Mitochondria transplantation for long-term tissue preservation

Abstract

Featured here are methods for improving tissue or organ preservation and utilization through mitochondrial transplantation.

Description

[0001] PATENT ATTORNEY DOCKET NO: 51789-002WO2

[0002] MITOCHONDRIAL TRANSPLANTATION FOR LONG-TERM TISSUE PRESERVATION

[0003] BACKGROUND OF THE INVENTION

[0004] End stage organ failures contribute to around 77,000 deaths annually in the United States and in many cases, there is no treatment other than transplant. To enhance the availability of tissues or organs for transplantation, tissues or organs can be procured from deceased cardiac donors. However, the warm ischemic time, or the period during which blood flow is halted, can cause damage to these tissues or organs, including mitochondrial injury. Mitochondrial damage can also occur during cold storage of organs. Current methods to address mitochondrial damage or injury are limited. Accordingly, there remains a need for exploring approaches for treating mitochondrial dysfunction so that currently discarded organs can be made transplantable and long-term tissue or organ preservation can be achieved.

[0005] SUMMARY OF THE INVENTION

[0006] In one aspect, the disclosure features a method of prolonging survival of tissue, the method comprising introducing into the tissue one or more exogenous mitochondria.

[0007] In another aspect, the disclosure features a method of prolonging survival of a cell, the method comprising introducing into the cell one or more exogenous mitochondria.

[0008] In another aspect, the disclosure features a method of transplanting an isolated tissue into a subject in need of replacement of the tissue, the method comprising: a) introducing, into a cell of the isolated tissue, one or more exogenous mitochondria; b) providing the subject with the tissue resulting from (a) in place of the subject’s endogenous tissue.

[0009] In another aspect, the disclosure features a method of transplanting an isolated tissue into a subject in need of replacement of the tissue, the method comprising providing the subject with the isolated tissue in place of the subject’s endogenous tissue, wherein prior to the providing, one or more exogenous mitochondria have been introduced into a cell of the isolated tissue.

[0010] In some embodiments, the tissue is a liver, a heart, a kidney, or a lung.

[0011] In some embodiments, the tissue is an isolated tissue.

[0012] In some embodiments, the method comprises introducing the exogenous mitochondria ex vivo.

[0013] In some embodiments, the cell is an endothelial cell. In some embodiments, the cell is a liver cell. In some embodiments, the liver cell is a hepatocyte.

[0014] In some embodiments, the tissue is obtained from a donor after cardiac death (DCD).

[0015] In some embodiments, the tissue is characterized by a warm ischemic time (WIT) of greater than 30 minutes. In some embodiments, the tissue is characterized by a WIT of greater than 45 minutes. In some embodiments, the tissue is characterized by a WIT of greater than 60 minutes. In some embodiments, the tissue is characterized by a WIT of from 30 minutes to 72 hours. In some embodiments, the tissue is characterized by a WIT of from 45 minutes to 48 hours. In some embodiments, the tissue is characterized by a WIT of from 60 minutes to 24 hours.

[0016] In some embodiments, the tissue has been subjected to static cold storage or subzero storage prior to the introducing of the one or more exogenous mitochondria. In some embodiments, the static cold storage or subzero storage comprises storage of the tissue at a temperature of about 4° C to about - 80° PATENT ATTORNEY DOCKET NO: 51789-002WO2

[0017] C. In some embodiments, the static cold storage or subzero storage comprises storage of the tissue at a temperature of about 4° C to about - 20° C. In some embodiments, the static cold storage or subzero storage further comprises storage of the tissue at the temperature for at least 24 hours. In some embodiments, the static cold storage or subzero storage comprises storage of the tissue at the temperature for at least 48 hours. In some embodiments, the static cold storage or subzero storage comprises storage of the tissue at the temperature for at least 72 hours. In some embodiments, the static cold storage or subzero storage comprises storage of the tissue at the temperature for at least 96 hours. In some embodiments, the static cold storage or subzero storage comprises storage of the tissue at the temperature for at least 144 hours. In some embodiments, the static cold storage or subzero storage further comprises storage of the tissue at the temperature for from about 24 hours to about 144 hours.

[0018] In some embodiments, subzero storage further comprises storage of the tissue at a temperature of about -196° C (e.g., using liquid nitrogen) for from about 1 month to about 3 years or more (e.g., 1 month, 3 months, 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, or 5 years).

[0019] In some embodiments, the one or more exogenous mitochondria are obtained from a population of cells selected from hepatocytes, hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells, or a combination thereof.

[0020] In some embodiments, the population of cells is of the same species as the cell into which the one or more mitochondria are introduced.

[0021] In some embodiments, the method further comprises providing the isolated tissue to a subject in need of replacement of the tissue. In some embodiments, the tissue is a liver.

[0022] In some embodiments, the subject is diagnosed as having a complication or disease selected from the group consisting of ischemic liver injury, primary non-function (PNF), early allograft dysfunction (EAD), small-for-size syndrome (SFSS), resection for malignant or benign disease, autoimmune hepatitis, cystic fibrosis, cholestatic liver diseases, primary sclerosing cholangitis, primary biliary cirrhosis, inborn errors of metabolism, metabolic diseases, sickle cell hepatopathy, erythropoietic protoporphyria (EPP) hepatopathy, congestive hepatopathy, metabolic dysfunction-associated steatotic liver disease (MAFLD), metabolic dysfunction-associated steatohepatitis (MASH), alcoholic liver disease, hemochromatosis, Alagille syndrome, Wilson's disease, viral hepatitis, alpha-1 antitrypsin deficiency, drug-induced liver injury (DILI), and liver cancer, cancer in the liver, liver metastases, cirrhosis, or a combination thereof.

[0023] In some embodiments, the subject is a human.

[0024] In some embodiments, the exogenous mitochondria are freshly isolated. In some embodiments, the exogenous mitochondria were stored prior to introduction into the tissue or cell. In some embodiments, the exogenous mitochondria are introduced at a dose of 2.5 to 150 pg mitochondria per mL of solution.

[0025] In another aspect, the disclosure features an ex vivo tissue transplant comprising an isolated tissue that comprises one or more heterologous mitochondria. In some embodiments, the tissue is an isolated liver. In some embodiments, the tissue transplant is obtained from a DCD. In some embodiments, the tissue is characterized by a warm ischemic time (WIT) of greater than 30 minutes. In some embodiments, the tissue is characterized by a WIT of greater than 45 minutes. In some embodiments, the tissue is characterized by a WIT of greater than 60 minutes. In some embodiments, the tissue is characterized by a WIT of from 30 minutes to 72 hours. In some embodiments, the tissue is PATENT ATTORNEY DOCKET NO: 51789-002WO2 characterized by a WIT of from 45 minutes to 48 hours. In some embodiments, the tissue is characterized by a WIT of from 60 minutes to 24 hours. In some embodiments, the tissue has been subjected to static cold storage or subzero storage prior to introducing the one or more heterologous mitochondria into the liver. In some embodiments, the one or more exogenous mitochondria are obtained from a population of cells selected from hepatocytes, hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells, or a combination thereof. In some embodiments, the heterologous mitochondria are freshly isolated. In some embodiments, the heterologous mitochondria were stored prior to introduction into the tissue. In some embodiments, the heterologous mitochondria are introduced at a dose of 2.5 to 150 pg per mL of solution.

[0026] BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1A - FIG. 1K show that a mitochondrial transplant improves hepatocyte and liver sinusoidal endothelial cell (LSEC) viability after cold storage. Images of mitochondrial uptake were captured by confocal microscopy (Nikon AXR), donor mitochondria were stained with MitoTrackerRed CMXROS (200 nm), and endogenous mitochondria were dyed with MitoTracker Green (100mM), and excess stain was washed off before imaging. In the image, the endogenous mitochondria appear as shading that fills each liver cell whereas the donor mitochondria appear as small, scattered dots within some of the recipient liver cells. (FIG. 1 A). Cell attachment was substantially increased with mitotherapy (mitochondrial transplantation) in hepatocytes. Images of mitochondrial uptake were captured by brightfield microscopy at 20x objective (FIG. 1 B). Hepatocytes, LSECs, stellate, and Kupffer liver cells were exposed to static cold storage (SCS) for 0, 1 , 3, or 5 days. Metabolic activity by PrestoBlue (Gokduman et al., Nanomedicine (Lond), 2018;13(11 ):1267-1284) was normalized to positive control. From left to right in the bar graph, for every time point, the cells studied are as follows: Hepatocytes, LSECs, stellate, and Kupffer liver cells (FIG. 1 C). ATP luminescence (Fu et al., Proc Natl Acad Sci U S A., 2013;110(18) :7288-93) for hepatocytes, LSECs, stellate, and Kupffer liver cells exposed to SCS for 0, 1 , 3, or 5 days. From left to right in the bar graph, for every time point, the cells studied are as follows: Hepatocytes, LSECs, stellate, and Kupffer liver cells (FIG. 1 D). Caspase 3 / 7 assay (Gramignoli et al., Cell Transplant, 2014;23(12):1545-56) for hepatocytes, LSECs, stellate, and Kupffer liver cells exposed to SCS for 0, 1 , 3, or 5 days. From left to right in the bar graph, for every time point, the cells studied are as follows: Hepatocytes, LSECs, stellate, and Kupffer liver cells (FIG. 1 E). Hepatocytes exposed to SCS for 2 hours (FIG. 1 F - FIG. 1 H). For hepatocytes, metabolic rate (FIG. 1 F) and ATP production (FIG. 1 G) following SCS were higher in the mitochondrial transplant than the negative control. Caspase 3 / 7, markers of cell death, were decreased after mitotherapy (FIG. 1 H). LSECs were cold stored for 0, 1 , or 3 days (FIG. 11 - FIG. 1 K). Normalized metabolic activity, % was increased in LSECs with mitotherapy after both 1 and 3 days of storage (FIG. 11). Normalized ATP levels, % were increased in LSECs with mitotherapy after both 1 and 3 days of storage (FIG. 1 J). Caspase production was decreased in LSECs with mitotherapy after both 1 and 3 days of storage (FIG. 1 K). n > 3 in all figures, error bars = 1 standard deviation.

[0028] FIG. 2A - FIG. 2G show that mitochondria can be transplanted by infusion during machine perfusion and leads to increased oxygen uptake and reduced edema. Schematic of liver perfusion and mitotherapy timing (FIG. 2A). Fluorescence images of mitochondrial transplant liver compared to the negative control demonstrate a qualitative increase of mitochondria uptake within the liver tissue and PATENT ATTORNEY DOCKET NO: 51789-002WO2 distribution of donor mitochondria to the periphery. Donor mitochondria were stained with fluorescent dye before transplantation and followed by a 2-hour machine perfusion to demonstrate persistence of donor mitochondria in tissue. The positive control shows all mitochondria stained, including endogenous mitochondria (FIG. 2B). Lactate consumption was significantly different between vehicle and mitotherapy groups (p=0.001 ) (FIG. 2C). The oxygen consumption was significantly different between the vehicle and mitotherapy groups (p=0.01 ) (FIG. 2F). Resistance followed a similar pattern (p=0.04) (FIG. 2D). Weight gain was below 20% for both groups, which is within expected range for transplantation (dashed line) (FIG. 2E). Potassium outflow (K+(mmol / L)) is shown for both groups (FIG. 2G). n > 4 in all figures, error bars = 1 standard deviation.

[0029] FIG. 3A - FIG. 3F show results of experiments in which mitochondria were transplanted with the purpose of alleviating warm ischemic injury in liver cells. Hepatocytes were exposed to 2 hours of warm ischemic conditions (FIG. 3A - FIG. 3C). ATP production in cells was compared between the no treatment condition and the mitotherapy condition (FIG. 3A). Metabolic activity was unchanged between hepatocytes of the no treatment condition and the mitotherapy condition (FIG. 3B). There was a decrease in caspase levels in hepatocytes treated with mitotherapy (FIG. 3C). LSECs were exposed to 2 hours of warm ischemic conditions (FIG. 3D - FIG. 3F). Slight increase in ATP levels in LSECs with mitotherapy (FIG. 3D). Metabolic activity in LSECs was compared between the no treatment condition and the mitotherapy condition (FIG. 3E). Caspase levels remained the same between groups (FIG. 3F). n > 2 in all figures, error bars = 1 standard deviation.

[0030] FIG. 4A - FIG. 4K show that mitochondria improve whole liver viability after warm ischemia. Livers were stored in warm ischemic conditions for 1 .5 hours (FIG. 4A - FIG. 4F). Lactate outflow during perfusion was compared between the vehicle and mitotherapy groups (FIG. 4A). Portal vein resistance was compared between the vehicle and mitotherapy groups (FIG. 4B). A substantial decrease in weight gain was observed in the mitotherapy group (FIG. 4C). An increase in oxygen uptake was observed in the mitotherapy group (FIG. 4D). A decrease in potassium outflow was observed in the mitotherapy group (FIG. 4E). Level of alanine transaminase (ALT), a liver injury marker, decreased in the mitotherapy group (FIG. 4F). Livers were stored in warm ischemic conditions for 2 hours (FIG. 4G - FIG. 4K). Lactate outflow during perfusion was compared between the vehicle and mitotherapy groups (FIG. 4G). Portal vein resistance was significantly decreased in the mitotherapy group (FIG. 4H). % Weight gain was above the transplant criteria for both groups (FIG. 4I). Oxygen uptake during perfusion was compared between the vehicle and mitotherapy groups (FIG. 4J). Potassium outflow slightly decreased in the mitotherapy group (FIG. 4K). n > 3 in all figures, error bars = 1 standard deviation.

[0031] FIG. 5A - FIG. 5D show mitochondrial isolation protocol design followed by results of isolated mitochondria characterization. Mitochondria were isolated from whole liver through a series of homogenization, centrifugation, and filtration (FIG. 5A). Mitochondrial respiratory health was tested using the gold standard technique, Mitochondrial Stress test (Seahorse, Agilent Technologies) to measure the oxygen consumption rate (OCR, pmol 02 / min) (FIG. 5B). As shown in the table in FIG. 5C, the respiratory profile was calculated. Ideal respiratory control ratio is between 4-5, which the mitochondria fell into. Electron microscopy was used to determine the structural integrity of the mitochondria. Both outer and inner membranes were observed as intact (FIG. 5D). PATENT ATTORNEY DOCKET NO: 51789-002WO2

[0032] FIG. 6A - FIG. 6D show mitochondrial uptake. Images of mitochondrial uptake were captured by fluorescent microscopy at 20x objective with stained donor mitochondria. Low dose=30 pg mitochondrial protein / well. High dose = 300pg mitochondrial protein / well (FIG. 6A). Mitochondrial uptake was quantified via plate reader fluorescence (Ex 579 / Em 599) before and after co-incubation. There was a significant increase in mitochondrial uptake during co-incubation over 90 minutes (p<0.0001 ), following a linear trend (FIG. 6B). Mitochondrial uptake was significantly impacted by temperature, with significantly reduced uptake at 4°C (p<0.01 ), which is the typical condition for whole organ SCS (FIG. 6C). Cells treated with the low dose (1 x) had 14% of the uptake of the high dose (10x) (FIG. 6D). FIG. 6D is a quantification of the fluorescence images of mitochondrial uptake shown in FIG. 6A.

[0033] DEFINITIONS

[0034] As used herein, the term “exogenous mitochondria” refers to any mitochondria that is not native to a cell, tissue, or organ in the body. Mitochondria can be isolated from a cell, tissue, or organ and then transferred into another cell, tissue, or organ in the same subject or in a different subject, thus, the mitochondria are exogenous in nature as they are not native to the tissue into which they are being transferred. In some embodiments, the exogenous mitochondria can be from a human source. In some embodiments, the exogenous mitochondria can be from a non-human mammalian source (e.g., porcine, monkey, bovine etc.). In some embodiments, the exogenous mitochondria can be obtained from a population of cells selected from hepatocytes, hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells, or a combination thereof. In some embodiments, the exogenous mitochondria can be obtained from skeletal muscles (e.g., rectus abdominis muscle, pectoralis major muscle, gastrocnemius muscle), fibroblasts, platelets, liver, or cardiac muscles. In some embodiments, the exogenous mitochondria can be mitochondria secreted extracellularly by many cell types, including mesenchymal stem cells (MSCs), astrocytes, neural stem cells, induced pluripotent stem cells (iPSCs), platelets, adipocytes, hepatocytes, cardiomyocytes, endothelial progenitor cells, osteoblasts, and various cell lines.

[0035] As used herein, the term “heterologous” refers to any biological product (for example, protein, organelle, ceil, tissue, or organ) that is derived from a different individual. In some embodiments, the mitochondria are heterologous mitochondria. In some embodiments, the heterologous mitochondria can be from a non-human mammalian source (e.g., porcine, monkey, bovine etc.) and can be transplanted into the cell, tissue, or organ of a human or a non-human mammal other than porcine.

[0036] As used herein, the term “isolated tissue” refers to a tissue or an organ that has been removed or isolated from the body of a subject and is present outside the body. A tissue or an organ (for example, heart, liver, kidney, lung, eye, muscle, brain tissue, skin etc.) can be procured from one individual and then transplanted into another individual. Once procured and before transplant, the tissue or organ is an isolated tissue or isolated organ.

[0037] As used herein, the term “isolated liver” refers to a liver that has been removed or isolated from the body of a subject and is present outside the body. A liver can be procured from one individual and then transplanted into another individual. Once procured and before transplant, the liver is an isolated liver.

[0038] As used herein, the term “prolonging survival” refers to increasing the length of time an isolated cell, tissue, or organ, which has been transplanted with exogenous mitochondria at any point between PATENT ATTORNEY DOCKET NO: 51789-002WO2 procurement and post-transplant into the recipient, will survive relative to an isolated cell, tissue, or organ that has not been transplanted with exogenous mitochondria. In some embodiments, the cell is an endothelial cell. In some embodiments, the cell is a liver cell. In some embodiments, the liver cell is a hepatocyte. In some embodiments, the tissue or organ is heart, liver, kidney, lung, eye, muscle, brain tissue, or skin.

[0039] As used herein, the term “subzero storage” refers to storage at a temperature below 0 °C. Subzero storage at a very low temperature, e.g., -196 °C (the temperature of liquid nitrogen), can result in cryopreservation or vitrification of a tissue or cell. As such, subzero storage may encompass supercooling, partial freezing cryopreservation, and vitrification techniques known to those skilled in the art.

[0040] As used herein, the term “xenotransplantation” refers to transplantation of a cell, tissue, or organ from one species to another.

[0041] DETAILED DESCRIPTION

[0042] The disclosure is directed towards advancing the therapeutic potential of mitochondria for tissue or organ (e.g., heart, liver, kidney, lung, eye, muscle, brain tissue, or skin) transplantation using both in vitro and in vivo models of liver injury, improving the utilization of extended warm ischemia tissues or organs, and extending the limits of tissue or organ preservation.

[0043] End stage organ failures contribute to several deaths annually in the United States and in many cases, there is no treatment other than transplant. For example, end stage liver disease contributes to around 77,000 deaths annually in the United States (Asrani et al., J Hepatol., 2019; 70(1 ):151 -171 ). There is no treatment other than transplant. While thousands of transplants take place every year in the US, at the end of 2018, 12,820 patients were still active on the waiting list for liver transplants (Kwong et al., Am J Transplant., 2020;20 Suppl s1 :193-299). To enhance the availability of tissues or organs for transplantation, many transplant centers are increasingly turning to extended criteria donor organs. One significant, yet underutilized group is deceased cardiac donors, also known as donors after cardiac death (DCD). However, the warm ischemic time, or the period during which blood flow is halted, can cause damage to these tissues or organs. For most transplant centers within the US, the current clinical maximum for warm ischemic time is >30 minutes for liver donation (Paterno et al., Liver Transpl., 2019;25(9):1342-1352). The main course of the ischemia-reperfusion cascade is facilitated by mitochondrial injury, but there is a significant gap in our knowledge about how to treat the injury to mitochondria, preventing the use of a large donor organ pool (Horvath et al., Int J Mol Sci . , 2021 ;22(6)) . Beyond sourcing tissues or organs, maintaining and distributing tissues or organs is also limited. The current method for storing and transporting organs is to place them in storage bags with preservation fluid on ice in a cooler. Historically, the time limit for cold storage of livers has been 12 hours. Extended criteria livers have a higher level of injury, which further limits how much preservation damage they can sustain (Maggi et al., Transplant Proc., 2014;46(7):2295-9). The ability to increase storage time or rescue suboptimal livers through advanced ex vivo treatment may increase the number of livers that are able to be transplanted. Mitochondrial damage during cold storage has been well documented to leading to decreased respiration and higher levels of cell death (Horvath et al., Int J Mol Sci., 2021 ;22(6); Vajdova et al., Hepatology, 2002;36(6):1543-52). PATENT ATTORNEY DOCKET NO: 51789-002WO2

[0044] Current methods to address mitochondrial damage are limited (Martins et al., Int J Med Sci., 2018;15(3):248-256; Saeb-Parsy et al., Trends Mol Med., 2021 ;27(2):185-198; Xu etal., Front Pharmacol., 2021 ;12:796207) and the critical and lasting damage to native mitochondria may not be recovered by such methods. Multiple studies, have demonstrated that recovery with machine perfusion (MP) improves extended criteria in animal models and human liver utilization, however, there is a limit to the extent of repair that MP alone can achieve (Bruinsma et al., Am J Transplant., 2014;14(6) :1400-9; Schon etal., Ann Surg., 2001 ;233(1 ):114-23; Tolboom etal., Transplantation, 2009;87(2):170-7; Tolboom et al., J Surg Res., 2012;175(1 ):149-56).

[0045] An innovative approach to treating mitochondrial dysfunction is using live, isolated mitochondrial transplantation. Treatment with exogenous mitochondria may be a more effective route to addressing mitochondrial damage and rescue widespread mitophagy. Mitochondrial transplant may rescue the mitochondrial network after the structural and functional damage induced by cold storage. The isolated cell, tissue, or organ can be transplanted with exogenous mitochondria at any point between procurement and post-transplant into the recipient. Exogenous mitochondria can be transplanted into the isolated cell, tissue, or organ after it has been removed or isolated from the donor. In some embodiments, the isolated cell, tissue, or organ can be transplanted with exogenous mitochondria post-transplant into the recipient. Alternatively, the donor cell, tissue, or organ can be transfused with exogenous mitochondria while the cell, tissue, or organ is still in the donor, that is, while the cell, tissue, or organ is still inside the body of the donor. The donor cell, tissue, or organ into which exogenous mitochondria has been introduced can be transplanted into an individual of the same species or the donor cell, tissue, or organ can be xenotransplanted, for example, the donor cell, tissue, or organ from a porcine, bovine, or monkey can be transplanted into a human. Mitochondrial transfer is a stress-relieving rescue mechanism to improve ATP content, decrease apoptosis, promote repair, and decrease oxidative stress (Hayakawa etal., Nature, 2016;535(7613):551 -555; Islam et al., Nat Med., 2012;18(5):759-65) and has improved ischemic damage in complex systems of hearts, kidneys, and livers in animal models (Blitzer et al., Ann Thorac Surg., 2020;109(3):711 -719; Jabbari et al., Biochim Biophys Acta Mol Basis Dis., 2020;1866(8):165809; Ko et al., J Cell Mol Med., 2020;24(17):10088-10099), and a pediatric clinical trial for cardiogenic shock (Guariento et al., The Journal of Thoracic and Cardiovascular Surgery, 2021 ;162(3):992-1001 ).

[0046] A tissue or an organ (for example, heart, liver, kidney, lung, eye, muscle, brain tissue, skin, etc.) can be procured from one individual and then transplanted into another individual. Once procured and before transplant, the tissue or organ is an isolated tissue or isolated organ. In some embodiments, the isolated tissue is obtained from a DCD. In isolated tissues, mitochondrial injury or other damage can happen during the warm ischemic time (WIT), or the period during which blood flow is halted. In some embodiments, the isolated tissue is characterized by a WIT of greater than 30 minutes. In some embodiments, the isolated tissue is characterized by a WIT of greater than 45 minutes. In some embodiments, the isolated tissue is characterized by a WIT of greater than 60 minutes. In some embodiments, the isolated tissue is characterized by a WIT of from 30 minutes to 72 hours. In some embodiments, the isolated tissue is characterized by a WIT of from 45 minutes to 48 hours. In some embodiments, the isolated tissue is characterized by a WIT of from 60 minutes to 24 hours. In some embodiments, the isolated tissue is characterized by a WIT of greater than 72 hours. In some embodiments, the isolated tissue is characterized by a WIT of from 72 hours to 96 hours. In some PATENT

[0047] ATTORNEY DOCKET NO: 51789-002WO2 embodiments, the isolated tissue is characterized by a WIT of from 96 hours to 120 hours. In some embodiments, the isolated tissue is characterized by a WIT of from 120 hours to 144 hours. In some embodiments, the isolated tissue is characterized by a WIT of from 30 minutes to 144 hours.

[0048] The isolated tissue can be subjected to static cold storage or subzero storage prior to the introducing of the one or more exogenous mitochondria. In some embodiments, the static cold storage or subzero storage comprises storage of the isolated tissue at a temperature of about 4° C to about - 196° C (the temperature of liquid nitrogen). In some embodiments, the static cold storage or subzero storage comprises storage of the isolated tissue at a temperature of about 4° C to about - 80° C. In some embodiments, the static cold storage or subzero storage comprises storage of the isolated tissue at a temperature of about 4° C to about - 20° C. In some embodiments, the static cold storage or subzero storage further comprises storage of the isolated tissue at the temperature for at least 24 hours. In some embodiments, the static cold storage or subzero storage comprises storage of the isolated tissue at the temperature for at least 48 hours. In some embodiments, the static cold storage or subzero storage comprises storage of the isolated tissue at the temperature for at least 72 hours. In some embodiments, the static cold storage or subzero storage comprises storage of the isolated tissue at the temperature for at least 96 hours. In some embodiments, the static cold storage or subzero storage comprises storage of the isolated tissue at the temperature for at least 144 hours. In some embodiments, the static cold storage or subzero storage further comprises storage of the isolated tissue at the temperature for from about 24 hours to about 144 hours.

[0049] In some embodiments, the subzero storage further comprises storage of the isolated tissue at - 196° C (e.g., using liquid nitrogen) and may encompass supercooling, partial freezing cryopreservation or vitrification. In some embodiments, the subzero storage further comprises storage of the isolated tissue at - 196° C for at least 1 month. In some embodiments, the subzero storage further comprises storage of the isolated tissue at - 196° C for at least 3 months. In some embodiments, the subzero storage further comprises storage of the isolated tissue at - 196° C for at least 6 months. In some embodiments, the subzero storage further comprises storage of the isolated tissue at - 196° C for at least 9 months. In some embodiments, the subzero storage further comprises storage of the isolated tissue at - 196° C for at least 1 year. In some embodiments, the subzero storage further comprises storage of the isolated tissue at - 196° C for at least 2 years. In some embodiments, the subzero storage further comprises storage of the isolated tissue at - 196° C for at least 3 years. In some embodiments, the subzero storage further comprises storage of the isolated tissue at - 196° C for about 1 month to about 3 years or more.

[0050] The currently disclosed study was performed to investigate the promise of mitochondrial transplant as a targeted option for mitochondria-driven pathologies. The objective of this study was to advance the therapeutic potential of mitochondria for tissue or organ (e.g., liver, heart, kidney, lung, eye, muscle, brain tissue, or skin) transplantation using both in vitro and in vivo models of liver injury, to improve the utilization of extended warm ischemia organs, and to extend the limits of tissue or organ preservation.

[0051] Beyond liver transplantation, mitochondrial transplantation can also be useful for heart, kidney, and lung transplantation. Further, successful mitochondrial transplantation would inform the ischemia reperfusion field, and help develop mitochondrial therapy applications to a wide range of conditions from aging to muscular dystrophy, blindness (Ng and Turnbull; J Neurol., 2016;263:179-191 ), and acute PATENT ATTORNEY DOCKET NO: 51789-002WO2 conditions such as stroke (Hayakawa et al., Nature, 2016;535(7613):551 -555), and traumatic brain injury (Kong et al., Mil Med Res., 2022;9:2), among others.

[0052] Source of mitochondria

[0053] Mitochondria can be isolated from a cell, tissue, or organ and then transferred into another cell, tissue, or organ in the same subject or in a different subject, thus, the mitochondria are exogenous in nature as they are not native to the tissue into which they are being transferred. In some embodiments, the exogenous mitochondria can be from a human source. In some embodiments, the exogenous mitochondria can be from a non-human mammalian source (e.g., porcine, monkey, bovine etc.). In some embodiments, the exogenous mitochondria can be obtained from a population of cells selected from hepatocytes, hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells, or a combination thereof. In some embodiments, the exogenous mitochondria can be obtained from skeletal muscles (e.g., rectus abdominis muscle, pectoralis major muscle, gastrocnemius muscle), fibroblasts, platelets, liver, or cardiac muscles. In some embodiments, the exogenous mitochondria can be mitochondria secreted extracellularly by many cell types, including mesenchymal stem cells (MSCs), astrocytes, neural stem cells, platelets, adipocytes, hepatocytes, cardiomyocytes, endothelial progenitor cells, osteoblasts, and various cell lines.

[0054] In some embodiments, the source of the mitochondria is the same as that of the source of the organ that will be transplanted. In some embodiments, the source of the mitochondria is independent of the source of the organ that will be transplanted. In some embodiments, the source of the mitochondria is independent of the source of the liver that will be transplanted. In some embodiments, the mitochondria can be isolated from the organ donor. In some embodiments, the mitochondria can be isolated from the organ donor’s muscle. In some embodiments, the mitochondria can be isolated from the organ donor’s liver. In some embodiments, the mitochondria can be isolated from the recipient. In some embodiments, the mitochondria can be isolated from the recipient’s liver. In some embodiments, the mitochondria can be isolated from the recipient’s muscle. In some embodiments, the mitochondria can be isolated from an individual who is neither the donor nor the recipient. In some embodiments, the mitochondria can be isolated from a different species and transplanted into a human’s isolated issue. In some embodiments, the mitochondria can be isolated from a pig or a monkey. In some embodiments, healthy mitochondria can be isolated from damaged tissue. In some embodiments, healthy mitochondria can be isolated from healthy tissue. In some embodiments, the mitochondria can be freshly isolated before transplant. In some embodiments, the mitochondria can be isolated and stored before transplant.

[0055] EXAMPLES

[0056] The following example is put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used and evaluated and is intended to be purely exemplary and is not intended to limit the scope of the disclosure.

[0057] Example 1. Investigation of the impact of mitochondrial transplantation on long-term liver preservation PATENT ATTORNEY DOCKET NO: 51789-002WO2

[0058] The aim of this study was to investigate the impact of mitochondrial transplantation on long-term liver preservation.

[0059] METHODS

[0060] Mitochondrial isolation and evaluation

[0061] Mitochondria were isolated from 200-300 mg of Lewis rat liver tissue and resuspended in isolation solution [210 mannitol, 70 mM sucrose, 10 mM K-HEPES, and 1 mM K-EGTA at pH 7.3 KOH, in DI water] (Preble et al., Journal of Visualized Experiments : JoVE, 2014(91 ):51682). The liver was sectioned into 50 mg portions and minced with a razor while in isolation solution. Each portion was homogenized in 5 mL of fresh isolation solution using a gentle MACS dissociator in C tubes (Miltenyi Biotec) and combined in 50 mL conicals with a final concentration of 5% BSA, added after homogenization to avoid froth. Next, this homogenate was centrifuged for 10 minutes at 800 g. Supernatant was strained through subsequent 40 urn, 40 urn, and 10 urn filters in 50 mL conicals. Next, the solution was spun down at 5,500 g for 10 minutes in 15 mL conicals. The fluffy part of the pellet was removed by careful mixing using a 1 mL pipet approximately 1 cm above the pellet, for 5 cycles, before aspiration of waste and replacement with 5 mL of fresh solution. A final centrifugation was performed at 5,500 g for 5 minutes. The crude mitochondrial pellet was resuspended in 1 mL of mitochondrial isolation solution. Total mitochondrial protein content was determined using a simple Bradford assay. Oxygen consumption ratio (OCR) and respiratory complex activity of donor mitochondria were analyzed using a Seahorse XF Mini Analyzer (Divakaruni et al., Current Protocols in Toxicology, 2014;60(1 ):25.2.1 - 25.2.16). Transmission electron microscopy was used to evaluate the ultrastructure of mitochondria as additional quality control (Franko et al., PLoS One., 2013;8(12):e82392).

[0062] Establishment of static cold storage (SCS) model and mitotherapy in vitro

[0063] Primary hepatocytes, liver sinusoidal endothelial cells (LSECs), stellate, and Kupffer cells were isolated from Lewis rats. Cells were kept at 37°C, 5% CO2 for 24 hours after seeding. Then, media was replaced with University of Wisconsin (UW) solution for SCS and cells were kept at 4°C for 24 hours. Cells were recovered with William’s E medium (WE) for hepatocytes, endothelial growth medium-2 (EGM2) for LSECs, and DMEM supplemented with 10% fetal bovine serum (FBS) for stellate and Kupffer cells and kept in 37°C, 5% CO2 for 24 hours before assays were performed. To transplant mitochondria to cells directly after rewarming, mitochondria were diluted to a concentration of 300 pg / mL in the cell’s respective media and co-incubated with the cells for 90 minutes followed by several wash steps with PBS and a final cell respective media (Caicedo et al., 2015). The vehicle control was mitochondria isolation solution added in same volume as mitochondria.

[0064] Establishment of warm ischemic model and mitotherapy in vitro

[0065] Cell media was replaced with PBS to deprive them of glucose. Then, the cells were placed in a sealed cell culture hypoxia chamber, and atmospheric gas was replaced with nitrogen gas for oxygen deprivation. Initially cells were incubated at 37°C for 2 hours. Recovery was completed with immediate mitochondrial treatment, or no treatment, for 90 minutes as described above, before a final media change and incubation in 37°C, 5% CO2. PATENT

[0066] ATTORNEY DOCKET NO: 51789-002WO2

[0067] Measurement of donor mitochondria uptake into cells

[0068] Mitochondria were incubated with 100 nM MitoTracker CMXRos fluorescent dye for 10 minutes at room temperature. Then, mitochondria were spun out of the solution at 5,500 g for 10 minutes before being resuspended in the cell’s respective media. Mitochondria were transplanted as described above. To visualize cell uptake, we performed fluorescent microscopy at 20x objective.

[0069] Establishment of SCS liver model and mitotherapy to livers during machine perfusion

[0070] The ex vivo perfusion protocol has been previously described (Tessier et al., Nat Commun., 2022;13(1 ):4008) in brief. Livers were procured from rats and the portal vein was cannulated. Directly after procurement, the liver was flushed with cold University of Wisconsin (UW) solution and placed in a culture dish in cold UW. On ice, the liver was transferred to a 4°C refrigerator for 72 hours covered in UW. After storage, the liver was then transferred to the perfusion basin that was warmed to 37°C with a WE- based media. To remove the UW solution, the livers were flushed with 30 mL of room temperature WE medium before attachment to the perfusion system. The portal vein cannula, previously secured in place, was attached to the inflow tubing and perfusion was initiated. A mechanical rotor pump was used to circulate the perfusing fluid. In the mitochondrial transplant group (n=4), donor mitochondria were injected directly into the portal vein cannula after 30 minutes of machine perfusion at a concentration of 1 x 109mitochondria per gram wet weight of tissue, an amount chosen based on previous groups evaluations (Orfany et al., J Vase Surg., 2020;71 (3):1014-1026). The negative control group (n=4) received 1 mL of vehicle. Perfusion was run for 4 hours and followed by end-point assessments.

[0071] Establishment of warm ischemic liver model and mitotherapy during machine perfusion

[0072] Livers were procured and flushed with room temperature PBS. They were placed in small plastic bags filled with PBS before being placed in a 37°C water bath for 1 .5 or 2 hours. Mitochondria were delivered as a post-warm ischemia (post-WI) treatment through the portal vein, as described above. The perfusion was run for 4 hours and followed by end-point assessments.

[0073] Measurement of perfusion metrics

[0074] Perfusate was collected every 30 minutes to be measured through blood gas analysis (Blood Gas Analyzer 500, Siemens) and for alanine transaminase / aspartate aminotransferase (ALT / AST) (Piccolo Xpress, Abaxis). Weight change was tracked before and after machine perfusion to monitor edema. Pressure through the portal vein was measured using a pressure monitor in line with the portal vein, which was later used to calculate resistance through the liver. At the end of perfusion, liver tissue biopsies were snap frozen for NMR to evaluate metabolite content and tissue biopsies were collected in formalin for histopathological assessment (H&E, Masson’s trichrome, TUNEL).

[0075] Measurement of donor mitochondria uptake into whole liver

[0076] Mitochondria were delivered in 1 mL of vehicle followed by two hours of machine perfusion at 37°C. The positive control group was flushed with 30 mL of 200 nm MitoTracker Red CMXRos which was allowed to incubate for 20 minutes before a 30 mL flush of saline. The negative control group was flushed with 30 mL of saline. To measure distribution of the mitochondrial injection, the fluorescence intensity of PATENT

[0077] ATTORNEY DOCKET NO: 51789-002WO2 donor mitochondria was imaged by the SPECTRAL AMI imaging system (Spectral Instruments) by the Mouse Imaging Program at MGH. Donor mitochondria were stained by 100 nm MitoTracker Red CMXRos.

[0078] Statistical analysis

[0079] All statistical analyses were performed using GraphPad Prism. The significance of differences was evaluated via tandem n-way ANOVA which accounts for interactions between independent variables such as cold storage duration and mitochondria treatment type and multi-way repeated measures ANOVA (MANOVA, which enables simultaneous consideration of multiple dependent variables, such as ATP and ROS levels). p<0.05 was used for significance unless otherwise noted.

[0080] RESULTS

[0081] Mitotherapy improves metabolic function in primary liver cells

[0082] Donor mitochondria were stained with MitoTrackerRed CMXROS for detection, with staining demonstrating successful uptake (FIG. 1 A). It has been previously demonstrated that after 24 hours of hypothermic storage in UW, rat hepatocytes display high viability one hour after rewarming, however, viability drops significantly during the first day of long-term culture with significant cell death at 3 days (Usta et al., PLoS One., 2013;8(7):e69334). The damage from hypothermic storage was quantified in additional liver cell types, with findings of universally decreased metabolic activity, ATP levels, and increased caspase expression, though each cell type had a varied injury profile (FIG. 1 C - FIG. 1 E). Hepatocytes were targeted as they are the majority cell type in livers and have clear damage with increasing cold storage. At 24 hours post-rewarming, hepatocyte viability dropped past 50% below fresh, healthy controls (FIG. 1 C - FIG. 1 E). Mitochondrial transplant substantially improved cell metabolic rate following SCS (FIG. 1 F - FIG. 1 H) and gross morphology (FIG. 1 B) compared to the negative control. Surprisingly, the cells were able to recover enough to match the metabolic rate of the positive control, which were kept in standard culture conditions throughout the experiment. Mitochondrial transplantation improved cell survival and attachment. A marked improvement in cell attachment was observed with mitochondrial transplant, demonstrated by increased density of cells (FIG. 1 B). Liver sinusoidal endothelial cells (LSECs) were determined as a critical group as they also displayed significant damage, including much higher caspase levels indicating substantial cell death (FIG. 1 E and FIG. 1 K). With mitotherapy, significant decrease of caspase activity was seen following cold storage (FIG. 1 K). An increase in metabolic activity and ATP production was also seen as significant (FIG. 1 1 - FIG. 1 J).

[0083] FIG. 1A - FIG. 1K show that a mitochondrial transplant improves hepatocyte and LSEC viability after cold storage. Images of mitochondrial uptake were captured by confocal microscopy (Nikon AXR), donor mitochondria were stained with MitoTrackerRed CMXROS (200 nm), and endogenous mitochondria were dyed with MitoTracker Green (1 OOmM), and excess stain was washed off before imaging. In the image, the endogenous mitochondria appear as shading that fills each liver cell whereas the donor mitochondria appear as small, scattered dots within some of the recipient liver cells. (FIG. 1 A). Cell attachment was substantially increased with mitotherapy in hepatocytes. Images of mitochondrial uptake were captured by brightfield microscopy at 20x objective (FIG. 1 B). Hepatocytes, LSECs, stellate, and Kupffer liver cells were exposed to static cold storage (SCS) for 0, 1 , 3, or 5 days. Metabolic activity PATENT

[0084] ATTORNEY DOCKET NO: 51789-002WO2 by PrestoBlue (Gokduman et al., Nanomedicine (Lond), 2018;13(11 ):1267-1284) was normalized to positive control. From left to right in the bar graph, for every time point, the cells studied are as follows: Hepatocytes, LSECs, stellate, and Kupffer liver cells (FIG. 1 C). ATP luminescence (Fu et al., Proc Natl Acad Sci U S A., 2013;110(18):7288-93) for hepatocytes, LSECs, stellate, and Kupffer liver cells exposed to SCS for 0, 1 , 3, or 5 days. From left to right in the bar graph, for every time point, the cells studied are as follows: Hepatocytes, LSECs, stellate, and Kupffer liver cells (FIG. 1 D). Caspase 3 / 7 assay (Gramignoli et al., Cell Transplant, 2014;23(12):1545-56) for hepatocytes, LSECs, stellate, and Kupffer liver cells exposed to SCS for 0, 1 , 3, or 5 days. From left to right in the bar graph, for every time point, the cells studied are as follows: Hepatocytes, LSECs, stellate, and Kupffer liver cells (FIG. 1 E). Hepatocytes exposed to SCS for 2 hours (FIG. 1 F - FIG. 1 H). For hepatocytes, metabolic rate (FIG. 1 F) and ATP production (FIG. 1 G) following SCS were higher in the mitochondrial transplant than the negative control. Caspase 3 / 7, markers of cell death, were decreased after mitotherapy (FIG. 1 H). LSECs were cold stored for 0, 1 , or 3 days (FIG. 11 - FIG. 1 K). Normalized metabolic activity, % was increased in LSECs with mitotherapy after both 1 and 3 days of storage (FIG. 11). Normalized ATP levels, % were increased in LSECs with mitotherapy after both 1 and 3 days of storage (FIG. 1 J). Caspase production was decreased in LSECs with mitotherapy after both 1 and 3 days of storage (FIG. 1 K). n > 3 in all figures, error bars = 1 standard deviation.

[0085] Mitotherapy alleviates cold storage injury in rat livers during ex vivo machine perfusion

[0086] Based on the success with hepatocytes, a preliminary assessment of mitotherapy was performed on rat liver stored for 72 hours in UW at 4°C and recovered during machine perfusion (5 hours, 37°C) (FIG. 2A). A clear qualitative increase in fluorescence intensity was observed from the negative control to the mitochondrial transplant group, demonstrating successful distribution across the liver (FIG. 2B). Following SCS conditions, livers that received mitochondrial transplant (n=4) were considerably improved overall compared to the negative control (n=4). Higher lactate production was seen in the mitotherapy group (FIG. 2C), which is the expected course of liver recovery from ischemia-reperfusion injury, which appears complete at recovering by five hours. These results also mimiced the lactate findings in a recent study of mitotherapy for renal injury in a DCD model (Rossi etal., Ann Surg., 2023). Mitotherapy demonstrated effects in an indirect metric of metabolic rate (oxygen consumption) (FIG. 2F) and resistance (FIG. 2D). Edema (weight gain) was below 20% for both groups, which is within acceptable limits according to transplantation criteria indicating minimized risk of complications related to excess weight gain post-transplant (FIG. 2E).

[0087] FIG. 2A - FIG. 2G show that mitochondria can be transplanted by infusion during machine perfusion and leads to increased oxygen uptake and reduced edema. Schematic of liver perfusion and mitotherapy timing (FIG. 2A). Fluorescence images of mitochondrial transplant liver compared to the negative control demonstrate a qualitative increase of mitochondria uptake within the liver tissue and distribution of donor mitochondria to the periphery. Donor mitochondria were stained with fluorescent dye before transplantation and followed by a 2-hour machine perfusion to demonstrate persistence of donor mitochondria in tissue. The positive control shows all mitochondria stained, including endogenous mitochondria (FIG. 2B). Lactate consumption was significantly different between vehicle and mitotherapy groups (p=0.001 ) (FIG. 2C). The oxygen consumption was significantly different between the vehicle and PATENT

[0088] ATTORNEY DOCKET NO: 51789-002WO2 mitotherapy groups (p=0.01 ) (FIG. 2F). Resistance followed a similar pattern (p=0.04) (FIG. 2D). Weight gain was below 20% for both groups, which is within expected range for transplantation (dashed line) (FIG. 2E). Potassium outflow (K+(mmol / L)) is shown for both groups (FIG. 2G). n > 4 in all figures, error bars = 1 standard deviation.

[0089] Mitotherapy improves metabolic function in primary liver cells

[0090] While metabolic activity (FIG. 3B) and ATP production (FIG. 3A) in hepatocytes were similar regardless of mitotherapy treatment, markers of cell death decreased in hepatocytes that received mitotherapy after two hours of warm ischemia (FIG. 3C). In contrast, while LSECs exhibited a slight increase in ATP levels (FIG. 3D), their cell death activity remained unchanged (FIG. 3F).

[0091] FIG. 3A - FIG. 3F show results of experiments in which mitochondria were transplanted with the purpose of alleviating warm ischemic injury in liver cells. Hepatocytes were exposed to 2 hours of warm ischemic conditions (FIG. 3A - FIG. 3C). ATP production in cells was compared between the no treatment condition and the mitotherapy condition (FIG. 3A). Metabolic activity was unchanged between hepatocytes of the no treatment condition and the mitotherapy condition (FIG. 3B). There was a decrease in caspase levels in hepatocytes treated with mitotherapy (FIG. 3C). LSECs were exposed to 2 hours of warm ischemic conditions (FIG. 3D - FIG. 3F). Slight increase in ATP levels in LSECs with mitotherapy (FIG. 3D). Metabolic activity in LSECs was compared between the no treatment condition and the mitotherapy condition (FIG. 3E). Caspase levels remained the same between groups (FIG. 3F). n > 2 in all figures, error bars = 1 standard deviation.

[0092] Mitotherapy alleviates warm ischemic injury in rat livers during ex vivo machine perfusion

[0093] Prior studies indicate that 30 mins is the current limit of warm ischemic time for liver transplant (Paterno et al., Liver Transpl., 2019;25(9):1342-1352), extended to 60 mins if followed by oxygenated machine perfusion (Schon et al., Ann Surg., 2001 ;233(1 ):114-23; Tolboom et al., Transplantation, 2009;87(2):170-7; Tolboom etal., J Surg Res., 2012;175(1 ):149-56). An attempt was made to extend these limits in rat livers exposed to 1 .5 hours and 2 hours of warm ischemic conditions. There were no significant differences in livers treated with mitotherapy or no treatment, however there was a substantial difference in weight gain, used to measure edema, which brought the mitotherapy group below the target threshold for transplantation, 20% (FIG. 4C). There were significant differences in livers treated with mitotherapy in portal vein resistance (p=0.0144) after 2 hours of warm ischemia (FIG. 4H).

[0094] FIG. 4A - FIG. 4K show that mitochondria improve whole liver viability after warm ischemia. Livers were stored in warm ischemic conditions for 1 .5 hours (FIG. 4A - FIG. 4F). Lactate outflow during perfusion was compared between the vehicle and mitotherapy groups (FIG. 4A). Portal vein resistance was compared between the vehicle and mitotherapy groups (FIG. 4B). A substantial decrease in weight gain was observed in the mitotherapy group (FIG. 4C). An increase in oxygen uptake was observed in the mitotherapy group (FIG. 4D). A decrease in potassium outflow was observed in the mitotherapy group (FIG. 4E). Level of alanine transaminase (ALT), a liver injury marker, decreased in the mitotherapy group (FIG. 4F). Livers were stored in warm ischemic conditions for 2 hours (FIG. 4G - FIG. 4K). Lactate outflow during perfusion was compared between the vehicle and mitotherapy groups (FIG. 4G). Portal vein resistance was significantly decreased in the mitotherapy group (FIG. 4H). % Weight gain was above the transplant criteria for both groups (FIG. 4I). Oxygen uptake during perfusion was compared between the PATENT ATTORNEY DOCKET NO: 51789-002WO2 vehicle and mitotherapy groups (FIG. 4J). Potassium outflow slightly decreased in the mitotherapy group (FIG. 4K). n > 3 in all figures, error bars = 1 standard deviation.

[0095] Mitochondria isolation, characterization, and uptake

[0096] Isolated mitochondria from whole Lewis rat livers were evaluated via respirometry and electron microscopy (FIG. 5A - FIG. 5D). Donor mitochondria were stained with MitoTrackerRed CMXROS for detection, with staining demonstrating successful uptake (FIG. 1 A) and dose-dependence of donor mitochondria incorporation into the cells (FIG. 6A).

[0097] FIG. 5A - FIG. 5D show mitochondrial isolation protocol design followed by results of isolated mitochondria characterization. Mitochondria were isolated from whole liver through a series of homogenization, centrifugation, and filtration (FIG. 5A). Mitochondrial respiratory health was tested using the gold standard technique, Mitochondrial Stress test (Seahorse, Agilent Technologies) to measure the oxygen consumption rate (OCR, pmol 02 / min) (FIG. 5B). As shown in the table in FIG. 5C, the respiratory profile was calculated. Ideal respiratory control ratio is between 4-5, which the mitochondria fell into. Electron microscopy was used to determine the structural integrity of the mitochondria. Both outer and inner membranes were observed as intact (FIG. 5D).

[0098] FIG. 6A - FIG. 6D show mitochondrial uptake. Images of mitochondrial uptake were captured by fluorescent microscopy at 20x objective with stained donor mitochondria. Low dose=30 pg mitochondrial protein / well. High dose = 300pg mitochondrial protein / well (FIG. 6A). Mitochondrial uptake was quantified via plate reader fluorescence (Ex 579 / Em 599) before and after co-incubation. There was a significant increase in mitochondrial uptake during co-incubation over 90 minutes (p<0.0001 ), following a linear trend (FIG. 6B). Mitochondrial uptake was significantly impacted by temperature, with significantly reduced uptake at 4°C (p<0.01 ), which is the typical condition for whole organ SCS (FIG. 6C). Cells treated with the low dose (1 x) had 14% of the uptake of the high dose (1 Ox) (FIG. 6D). FIG. 6D is a quantification of the fluorescence images of mitochondrial uptake shown in FIG. 6A.

[0099] SPECIFIC EMBODIMENTS

[0100] The present disclosure is exemplified by the specific embodiments below.

[0101] 1 . A method of prolonging survival of tissue, the method comprising introducing into the tissue one or more exogenous mitochondria.

[0102] 2. A method of prolonging survival of a cell, the method comprising introducing into the cell one or more exogenous mitochondria.

[0103] 3. A method of transplanting an isolated tissue into a subject in need of replacement of the tissue, the method comprising: a) introducing, into a cell of the isolated tissue, one or more exogenous mitochondria; b) providing the subject with the tissue resulting from (a) in place of the subject’s endogenous tissue.

[0104] 4. A method of transplanting an isolated tissue into a subject in need of replacement of the tissue, the method comprising providing the subject with the isolated tissue in place of the subject’s endogenous tissue, wherein prior to the providing, one or more exogenous mitochondria have been introduced into a cell of the isolated tissue. PATENT ATTORNEY DOCKET NO: 51789-002WO2

[0105] 5. The method of any one of embodiments 1 , 3, and 4, wherein the tissue is a liver, a heart, a kidney, or a lung.

[0106] 6. The method of embodiment 1 , wherein the tissue is an isolated tissue.

[0107] 7. The method of any one of embodiments 1 -6, wherein the method comprises introducing the exogenous mitochondria ex vivo.

[0108] 8. The method of any one of embodiments 2, 3, and 4, wherein the cell is an endothelial cell.

[0109] 9. The method of any one of embodiments 2, 3, and 4, wherein the cell is a liver cell.

[0110] 10. The method of embodiment 8, wherein the liver cell is a hepatocyte.

[0111] 11 . The method of any one of embodiments 1 and 3-7, wherein the tissue is obtained from a donor after cardiac death (DCD).

[0112] 12. The method of any one of embodiments 1 and 3-7, wherein the tissue is characterized by a warm ischemic time (WIT) of greater than 30 minutes.

[0113] 13. The method of embodiment 12, wherein the tissue is characterized by a WIT of greater than 45 minutes.

[0114] 14. The method of embodiment 13, wherein the tissue is characterized by a WIT of greater than 60 minutes.

[0115] 15. The method of any one of embodiments 1 and 3-7, wherein the tissue is characterized by a WIT of from 30 minutes to 72 hours.

[0116] 16. The method of embodiment 15, wherein the tissue is characterized by a WIT of from 45 minutes to 48 hours.

[0117] 17. The method of embodiment 16, wherein the tissue is characterized by a WIT of from 60 minutes to 24 hours.

[0118] 18. The method of any one of embodiments 1 and 3-17, wherein the tissue has been subjected to static cold storage or subzero storage prior to the introducing of said one or more exogenous mitochondria.

[0119] 19. The method of embodiment 18, wherein the static cold storage or subzero storage comprises storage of the tissue at a temperature of about 4° C to about - 80° C.

[0120] 20. The method of embodiment 19, wherein the static cold storage or subzero storage comprises storage of the tissue at a temperature of about 4° C to about - 20° C.

[0121] 21 . The method of embodiment 20, wherein the static cold storage or subzero storage further comprises storage of the tissue at said temperature for at least 24 hours.

[0122] 22. The method of embodiment 21 , wherein the static cold storage or subzero storage comprises storage of the tissue at said temperature for at least 48 hours.

[0123] 23. The method of embodiment 22, wherein the static cold storage or subzero storage comprises storage of the tissue at said temperature for at least 72 hours.

[0124] 24. The method of embodiment 23, wherein the static cold storage or subzero storage comprises storage of the tissue at said temperature for at least 96 hours.

[0125] 25. The method of embodiment 24, wherein the static cold storage or subzero storage comprises storage of the tissue at said temperature for at least 144 hours. PATENT ATTORNEY DOCKET NO: 51789-002WO2

[0126] 26. The method of embodiment 19, wherein the static cold storage or subzero storage further comprises storage of the tissue at said temperature for from about 24 hours to about 144 hours.

[0127] 27. The method of any one of embodiments 1 -26, wherein the one or more exogenous mitochondria are obtained from a population of cells selected from hepatocytes, hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells, or a combination thereof.

[0128] 28. The method of embodiment 18, wherein the population of cells is of the same species as the cell into which the one or more mitochondria are introduced.

[0129] 29. The method of embodiment 1 , further comprising providing the isolated tissue to a subject in need of replacement of the tissue.

[0130] 30. The method of embodiment 29, wherein the tissue is a liver.

[0131] 31 . The method of any one of embodiments 3-30, wherein the subject is diagnosed as having a complication or disease selected from the group consisting of ischemic liver injury, primary non-function (PNF), early allograft dysfunction (EAD), small-for-size syndrome (SFSS), resection for malignant or benign disease, autoimmune hepatitis, cystic fibrosis, cholestatic liver diseases, primary sclerosing cholangitis, primary biliary cirrhosis, inborn errors of metabolism, metabolic diseases, sickle cell hepatopathy, erythropoietic protoporphyria (EPP) hepatopathy, congestive hepatopathy, metabolic dysfunction-associated steatotic liver disease (MAFLD), metabolic dysfunction-associated steatohepatitis (MASH), alcoholic liver disease, hemochromatosis, Alagille syndrome, Wilson's disease, viral hepatitis, alpha-1 antitrypsin deficiency, drug-induced liver injury (DILI), and liver cancer, cancer in the liver, liver metastases, cirrhosis, or a combination thereof.

[0132] 32. The method of any one of embodiments 1 -31 , wherein the subject is a human.

[0133] 33. The method of any one of embodiments 1 -32, wherein the exogenous mitochondria are freshly isolated.

[0134] 34. The method of any one of embodiments 1 -32, wherein the exogenous mitochondria were stored prior to introduction into the tissue or cell.

[0135] 35. The method of any one of embodiments 1 -32, wherein the exogenous mitochondria are introduced at a dose of 2.5 to 150 pg per mL of solution.

[0136] 36. An ex vivo tissue transplant comprising an isolated tissue that comprises one or more heterologous mitochondria.

[0137] 37. The ex vivo tissue transplant of embodiment 36, wherein the tissue is an isolated liver.

[0138] 38. The ex vivo tissue transplant of embodiment 36 or 37, wherein the tissue transplant is obtained from a DCD.

[0139] 39. The ex vivo tissue transplant of any one of embodiments 36-38, wherein the tissue is characterized by a warm ischemic time (WIT) of greater than 30 minutes.

[0140] 40. The ex vivo tissue transplant of embodiment 39, wherein the tissue is characterized by a WIT of greater than 45 minutes.

[0141] 41 . The ex vivo tissue transplant of embodiment 40, wherein the tissue is characterized by a WIT of greater than 60 minutes.

[0142] 42. The ex vivo tissue transplant of any one of embodiments 36-38, wherein the tissue is characterized by a WIT of from 30 minutes to 72 hours. PATENT

[0143] ATTORNEY DOCKET NO: 51789-002WO2

[0144] 43. The ex vivo tissue transplant of embodiment 42, wherein the tissue is characterized by a WIT of from 45 minutes to 48 hours.

[0145] 44. The ex vivo tissue transplant of embodiment 43, wherein the tissue is characterized by a WIT of from 60 minutes to 24 hours.

[0146] 45. The ex vivo tissue transplant of any one of embodiments 36-38, wherein the tissue has been subjected to static cold storage or subzero storage prior to introducing said one or more heterologous mitochondria into the liver.

[0147] 46. The ex vivo tissue transplant of any one of embodiments 36-38, wherein the one or more exogenous mitochondria are obtained from a population of cells selected from hepatocytes, hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells, or a combination thereof.

[0148] 47. The ex vivo tissue transplant of any one of embodiments 36-38, wherein the heterologous mitochondria are freshly isolated.

[0149] 48. The ex vivo tissue transplant of any one of embodiments 36-38, wherein the heterologous mitochondria were stored prior to introduction into the tissue.

[0150] 49. The ex vivo tissue transplant of any one of embodiments 36-38, wherein the heterologous mitochondria are introduced at a dose of 2.5 to 150 pg per mL of solution.

[0151] Additional embodiments

[0152] All references cited in this specification, including, database-accessioned information (e.g., in GENBANK, UNIPROT, PUBMED), are herein incorporated by reference as though each reference was specifically and individually indicated to be incorporated by reference. The citation of any reference is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such reference by virtue of prior invention.

[0153] It will be understood that each of the elements described above, or two or more together may also find a useful application in other types of methods differing from the type described above. Without further analysis, the foregoing will so fully reveal the gist of the present disclosure that others can, by applying current knowledge, readily adapt it for various applications without omitting features that, from the standpoint of prior art, fairly constitute essential characteristics of the generic or specific aspects of this disclosure set forth in the appended claims. The foregoing embodiments are presented by way of example only.

Claims

PATENTATTORNEY DOCKET NO: 51789-002WO2CLAIMSWhat is claimed is:1 . A method of prolonging survival of tissue, the method comprising introducing into the tissue one or more exogenous mitochondria.

2. A method of prolonging survival of a cell, the method comprising introducing into the cell one or more exogenous mitochondria.

3. A method of transplanting an isolated tissue into a subject in need of replacement of the tissue, the method comprising: a) introducing, into a cell of the isolated tissue, one or more exogenous mitochondria; b) providing the subject with the tissue resulting from (a) in place of the subject’s endogenous tissue.

4. A method of transplanting an isolated tissue into a subject in need of replacement of the tissue, the method comprising providing the subject with the isolated tissue in place of the subject’s endogenous tissue, wherein prior to the providing, one or more exogenous mitochondria have been introduced into a cell of the isolated tissue.

5. The method of any one of claims 1 , 3, and 4, wherein the tissue is a liver, a heart, a kidney, or a lung.

6. The method of claim 1 , wherein the tissue is an isolated tissue.

7. The method of claim 1 , wherein the method comprises introducing the exogenous mitochondria ex vivo.

8. The method of any one of claims 2, 3, and 4, wherein the cell is an endothelial cell.

9. The method of any one of claims 2, 3, and 4, wherein the cell is a liver cell.

10. The method of claim 8, wherein the liver cell is a hepatocyte.11 . The method of claim 1 , wherein the tissue is obtained from a donor after cardiac death (DCD).

12. The method of claim 1 , wherein the tissue is characterized by a warm ischemic time (WIT) of greater than 30 minutes.

13. The method of claim 12, wherein the tissue is characterized by a WIT of greater than 45 minutes.

14. The method of claim 13, wherein the tissue is characterized by a WIT of greater than 60 minutes.PATENT ATTORNEY DOCKET NO: 51789-002WO215. The method of claim 1 , wherein the tissue is characterized by a WIT of from 30 minutes to 72 hours.

16. The method of claim 15, wherein the tissue is characterized by a WIT of from 45 minutes to 48 hours.

17. The method of claim 16, wherein the tissue is characterized by a WIT of from 60 minutes to 24 hours.

18. The method of claim 1 , wherein the tissue has been subjected to static cold storage or subzero storage prior to the introducing of said one or more exogenous mitochondria.

19. The method of claim 18, wherein the static cold storage or subzero storage comprises storage of the tissue at a temperature of about 4° C to about - 80° C.

20. The method of claim 19, wherein the static cold storage or subzero storage comprises storage of the tissue at a temperature of about 4° C to about - 20° C.21 . The method of claim 20, wherein the static cold storage or subzero storage further comprises storage of the tissue at said temperature for at least 24 hours.

22. The method of claim 21 , wherein the static cold storage or subzero storage comprises storage of the tissue at said temperature for at least 48 hours.

23. The method of claim 22, wherein the static cold storage or subzero storage comprises storage of the tissue at said temperature for at least 72 hours.

24. The method of claim 23, wherein the static cold storage or subzero storage comprises storage of the tissue at said temperature for at least 96 hours.

25. The method of claim 24, wherein the static cold storage or subzero storage comprises storage of the tissue at said temperature for at least 144 hours.

26. The method of claim 19, wherein the static cold storage or subzero storage further comprises storage of the tissue at said temperature for from about 24 hours to about 144 hours.

27. The method of claim 1 , wherein the one or more exogenous mitochondria are obtained from a population of cells selected from hepatocytes, hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells, or a combination thereof.

28. The method of claim 18, wherein the population of cells is of the same species as the cell into which the one or more mitochondria are introduced.

29. The method of claim 1 , further comprising providing the isolated tissue to a subject in need of replacement of the tissue.

30. The method of claim 29, wherein the tissue is a liver.PATENTATTORNEY DOCKET NO: 51789-002WO231 . The method of claim 3, wherein the subject is diagnosed as having a complication or disease selected from the group consisting of ischemic liver injury, primary non-function (PNF), early allograft dysfunction (EAD), small-for-size syndrome (SFSS), resection for malignant or benign disease, autoimmune hepatitis, cystic fibrosis, cholestatic liver diseases, primary sclerosing cholangitis, primary biliary cirrhosis, inborn errors of metabolism, metabolic diseases, sickle cell hepatopathy, erythropoietic protoporphyria (EPP) hepatopathy, congestive hepatopathy, metabolic dysfunction- associated steatotic liver disease (MAFLD), metabolic dysfunction-associated steatohepatitis (MASH), alcoholic liver disease, hemochromatosis, Alagille syndrome, Wilson's disease, viral hepatitis, alpha-1 antitrypsin deficiency, drug-induced liver injury (DILI), and liver cancer, cancer in the liver, liver metastases, cirrhosis, or a combination thereof.

32. The method of claim 1 , wherein the subject is a human.

33. The method of claim 1 , wherein the exogenous mitochondria are freshly isolated.

34. The method of claim 1 , wherein the exogenous mitochondria were stored prior to introduction into the tissue or cell.

35. The method of claim 1 , wherein the exogenous mitochondria are introduced at a dose of 2.5 to 150 pg per mL of solution.

36. An ex vivo tissue transplant comprising an isolated tissue that comprises one or more heterologous mitochondria.

37. The ex vivo tissue transplant of claim 36, wherein the tissue is an isolated liver.

38. The ex vivo tissue transplant of claim 36, wherein the tissue transplant is obtained from a DCD.

39. The ex vivo tissue transplant of claim 36, wherein the tissue is characterized by a warm ischemic time (WIT) of greater than 30 minutes.

40. The ex vivo tissue transplant of claim 39, wherein the tissue is characterized by a WIT of greater than 45 minutes.41 . The ex vivo tissue transplant of claim 40, wherein the tissue is characterized by a WIT of greater than 60 minutes.

42. The ex vivo tissue transplant of claim 36, wherein the tissue is characterized by a WIT of from 30 minutes to 72 hours.

43. The ex vivo tissue transplant of claim 42, wherein the tissue is characterized by a WIT of from 45 minutes to 48 hours.PATENTATTORNEY DOCKET NO: 51789-002WO244. The ex vivo tissue transplant of claim 43, wherein the tissue is characterized by a WIT of from 60 minutes to 24 hours.

45. The ex vivo tissue transplant of claim 36, wherein the tissue has been subjected to static cold storage or subzero storage prior to introducing said one or more heterologous mitochondria into the liver.

46. The ex vivo tissue transplant of claim 36, wherein the one or more exogenous mitochondria are obtained from a population of cells selected from hepatocytes, hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells, or a combination thereof.

47. The ex vivo tissue transplant of claim 36, wherein the heterologous mitochondria are freshly isolated.

48. The ex vivo tissue transplant of claim 36, wherein the heterologous mitochondria were stored prior to introduction into the tissue.

49. The ex vivo tissue transplant of claim 36, wherein the heterologous mitochondria are introduced at a dose of 2.5 to 150 pg per mL of solution.

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