implantation. a method comprising, consisting essentially of, or consisting of exposing the In a further embodiment, the chelate solution further comprises dissolving the chelate by a citric acid method. Sodium citrate solution or ethylenediaminetetraacetic acid (EDTA) solution or sodium citrate A solution comprising or consisting essentially of sodium EDTA and In various embodiments, the method further comprises, for example, degrading the semipermeable membrane prior to implantation. In various embodiments, the method further comprises the steps of: The organ(s) or organ(s) may be, for example, in a gel, in a semipermeable membrane, or in a gel and a semipermeable membrane. inflammatory factors compared to tissues or organs stored without encapsulation in a membrane In a further embodiment, the inflammatory factor(s) reduces the release of monoacid one or more of nitric oxide, cytokines, or nitric oxide and cytokines The present invention may comprise, consist essentially of, or further consist of one or more of both of the following: In embodiments, the tissue(s) or organ(s) are / are kept viable, e.g., for transplantation. Additionally or alternatively, the tissue(s) or organ(s) may be pre-encapsulated prior to the encapsulation step. at least about 60%, or at least about 65%, or at least about 7 0%, or at least about 75%, or at least about 80%, or at least about 85 % or higher. In various embodiments, the method further comprises: Before injection, a buffer solution (such as saline) is added to the alginate gel or the culture chamber or In a further embodiment, the method further comprises injecting the solution into an alginate gel of 0.1% by volume. Salt solution) to reduce or remove soluble calcium or barium. In an embodiment of the present invention, the encapsulated tissue(s) or organ(s) (e.g., the cornea) may be For example, in one embodiment, the cells are stored for at least about two weeks prior to implantation.

[0008] In another aspect, the present disclosure provides a tissue transport system. The system comprises, in part, or entirely formed of 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, said culture chamber comprising: a tissue chamber configured to contain a calcium solution or a barium solution or both; When added through the inlet, the alginate solution gels and encapsulates the tissue in the culture chamber. A transport box or storage container configured to contain a culture chamber; and a transport box or An optional temperature sensor is disposed in the storage container and configured to measure the temperature of the incubation chamber. Various In embodiments, the tissue chamber is enclosed. In various embodiments, the culture chamber is enclosed. In various embodiments, the alginate solution is a highly purified alginate (UPAL) solution. In this embodiment, the tissue chamber comprises an enclosure configured to encapsulate the tissue by a semi-permeable membrane. In various embodiments, the semipermeable membrane is at least about 250 pm, e.g. , about 0.22 μm to about 1000 μm and the range therebetween (0.22 μm to about 100 μm, or from about 100 μm to about 1000 μm. The semipermeable membrane has a mesh opening rate of about 1% to about 50% and any range or percentage therebetween (e.g. Additionally or alternatively, the semipermeable membrane has a molecular weight cut off (MWCO) of about In various embodiments, the alginate has a molecular weight of about 50 kDa or about 200,000 kDa. The solution may contain from about 0.5% to about 2.0% alginate and any range or percentage therebetween, as needed. Depending on the composition, the composition may contain, consist essentially of, or further comprise about 1.2% alginate. In various embodiments, the transport box or storage container or incubation chamber comprises a transport box. Keep the temperature in the storage container or incubation room below 10°C (for example, about 0°C to about 4°C and below). or about 4° C. Both the thermal insulating component and the cooling component In various embodiments, the system further comprises a semi-permeable membrane or a culture chamber or a storage vessel. CO2 levels, or O2 levels, or humidity, or any combination of these In various embodiments, the shipping box or storage container does not include a sensor or controller. The vessel is sealed against the continuous flow of gas or liquid.

[0009] In some embodiments, the tissue culture device is an inlet, Inlet constructed to allow the inflow of fluid through the outlet an outlet constructed to permit the exit of fluid through said outlet; an outer chamber defining a trough, the outer chamber being surrounded by said outer chamber (e.g., The inner chamber is at least partially enclosed within the outer chamber. and the inner chamber has at least one wall formed from a semipermeable membrane, the inner chamber being surrounded by The tissue culture device is constructed to hold tissue within an internal chamber. Accordingly, the present invention provides an enclosed storage vessel, an outer chamber disposed within an interior volume defined by the storage vessel, and In various embodiments, the tissue culture The storage device is operably connected to the storage vessel and measures the temperature within the storage vessel. In various embodiments, the tissue further comprises a temperature sensor configured to In various embodiments, the fluid is an alginate solution, calcium carbonate, or the like. at least one of the following: sodium solution, barium solution, saline, culture medium, or storage medium In various embodiments, the semi-permeable The 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 an embodiment, the semipermeable membrane has a thickness in the range of about 0.22 μm to about 100 μm and ranges therebetween (about 1 00 μm to about 1000 μm, or about 0.22 μm to about 1000 μm, etc. In various embodiments, the semipermeable membrane has a mesh opening rate of about 1% to about 50% and ranges therebetween. In various embodiments, the tissue culture device has a range or percentage (e.g., about 2%). At least one of an insulating component or a cooling component disposed within the storage vessel. and wherein at least one of said insulating or cooling components is The temperature within the storage vessel and / or outer chamber is configured to be maintained below 10°C.

[0010] In one embodiment, the tissue culture device disclosed herein and optionally instructions for use. The kits disclosed herein comprise or consist essentially of: In some embodiments, the kits disclosed herein further comprise one or more of the following: or any other hydrogel monomer as required, calcium solution, barium solution A solution, calcium salt, barium salt, solvent, wash, or 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 provides a method for the encapsulation of tissue to be transplanted in an alginate gel for long-term storage or Improved tissue quality and viability after transport, or after both storage and transport Based on discovery.

[0020] (definition) It will be understood that the section and subsection headings used herein are for organizational purposes only. and the purpose is to limit or separate or both limit and separate the subject matter of the invention described. shall not be construed as construed otherwise.

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

[0022] The practice of the present technology requires, unless otherwise indicated, knowledge of tissue culture, immunology, and the like, all of which are within the skill of the art. The present invention will employ conventional techniques in biology, molecular biology, microbiology, cell biology, and recombinant DNA. For example, Green and Sambrook eds. (2012) Mole cular Cloning:A Laboratory Manual,4 th ed ition;the series Ausubel et al.eds.(2015 )Current Protocols in Molecular Biology; the series Methods in Enzymology(Academi c Press,Inc.,N.Y.);MacPherson et al.(201 5)PCR 1:A Practical Approach(IRL Press a t Oxford University Press);MacPherson et al.(1995)PCR 2:A Practical Approach;McP herson et al.(2006)PCR:The Basics(Garlan d Science);Harlow and Lane eds.(1999)Ant ibodies,A Laboratory Manual;Greenfield e d.(2014)Antibodies,A Laboratory Manual;F reshney(2010)Culture of Animal Cells:A M anual of Basic Technique,6 th edition;Gai t ed.(1984)Oligonucleotide Synthesis;U.S .Pat.No.4,683,195;Hames and Higgins eds. (1984)Nucleic Acid Hybridization;Anderso n(1999)Nucleic Acid Hybridization;Herdew ijn ed.(2005)Oligonucleotide Synthesis:M ethods and Applications;Hames and Higgin s eds.(1984)Transcription and Translatio n;Buzdin and Lukyanov ed.(2007)Nucleic A cids Hybridization:Modern Applications;I mmobilized Cells and Enzymes(IRL Press(1 986));Grandi ed.(2007)In Vitro Transcrip tion and Translation Protocols,2 nd editi on;Guisan ed.(2006)Immobilization of Enz ymes and Cells;Perbal(1988)A Practical G uide to Molecular Cloning,2 nd edition;Mi ller and Calos eds,(1987)Gene Transfer V ectors for Mammalian Cells(Cold Spring H arbor Laboratory);Makrides ed.(2003)Gene Transfer and Expression in Mammalian Ce lls;Mayer and Walker eds.(1987)Immunoche mical Methods in Cell and Molecular Biol ogy(Academic Press,London);Lundblad and Macdonald eds.(2010)Handbook of Biochemi stry and Molecular Biology,4 th edition;お よびHerzenberg et al.eds(1996)Weir’s Handb ook of Experimental Immunology,5 th edited on; and more recent versions 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" includes the plural unless the context clearly indicates otherwise. For example, a reference to "the method" includes one of the types described herein. The above methods, one or more steps, and both one or more methods and one or more steps are included. In most cases, these will be obvious to those of ordinary skill in the art upon reading this disclosure and so forth.

[0024] The term "comprising" means "including," "including Used interchangeably with "containing" or "characterized by" and means inclusive or or open-ended terms and do 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" refers to the specified materials or steps as well as any other steps set forth in the claims. The scope of the claims should not be limited to those that do not materially affect the basic and novel characteristics of the invention. The present disclosure provides composition and method embodiments that fall within the scope of each of these phrases. Thus, any composition or method that includes a recited element or step is intended to be a composition or method. contemplates certain embodiments in which the method consists essentially of or consists of the elements or steps. .

[0025] "As necessary" or "as needed" means that the situation described thereafter will not occur. This means that the situation does not necessarily have to be Including cases where it doesn't happen.

[0026] As used herein, "and / or" means one of the associated listed items. All possible combinations of the above are also possible when interpreted as options. It refers to and is inclusive of the absence of ("or").

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

[0028] As used herein, the term "subject" refers to any individual on whom the subject method is performed. Generally, a subject is a human, but as will be appreciated by those of skill in the art, a subject may refer to any number of different The may be an animal. Thus, other animals (rodents (mouse, rat, hamster) animals (including cats, dogs, rabbits, livestock (cattle, horses, goats, sheep, etc.) animals, sport animals, pets, etc., and primates (monkeys, chimpanzees, etc.) Mammals such as reptiles, orangutans, and gorillas are affected. falls within the scope of the subject definition.

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

[0030] As used herein, the terms "sample" and "biological sample" refer to any sample provided by this disclosure. In one embodiment, the biological sample of the present disclosure is a tissue sample. 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 interchangeably. indicates that the decrease is below the detection level of a particular assay (e.g., about 5%, about 10% of the baseline level). , 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% As such, the terms are used interchangeably because they are recognized as being related to the expression level or activity of the Whether the assay is "degraded" below detection levels or completely "inhibited" is always a question. It may not be clear. Nevertheless, it can be clearly determined after treatment according to the present method. It would be.

[0032] Chondrocytes, as used herein, refer to cells in cartilage. The cells produce a cartilaginous matrix, which is composed mainly of collagen and proteoglycans (PGs), Mesenchymal stem cells are the naturally occurring precursors of chondrocytes. Other stem cells are preferred. Chondrocytes may be induced under suitable conditions, non-limiting examples of which are provided in the Examples.

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

[0034] As used herein, the term "organ" refers to an organ that is specialized to perform a particular function. Refers to a group of tissues that structurally form a functional unit (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 includes articular cartilage and subchondral bone regions. In some embodiments, the osteochondral tissue consists essentially of, or further consists of, , comprising, consisting essentially of, or further consisting of an osteochondral core. Alternatively, osteochondral tissue is obtained from an animal (human or non-human) and trimmed if necessary. In a further embodiment, the osteochondral tissue has a bone to cartilage thickness ratio of about 20:1 to about 1. In yet further embodiments, for example, the osteochondral tissue comprises or comprises: The thickness to thickness ratio is about 4:1.

[0036] As used herein, articular cartilage refers to the hyaline cartilage on the articular surfaces of bones and synovial joints. It is located inside the joint cavity of the joint and is filled with synovial fluid produced by the synovial membrane that lines the walls of the joint cavity. is.

[0037] As used herein, fibrocartilage tissue is a mixture of white fibrous tissue and soft fibrous tissue in varying proportions. Refers to a mixture of bone tissue (e.g., fibrocartilaginous symphysis (pubic symphysis), annulus fibrosus (annul us fibrosis of intervertebral disc), or chest Symphysis of the knee joint, etc.); labrum of the shoulder joint; labrum of the hip joint; meniscus (medial meniscus of the knee joint) meniscus and lateral meniscus); or tendons and ligaments attached to bone In some embodiments, the fibrocartilage tissue disclosed herein is attached to the meniscus. Refers to the plate (e.g., medial or lateral meniscus or both).

[0038] As used herein, a tendon is a fiber that connects muscles to bones and can withstand tension. In some embodiments, the tendon disclosed herein refers to the patellar tendon. Additionally or alternatively, the tendon disclosed herein refers to the Achilles tendon. In one embodiment, the tendons used herein are human tendons. The tendons used are tendons of non-human animals.

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

[0040] As used herein, the terms "encapsulation," "embedding," or grammatical variations thereof The mold may be at least partially (e.g., at least 99% of the surface of the structure, or at least 9 8%, or at least 97%, or at least 96%, or at least 95%, or or at least 90%, or at least 85%, or at least 80%, or at least at least 75%, or at least 70%, or at least 65%, or at least 6 0%, or at least 55%, or at least 50%, or at least 45%, or or at least 40%, or at least 35%, or at least 30%, or less at least 25%, or at least 20%, or at least 15%, or at least 1 0%, or at least 5%), and in some embodiments completely (e.g., the surface of the structure approximately 100% of the total area of ​​the structure, and It refers to the orientation or position of a structure or component, such as the area that is (directly or indirectly) surrounded by it. In some embodiments, the term "encapsulation" or grammatical variants thereof may refer to one or more other structures. A structure or configuration that is completely surrounded by an object or one or more other components In some embodiments, the term "encapsulation" refers to at least One non-limiting example is an Al The tissue is surrounded by an alginate gel, or an alginate gel and a semipermeable membrane, and is called encapsulated tissue.

[0041] The term "exposure" or its grammatical variants refers to contact (the interaction between two or more components) In various embodiments, the term "exposure" refers to As used herein, one non-limiting example is the culture of a tissue. Contact with the culture medium (e.g., encasing or immersing the tissue in the culture medium) or Qualitatively, or via a method consisting of (tissue encapsulated in alginate gel, or encapsulating tissue in alginate gel and semipermeable membrane, or using any encapsulation. A further non-limiting example is exposure of a culture medium to a tissue in a chamber. Tissue (encapsulated in alginate gel or encapsulated in alginate gel and semipermeable membrane) Incubation of tissue containing cells in culture medium (e.g., cellularized tissue, or tissue without any encapsulation) Addition of culture medium into the chamber (e.g., the first medium present in the chamber, if any) At least a portion of the solution (e.g., about 50%, about 60%, about 70%, about 80%, about 90%) %, or more) or complete (i.e., about 100%) substitution) It is exposure via a method that essentially consists of or consists of.

[0042] As used herein, the term conserved or grammatical variants thereof refer to a particular Duration (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 4 weeks, at least 5 weeks , at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, Stored cells, tissues, organs, or tissues for a period of time (e.g., 10 weeks or longer) or while maintaining the viability of other components (e.g., approximately 100% of the viability before storage, or or at least 99%, or at least 98%, or at least 97%, or at least at least 96%, or at least 95%, or at least 90%, or at least 8 5%, or at least 80%, or at least 75%, or at least 70%, or or at least 65%, or at least 60%, or at least 55%, or less at least 50%, or at least 45%, or at least 40%, or at least 3 5%, or at least 30%, or at least 25%, or at least 20%, or or at least 15%, or at least 10%, or at least 5% survival rate Methods for assessing the viability of tissues or organs are available to those of skill in the art. (see, for example, the methods disclosed in the Examples).

[0043] As used herein, culture medium refers to a medium that, for example, supports the growth of cells or Used in the culture of cells, tissues or organs to maintain viability or to maintain tissue viability In some embodiments, the culture medium comprises or is selected from the following: Consists of or further comprises: one or more of the amino acid(s) or One or more carbohydrates as an energy source or one of the trace elements or one or more of the vitamins, or one or more of the salts, or any other possible additives. one or more of the additional components (e.g., cell growth and / or productivity and / or affect the quality of the product), or any combination of these. In one embodiment, the culture medium is Dulbecco's modified essential medium (DMEM), or Ham's F-1 2 medium (F12), or Roswell Park Memorial Institute (RPMI) 1640 medium, or may comprise, consist essentially of, or further consist of any combination thereof. In yet another embodiment, the culture medium is DMEM / F12 (a 1:1 mixture of DMEM and F12). ).

[0044] In some embodiments, complete media contains additional components that may contribute to cell growth. One or more of the following (one or more of the growth factors, one or more of the hormones, one or more of the proteins) one or more of a serum, one or more of a serum substitute, etc. In a further embodiment, the complete medium is supplemented with serum, such as fetal bovine serum (FBS). Additionally or alternatively, a complete medium refers to a culture medium further comprising about 1% to about 20% ( About 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or 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%, etc.

[0045] As used herein, semi-permeable membranes, also called dialysis membranes, are membranes that are capable of filtering certain particles (molecules or ions) by osmosis, and sometimes by diffusion, passive transport, or active transport. by more specialized processes that facilitate the selection of molecules by size or molecular weight. It refers to a type of biological or synthetic polymeric membrane that allows particles to pass through it in quantity.

[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 a further embodiment, 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 thickness of at least about 250 picometers. torr (referred to herein as "pm"), at least about 300 pm, at least about 50 0 pm, at least about 750 pm, at least about 1 nm, at least about 20 nm, at least At least about 50 nm, at least about 100 nm, at least about 150 nm, at least about 20 0 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 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 At least about 300 μm, at least about 400 μm, at least about 500 μm, at least about 60 0 μm, at least about 700 μm, at least about 800 μm, at least about 900 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 at least about 2200 μm, at least about 2300 μm, at least about 2400 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 4 000 μm, at least about 5000 μm, at least about 6000 μm, at least about 70 00 μm, at least about 8000 μm, at least about 9000 μm, at least about 10 m m or greater and any range of pore sizes therebetween. The semipermeable membranes disclosed herein have a viscosity of 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 600nm, up to about 700nm, up to about 800nm, up to about 900nm, up to about 1μm Up to about 20 μm, up to about 50 μm, up to about 100 μm, up to about 150 μm, up to about 200 up to about 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 1 Up to 100μm, up to about 1200μm, up to about 1300μm, up to about 1400μm, up to about 1 Up to 500μm, up to about 1600μm, up to about 1700μm, up to about 1800μm, up to about 1 Up to 900μm, up to about 2000μm, up to about 2100μm, up to about 2200μm, up to about 2 Up to 300μm, up to about 2400μm, up to about 2500μm, up to about 2600μm, up to about 2 Up to 700μm, up to about 2800μm, up to about 3900μm, up to about 3000μm, up to about 4 Up to about 5000 μm, up to about 6000 μm, up to about 7000 μm, up to about 8 000 μm, up to about 9000 μm, up to about 10 mm, or more In some embodiments, the semipermeable membranes disclosed herein have pore sizes of about 0.22 μm to about 1 000μm, and ranges or sizes therebetween (approximately 0.22μm to approximately 100μm and or about 100 μm to about 1000 μm and any range therebetween). In some embodiments, the semipermeable membranes disclosed herein have pore sizes of about 100 μm to about 1000 μm. μm and the range between them (100 μm to about 200 μm, 100 μm to about 300 μm, 10 0μm~about 400μm, 100μm~about 500μm, 100μm~about 600μm, 100 μm to about 700 μm, 100 μm to about 800 μm, or about 100 μm to about 900 μm In various embodiments, the skilled artisan will be able to determine the components (e.g., of the culture medium (e.g., complete medium) One or more of the proteins (or proteins) can cross the membrane, but the other proteins (or proteins) can be trapped in the cell, tissue (e.g., capsule) The present invention relates to a method for treating a vascular endothelial cell, such as a vascular endothelial cell, ... The pore size of the semipermeable membrane disclosed is selected.

[0047] In some embodiments, the molecular weight cut-off (MWCO) describes the permeability of a semipermeable membrane. The molecular weight of the particles is then measured, and more than 90% of the particles with the lowest molecular weight are retained by the membrane. In various embodiments, the term refers to a quantity (such as Daltons (Da) or kilodaltons (kDa)). The MWCO of the semipermeable membrane is selected or determined by selecting the pore size. In various embodiments, the pore size of the semipermeable membrane is selected or determined by selecting the MWCO. In this embodiment, the MWCO is selected based on the solution and its concentration used herein. The solution may be selected from a variety of options (e.g., calcium, barium, or chelate solutions). In the present case, the skilled artisan will be able to determine whether one or more of the components of the culture medium (such as a complete medium) is membrane-bound. can pass through cells, tissues (e.g., encapsulated tissues), or alginate. The MWCO of the semipermeable membrane disclosed herein is selected so that one or more of the gels cannot pass through. In some embodiments, the MWCO of the semipermeable membranes disclosed herein is about 1 kDa to about 1 kDa. About 10,000,000 kDa and ranges therebetween (e.g., about 1 kDa to about 2,000 In some embodiments, the MWCO of the semipermeable membrane disclosed herein is 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 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 Da, 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, 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 10 0,000 kDa, at least about 200,000 kDa, at least about 300,000 kDa Da, 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, 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 a, 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 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 may 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 500 kDa, up to about 600 kDa, up to about 700 kDa, up to about 800 kDa, Up to about 900kDa, up to about 1,000kDa, up to about 2,000kDa, up to about 3,000 Up to about 4,000kDa, Up to about 5,000kDa, Up to about 6,000kDa Up to about 7,000 kDa, up to about 8,000 kDa, up to about 9,000 kDa, 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, Up to about 40,000kDa, up to about 200,000kDa, up to about 300,000kDa Up to 0,000kDa, Up to about 500,000kDa, Up to about 600,000kDa, Up to about Up to 700,000kDa, Up to about 800,000kDa, Up to about 900,000kDa , up to about 1,000,000 kDa, up to about 2,000,000 kDa, up to about 3,000, Up to approx. 4,000,000 kDa, Up to approx. 5,000,000 kDa , up to about 6,000,000 kDa, up to about 7,000,000 kDa, up to about 8,000, 000kDa, up to about 9,000,000kDa, up to about 10,000,000kDa In some embodiments, the MWC of the semipermeable membranes disclosed herein is at or above 100 nm. O 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 can selectively transport certain fluids through it. In contrast, a completely semipermeable membrane can transport fluids through it without restriction, whereas an alginate gelatinous membrane can transport fluids through it without restriction. Such a completely semipermeable membrane may be formed by a mesh (e.g., medical grade mesh) Thus, in some embodiments, the mesh opening size is determined by the pore size or refers to the average and can be used interchangeably with membrane pore size.

[0049] In some embodiments, the semi-permeable membrane is about 1% to about 50% and any range or percentage therebetween. In some embodiments, the mesh has an aperture ratio (also referred to herein as mesh aperture ratio). In the present invention, the semipermeable membrane has a mesh opening rate of at least about 0.1%, at least about 0.2%, or 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 less Additionally or alternatively, the semipermeable membrane has an opening ratio of less than about 5%, less than about 10%, or at least about 45%. %, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, Less than 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 In some embodiments, the semipermeable membrane has a mesh opening rate of about 1%, about 2% or less. %, 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 5 It has 0%.

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

[0051] As used herein, percentage (%) refers to the percentage of a component in a mixture (solid or liquid). Percentages (%) are used to indicate the concentration of an element. In some embodiments, percentages (%) refer to mass ratios or In other embodiments, the percentages (%) refer to volume ratios. In some embodiments, percentage (%) refers to molar ratio. In some embodiments, x% of a composition in solution An element refers to x grams of a component in 100 mL of solution.

[0052] As used herein, a chelating solution is a solution containing a chelating agent (such as calcium chelating agent). Non-limiting examples of chelating agents include edetic acid, citric acid, ede Disodium edetate anhydrous, calcium disodium edetate anhydrous, ethylenediamine tetraacetate Acetic acid (EDTA), or ethylene glycol-bis(β-aminoethyl ether)-N In some embodiments, chelates may include ethyl esters of N,N',N'-tetraacetic acid (EGTA). The inhibitors are 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, and the solution may contain additional components (e.g., physiological salt water, etc.).

[0054] An inflammatory response, which is used interchangeably herein with inflammation, is an inflammation caused by bacteria, trauma, toxins, heat, or This occurs when tissue is damaged by any other cause (such as ex vivo storage) During an inflammatory response, tissues react with inflammatory factors (histamine, bradykinin, ceramide, etc.) As used herein, the term "antibody" refers to a hormone that releases hormones, such as hormones, ... and hormone receptors (e.g., steroids, cytokines, nitric oxide, etc.). Cytokines (interleukin (IL)-1, IL-1β, IL-6, and tumor necrosis Tumor necrosis factor-α (TNFα) and other immune cells (such as activated macrophages), tissues, or It is produced by organs or cartilage cells.

[0055] As used herein, the term "allograft" refers to a transplant that is of the same species as the recipient, but Allograft tissue refers to tissue transplants derived from donors who are not genetically identical. The tissue can be removed from the donor by known techniques. For example, the general angiography of allografts. Surgical or other physical interventions include extraction, resection, amputation, transplantation, microsurgery, general surgery, These may include, but are not limited to, surgical procedures, laser surgery, robotic surgery, or autopsy. Not limited to.

[0056] Unless otherwise defined, all technical and scientific terms used herein are defined by the The present disclosure has the meaning commonly understood by those skilled in the art. Any methods and materials similar or equivalent to those described herein may be used in the practice or testing of this disclosure. However, preferred methods and materials are described herein. EMBODIMENTS FOR CARRYING OUT THE DISCLOSURE

[0057] Sources of tissue or allografts include all organism types (human, porcine, ovine, bovine, canine, and horses, etc. In one embodiment, the source of the tissue or allograft is human. Potential tissue or allograft sources include eye, brain, heart, kidney, liver, intestine, bone, cartilage, skin, Lung, thyroid, stomach, ligament, tendon tissue, or any other tissue or cell that may require a transplant. In various embodiments, the source of the cells may include, but is not limited to, an allograft. Or the tissue may be a spine, a scapula, a humerus, a radius, an ulna, a pelvis, a femur, a tibia, a fibula, The bones of the patella, talus, phalanges, or temporomandibular joint, or any combination of these In various embodiments, the allograft or tissue may include cartilage or meniscus tissue. Although the description herein may refer to allograft tissue, Those skilled in the art will recognize that other tissues may be used in the methods described herein.

[0058] In some embodiments, the capsule is encapsulated by a gel, a semipermeable membrane, or both a gel and a semipermeable membrane. A cellular tissue is one that has a dimension greater than about 1 millimeter (mm) in any direction of the tissue (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 longer). Tissue encapsulated by a gel, a semipermeable membrane, or both a gel and a semipermeable membrane is All dimensions are approximately 20 cm or less (e.g., approximately 15 cm or less, approximately 10 cm or less, Approximately 5 cm or less, approximately 4 cm or less, approximately 3 cm or less, approximately 2 cm or less, approximately 1 cm or less, or shorter).

[0059] Osteochondral allografts (OCA) are currently stored at 4°C and are maintained for 28 days after donor harvest. The disease testing protocol takes 2 weeks to implement, making it a good time for transplantation. is limited to 14 days, thereby significantly limiting availability to potential recipients. This study is intended to provide an understanding of the shelf life of OCA and its clinical use and clinical outcomes. This was done to find improved storage conditions that would increase the chances of improvement.

[0060] High-purity alginate (UPAL) is highly purified and has low endotoxin toxicity ( (less than 1 / 10,000 compared to conventional alginates). In some embodiments, the UP AL is a method to precipitate alginate by adding acid to conventional alginate, resulting in insoluble alginate. The method of the present invention further comprises, or consists essentially of, a step of isolating the It is purified from arginate. For example, Igarashi, et al., Cartila ge 2012:70. As a scaffolding material, UPAL has been shown to Enhances the repair of osteochondral defects (Igarashi, et al., Cartilage 2 012:70; and Baba, et al., Am J Sports Med. 20 18;1970), as an injectable material into joints to slow the progression of osteoarthritis (Ts ukuda,et al.,J Biomed Mater Res A.2015:3 441) Alginate is similar to the matrix of articular cartilage, and alginic acid In the form of beads, it is preferable as an embedding material for three-dimensional culture (Chiba, et al. .,Spine J.2007:694), UPAL is the best way to prevent the decomposition of OCA. It is suggested that it can be expected to act as a suitable storage medium. It is believed 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 determine the efficacy and safety of OCA obtained from porcine knees for 4 weeks. To evaluate the effect of UPAL on cell viability and cartilage matrix status after storage. .

[0061] Alginic acid, also called algin, is a polysaccharide widely distributed in the cell walls of brown algae. It is hydrophilic and forms a viscous gum when hydrated. Sodium and calcium The salts thereof with metals such as glycerol and glycerol are known as alginates. Gluconate (G), mannuronate (M), or glucuronate-mannuronate block? The strength of the gel formed is determined by the ratio of sugars. In this embodiment, the M / G ratio of the alginic acid and / or its salt is usually about 0.2 to about 4.0, about 0.4 to about 3.0, and about 0.5 to about 3.0. Natural product derived polymeric materials are generally classified into various classes. Since it is an aggregate of low molecular weight molecules, it is measured as a molecular weight distribution having a certain range. The molecular weight of alginate was determined by SEC-MALS (size exclusion chromatography with multi-angle light scattering detector). Standard calculation method (ASTM F2605-16(2016), A It can be measured by the STM International lab. In an embodiment, the molecular weight of the alginic acid and / or its salts used is usually determined by SEC- When measured by the MALS method, it is about 10,000 to about 2,000,000, about 15,0 00 to 1,500,000, 20,000 to 1,000,000, or or a range selected from within the range of about 25,000 to about 500,000 It is.

[0062] It is well known that high survival rates of chondrocytes result in favorable in vivo function (Pal lante, et al., Am J Sports Med.2012:1814). Encapsulation of OCA with UPAL gel significantly improved the surface area compared to other groups. The survival rate of chondrocytes was significantly increased. The survival rate of the middle section and The total thickness also tended to be higher than in the other groups. These results suggest that the surface area was weak. and that cell viability in the surface compartment is sensitive to its storage conditions. (Pallante, et al., Am J Sports Med. 20 09:24S). No structural changes were observed in the UPAL gel encapsulation during storage. In addition, the maintenance of cell viability and matrix content in alginate gels, as well as Polyglycan (PG) synthesis was suppressed by low total nitric oxide (NO) concentrations in the storage medium. Embedding in alginate gel also improves the function of the tissue during transport to the clinical site. The use of UPAL gel encapsulation provides some protection to the tissue from mechanical damage. can be effective in improving the quality of OCA after 28 days of storage. The use of AL gel improves clinical outcomes after osteochondral allograft transplantation and reduces the risk of osteochondral allograft failure during storage. Cell death may be minimal.

[0063] As illustrated in the examples disclosed herein, osteochondral allografts (OCs) were implanted in UPAL gel. A) was embedded (i.e., encapsulated) to increase cell viability after 28 days or 6 weeks of storage. The survival rate and matrix composition are improved. Matrix synthesis is improved compared to control-stored OCA. Furthermore, cell viability with alginate was significantly higher than without alginate. The cell viability of the grafted tissue was higher than that of the control culture. The cell viability was significantly higher than that of tissue stored in medium alone. The UPAL membrane group with tissue embedded in the semipermeable membrane showed higher cell viability than all other test conditions. Without wishing to be bound by theory, the conditions in the UPAL membrane group were It provides excellent access to nutrients through the membrane and also provides physical protection during storage. Furthermore, embedding in a single alginate gel protects tissue from mechanical damage during storage. At the end of the storage period, the tissue containing the alginate gel is able to absorb the medium. The chelating solution is removed from the device or system by replacing it with the chelating solution. Any remaining residues surrounding the tissue may be removed by exposure to EDTA or sodium citrate. The alginate gel that is being applied is also dissolved; dissolution may be performed in the operating room immediately prior to implantation. Embedding in acetic acid gel protects the tissue somewhat from mechanical damage during transportation to the clinical site. Be protected.

[0064] There are several methods available for preserving cartilage tissue, but the methods and equipment disclosed herein are The device or system has an advantage or advantages over other methods, for example during transportation. For example, one such advantage over liquid storage systems is the ability to store the cells by embedding them in alginate gel. Improved tissue storage conditions; protecting tissue from shock, vibration, or shaking during transport to the hospital. Protects tissue, thereby preventing tissue damage during transport; improves cell viability and suppression of inflammatory factors (such as cytokines and nitric oxide); and It may be noted that the storage and transportation stages can be covered by an enclosed system. Therefore, the present invention is not limited to the above. Preserving the allograft tissue improves the maintenance of cells in the tissue. The semipermeable membrane may be a partially semipermeable membrane or a completely semipermeable membrane. For example, a partially semipermeable membrane may be It may be possible to selectively transport certain fluids through the membrane. The permeable membrane can transport fluid through it without restriction, but the passage of the alginate gel and tissue Such a completely semipermeable membrane may be made of a mesh (e.g., a medical grade mesh). ). Modification of storage conditions improves the quality of tissues (such as osteochondral allografts) disclosed herein will be done.

[0065] Thus, in one aspect, the present disclosure provides a method, system, or device for tissue preservation. The method includes the steps of encapsulating tissue in a UPAL gel and subjecting the encapsulated tissue to For example, the tissue may be stored for about 24 hours to about 8 weeks prior to transplantation. In various embodiments, the tissue is a tissue other than osteochondral tissue as disclosed herein. It is an organization.

[0066] An unexpected advantage of the procedures disclosed herein in any aspect or embodiment is the ability to The material can be kept viable and sterile for extended periods of time compared to prior art methods. For example, typically in the prior art, the allograft is extracted from the donor. When prepared, the tissue was stored on ice or at approximately 4°C. tended to remain viable for approximately 21 to 28 days. The procedure described herein surprisingly and unexpectedly increases the survival rate of allograft tissue. Tissues prepared and stored according to the procedure described in showed long-term storage compared to storage at 4 °C. Continue to survive. Long-term means 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 to 2 days prior to use (e.g., implantation). The fruit is stored for 1, 7, 14, 28 days, 4, 6, or 8 weeks. In embodiments, at least about 10% of the cells of the tissue are erythrocytes or cerebrospinal fluid (ECF) cells ... 40% remain viable after storage.

[0067] In another aspect, the present disclosure relates to a method for the treatment of osteochondral tissue or other tissue(s) disclosed herein. The present invention provides a method for storing a human organ or organs, for example, prior to transplantation. Obtaining osteochondral tissue or other tissue(s) or organ(s), Cartilage tissue or other tissue(s) or organ(s) are mixed with a semi-solid medium containing an alginate solution. A step of placing the semi-permeable membrane chamber in a culture chamber, and a step of placing the calcium solution or the barrier solution in the culture chamber. Addition of calcium solution or both to the culture chamber, thereby allowing the growth of osteochondral tissue or other tissues (complexes) The method includes the steps of encapsulating a plurality of cells or organs in an alginate gel, adding the cells to the gel, and culturing the cells. The method includes the steps of injecting nutrient medium into the culture chamber and, if necessary, placing the culture chamber in a storage container. In various embodiments, the composition may include (e.g., comprises, consists essentially of, or consists of). The encapsulated osteochondral tissue remains viable for implantation up to 8 weeks after the obtaining procedure.

[0068] Embedded in an alginate gel and optional semipermeable membrane to improve cell retention in tissue Methods, materials, compositions, systems, or devices relating to the preservation of allograft tissue are provided herein. In some embodiments, the method, material, formulation, system, or apparatus It also provides 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. Other methods of preserving tissue(s) or organ(s) disclosed herein are provided. In some embodiments, the tissue(s) or organ(s) is / are osteochondral tissue (cartilage of bone). and a thickness ratio of about 20:1 to about 1:10 and ranges therebetween, for example, about 4:1. (e.g., osteochondral core trimmed to fit the bone), articular cartilage without any subchondral bone, fibrous Cartilage (meniscus, e.g., medial or lateral meniscus or both), tendons (patella tendon or Achilles tendon), the cornea, or the cornea with vitreous.

[0070] In some embodiments, the method comprises the steps of: encapsulating organ(s) in alginate gel and forming gel-encapsulated osteochondral tissue or other suitable tissue(s) or organ(s) to the culture medium. In some embodiments, In the form, the gel-encapsulated tissue is immersed in the gel for a period selected from about 24 hours to about 8 weeks prior to implantation. and ranges therebetween, from about 28 days to at least about 6 weeks, for example, from about 24 hours to 8 weeks. Additionally or alternatively, the method may involve culturing osteochondral tissue or other suitable tissue. The appropriate tissue(s) or organ(s) are encapsulated in an alginate gel. The culture medium is then cultured to obtain the cultured osteochondral tissue or other suitable tissue(s) or organ(s). At least about 2 weeks (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 The method includes (e.g., includes or includes exposing the subject to (e.g., for a period of 1 week or longer) In some embodiments, the method comprises the steps of: or other suitable tissue(s) or organ(s) encapsulated in an alginate gel. and encapsulating the osteochondral tissue or other suitable tissue(s) or organ(s). ) in the 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 6 weeks, 2 weeks to 7 weeks, 2 weeks to 8 weeks, 2 weeks to 9 weeks, 2 weeks 10 weeks, 3 weeks to 4 weeks, 3 weeks to 5 weeks, 3 weeks to 6 weeks, 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, 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 each of the above or consisting essentially of, or (Further consisting of).

[0071] In various embodiments, the method includes encapsulating the tissue(s) or organ(s) in a semipermeable membrane. In various embodiments, the semipermeable membrane has a thickness of at least about 250 pm. (About 0.22 μm to about 100 μm and the range therebetween, about 0.22 μm to about 1000 μm and the range therebetween, or from about 100 μm to about 1000 μm and the range therebetween) Additionally or alternatively, the semipermeable membrane has a pore size of about 50 molecular weight cut off (MWCO). In some embodiments, the semipermeable membrane has a molecular weight of about 200,000 kDa or about 200,000 kDa. The mesh opening ratio is about 1% to about 50% and any range or percentage therebetween (e.g., about 2%). In various embodiments, the tissue is encapsulated prior to, during, or after encapsulation in a semipermeable membrane. After capsule formation, the particles are encapsulated in a gel.

[0072] In various embodiments, the alginate gel is a purified gel or a highly purified alginate. alginate gel (UPAL). In various embodiments, the alginate gel is about 0.5% ~ about 2.0% alginate and ranges or percentages therebetween (e.g., about 1.2% alginate In various embodiments, UPAL comprises or consists essentially of about 0.05 g of UPAL. Gel at concentrations ranging from 0.5% to about 2.0% and ranges or percentages therebetween (e.g., about 1.2%) It is present in the

[0073] Alginate is a natural polysaccharide that is found in 30-60% of brown algae (such as kelp). Gynates consist essentially of or consist of D-mannuronic acid (M) and L-guluronic acid (G). It is a heteropolymer aggregate consisting of or consisting of a calicopolymer consisting of M and G. The carboxyl group has a high ion exchange capacity and easily binds to various cations. See the example of the cation moiety. The physical properties of alginates are determined by the presence of different types of cations. In particular, the cross-linking of alginate with calcium ions A water-insoluble hydrated gel is formed. 4 ~10 6 Argina 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 It is a water-insoluble white powder. The purification process of alginate from raw materials includes: , consisting essentially of, or consisting of: drying and grinding; washing and swelling; extracting; filtering filtering; solidification and precipitation; and drying of alginates (wet). Foods (e.g. thickeners, gels) Agents for improving noodle quality, pet food, farmed fish feed, binders, and thickeners. etc.), cosmetics, textile printing pastes, etc., as well as in the pharmaceutical field (e.g. tablet disintegration It is widely used in the fields of medicine, and as a stomach wall protectant. Alginate is the main component of impression materials used in industry. It also has excellent cartilage formation ability. has.

[0074] Highly purified alginic acid (UPAL) has been developed. Conventional alginates have low endotoxin levels. The endotoxin levels are so high that it cannot be used clinically except for oral administration or as a wound dressing. High levels of acetylcholine induce an inflammatory response in vivo. This adverse effect was observed in alginate-based Mochida has developed an endotoxin level reagent called UPAL. UPAL has developed a highly purified alginate with extremely low levels of endotoxins. Eliminates the in vivo inflammatory response that may be associated with alginate-based materials, allowing for clinical application For example, UPAL gel enhances cell proliferation of bone marrow stromal cells (BMSCs). For example, see Igarashi, Iwasaki, et al. JBMR A, 2010. Various tests have demonstrated that UPAL gel promotes the regeneration of hyaline-like cartilage. Candidates who wish to use sodium alginate in the field of regenerative medicine or cell therapy Clinical research using this method is currently being conducted.

[0075] In various embodiments, alginate (ALG) as used herein refers to a variety of molecules. Amount(10 4 ~10 6 (Da and any values ​​therebetween). The higher the pH, the firmer and more elastic the alginate gel will be. In practicing the disclosed methods, one is prepared to select an alginate of appropriate molecular weight. There will be.

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

[0077] In various embodiments, the encapsulated osteochondral tissue or other suitable tissue(s) may also be Or the organ(s) must be stored for approximately 28 days (approximately 4 weeks) before transplantation or approximately 6 weeks before transplantation. In some embodiments, the encapsulated tissue(s) or organ(s) are stored. For example, at least about 2 weeks (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, In some embodiments, the composition is stored for at least about 10 weeks, or longer. The encapsulated tissue(s) or organ(s) may be incubated for, e.g., about 2 weeks to about 3 days prior to implantation. 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 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, 3 weeks to 9 weeks, 3 weeks to 10 weeks, 4 weeks to 5 weeks, 4 weeks or more 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 and up 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 ~9 weeks, ~10 weeks, ~7 weeks, ~8 weeks, ~6 weeks The mixture is stored for a period ranging from about 1 to about 9 weeks, from about 6 to about 10 weeks, or between each of the aforementioned periods. In various embodiments, the encapsulated cornea is stored for at least about two weeks prior to implantation. .

[0078] In various embodiments, the culture medium is replenished following exposure of the gel-encapsulated tissue to the culture medium. In various embodiments, the culture medium is not Refills 1 or less, or 2 or less, or 3 or less, or 4 or less, or 5 or less will be done.

[0079] In various embodiments, the encapsulated tissue(s) or organ(s) may be heated to below 10° C. For example, from about 0°C to about 4°C and any range therebetween, or at about 4°C.

[0080] In various embodiments, the encapsulated tissue(s) or organ(s) can be artificially controlled. Controlled CO2 levels, or artificially controlled O2 levels, or artificially controlled The material is not stored at elevated temperatures, elevated humidity, or any combination thereof.

[0081] In various embodiments, the method further comprises exposing the alginate gel to a culture medium, and optionally Prior to implantation, for example, the encapsulated tissue(s) or organ(s) may be exposed to a chelating solution. Dissolved by a process that comprises, consists essentially of, or consists of exposing In a further embodiment, the chelate solution is a sodium citrate solution or or ethylenediaminetetraacetic acid (EDTA) solution or sodium citrate and EDTA The present invention may comprise, consist essentially of, or further consist of a solution comprising both of

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

[0083] In various embodiments, the encapsulated tissue(s) or organ(s) can be, for example, Incorporation of carboxylates into the alginate gel, into the semipermeable membrane, or into both the alginate gel and the semipermeable membrane Release of inflammatory factor(s) compared to tissues or organs stored without encapsulation In a further embodiment, the inflammatory factor(s) is / are a cytokine. one or more of, or nitric oxide, or nitric oxide and cytokines Comprising, consisting essentially of, or further consisting of both.

[0084] In various embodiments, the encapsulated tissue(s) or organ(s) prior to transplantation may be Prior to the encapsulation step or the obtaining step (i.e., obtaining tissue(s) or organ(s) from a subject, etc. (c) obtaining a cell viability after the step (c), At least about 60%, or at least about 65%, or at least about 70% of the rate, is at least about 75%, or at least about 80%, or at least about 85%, or Maintain at least 90%, or at least 95% or higher cell viability.

[0085] In various embodiments, the encapsulated tissue(s) or organ (e.g., The cells (or cells) are washed with a buffer, such as saline or culture medium.

[0086] In one aspect, the present disclosure provides a method for storing osteochondral tissue. Methods for storing the tissue(s) or organ(s) disclosed herein are provided. In some embodiments, the tissue(s) or organ(s) are treated with osteochondral tissue (e.g., osteochondral tissue) prior to transplantation. The thickness of the bone to the cartilage is adjusted to have a ratio of about 20:1 to about 1:10, for example, about 4:1. Rimmed osteochondral core, etc.), articular cartilage without any subchondral bone, fibrocartilage tissue (Meniscus, e.g., medial or lateral meniscus, or both medial and lateral meniscus, etc.) ), tendon (such as the patellar tendon or Achilles tendon), cornea, or cornea with vitreous body. do.

[0087] In some embodiments, the method comprises the steps of: (a) administering to the patient a portion of osteochondral tissue or other suitable tissue to be transplanted; Optionally, obtaining the resulting osteochondral tissue or other suitable organ(s), The appropriate tissue(s) or organ(s) is placed in a semi-permeable chamber containing an alginate solution. a step of inserting the semipermeable membrane chamber into the culture chamber, and a step of introducing a calcium solution or a barium solution or adding both to the culture chamber, thereby growing osteochondral tissue or other suitable tissue. The tissue(s) or organ(s) are encapsulated in an alginate gel. Step 2: Injecting a culture medium into the alginate gel or into the culture chamber, or incubating the alginate gel and optionally placing the incubation chamber in a storage container (e.g., , comprising, or consisting essentially of, or further consisting of.

[0088] In some embodiments, the encapsulated osteochondral tissue or other suitable tissue(s) ) or organ(s) for at least about 2 weeks (at least about 4 weeks or In some embodiments, the encapsulated The osteochondral tissue or other suitable tissue(s) or organ(s) may be cultured for 8 days after the step of obtaining the tissue(s). In some embodiments, the encapsulated osteochondral tissue remains viable for up to a week for implantation. The tissue or other suitable tissue(s) or organ(s) may be incubated for at least about two weeks ( 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 bone marrow remains viable for transplantation (e.g., for a period of time). Bone 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 3 weeks to 5 weeks, 3 weeks to 6 weeks, 3 weeks to 7 weeks, 3 weeks to 8 weeks 3 weeks to 9 weeks, 3 weeks to 10 weeks, 4 weeks to 5 weeks, 4 weeks to 6 weeks 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 1 0 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, 5 weeks to 10 weeks, 6 weeks to 7 weeks, 6 weeks to 8 weeks, 6 weeks and up The cells remain viable for transplantation for about 9 weeks, or for about 6 weeks to about 10 weeks. The encapsulated corneas remain viable for transplantation for at least approximately two weeks.

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

[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 semi-permeable membrane chamber. In various embodiments, the semipermeable membrane has a thickness of at least about 250 pm (about 0.22 μm to About 100 μm and the range therebetween, about 0.22 μm to about 1000 μm and the range therebetween or about 100 μm to about 1000 μm and ranges therebetween. Alternatively, the semipermeable membrane may have a molecular weight cut off (MWCO) of about 50 kDa or about 20 In some embodiments, the semipermeable membrane has a mesh opening rate of about 1%. having / including up to about 50% and any range or percentage therebetween (e.g., about 2%).

[0092] In various embodiments, the alginate solution is a high purity alginate solution. In this embodiment, the alginate solution contains about 0.5% to about 2.0% alginate and any range therebetween. or percentage, optionally comprising or consisting essentially of about 1.2% alginate. .

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

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

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

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

[0097] In various embodiments, the method includes, for example, encapsulating tissue(s) or organ(s). The alginate gel is dissolved prior to implantation by a method including exposing the alginate gel to a chelating solution. In a further embodiment, the chelating solution is a sodium citrate solution. or ethylenediaminetetraacetic acid (EDTA) solution or sodium citrate and EDT A solution comprising, consisting essentially of, or further consisting of both A and B. Alternatively, the alginate gel can be removed mechanically, 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, the tissue(s) advanced using the methods disclosed herein are or organ(s) (e.g., those that remain viable for transplantation at the time of donation) may be , in an alginate gel, or in a semipermeable membrane, or in both an alginate gel and a semipermeable membrane of inflammatory factor(s) compared to tissues or organs stored without encapsulation In a further embodiment, the inflammatory factor(s) is / are nitric oxide, is associated with one or more of the cytokines, or nitric oxide and one or more of the cytokines comprising, consisting essentially of, or further consisting of both of the above.

[0100] In various embodiments, the tissue(s) processed using the methods disclosed herein are The organ(s) (e.g., those that remain viable for transplantation at the time of donation) are encapsulated Prior to the extraction step or the obtaining step (i.e., for example, the extraction of tissue(s) or organ(s) from a subject), (including, consisting essentially of, or consisting of a step (a step or steps) of obtaining a cell viability after the ... 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 less maintain at least about 90%, or at least about 95% or higher cell viability .

[0101] In various embodiments, the method includes adding a buffer solution (such as saline) to the agarose prior to injection of culture medium. and further injecting the alginate gel into the culture chamber or into the alginate gel in the culture chamber. In a further embodiment, the soluble calcium or or reduce or eliminate barium.

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

[0103] In another aspect, the present disclosure provides a tissue transport system. For example, FIG. 1 illustrates a tissue culture device that may form part of the system. an inlet constructed to permit the entry of a fluid therethrough; a fluid inlet and an outlet, the fluid flow passing through said outlet being controlled by said inlet and outlet being connected to said inlet and outlet; An outer chamber (herein referred to as the "culture chamber") defining an outlet constructed to allow for the exit of In some embodiments, instead of having separate inlets and outlets, Additionally, the outer chamber may include a single valve that may be selectively used to provide fluid communication between the interior and exterior of the outer chamber. In some embodiments, the fluid may include an alginate solution, a calcium solution, , barium solution, saline, culture medium, or storage medium. The outer chamber is enclosed (i.e., defines a closed interior volume).

[0104] The tissue culture device also includes an inner chamber (also referred to herein as the "tissue chamber") disposed within an outer chamber. The interior chamber includes at least one wall formed from a semipermeable membrane (e.g., In various embodiments, the semipermeable membrane comprises a wall portion, a cap, or a bottom portion formed from the semipermeable membrane. may include, consist essentially of, or consist of a mesh. In form, the semi-permeable membrane can be a bag strainer structure or a cell strainer-like structure. For example, the inner chamber may be configured such that tissue is placed into a bag and then the bag is placed into the outer chamber. In some embodiments, the inner part may include a bag formed from a semi-permeable membrane or mesh. The chamber has walls formed from a non-porous material (e.g., a plastic, polymer, or metal). and the cap of the inner chamber may be formed at least in part from a semi-permeable membrane. (i.e., define a closed interior volume) and are adapted to hold or contain tissue within the interior chamber. (e.g., for tissue storage or transport) and surround or In some embodiments, the tissue to be preserved / transported is encapsulated in an alginate gel. After the tissue is placed in the inner chamber, an alginate solution is added to the inner chamber. 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, calcium solution or barium solution or both are introduced through the inlet. In another embodiment, both the calcium solution and the barium solution are communicated into the outer chamber. Before the inner chamber is placed into the outer chamber, a solution (e.g., a calcium solution and / or a barium solution) may be added. The tissue is then encapsulated in a liquid-filled container (by immersing the inner chamber in the liquid). When an alginate or other polymer network that is encapsulated is formed within the inner chamber, the inner chamber is encapsulated within the outer chamber. In some embodiments, the tissue within the inner chamber is placed in an alginate solution, calcium The tissue may be immersed in a solution, such as one or more of a solution, a barium solution, or a culture medium. In certain embodiments, the solution may be removed or replaced without expelling tissue from the internal chamber.

[0105] Although alginate gels are generally described herein, it is understood that the tissue may be cross-linked with any other cross-linked gel (e.g., For example, hydrogels or hydrophilic polymer networks, e.g., agarose, methylcellulose, Hyaluronan, elastin-like polypeptides, polyethylene glycol (PEG), poly( 2-Hydroxyethyl methacrylate (PHEMA), 2-Hydroxyethyl methacrylate acrylate (HEMA), methacrylic acid (MAA), PEG-PEGMA, carboxymethyl Cellulose (CMC), Polyvinylpyrrolidone (PVP), Acrylamide / Acrylic Acid Copolymer, linear cationic polyallyl ammonium chloride, poly(N-isopropyl acrylate) Poly(phenylene amide) (PNIPAM), chitosan, acrylate-modified PEG and acrylate Modified hyaluronic acid, amine end functionalized 4-arm star PEG, any other suitable hydrogel It should be appreciated that the polymer may be encapsulated in a polymeric or inorganic salt (e.g., a cellulose or polymer, or a combination thereof). In some embodiments, a gel as used herein is, for example, a gel that is formed by contacting a solution with the solution. In vivo partial or complete fusion of the chelating agent with the chelate solution (e.g., dissolving alginate gel in chelate solution) Additionally or alternatively, the gel as used herein may include a gel containing Culture medium, calcium solution, barium solution, or chelating solution can flow through the A gel (e.g., an alginate gel or any of the gels described herein) that encapsulates the tissue. Other gels or polymers) are used to keep the tissue placed in the chamber until it is used. Further, the semipermeable membrane or membrane forming at least a portion of the inner chamber The solution may be a fluid (e.g., calcium solution, barium solution, saline, culture medium, or Fluid flows through a semipermeable membrane or mesh so that the storage medium (storage medium) communicates with the interior volume of the inner chamber. Alginate gel or any other gel that encapsulates the tissue while allowing it to be Alternatively, it prevents the polymer from leaking out of the inner chamber.

[0106] In various embodiments, the gels disclosed herein, such as alginate gels, are Expose the gel to a chelating solution (such as an EDTA solution or a sodium citrate solution) disclosed in The composition may be dissolved by a process comprising, consisting essentially of, or consisting of the step of: Additionally or alternatively, the gels disclosed herein may be resistant to exposure to excess saline. The composition may be dissolved by a process comprising, consisting essentially of, or consisting of the steps of:

[0107] In some embodiments, the semi-permeable membrane allows fluid to pass from the inner chamber to the alginate while communicating with the inner chamber. Porosity (e.g., pores) constructed to prevent leakage of the acid solution or alginate gel The diameter is at least about 250 pm, for example, 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) In some embodiments, the inner chamber may include a dialysis cassette (e.g., SIGMA AL Dialysis cassettes available from DRICH® under the trade name SLIDE-A-LYZER It may include (

[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 is then mixed with calcium or barium solution or both. The gel can be formed by adding calcium solution or barium solution or both by injecting saline or culture medium ( For example, calcium or barium solution or both may be added to the outlet chamber. The liquid is washed (by discharging it from the outlet).

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

[0110] The reservoir can be stored for up to 4-8 weeks or transported to a hospital for transplantation. They can be stored for up to 8 weeks and transported to the hospital for transplantation. The medium is replaced with lysis buffer for a few minutes. Afterwards, the inner chamber is removed and the cells are placed in alginate gel. The encapsulated tissue is then transferred to an EDTA or sodium citrate solution to form an alginate gel. Once the alginate gel has dissolved, the tissue is ready for implantation into the subject. The tissue is collected and washed with saline.

[0111] 4B-4F are diagrams illustrating another embodiment of the tissue culture device 100 and the tissue culture device 10 1 is a schematic diagram of various components of the tissue culture device 100. The tissue culture device 100 includes an outer chamber 110 or A culture chamber and an inner chamber 120 or tissue chamber disposed at least partially inside the outer chamber 110. include.

[0112] As shown in FIG. 4D, the outer chamber 110 includes a fluid storage portion 111 and an upper end of the fluid storage portion 111. The fluid containment portion 111 includes an outer chamber cap 112 connected to the inner chamber 120. and receive fluids (e.g., culture medium, saline, calcium solution, washing solution, calcium chloride ... Define an internal volume constructed to contain the sample (e.g., the rate solution). As shown in Figure 4B-4D In particular, the fluid containment portion 111 may define a circular cross-section. 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 made of a transparent or translucent material (e.g., , acrylic, plastic, glass, etc.

[0113] The outer chamber cap 112 is removably or fixedly connected to the upper end of the fluid storage section 111. In some embodiments, the outer chamber cap 112 may be secured to the outer chamber 112 by, for example, threads, a friction fit, or the like. 10, the housing 111 may be secured to the housing 111 by a locking, snap-fit, interference fit, or any other suitable attachment mechanism. The outer chamber cap 112 has an opening 114 extending therethrough. The opening 114 is configured to receive at least a portion of the interior chamber 120 therethrough. An inlet 115 for communicating fluid into the fluid containment 111, and An outlet 117 for communicating fluid to the exterior of the portion 111 passes through the outer chamber cap 112. In some embodiments, the inlet 115 and the outlet 117 are defined by the opening 114. and the outer periphery of the outer chamber cap 112. Threads 116 are defined on the inner wall of the opening 114 and are mated with corresponding fittings defined on the interior chamber 120. It may be configured to mate with a mating thread.

[0114] As shown in FIGS. 4E to 4F, the inner chamber 120 includes a tissue storage section 121 and a tissue storage section 121. The tissue storage section 121 includes an inner chamber cap 122 connected to the upper end of the tissue storage section 121. an interior volume constructed to hold a tissue (e.g., any of the tissues described herein); In some embodiments, at least a portion of the tissue containment portion 121 is defined by a semi-permeable membrane (e.g. For example, any of the semipermeable membranes or meshes, such as medical grade meshes, described herein. For example, only the bottom of the tissue storage section 121 is 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 Semipermeable membrane (e.g., about 80%, at least about 90%, at least about 95%, or more) or only the walls of the tissue storage portion 121 may be formed completely or at least partially from 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. or both the walls and the bottom of the tissue storage compartment 121 may be 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) formed from a semipermeable membrane. In some embodiments, the tissue placed in the tissue storage section 121 may be aliquotted in alginate. Encapsulated in an acid gel.

[0115] The inner chamber cap 124 is connected to the upper end of the tissue storage section 121. For example, The series of threads 123 may be defined on the inner surface of the upper end of the tissue containment portion 121. 124 is a protrusion 124 protruding from the bottom of the inner chamber cap 124 toward the tissue storage section 121. 26. A series of mating threads 125 are defined radially on the outer surface of protrusion 121. 2, a first set of threads 123 and a second set of threads 124 for coupling to the tissue storage section 121. It can fit.

[0116] A second set of threads 127 are radially arranged on the outer surface of the upper end of the tissue containment section 121. 10 and configured to mate with threads 116 defined on the inner wall of the opening 114. The tissue containment section 121 has a second series of threads to secure the inner chamber 120 to the outer chamber 110. 127 engages with the threads 116. In some embodiments, the tissue containment device is configured to be inserted into the tissue containment device 111. When the portion 121 is disposed inside the fluid storage portion 111, the cap 124 passes through the opening 114. The inner chamber cap 110 is secured to the outer chamber 110 so that it cannot be inserted through the inner chamber cap 110 and remains positioned outside the outer chamber 110. The cross-sectional width (e.g., diameter) of 24 is greater than the cross-sectional width (e.g., diameter) of opening 114. In some embodiments, when the tissue storage portion 121 is removed from the outer chamber cap 112, the tissue In order to prevent the inner chamber cap 124 from coming off the storage portion 121, the inner chamber cap 124 A first set of threads 123 coupled to mating threads 125 of the opening 114 are threaded with threads 116 of the opening 114. In operation, the user may rotate the internal To connect the chamber 120 to the outer chamber 110 or to disconnect the inner chamber 120 from the outer chamber 110, an inner chamber cap is provided. The inner chamber 120 may be held by a cap 124. The tissue culture device 100 is As described, it is placed in a storage container or box for shipping.

[0117] In one embodiment, a semipermeable membrane (also referred to herein as a tissue chamber or semipermeable membrane chamber) is provided. comprising, consisting essentially of, or including an outer chamber (also referred to herein as the culture chamber) which contains an inner chamber; The present invention provides a system comprising: In embodiments, the walls of the interior chamber are 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%, The membrane may be formed from a semipermeable membrane (such as at least about 95%, or more).

[0118] In some embodiments, the semipermeable membrane has a thickness of at least about 250 pm, e.g., about 0.22 μm. m to about 100 μm and ranges therebetween, about 0.22 μm to about 1000 μm and ranges therebetween The pore size ranges from about 100 μm to about 1000 μm and ranges therebetween.

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

[0120] In some embodiments, the MWCO of the semipermeable 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) is filled with an alginate solution After filling the container with water, the container can be closed and secured in an outer container (also referred to herein as the outer chamber). Additionally or alternatively, the inner container / chamber can be used to retrieve tissue in the operating room for transplantation. The outer container / chamber can be removed for storage.

[0122] In various embodiments, the outer container (also referred to herein as the outer chamber) contains a crosslinking solution (i.e. i.e. calcium solution, barium solution, or both (e.g. calcium chloride solution or or barium chloride solution) or dissolving solution (i.e. That is, a chelating solution (e.g., EDTA solution or sodium citrate solution or ED A solution that dissolves alginate gel, such as a solution containing both TA and sodium citrate The inner vessel (also referred to herein as the inner chamber) includes an inlet and an outlet for injection. The gelation of alginate or dissolution of alginate can be performed aseptically without opening the tube (which is used for the It can be done.

[0123] In one embodiment, a newly designed endothelial cell is provided with an alginate gel and an optional semipermeable membrane. Provided herein is an embedded system or device.

[0124] In some embodiments, the system or device is mechanically In some embodiments, the tissue is treated with an alginate. The device or system is embedded in the

[0125] In some embodiments, the system or device is mechanically In some embodiments, the tissue is treated with alginate, providing some protection from injury. The tissue is then embedded in a device or system at 4°C and stored at 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) may be used. In some embodiments, the tissue is embedded, stored, and transferred using a device or system. After the device or system is placed in the alginate solution and calcium solution are added to the device or system. In some embodiments, the tissue is placed in a semi-permeable membrane chamber. Alginate solution, calcium solution (or barium solution or barium salts) and a solution comprising or consisting essentially of a calcium salt) is added to the chamber. In some embodiments, the alginate is a calcium or barium solution, or is gelled by adding both to the chamber. After gelation, the alginate gel can be infused with saline or culture medium to remove excess calcium. or by removing barium (i.e., soluble calcium or barium) Then, it is washed.

[0127] In some embodiments, the semi-permeable membrane chamber (i.e., the inner chamber) is separated from the culture chamber (i.e., the outer chamber). In some embodiments, the two bilayered channels are The members are housed in an outer box (i.e., a storage container or box) equipped with a temperature sensor. It is placed in.

[0128] In some embodiments, the tissue is then cultured for at least about two weeks (at least about four 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 organization may In some embodiments, the tissue is then stored for about 2 weeks to 10 weeks. 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 or more 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 ~4 weeks, ~5 weeks, ~6 weeks, ~7 weeks, ~3 weeks ~8 weeks, ~9 weeks, ~10 weeks, ~5 weeks, ~4 weeks 4 weeks to 6 weeks, 4 weeks to 7 weeks, 4 weeks to 8 weeks, 4 weeks to 9 weeks, 4 weeks 10 weeks, 5 weeks to 6 weeks, 5 weeks to 7 weeks, 5 weeks to 8 weeks, 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 The product may be stored for 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 4-8 weeks.

[0129] In some embodiments, the outer box with the two chambers is transported to the hospital. In some embodiments, after arrival at the hospital, the alginate gel is applied in the operating room. It is dissolved in the system using sodium citrate or EDTA. In one embodiment, in the operating room, the storage medium (e.g., culture medium) is mixed with the lysis buffer for several minutes. In some embodiments, the semipermeable membrane chamber is disassembled and replaced with an alginate gel. The tissue is transferred to an EDTA solution to dissolve the alginate gel. The tissue is removed from the alginate gel and washed with saline for implantation.

[0130] The tissue transport system offers several advantages over standard liquid-based tissue storage systems: Tissue preservation conditions are improved by embedding the tissue in alginate gel. are protected from shock, vibration, or shaking during transport of the tissue to the hospital. In addition, as shown herein, the present invention can be used to prevent tissue damage during transportation. , improved cell viability, and suppressed inflammatory factors (e.g., cytokines and nitric oxide). A single enclosed system covers the storage and transportation stages.

[0131] In some embodiments, the tissue transport system and / or tissue culture device are For example, the kit may be provided in an outer chamber (e.g., outer chamber 110 or the like shown in FIG. 4A). 4A) and an inner chamber (e.g., inner chamber 120 or the inner chamber depicted in FIG. 4A). may include (e.g., may include, consist essentially of, or consist of) a device The kit may also include insulation and / or cooling components, and a temperature sensor. The device may include a storage container or box (e.g., as shown in FIG. 4A) that may The kit may also include an alginate solution or any other hydrogel solution. containers or vials containing calcium or barium solutions or both or vials, or containers or vials containing calcium salts or barium salts or both , a washing solution (e.g., saline or any other solution described herein) and / or or a container filled with a culture medium (e.g., any of the culture media described herein). The kit may include (e.g., may further include) a vial. Written or downloadable instructions for using the tissue culture device are available. In various embodiments, the kits disclosed herein may include the tissue The present invention comprises or consists essentially of a culture device and optionally instructions for use. In embodiments, the kits disclosed herein further comprise one or more of the following: phosphate or any other hydrogel monomer solution as required, calcium solution, sodium solutions, calcium salts, barium salts, solvents, wash solutions, or culture media. EXAMPLES

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

[0133] method Explant preparation and storage conditions - Osteochondral cores (n=25) were cultured in meat samples within 48 hours after slaughter. The samples were taken from 10 knees of 5 mature sows obtained from a slaughterhouse. The core is drilled internally or externally by coring with a specially ordered 4.0mm bit drill. The core bones were harvested from either of the lateral femoral condyles. Prior to storage, the core bones were The subchondral bone was subjected to pulsed lavage with air. The bone marrow components were removed by rinsing with normal saline. The cores were stored overnight in DMEM / F12 with 10% fetal bovine serum (FBS) and then Place them in individual sterile 5 mL culture tubes and incubate at 4 °C according to the following conditions assigned to each core: (Figure 1): (1) Control group - 1.5 mL complete medium (DMEM / F12 + 10% F (2) UPAL solution (sol) group - stored in 1.5 ml U of complete medium (3) UPAL gel group - stored in PAL solution (1.2%); (4) UPAL gel group - dissolved in normal saline The cells were encapsulated in 1.2% UPAL gel and stored in 1.5 ml complete medium. After storage for 3 days, each sample was warmed at 37°C for 24 hours, and then the full thickness cartilage was removed from the bone. The following evaluations were carried out.

[0134] Chondrocyte viability - Chondrocyte viability across all full-thickness cartilage depths (n=4) was measured using LIV Analyzed using the E / DEAD kit and imaged using confocal laser microscopy. For the vertical profile, the biopsy data were obtained for the entire cartilage thickness and for the following cartilage regions: Survival rates were calculated: superficial (upper 15%), mid (35%), and deep (50%).

[0135] Proteoglycan (PG) content - The 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 The incorporation of S-sulfate into sulfated PG (n=6) was As an index of G synthesis, the rapid Alcian blue filtration assay (Masuda, et al., Anal Biochem. 1994:167).

[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 The remaining amount of S-PG was examined 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 were analyzed for total NO (n=6) and and MMP-3 (n=4) concentrations.

[0139] Histology. Vertical midsections (5 μm) of each OCA (n=7) were stained with hematoxylin and The tissue was stained with either eosin or safranin-O. Histological sections were analyzed by independent observers using a modified O'Driscoll histological scoring system. The sections were cut using the Spine system (Aota, et al., Spine 2005:722). It was rated.

[0140] Statistical Analysis. All data are expressed as mean ± standard deviation (SD). Data were analyzed for comparison. To verify the statistical significance of the differences, a two-way repeated measures analysis of variance (ANOVA) or a one-way ANOVA was used. A P value of 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 the survival rate in the control group (18.6 ± 10.7%, p < 0.05; Figure 2).

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

[0143] In the PG synthesis-UPAL gel group (33192.8±15906.4CPM / mg) PG synthesis was significantly higher than in the control group (12748±2699 CPM / mg, p<0.05) on day 28. The results were significantly higher on the 28th day. 35 S incorporation was significantly improved in all groups compared to day 1. There was a significant decrease in the BP-related cholesterol level (2073811±496258 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 (MM) in the storage medium P-3) concentration-total NO production in the control group (3.8 ± 2.4 μM / mg) at 28 days UPAL solution group (1.0±0.8μM / mg, p<0.01) and UPAL gel group ( 1.5±0.8μM / mg, p<0.01). No significant differences in MMP-3 levels were observed among the three conditions.

[0146] Histology: In the control group, the cartilage matrix was poorly stained with Safranin-O and was not densely cellular. In contrast, the OCA in the UPAL gel group showed sufficient staining of the matrix and showed cellular staining. The total histological score in the UPAL gel group (13.9 ± 1 .1) was significantly higher than the control group (12.0±1.3, p<0.01; Figure 3).

[0147] Example 2 Example 1 shows the effect of using ultrapure alginate (UPAL) gel encapsulation on the solubility of soluble alginate after 28 days of storage. The results showed that the method is effective in improving the quality of osteochondral grafts. In order to improve the stability of the product, it is important to extend the storage period. First, in Example 2, the product was stored for 6 weeks. The present application has demonstrated that the storage period of cartilage can be extended for up to 10 days. Second, the use of UPAL to improve storage conditions To investigate the effect of different gelation conditions of UPAL on cell viability during extended storage, The endpoints of this study were similar to those of the current tissue preservation protocol. It should be noted that the storage period for viable tissue is 1.5 times longer (6 weeks). The longer the storage time, the greater the availability of graft tissue. The use of glycerol is advantageous for maintaining cell viability and tissue structure during storage and also reduces the need for glycerol during transportation. Provides physical protection as a filler material.

[0148] method Preparation and storage conditions of the explants - Osteochondral cores, menisci, patellar tendons, and corneas were prepared from juvenile mini- Osteochondral cores were extracted from 6-month-old pigs under sterile conditions using a custom-made 4.0 mm drill. The femoral condyle was sampled from either the medial or lateral femoral condyle by coring with a Lubit. The subchondral bone was subjected to pulsed lavage with air to remove bone marrow components, followed by normal The bone was thoroughly rinsed with saline. Prior to storage, the core bones were The tissues from the medial and lateral menisci were trimmed to a ratio of 4:1. The patellar tendon was punched out using a 4 mm knife and aseptically dissected and cut into 5 x 10 mm pieces. In addition, the corneal tissue with the vitreous body was incised to prepare graft tissue of 5 × 10 mm. The explant tissue was stored overnight in DMEM / F12 with 10% fetal bovine serum (FBS), They were then placed in individual sterile 5 mL culture tubes and incubated according to the following conditions assigned to each core: Stored at 4°C (Figure 5): (1) Control group - 3 mL complete medium (DMEM / F12 + 10% F (2) UPAL solution (sol) group - 3 ml UPA dissolved in complete medium (3) UPAL gel-coated group - stored in 1.2% UPAL gel Approximately 1 ml of calcium solution was cross-linked and washed with normal saline. (4) UPAL membrane group, encapsulated in 3 ml complete medium; and (5) UPAL membrane group. Tissues prepared as described in the UPAL gel coating group were coated with 50 kDa cut-off half-mask. A dialysis device equipped with a permeable membrane (Tube-O-DIALYZER (trademark), G-Bioscience) The grafts were placed in a 1.2% alginate solution (Bon Applied Pharmacopoeia, MO, USA) and filled with the 1.2% alginate solution. The tube containing the gel was immersed in 0.1M CaCl2 solution for 30 minutes to promote gelation. The cartilage was washed with saline and placed in a 50 ml culture tube filled with 40 ml of complete medium. The meniscus and Achilles tendon were stored for 6 weeks, whereas the corneal tissue was stored for 2 weeks (total (4°C).

[0149] After 6 weeks of storage, each sample was incubated at 37°C for 24 hours, and then the tissue was soaked in 30 mM ED The alginate gel was dissolved in TA solution for 5 minutes. The cell viability was evaluated using the LIVE / DEAD The plates were analyzed using a kit and imaged using confocal laser scanning microscopy. Viability was assessed for the profiles using the Otsu binarization method followed by Image-J Pro. The survival rate was calculated using the particle analysis function of Gram. The survival rate was calculated as the survival rate at the end point / the original survival rate. It was expressed as cell viability.

[0150] Statistical Analysis. All data are expressed as mean ± standard error (SE). Data were analyzed using multiple One-way analysis of variance (ANOVA) adjusted by Bonferroni correction for Statistical analysis was performed using the Kruskal-Wallis test. P values ​​less than 0.05 were considered statistically insignificant. I consider there to be a difference.

[0151] result Articular cartilage: At 6 weeks, the survival rate of chondrocytes in the surface area of ​​the UPAL membrane group was higher than that of the medium group. At 6 weeks, the UPALsol and UPA The survival rate of chondrocytes in the L-gel-coated group tended to be higher, but the sample size was No statistical significance was observed due to size.

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

[0153] As seen in the patellar tendon-meniscus, cell viability within the patellar tendon was significantly improved after 6 weeks of culture. The UPAL gel coating group and the UPAL membrane group showed a significant decrease in the The cell viability was significantly higher than that in the medium group (Figure 6C).

[0154] Cornea - After 2 weeks, the cell viability of corneal tissue in the UPAL membrane group was significantly higher than that in the medium group. 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 are defined by the It has the meaning commonly understood by one of ordinary skill in the art.

[0156] The technology illustratively described herein is not limited to the technology specifically disclosed herein. is properly performed in the absence of any component(s), limit(s) Thus, for example, the terms "comprising," "including," "including," "containing," etc. shall be interpreted broadly and without limit. Moreover, the terms and expressions used herein are for purposes of illustration and not of limitation. The terms and expressions are used for the purpose of clarity and the use of such terms and expressions is at the discretion of the user. The present invention is not intended to exclude any equivalents or parts thereof, but to be limited to the present invention as set forth in the claims. It will be appreciated that various modifications are possible within the scope of the technology.

[0157] Thus, the materials, methods, and examples provided herein represent preferred embodiments. These are examples and should be understood as not intended to limit the scope of the present technology. It is.

[0158] The present technology is described broadly and generically herein. Each of the narrower species and subinclusive groups contained herein also form part of the present technology. This includes the disclaimer, regardless of whether the excerpted material is specifically cited in this specification. or a generic description of the technology including a negative limitation excluding any subject matter from the genus.

[0159] Furthermore, when features or aspects of the present technology are described in Markush groups, Merchant agrees that the technology does not involve any individual member or members of the Markush family of companies. It will be appreciated that subgroups of bars may also be described.

[0160] All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety. All rights reserved. In all cases, the present specification, including definitions, will control.

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

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

Claims

1. Osteochondral tissue, articular cartilage without any subchondral bone, fibrocartilage, tendon, or cornea A method for preserving tissue selected from the group consisting of encapsulating said tissue in an alginate gel. and exposing the gel-encapsulated tissue to culture medium.

2. The gel encapsulated tissue is allowed to stand for a period selected from about 24 hours to about 8 weeks, for about 28 days; The method of claim 1, wherein the cell is placed in a culture medium for at least about six weeks, and any range therebetween.

2. The method according to claim 1.

3. The method of claim 1 or 2, further comprising the step of encapsulating the tissue in a semipermeable membrane. Law.

4. The semipermeable membrane has a pore size of at least about 250 μm and a pore size of about 100 μm to about 1000 μm. , pore size of about 0.22 μm to about 1000 μm, or pore size of about 0.22 μm to about 100 μm , with a molecular weight cut-off (MWCO) of about 50 kDa or about 200,000 kDa, or 4. The mesh of claim 3, having one or more characteristics of a mesh open rate of about 1% to about 50%. method.

5. 3. The method of claim 2, wherein the tissue is encapsulated in a gel after being encapsulated in the semipermeable membrane.

5. The method according to claim 4.

6. 3. The method of claim 2, wherein the tissue is encapsulated in a gel prior to encapsulation in the semipermeable membrane.

5. The method according to claim 4.

7. The osteochondral tissue has a bone to cartilage thickness ratio of about 20:1 to about 1:10, or about 4: 1 to 5, comprising an osteochondral core trimmed to have a diameter of 1:1 and a range therebetween.

7. The method according to any one of claims 6 to 6.

8. 7. The method of claim 1, wherein the fibrocartilage tissue comprises a meniscus, a medial meniscus, or a lateral meniscus.

2. The method according to claim 1.

9. The method according to any one of claims 1 to 6, wherein the tendon is the patellar tendon or the Achilles tendon.

10. The method according to any one of claims 1 to 6, wherein the cornea is a cornea having a vitreous body.

11. The alginate gel according to any one of claims 1 to 10, wherein the alginate gel is a highly purified alginate (UPAL) gel. The method according to any one of claims 1 to 5.

12. The UPAL is at a concentration of about 0.5% to about 2.0% or about 1.2% and ranges therebetween. The method of claim 11 , wherein

13. The method according to any one of claims 1 to 12, wherein the culture medium is a complete medium.

14. the culture medium is not replenished following exposure of the gel-encapsulated tissue to the culture medium; The method according to any one of claims 1 to 13.

15. The tissue is incubated at a temperature selected from below about 10°C, between about 0°C and about 4°C, or at about 4°C and ranges therebetween. The method of any one of claims 1 to 14, further comprising exposing the material to a temperature selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 。

16. The encapsulated tissue was then incubated in an artificially controlled CO ２ Levels, artificially controlled O ２ R 3. The method according to claim 1, wherein the storage is not performed using one or more of the following:

16. The method according to any one of claims 15 to 15.

17. 10. The method of claim 1, further comprising the step of dissolving the alginate gel after exposure to the culture medium.

17. The method according to any one of claims 1 to 16.

18. The alginate gel is dissolved in a sodium citrate solution or ethylenediaminetetraacetate solution. Diaminetetraacetic acid (EDTA) solution or a solution containing both sodium citrate and EDTA is dissolved by a method comprising exposing the compound to a chelating solution optionally selected from 20. The method of claim 17.

19. The method according to any one of claims 2 to 18, further comprising the step of degrading the semipermeable membrane after exposure to the culture medium. The method according to any one of claims 1 to 5.

20. The encapsulated tissues are optionally compared to tissues stored without encapsulation. and reducing the release of inflammatory factors selected from cytokines or nitric oxide as appropriate. The method according to any one of claims 1 to 19, wherein the

21. The pre-implant encapsulated tissue has a cell viability of at least about 6% prior to the encapsulation step. 0%, 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 cell viability The method of any one of claims 1 to 20, wherein the rate is maintained.

22. 1. A tissue transport system comprising: (a) A membrane that is partially or entirely made of a semipermeable membrane and that is connected to the tissue to be transported and to the alginate solution. a tissue chamber configured to contain a fluid, the tissue chamber being enclosed; (b) a culture chamber having an inlet and an outlet and configured to contain the tissue chamber, A calcium solution or a barium solution or both are added through the inlet. The culture chamber is then sealed with a gelling solution of glycerol, encapsulating the tissue. and, (c) a transport box or storage container configured to contain the incubation chamber; (d) optionally, placing the container in the transport box or the storage container; a temperature sensor configured to measure a temperature of , a tissue transport system.

23. 23. The system of claim 22, wherein the alginate solution is a high purity alginate solution.

24. The tissue chamber is configured to encapsulate the tissue by the semipermeable membrane.

24. The system of claim 22 or 23, wherein the tissue chamber is a separate tissue chamber.

25. The semipermeable membrane has a pore size of at least about 250 μm and a pore size of about 100 μm to about 1000 μm. , a pore size of about 0.22 μm to about 1000 μm, or about 0.22 μm to about 100 μm, The molecular weight cut-off (MWCO) is about 50 kDa or about 200,000 kDa, or Any of claims 22 to 24, wherein the aperture ratio is one or more of about 1% to about 50%.

13. The system according to claim 1 .

26. The alginate solution may be from about 0.5% to about 2.0% alginate, optionally from about 1.2 % alginate and ranges therebetween. Stem.

27. The transport box or the storage container or the incubation chamber is The temperature in the storage container or in the culture chamber is kept below 10° C., and if necessary, from about 0° C. to about 4° C. and an insulating or cooling component configured to maintain the temperature at about 4° C. as needed. The method according to any one of claims 22 to 26, further comprising both an insulating component and a cooling component. The system described.

28. The system further comprises: ２ Level, or the above O ２ level, or said humidity, or any combination thereof. The system according to any one of claims 22 to 27, which does not include a sensor or controller. Tem.

29. The transport box or the storage container is sealed against continuous flow of gas or liquid. The system according to any one of claims 22 to 28,

30. 1. A tissue culture device comprising: An inlet constructed to allow the inflow of a fluid and the flow of the fluid therethrough an outer chamber defining an outlet constructed to allow for the exit of the outer chamber; The outer chamber, an inner chamber disposed at least in part within the outer chamber, the inner chamber being formed from a semipermeable membrane; a wall formed of a tissue support and surrounding the chamber, the wall supporting the tissue support and the wall supporting the tissue support. and an inner chamber, Optionally, an enclosed storage vessel, wherein the outer chamber, and thus the inner chamber, a storage vessel disposed within an interior volume defined by the vessel; 1. A tissue culture device comprising:

31. a temperature sensor operatively connected to the storage vessel and adapted to measure a temperature within the storage vessel; The tissue culture device of claim 30 , further comprising a temperature sensor configured in the

32. 27. The tissue culture medium of claim 26, wherein the tissue is encapsulated by an alginate gel. Cultivation device.

33. The fluid may be an alginate solution, a calcium solution, a barium solution, a physiological saline solution, a culture medium, 33. The method according to claim 30, further comprising at least one of: Mount the tissue culture device.

34. The semi-permeable membrane has a molecular weight cut-off (MW) of about 50 kDa or about 200,000 kDa. The tissue culture device according to any one of claims 30 to 33, comprising a cellular membrane (C0).

35. Any one of claims 30 to 33, wherein the semipermeable membrane has a pore size of at least about 250 pm. Item 1. The tissue culture device described in item 1.

36. The semipermeable membrane has a thickness of about 0.22 μm to about 100 μm, or about 100 μm to about 1,000 μm. 30 to 33, wherein the pore size is in the range of about 0.22 μm to about 1,000 μm.

2. The tissue culture device according to any one of claims 1 to 11.

37. Any of claims 30 to 36, wherein the semipermeable membrane has a mesh opening rate of about 1% to about 50%.

2. The tissue culture device according to claim 1.

38. At least one of an insulating component or a cooling component disposed within the storage vessel. and wherein the at least one of the thermal insulation component or the cooling component comprises: The storage container and / or the outer chamber are configured to maintain a temperature below 10° C. The tissue culture device according to any one of claims 30 to 37.

39. A tissue culture device according to any one of claims 30 to 38, and optionally instructions for use. Kit includes instructions and instructions.

40. Alginate or any other hydrogel monomer, optionally in solution calcium solution, barium solution, calcium salt, barium salt, solvent, cleaning solution, or 40. The kit of claim 39, further comprising one or more of a culture medium.

Images