JP2024063218A - Method to decellularize isolated organ or portion thereof - Google Patents

Abstract

To provide methods to decellularize an isolated organ or portion thereof.SOLUTION: A method to decellularize an isolated organ or portion thereof comprises contacting an isolated organ or portion thereof with a liquid, wherein the liquid is at least partially degassed, and the liquid comprises a detergent, a radical-generating compound, or both of them.SELECTED DRAWING: None

Description

CROSS-REFERENCE This application claims priority to U.S. Provisional Patent Application No. 62/821,620, filed March 21, 2019, which is incorporated by reference in its entirety.

Disclosed herein are methods that can include contacting an isolated organ or portion thereof with a liquid. In some embodiments, (a) the liquid can be at least partially degassed; (b) the liquid can include a detergent and/or a radical-generating compound; (c) the isolated organ or portion thereof can be from a mammal that has been fed within about 10 hours prior to removal of the isolated organ or portion thereof; or (d) any combination of (a)-(c). In some embodiments, the method can include at least two of (a), (b), or (c). In some embodiments, the method can include (a), (b), and (c). In some embodiments, the isolated organ or portion thereof can be from a mammal. In some embodiments, the mammal can be from a non-human mammal. In some embodiments, the non-human mammal can be a pig, sheep, goat, cow, dog, cat, or monkey. In some embodiments, the mammal can be a human mammal. In some embodiments, the isolated organ or part thereof may be a liver, lung, heart, kidney, bladder, pancreas, spleen, uterus, any part thereof, or any combination thereof. In some embodiments, the contacting may include (i) perfusing at least a portion of the isolated organ or part thereof with a liquid, (ii) injecting at least a portion of the isolated organ or part thereof with a liquid, (iii) spraying at least a portion of the isolated organ or part thereof with a liquid, (iv) submerging the isolated organ or part thereof in a liquid, or (v) any combination thereof. In some embodiments, the isolated organ or part thereof may be cannulated before, during, or after contacting. In some embodiments, the liquid may include an at least partially degassed decellularization medium. In some embodiments, contacting with an at least partially degassed decellularization medium may form an at least partially decellularized isolated organ or part thereof. In some embodiments, the at least partially decellularized isolated organ or part thereof may include an extracellular matrix. In some embodiments, the extracellular matrix may include a vasculature bed. In some embodiments, after contact, the vasculature bed may remain at least partially intact. In some embodiments, the liquid may be at least partially degassed. In some embodiments, the extracellular matrix may have increased compressibility compared to an otherwise comparable extracellular matrix produced by contact with a non-degassed liquid for a comparable period of time. In some embodiments, the method may include at least (a). In some embodiments, the isolated organ or portion thereof may be contacted with the at least partially degassed liquid for at least about 2 hours. In some embodiments, the dissolved gas in the at least partially degassed liquid may have a concentration of less than about 1 milligram (mg) per liter (L). In some embodiments, the dissolved gas may be selected from the group consisting of oxygen, nitrogen, carbon monoxide, carbon dioxide, a noble gas, and any combination thereof. In some embodiments, the isolated organ or portion thereof after contact may contain fewer air emboli, fewer microbubbles, less pigmentation, or any combination thereof, compared to an otherwise comparable isolated organ or portion thereof produced by contacting with an otherwise comparable liquid that is not at least partially degassed for a comparable period of time. In some embodiments, the at least partially degassed liquid may contain at least partially degassed decellularization medium, thereby forming an at least partially decellularized isolated organ or portion thereof. In some embodiments, the at least partially decellularized isolated organ or portion thereof after contact with the at least partially degassed decellularization medium may contain fewer cells than an otherwise comparable isolated organ or portion thereof that was decellularized with an otherwise comparable non-degassed decellularization medium. In some embodiments, the time sufficient to produce an at least partially decellularized isolated organ or portion thereof with an at least partially degassed decellularization medium may be less than the time sufficient to produce a substantially decellularized isolated organ or portion thereof with a non-degassed decellularization medium. In some embodiments, the method may include at least (b). In some embodiments, the radical generating compound may be of the formula R ¹ -O-O-R ² , where R ¹ and R ² may independently be H, C ₁ -C ₆ alkyl, substituted C ₁ -C ₆ alkyl, aryl, substituted aryl, benzyl, substituted benzyl, C(═O)A ¹ , C(═O)OA ² , where A ¹ and A ² may independently be H, C ₁ -C ₆ alkyl or substituted C ₁ -C ₆ alkyl. In some embodiments, R ¹ may be H and R ² may be C(═O)CH _3. In some embodiments, the radical generating compound may be a peracid. In some embodiments, the peracid may include: peroxyacetic acid, peroxyoctanoic acid, sulfoperoxycarboxylic acid, peroxysulfonated oleic acid, peroxyformic acid, peroxyoxalic acid, peroxypropanoic acid, peroxybutanoic acid, peroxypentanoic acid, peroxyhexanoic acid, peroxyadipic acid, peracetic acid, perlactic acid, peroxycitric acid, peroxybenzoic acid, or any combination thereof. In some embodiments, the peracid may include peracetic acid. In some embodiments, the peracid is at least about 10 ppm, 25 ppm, 50 ppm, 75 ppm, 90 ppm, 100 ppm, 125 ppm, 150 ppm, 175 ppm, 200 ppm, 225 ppm, 250 ppm, 275 ppm, 300 ppm, 325 ppm, 350 ppm, 375 ppm, 400 ppm, 425 ppm, 450 ppm, 475 ppm, 500 ppm, 525 ppm, 550 ppm, 575 ppm, 600 ppm, The amount of decellularized organ or portion thereof may be present in an amount including 1000 ppm, 625 ppm, 650 ppm, 675 ppm, 700 ppm, 725 ppm, 750 ppm, 775 ppm, 800 ppm, 900 ppm, 1000 ppm, 1200 ppm, 1400 ppm, 1500 ppm, 1700 ppm, 2000 ppm, 2200 ppm, 2500 ppm, 2750 ppm, 3000 ppm, 3200 ppm, 3500 ppm, 3750 ppm, or 4000 ppm. In some embodiments, the contacting can produce an at least partially decellularized isolated organ or portion thereof. In some embodiments, the at least partially decellularized isolated organ or portion thereof may comprise a reduced amount of cells compared to an otherwise comparable decellularized isolated organ or portion thereof contacted with an otherwise comparable liquid without peroxy acid for a comparable period of time. In some embodiments, the reduced amount of cells may comprise a reduction in cells from the isolated organ or portion thereof of at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5%. In some embodiments, the contacting may comprise perfusion. In some embodiments, the flow rate of the perfusion may be increased compared to the flow rate of the otherwise comparable liquid without peroxy acid. In some embodiments, the flow rate may be increased without substantially compromising the integrity of the extracellular matrix of the isolated organ or portion thereof. In some embodiments, the method may comprise at least (c). In some embodiments, the isolated organ or portion thereof may comprise a reduced particulate level as compared to an otherwise comparable isolated organ or portion thereof obtained from an animal that has not been fed within about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, or about 1 hour prior to removal of the isolated organ or portion thereof from the animal. In some embodiments, the reduced particulate level may be reduced by at least about: 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1% as compared to an otherwise comparable isolated organ or portion thereof obtained from an animal that has not been fed within about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, or about 1 hour prior to removal of the isolated organ or portion thereof from the animal. In some embodiments, the reduced particulate level may be reduced by visual inspection,
It can be determined by determining outflow, determining turbidity, microscopic analysis, or any combination thereof. In some embodiments, the method can further include washing the isolated organ or portion thereof with a washing medium prior to the contacting. In some embodiments, the washing medium can include a hypertonic solution. In some embodiments, the hypertonic solution can include saline. In some embodiments, the washing medium can include a disinfectant. In some embodiments, the method can further include contacting the isolated organ or portion thereof with a second liquid. In some embodiments, contacting with the second liquid can occur after contacting with the liquid. In some embodiments, the second liquid can be an at least partially degassed liquid. In some embodiments, the second liquid can include a washing medium. In some embodiments, the isolated organ or portion thereof can include less than about 1% by weight of detergent after contacting the isolated organ or portion thereof with the second liquid. In some embodiments, after contacting with the second liquid, blood can flow through a larger volume of vasculature of the isolated organ or portion thereof compared to an otherwise equivalent isolated organ or portion thereof produced using a non-degassed second liquid. In some embodiments, the method can further include contacting the at least partially decellularized isolated organ or portion thereof with a regeneration medium. In some embodiments, the regeneration medium can be an at least partially degassed cell regeneration medium. In some embodiments, contact with the at least partially degassed cell regeneration medium can result in the formation of an at least partially recellularized organ or portion thereof. In some embodiments, the at least partially degassed cell regeneration medium can include a population of regenerative cells. In some embodiments, contact with the at least partially degassed cell regeneration medium can result in a greater degree of recellularization compared to an otherwise equivalent harvested organ or portion thereof produced using a non-degassed cell regeneration medium.

Also disclosed herein, in some embodiments, is an at least partially decellularized harvested organ or portion thereof produced by the methods described herein.

Also disclosed herein, in some embodiments, is an at least partially recellularized isolated mammalian organ or portion thereof produced by the methods described herein.

Also disclosed herein, in some embodiments, is a system. The system described herein can include: (a) an at least partially decellularized harvested organ or part produced by the methods described herein; (b) a device for transferring fluids; (c) a cannula; or (d) a port. In some embodiments, the system can include any combination of (a), (b), (c), and (d), including at least two, three, or all of (a), (b), (c), and (d). In some embodiments, the system can further include a degassed decellularization medium. In some embodiments, the system can further include a degassed cleaning medium.

Also disclosed herein, in some embodiments, are kits. The kits described herein can include an at least partially decellularized harvested organ or portion thereof produced by the methods described herein. In some embodiments, the kit can further include (a) at least one cannula, (b) liquid, (c) instructions for recellularizing the at least partially decellularized harvested organ or portion thereof, (d) instructions for introducing the at least partially recellularized harvested organ or portion thereof in a subject, or (e) any combination thereof. In some embodiments, the at least partially decellularized harvested organ or portion thereof can be in packaging.

Figure 1 is a schematic showing the initial preparation for cardiac decellularization. The aorta, pulmonary artery, and superior vena cava are cannulated (A, B, C, respectively), and the inferior vena cava, brachiocephalic trunk, left common carotid artery, and left subclavian artery are ligated. Arrows indicate the direction of antegrade and retrograde perfusion. FIG. 2 shows decellularization of an adult porcine heart for about 48 hours. A 6-month old porcine heart undergoing perfusion decellularization for 48 hours. At least some native structure and at least some vasculature can be preserved after decellularization. After 48 hours, the heart can be at least partially decellularized. Figure 3 shows decellularization of an adult porcine liver over a 24 hour period. A 6-month old porcine liver undergoing perfusion decellularization for 24 hours. The native structure and vasculature remain preserved following decellularization. FIG. 4A shows porcine liver after decellularization with a microparticle percentage of 0-5%. FIG. 4B shows a porcine liver after decellularization with a particulate percentage of 20%. FIG. 4C shows a porcine liver after decellularization with a microparticle percentage of 40-50%. FIG. 4D shows a porcine liver after decellularization with a microparticle percentage of 50-60%. FIG. 4E shows a porcine liver after decellularization with a microparticle percentage of 60%. FIG. 4F shows a porcine liver after decellularization with a microparticle percentage of 70%. FIG. 4G shows a porcine liver after decellularization with a particulate percentage of 80%. FIG. 4H shows a porcine liver after decellularization with a microparticle percentage of 95-100%. FIG. 5 shows the elution of microparticles into the tubing connected to the liver portal vein cannula, and visible microparticles undergo perfusion decellularization when perfusion can be stopped for 10 minutes after approximately 24 hours of perfusion decellularization. Figure 6A shows decellularization of an adult porcine lung over 48 hours. A 6-month-old porcine lung undergoing perfusion decellularization for 48 hours. Native structure and vasculature can be preserved after decellularization. FIG. 6B shows decellularization after 48 hours, indicating that the lung can be completely decellularized. 7A shows decellularization of an adult porcine kidney over a 48 hour period. A 6-month-old porcine kidney undergoing perfusion decellularization over a 48 hour period. Native structure and vasculature can be preserved following decellularization. FIG. 7B shows decellularization after 48 hours, demonstrating that the kidney can be completely decellularized. FIG. 8A shows the absorbance profile of the solution surrounding the liver with visible particulates (L281) after a 30 minute recirculating water rinse. FIG. 8B shows the effluent absorbance profile in the solution surrounding the liver with visible particulate L274 after a 15 minute recirculating water rinse. 9 shows the treatment of native and decellularized kidney scaffolds with a DNA-binding fluorescent stain. Examination was performed by immunofluorescence microscopy. 10A shows an increase in flow rate after a radical-generating compound (e.g., peracetic acid) can be introduced into the perfusion decellularization process. The increase in flow rate can occur after addition of a radical-generating compound during perfusion decellularization of a porcine liver at a set pressure of 12 mmHg. The decellularization device allows a defined pressure to be specified, and the flow rate can be adjusted to maintain the defined pressure. 10B shows an increase in flow rate after a radical-generating compound (e.g., peracetic acid) can be introduced into the perfusion decellularization process. The increase in flow rate can occur after addition of a radical-generating compound during perfusion decellularization of a porcine liver at a set pressure of 12 mmHg. The decellularization device allows a defined pressure to be specified, and the flow rate can be adjusted to maintain the defined pressure. 10C shows an increase in flow rate after a radical-generating compound (e.g., peracetic acid) can be introduced into the perfusion decellularization process. The increase in flow rate can occur after addition of a radical-generating compound during perfusion decellularization of a porcine liver at a set pressure of 12 mmHg. The decellularization device allows a defined pressure to be specified, and the flow rate can be adjusted to maintain the defined pressure. FIG. 11 shows a plot of normalized peak load force values for samples that can be stretched and relaxed in 4% strain increments. FIG. 12A depicts endothelial seeding used in histological analysis. FIG. 12B shows representative images of the bioreactor, tissue structure after 21 days, and exemplary vascular surfaces. FIG. 13 shows immunofluorescence staining, which demonstrates the distribution and engraftment of native kidney cells throughout the kidney in place. FIG. 14 depicts the improvement of perfusion flow rate at constant pressure by addition of the radical generating compound PAA during the decellularization process workflow.

INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. U.S. Patent Nos. 10,213,525 and 10,220,056 are herein incorporated by reference in their entireties.

The novel features of the exemplary embodiments are set forth in detail in the appended claims. The features and advantages will be better understood by reference to the following detailed description that sets forth exemplary embodiments in which the exemplary embodiments are utilized, and the accompanying drawings.

SUMMARY In some embodiments, disclosed herein are methods of substantially decellularizing an isolated organ, or a portion thereof. In some embodiments, the decellularization method may include perfusing the isolated organ, or a portion thereof, with a degassed cell disruption medium at a pressure of less than about 700 torr and at about 15° C. to about 95° C. prior to perfusion, thereby substantially removing dissolved gas from the degassed cell disruption medium. In some embodiments, the degassed cell disruption medium may include a detergent. In some embodiments, the detergent may include sodium dodecyl sulfate, or polyethylene glycol, or Triton-X100. In some embodiments, the amount of microbubbles during perfusion may be reduced as compared to an otherwise equivalent cell disruption medium that includes greater than about 8 parts per million (ppm) oxygen when stored at a temperature of about 25° C. In some embodiments, the degassed cell disruption medium may be connected to a vacuum pump for about 10 minutes prior to perfusion. In some embodiments, the degassed cell disruption medium may be at a temperature of about 4° C. to about 95° C. prior to perfusion. In some embodiments, the dissolved gas may include oxygen or nitrogen. In some embodiments, the time sufficient to produce an at least partially decellularized isolated organ or portion thereof with a degassed cell disruption medium may be less than the time to produce an at least partially decellularized isolated organ or portion thereof with a non-degassed cell disruption medium. In some embodiments, the volume of degassed cell disruption medium that may be sufficient to produce an at least partially decellularized isolated organ or portion thereof may be less than the volume of non-degassed cell disruption medium required to produce an at least partially decellularized isolated organ or portion thereof. In some embodiments, the weight of degassed cell disruption medium that may be sufficient to produce an at least partially decellularized isolated organ or portion thereof may be less than the weight of non-degassed cell disruption medium required to produce an at least partially decellularized isolated organ or portion thereof. In some embodiments, the at least partially decellularized isolated organ or portion thereof may comprise an extracellular matrix. In some embodiments, the extracellular matrix may comprise fibronectin, fibrillin, laminin, elastin, a protein of the collagen family, glycosaminoglycan, ground substance, reticular fibers, or thrombospondin. In some embodiments, the collagen family protein can include collagen I, collagen II, collagen III, or collagen IV. In some embodiments, the at least partially decellularized isolated organ or portion thereof can include a vasculature bed. In some embodiments, the isolated organ or portion thereof can be from a mammal. In some embodiments, the mammal can be a rodent, a pig, a rabbit, a cow, a sheep, a dog, a cat, or a human. In some embodiments, the isolated organ or portion thereof can be a heart, a liver, a lung, a brain, a pancreas, a spleen, a kidney, a uterus, or a bladder. In some embodiments, the perfusion can be antegrade. In some embodiments, the perfusion can be retrograde. In some embodiments, the degassed cell disruption medium can be recirculated during perfusion.

Also disclosed herein is a harvested organ or portion thereof that can include an extracellular matrix, and can be cannulated with one or more cavities, blood vessels, and/or ducts. In some embodiments, a degassed cell disruption medium can be perfused to obtain an at least partially decellularized cannulated harvested organ or portion. In some embodiments, the at least partially decellularized cannulated organ or portion can include an at least partially decellularized extracellular matrix, which can include an exterior surface and a vascular tree. In some embodiments, the degassed cell disruption medium can include less than about 8 parts per million (ppm) oxygen when stored at a temperature of about 25° C. In some embodiments, the degassed cell disruption medium can increase cell removal in a shorter time compared to an otherwise equivalent cell disruption medium that includes more than about 8 ppm oxygen when stored at a temperature of about 25° C. In some embodiments, the degassed cell disruption medium can include a detergent. In some embodiments, the detergent can include sodium dodecyl sulfate, or polyethylene glycol, or Triton-X100. In some embodiments, the amount of microbubbles during perfusion may be reduced as compared to an otherwise equivalent cell disruption medium containing greater than about 8 parts per million (ppm) oxygen when stored at a temperature of about 25° C. In some embodiments, the degassed cell disruption medium may be connected to a vacuum pump for about 10 minutes prior to perfusion. In some embodiments, the degassed cell disruption medium may be at a temperature of about 4° C. to about 95° C. prior to perfusion. In some embodiments, the dissolved gas may comprise oxygen or nitrogen. In some embodiments, the time sufficient to produce an at least partially decellularized isolated organ or portion thereof with a degassed cell disruption medium may be less than the time to produce an at least partially decellularized isolated organ or portion thereof with a non-degassed cell disruption medium. In some embodiments, the volume of degassed cell disruption medium that may be sufficient to produce an at least partially decellularized isolated organ or portion thereof may be less than the volume of non-degassed cell disruption medium required to produce an at least partially decellularized isolated organ or portion thereof. In some embodiments, the weight of degassed cell disruption medium that may be sufficient to produce an at least partially decellularized isolated organ or portion thereof may be less than the weight of non-degassed cell disruption medium required to produce an at least partially decellularized isolated organ or portion thereof. In some embodiments, the at least partially decellularized isolated organ or portion thereof may comprise an extracellular matrix. In some embodiments, the extracellular matrix may comprise fibronectin, fibrillin, laminin, elastin, a collagen family protein, glycosaminoglycan, ground substance, reticular fibers, or thrombospondin. In some embodiments, the collagen family protein may be collagen I, collagen II, collagen III, or collagen IV. In some embodiments, the at least partially decellularized isolated organ or portion thereof may comprise a vasculature bed. In some embodiments, the isolated organ or portion thereof may be from a mammal. In some embodiments, the mammal may be a rodent, a pig, a rabbit, a cow, a sheep, a dog, a cat, or a human. In some embodiments, the isolated organ or part thereof may be the heart, liver, lung, brain, pancreas, spleen, kidney, uterus, or bladder. In some embodiments, the perfusion may be antegrade. In some embodiments, the perfusion may be retrograde. In some embodiments, the degassed cell disruption medium may be recirculated during perfusion.

Also disclosed herein is a composition that can include an at least partially decellularized isolated mammalian organ or portion thereof. In some embodiments, the isolated organ or portion thereof can have an at least partially decellularized extracellular matrix having an exterior surface and a vascular tree. In some embodiments, the at least partially decellularized extracellular matrix of the isolated mammalian organ or portion thereof can include a cell disruption medium that includes less than about 8 parts per million (ppm) oxygen when stored at a temperature of about 25°C.

Also disclosed herein is a composition that can include a decellularized isolated mammalian organ or portion thereof perfused with a cell disruption medium. In some embodiments, the cell disruption medium can include a dissolved oxygen concentration of less than about 8 parts per million (ppm). In some embodiments, the decellularized isolated mammalian organ or portion thereof can include up to about 10% native cells after perfusion with a cell disruption medium for up to 10 hours. In some embodiments, the system can include an at least partially decellularized isolated mammalian organ or portion thereof. In some embodiments, the system can include an isolated decellularized isolated mammalian organ or portion thereof. In some embodiments, the system can include a peristaltic pump. In some embodiments, the system can include a kit that can include a decellularized isolated mammalian organ or portion thereof, at least one cannula, a reagent, instructions for recellularizing the decellularized isolated mammalian organ or portion thereof, and/or instructions for introducing the decellularized isolated mammalian organ or portion thereof into a subject. In some embodiments, the kit can be in packaging. In some embodiments, the packaging may be sterile.

Also disclosed herein is a method of providing an at least partially decellularized isolated organ or portion thereof, where the at least partially decellularized isolated organ or portion thereof can include a vasculature bed, and the at least partially decellularized isolated organ or portion thereof can be substantially free of endothelial cells. In some cases, the method can include generating an at least partially recellularized organ or portion thereof, where generating can include perfusing the at least partially decellularized isolated organ or portion thereof with a degassed cell regeneration medium; the degassed cell regeneration medium can include less than about 8 parts per million (ppm) of oxygen when stored at a temperature of about 25° C. In some embodiments, the degassed cell regeneration medium can include a population of cells. In some embodiments, the population of cells can include totipotent cells, pluripotent cells, multipotent cells, or any combination thereof. In some embodiments, the regenerative cell population can include undifferentiated cells, partially differentiated cells, fully differentiated cells, or a combination thereof. In some embodiments, the regenerative cell population can be independently from a mammal. In some embodiments, the mammal may be a rodent, a pig, a rabbit, a cow, a sheep, a dog, or a human. In some embodiments, the regenerative cell population may be embryonic stem cells, umbilical cord blood cells, tissue-derived stem or progenitor cells, bone marrow-derived stem or progenitor cells, blood-derived stem or progenitor cells, mesenchymal stem cells (MSCs), cells derived from skeletal muscle, multipotent adult progenitor cells (MAPCs), cardiomyocyte stem cells (CSCs), multipotent adult cardiac-derived stem cells, cardiac fibroblasts, cardiac microvasculature endothelial cells, aortic endothelial cells, bone marrow mononuclear cells (BM-MNCs), endothelial progenitor cells (EPCs), or any combination thereof. In some embodiments, the regenerative cell population may comprise at least about 1,000 regenerative cells. In some embodiments, the amount of degassed cell regeneration medium during perfusion may comprise at least about 1,000 regenerative cells per mg of substantially decellularized isolated organ or portion thereof. In some embodiments, the degassed cell generation medium may comprise growth factors. In some embodiments, the growth factor may be VEGF, DKK-1, FGF, BMP-1, BMP-4, SDF-1, IGF, HGF, or any combination thereof. In some embodiments, the degassed cell production medium may include an immunomodulatory agent. In some embodiments, the immunomodulatory agent may be a cytokine, a glucocorticoid, an IL2R antagonist, a leukotriene antagonist, or any combination thereof. In some embodiments, the at least partially decellularized isolated organ or portion thereof may further include an extracellular matrix. In some embodiments, the extracellular matrix may include fibronectin, fibrillin, laminin, elastin, a collagen family protein, a glycosaminoglycan, a ground substance, a reticular fiber, or a thrombospondin. In some embodiments, the collagen family protein may be collagen I, collagen II, collagen III, or collagen IV. In some embodiments, the at least partially decellularized isolated organ may be from a mammal. In some embodiments, the mammal may be a rodent, a pig, a rabbit, a cow, a sheep, a dog, a cat, or a human. In some embodiments, the at least partially recellularized organ or portion thereof can be a heart, liver, lung, brain, pancreas, spleen, kidney, uterus, or bladder. In some embodiments, the perfusion can be antegrade. In some embodiments, the perfusion can be retrograde.

Also disclosed herein is an at least partially recellularized explant or portion thereof from a mammal that can contain a cell regeneration medium having less than about 8 parts per million (ppm) oxygen when stored at a temperature of about 25°C.

Also disclosed herein, in some embodiments, are compositions and methods for decellularization of whole excised organs and decellularization of portions of whole excised organs. Also disclosed herein, in some embodiments, are improved decellularization methods and compositions. In some embodiments, the improved decellularization compositions and methods disclosed herein may result in more efficient decellularization and improved recellularization of the compositions described herein.

Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms "a,""an," and "the" are intended to include the plural forms as well unless the context clearly indicates otherwise. Furthermore, as used herein, the terms "including,""includes,""having,""has,""with," or variations thereof, include, "comprise,""includes,""having,""has,""with ...
To the extent that it means "rising."

The term "about" in connection with a reference numerical value and its grammatical equivalents and grammatical equivalents used herein can include a range of values of ±10% from that value. For example, the amount "about 10" includes the amounts 9 to 11. The term "about" in connection with a reference numerical value can also include a range of values of ±10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value.

As used herein, the term "substantially" can refer to a value approaching 100% of a given value. In some embodiments, the term can refer to an amount that can be at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of the total amount. In some embodiments, the term can refer to an amount that can be about 100% of the total amount.

The term "decellularized" or "decellularization" as used herein can refer to a biological structure (e.g., an excised organ or part thereof, or tissue) from which cellular and tissue contents have been removed, leaving behind an intact acellular infrastructure. An organ, such as a kidney, can be composed of various differentiated tissues. The differentiated tissue structure or parenchyma of an organ can provide a specific function associated with the organ. The supporting fibrous network of an excised organ can be the interstitium. Most organs can have an interstitial framework composed of undifferentiated connective tissue that supports differentiated tissues. The process of decellularization at least partially removes the cellular portion of the tissue, leaving behind a complex three-dimensional meshwork of extracellular matrix (ECM). The ECM infrastructure can be composed primarily of collagen, but can also include cytokines, proteoglycans, laminin, fibrillin, and other proteins secreted by cells. The at least partially decellularized structure can provide a biocompatible substrate onto which different cell populations can be infused or used to implant, for example, but not limited to, acellular medical devices such as wound healing matrices, fistula matrices, void fillers, dermal fillers, soft tissue reinforcements, or other substrates that allow cell infiltration and remodeling after implantation or application. The decellularized biological structure can be rigid or semi-rigid and can change its shape. Examples of decellularized harvested organs include, but are not limited to, the heart, kidney, liver, lung, pancreas, brain, bone, spleen, and parenchymal organs such as the bladder, uterus, ureter, and urethra.

The term "effective amount" or "therapeutically effective amount" can refer to an amount of a composition, e.g., a composition including cells, such as regenerative cells, that may be sufficient to provide a desired activity upon introduction into an isolated organ or portion thereof as disclosed herein.

As used herein, the term "functional" and its grammatical equivalents can refer to the ability to operate for an intended purpose, have an intended purpose, or perform an intended purpose. Functional can include any percentage from baseline up to 100% of the intended purpose. For example, functional can include or include about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to about 100% of the intended purpose. In some embodiments, the term functional can mean more than or more than about 100% of normal function, for example, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, or up to about 1000% of the intended purpose.

The term "recipient" and its grammatical equivalents as used herein can refer to a subject. The subject can be a human or a non-human animal. The recipient can also be in need thereof, such as in need of treatment for a disease, such as cancer. In some embodiments, the recipient can be in need of prophylactic therapy. The recipient can be in need thereof in other cases.

As used herein, the term "subject" and its grammatical equivalents can refer to a human or a non-human. The subject can be a mammal. The subject can be a human mammal of male or female biological sex. The subject can be of any age. The subject can be an embryo. The subject can be of age from a newborn to about 100 years of age. The subject can be in need thereof. The subject can be suffering from a disease such as cancer. The subject can be pre-menopausal, menopausal, or induced menopause.

The terms "treatment" or "treating" and their grammatical equivalents may refer to the medical management of a subject with the goal of curing, ameliorating, or stabilizing a disease, condition, or disorder. Treatment may include active treatment, i.e., treatment specifically directed to ameliorating a disease, condition, or disorder. Treatment may include causal treatment, i.e., treatment directed to removing the cause of the associated disease, condition, or disorder. In addition, such treatment may include palliative treatment, i.e., treatment designed for the relief of symptoms, rather than curing the disease, condition, or disorder. Treatment may include preventative treatment, i.e., treatment aimed at minimizing or partially or completely inhibiting the progression of a disease, condition, or disorder. Treatment may include supportive treatment, i.e., treatment employed to complement another specific therapy directed to ameliorating a disease, condition, or disorder. In some embodiments, the condition may be pathological. In some embodiments, the treatment may not completely cure, ameliorate, or stabilize the disease, condition, or disorder.

The terms "prevent" or "preventing," and their grammatical equivalents, can refer to prophylactic medical management of a subject. Such prophylactic medical management can include prophylactic management prior to the onset of a disease, condition, or disorder to prevent the onset of the disease, condition, or disorder.

In some embodiments, disclosed herein are compositions and methods for decellularization of whole organs and decellularization of parts of whole organs. Also disclosed herein, in some embodiments, are improved decellularization methods utilizing the methods and compositions. Also disclosed herein, in some embodiments, are improved decellularization compositions and methods disclosed herein that can result in more efficient decellularization and improved recellularization.

Preferred Organs and Parts Disclosed herein in some embodiments are compositions that may include an isolated organ or part thereof. Also disclosed herein in some embodiments are modified isolated organs or parts thereof. In some embodiments, the isolated organ or part thereof may be part of an isolated organ system of the body. In some embodiments, the isolated organ system may include, without limitation, the integumentary system, the muscular system, the skeletal system, the nervous system, the circulatory system, the lymphatic system, the respiratory system, the endocrine system, the urinary/excretory system, the reproductive system, and the digestive system. In some embodiments, the isolated organ or part thereof may be from the musculoskeletal system. In some embodiments, the isolated organ or part thereof from the musculoskeletal system may include bones, joints, ligaments, tendons, and muscles. In some embodiments, the isolated organ or part thereof may be from the digestive system. In some embodiments, the digestive system may include the tongue, teeth, salivary glands (parotid, submandibular, sublingual), pharynx, esophagus, stomach, small intestine (duodenum, jejunum, and ileum), large intestine, liver, gallbladder, or pancreas. In some embodiments, the isolated organ or portion thereof may include a respiratory system. In some embodiments, the respiratory system may include lungs, diaphragm, bronchi, trachea, larynx, pharynx, or nasal cavity. In some embodiments, the isolated organ or portion thereof may include a urinary system. In some embodiments, the urinary system may include kidneys, ureters, bladder, or urethra. In some embodiments, the isolated organ or portion thereof may include a reproductive system. In some embodiments, the reproductive system may include ovaries, fallopian tubes, uterus, vagina, vulva, clitoris, placenta, testes, epididymis, vas deferens, seminal vesicles, prostate, bulbourethral glands, penis, or scrotum. In some embodiments, the isolated organ or portion thereof may include an endocrine gland. In some embodiments, the endocrine gland may include a pituitary gland, pineal gland, thyroid gland, parathyroid gland, adrenal gland, or pancreas. In some embodiments, the isolated organ or portion thereof may include a circulatory system. In some embodiments, the circulatory system may include a cardiovascular system or a lymphatic system. In some embodiments, the cardiovascular system may include the heart, arteries, veins, or capillaries. In some embodiments, the lymphatic system may include lymphatic vessels, lymph nodes, bone marrow, thymus, or spleen. In some embodiments, the isolated organ or portion thereof may be from the nervous system. In some embodiments, the nervous system may include the central nervous system, the peripheral nervous system, or a sensory organ. In some embodiments, the central nervous system may include the brain, cerebrum, diencephalon, midbrain, pons, medulla oblongata, cerebellum, spinal cord, ventricular system (choroid plexus). In some embodiments, the peripheral nervous system may include nerves such as cranial nerves, spinal nerves, ganglia, and enteric nervous system. In some embodiments, the sensory organ may include the eye (cornea, iris, ciliary body, lens, or retina), ear (earlobe, tympanic membrane, middle ear, cochlea, semicircular canals, and vestibule of the ear), olfactory epithelium, or tongue. In some embodiments, the isolated organ or portion thereof may include the integumentary system. In some embodiments, the integumentary system may include mammary gland, skin, or subcutaneous tissue. In some embodiments, the isolated organ may be a solid organ. In some embodiments, the solid organ may have a solid tissue consistency, and is not hollow like the organs of the gastrointestinal tract, and is not liquid like blood. In some embodiments, the solid organ may include the heart, kidney, liver, lung, and pancreas. In some embodiments, the solid organ may have three major components: extracellular matrix (ECM), cells embedded therein, and a vasculature bed. In some embodiments, the decellularization of solid organs described herein may remove most or all of the cellular components while substantially preserving the extracellular matrix (ECM) and the vasculature bed. In some embodiments, the decellularization may include decellularizing about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or up to about 100% of the isolated organ. In some embodiments, decellularization may include decellularizing from about 50%-60%, 50-70%, 60-80%, 60-85%, 70-90%, 70-95%, 75-96%, 75-97%, 80-98%, 80-99%, or about 75-100% of the harvested organ.

In some embodiments, the isolated organ or portion thereof may be human. In some embodiments, the isolated organ or portion thereof may be non-human. The methods described herein may include providing a perfused decellularized ECM of a non-human mammalian organ. In some examples, the non-human mammalian organ may be from a larger animal. The perfused decellularized ECM may include an intact exterior surface. The perfused decellularized ECM may include a decellularized ECM vascular tree that may include an intact vasculature bed. In some cases, the perfused decellularized ECM may retain at least a portion, or a majority, of the fluid introduced into the decellularized ECM vascular tree.

In some embodiments, the isolated organ or portion thereof may be allogenic, autologous, or xenogenic. In some embodiments, the isolated organ or portion thereof may be from an adult or pediatric donor. In some embodiments, the isolated organ or portion thereof may include synthetic components. In some embodiments, the non-human isolated organ donor may be from a non-human mammal. In some embodiments, the non-human isolated organ may be from a non-human primate. In some embodiments, the non-human primate may be a baboon or chimpanzee. In some embodiments, the non-human isolated organ or portion thereof may be from a rodent, pig, rabbit, cow, sheep, dog, cat, cow, or monkey. In some embodiments, a gnotobiotic mammal may be utilized to provide the isolated organ or portion thereof. In some embodiments, the gnotobiotic mammal may be devoid of a microbiota. In some embodiments, the gnotobiotic mammal may be surgically delivered under sterile conditions. In some embodiments, the gnotobiotic mammal may be housed in an isolation barrier. In some embodiments, a gnotobiotic mammal may be about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 100% devoid of microbiota compared to an otherwise comparable non-gnotobiotic mammal. Examples of zoonotic pathogens that may be substantially devoid of in a gnotobiotic mammal may be rabies, monkeypox virus, Brucella/Suis, Mycobacterium spp., Trypanosoma cruzi, Ascaris spp., larvae, Toxoplasma gondii, EMCV, measles, rubella, hepatocystiis kochi, influenza, African swine fever, swine vesicular disease virus, to name a few.

In some embodiments, the isolated organ or portion thereof can be extracted from a non-human animal. In some embodiments, the non-human animal can be a fasted non-human animal. In some embodiments, the non-human animal can be a non-fasted non-human animal. In some embodiments, the non-fasted animal can be a non-human animal that has been fed within about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, or about 1 hour prior to removing the isolated organ or portion thereof from the animal.

The harvested organ or portion thereof may contain reduced levels of particulates before, during, or after decellularization. For example, the decellularization methods described herein may proceed with lower generation of particulates. Thus, the decellularization process may proceed, e.g., faster, more efficiently, or to a greater extent, as compared to an otherwise comparable decellularization method that has a higher generation of particulates.

In some embodiments, the isolated organ or portion thereof may comprise a reduced particulate level compared to an otherwise comparable isolated organ or portion thereof produced from an animal that has not been fed within about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, or about 1 hour prior to removal of the isolated organ or portion thereof from the animal. In some embodiments, the reduced particulate level may be reduced by at least about: 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1% compared to an otherwise comparable isolated organ or portion thereof produced from an animal that has not been fed within about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, or about 1 hour prior to removal of the isolated organ or portion thereof from the animal.

Organ Decellularization Also disclosed herein, in some embodiments, are compositions and methods of decellularization that can include a decellularized harvested organ. In some embodiments, the harvested organ can undergo decellularization. In some embodiments, decellularization can refer to a process that includes isolating the extracellular matrix (ECM) of a tissue from its native cells. A harvested organ or tissue that is at least partially decellularized can include extracellular matrix (ECM) components of all or most of the region of the harvested organ or part thereof, or tissue, including ECM components of the vascular tree. In some embodiments, the ECM components can include: fibronectin, fibrillin, laminin, elastin, members of the collagen family (e.g., collagen I, III, and IV), glycosaminoglycans, proteoglycans, cytokines, heparin sulfate, matrix, reticular fibers, or thrombospondin, or any combination thereof, which can remain organized as a defined structure, such as a basement membrane. In some embodiments, decellularization can refer to the reduction or absence of detectable myofilaments, endothelial cells, smooth muscle cells, and nuclei in tissue sections using standard histological staining procedures. Residual cellular debris can also be removed from decellularized harvested organs or tissues. Organ decellularization can be performed using a variety of techniques, such as perfusion decellularization, immersion decellularization, physical treatment, chemical treatment, enzymatic treatment, and combinations thereof.

Perfusion Decellularization Also disclosed herein, in some embodiments, are methods that may include perfusion-based decellularization of solid organs or portions thereof. In some embodiments, perfusion decellularization may enable decellularization via the native vasculature, tubes, cavities, and combinations thereof, such that large organs or portions thereof may be rapidly decellularized while keeping intact the vascular conduits important for reconstructing tissue. In some embodiments, perfusion decellularization allows rapid access to entire organs via the native vasculature by cannulating the vasculature and perfusing a mild detergent solution through the native blood vessels. Because organs are densely packed with capillaries, most cells may be located within 50-100 micrometers (μm) of capillaries, resulting in an exponential increase in the effective surface area of the detergent and a decrease in the time to dissolve cellular material during evacuation via the venous system, as opposed to via the isolated organ wall or capsule as occurs in immersion-based decellularization. Perfusion decellularization of ECM from isolated organs or tissues can preserve native microstructures, e.g., intact blood vessels and/or microvasculature, compared to other decellularization techniques, such as immersion-based decellularization. For example, perfused decellularized ECM from an organ or tissue can preserve collagen content and other binding and signaling factors and vasculature, thus providing a niche environment with native cues for functional differentiation of introduced cells or maintenance of cellular function. In some embodiments, perfused decellularized ECM from an organ or tissue can be perfused with cells and/or media using the vasculature of the perfused decellularized ECM under appropriate conditions, including appropriate pressure and flow to mimic conditions normally found in an organism. Normal pressure for human-sized organs can be from about 5 mmHg to about 200 mmHg, with the resulting flow rate depending on the diameter of the inflow perfusion vessel. In some embodiments, normal pressure for a human-sized organ can be from 5 mmHg, 10 mmHg, 15 mmHg, 20 mmHg, 25 mmHg, 30 mmHg, 40 mmHg, 50 mmHg, 60 mmHg, 80 mmHg, 100 mmHg, 120 mmHg, 140 mmHg, 160 mmHg, 180 mmHg, or up to about 200 mmHg. For a normal human heart, the resulting perfusion rate can be from about 20 mL/min/100 g to about 200 mL/min/100 g. Using such a system for seeded cells, higher seeding concentrations can be achieved, e.g., from about 5× up to about 1000× higher, than can be achieved under 2D cell culture conditions, and unlike 2D culture systems, the ECM environment allows for further functional differentiation of cells, e.g., differentiation of progenitor cells into cells exhibiting organ- or tissue-specific phenotypes with sustained function. Perfusion decellularization may include cannulation of the isolated organ or portion thereof. In some embodiments, at least one cannulation may be introduced into the isolated organ or portion thereof. In some cases, at least two cannulations may be introduced into the isolated organ or portion thereof. In some embodiments, about 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 cannulations may be introduced into the isolated organ or portion thereof. In some embodiments, the cannula may be part of a cannulation system. In some embodiments, the cannulation system may include a hollow tube appropriately sized for introduction into a vessel, duct, cavity, or any combination thereof of the isolated tissue, organ, or portion thereof. In some embodiments, the cannula may have a diameter appropriate for the animal; for example, the cannula may have a gauge. In some embodiments, the gauge may be 1-75. In some embodiments the gauge is 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, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
The cannula may be of 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 gauge. In some embodiments, the cannula may have a gauge of about 16. In some embodiments, the cannula may have a gauge of about 23. In some embodiments, the cannula may have a gauge of about 27. In some embodiments, the cannula may have a gauge of about 30. In some embodiments, the cannula may have a gauge of about 34. In some embodiments, the cannula may have a gauge of 16 gauge to 34 gauge. In some embodiments, the cannula may have a gauge of 30 gauge to 50 gauge. In some embodiments, the cannula may have a gauge of 30 gauge to 50 gauge. In some embodiments, the type of "cannula" can vary. In some embodiments, a typical cannula used for blood draws or IVs can be about 14-26 gauge. In some embodiments, the cannula can include a barbed connector with a luer fitting or a two-way barbed tubing to tubing connector. In some embodiments, the barb end can vary from about 1/32" to about 5/8" or more; for example, cannula size for a 6 month porcine liver can be 5/8" in diameter. In some embodiments, pigs can use cannulas from 1/16" to about 1/4". In some embodiments, the cannulas can have a gauge of about 1/16" to about 1". In some embodiments, the isolated organ or portion thereof can include at least a 34 gauge cannula. In some embodiments, disclosed herein can be an isolated organ or portion thereof that includes at least a 50 gauge cannula. In some embodiments, disclosed herein can be an isolated organ or portion thereof that includes at least a 16 gauge cannula. In some embodiments, disclosed herein can be an isolated organ or portion thereof that includes at least a 20 gauge cannula. In some embodiments, at least one blood vessel, duct, and/or cavity can be cannulated in the isolated organ. In some embodiments, the perfusion device or cannulation system includes a holding container for a solution (e.g., cell disruption medium) and one or more A mechanism (e.g., pump, air pressure, gravity) can be included to move fluids through the isolated organ via the cannula. In some embodiments, isolated organs or tissues during decellularization and/or recellularization can be maintained in a sterile state using various techniques, such as air flow control and filtering and/or perfusion with, for example, antibiotics, antifungals or other antimicrobial agents to prevent undesired microbial growth. In some embodiments, the systems described herein can have the ability to monitor certain perfusion characteristics (e.g., pressure, volume, flow pattern, temperature, gases, pH), mechanical forces (e.g., ventricular wall motion and tension), and electrical stimulation (e.g., pacing). In some embodiments, the vascular bed can change during decellularization and recellularization (e.g., vascular resistance, volume). In some embodiments, a pressure-regulated perfusion device or cannulation system can be advantageous to avoid or reduce fluctuations. In some embodiments, the effectiveness of perfusion can be evaluated in the effluent and in tissue sections. In some embodiments, perfusion volume, flow pattern, temperature, _O2 and _CO2 partial pressures, and pH can be monitored using standard methods. In some embodiments, sensors can be used to monitor the system (e.g., bioreactor) and/or the isolated tissue, organ, or portion thereof. In some embodiments, sonomicrometry, micromanometry, and/or conductance measurements can be used to obtain pressure-volume or preload recruitable stroke work information regarding myocardial wall motion and activity. In some embodiments, for example, sensors can be used to monitor the pressure of fluid moving through the cannulated organ or tissue; the ambient temperature in the system and/or the temperature of the isolated organ or tissue; the pH and/or flow rate of fluid moving through the cannulated organ or tissue; and/or the biological activity of the recellularized organ or tissue. In some embodiments, in addition to having sensors to monitor such characteristics, a system for decellularization and/or recellularization of an isolated organ or tissue can also include means for maintaining or adjusting such characteristics. In some embodiments, the means for maintaining or adjusting such characteristics can include features such as a thermometer, a thermostat, electrodes, pressure sensors, overflow valves, valves for altering the flow rate of liquids, valves for opening and closing fluid connections to solutions used to alter the pH of the solutions, balloons, external pacemakers, and/or ventricular chamber compliance. In some embodiments, the chamber, reservoir, or tubing can be fitted with a water jacket to help ensure stable conditions (e.g., temperature). In some embodiments, the method of decellularization can include providing an isolated organ or portion thereof, cannulating the isolated organ or portion thereof, and perfusing the cannulated isolated organ or portion thereof with a solution or medium via cannulation. In some embodiments, the cannulation occurs in a cavity, blood vessel, tube, or combination thereof. In some embodiments, about 1-3, about 1-5, about 2-3, about 2-5, about 1-8 solutions can be utilized for organ perfusion. In some embodiments, the solution can be perfused at least twice. In some embodiments, the solution can be perfused through the isolated organ or portion thereof at least 3, 4, 5, 6, 7, 8, 9, or up to 10 times. In some embodiments, a variety of solutions and media can be used during recellularization. In some embodiments, the solution can be selected from the group consisting of a cell disruption media, a cleaning solution, a disinfecting solution, or a combination thereof. In some embodiments, the cell disruption media can be a solution that can include at least one cleaning agent, Table 1. In some embodiments, at least one of sodium dodecyl sulfate (SDS), polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether (Triton X), NP-40, Brij, polyoxyethylene sorbitan monolaurate (Tween), octyl glucoside, octyl thioglucoside, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), a salt thereof, or a modification thereof can be utilized during decellularization. In some embodiments, the detergent can be an amphipathic molecule that can include both a non-polar "tail" and a polar "head" with aliphatic or aromatic character. In some embodiments, the ionic nature of the polar head group can form the basis of broad classifications of detergents; they can be ionic (charged, either anionic or cationic), nonionic (uncharged), or zwitterionic (having both positively and negatively charged groups, but with a net charge of zero). In some embodiments, detergents can be denaturing or non-denaturing with respect to protein structure. In some embodiments, denaturing detergents are anionic, such as sodium dodecyl sulfate (SDS), or ethyltrimethylammonium bromide.

In some embodiments, these detergents may be cationic, such as tetracycline, ... In some embodiments, the concentration of PBS is about 0.5%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5
%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to about 100%. In some embodiments, the cleaning solution can be supplemented with a drug. In some embodiments, the drug can be an antibiotic, DNase I, or a disinfectant. In some embodiments, the cleaning solution can include saline. In some embodiments, the cleaning solution can be 0.9% NaCl, 0.55% PBS, and 100% PBS, saline, and any combination thereof. In some embodiments, the cell disruption medium can be a degassed cell disruption medium. In some embodiments, a "degassed" cell disruption medium can refer to a disruption medium as described herein from which dissolved gas has been substantially removed. In some embodiments, the dissolved gases may include oxygen, nitrogen, air, carbon dioxide, carbon monoxide, argon, hydrogen sulfide, methane, ethylene, ethane, chlorine, hydrogen, helium, ammonia, sulfur dioxide, or any combination thereof, hi some embodiments, the degassed medium may contain less dissolved gases than the non-degassed medium. In some embodiments, the degassing medium is about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5%, 25%, 25.5% , 26%, 26.5%, 27%, 27.5%, 28%, 28.5%, 29%, 29.5%, 30%, 30.5%, 31%, 31.5%, 32%, 32.5%, 33%, 33.5%, 34%, 34.5%, 35%, 35.5%, 36%, 36.5%, 37%, 37.5%, 38%, 38. 5%, 39%, 39.5%, 40%, 40.5%, 41%, 41.5%, 42%, 42.5%, 43%, 43.5%, 44%, 44.5%, 45%, 45.5%, 46%, 46.5%, 47%, 47.5%, 48%, 48.5%, 49%, 49.5%, 50%, 50.5%, 51%, 51 .5%, 52%, 52.5%, 53%, 53.5%, 54%, 54.5%, 55%, 55.5%, 56%, 56.5%, 57%, 57.5%, 58%, 58.5%, 59%, 59.5%, 60%, 60.5%, 61%, 61.5%, 62%, 62.5%, 63%, 63.5%, 64%, 64.5%, 65%, 65.5%, 66%, 66.5%, 67%, 67.5%, 68%, 68.5%, 69%, 69.5%, 70%, 70.5%, 71%, 71.5%, 72%, 72.5%, 73%, 73.5%, 74%, 74.5%, 75%, 75.5%, 76%, 76.5%, 77% , 77.5%, 78%, 78.5%, 79%, 79.5%, 80%, 80.5%, 81%, 81.5%, 82%, 82.5%, 83%, 83.5%, 84%, 84.5%, 85%, 85.5%, 86%, 86.5%, 87%, 87.5%, 88%, 88.5%, 89%, 89.5%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 99.9% dissolved gas content of the non-degassed medium. In some embodiments, the degassing medium can contain an amount of gas at a given temperature; for example, the degassing medium can contain an amount of gas of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6 ,4.7,4.8,4.9,5,5.1,5.2,5.3,5.4,5.5,5.6,5.7,5.8,5.9,6,6.1,6.2,6.3,6.4,6.5,6.6,6.7,6.8,6.9,7,7.1,7.2,7.3,7.4,7.5,7.6,7.7,7.8,7.9,8,8.1,8.2,8.3,8.4,8.5,8.6,8.7,8.8,8.9,9,9.1,9.2,9.3,9.4,9.5,9.6,9.7,9.8,9.9,10,10.1,10.2,10.3,10.4,1
0.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13 .1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 1 5.7, 15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1, 18.2, 18 .3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20, 20.1, 20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 2 0.9, 21, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 22, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7, 22.8, 22.9, 23, 23.1, 23.2, 23.3, 23.4, 23 .5, 23.6, 23.7, 23.8, 23.9, 24, 24.1, 24.2, 24.3, 24.4, 24.5, 24.6, 24.7, 24.8, 24.9, 25, 25.1, 25.2, 25.3, 25.4, 25.5, 25.6, 25.7, 25.8, 25.9, 26, 26. 1, 26.2, 26.3, 26.4, 26.5, 26.6, 26.7, 26.8, 26.9, 27, 27.1, 27.2, 27.3, 27.4, 27.5, 27.6, 27.7, 27.8, 27.9, 28, 28.1, 28.2, 28.3, 28.4, 28.5, 28.6, 28 .7, 28.8, 28.9, 29, 29.1, 29.2, 29.3, 29.4, 29.5, 29.6, 29.7, 29.8, 29.9, 30, 30.1, 30.2, 30.3, 30.4, 30.5, 30.6, 30.7, 30.8, 30.9, 31, 31.1, 31.2, 31. 3, 31.4, 31.5, 31.6, 31.7, 31.8, 31.9, 32, 32.1, 32.2, 32.3, 32.4, 32.5, 32.6, 32.7, 32.8, 32.9, 33, 33.1, 33.2, 33.3, 33.4, 33.5, 33.6, 33.7, 33.8, 33 .9, 34, 34.1, 34.2, 34.3, 34.4, 34.5, 34.6, 34.7, 34.8, 34.9, 35, 35.1, 35.2, 35.3, 35.4, 35.5, 35.6, 35.7, 35.8, 35.9, 36, 36.1, 36.2, 36.3, 36.4, 36. 5, 36.6, 36.7, 36.8, 36.9, 37, 37.1, 37.2, 37.3, 37.4, 37.5, 37.6, 37.7, 37.8, 37.9, 38, 38.1, 38.2, 38.3, 38.4, 38.5, 38.6, 38.7, 38.8, 38.9, 39, 39.1 , 39.2, 39.3, 39.4, 39.5, 39.6, 39.7, 39.8, 39.9, 40, 40.1, 40.2, 40.3, 40.4, 40.5, 40.6, 40.7, 40.8, 40.9, 41, 41.1, 41.2, 41.3, 41.4, 41.5, 41.6, 41. 7, 41.8, 41.9, 42, 42.1, 42.2, 42.3, 42.4, 42.5, 42.6, 42.7, 42.8, 42.9, 43, 43.1, 43.2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44, 44.1, 44.2, 44.3 , 44.4, 44.5, 44.6, 44.7, 44.8, 44.9, 45, 45.1, 45.2, 45.3, 45.4, 45.5, 45.6, 45.7, 45.8, 45.9, 46, 46.1, 46.2, 46.3, 46.4, 46.5, 46.6, 46.7, 46.8, 46. 9, 47, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, 48, 48.1, 48.2, 48.3, 48.4, 48.5, 48.6, 48.7, 48.8, 48.9, 49, 49.1, 49.2, 49.3, 49.4, 49.5 ,49.6,49.7,49.8,49.9,50,50.1,50.2,50.3,50.4,50.5,50.6,50.7,50.8,50.9,51,51.1,51.2,51.3,51.4,51.5,51.6,51.7,51.8,51.9,52,52.1,
52.2, 52.3, 52.4, 52.5, 52.6, 52.7, 52.8, 52.9, 53, 53.1, 53.2, 53.3, 53.4, 53.5, 53.6, 53.7, 53.8, 53.9, 54, 54.1, 54.2, 54.3, 54.4, 54.5, 54.6, 54.7 , 54.8, 54.9, 55, 55.1, 55.2, 55.3, 55.4, 55.5, 55.6, 55.7, 55.8, 55.9, 56, 56.1, 56.2, 56.3, 56.4, 56.5, 56.6, 56.7, 56.8, 56.9, 57, 57.1, 57.2, 57.3, 57.4, 57.5, 57.6, 57.7, 57.8, 57.9, 58, 58.1, 58.2, 58.3, 58.4, 58.5, 58.6, 58.7, 58.8, 58.9, 59, 59.1, 59.2, 59.3, 59.4, 59.5, 59.6, 59.7, 59.8, 59.9 ,60,60.1,60.2,60.3,60.4,60.5,60.6,60.7,60.8,60.9,61,61.1,61.2,61.3,61.4,61.5,61.6,61.7,61.8,61.9,62,62.1,62.2,62.3,62.4,62.5,
62.6, 62.7, 62.8, 62.9, 63, 63.1, 63.2, 63.3, 63.4, 63.5, 63.6, 63.7, 63.8, 63.9, 64, 64.1, 64.2, 64.3, 64.4, 64.5, 64.6, 64.7, 64.8, 64.9, 65, 65.1, 65.2, 65.3, 65.4, 65.5, 65.6, 65.7, 65.8, 65.9, 66, 66.1, 66.2, 66.3, 66. 4, 66.5, 66.6, 66.7, 66.8, 66.9, 67, 67.1, 67.2, 67.3, 67.4, 67.5, 67.6, 67.7, 67.8, 67.9, 68, 68.1, 68.2, 68.3, 68.4, 68.5, 68.6, 68.7, 68.8, 68.9, 69, 69.1, 69.2, 69.3, 69.4, 69.5, 69.6, 69.7, 69.8, 69.9, 70, 70.1, 70.2, 7 0.3, 70.4, 70.5, 70.6, 70.7, 70.8, 70.9, 71, 71.1, 71.2, 71.3, 71.4, 71.5, 71.6, 71.7, 71.8, 71.9, 72, 72.1, 72.2, 72.3, 72.4, 72.5, 72.6, 72.7, 72.8, 72.9, 73, 73.1, 73.2, 73.3, 73.4, 73.5, 73.6, 73.7, 73.8, 73.9, 74, 74.1 ,74.2,74.3,74.4,74.5,74.6,74.7,74.8,74.9,75,75.1,75.2,75.3,75.4,75.5,75.6,75.7,75.8,75.9,76,76.1,76.2,76.3,76.4,76.5,76.6,76.7,76.8,76.9,77,77.1,77.2,77.3,77.4,77.5,77.6,77.7,77.8,77.9, 78, 78.1, 78.2, 78.3, 78.4, 78.5, 78.6, 78.7, 78.8, 78.9, 79, 79.1, 79.2, 79.3, 79.4, 79.5, 79.6, 79.7, 79.8, 79.9, 80, 80.1, 80.2, 80.3, 80.4, 80.5, 80.6, 80.7, 80.8, 80.9, 81, 81.1, 81.2, 81.3, 81.4, 81.5, 81.6, 81.7, 81. 8, 81.9, 82, 82.1, 82.2, 82.3, 82.4, 82.5, 82.6, 82.7, 82.8, 82.9, 83, 83.1, 83.2, 83.3, 83.4, 83.5, 83.6, 83.7, 83.8, 83.9, 84, 84.1, 84.2, 84.3, 84.4, 84.5, 84.6, 84.7, 84.8, 84.9, 85, 85.1, 85.2, 85.3, 85.4, 85.5, 85.6, 8 5.7, 85.8, 85.9, 86, 86.1, 86.2, 86.3, 86.4, 86.5, 86.6, 86.7, 86.8, 86.9, 87, 87.1, 87.2, 87.3, 87.4, 87.5, 87.6, 87.7, 87.8, 87.9, 88, 88.1, 88.2, 88.3, 88.4, 88.5, 88.6, 88.7, 88.8, 88.9, 89, 89.1, 89.2, 89.3, 89.4, 89.5 ,89.6,89.7,89.8,89.9,90,90.1,90.2,90.3,90.4,90.5,90.6,90.7,90.8,90.9,91,91.1,91.2,91.3,91.4,91.5,91.6,91.7,91.8,91.9,92,92.1,92.2,92.3,92.4,92.5,92.6,92.7,92.8,92.9,93,93.1,93.2,93.3,93
． 4, 93.5, 93.6, 93.7, 93.8, 93.9, 94, 94.1, 94.2, 94.3, 94.4, 94.5, 94.6, 94.7, 94.8, 94.9, 95, 95.1, 95.2, 95.3, 95.4, 95.5, 95.6, 95.7, 95.8, 95.9, 96, 96.1, 96.2, 96.3, 96.4, 96.5, 96.6, 96.7, 96.8, 96. 9, 97, 97.1, 97.2, 97.3, 97.4, 97.5, 97.6, 97.7, 97.8, 97.9, 98, 98.1, 98.2, 98.3, 98.4, 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, or less than 100 ppb of gas at 25° C. In some embodiments, the degassing medium is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, The degassed medium may contain less than 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 ppm gas at 25° C. In some embodiments, the degassed medium may be prepared by placing the medium under reduced pressure (e.g., under a vacuum or vacuum pump). In some embodiments, the "reduced pressure" can be a pressure less than atmospheric pressure; for example, the "reduced pressure" can be a pressure less than 760 torr. In some embodiments, the reduced pressure can be a vacuum, a medium vacuum, a high vacuum, or an ultra-high vacuum. In some embodiments, the reduced pressure can be about 760, 759, 758, 757, 756, 755, 754, 753, 752, 751, 750, 749, 748, 747, 746, 745, 744, 743, 742, 741, 740, 739, 738, 737, 736, 735, 734, 733, 732, 731, 730, 729, 728, 727, 726, 725, 724, 723, 722, 721, 720, 719, 718, 717, 716, 715, 714, 713, 712, 711, 710, 709, 708, 707, 706, 705, 704, 703, 702, 701, 700, 699, 698 , 697, 696, 695, 694, 693, 692, 691, 690, 689, 688, 687, 686, 685, 684, 683, 682, 681, 680, 679, 678, 677, 676, 675, 674, 673, 672, 671, 670, 669, 668, 667, 666, 665, 66 4, 663, 662, 661, 660, 659, 658, 657, 656, 655, 654, 653, 652, 651, 650, 649, 648, 647, 646, 645, 644, 643, 642, 641, 640, 639, 638, 637, 636, 635, 634, 633, 632, 631, 63 0, 629, 628, 627, 626, 625, 624, 623, 622, 621, 620, 619, 618, 617, 616, 615, 614, 613, 612, 611, 610, 609, 608, 607, 606, 605, 604, 603, 602, 601, 600, 599, 598, 597, 5 96, 595, 594, 593, 592, 591, 590, 589, 588, 587, 586, 585, 584, 583, 582, 581, 580, 579, 578, 577, 576, 575, 574, 573, 572, 571, 570, 569, 568, 567, 566, 565, 564, 563, 5 62, 561, 560, 559, 558, 557, 556, 555, 554, 553, 552, 551, 550, 549, 548, 547, 546, 545, 544, 543, 542, 541, 540, 539, 538, 537, 536, 535, 534, 533, 532, 531, 530, 529, 528, 527, 526, 525, 524, 523, 522, 521, 520, 519, 518, 517, 516, 515, 514, 513, 512, 511, 510, 509, 508, 507, 506, 505, 504, 503, 502, 501, 500, 499, 498, 497, 496, 495,
494, 493, 492, 491, 490, 489, 488, 487, 486, 485, 484, 483, 482, 481, 480, 479, 478, 477, 476, 475, 474, 473, 472, 471, 470, 469, 468, 467, 466, 465, 464, 463, 462, 461, 460, 459, 458, 457, 456, 455, 454, 453, 452, 451, 450, 449, 448, 447, 446, 445, 44 4, 443, 442, 441, 440, 439, 438, 437, 436, 435, 434, 433, 432, 431, 430, 429, 428, 427, 426, 425, 424, 423, 422, 421, 420, 419, 418, 417, 416, 415, 414, 413, 412, 411, 410, 409, 408, 407, 406, 405, 404, 403, 402, 401, 400, 399, 398, 397, 396, 395, 394, 3 93, 392, 391, 390, 389, 388, 387, 386, 385, 384, 383, 382, 381, 380, 379, 378, 377, 376, 375, 374, 373, 372, 371, 370, 369, 368, 367, 366, 365, 364, 363, 362, 361, 360, 359, 358, 357, 356, 355, 354, 353, 352, 351, 350, 349, 348, 347, 346, 345, 344, 343, 342, 341, 340, 339, 338, 337, 336, 335, 334, 333, 332, 331, 330, 329, 328, 327, 326, 325, 324, 323, 322, 321, 320, 319, 318, 317, 316, 315, 314, 313, 312, 311, 310, 309, 308, 307, 306, 305, 304, 303, 302, 301, 300, 299, 298, 297, 296, 295, 294, 293, 292 , 291, 290, 289, 288, 287, 286, 285, 284, 283, 282, 281, 280, 279, 278, 277, 276, 275, 274, 273, 272, 271, 270, 269, 268, 267, 266, 265, 264, 263, 262, 261, 260, 259, 258, 257, 256, 255, 254, 253, 252, 251, 250, 249, 248, 247, 246, 245, 244, 243, 242, 2 41, 240, 239, 238, 237, 236, 235, 234, 233, 232, 231, 230, 229, 228, 227, 226, 225, 224, 223, 222, 221, 220, 219, 218, 217, 216, 215, 214, 213, 212, 211, 210, 209, 208, 207, 206, 205, 204, 203, 202, 201, 200, 199, 198, 197, 196, 195, 194, 193, 192, 191, 190, 189, 188, 187, 186, 185, 184, 183, 182, 181, 180, 179, 178, 177, 176, 175, 174, 173, 172, 171, 170, 169, 168, 167, 166, 165, 164, 163, 162, 161, 160, 159, 158, 157, 156, 155, 154, 153, 152, 151, 150, 149, 148, 147, 146, 145, 144, 143, 142, 141, 140 , 139, 138, 137, 136, 135, 134, 133, 132, 131, 130, 129, 128, 127, 126, 125, 124, 123, 122, 121, 120, 119, 118, 117, 116, 115, 114, 113, 112, 111, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86,
85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42 , 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19 ^, 18, 17, 16 ^{, 15} , 14, ¹³ , 12, 11, 10, 9, 8, 7, 6, 5, 4, ³ , 2, or 1 torr. In some embodiments, the reduced pressure is less than about 1×10 ⁰ , 1×10 ^-1 , 1×10 ^-2 , 1×10 -3 , 1×10 ^-4 , 1×10 -5 , 1×10 ^-6 , 1×10 ^-7 , 1×10 -8 , 1×10 -9 , 1×10 ^-10 , 1×
^10-11 , or a pressure of less than ^1x10-12 torr. In some embodiments, the degassed medium can be prepared by heating the degassed medium. In some embodiments, the degassed medium can be prepared at a temperature of about 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, °C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, or 100°C. In some embodiments, the degassing medium is from about 25°C to about 27°C, from about 25°C to about 28°C, from about 25°C to about 29°C, from about 25°C to about 30°C, from about 25°C to about 31°C, from about 25°C to about 32°C, from about 25°C to about 33°C, from about 25°C to about 34°C, from about 25°C to about 35°C, from about 25°C to about 36°C, from about 25°C to about 37°C, from about 25°C to about 38°C, from about 25°C to about 39°C, from about 25°C to about 40°C, from about 25°C to about 41°C, from about 25°C to about 42°C, from about 25°C to about 43°C, from about 25°C to about 44°C, from about 25°C to about 45°C, from about 25°C to about 46°C, from about 25°C to about 47°C, from about 25°C to about 48°C, from about 25°C to about 49°C, from about 25°C to about 50°C, from about 25°C to about 51°C, from about 25°C to about 52°C, from about 25°C to about 53°C, from about 25°C to about 54°C, from about 25°C to about 55°C, from about 25°C to about 56°C, from about 25°C to about 57°C, from about 25°C to about 58°C, from about 25°C to about 59°C, from about 2 °C to about 44°C, about 25°C to about 45°C, about 25°C to about 46°C, about 25°C to about 47°C, about 25°C to about 48°C, about 25°C to about 49°C, about 25°C to about 50°C, about 25°C to about 51°C, about 25°C to about 52°C, about 25°C to about 53°C, about 25°C to about 54°C, about 25°C to about 55°C, about 25°C to about 56°C, about 25°C to about 57°C, about 25°C to about 58°C, about 25°C to about 59°C, about 25°C to about 60°C, about 25°C to about 61°C, about 25°C to about 62°C, about 25°C to about 63°C, about 25°C to about 64°C, about 25°C to about 65°C, about 25°C to about 66°C, about 25°C to about 67°C, about 25°C to about 68°C, about 25°C to about 69°C, about 25°C to about 70°C, about 25°C to about 71°C, about 25°C to about 72°C, about 25°C to about 73°C, about 25°C to about 74°C, about 25°C to about 75°C, about 25°C to about 76°C, about 25°C to about 77°C, about 25°C to about 78°C, about 25°C to about 79°C, about 25°C to about 80°C, about 25°C to about 81°C, about 25°C to about 82°C , from about 25°C to about 83°C, from about 25°C to about 84°C, from about 25°C to about 85°C, from about 25°C to about 86°C, from about 25°C to about 87°C, from about 25°C to about 88°C, from about 25°C to about 89°C, from about 25°C to about 90°C, from about 25°C to about 91°C, from about 25°C to about 92°C, from about 25°C to about 93°C, from about 25°C to about 94°C, from about 25°C to about 95°C, from about 25°C to about 96°C, from about 25°C to about 97°C, from about 25°C to about 98°C, from about 25°C to about 99°C, from about 25°C to about 100°C. In some embodiments, the degassing medium is cooled to about 50° C., about 50° C. to about 52° C., about 50° C. to about 53° C., about 50° C. to about 54° C., about 50° C. to about 55° C., about 50° C. to about 56° C., about 50° C. to about 57° C., about 50° C. to about 58° C., about 50° C. to about 59° C., about 50° C. to about 60° C., about 50° C. to about 61° C., about 50° C. to about 62° C., about 50° C. to about 63° C., about 50° C. to about 64° C., about 50° C. to about 65° C., about 50° C. to about 66° C., about 50° C. to about 67° C., about 50° C. to about 68° C., about 50° C. to about 69° C., about 50° C. to about 70° C., about 50° C. to about 71° C., about 50° C. to about 72° C., about 50° C. to about 73° C., about 50° C. to about 74° C., about 50° C. to about 75° C., about 50° C. to about 76° C., about 50° C. to about 77° C., about 50° C. to about 78° C., about 50° C. to about 79° C., about 50° C. to about 80° C., about 50° C. to about 81° C., about 50° C. to about °C to about 63 °C, about 50 °C to about 64 °C, about 50 °C to about 65 °C, about 50 °C to about 66 °C, about 50 °C to about 67 °C, about 50 °C to about 68 °C, about 50 °C to about 69 °C, about 50 °C to about 70 °C, about 50 °C to about 71 °C, about 50 °C to about 72 °C, about 50 °C to about 73 °C, about 50 °C to about 74 °C, about 50 °C to about 75 °C, about 50 °C C. to about 76.degree. C., about 50.degree. C. to about 77.degree. C., about 50.degree. C. to about 78.degree. C., about 50.degree. C. to about 79.degree. C., about 50.degree. C. to about 80.degree. C. to about 81.degree. C., about 50.degree. C. to about 82.degree. C. to about 83.degree. C., about 50.degree. C. to about 84.degree. C. to about 85.degree. C., about 50.degree. C. to about 86.degree. C. to about 87.degree. C., about 50.degree. C. to about 88.degree. C. to about 89.degree. C., about 50.degree. C. to about 90.degree. C. to about 91.degree. C., about 50.degree. C. to about 92.degree. C. to about 93.degree. C., about 50.degree. C. to about 94.degree. C. to about 95.degree. C. to about 96.degree. C., about 50.degree. C. to about 97.degree. C., about 50.degree. C. to about 98.degree. C., about 50.degree. C. to about 99.degree. C., or about 50.degree. C. to about 100.degree. In some embodiments, the medium can be placed under reduced pressure, elevated pressure, or both for a given period of time to reduce the concentration of dissolved gases, hi some embodiments, the medium can be placed under reduced pressure, elevated pressure, or both for at least 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, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 minutes. In some embodiments, the medium can be placed under reduced pressure, elevated pressure, or both for at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, or 24 hours. In some embodiments, a degassed medium (e.g., a degassed decellularization or recellularization medium) may function more efficiently than a non-degassed medium when perfused into a harvested organ; for example, decellularization using a degassed medium may require a smaller volume of degassed medium to achieve an equivalent amount of decellularization compared to a non-degassed medium. In some embodiments, decellularization using a degassed medium may require less time to achieve a given amount of decellularization compared to the time required to achieve an equivalent amount of decellularization using a non-degassed medium. In some embodiments, perfusion with a degassed medium produces fewer microbubbles than perfusion with a non-degassed medium. In some embodiments, a disinfectant solution may be utilized during decellularization. In some embodiments, the disinfectant solution may include any number of agents, such as an antibiotic, a disinfectant, or a combination thereof. In some embodiments, antibiotics that can be used in the decellularization solution can be selected from the group consisting of: actinomycin, ampicillin, carbenicillin, cefotaxime, fosmidomycin, gentamicin, kanamycin, neomycin, amphotericin, penicillin, polymyxin, streptomycin, broad spectrum antibiotics, and combinations thereof. In some embodiments, any concentration of antibiotic can be introduced into the disinfectant solution. In some embodiments, suitable concentrations of antibiotics can be: 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or up to about 60%. In some embodiments, suitable concentrations of antibiotics can be: 0.5 U/ml, 1 U/ml, 5 U/ml, 10 U/ml, 20 U/ml, 30 U/ml, 40 U/ml, 50 U/ml, 60 U/ml, 70 U/ml, 80 U/ml, 90 U/ml, 100 U/ml, 110 U/ml, 120 U/ml, 130 U/ml, 140 U/ml, 150 U/ml, 160 U/ml, 170 U/ml, 180 U/ml, 190 U/ml, 200 U/ml, 300 U/ml, 400 U/ml, 500 U/ml, 600 U/ml, 700 U/ml, 800 U/ml, 900 U/ml, 1000 U/ml, and up to about 1500 U/ml. In some embodiments, a suitable concentration of antibiotic may be: 0.5 μg/ml, 1 μg/ml, 1.5 μg/ml, 2 μg/ml, 2.5 μg/ml, 3 μg/ml, 3.5 μg/ml, 4 μg/ml, 4.5 μg/ml, 5 μg/ml, 5.5 μg/ml, 6 μg/ml, 6.5 μg/ml, 7 μg/ml, 7.5 μg/ml, 8 μg/ml, 8.5 μg/ml, 9 μg/ml, 9.5 μg/ml, 10 μg/ml, 15 μg/ml, 20 μg/ml, 25 μg/ml, 30 μg/ml, 35 μg/ml, 40 μg/ml, 45 μg/ml, 50 μg/ml, or up to about 60 μg/ml. In some embodiments, the antibiotics may be 1% benzalkonium chloride, 100 U/ml penicillin-G, 100 U/ml streptomycin, 50 μg/ml gentamicin, and 0.25 μg/ml amphotericin B. In some embodiments, generally moderate concentrations of neutral (i.e., non-ionic) detergents can compromise cell membrane integrity, thereby facilitating cell lysis and extraction of soluble proteins, often in their native form. In some embodiments, using certain buffer conditions, various detergents effectively penetrate between membrane bilayers at sufficient concentrations to form mixed micelles with isolated phospholipids and membrane proteins. In some embodiments, denaturing detergents such as SDS can bind both membrane (hydrophobic) and non-membrane (water-soluble, hydrophilic) proteins at concentrations below the CMC (i.e., as monomers). In some embodiments, the reaction can be driven at equilibrium until saturation, and thus the free concentration of monomers can determine the detergent concentration. In some embodiments, SDS binding can be cooperative (i.e., binding of one molecule of SDS increases the probability that another molecule of SDS will bind to the protein), turning most proteins into rigid rods whose length can be proportional to molecular weight. In some embodiments, non-denaturing detergents such as Triton X-100 have a rigid and bulky non-polar head that does not penetrate water-soluble proteins; as a result, they generally do not disrupt the native interactions and structure of water-soluble proteins and do not have cooperative binding properties. In some embodiments, the primary effect of non-denaturing detergents can be to associate with hydrophobic portions of membrane proteins, thereby conferring miscibility to them. In some embodiments, the system for generating isolated organs or parts thereof or tissues can be controlled by a computer-readable storage medium in combination with a programmable processor (e.g., a computer-readable storage medium as used herein has stored thereon instructions for causing a programmable processor to perform certain steps). In some embodiments, for example, such a storage medium can be in combination with a programmable processor to receive and process information from one or more sensors. In some embodiments, such a storage medium in combination with a programmable processor can also transmit information and instructions back to the bioreactor and/or the isolated organ or tissue. In some embodiments, the isolated organ or tissue undergoing recellularization can be monitored for biological activity. In some embodiments, the biological activity can be the activity of the isolated organ or part thereof or tissue itself, e.g., in the case of cardiac tissue, the electrical activity, mechanical activity, mechanical pressure, contractility, and/or wall stress of the isolated organ or tissue. In some embodiments, the biological activity of cells attached to or transplanted on the isolated organ or part thereof or tissue can be monitored for, e.g., ion transport/exchange activity, cell division, and/or cell viability. In some embodiments, it can be useful to simulate an active load on the isolated organ or part thereof during recellularization. In some embodiments, the computer readable storage medium in combination with a programmable processor can be used to adapt the necessary components to monitor and maintain an active load on the isolated organ or tissue. In some embodiments, the weight of the isolated organ or part thereof or tissue can be entered into a computer readable storage medium as described herein, which in combination with a programmable processor can calculate the exposure time and perfusion pressure for that particular organ or tissue. In some embodiments, such a storage medium can record the preload and afterload (pre-perfusion and post-perfusion pressures, respectively) and flow rates. In some embodiments, for example, a computer readable storage medium in combination with a programmable processor can regulate the perfusion pressure, the direction of perfusion, and/or the type of perfusion solution via one or more pump and/or valve controls. In some embodiments, perfusion decellularization of the isolated organ or part thereof can be about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or up to about 100% more effective than a non-perfusion based decellularization system. In some embodiments, decellularization of the isolated organ or part thereof can be determined using a variety of means. In some embodiments, decellularization can be determined by histological examination. In some embodiments, histological examination may show a lack or reduction in cellular material, nuclei, and combinations thereof within an excised organ or portion thereof that has been at least partially decellularized, while retaining overall structures such as lobules and central veins. In some embodiments, decellularization may be determined by immunohistochemical staining. In some embodiments, immunohistochemical staining may show a lack of cellular factors such as galactosyl-α(1,3) galactose (α-Gal) following perfusion decellularization. In some embodiments, decellularization may be determined using DNA quantification. In some embodiments, DNA quantification may include assays such as PicoGreen, qPCR, and Quant-IT. In some embodiments, DNA quantification assays may determine the amount of DNA reduced in an excised organ or portion thereof. In some embodiments, decellularization may be determined by any of the following methods: histological structure with H&E staining, immunofluorescence of cellular structures, immunohistochemical staining of cell membrane associated proteins, nuclear staining, e.g., Dapi, or any combination thereof. In some embodiments, the perfusion-based decellularized harvested organ or part thereof preserves the native scaffold containing the appropriate microenvironment required for the introduction of organ-specific cells, along with an intact vascular network for reconnection to an outer capsule capable of maintaining the subject's blood supply and physiological blood pressure. In some embodiments, these components may be important for the subsequent use of perfusion recellularization, which also uses perfusion to repopulate the at least partially decellularized harvested organ or part thereof with blood vessels and organ-specific regenerative cells, where they can migrate to the appropriate microenvironment (via associated signaling protein markers that remain within the perfused decellularized scaffold) as the at least partially decellularized harvested organ or part thereof grows and matures under normal physiological conditions in a bioreactor. In some embodiments, the resulting at least partially recellularized harvested organ or part thereof can then be transplanted utilizing techniques comparable to current organ transplants. In some embodiments, the scaffold created by perfusion decellularization may be capable of receiving and incorporating a variety of cells. In some embodiments, the decellularization process can generate particulates. In some embodiments, particulates can refer to residual comments from decellularization. In some embodiments, particulates can be formed by insoluble interactions between native proteins and detergents. In some embodiments, particulates can be immiscible in aqueous solutions. In some embodiments, insoluble white particulates are seen during decellularization of whole organs with SDS or other detergents. In some embodiments, particulates are formed during decellularization and then become trapped in the organ, which can negatively affect the use of the decellularized matrix for acellular products or the use of the decellularized harvested organ part or matrix for recellularization. In some embodiments, the method can reduce particulate formation by using saline solutions, non-fasting mammals, and combinations thereof. In some embodiments, reducing particulate formation can reduce the cytotoxicity of the decellularized matrix to introduced cells, for example, during recellularization. In some embodiments, particulate reduction can be achieved by using solutions, controlling the dietary habits of the mammal, or combinations thereof.

Immersion Decellularization Also disclosed herein, in some embodiments, is a method of immersion-based decellularization of an isolated organ or a portion thereof. In some embodiments, an entire organ or a portion thereof can be decellularized by removing all cellular and tissue content from the organ. In some embodiments, decellularization can include a series of sequential extractions. In some embodiments, a first step can include removal of cellular debris and solubilization of cell membranes. In some embodiments, this can be followed by solubilization of nuclear cytoplasmic and nuclear components. In some embodiments, an isolated organ can be decellularized by removing cell membranes and cell debris surrounding the isolated organ using gentle mechanical disruption methods. In some embodiments,
Gentle mechanical disruption methods can disrupt cell membranes. In some embodiments, the decellularization process can avoid damaging or disturbing the complex substructure of the biological structure. In some embodiments, gentle mechanical disruption methods can include scraping the surface of the isolated organ or part thereof, agitating the isolated organ or part thereof, or stirring the isolated organ or part thereof in a suitable volume of fluid, such as distilled water. In some embodiments, gentle mechanical disruption methods can disrupt cell membranes and can include magnetically stirring the isolated organ or part thereof in a suitable volume of distilled water (e.g., using a magnetic stir bar and magnetic plate) until cellular debris is removed from the isolated organ or part thereof. In some embodiments, after removing the cell membrane, nuclear or cytoplasmic biological structural components can be removed. In some embodiments, this can be done by solubilizing the cell or nuclear components without disrupting the substructure. In some embodiments, non-ionic detergents or detergents can be used to solubilize the nuclear components. In some embodiments, examples of non-ionic detergents or surfactants include, but are not limited to, Triton X-100, Triton N-101, Triton X-114, Triton X-405, Triton N-102, Triton N-101 ...3, Triton N-104, Triton N-105, Triton N-106, Triton N-107, Triton N-108, Triton N-109, Triton N-114, Triton N-115, Triton N-116, Triton N-117, Triton N-118, Triton N-119, Triton N-120, Triton N-121, Triton N-122, Triton N-123, Triton N-124, Triton N-125, Triton N-126, Tri
Triton series detergents available from Rohm and Haas, Philadelphia, Pa., including Triton DF-16, X-705, and Triton DF-16; Tween series detergents, such as monolaurate (Tween 20), monopalmitate (Tween 40), monooleate (Tween 80), and polyoxyethylene-23-lauryl ether (Brij. 35), polyoxyethylene ether W-1 (Polyox), sodium cholate, deoxycholate, CHAPS, saponin, n-decyl β-D-glucopyranoside, n-heptyl β-D-glucopyranoside, n-octyl α-D-glucopyranoside, Nonidet P-40, or any combination thereof.

Physical Treatment Also disclosed herein, in some embodiments, is a method of at least partially decellularizing an isolated organ or a portion thereof by physical treatment. In some embodiments, physical treatment can be used to lyse, kill, and remove cells from the ECM or a portion thereof. In some embodiments, physical treatment can utilize temperature, force, pressure, and electrical disruption. In some embodiments, temperature methods can be used with a quick-freezing mechanism. In some embodiments, for example, by freezing the tissue, fine ice can form around the cell membrane and lyse the cells. In some embodiments, after lysing one or more cells, the tissue can be further exposed to a liquefying chemical to break down and wash away any remaining or undesirable components. In some embodiments, temperature methods can preserve the physical structure of the ECM scaffold. In some embodiments, the isolated organ or portion thereof and tissue can be decellularized at a suitable temperature. In some embodiments, the suitable temperature may be about 4°C, 8°C, 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, 45°C, 50°C, 55°C, 60°C, or up to about 70°C. In some embodiments, the physical treatment may also include the use of pressure. In some embodiments, pressure decellularization may include the controlled use of hydrostatic pressure applied to the tissue, isolated organ, or portion thereof. In some embodiments, pressure decellularization may be performed at elevated temperatures to avoid the formation of unmonitored ice crystals. In some embodiments, electrical disruption of the isolated organ or portion thereof may be performed. In some embodiments, electrical disruption may be performed to lyse cells contained within the tissue or isolated organ or portion thereof. In some embodiments, micropores may be formed in cell membranes by exposing the tissue, isolated organ, or portion thereof to an electrical pulse. In some embodiments, one or more cells may die after such an applied stimulus disrupts homeostatic electrical balance. In some embodiments, this electrical process may be demonstrated as non-thermal irreversible electroporation (NTIRE). In some embodiments, sonication may also be used for decellularization or to enhance perfusion decellularization.

Chemical and Enzymatic Treatments Also disclosed herein, in some embodiments, are methods of chemical treatment of a harvested organ or a portion thereof to achieve at least partial decellularization. In some embodiments, chemicals and/or salts thereof for use in chemical treatment can be selected for decellularization depending on the thickness, extracellular matrix composition, intended use of the tissue or harvested organ, or any combination thereof. In some embodiments, enzymes may not be used for collagenous tissues, for example, because enzymes may destroy connective tissue fibers. In some embodiments, enzymes may be a viable option for decellularization when collagen is not present in high concentrations or is not needed in the tissue. In some embodiments, chemicals or salts thereof that can be used to kill and remove cells can be, but are not limited to, acids, alkaline treatments, ionic detergents, non-ionic detergents, zwitterionic detergents, or any combination thereof. In some embodiments, the one or more chemicals can include a cell disruption medium. In some embodiments, the cell disruption medium can include at least one detergent, such as sodium dodecyl sulfate (SDS), polyethylene glycol (PEG), or Triton X. In some embodiments, the detergent can effectively act to dissolve cell membranes and expose the contents to further degradation. In some embodiments, for example, after SDS dissolves cell membranes, endonucleases and/or exonucleases can degrade genetic contents while solubilizing other components of the cells and washing them out of the matrix. In some embodiments, alkaline and/or acid treatments can degrade nucleic acids and solubilize cytoplasmic inclusions, and can be mixed with detergents. In some embodiments, one or more cell disruption media can be used to decellularize the isolated organ or tissue. In some embodiments, the cell disruption media can include at least one detergent, such as SDS, PEG, or Triton X. In some embodiments, the cell disruption media can include water, such that the media can be osmotically incompatible with the cells. Alternatively, in some embodiments, the cell disruption media can include a buffer (e.g., PBS) for osmotic compatibility with the cells. In some embodiments, the cell disruption medium can also include enzymes, such as, but not limited to, one or more collagenases, one or more dispases, one or more DNases, one or more proteases, and any combination thereof. In some embodiments, the cell disruption medium can additionally or alternatively include one or more enzyme inhibitors (e.g., protease inhibitors, nuclease inhibitors, and/or collagenase inhibitors). In some embodiments, the cell disruption medium can include water such that the medium can be osmotically incompatible with the cells. In some embodiments, the cell disruption medium can alternatively include a buffer (e.g., PBS) for osmotic compatibility with the cells. In some embodiments, the cell disruption medium can also include enzymes such as one or more collagenases, one or more dispases, one or more DNases, or proteases, such as trypsin. In some embodiments, the cell disruption medium can additionally or alternatively include one or more enzyme inhibitors (e.g., protease inhibitors, nuclease inhibitors, and/or collagenase inhibitors). In some embodiments, a non-ionic detergent such as Triton X-100 can be utilized. In some embodiments, Triton X-100 can disrupt interactions between lipids or between lipids and proteins. In some embodiments, Triton X-100 may not disrupt protein-protein interactions, which can be beneficial for keeping the ECM intact. In some embodiments, EDTA can be utilized. In some embodiments, EDTA can be a chelating agent that binds calcium, which can be a component for proteins to interact with each other. In some embodiments, by making calcium unavailable, EDTA can prevent endogenous proteins between cells from binding to each other. In some embodiments, EDTA can be used with trypsin, an enzyme that acts as a protease to break existing bonds between endogenous proteins of adjacent cells in a tissue. In some embodiments, the cleaning agent is applied for about 10 minutes, 30 minutes, 60 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, 75 hours, 76 hours, 77 hours, 78 hours, 79 hours, 80 hours, 81 hours, 82 hours, 83 hours, 84 hours, 85 hours, 86 hours, 87 hours, 88 hours, 89 hours, 90 hours, 91 hours, 92 hours, 93 hours, 94 hours, 95 hours, 96 hours, 97 hours, 98 hours, 99 hours It can be administered for 2 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 70 hours, 80 hours, 90 hours, or up to about 100 hours. In some embodiments, depending on the size and/or weight of the isolated organ or portion thereof, the chemical treatment, such as a detergent, can be contacted with the isolated organ or portion thereof for from about 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, to about 20 hours per gram of isolated parenchymal organ or tissue containing the cell disruption medium. In some embodiments involving washing, the isolated organ can be perfused for up to about 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 70 hours, 80 hours, 90 hours, or up to about 100 hours. In some embodiments, the isolated organ or part thereof can be perfused for about 12 to about 72 hours per gram of tissue. In some embodiments, the perfusion can be adjusted to physiological conditions, including pulse flow, rate, pressure, and any combination thereof. In some embodiments, the isolated organ, part thereof, or tissue can be contacted sequentially with at least two different cell disruption media. In some embodiments, for example, a first cell disruption media can include an anionic detergent, such as SDS, and a second cell disruption media can include an ionic detergent, such as Triton X. In some embodiments, after contact with at least one cell disruption media, such as perfusion, the cannulated isolated organ or tissue can be perfused with, for example, a washing solution or a solution containing one or more enzymes. In some embodiments, alternating the direction of perfusion (e.g., antegrade and retrograde) can help effectively decellularize the isolated organ, part thereof, or tissue. In some embodiments, decellularization can decellularize the isolated organ or part thereof from the inner portion toward the outer portion, resulting in little damage to the ECM. In some embodiments, the sequential method of decellularization may include contacting the isolated organ or portion thereof with a cell disruption medium, such as SDS detergent, followed by a washing step, followed by the addition of one or more chemicals, followed by contact with a detergent, and finally at least one washing step. In some embodiments, the sequential method of decellularization may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or up to 15 contact steps with any of the media or solutions disclosed herein. In some embodiments, the buffers disclosed herein may be at a concentration of about 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or up to about 100%. In some embodiments, the isolated organ, tissue, or portion thereof (e.g., liver, lung, kidney, heart, bladder, pancreas, spleen, uterus, or portion thereof) can be pretreated or flushed with a flushing solution prior to decellularization. In some embodiments, pretreatment or flushing of the isolated organ, tissue, or portion thereof with a solution can be used to remove blood and/or reduce blood clot formation. In some embodiments, the isolated organ, tissue, or portion thereof can be flushed one, two, three, four, five, or more times. In some embodiments, flushing occurs over a period of time. In some embodiments, flushing of the isolated organ, tissue, or portion thereof can range from minutes to days, minutes to hours, or hours (e.g., about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 10 hours, 24 hours, 48 hours, or more). In some embodiments, the flushing solution can be a hypertonic solution. In some embodiments, a hypertonic solution can refer to a solution that has a solute concentration that is higher than the solute concentration inside a cell. In some embodiments, a hypertonic solution includes sodium chloride and a second sodium source, such as sodium acetate. In some embodiments, a hypertonic solution includes a sodium source and a chloride source. In some embodiments, the ratio of sodium to chloride can be about 1:0.7 to 1:1. In some embodiments, a hypertonic solution can include NaCl, KCl, NaHCO ₃ , Na ₂ SO ₄ , Na ₂ HPO ₄ , MgCl ₂ , Na acetate, Na lactate, saline, 4x saline, 5x saline, or any combination thereof. In some embodiments, a hypertonic solution can be saline. In some embodiments, a hypertonic solution can be 5x saline. In some embodiments, the hypertonic solution is about 0.1% NaCl, 0.2% NaCl, 0.5% NaCl, 0.7% NaCl, 0.8% NaCl, 0.9% NaCl, 1% NaCl, 1.5% NaCl, 2% NaCl, 2.5% NaCl, 3% NaCl, 3.5% NaCl, 4% NaCl, 4.5% NaCl, 5% NaCl, 5.5% NaCl, 6% NaCl, 6.5% NaCl, 7% NaCl, 8% NaCl, 9% NaCl, 10% NaCl, 11% NaCl, 12% NaCl, 13% NaCl, 14% NaCl, 15% NaCl, 16% NaCl, 17% NaCl, 18% NaCl, 19% NaCl, 20% NaCl, 21% NaCl, 22% NaCl, 23% NaCl, 24% NaCl, 25% NaCl, 26% NaCl, 27% NaCl, 28% NaCl, 29% NaCl, 30% NaCl, 31% NaCl, 32% NaCl, 33% NaCl, 34% NaCl, 35% NaCl, 36% NaCl, 37% NaCl, 38% NaCl, 39% NaCl, 40% NaCl, 40% NaCl, 41% NaCl, 42% NaCl, 43% NaCl, 44% NaCl, 45% NaCl, 46% NaCl, 47% NaCl, 48 ...9% NaCl, aCl, 8% NaCl, 9% NaCl, 10% NaCl, 12% NaCl, 15% NaCl, 20% NaCl, 23% NaCl, or 25% NaCl, about 0.1% NaCl, 0.2% NaCl, 0.5% NaCl, 0.7% NaCl, 0.8% NaCl, 0.9% NaCl, 1% NaCl, 1.5% NaCl, 2% NaCl, 2.5% NaCl, 3% NaCl, 3.5% NaCl , 4% NaCl, 4.5% NaCl, 5% NaCl, 5.5% NaCl, 6% NaCl, 6.5% NaCl, 7% NaCl, 8% NaCl, 9% NaCl, 10% NaCl, 12% NaCl, 15% NaCl, 20% NaCl, 23% NaCl, or 25% NaCl or more, or about 0.1% NaCl, 0.2% NaCl, 0.5% NaCl, 0.7% NaCl, 0.8% NaCl, 0.9% NaCl, 1% NaCl, 1.5% NaCl, 2% NaCl, 2.5% NaCl, 3% NaCl, 3.5% NaCl, 4% NaCl, 4.5% NaCl, 5% NaCl, 5.5% NaCl, 6% NaCl, 6.5% NaCl, 7% NaCl, 8% NaCl, 9% NaCl, 10% NaCl, 12% NaCl, 15% NaCl, 20% NaCl, 23% NaCl, or 25%

NaCl. In some embodiments, the hypertonic solution may include other components such as KHCO ₃ , K-acetate, K-lactate, MgSO ₄ , or K ₂ HPO _4. In some embodiments, the saline solution may have a 10× formulation, which can be diluted with water to obtain various hypertonic solutions. In some embodiments, the hypertonic solution may range from about 1.01× to about 10× buffer. In some embodiments, the buffer may be a saline solution. In some embodiments, the isolated organ, tissue, or any portion thereof may be washed with a hypertonic solution, a hypotonic solution, or both. In some embodiments, the isolated organ, tissue, or portion thereof may be disinfected before and/or after decellularization. In some embodiments, disinfection may be performed before decellularization. In some embodiments, disinfection may be performed after decellularization. In some embodiments, disinfection may be performed after the isolated organ, tissue, or portion thereof may be flushed with a flushing solution. In some embodiments, disinfection may be performed without flushing the isolated organ, tissue, or portion thereof with a flushing solution. In some embodiments, disinfection can be performed before the isolated organ, tissue, or part thereof can be washed with a washing solution. In some embodiments, disinfection of the isolated organ, tissue, or part thereof can be performed, for example, by placing the isolated organ, tissue, or part thereof in a disinfecting bath containing a disinfecting solution. In some embodiments, disinfection can also be performed by perfusing or immersing the isolated organ, tissue, or part thereof with a disinfecting solution. In some embodiments, disinfection is performed by submerging the isolated tissue, organ, or part thereof in a disinfecting solution. In some embodiments, the disinfecting solution can include an acid, a peracid, hydrogen peroxide, peroxide, acetic acid, peracetic acid, and peroxyacetic acid. In some embodiments, the disinfecting solution can include one or more of peracid, hydrogen peroxide, acetic acid, peracetic acid (PAA), saline, SDS, or sodium hydroxide (NaOH). In some embodiments, the solution used for disinfection can include an acid (e.g., a peracid), hydrogen peroxide, a compound containing hydrogen peroxide, a compound containing a peracid, hydrogen peroxide covalently bound to an organic moiety, saline, and/or a sodium-containing solution. In some embodiments, the disinfecting solution can include saline. In some embodiments, the disinfectant solution may include sodium. In some embodiments, the disinfectant solution may include NaCl (e.g., 0.1% NaCl, 0.2% NaCl, 0.5% NaCl, 0.7% NaCl, 0.8% NaCl, 0.9% NaCl, 1% NaCl, 1.5% NaCl, 2% NaCl 2.5% NaCl, 3% NaCl, 3.5% NaCl, 4% NaCl, 4.5% NaCl, 5% NaCl, 5.5% NaCl, 6% NaCl, 6.5% NaCl, 7% NaCl, 8% NaCl, 9% NaCl, 10% NaCl, 12% NaCl, 15% NaCl, 20% NaCl, 23% NaCl, or 25% NaCl). In some embodiments, the disinfectant solution may include saline and an acid. In some embodiments, the disinfectant solution may include saline and a peracid (e.g., peracetic acid). In some embodiments, the disinfectant solution may include 0.9% NaCl and 600 ppm peracetic acid. In some embodiments, the saline solution may be 1× saline, 2× saline, 5× saline, 7× saline, 10× saline, 12× saline, 15× saline. In some embodiments, the disinfectant solution may include saline and a peracid (e.g., peracetic acid). In some embodiments, the saline solution may be 1× saline. In some embodiments, the acid or peracid in the disinfectant solution may range from about 25 parts per million (ppm) to about 4000 ppm. In some embodiments, the acid or peracid in the disinfectant solution may range from about 500 ppm to about 700 ppm (500 ppm to 700 ppm), 600 ppm to 650 ppm, 250 ppm to 700 ppm, 250 ppm to 800 ppm, 550 ppm to 1000 ppm, 600 ppm to 700 ppm, 550 ppm to 2000 ppm, 550 ppm to 3000 ppm, 550 ppm to 4000 ppm, 1000 ppm to 2000 ppm, 2000 ppm to 3000 ppm, or 3000 ppm to 4000 ppm. In some embodiments, the acid or peracid is at about 10 ppm, 25 ppm, 50 ppm, 75 ppm, 90 ppm, 100 ppm, 125 ppm, 150 ppm, 175 ppm, 200 ppm, 225 ppm, 250 ppm, 275 ppm, 300 ppm, 325 ppm, 350 ppm, 375 ppm, 400 ppm, 425 ppm, 450 ppm, 475 ppm, 500 ppm, 525 ppm, 550 ppm, 575 ppm, 600 ppm, 625 ppm, 650 ppm, 67 5 ppm, 700 ppm, 725 ppm, 750 ppm, 775 ppm, 800 ppm, 900 ppm, 1000 ppm, 1200 ppm, 1400 ppm, 1500 ppm, 1700 ppm, 2000 ppm, 2200 ppm, 2500 ppm, 2750 ppm, 3000 ppm, 3200 ppm, 3500 ppm, 3750 ppm, or 4000 ppm, and at least about 10 ppm, 25 ppm, 50 ppm, 75 ppm, 90 ppm, 100 ppm, 125 ppm , 150 ppm, 175 ppm, 200 ppm, 225 ppm, 250 ppm, 275 ppm, 300 ppm, 325 ppm, 350 ppm, 375 ppm, 400 ppm, 425 ppm, 450 ppm, 475 ppm, 500 ppm, 525 ppm, 550 ppm, 575 ppm, 600 ppm, 625 ppm, 650 ppm, 675 ppm, 700 ppm, 725 ppm, 750 ppm, 775 ppm, 800 ppm, 900 ppm, 1000 ppm, 1200 ppm, 1400 ppm, 1500 ppm, 1700 ppm, 2000 ppm, 2200 ppm, 2500 ppm, 2750 ppm, 3000 ppm, 3200 ppm, 3500 ppm, 3750 ppm, or 4000 ppm, or at about 10 ppm, 25 ppm, 50 ppm, 75 ppm, 90 ppm, 100 ppm, 125 ppm, 150 ppm, 175 ppm, 200 ppm, 225 ppm, 250 ppm, 275 ppm, 300 ppm, 325 ppm, 350 ppm, 375
ppm, 400 ppm, 425 ppm, 450 ppm, 475 ppm, 500 ppm, 525 ppm, 550 ppm, 575 ppm, 600 ppm, 625 ppm, 650 ppm, 675 ppm, 700 ppm, 725 ppm, 750 ppm, 775 ppm, 800 ppm, 900 ppm, 1000 ppm, 1200 ppm, 1400 ppm, 1500 ppm, 1700 ppm, 2000 ppm, 2200 ppm, 2500 ppm, 2750 ppm, 3000 ppm, 3200 ppm, 3500 ppm, 3750 ppm, or up to 4000 ppm. In some embodiments, the acid or peracid (e.g., peracetic acid) can be about 600 ppm. In some embodiments, the acid or peracid (e.g., peracetic acid) can be about 50 ppm. In some embodiments, the disinfectant solution can have a pH of about 4 to about 10 (e.g., a pH of 6-7, 5-8, 5-10, 6-8, and 5.5-9). In some embodiments, the disinfectant solution has a pH of about 5.00 to about 7.50, about 6.00 to about 8.00, about 6.10 to about 7.00, about 4.50 to about 9.00, or about 6.00 to about 6.50. In some embodiments, the disinfectant solution is about 4.00, 4.50, 5.00, 5.50, 5.80, 5.90, 6.00, 6.05, 6.10, 6.11, 6.12, 6.13, 6.14, 6.15, 6.16, 6.17, 6.18, 6.19, 6.20, 6.30, 6.40, 6.41, 6.42, 6.43, 6.44, 6.45, 6.50, 6.60, 6.90, 7.00, 7.50, 8.00, 8.50, 9.0, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, or 14, at least about 4.00, 4.50, 5.00, 5.50, 5.80, 5.90, 6.00, 6.05, 6.10, 6.11, 6.12, 6.13, 6.14, 6.15, 6.16, 6.17, 6.18, 6.19, 6.20, 6.30, 6.40, 6.41, 6.42, 6.43, 6.44, 6.45, 6.50, 6.60, 6.90, 7.00, 7.50, 8.00, 8.50, 9.0, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, or 14, or about 4.00, 4.50, 5.00, 5.50, 5.80, 5.90, 6.00, 6.05, 6.10, 6.11, 6.1 2, 6.13, 6.14, 6.15, 6.16, 6.17, 6.18, 6.19, 6.20, 6.30, 6.40, 6.41, 6.42, 6.43, 6.44, 6.45, 6.50, 6.60, 6.90, 7.00, 7.50, 8.00, 8.50, 9.0, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, or 14. In some embodiments, disinfection of the isolated organ, tissue, or portion thereof can range from minutes, to days, to weeks, to months, or longer. In some embodiments, disinfection lasts for at least about 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, 22 hours, 24 hours, 27 hours, 30 hours, 34 hours, 40 hours, 44 hours, 48 hours, or more. The reaction can be carried out for 5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, 22 hours, 24 hours, 27 hours, 30 hours, 34 hours, 40 hours, 44 hours, 48 hours or more, or for up to about 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, 22 hours, 24 hours, 27 hours, 30 hours, 34 hours, 40 hours, 44 hours, 48 hours or more. In some embodiments, disinfection can be carried out for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 30 days, or more, at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 30 days, or more, or up to about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 30 days, or more. In some embodiments, sterilization can be carried out for about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 7 weeks, 8 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or more, at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 7 weeks, 8 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or more, or up to about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 7 weeks, 8 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or more.

In some embodiments, the isolated organ, tissue, or portion thereof may be decellularized using a decellularization solution that includes an acid (e.g., acetic acid) or a radical-generating compound. The radical-generating compound may be a compound that may be decomposable into radicals under mild conditions. The radical-generating compound may include a compound of formula R ¹ -O-O-R ² , where R ¹ and R ² are independently H, C ₁ -C ₆ alkyl, substituted C ₁ -C ₆ alkyl, aryl, substituted aryl, benzyl, substituted benzyl, C(=O)A ¹ , C(=O)OA ² , where A ₁ and A ₂ may be independently H, C ₁ -C ₆ alkyl, or substituted C ₁ -C ₆ alkyl. In some embodiments, the radical-generating compound may be a compound of formula R ¹ -O-O-R ² , where R ¹ may be H and R ² may be C(=O)CH _3. In some cases, the radical-generating compound may be a peracid as disclosed herein.

In some embodiments, decellularization of the isolated organ, tissue, or portion thereof can be performed after disinfection. In some embodiments, decellularization of the isolated organ, tissue, or portion thereof can be performed before disinfection. In some embodiments, decellularization of the isolated organ, tissue, or portion thereof can be performed after flushing. In some embodiments, decellularization of the isolated organ, tissue, or portion thereof can be performed before flushing. In some embodiments, decellularization of the isolated organ, tissue, or portion thereof can be performed after flushing and disinfection. In some embodiments, decellularization of the isolated organ, tissue, or portion thereof can be performed before flushing and disinfection. In some embodiments, the solution used for decellularization can include a detergent (e.g., sodium dodecyl sulfate (SDS)). In some embodiments, the solution used for decellularization can include a detergent and an acid, such as peracid. In some embodiments, the solution used for decellularization can include an acid, a peracid, hydrogen peroxide, a compound comprising hydrogen peroxide, a compound comprising a peracid, hydrogen peroxide covalently bound to an organic moiety, saline, and/or a sodium-containing solution. In some embodiments, the detergent (e.g., SDS) may be present in an amount of about 0.1%, 0.2%, 0.3%, 0.5%, 0.6%, 0.8%, 0.9%, 1%, 5%, 10%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 65%, 70%, 80%, 90%, or more. In some embodiments, the detergent may be present at 0.6%. In some embodiments, the detergent may be present at 0.9%. In some embodiments, the detergent may be commercially available. In some embodiments, the solution used for decellularization may include a detergent and a peracid (e.g., SDS and peracetic acid (PAA)). In some embodiments, the decellularization solution may have a pH of about 4 to about 10 (e.g., a pH of 6-7, 5-8, 5-10, 6-8, and 5.5-9). In some embodiments, the decellularization solution has a pH of about 5.00 to about 7.50, about 6.00 to about 8.00, about 6.10 to about 7.00, about 4.50 to about 9.00, or about 6.00 to about 6.50. In some embodiments, the decellularization solution comprises about 4.00, 4.50, 5.00, 5.50, 5.80, 5.90, 6.00, 6.05, 6.10, 6.11, 6.12, 6.13, 6.14, 6.15, 6.16, 6.17, 6.18, 6.19, 6.20, 6.30, 6.40, 6.41, 6.42, 6.43, 6.44, 6.45, 6.50, 6.60, 6.90, 7.00, 7.50, 8.00, 8.50, 9.0, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, or 14, at least about 4.00, 4.50, 5.00, 5.50, 5.80, 5.90, 6.00, 6.05, 6.10, 6.11, 6.12, 6.13, 6.14, 6.15, 6.16, 6.17, 6.18, 6.19, 6.20, 6.30, 6.40, 6.41, 6.42, 6.43, 6.44, 6.45, 6.50, 6.60, 6.90, 7.00, 7.50, 8
0.00, 8.50, 9.0, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, or 14, or about 4.00, 4.50, 5.00, 5.50, 5.80, 5.90, 6.00, 6.05, 6.10, 6.11, 6.12, 6.13, 6.14, 6.15, 6.16, 6.17, 6.18, 6.19, 6.20, 6.21, 6.22, 6.23, 6.24, 6.25, 6.26, 6.27, 6.28, 6.29, 6.30, 6.31, 6.32, 6.33, 6.34, 6.35, 6.36, 6.37, 6.38, 6.39, 6.40, 6.41, 6.42, 6.43, 6.44, 6.45, 6.46, 6.47, 6.48, 6.49, 6.50, 6.51, 6.52, 6.53, 6.54, 6.55, 6.56, 6.57, 6.58, 6.59, 6.60, 6.61, 6.62, 6.63, 6.64, 6.65, 6.66, 6.67, 6.68, 6.69, 6.70, 6.71, 6.72, 6.73, 6.74, 6.7 .18, 6.19, 6.20, 6.30, 6.40, 6.41, 6.42, 6.43, 6.44, 6.45, 6.50, 6.60, 6.90, 7.00, 7.50, 8.00, 8.50, 9.0, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, or 14. In some embodiments, decellularization of the isolated organ, tissue, or portion thereof can take from minutes, to days, to weeks, to months, or longer. In some embodiments, decellularization may take place over at least about 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, 22 hours, 24 hours, 27 hours, 30 hours, 34 hours, 40 hours, 44 hours, 48 hours, or more. 5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, 22 hours, 24 hours, 27 hours, 30 hours, 34 hours, 40 hours, 44 hours, 48 hours, or more, or up to about 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, 22 hours, 24 hours, 27 hours, 30 hours, 34 hours, 40 hours, 44 hours, 48 hours, or more. In some embodiments, decellularization may be carried out for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 30 days, or more, at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 30 days, or more, or up to about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 30 days, or more. In some embodiments, disinfection may be performed for about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 7 weeks, 8 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or more, at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 7 weeks, 8 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or more, or up to about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 7 weeks, 8 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or more. In some embodiments, decellularization may include at least one washing step, at least one flushing step, at least one disinfection step, and at least one decellularization step. In some embodiments, the peracid solution may be a peroxide solution. In some embodiments, examples of peracids include, but are not limited to, peroxyacetic acid, peroxyoctanoic acid, sulfoperoxycarboxylic acid, peroxysulfonated oleic acid, peroxyformic acid, peroxyoxalic acid, peroxypropanoic acid, peroxybutanoic acid, peroxypentanoic acid, peroxyhexanoic acid, peroxyadipic acid, perlactic acid, peroxycitric acid, peroxybenzoic acid, and any combination thereof. In some embodiments, the peracid can be a mixture of hydrogen peroxide and acetic acid. In some embodiments, the peracid can be peracetic acid (PAA). In some embodiments, the peracid (e.g., peracetic acid) can be present in a range of about 0.1% to about 1.0%, about 0.1% to about 15%, about 0.1% to about 5.0%, about 0.1% to about 10%, or about 0.1% to about 0.5%. In some embodiments, the solution comprising peracid (e.g., peracetic acid) can comprise about 0.1% peracid. In some embodiments, the concentration of peracid in a solution (e.g., a decellularization solution or a disinfectant solution or another solution used during the decellularization process) is about 0.1%, 0.3%, 0.5%, 0.8%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10% or 11%.
0.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 18%, 20%, 23%, 25%, or more, at least about 0.1%, 0.3%, 0.5%, 0.8%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, or more. %, 15%, 15.5%, 18%, 20%, 23%, 25% or more, or up to about 0.1%, 0.3%, 0.5%, 0.8%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 18%, 20%, 23%, 25% or more. In some embodiments, the acid or peracid in a solution (e.g., a decellularization solution or disinfectant solution or another solution used during the decellularization process) can range from about 25 parts per million (ppm) to about 4000 ppm. In some embodiments, the acid or peracid in a solution (e.g., a decellularization solution or disinfectant solution or another solution used during the decellularization process) can be in the range of about 500 ppm to about 700 ppm (500 ppm to 700 ppm), 600 ppm to 650 ppm, 250 ppm to 700 ppm, 250 ppm to 800 ppm, 550 ppm to 1000 ppm, 600 ppm to 700 ppm, 550 ppm to 2000 ppm, 550 ppm to 3000 ppm, 550 ppm to 4000 ppm, 1000 ppm to 2000 ppm, 2000 ppm to 3000 ppm, or 3000 ppm to 4000 ppm. In some embodiments, the acid or peracid in a solution (e.g., a decellularization solution or a disinfectant solution or another solution used during the decellularization process) is at about 10 ppm, 25 ppm, 50 ppm, 75 ppm, 90 ppm, 100 ppm, 125 ppm, 150 ppm, 175 ppm, 200 ppm, 225 ppm, 250 ppm, 275 ppm, 300 ppm, 325 ppm, 350 ppm, 375 ppm, 400 ppm, 425 ppm, 450 ppm, 475 ppm, 500 ppm, 525 ppm, 550 ppm, 575 ppm, 600 ppm, 625 ppm, , 650 ppm, 675 ppm, 700 ppm, 725 ppm, 750 ppm, 775 ppm, 800 ppm, 900 ppm, 1000 ppm, 1200 ppm, 1400 ppm, 1500 ppm, 1700 ppm, 2000 ppm, 2200 ppm, 2500 ppm, 2750 ppm, 3000 ppm, 3200 ppm, 3500 ppm, 3750 ppm, or 4000 ppm, at least about 10 ppm, 25 ppm, 50 ppm, 75 ppm, 90 ppm, 100 ppm, 125 ppm, 150 ppm, 175 ppm, 200 ppm, 225 ppm, ppm, 250 ppm, 275 ppm, 300 ppm, 325 ppm, 350 ppm, 375 ppm, 400 ppm, 425 ppm, 450 ppm, 475 ppm, 500 ppm, 525 ppm, 550 ppm, 575 ppm, 600 ppm, 625 ppm, 650 ppm, 675 ppm, 700 ppm, 725 ppm, 750 ppm, 775 ppm, 800 ppm, 900 ppm, 1000 ppm, 1200 ppm, 1400 ppm, 1500 ppm, 1700 ppm, 2000 ppm, 2200 ppm, 2500 ppm, 2750 ppm, 30 00 ppm, 3200 ppm, 3500 ppm, 3750 ppm, or 4000 ppm, or about 10 ppm, 25 ppm, 50 ppm, 75 ppm, 90 ppm, 100 ppm, 125 ppm, 150 ppm, 175 ppm, 200 ppm, 225 ppm, 250 ppm, 275 ppm, 300 ppm, 325 ppm, 350 ppm, 375 ppm, 400 ppm, 425 ppm, 450 ppm, 475 ppm, 500 ppm, 525 ppm, 550 ppm, 575 ppm, 600 ppm, 625 ppm, 650 ppm, 675 ppm, 700 ppm,
The acid or peracid (e.g., peracetic acid) may be about 600 ppm. In some embodiments, the acid or peracid (e.g., peracetic acid) may be about 50 ppm. In some embodiments, the acid or peracid may be added to the solution to increase the flow rate. In some embodiments, the increased flow rate may be maintained in subsequent steps that do not use acid or peracid. In some embodiments, the flow rate can be increased by adding peracid to the solution during perfusion decellularization, and can be maintained (or not substantially decreased) during a subsequent perfusion without the use of peracid. In some embodiments, the flow rate can be adjusted at a predetermined pressure. In some embodiments, the predetermined pressure can be about 1 mmHg, 2 mmHg, 3 mmHg, 4 mmHg, 5 mmHg, 6 mmHg, 7 mmHg, 8 mmHg, 9 mmHg, 10 mmHg, 11 mmHg, 12 mmHg, 13 mmHg, 14 mmHg, 15 mmHg, 16 mmHg, 17 mmHg, 18 mmHg, 19 mmHg, 20 mmHg, 21 mmHg, 22 mmHg, 24 mmHg, 26 mmHg, 28 mmHg, 29 mmHg, 30 mmHg, 31 mmHg, 32 mmHg, 33 mmHg, 34 mmHg, 35 mmHg, 36 mmHg, 37 mmHg, 38 mmHg, 39 mmHg, 40 mmHg, 41 mmHg, 42 mmHg, 43 mmHg, 44 mmHg, 45 mmHg, 46 mmHg, 47 mmHg, 48 mmHg, 49 mmHg, 50 mmHg, 51 mmHg, 52 mmHg, 53 mmHg, 54 mmHg, 55 mmHg, 56 mmHg, 57 mmHg, 58 mmHg, 59 mmHg, 60 mmHg, 61 mmHg, 62 mmHg, 63 mmHg, 64 mmHg, 65 mmHg, 66 mmHg, 67 mmHg mmHg, 23mmHg, 24mmHg, 25mmHg, 26mmHg, 27mmHg, 28mmHg, 29mmHg, 30mmHg, 35mmHg, 40mmHg, 45mmHg, 50mmHg, 55mmHg, 60mmHg, 65mmHg, 70mmHg, 75mmHg, 80mmHg, 90mmHg, 100mmHg, 150mmHg, 175mmHg , 200 mmHg, 250 mmHg, 300 mmHg, 350 mmHg, 400 mmHg, 450 mmHg, 500 mmHg, 550 mmHg, 600 mmHg, 700 mmHg, 800 mmHg, 900 mmHg, 1000 mmHg, or more, at least about 1 mmHg, 2 mmHg, 3 mmHg, 4 mmHg, 5 mmHg, 6 mmHg, 7 mmHg, Hg, 8mmHg, 9mmHg, 10mmHg, 11mmHg, 12mmHg, 13mmHg, 14mmHg, 15mmHg, 16mmHg, 17mmHg, 18mmHg, 19mmHg, 20mmHg, 21mmHg, 22mmHg, 23mmHg, 24mmHg, 25mmHg, 26mmHg, 27mmHg, 28mmHg, 29mmHg, 30mmHg , 35mmHg, 40mmHg, 45mmHg, 50mmHg, 55mmHg, 60mmHg, 65mmHg, 70mmHg, 75mmHg, 80mmHg, 90mmHg, 100mmHg, 150mmHg, 175mmHg, 200mmHg, 250mmHg, 300mmHg, 350mmHg, 400mmHg, 450mmHg, 500mmHg, 55 0 mmHg, 600 mmHg, 700 mmHg, 800 mmHg, 900 mmHg, 1000 mmHg or more, or about 1 mmHg, 2 mmHg, 3 mmHg, 4 mmHg, 5 mmHg, 6 mmHg, 7 mmHg, 8 mmHg, 9 mmHg, 10 mmHg, 11 mmHg, 12 mmHg, 13 mmHg, 14 mmHg, 15 mmHg, 16 mmHg , 17mmHg, 18mmHg, 19mmHg, 20mmHg, 21mmHg, 22mmHg, 23mmHg, 24mmHg, 25mmHg, 26mmHg, 27mmHg, 28mmHg, 29mmHg, 30mmHg, 35mmHg, 40mmHg, 45mmHg, 50mmHg, 55mmHg, 60mmHg, 65mmHg, 70mmHg, 75mmHg g, 80 mmHg, 90 mmHg, 100 mmHg, 150 mmHg, 175 mmHg, 200 mmHg, 250 mmHg, 300 mmHg, 350 mmHg, 400 mmHg, 450 mmHg, 500 mmHg, 550 mmHg, 600 mmHg, 700 mmHg, 800 mmHg, 900 mmHg, 1000 mmHg, or more. In some embodiments, the predetermined pressure can be from about 5 mmHg to about 20 mmHg, from about 10 mmHg to about 30 mmHg, from about 5 mmHg to about 50 mmHg, or from about 8 mmHg to about 100 mmHg. In some embodiments, the flow rate is about 50 mL/min, 100 mL/min, 150 mL/min, 200 mL/min, 250 mL/min, 300 mL/min, 350 mL/min, 400 mL/min, 450 mL/min, 500 mL/min, 550 mL/min, 600 mL/min, 650 mL/min, 700 mL/min, 750 mL/min, 800 mL/min, 900 mL/min, 1000 mL/min, 150 ... 000 mL/min or more, at least about 50 mL/min, 100 mL/min, 150 mL/min, 200 mL/min, 250 mL/min, 300 mL/min, 350 mL/min, 400 mL/min, 450 mL/min, 500 mL/min, 550 mL/min, 600 mL/min, 650 mL/min, 700 mL/min, 750 mL/min, 800 mL/min, 900 mL/min, 1000 mL/min, 1500 mL/min
L/min, 2000 mL/min, or more, or up to about 50 mL/min, 100 mL/min, 150 mL/min, 200 mL/min, 250 mL/min, 300 mL/min, 350 mL/min, 400 mL/min, 450 mL/min, 500 mL/min, 550 mL/min, 600 mL/min, 650 mL/min, 700 mL/min, 750 mL/min, 800 mL/min, 900 mL/min, 1000 mL/min, 1500 mL/min, 2000 mL/min, or more. In some embodiments, the flow rate can be increased above physiological rates. In some embodiments, the flow rate can be increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95% or more above physiological rates, hi some embodiments, the flow rate can be increased by about 500 mL/min, 1000 mL/min, 1500 mL/min, 2000 mL/min, 2500 mL/min, or more, at least about 500 mL/min, 1000 mL/min, 1500 mL/min, 2000 mL/min, 2500 mL/min, or more, or to about 500 mL/min, 1000 mL/min, 1500 mL/min, 2000 mL/min, 2500 mL/min, or more. In some embodiments, the flux can be measured by chromatography, such as liquid chromatography (LC), gas chromatography (GC), or supercritical fluid chromatography (SFC). In some embodiments, the decellularization process can last for about 10, 8, 7, 5, 4, 3, 2, 1, or less than 1 day, at least about 10, 8, 7, 5, 4, 3, 2, 1, or less than 1 day, or up to about 10, 8, 7, 5, 4, 3, 2, 1, or less than 1 day. In some embodiments, the process is carried out for about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 15 hours, 18 hours, 20 hours, 24 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, or more than about 100 hours, at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 15 hours, 18 hours, 20 hours, 24 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, or more than about 100 hours. The decellularization process may last for up to about 1 to about 10, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more than about 100 hours, or for up to about 1, 2, 3, 4, 5, 6, 7, 10, 15, 18, 20, 24, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more than about 100 hours. In some embodiments, the decellularization process comprises, for example, perfusing or immersing or submerging the isolated organ, tissue, or portion thereof with a detergent solution for about 1 to about 10, about 1 to about 20, about 1 to about 7, about 1 to about 15, about 1 to about 2, about 1 to about 5, or about 1 to about 6 phases or cycles. In some embodiments, the decellularization process may involve, for example, incubating the isolated organ, tissue, or portion thereof with the detergent solution for a total of about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, or more, including at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, or more. In some embodiments, the decellularization process includes perfusing, immersing, or submerging the isolated organ, tissue, or portion thereof in a detergent solution for, for example, a total of about 20 to about 50 hours, about 10 to about 80 hours, or about 30 to about 50 hours. In some embodiments, the decellularization process comprises perfusing or immersing or submerging the harvested organ, tissue, or portion thereof with a peracid (e.g., PAA) solution for about 1 to about 10, about 1 to about 20, about 1 to about 7, about 1 to about 15, about 1 to about 2, about 1 to about 5, about 1 to about 3, about 1 to about 2, or about 1 to about 6 phases or cycles. In some embodiments, the decellularization process may involve, for example, treating a harvested organ, tissue, or portion thereof with a peracid solution for a total of about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, or more, including at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, or more. 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more hours, or up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more hours. In some embodiments, the decellularization process includes, for example, perfusing, immersing, or submerging the isolated organ, tissue, or portion thereof, with a peracid solution for a total of about 2 to about 10 hours, about 30 to about 7 hours, or about 1 to about 15 hours. In some embodiments, the detergent solution and the peracid solution overlap. In some embodiments, the detergent solution may include a peracid solution. In some embodiments, the decellularization process includes washing with water. In some embodiments, the decellularization process includes washing the isolated organ, tissue, or portion thereof with water for a total of about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, or more, including at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, or more. In some embodiments, the method may include perfusing, soaking, or submerging for up to about 1 hour, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, or more, or up to about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, or more. In some embodiments, the decellularization process may include perfusing or immersing or submerging the isolated organ, tissue, or portion thereof with water for about 1 to about 10, about 1 to about 20, about 1 to about 7, about 1 to about 15, about 1 to about 2, about 1 to about 5, about 1 to about 3, about 1 to about 2, or about 1 to about 6 phases or cycles. In some embodiments, the water washes may include PBS (e.g., about 5% PBS to about 60% PBS). In some embodiments, the water may be specific to the isolated organ, tissue, or portion thereof (e.g., hepatic water for liver decellularization). In some embodiments, the decellularized harvested organ, tissue, or portion thereof may comprise up to about 50% natural cells, 40% natural cells, 35% natural cells, 30% natural cells, 20% natural cells, 10% natural cells, 5% natural cells, 3% natural cells, 2% natural cells, 1% natural cells, or about 0% natural cells.

Also disclosed herein, in some embodiments, is a method of decellularization of an isolated organ, tissue, or portion thereof that does not compromise matrix integrity. In some embodiments, the addition of acid or peracid to the decellularization process can result in increased flow rates and decreased time required for decellularization. In some embodiments, the addition of acid or peracid to the decellularization process can result in increased flow rates that can be maintained after the peracid is removed from the solution or after the use of a non-peracid solution. In some embodiments, the decellularization process can result in purer decellularization (e.g., increased cell removal in less time). In some embodiments, the decellularization process can result in purer decellularization (e.g., reduced residual DNA) as described herein. In some embodiments, the addition of peracid to the solution or increased flow rates during decellularization does not affect adhesion properties. In some embodiments, the addition of peracid (e.g., PAA) during decellularization does not affect cell adhesion during recellularization of the decellularized isolated organ, tissue, or portion thereof. In some embodiments,
Decellularization using peracid may result in complete removal of cells. In some embodiments, decellularization using peracid may not affect the stiffness or flexibility of the extracellular matrix. In some embodiments, decellularization may be by perfusion. In some embodiments, decellularization does not include agitation. In some embodiments, decellularization using peracid may not compromise the integrity of the matrix (e.g., ECM integrity). In some embodiments, decellularization using peracid may not compromise the flexibility, elasticity, or stiffness of the matrix (e.g., ECM). In some embodiments, decellularization using peracid may not result in purer decellularization. In some embodiments, decellularization using peracid may not affect the surface properties of the isolated organ, tissue, or portion thereof. In some embodiments, decellularization using peracid does not affect the metabolic activity of the isolated organ, tissue, or portion thereof. In some embodiments, the decellularized isolated organ, tissue, or portion thereof can be introduced into a subject (e.g., a human). In some embodiments, the decellularized harvested organ, tissue, or portion thereof can be introduced into a subject after recellularization. In some embodiments, the decellularized harvested organ, tissue, or portion thereof can be introduced into a subject prior to recellularization. In some embodiments, human cells (e.g., stem cells, progenitor cells, regenerative cells, or harvested organ-specific cells) can be introduced into an at least partially decellularized non-human mammal. Also disclosed herein, in some embodiments, are methods or compositions (e.g., at least partially decellularized or recellularized harvested organ, tissue, or portion thereof) that can provide improved rejection rates compared to alternative decellularization methods or compositions resulting from alternative methods. In some embodiments, the rejection rate can be less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% or up to about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5%. In some embodiments, the rejection rate can be reduced if the isolated organ, tissue, or portion thereof can be flushed or pretreated prior to decellularization. In some embodiments, the rejection rate can be reduced if the isolated organ, tissue, or portion thereof can be disinfected prior to decellularization. In some embodiments, the rejection rate can be reduced if the isolated organ, tissue, or portion thereof can be decellularized with a solution containing peracid, or an acid, or a peroxide.

Organ Recellularization Also disclosed herein, in some embodiments, is a method of at least partially recellularizing an at least partially decellularized harvested organ and portions thereof. In some embodiments, the harvested organ or tissue can be generated by contacting an at least partially decellularized harvested organ or tissue described herein with a population of cells. In some embodiments, the population of cells can include regenerative cells. In some embodiments, the regenerative cells described herein can be any cell used to recellularize an at least partially decellularized tissue, harvested organ, or portions thereof. In some embodiments, the regenerative cells can be totipotent, pluripotent, or multipotent cells, and can be uncommitted or committed. In some embodiments, the regenerative cells can also be single-lineage cells. In some embodiments, the regenerative cells can be undifferentiated, partially differentiated, or fully differentiated. In some embodiments, the regenerative cells can include "adult" derived stem cells, including embryonic stem cells, progenitor cells, precursor cells, umbilical cord cells, and fetal stem cells, or any combination thereof. In some embodiments, regenerative cells that can be used to recellularize a harvested organ or portion thereof disclosed herein can be, but are not limited to, embryonic stem cells, umbilical cord blood cells, tissue-derived stem or progenitor cells, bone marrow-derived stem or progenitor cells, blood-derived stem or progenitor cells, induced pluripotent stem cells (iPSCs), adipose tissue-derived stem or progenitor cells, mesenchymal stem cells (MSCs), skeletal muscle-derived cells, or multipotent adult progenitor cells (MAPCs).
Additional regenerative cells that can be used include tissue-specific stem cells, including cardiomyocyte stem cells (CSCs), pluripotent adult cardiac derived stem cells, cardiac fibroblasts, cardiac microvasculature endothelial cells, or aortic endothelial cells. In some embodiments, bone marrow derived stem cells, such as bone marrow mononuclear cells (BM-MNCs), endothelial or vascular stem or progenitor cells, and peripheral blood derived stem cells, such as endothelial progenitor cells (EPCs), can also be used as regenerative cells. In some embodiments, the number of regenerative cells that can be introduced into an at least partially decellularized isolated organ or portion thereof to generate an isolated organ or tissue can depend on the size, weight, or type of isolated tissue, organ, or portion thereof, or the type or developmental stage of regenerative cells that can be used. In some embodiments, different types of cells can have different tendencies with respect to the population density that such cells reach. In some embodiments, different isolated tissues, organs, or portions thereof can be recellularized at different densities. In some embodiments, an at least partially decellularized harvested organ or tissue can be "seeded" with at least about 1,000 (e.g., at least 10,000, 100,000, 1,000,000, 10,000,000, or 100,000,000) regenerative cells; or can have attached thereto 1,000 cells/mg or about 1,000 cells/mg of tissue (wet weight, i.e., before decellularization) to 10,000,000 cells/mg or about 10,000,000 cells/mg of tissue (wet weight). In some embodiments, regenerative cells can be introduced ("seeded") into an at least partially decellularized harvested organ or tissue by injection into one or more locations. In some embodiments, at least one type of cell (i.e., a cocktail of cells) can be introduced into an at least partially decellularized harvested organ or portion thereof. In some embodiments, a cocktail or population of cells can be injected into multiple locations in the at least partially decellularized isolated organ or tissue, or different cell types can be injected into different parts of the at least partially decellularized isolated organ or part thereof. In some embodiments, in addition to injection, the regenerative cells, population of cells, or cocktail of cells can be introduced by perfusion into the cannulated decellularized isolated organ or part thereof. In some embodiments, the regenerative cells can be perfused into the at least partially decellularized isolated organ using a perfusion medium, which can then be converted into a proliferation and/or differentiation medium to induce the growth and/or differentiation of the regenerative cells. In some embodiments, during recellularization, the isolated organ or tissue can be maintained under conditions that allow one or more regenerative cells to proliferate, multiply, differentiate, and any combination thereof, in the at least partially decellularized isolated organ or part thereof. In some embodiments, these conditions can include, but are not limited to, appropriate temperature, pressure, electrical activity, mechanical activity, force, an appropriate amount of _O2 and/or _CO2 , an appropriate amount of humidity, sterile or near-sterile conditions, and any combination thereof. In some embodiments, during recellularization, the at least partially decellularized isolated organ or tissue and the regenerative cells attached thereto can be maintained in a suitable environment. In some embodiments, for example, the regenerative cells can require nutrients (e.g., nutrients and/or a carbon source such as glucose), exogenous hormones or growth factors, and/or a specific pH. In some embodiments, the growth factors can be at least one of VEGF, DKK-1, FGF, BMP-1, BMP-4, SDF-1, IGF, HGF, immunomodulators such as cytokines, glucocorticoids, IL2R antagonists, leukotriene antagonists, or any combination thereof. In some embodiments, recellularization can be improved by removing or reducing particulates in the isolated organ or portion thereof. In some embodiments, engraftment, seeding, proliferation, survival, differentiation, and any combination thereof of cells introduced into the at least partially decellularized isolated organ or portion thereof can be improved by reducing the particulate percentage of the isolated organ or portion thereof. In some embodiments, the amount or percentage of engraftment, seeding, proliferation, survival, differentiation, and any combination thereof of cells introduced into an at least partially decellularized isolated organ or portion thereof can be improved by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or up to about 100%. In some embodiments, introducing a population of cells into an at least partially decellularized isolated organ or portion thereof disclosed herein can improve the viability (%) of the population of cells compared to a comparable population of cells that can be introduced into an otherwise comparable isolated solid organ or portion thereof that may not have reduced levels of microparticles. In some embodiments, the microparticles can increase the cytotoxicity (%), thereby decreasing the viability (%) of the cells. In some embodiments, the viability can be about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% in a population of cells introduced into an isolated organ or portion thereof that includes reduced levels of microparticles. In some embodiments, the regenerative cells disclosed herein can be allogeneic to a harvested organ or portion thereof that has been at least partially decellularized (e.g., a human decellularized harvested organ or tissue seeded with human regenerative cells), or the regenerative cells can be xenogenic to a harvested organ or portion thereof that has been at least partially decellularized (e.g., a porcine decellularized harvested organ or tissue seeded with human regenerative cells). As used herein, "allogenic" refers to cells obtained from the same species (e.g., autologous (i.e., autologous) or related or unrelated individuals) from which the harvested organ or tissue is derived, while "xenogenic" as used herein refers to cells obtained from a different species than that from which the harvested organ or tissue is derived.

Uses of harvested organs and parts thereof Also disclosed herein in some embodiments are methods and uses of decellularized and recellularized harvested organs or parts thereof in various applications. In some embodiments, the harvested organ or parts thereof can be transplanted into a subject. In some embodiments, the compositions described herein, such as harvested organs or parts thereof, can be transplanted into a subject with a disease. In some embodiments, relevant diseases that may require harvested organ transplantation include, but are not limited to, harvested organ failure, cardiomyopathy, cirrhosis, chronic obstructive pulmonary disease, pulmonary edema, biliary atresia, emphysema and pulmonary hypertension, coronary heart disease, valvular heart disease, congenital heart disease, coronary artery disease, pancreatitis, cystic fibrosis, diabetes, hepatitis, hypertension, idiopathic pulmonary fibrosis, polycystic kidney disease, short bowel syndrome, injury, congenital defect, genetic disease, autoimmune disease, and any combination thereof. In some embodiments, the implant can be used to replace or augment existing tissue, for example, to treat a subject with kidney failure by replacing a subject's failing kidney with an exogenous or engineered kidney. In some embodiments, a subject can be monitored for amelioration of kidney damage after transplantation of an exogenous kidney. In some embodiments, the decellularized harvested organ or portion thereof disclosed herein is available for transplantation into a subject. In some embodiments, a composition disclosed herein, such as a harvested solid organ or portion thereof, can have about 1% to about 100% of its original functionality upon decellularization. In some embodiments, a composition disclosed herein, such as a harvested solid organ or portion thereof, can have about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or up to about 100% of its original functionality upon decellularization. In some embodiments, certain harvested organs or portions thereof may be suitable for transplantation if they function less well than their native counterparts. In some embodiments, for example, the liver and kidneys may require from approximately about 20% of total organ function to rescue a human from liver failure or to relieve them from dialysis. In some embodiments, livers and kidneys may require approximately 20-30%, 30-40%, 20-50%, 20-60%, 40-60% of total organ function to be suitable for transplantation. In some embodiments, the harvested organ can function equivalently to its native counterpart. In some embodiments, for example, the heart may be more complex in that it may require approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or up to approximately 100% of function upon transplantation. In some embodiments, harvested organs such as the pancreas or lungs may function at 15%, 20%, 30%, 40%, or 50% of their native function and be transplantable. In some embodiments, the pancreas may function from 10% or more and be transplantable. In some embodiments, the harvested organs disclosed herein can be used as accessory organs to support native functions. In some embodiments, the lifespan of a subject may be extended following transplantation of a composition such as a harvested organ or portion thereof disclosed herein. In some embodiments, for example, the lifespan of a subject can be extended by about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 30 years, 40 years, 50 years, 60 years, 70 years, 80 years, 90 years, or up to about 100 years after transplantation. In some embodiments, transplantation of a composition such as a harvested organ or a portion thereof disclosed herein can reduce the need for secondary treatments in a subject. In some embodiments, secondary treatments can refer to dialysis, pacemakers, respiratory machines, and combinations thereof. In some embodiments, the at least partially decellularized harvested organ or a portion thereof or the at least partially recellularized harvested organ or a portion thereof can be used in vitro to screen a wide variety of compounds for efficacy and cytotoxicity of pharmaceuticals, chemicals, growth factors, or regulators. In some embodiments, the cultures can be maintained in vitro and exposed to the compound to be tested. In some embodiments, the activity of a cytotoxic compound can be measured by its ability to damage or kill cells in culture. In some embodiments, this can be easily assessed by vital staining techniques. In some embodiments, the effect of growth or regulatory factors can be assessed by analyzing the cellular content of the matrix, for example, by total cell number and cell percentage. In some embodiments, this can be accomplished using standard cytological and/or histological techniques, including the use of immunocytochemical techniques with antibodies that define type-specific cellular antigens. In some embodiments, the effect of various drugs on normal cells cultured on the reconstructed artificial isolated organ can be evaluated. In some embodiments, blood can be filtered using decellularized and recellularized isolated organs or parts thereof that can be used in vitro to filter aqueous solutions, such as a reconstructed artificial kidney. In some embodiments, the use of a reconstructed kidney provides a system with morphological characteristics similar to the in vivo kidney product, which in some embodiments may be suitable for hemodialysis or hemofiltration to remove water and low molecular weight solutes from the blood. In some embodiments, the artificial kidney can be maintained in vitro and exposed to blood that can be infused into the luminal side of the artificial kidney. In some embodiments, the treated aqueous solution can be collected from the abluminal side of the modified kidney. In some embodiments, the efficiency of filtration can be assessed by measuring the ionic or metabolic waste content of the filtered and unfiltered blood. In some embodiments, the decellularized and recellularized harvested organ or portion thereof can be used as a vehicle to introduce genes or gene products in vivo to aid or improve transplant outcomes, or for use in gene therapy. In some embodiments, cultured cells, such as endothelial cells, can be engineered to express gene products. In some embodiments, the cells can be engineered to express gene products under transient or inducible control, or as chimeric fusion proteins anchored to the endothelial cells, e.g., as chimeric molecules composed of the intracellular and/or transmembrane domains of a receptor or receptor-like molecule fused to a gene product as an extracellular domain. In some embodiments, one or more endothelial cells can be engineered to express genes that the patient may be deficient in or that would have a therapeutic effect. In some embodiments, the gene of interest engineered into the endothelial or parenchymal cells can be associated with the disease being treated. In some embodiments, for example, in the case of kidney damage, the endothelial or cultured kidney cells can be engineered to express a gene product that can ameliorate the kidney damage. In some embodiments, at least two cell populations can be introduced into an isolated organ or part thereof that has been at least partially decellularized. In some embodiments, isolated organs that can be engineered include, but are not limited to, the heart, kidney, liver, pancreas, spleen, bladder, uterus, ureter, urethra, skeletal muscle, small and large intestine, esophagus, stomach, brain, spinal cord, and bone. Other embodiments and uses will be apparent to one of skill in the art in light of the uses and practices disclosed herein.

In some embodiments, a decellularized harvested organ or a portion thereof can be used as the acellular composition. Disclosed herein can be a cell-free or substantially cell-free harvested organ or a portion thereof.

In some embodiments, the method of transplanting a harvested organ may include compressing the harvested organ or a portion thereof. The ability to compress the harvested organ or a portion thereof may be affected by the particulate percentage. A harvested organ or a portion thereof containing more than 35% particulates may be more difficult to compress than a harvested organ or a portion thereof containing 35% or less than 35% particulates. A harvested organ or a portion thereof containing more than 50% particulates may be more difficult to compress than a harvested organ or a portion thereof containing 50% or less than 50% particulates.

Kits Disclosed herein can be kits that include an at least partially decellularized mammalian isolated organ or part thereof. In some embodiments, the kit can include an at least partially decellularized isolated organ or part thereof and instructions for its use. In some embodiments, the kit can include a sterile container that can include an at least partially decellularized isolated organ or part thereof; such a container can be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container known in the art. Such a container can be made of plastic, glass, laminated paper, metal foil, or other material suitable for holding an isolated organ or part thereof. In some embodiments, the delivery vehicle composition can be dehydrated, stored, and then reconstituted such that a substantial portion of the internal contents can be retained. In some embodiments, the kit can include a compressed packaging that includes an at least partially decellularized isolated organ or part thereof. For example, a sterile pouch that includes an isolated organ or part thereof can be degassed or otherwise compressed into a sheet.

In some embodiments, disclosed herein can be a system comprising any of the compositions disclosed herein. In some embodiments, the system can include at least one of a pump, a housing, a tubing, an incubator, a motor, a computer, a storage medium, or any combination thereof.

SPECIFIC EMBODIMENTS Several methods and systems are disclosed herein, and specific exemplary embodiments of these methods and systems are disclosed below.
EMBODIMENT 1
1. A method comprising contacting an isolated organ or portion thereof with a liquid, wherein: (a) the liquid can be at least partially degassed; (b) the liquid can comprise peracid; (c) the isolated organ or portion thereof can be from a mammal that has been fed within about 10 hours prior to removing the isolated organ or portion thereof; or (d) any combination of (a)-(c).
EMBODIMENT 2
2. The method of embodiment 1, which can include at least two of (a), (b), or (c).
EMBODIMENT 3
3. The method of embodiment 1 or embodiment 2, which may include (a), (b), and (c).
EMBODIMENT 4
4. The method according to any one of embodiments 1 to 3, wherein the isolated organ or part thereof may be from a mammal.
EMBODIMENT 5
5. The method of embodiment 4, wherein the mammal can be a non-human mammal.
EMBODIMENT 6
6. The method of embodiment 5, wherein the non-human mammal can be a pig, sheep, goat, cow, dog, cat, or monkey.
EMBODIMENT 7
5. The method of embodiment 4, wherein the mammal may be a human mammal.
EMBODIMENT 8
The method according to any one of embodiments 1 to 7, wherein the isolated organ or part thereof can be a liver, a lung, a heart, a kidney, a bladder, a pancreas, a spleen, a uterus, any part thereof, or any combination thereof.
EMBODIMENT 9
The method according to any one of the preceding embodiments, wherein the contacting can include perfusing at least a portion of the isolated organ or part thereof with a liquid, injecting at least a portion of the isolated organ or part thereof with a liquid, spraying at least a portion of the isolated organ or part thereof with a liquid, submerging the isolated organ or part thereof in a liquid, or any combination thereof.
EMBODIMENT 10
10. The method of embodiment 9, which may comprise perfusing at least a portion of the isolated organ or part thereof with the liquid.
EMBODIMENT 11
11. The method of embodiment 10, wherein the isolated organ or part thereof can be perfused with at least about 10 liters of said liquid.
EMBODIMENT 12
11. The method of embodiment 10, wherein the liquid can be continuously perfused into the isolated organ or part thereof.
EMBODIMENT 13
11. The method of embodiment 10, wherein the perfusion can be retrograde.
EMBODIMENT 14
11. The method of embodiment 10, wherein the perfusion can be antegrade.
Embodiment 15. The method of embodiment 10, wherein the liquid can be recirculated during perfusion.
EMBODIMENT 16
16. The method according to any one of the preceding embodiments, wherein the isolated organ or part thereof can be cannulated before, during or after contacting.
EMBODIMENT 17
17. The method according to any one of the preceding embodiments, wherein the liquid can comprise an at least partially degassed decellularization medium, thereby forming an at least partially decellularized harvested organ or part thereof.
EMBODIMENT 18
18. The method of embodiment 17, wherein the at least partially decellularized harvested organ or portion thereof can comprise an extracellular matrix.
EMBODIMENT 19
20. The method of embodiment 18, wherein the extracellular matrix can comprise: fibronectin, fibrillin, laminin, elastin, collagen family proteins, glycosaminoglycans, ground substance, reticular fibers, thrombospondin, or any combination thereof.
EMBODIMENT 20
20. The method of embodiment 18, wherein the extracellular matrix can comprise a protein of the collagen family, and the protein of the collagen family can be collagen I, collagen II, collagen III, or collagen IV.
EMBODIMENT 21
21. The method of any one of embodiments 18-20, wherein the extracellular matrix can comprise a vasculature bed.
EMBODIMENT 22
22. The method of embodiment 21, wherein the vasculature bed can be intact.
EMBODIMENT 23
23. The method of any one of embodiments 18-22, wherein the liquid can be at least partially degassed, and the extracellular matrix has increased compressibility compared to an otherwise equivalent extracellular matrix produced using a non-degassed liquid.
EMBODIMENT 24
24. The method of any one of embodiments 1 to 23, which may include at least (a).
EMBODIMENT 25
25. The method of embodiment 24, wherein the isolated organ or part thereof can be contacted with the at least partially degassed liquid for at least about 2 hours.
EMBODIMENT 26
25. The method of embodiment 24, wherein the dissolved gas in the at least partially degassed liquid has a concentration of less than about 1 milligram (mg) per liter (L).
EMBODIMENT 27
27. The method of embodiment 26, wherein the concentration of the dissolved gas can be measured using static headspace gas chromatography or dynamic luminescence quenching.
EMBODIMENT 28
27. The method of embodiment 26, wherein the dissolved gas can be selected from the group consisting of oxygen, nitrogen, carbon monoxide, carbon dioxide, a noble gas, and any combination thereof.
EMBODIMENT 29
25. The method of embodiment 24, wherein prior to contacting, the liquid can be exposed to an air pressure of less than about 700 torr, thereby forming an at least partially degassed liquid.
EMBODIMENT 30
25. The method of embodiment 24, wherein prior to contacting, the liquid can be exposed to an air pressure of less than about 1 torr, thereby forming an at least partially degassed liquid.
EMBODIMENT 31
30. The method of embodiment 29, wherein the liquid can be exposed to an air pressure of less than about 700 torr for at least about 10 minutes prior to contacting, thereby forming an at least partially degassed liquid.
EMBODIMENT 32
25. The method of embodiment 24, wherein prior to the contacting, the liquid can be exposed to a temperature of from about 10° C. to about 100° C. for at least 2 hours, thereby forming an at least partially degassed liquid.
EMBODIMENT 33
25. The method of embodiment 24, wherein prior to the contacting, the liquid can be exposed to ultrasonic treatment for at least about 1 second, thereby forming an at least partially degassed liquid.
EMBODIMENT 34
25. The method of embodiment 24, wherein prior to contacting, the liquid can be sparged for at least about 1 second to form an at least partially degassed liquid.
EMBODIMENT 35
35. The method of embodiment 34, wherein the liquid can be sparged with a chemically inert gas.
EMBODIMENT 36
36. The method of embodiment 35, wherein the chemically inert gas may comprise nitrogen, argon, helium, or any combination thereof.
EMBODIMENT 37
25. The method of embodiment 24, wherein prior to the contacting, the liquid can be contacted with a gas-liquid separation membrane, thereby forming an at least partially degassed liquid.
EMBODIMENT 38
25. The method of embodiment 24, wherein prior to the contacting, the liquid can be contacted with a reducing agent, thereby forming an at least partially degassed liquid.
EMBODIMENT 39
39. The method of embodiment 38, wherein the reducing agent can be ammonium sulfite.
EMBODIMENT 40
25. The method of embodiment 24, wherein prior to the contacting, the liquid can be degassed by freeze-pump-thaw, thereby forming an at least partially degassed liquid.
EMBODIMENT 41
25. The method of embodiment 24, wherein the isolated organ or portion thereof after contacting may comprise (a) less air emboli, (b) less microbubbles, (c) less pigmentation, or (d) any combination thereof, compared to an otherwise equivalent isolated organ or portion thereof produced by contacting with an otherwise equivalent liquid for an equivalent period of time that has not been at least partially degassed.
EMBODIMENT 42
25. The method of embodiment 24, wherein the at least partially degassed liquid can comprise an at least partially degassed decellularization medium, thereby forming an at least partially decellularized harvested organ or portion thereof.
EMBODIMENT 43
43. The method of embodiment 42, wherein the at least partially decellularized isolated organ or portion thereof after said contact with the at least partially degassed decellularization medium comprises fewer cells than an otherwise equivalent isolated organ or portion thereof that was decellularized with an otherwise equivalent non-degassed decellularization medium for a comparable period of time.
EMBODIMENT 44
43. The method of embodiment 42, wherein the time sufficient to produce an at least partially decellularized isolated organ or portion thereof with the at least partially degassed decellularization medium may be less than the time sufficient to produce a substantially decellularized isolated organ or portion thereof with a non-degassed decellularization medium.
EMBODIMENT 45
43. The method of embodiment 42, wherein the at least partially degassed decellularization medium can comprise an acid, a base, a hypertonic solution, a hypotonic solution, a detergent, a surfactant, an alcohol, a biological reagent, a salt, or any combination thereof.
EMBODIMENT 46
46. The method of embodiment 45, wherein the at least partially degassed decellularization medium can comprise a surfactant, and the surfactant can comprise an ionic surfactant.
EMBODIMENT 47
47. The method of embodiment 46, wherein the ionic surfactant may comprise an alkyl sulfate.
EMBODIMENT 48
48. The method of embodiment 47, wherein the alkyl sulfate can comprise sodium dodecyl sulfate.
EMBODIMENT 49
48. The method of embodiment 47, wherein the alkyl sulfate can comprise: sodium dodecyl sulfate, ammonium dodecyl sulfate, sodium lauryl sulfate, sodium octyl sulfate, sodium 2-ethylhexyl sulfate, lithium dodecyl sulfate, potassium lauryl sulfate, or any combination thereof.
EMBODIMENT 50
50. The method according to any one of the preceding embodiments, further comprising cannulating the isolated organ or portion thereof, thereby producing a cannulated isolated organ or portion thereof.
EMBODIMENT 51
51. The method of embodiment 50, wherein the liquid can be perfused into the cannulated isolated organ or part thereof.
EMBODIMENT 52
52. The method of embodiment 51, wherein the fluid perfusing through the isolated organ or portion thereof can be at a pressure of about 10 MPa to about 1000 MPa.
EMBODIMENT 53
53. The method of embodiment 51 or 52, wherein the fluid perfusing through the isolated organ or part thereof can be at a flow rate of about 100 milliliters (mL) per minute to about 100 liters (L) per minute.
EMBODIMENT 54
24. The method of any one of embodiments 1 to 23, which may include at least (b).
EMBODIMENT 55
55. The method of embodiment 54, wherein the peracid can comprise peroxyacetic acid, peroxyoctanoic acid, sulfoperoxycarboxylic acid, peroxysulfonated oleic acid, peroxyformic acid, peroxyoxalic acid, peroxypropanoic acid, peroxybutanoic acid, peroxypentanoic acid, peroxyhexanoic acid, peroxyadipic acid, peracetic acid, perlactic acid, peroxycitric acid, peroxybenzoic acid, or any combination thereof.
EMBODIMENT 56
56. The method of embodiment 55, wherein the peracid can include peracetic acid.
EMBODIMENT 57
55. The method of embodiment 54, wherein the peracid may comprise a peroxyacid.
EMBODIMENT 58
58. The method of embodiment 57, wherein the peroxyacid can be present in the liquid at a concentration of about 0.1% to about 1.0% by weight.
EMBODIMENT 59
58. The method of embodiment 57, wherein the peroxyacid can be present in the liquid at a concentration of about 0.1% to about 1.0% by volume.
EMBODIMENT 60
58. The method of embodiment 57, wherein the peroxyacid can be present in the liquid at a concentration of from about 40 parts per million (ppm) to about 700 ppm.
EMBODIMENT 61
51. The method of any one of embodiments 54-50, wherein said contacting produces an at least partially decellularized harvested organ or portion thereof.
EMBODIMENT 62
62. The method of embodiment 61, wherein the at least partially decellularized harvested organ or portion thereof can comprise an extracellular matrix comprising an outer surface and a vascular tree.
EMBODIMENT 63
63. The method according to any one of embodiments 54-62, further comprising cannulating the isolated organ or part thereof, thereby producing a cannulated isolated organ or part thereof.
EMBODIMENT 64
64. The method of embodiment 63, wherein the liquid can be perfused into the cannulated isolated organ or part thereof.
EMBODIMENT 65
64. The method according to any one of embodiments 61 to 63, wherein the at least partially decellularized harvested organ or portion thereof can comprise a reduced amount of cells compared to an otherwise equivalent decellularized harvested organ or portion thereof contacted with an otherwise equivalent liquid that does not contain peracid for an equivalent period of time.
EMBODIMENT 66
66. The method of embodiment 65, wherein the reduced amount of cells may comprise a reduction of at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% of cells from the removed organ or portion thereof.
EMBODIMENT 67
65. The method of embodiment 64, wherein the flow rate of the perfusion can be increased compared to the flow rate of the perfusion of an otherwise equivalent liquid that does not contain peracid.
EMBODIMENT 68
68. The method of embodiment 67, wherein the flow rate can be increased without substantially compromising the integrity of the extracellular matrix of the isolated organ or portion thereof.
69. The Method of Claim 69
68. The method of embodiment 67, wherein the flow rate can be increased above physiological rates.
EMBODIMENT 70
70. The method of embodiment 69, wherein the flow rate can be increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95% or 99% above physiological rates.
EMBODIMENT 71
24. The method of any one of embodiments 1 to 23, which may include at least (c).
EMBODIMENT 72
72. The method of embodiment 71, wherein the isolated organ or portion thereof can comprise an extracellular matrix comprising an outer surface and a vascular tree.
Embodiment 73. The method of embodiment 72, further comprising cannulating the isolated organ or portion thereof, thereby producing a cannulated isolated organ or portion thereof.
EMBODIMENT 74
74. The method of embodiment 73, wherein the liquid can be perfused into the cannulated isolated organ or part thereof.
EMBODIMENT 75
75. The method according to any of embodiments 71 to 74, wherein the isolated organ or part thereof can subsequently be contacted with a washing medium.
EMBODIMENT 76
76. The method of embodiment 75, wherein said contacting with the cleaning medium can be by perfusion, injection, immersion, or any combination thereof.
EMBODIMENT 77
77. The method of embodiment 76, wherein said contacting with said washing medium can be by perfusion.
EMBODIMENT 78
77. The method of embodiment 76, wherein the contacting with the cleaning medium can be by immersion.
EMBODIMENT 79
79. The method of any one of embodiments 75-78, wherein the washing medium may comprise a pH buffer.
EMBODIMENT 80
80. The method of embodiment 79, wherein the pH buffer can comprise phosphate.
EMBODIMENT 81
81. The method according to any one of embodiments 75 to 80, wherein the washing medium may comprise saline.
EMBODIMENT 82
82. The method of embodiment 81, wherein the saline solution may be at a concentration of about 0.1% to about 10%.
EMBODIMENT 83
83. The method of any one of embodiments 71-82, wherein the isolated organ or portion thereof can comprise reduced particulate levels compared to an otherwise equivalent isolated organ or portion thereof produced from an animal that has not been fed within about 10 hours prior to removing the isolated organ or portion thereof from the animal.
EMBODIMENT 84
The method of embodiment 83, wherein the reduced particulate levels can be reduced by at least about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1% as compared to an otherwise comparable isolated organ or part thereof produced from an animal that was not fed within about 10 hours prior to removal of the isolated organ or part thereof from the animal.
EMBODIMENT 85
85. The method of embodiment 83 or 84, wherein the reduced particulate level can be determined by visual inspection, by assessing the effluent, by assessing turbidity, by microscopic analysis, or any combination thereof.
EMBODIMENT 86
86. The method according to any one of the preceding embodiments, further comprising washing the isolated organ or part thereof with a washing medium prior to contacting.
EMBODIMENT 87
87. The method of embodiment 86, wherein the washing medium can be a hypertonic solution.
EMBODIMENT 88
88. The method of embodiment 87, wherein the hypertonic solution may comprise saline.
EMBODIMENT 89
87. The method of embodiment 86, wherein the cleaning medium may comprise a disinfectant solution.
EMBODIMENT 90
The method of embodiment 8940, wherein washing with the disinfectant solution may include placing the isolated organ or a part thereof in a disinfectant bath containing the disinfectant solution.
EMBODIMENT 91
91. The method of embodiment 89 or 90, wherein the disinfecting solution can include: peracid, hydrogen peroxide, acetic acid, peracetic acid (PAA), saline, sodium dodecyl sulfate, sodium hydroxide (NaOH), or any combination thereof.
EMBODIMENT 92
91. The method of embodiment 89 or 90, wherein the disinfecting solution may comprise peroxyacid.
EMBODIMENT 93
93. The method of embodiment 92, wherein the peroxyacid can include: peroxyacetic acid, peroxyoctanoic acid, sulfoperoxycarboxylic acid, peroxysulfonated oleic acid, peroxyformic acid, peroxyoxalic acid, peroxypropanoic acid, peroxybutanoic acid, peroxypentanoic acid, peroxyhexanoic acid, peroxyadipic acid, peracetic acid, perlactic acid, peroxycitric acid, peroxybenzoic acid, or any combination thereof.
EMBODIMENT 94
93. The method of embodiment 92, wherein the peroxyacid can be peracetic acid.
EMBODIMENT 95
95. The method of any one of embodiments 92-94, wherein the peroxyacid can be present at a concentration of about 0.1% to about 1.0% by weight.
EMBODIMENT 96
95. The method of any one of embodiments 92-94, wherein the peroxyacid can be present in a concentration of about 0.1% to about 1.0% by volume.
EMBODIMENT 97
95. The method of any one of embodiments 92-94, wherein the peroxyacid can be present at a concentration from about 40 ppm to about 700 ppm.
EMBODIMENT 98
98. The method according to any one of the preceding embodiments, wherein after said contacting with said liquid, the isolated organ or part thereof is further contacted with a second liquid.
EMBODIMENT 99
99. The method of embodiment 98, wherein the second liquid can be an at least partially degassed liquid.
EMBODIMENT 100
100. The method of embodiment 99, wherein the second liquid may comprise a cleaning medium.
EMBODIMENT 101
101. The method of embodiment 100, wherein the cleaning medium may comprise distilled water.
EMBODIMENT 102
101. The method of embodiment 100, wherein the washing medium may include: distilled water, reverse osmosis water, filtered water, HEPC-treated water, purified water, deionized water, saline, or any combination thereof.
EMBODIMENT 103
100. The method of embodiment 99, wherein the at least partially decellularized isolated organ or portion thereof comprises less than about 1% by weight of detergent after contacting the at least partially decellularized isolated organ or portion thereof with the second liquid.
EMBODIMENT 104
100. The method of embodiment 99, wherein the at least partially decellularized isolated organ or portion thereof comprises less than about 0.1% by weight of detergent after contacting the at least partially decellularized isolated organ or portion thereof with the second liquid.
EMBODIMENT 105
100. The method of embodiment 99, wherein after contact with the second liquid, blood flows through a greater volume of the vasculature of the at least partially decellularized isolated organ or portion thereof compared to an otherwise comparable at least partially decellularized isolated organ or portion thereof produced using a non-degassed second liquid.
EMBODIMENT 106
107. The method according to any one of embodiments 1 to 106, further comprising contacting the at least partially decellularized harvested organ or part thereof with a regeneration medium.
EMBODIMENT 107
107. The method of embodiment 106, wherein the regeneration medium can be an at least partially degassed cell regeneration medium.
EMBODIMENT 108
108. The method of embodiment 107, wherein said contact with said at least partially degassed cell regeneration medium forms an at least partially recellularized organ or part thereof.
EMBODIMENT 109
108. The method of embodiment 107, wherein the at least partially degassed cell regeneration medium can contain a population of regenerative cells.
EMBODIMENT 110
110. The method of embodiment 109, wherein the regenerative cell population can comprise totipotent cells, pluripotent cells, multipotent cells, or any combination thereof.
EMBODIMENT 111
110. The method of embodiment 109, wherein the regenerative cell population can comprise undifferentiated cells, partially differentiated cells, fully differentiated cells, or any combination thereof.
EMBODIMENT 112
110. The method of embodiment 109, wherein said regenerative cell population comprises embryonic stem cells, umbilical cord blood cells, induced pluripotent stem cells (iPSCs), tissue-derived stem or progenitor cells, bone marrow-derived stem or progenitor cells, blood-derived stem or progenitor cells, mesenchymal stem cells (MSCs), skeletal muscle-derived cells, multipotent adult progenitor cells (MAPCs), cardiomyocyte stem cells (CSCs), multipotent adult cardiac-derived stem cells, cardiac fibroblasts, cardiac microvasculature endothelial cells, aortic endothelial cells, bone marrow mononuclear cells (BM-MNCs), endothelial progenitor cells (EPCs), or any combination thereof.
EMBODIMENT 113
113. The method of any one of embodiments 109-112, wherein said regenerative cell populations can be independently derived from a mammal.
EMBODIMENT 114
The method of embodiment 113, wherein the mammal may be a rodent, a pig, a rabbit, a cow, a sheep, a dog, or a human.
EMBODIMENT 115
115. The method of any one of embodiments 109-114, wherein said population of regenerative cells can comprise at least about 1,000 cells.
EMBODIMENT 116
115. The method of any one of embodiments 109-114, wherein the amount of said regenerative cell population can be at least about 1,000 regenerative cells per milligram (mg) of said at least partially decellularized harvested organ or portion thereof.
EMBODIMENT 117
108. The method of embodiment 107, wherein the at least partially degassed cell regeneration medium can include growth factors.
EMBODIMENT 118
118. The method of embodiment 117, wherein said growth factors can include: VEGF, DKK-1, FGF, BMP-1, BMP-4, SDF-1, EGF, IGF, HGF, or any combination thereof.
EMBODIMENT 119
108. The method of embodiment 107, wherein the at least partially degassed cell regeneration medium can include an immunomodulatory agent.
EMBODIMENT 120
120. The method of embodiment 119, wherein the immunomodulatory agent can include: a cytokine, a glucocorticoid, an IL-2R antagonist, a leukotriene antagonist, or any combination thereof.
EMBODIMENT 121
108. The method of embodiment 107, wherein said contact with said at least partially degassed cellular regeneration medium results in a greater degree of recellularization than an otherwise equivalent isolated organ or portion thereof produced using a non-degassed cellular regeneration medium.
EMBODIMENT 122
109. The method of embodiment 108, wherein said at least partially recellularized organ or portion thereof can comprise at least one human cell.
EMBODIMENT 123
An at least partially decellularized harvested organ or part thereof produced by the method according to any one of embodiments 1 to 105.
EMBODIMENT 124
124. An at least partially decellularized isolated organ or part thereof according to embodiment 123, wherein the isolated organ or part thereof comprises an at least partially degassed solution.
EMBODIMENT 125
124. A method for reducing cellular cytotoxicity during recellularization of an at least partially decellularized harvested organ or part thereof, comprising introducing a cell population into the at least partially decellularized harvested organ or part thereof according to embodiment 123.
EMBODIMENT 126
123. An at least partially recellularized mammalian isolated organ or part thereof produced by the method according to any one of embodiments 106 to 122.
EMBODIMENT 127
127. An at least partially recellularized mammalian isolated organ or portion thereof as described in embodiment 126, comprising a cell regeneration medium having less than about 8 parts per million (ppm) oxygen when stored at a temperature of about 25°C.
EMBODIMENT 128
123. A system comprising: (a) an at least partially decellularized harvested organ or part produced by the method of any one of embodiments 1 to 122; (b) a device for transferring fluids; (c) a cannula; and (d) a port.
EMBODIMENT 129
The system of embodiment 128, which may further comprise a degassed decellularization medium.
EMBODIMENT 130
129. The system of embodiment 128, which may further comprise a degassed cleaning medium.
EMBODIMENT 131
The system of embodiment 128, wherein the system may further include an at least partially closed container.
EMBODIMENT 132
The system of embodiment 128, wherein a flow of at least partially degassed solution can pass through the isolated organ or part thereof at a flow rate of at or about 100 ml per minute to at or about 100 liters per minute.
EMBODIMENT 133
The system of embodiment 128, wherein the at least partially degassed solution can be passed through the isolated organ or part thereof at a flow rate of at or about 100 ml per minute to at or about 100 liters per minute.
EMBODIMENT 134
129. The system of embodiment 128, further comprising a peristaltic pump.
EMBODIMENT 135
123. A kit comprising: (a) the at least partially decellularized harvested organ or portion thereof produced by the method of any one of embodiments 1 to 122; (b) at least one cannula; (c) a reagent; (d) instructions for recellularizing the at least partially decellularized harvested organ or portion thereof; (e) instructions for introducing the at least partially recellularized harvested organ or portion thereof into a subject; or (f) any combination thereof.
EMBODIMENT 133
The kit of embodiment 135, wherein the at least partially decellularized harvested organ or part thereof may be in packaging.
EMBODIMENT 134
The kit of embodiment 136, wherein the packaging may be sterile.
EMBODIMENT 135
A kit comprising an at least partially decellularized harvested organ or part thereof according to embodiment 135.

Example 1: Perfusion decellularization of isolated solid organs using degassed media Part 1: Cannulation The heart is systemically heparinized with 400 U heparin per kg of donor, e.g., pig, dog, human, primate, and sheep. After heparinization, the heart and adjacent large vessels are carefully removed. The heart is then perfused with a saline solution (
0.9%) and kept at 5° C. until further processing. Under sterile conditions, connective tissue can be removed from the heart and great vessels. The inferior vena cava and left and right pulmonary veins can be ligated distal to the right and left atria using monofilament non-absorbable ligation techniques.

The heart can be attached to the decellularization device for perfusion (Figure 1). The descending thoracic aorta can be cannulated to allow retrograde coronary perfusion (Figure 1, cannula A). Branches of the thoracic arteries (e.g. brachiocephalic, left common carotid, left subclavian) are ligated. The pulmonary artery can be cannulated before bifurcating into the left and right pulmonary arteries (Figure 1, cannula B). The superior vena cava can be cannulated (Figure 1, cannula C). This configuration allows both retrograde and antegrade coronary perfusion. If positive pressure can be applied to the aortic cannula (A), perfusion occurs from the coronary arteries through the capillary beds and down the coronary venous system to the right atrium and superior vena cava (C). Perfusion can occur from the right atrium, coronary sinus, and coronary veins, through the capillary beds, to the coronary artery and aortic cannula (A) while applying positive pressure to the superior vena cava cannula (C).

Part II: Basic Decellularization Protocol After mounting the heart on the decellularization apparatus, antegrade perfusion can be initiated with cold heparinized calcium-free phosphate buffer containing 1-5 mM adenosine per L of perfusion to restore constant coronary flow. Prior to perfusion, the buffer can be placed under high vacuum for 30 min with vigorous stirring using a stir bar at 25° C. for 30 min.

Coronary blood flow can be assessed by measuring coronary perfusion pressure and flow and calculating coronary resistance. After 15 minutes of stable coronary blood flow, the detergent-based decellularization process can be initiated utilizing a degassed decellularization solution. The heart can be perfused antegradely with a degassed detergent solution. After perfusion, the heart can be flushed retrogradely with a degassed buffer (e.g., PBS). The heart can then be perfused with a degassed PBS buffer containing antibiotics, followed by a degassed PBS buffer containing DNase I. The heart can then be perfused with 1% benzalkonium chloride to reduce and prevent future microbial contamination, and then with PBS to wash any remaining cellular components, enzymes, or detergents from the isolated organ.

Exemplary decellularized solid organs are shown in Figure 2 (pig heart) and Figure 3 (pig liver).

Decellularization with degassed buffer produces fewer microbubbles than decellularization with non-deregassed buffer.

PEG decellularization The heart is washed in 200 ml of PBS containing 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 μg/ml amphotericin B without recirculation. The heart is then decellularized with 35 ml of degassed polyethylene glycol (PEG; 1 g/ml) for up to 30 minutes with manual recirculation. Degassed polyethylene glycol was prepared by placing the polyethylene glycol solution under a vacuum pump as described above. The isolated organ was then washed with 500 ml of degassed PBS for up to 24 hours using a pump for recirculation. The washing step can be repeated at least twice, each time for at least 24 hours. The heart is exposed to 35 ml of DNase I (70 U/ml) for at least 1 hour with manual recirculation. The isolated organ is washed again with 500 ml of degassed PBS for at least 24 hours.

Decellularization with degassed buffer results in more efficient decellularization when delivered by pump for 30 minutes compared to using non-degassed buffer.

Triton X and trypsin decellularization The heart is washed in 200 ml of PBS containing 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 μg/ml amphotericin B for at least about 20 minutes without recirculation. The heart is then decellularized with a degassed solution of 0.05% trypsin in water for 30 minutes, followed by perfusion for about 6 hours with 500 ml of degassed PBS containing 5% Triton-X and 0.1% ammonium hydroxide. Both solutions are prepared by dissolving the detergent in previously degassed water. The heart is perfused for about 1 hour with degassed deionized water, then with degassed PBS for 12 hours. The heart is then washed three times, each time for 24 hours, in 500 ml of degassed PBS, using a pump for recirculation. Hearts are perfused with 35 ml of DNase I (70 U/ml) for 1 h with manual recirculation and washed twice in 500 ml of PBS, each time for at least about 24 h, using a recirculation pump.

Decellularization with degassed buffer results in more efficient decellularization when delivered by pump for 30 minutes compared to using non-degassed buffer.

SDS Decellularization The heart is washed in 200 ml of PBS containing 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 μg/ml amphotericin B for at least about 20 minutes without recirculation. The heart is decellularized in 4000 ml of degassed water containing 1% SDS for at least about 6 hours using a recirculation pump. The degassed water was prepared by heating deionized water to about 65° C. under low vacuum for about 1 hour. The heart is then washed in degassed deionized water for about 1 hour and degassed PBS for about 12 hours. The heart is washed three times with 500 ml of degassed PBS using a recirculation pump for at least about 24 hours each time. The heart can then be perfused with 35 ml of DNase I (70 U/ml) for approximately 1 hour with manual recirculation and washed three times with 500 ml of degassed PBS for at least approximately 24 hours each time using a recirculation pump.

The liver is washed in 2000 ml of PBS, disinfected with PAA, and perfused with 2000 ml of degassed 0.9% saline. The liver is decellularized with a total of about 10 L of degassed water containing about 0.6% SDS for at least about 6 hours using a series of single flow-through pumps and recirculating baths. The degassed water was prepared by passing the degassed water through a vacuum filter designed to degas the solution. The liver is then washed with degassed deionized water for about 1 hour and degassed PBS for about 12 hours. The liver is washed three times with 2000 ml of degassed PBS, each time for at least about 2 hours, using a recirculating pump.

Decellularization with degassed buffer produces fewer microbubbles than decellularization with non-deregassed buffer.

Triton X decellularization The heart is washed with 200 ml of PBS containing 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 μg/ml amphotericin B for at least about 20 minutes without recirculation. The heart is then decellularized with 500 ml of degassed water containing 5% Triton X and 0.1% ammonium hydroxide for at least 6 hours using a recirculation pump. The degassing buffer is prepared as described above for PEG decellularization. The heart is then perfused with degassed deionized water for about 1 hour, then with degassed PBS for about 12 hours. The heart is washed by perfusing with 500 ml of degassed PBS three times, each time for at least 24 hours, using a recirculation pump. The heart is then perfused with 35 ml of DNase I (70 U/ml) with recirculation for approximately 1 hour and washed three times in 500 ml of degassed PBS for approximately 24 hours each time.

For initial experiments, the decellularization apparatus can be placed in a laminar flow hood. Hearts are perfused at a coronary perfusion pressure of 60 cm _H2O . Although not required, hearts described in the above experiments are mounted in a decellularization chamber and completely submerged and perfused with degassed PBS containing antibiotics for 72 hours in recirculation mode at a continuous flow of 5 ml/min to wash out as many cellular components and detergents as possible.

Successful decellularization can be defined by the absence of myofilaments and nuclei in tissue sections. Successful preservation of vasculature can be assessed by perfusing the tissue sections with 2% Evans Blue prior to embedding. Highly efficient decellularization can be performed by first perfusing the heart antegrade with an ionic detergent (1% sodium dodecyl sulfate (SDS), approx. 0.03 M) dissolved in deionized H ₂ O at constant coronary perfusion pressure, followed by antegrade perfusion with a non-ionic detergent (1% Triton X-100) to remove the SDS and presumably regenerate extracellular matrix (ECM) proteins. Intermittently, the heart can be perfused retrogradely with phosphate buffer to clear occluded capillaries and small blood vessels.

Example 2: Evaluation of decellularized isolated organs To demonstrate the intact vasculature of decellularization, at least partially decellularized hearts can be stained by Langendorff perfusion with Evans Blue to stain vascular basement membranes and to quantify macrovascular and microvascular density. Additionally, polystyrene particles can be perfused into the heart to quantify coronary vascular volume, levels of vascular leakage, and the distribution of perfusion can be evaluated by analyzing coronary effluent and tissue sections. A combination of three criteria: 1) uniform distribution of polystyrene particles, 2) significant changes in leakage at a certain level, and 3) microvascular density is evaluated and compared to isolated non-decellularized hearts.

Fiber alignment was measured using the polarized light microscopy technique of Tower et al. (2002)
The mechanical properties of the at least partially decellularized ECM during Langendorff perfusion can be recorded (compliance, elasticity, burst pressure) and compared to freshly isolated hearts.

Histological Evaluation Representative samples of both native and decellularized kidney, heart, and liver are processed for hematoxylin and eosin (H&E) to assess cellular content and tissue structure. Samples are fixed in 10% neutral buffered formalin solution for 24 hours at 4°C. They are subsequently washed in distilled water, dehydrated in graded alcohols, embedded in paraffin, and sectioned (5 mm) for staining. Tissue slides were stained with H&E according to standard protocols. To exclude the presence of nuclear material, native kidney and decellularized kidney scaffolds are processed for DNA-binding fluorescent staining with DAPI (40,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA)) stain. Examination of stained sections was performed under an immunofluorescence microscope (Olympus, Japan), FIG. 9.

Scanning Electron Microscopy (SEM)
For evaluation of the ultrastructure after the decellularization step, native and decellularized kidney specimens are processed for scanning electron microscopy. Specimens are washed with distilled water and fixed in cold 2.5% glutaraldehyde for 2 hours at 48°C. After washing with PBS to remove residual glutaraldehyde, the specimens are fixed in increasing concentrations of ethanol at room temperature for 20-30 minutes (
The samples are dehydrated using 100% ethanol (30%, 50%, 70%, 90%, and 100% ethanol), critical point dried using _CO2 , and finally mounted on aluminum stubs using adhesive carbon taps for imaging with a scanning electron microscope (Carl Zeiss, Oberkochen, Germany).

Vascular Network Imaging To assess vascular network and renal capsule integrity, radio-opaque contrast solution (Omnipaque 350, GE Healthcare, Piscataway, NJ) can be perfused into native and decellularized kidneys via the renal artery, and contrast radiography (KMC-950, Komed, Korea) can be used 10 minutes after contrast agent perfusion.

Quantification of Extracellular Matrix (ECM) Components To check the effect of the decellularization process on ECM components, samples of native and decellularized kidney tissue are digested overnight at 56°C in PBS containing 50 mgmL proteinase K. Lysates are then heat inactivated at 90°C for 10 min and centrifuged at 13,000 x g for 10 min. Supernatants are collected and analyzed for protein concentration using the BCA Protein Assay (Thermo Scientific, Rockford, IL). GAG, collagen, and elastin levels are analyzed using the Blyscan sGAGs Assay Kit (Biocolor Ltd, Carrickfergus, UK), Sircol Soluble Collagen Assay Kit (Biocolor Ltd, Carrickfergus, UK), and the ELISA Kit (ELISA Kit).
The elastin activity is quantified in lysates using the Fastin Elastin Assay Kit (Biocolor Ltd), and Fastin Elastin Assay Kit (Biocolor Ltd), respectively, according to the manufacturer's instructions.

Mechanical Testing Myocardial tissue crosses can be cut from rat left ventricles such that the central section can comprise approximately 5 mm x 5 mm, with the axes of the cross aligned with the circumferential and longitudinal directions of the heart. The initial thickness of the tissue crosses is measured in micrometers. Crosses are also cut from decellularized rat left ventricular tissue in the same orientation and with the same central section size. Additionally, the mechanical properties of fibrin gel, another tissue engineering scaffold used in vascular and cardiac tissue engineering, are tested. Fibrin gel is cast into a cruciform mold with a final concentration of 6.6 mg of fibrin per ml. All samples are mounted via clamps to a biaxial mechanical tester (Instron Corporation, Norwood, Mass.), submerged in PBS, and equibiaxially stretched to 40% strain. To accurately examine static passive mechanical properties, samples are stretched in 4% strain increments and allowed to relax at each strain value for at least 60 seconds. Figure 11 shows a plot of the peak load of the samples. Force is converted to engineering stress by normalizing the force value by the cross-sectional area (5 mm x initial thickness) in the specified axis. Engineering stress can be calculated as displacement normalized by the initial length. To compare data between the two axes and between groups of samples, the tangent modulus can be calculated as follows: [T(ε=40% strain)-T(ε=36% strain)]/4% strain, where T is the engineering stress and E is the engineering strain. Tangent modulus values are averaged and compared between the two axes (circumferential and longitudinal) and between groups.

Example 3: Perfusion of isolated parenchymal organs using degassed media Recellularized human umbilical vein endothelial cells (HUVECs)
Perfusion recellularization of isolated parenchymal organs can be performed by perfusing cells into an at least partially decellularized porcine heart by coronary perfusion. The at least partially decellularized porcine heart is transferred to an isolated organ chamber and incubated under cell culture conditions (5% _CO2 , 60% humidity, 3 h).
The heart can be continuously perfused with oxygenated cell culture medium at 40° C. (37° C.). 120×10 ⁶ PKH-labeled HUVECs (suspended in 50 ml of degassed endothelial cell growth medium) are injected at 40 cm H ₂ O coronary perfusion pressure. The degassed endothelial cell growth medium can be perfused by storing the endothelial cell growth medium as described in Example 1. The coronary effluent can be stored and the cells can be counted. The effluent can then be recirculated and perfused again to deliver the maximum number of cells. The recirculation can be repeated twice. After the third passage, approximately 90×10 ⁶ cells are retained in the heart. The heart can be continuously perfused with 500 ml of recirculated oxygenated endothelial cell culture medium for 120 hours. The heart can then be removed and embedded for cryosectioning.

Regenerative Cells Perfusion recellularization of isolated solid organs can be performed by perfusing regenerative cells (embryonic stem cells, umbilical cord cells, autologous stem or progenitor cells, bone marrow derived cells, blood derived cells, induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), skeletal muscle derived cells, multipotent adult progenitor cells (MAPCs), cardiac stem cells (CSCs), multipotent adult cardiac derived stem cells) into an at least partially decellularized porcine heart by coronary perfusion. The at least partially decellularized porcine heart can be transferred to an isolated organ chamber and continuously perfused with oxygenated cell culture medium under cell culture conditions (5% _CO2 , 60% humidity, 37°C). ^120x106 regenerative cells (suspended in 50 ml cell growth medium) are injected at a coronary perfusion pressure of 40 _cmH2O . The coronary effluent can be saved and the regenerative cells counted. The effluent can then be recirculated and perfused again to deliver the maximum number of cells. The recirculation can be repeated twice. After the third passage, approximately ^90x106 cells are retained within the heart. The heart can be continuously perfused with 500 ml of recirculating oxygenated endothelial cell culture medium for 120 hours. The heart can then be removed and embedded for cryosectioning.

Example 4: Reduction of particulate formation During perfusion decellularization of whole or part of an excised organ with some detergents such as SDS, the presentation of insoluble white particulates is seen. Particulates may form and then become trapped in the excised organ or part of it during decellularization. Reduction of particulate formation may include a significant reduction in the amount of trace detergent or detergent remaining after decellularization. Reduction of particulate formation may increase the ability to recellularize the excised organ. Reduction of particulate formation may reduce the cytotoxicity of the decellularized matrix to the introduced cells. Particulates may be formed by insoluble interactions between native proteins and detergents. Reduction of particulates may be performed by using solutions, controlling the mammal's dietary habits, or a combination thereof.

During isolated organ decellularization, isolated organs were immediately treated with detergent. The addition of saline was found to inhibit or prevent particulate formation. After removal from the mammal, isolated organs were flushed with 0.9% saline or PBS and then disinfected with peracetic acid. Isolated organs were primed with 0.9% saline before decellularization with detergent and placed in a 0.9% saline or PBS bath before detergent perfusion.

In particular, the porcine liver was washed with 0.9% saline or PBS, followed by a peracetic acid wash. The washed liver was then submerged in a 0.9% saline or PBS bath. For decellularization, the liver was decellularized with 5000 ml of water containing 1% SDS for at least about 6 hours using a recirculation pump. The liver was then washed with deionized water for about 1 hour and with PBS for about 12 hours. The liver was washed three times with 500 ml of PBS, each time for at least about 24 hours, using a recirculation pump. The liver was then perfused with 35 ml of DNase I (70 U/ml) for about 1 hour with manual recirculation, and washed three times with 500 ml of PBS, each time for at least about 24 hours, using a recirculation pump.

Non-fasted mammals Particulate formation can be reduced in mammals by ensuring that the mammal has been fed for the last 8 hours prior to euthanasia.

After 4 hours of feeding, the pigs were euthanized and their livers were harvested. The pig livers were washed in 200 ml of PBS containing 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 μg/ml amphotericin B for at least about 20 minutes without recirculation. The livers were decellularized with 500 ml of water containing 1% SDS for at least about 6 hours using a recirculation pump. The livers were then washed with deionized water for about 1 hour and PBS for about 12 hours. The livers were washed three times with 500 ml of PBS, each time for at least about 24 hours, using a recirculation pump. The livers were washed three times with 500 ml of PBS, each time for at least about 24 hours, using a recirculation pump. When pigs were fasted or had limited access to food, a high percentage of decellularized livers were observed to contain trace to large amounts of particulates. Harvesting from non-fasted or recently fed animals significantly dropped particulate formation from 79% to 4.6% per decellularized lot, Table 2. % particulates refers to the amount of total grafted tran that contained particulates upon visual inspection. For DL-071116 lot, 11 of 30 grafted tran had particulates, representing 37% of the lot.

Whole livers were removed from fasted or non-fasted animals, perfused decellularized with SDS, and the level of microparticle formation was visually quantified. Livers removed from non-fasted animals showed very low levels of microparticle formation compared to fasted animals.

Non-fasted mammals + saline bath Livers collected from slaughterhouses under standard conditions of regular fasting were washed with 0.9% saline or PBS followed by peracetic acid disinfection. The washed liver was then submerged in a 0.9% saline or PBS bath and transferred to a 0.9% saline solution bath until decellularization was initiated, with the liver remaining in the initial 0.9% saline bath. For decellularization, the liver was decellularized with at least 1000 ml of water containing 0.6% SDS using a first single flow pump through the liver for at least about 2 hours, then transferred to a new bath containing at least 3000 ml of water containing 0.6% SDS with recirculation for at least about 6 hours. The liver was then washed with deionized water for about 1 hour and with PBS for about 12 hours. The liver was washed three times with 500 ml of PBS using a recirculation pump for at least about 24 hours each time. The liver was then perfused with 35 ml of DNase I (70 U/ml) for approximately 1 hour with manual recirculation and washed three times with 500 ml of PBS for at least approximately 24 hours each time using a recirculation pump. Placing livers harvested at the start of decellularization with an aqueous solution containing 0.6% SDS in a 0.9% saline bath significantly reduced particulate formation (Table 3).

Microparticle formation was visually detected in less than 3% of livers when the liver was in a 0.9% saline bath at the start of decellularization, compared to more than 40% when no saline bath was used.

Liver recellularization HUVEC cell culture and seeding of decellularized liver constructs: Human umbilical vein endothelial cells were cultured in EGM-2 media without antibiotics in tissue culture flasks at 37°C and 5% _CO2 and passaged with 0.25% trypsin at 90-100% confluency according to the manufacturer's protocol. The highest passage used for seeding the liver grafts was passage 11. A decellularized porcine liver utilizing degassed decellularization was placed in a custom bioreactor containing 800 ml of culture media, connected to the perfusion inlet via the suprahepatic vena cava and perfused at 12 mmHg with culture media before seeding. HUVECs were resuspended in 100 ml of media and seeded via the suprahepatic vena cava. The injected cell suspension was left under static conditions for 1 hour and then perfusion was resumed. After 24 hours, perfusion was changed from the suprahepatic vena cava to the portal vein and the seeding protocol was repeated. Re-endothelialized grafts were maintained in a continuous perfusion loop with metabolites (glucose, lactate, glutamine, glutamate and ammonia) monitored daily in media samples collected using a BioProfile FLEX Analyzer. Culture medium was exchanged and the volume increased depending on the rate of glucose depletion in the circulating medium to ensure 24-hour glucose levels were above 500 mg/L.

Histological Analysis: Tissue samples analyzed in this study were perfused with PBS and fixed in 10% neutral buffered formalin (VWR16004-128). Fixed tissues were paraffin embedded, sectioned, and stained using standard histological techniques. Figure 12A shows a representation of the endothelial seeding used in the histological analysis. Figure 12B shows representative images of the bioreactor, tissue structure after 21 days, and exemplary vessel surfaces.

Recellularization of kidneys with total liver resection Decellularized porcine kidneys utilizing degassed decellularization were placed in a custom bioreactor containing 800 ml of culture medium, connected to a perfusion inlet via the renal artery, and perfused at 12 mmHg with culture medium prior to seeding. The kidney resection was resuspended in 100 ml of medium and seeded through the ureter. The infused cell suspension was left under static conditions for 1 hour, and then perfusion was resumed. The recellularized kidney was maintained in a continuous perfusion loop with metabolites (glucose, lactate, glutamine, glutamate, and ammonia) monitored daily in medium samples collected using a BioProfile FLEX analyzer. Culture medium was replaced and the volume was increased depending on the rate of glucose depletion in the circulating medium to ensure that 24-hour glucose levels were above 500 mg/L. As shown in FIG. 13, immunofluorescence staining shows the distribution and engraftment of native kidney cells throughout the kidney in the proper position.

Example 5: Decellularization of Harvested Organs Harvested organs, such as liver, spleen, lungs, kidneys, and heart, are flushed with a hypertonic solution (e.g., 5x saline). The harvested organs can then be disinfected. For example, the harvested organs can be placed in a disinfecting bath containing either peracid alone or a solution of peracid and saline. Specifically, the disinfecting bath can include saline, peracid (e.g., peracetic acid), and NaOH and can have a pH of 6-7. The disinfecting bath can include a disinfecting solution that can be made, for example, by adding 2 liters of 10x saline (9% NaCl) to 18 liters of water to make 1x saline (0.9% NaCl), and adding 80 ml of 15% peracetic acid and 51 ml of 5N NaOH to make a final concentration of 600 ppm of PAA added in 0.9% NaCl at a pH of 6.12. The isolated organs were exposed to different concentrations of peracid (e.g., 50 parts per million (ppm), 100 ppm, 150 ppm, 200 ppm, 250 ppm, 300 ppm, 350 ppm, 40 ppm, 50 ppm, 60 ppm, 70 ppm, 80 ppm, 90 ppm, ...100 ppm, 150 ppm, 200 ppm, 250 ppm, 300 ppm, 350 ppm, 40 ppm, 50 ppm, 50 ppm, 60 ppm, 70 ppm, 80 ppm, 100 ppm, 150 ppm, 200 ppm, 250 ppm, 300 ppm, 350 ppm, 40 ppm, 50 ppm, 100 ppm, 150 ppm, 200 ppm, 250 ppm, 300 ppm, 350 The isolated organ may be placed in a disinfection bath with a detergent concentration of 0 ppm, 450 ppm, 500 ppm, 550 ppm, 600 ppm, 650 ppm, 700 ppm, 750 ppm, 800 ppm, 850 ppm, 900 ppm, 1000 ppm, 1200 ppm, 1400 ppm, 1500 ppm, 1700 ppm, 1800 ppm, 1900 ppm, 2000 ppm, or greater than 2000 ppm. After removing the isolated organ from the disinfection bath, the isolated organ is decellularized. The isolated organ may be decellularized with a solution containing a detergent (e.g., sodium dodecyl sulfate (SDS)) or a solution containing a detergent and peracid. The SDS in the decellularization solution may be, for example, in the range of about 0.2% to 2%. In some embodiments, decellularization can be perfused with 0.6% SDS or 0.9% SDS. The peracid present in the solution during decellularization can range from about 25 ppm to 3000 ppm. In some embodiments, the peracid can be present at 50 ppm, 100 ppm, 200 ppm, 400 ppm, 500 ppm, 600 ppm, 650 ppm, 700 ppm, 1000 ppm, 1500 ppm, or 2000 ppm. In some embodiments, the isolated organ can be perfused with a solution comprising PAA and SDS.

Briefly, the perfusable liver was flushed with 5x saline and then placed in a disinfecting bath containing 600 ppm peracetic acid (PAA). Peracetic acid (PAA) was added to 1x saline solution (0.9% NaCl) to create a final solution containing 600 ppm PAA at a pH of 6.12. After removing the liver from the disinfecting bath, the liver was perfused with a solution containing PAA and SDS for a period of time (e.g., about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 16 hours, 20 hours, 24 hours, 30 hours, 35 hours, 40 hours, 45 hours, 46 hours, 47 hours, 48 hours, 49 hours, 50 hours, or more). The PAA in the solutions used for perfusion decellularization of the liver ranged from about 50 ppm to 3000 ppm.

Example 6: Complete decellularization of isolated organs with radical generating compounds The use of radical generating compounds in solution during decellularization of isolated organs affected the flow rate and the decellularization process of isolated organs without compromising the integrity matrix (FIGS. 10A-10C). A liver from a pig was obtained and cannulated. The native vasculature of the pig liver was cannulated and perfused with a mild detergent solution to decellularize the liver. The detergent solution further contained a radical generating compound (e.g., peracetic acid). After introducing the detergent solution containing the radical generating compound into the isolated organ at a set pressure (e.g., 12 mmHg), an increase in flow rate (e.g., to at least 500 mL/min, 1000 mL/min, 1500 mL/min, 2000 mL/min, 2500 mL/min, or more) and a decrease in the decellularization process were observed (FIGS. 10A-10C). This experiment demonstrated that the use of a radical-generating compound (e.g., peracetic acid) at a concentration of about 50-2000 ppm increased the flow rate during perfusion decellularization at a given pressure compared to an otherwise equivalent decellularization process without the radical-generating compound. FIG. 14 shows the improvement in flow rate by incorporating a radical-generating compound into the decellularization workflow. As shown in FIG. 14, the overall flow rate of each step in the decellularization workflow improved after the addition of the radical-generating compound. This improvement in the workflow propagated throughout the decellularization workflow, even to perfusion steps that did not contain the radical-generating compound. In fact, this increase in flow rate at a set pressure occurred during perfusion with a radical-generating compound (e.g., PAA) and was maintained in subsequent perfusion steps, even those that did not contain the radical-generating compound. This increase in flow rate resulted in a more complete decellularization of the explanted organ without affecting the matrix integrity.

Overall, the results showed that more cells were removed in a shorter period of time, and that increasing the flow rate resulted in a purer and more complete decellularization of the isolated organ. Isolated organs are densely packed with capillaries, and most cells are located in close proximity to the capillaries. Thus, perfusing a cannulated isolated organ with a solution containing a radical-generating compound and then increasing the flow rate increased the effective surface area of the detergent and reduced the time required to expel native cellular material through the venous system. The results also showed that increasing the flow rate or adding a radical-generating compound during decellularization did not significantly affect or impair the integrity matrix.

Example 7: Radical generating compounds improve isolated organ decellularization Isolated mammalian livers were placed in a reservoir bin containing SDS for about 1-24 hours. The livers were then placed in a reservoir bin containing liver SDS for about 1-10 hours and then placed in a bin containing water ( _H2O ). The livers were removed from the water bin and placed in a bin containing a radical generating compound (PAA) followed by a bin containing a solution of PAA in SDS. The PAA ranged from about 0.1% to about 30%. The PAA in SDS ranged from about 25 ppm to 3000 ppm. The livers were then placed in SDS followed by further steps including washing with liver _H2O , _H2O , _H2O and PBS, further treatment with PAA, and further washing. The duration of SDS perfusion ranged from about 10-100 hours (eg, 25-60 hours), and the duration of PAA perfusion ranged from about 1-20 hours (eg, 2-10 hours).

Example 8: Quantification of radical generating compounds Improved decellularization Excised mammalian livers were decellularized in the presence or absence of the radical generating compound PAA as described in Example 7. To quantify the improvement of the decellularization process after the addition of PAA, the residual concentration of DNA was determined from the tips of the decellularized lobes. Approximately 10-30 mg of tissue was collected and digested with proteinase K in buffer at 60°C for 8-30 hours. Chloroform was added in a ratio of 2:5 to tissue and the mixture was centrifuged at 14,000 x g for 10 minutes. The supernatant was collected and DNA stripping solution was added to the supernatant. The suspension was incubated at 60°C for 5-10 minutes. After adding precipitation solution and centrifuging at 14,000 x g for 5 minutes, the supernatant was collected. DNA was precipitated using 100% isopropanol and mussel glycogen followed by centrifugation at 14,000 x g for 5 minutes. The pellet was washed with 70% ethanol and dissolved in Tris buffer. The amount of DNA in each sample was quantified using a Qubit™ dsDNA BR Assay Kit and a Qubit® 2.0 Fluorimeter.

Table 4 above shows the residual DNA concentration after decellularization. Evaluation of the residual DNA content, an indication of quantification of the amount and/or completeness of decellularization, showed a 10-fold reduction in residual DNA with the addition of PAA. This therefore indicates that the addition of a radical generating compound improved the completeness of the decellularization process.

While exemplary embodiments have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many modifications, changes, and substitutions will occur to those skilled in the art. It will be understood that various modifications to the embodiments described herein can be used. The following claims define the scope of the disclosure, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims (62)

1. A method comprising contacting an isolated organ or part thereof with a liquid, comprising:
(a) the liquid is at least partially degassed;
(b) the liquid comprises a cleaning agent and a radical-producing compound;
(c) the isolated organ or part thereof is from a mammal that was fed within about 10 hours prior to removal of the isolated organ or part thereof; or (d) any combination of (a)-(c). The method of claim 1, comprising at least two of (a), (b) or (c). The method of claim 1 or claim 2, comprising (a), (b), and (c). The method according to any one of claims 1 to 3, wherein the isolated organ or part thereof is from a mammal. The method of claim 4, wherein the mammal is a non-human mammal. The method of claim 5, wherein the non-human mammal is a pig, sheep, goat, cow, dog, cat, or monkey. The method of claim 4, wherein the mammal is a human mammal. The method according to any one of claims 1 to 7, wherein the removed organ or part thereof is the liver, lung, heart, kidney, bladder, pancreas, spleen, uterus, any part of these, or any combination thereof. The method according to any one of claims 1 to 8, wherein the contacting includes (i) perfusing at least a portion of the isolated organ or part thereof with a liquid, (ii) injecting at least a portion of the isolated organ or part thereof with a liquid, (iii) spraying at least a portion of the isolated organ or part thereof with a liquid, (iv) submerging the isolated organ or part thereof in a liquid, or (v) any combination thereof. The method according to any one of claims 1 to 9, wherein a cannula is inserted into the isolated organ or a part thereof before, during, or after the contact. The method according to any one of claims 1 to 10, comprising (a) and wherein the liquid comprises an at least partially degassed decellularization medium, thereby forming an at least partially decellularized harvested organ or part thereof. The method according to claim 11, wherein the at least partially decellularized isolated organ or part thereof comprises an extracellular matrix. The method of claim 12, wherein the extracellular matrix comprises a vasculature bed. The method of claim 13, wherein the vasculature bed remains at least partially intact after the contacting. 15. The method of any one of claims 12 to 14, wherein the extracellular matrix has an increased compressibility compared to an otherwise equivalent extracellular matrix produced by contact with an otherwise equivalent non-degassed liquid for an equivalent period of time. The method according to any one of claims 1 to 15, comprising (a). The method of claim 16, wherein the isolated organ or part thereof is contacted with the at least partially degassed liquid for at least about 2 hours. The method of claim 16, wherein the dissolved gas in the at least partially degassed liquid has a concentration of less than about 1 milligram (mg) per liter (L). 19. The method of claim 18, wherein the dissolved gas is selected from the group consisting of oxygen, nitrogen, carbon monoxide, carbon dioxide, a noble gas, and any combination thereof. 17. The method of claim 16, wherein the isolated organ or portion thereof after said contacting comprises (a) less air emboli, (b) less microbubbles, (c) less pigmentation, or (d) any combination thereof, compared to an otherwise equivalent isolated organ or portion thereof that has not been at least partially degassed and that has been contacted with an otherwise equivalent liquid for an equivalent period of time. 17. The method of claim 16, wherein the at least partially degassed liquid comprises an at least partially degassed decellularization medium, thereby forming an at least partially decellularized harvested organ or portion thereof. 22. The method of claim 21, wherein the at least partially decellularized isolated organ or portion thereof after said contact with the at least partially degassed decellularized medium contains fewer cells than an otherwise equivalent at least partially decellularized isolated organ or portion thereof contacted with an otherwise equivalent non-degassed decellularized medium for a comparable period of time. 22. The method of claim 21, wherein the time sufficient to produce the at least partially decellularized isolated organ or portion thereof with the at least partially degassed decellularization medium is less than the time to produce an otherwise equivalent at least partially decellularized isolated organ or portion thereof with an otherwise equivalent non-degassed decellularization medium. The method according to any one of claims 1 to 15, comprising (b). 25. The method of claim 24, wherein the radical generating compound is of the formula R ¹ -O-O-R ² , where R ¹ and R ² are independently H, C ₁ -C ₆ alkyl, substituted C ₁ -C ₆ alkyl, aryl, substituted aryl, benzyl, substituted benzyl, C(=O)A ¹ , C(=O)OA ² , and A ¹ and A ² are independently H, C ₁ -C ₆ alkyl or substituted C ₁ -C ₆ alkyl. 26. The method of claim 25, wherein R ¹ is H and R ² is C(=O)CH ₃ . 25. The method of claim 24, wherein the radical-generating compound is a peracid. 28. The method of claim 27, wherein the peracid comprises peroxyacetic acid, peroxyoctanoic acid, sulfoperoxycarboxylic acid, peroxysulfonated oleic acid, peroxyformic acid, peroxyoxalic acid, peroxypropanoic acid, peroxybutanoic acid, peroxypentanoic acid, peroxyhexanoic acid, peroxyadipic acid, peracetic acid, perlactic acid, peroxycitric acid, peroxybenzoic acid, or any combination thereof. 27. The method of claim 26, wherein the peracid comprises peracetic acid. The peracid is at least about 10 ppm, 25 ppm, 50 ppm, 75 ppm, 90 ppm, 100 ppm, 125 ppm, 150 ppm, 175 ppm, 200 ppm, 225 ppm, 250 ppm, 275 ppm, 300 ppm, 325 ppm, 350 ppm, 375 ppm, 400 ppm, 425 ppm, 450 ppm, 475 ppm, 500 ppm, 525 ppm, 550 ppm, 575 ppm, 600 ppm, 625 ppm, 650 ppm The method of any one of claims 24 to 29, wherein the cations are present in the liquid in an amount including 0.01 ppm, 675 ppm, 700 ppm, 725 ppm, 750 ppm, 775 ppm, 800 ppm, 900 ppm, 1000 ppm, 1200 ppm, 1400 ppm, 1500 ppm, 1700 ppm, 2000 ppm, 2200 ppm, 2500 ppm, 2750 ppm, 3000 ppm, 3200 ppm, 3500 ppm, 3750 ppm, or 4000 ppm. The method according to any one of claims 24 to 30, wherein the contact produces an at least partially decellularized isolated organ or part thereof. 32. The method of claim 31, wherein the at least partially decellularized isolated organ or portion thereof comprises a reduced amount of cells compared to the amount of cells in an otherwise equivalent at least partially decellularized isolated organ or portion thereof that has been contacted with an otherwise equivalent liquid that does not contain peroxyacid for an equivalent period of time. 33. The method of claim 32, wherein the reduced cell mass comprises a reduction in cells of at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% compared to the cell mass in the otherwise comparable at least partially decellularized harvested organ or portion thereof. The method of any one of claims 24 to 33, wherein the contacting comprises perfusion, and the flow rate of the perfusion is increased compared to the flow rate of the perfusion of an otherwise equivalent liquid that does not comprise the peracid. The method of claim 34, wherein the flow rate is increased without substantially compromising the integrity of the extracellular matrix of the isolated organ or portion thereof. The method according to any one of claims 1 to 15, further comprising (c), wherein the liquid comprises a decellularization medium, thereby at least partially decellularizing the isolated organ or a portion thereof. 37. The method of claim 36, wherein the decellularization proceeds by reducing the level of microparticles in the isolated organ or portion thereof compared to the level of microparticles produced in an otherwise equivalent decellularization of an otherwise equivalent isolated organ or portion thereof removed from an animal that has not been fed within about 10 hours prior to removing the isolated organ or portion thereof from the animal. 38. The method of claim 37, wherein the microparticle levels are reduced by at least about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1% compared to the microparticle levels produced in the otherwise equivalent decellularization. The method of claim 37 or 38, wherein the reduced particulate levels are determined by visual inspection, flow assessment, turbidity assessment, microscopic analysis, or any combination thereof. The method according to any one of claims 1 to 39, further comprising washing the isolated organ or part thereof with a washing medium prior to the contacting. The method of claim 40, wherein the washing medium comprises a hypertonic solution. The method of claim 41, wherein the hypertonic solution comprises saline. The method of claim 40, wherein the cleaning medium comprises a disinfectant solution. The method according to any one of claims 1 to 43, further comprising contacting the isolated organ or a part thereof with a second liquid. The method of claim 44, wherein the contact with the second liquid occurs after the contact with the liquid. The method of claim 44 or 45, wherein the second liquid is an at least partially degassed liquid. The method of any one of claims 44 to 46, wherein the second liquid comprises a cleaning medium. The method according to any one of claims 44 to 47, wherein the isolated organ or part thereof contains less than about 1% by weight of cleaning agent after contact of the isolated organ or part thereof with the second liquid. 47. The method of claim 46, wherein after said contact with the second liquid, blood flows through a greater volume of the vasculature of the at least partially decellularized isolated organ or portion thereof compared to a volume of vasculature of an otherwise equivalent at least partially decellularized isolated organ or portion thereof produced using an otherwise equivalent non-degassed second liquid. The method according to any one of claims 11 to 49, further comprising contacting the at least partially decellularized isolated organ or part thereof with a regeneration medium. The method of claim 50, wherein the regeneration medium is an at least partially degassed cell regeneration medium. 52. The method of claim 51, wherein said contact with said at least partially degassed cell regeneration medium forms an at least partially recellularized organ or part thereof. 52. The method of claim 51, wherein the at least partially degassed cell regeneration medium comprises a population of regenerative cells. 52. The method of claim 51, wherein said contact with the at least partially degassed cell regeneration medium results in a greater degree of recellularization than the degree of recellularization of an otherwise comparable at least partially decellularized organ or portion contacted with an otherwise comparable non-degassed cell regeneration medium. An at least partially decellularized harvested organ or part thereof produced by the method according to any one of claims 1 to 49. 55. An at least partially recellularized mammalian isolated organ or part thereof produced by the method of any one of claims 50 to 54. A system comprising: (a) an at least partially decellularized harvested organ or part produced by the method of any one of claims 1 to 49; (b) a device for transferring fluids; (c) a cannula; and (d) a port. The system of claim 57, further comprising a degassed decellularization medium. The system of claim 57, further comprising a degassed cleaning medium. A kit comprising an at least partially decellularized harvested organ or a part thereof produced by the method according to any one of claims 1 to 49 in a container. 61. The kit of claim 60, further comprising: (a) at least one cannula; (b) a liquid; (c) instructions for recellularizing the at least partially decellularized harvested organ or portion thereof; (d) instructions for introducing the at least partially recellularized harvested organ or portion thereof in a subject; or (e) any combination thereof. The kit according to claim 60 or 61, wherein the container is packaging.

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