Composition for refrigerated storage of isolated mitochondria and its uses

Abstract

The present invention relates to a composition for preserving isolated mitochondria and uses thereof. The storage composition of the present invention can maintain the activity of mitochondria isolated from cells, tissues, plasma, etc. for a long period of time, and can be used in a variety of ways.

Description

[Technical Field]

[0001] The present invention relates to a composition for preserving isolated mitochondria and uses thereof. [Background technology]

[0002] Mitochondria are essential organelles for the survival of eukaryotic cells, responsible for the synthesis and regulation of adenosine triphosphate (ATP), an energy source. Mitochondria are crucial organelles involved in the regulation of various metabolic pathways in the body, including cell signaling, differentiation, and cell death, as well as the cell cycle and cell growth. Recently, the presence of mitochondria released from cells in the blood has been confirmed.

[0003] On the other hand, if mitochondrial function is impaired, various diseases can be caused. Diseases caused by mitochondrial dysfunction include, for example, multiple sclerosis, encephalomyelitis, peripheral neuropathy, Reye's syndrome, MELAS syndrome, migraine, psychiatry, Alzheimer's disease, Parkinson's disease, optic atrophy, myopathy, myoatrophy, myoglobinuria, renal failure, hepatic failure, neutropenia, thrombocytopenia, sensorineural hearing loss, epilepsy, Huntington's disease, and others. It can also be a direct or indirect cause of various diseases, such as cerebrospinal fluid (CEF) and cerebrospinal fluid (CEF).

[0004] As a method for treating these diseases, transplantation of mitochondria isolated from healthy cells into the patient's cells, or transplantation of cells with increased activity by introducing mitochondria into the patient has been proposed.

[0005] Therefore, in order to apply mitochondria as a therapeutic agent as described above, there has been increasing interest in techniques for isolating mitochondria or effectively storing / preserving isolated mitochondria.

[0006] Therefore, the present inventors developed a technique that enables isolated mitochondria to be stored for a long period of time while maintaining their activity, and completed the present invention. Summary of the Invention [Problem to be solved by the invention]

[0007] One embodiment is a mixture of 1, HEPES (Hydroxyethyl piperazine ethanol sulfonic acid), a chelating agent, and magnesium ions (Mg 2+ The present invention provides a composition for storing isolated mitochondria, comprising one or more selected from the group consisting of:

[0008] Another embodiment provides a method for storing isolated mitochondria, comprising: 1) providing isolated mitochondria from a mitochondria-containing sample; 2) mixing the isolated mitochondria and the storage composition; and 3) refrigerating or freezing the mixture.

[0009] Yet another embodiment provides a pharmaceutical composition for treating or preventing a disease caused by a mitochondrial functional defect, comprising isolated mitochondria.

[0010] Yet another embodiment provides a method for treating or preventing a disease caused by mitochondrial dysfunction, comprising the step of administering to an individual the pharmaceutical composition for treating or preventing a disease caused by mitochondrial dysfunction.

[0011] Yet another embodiment provides a mitochondrial transplant composition comprising isolated mitochondria.

[0012] Yet another embodiment provides a hydrogel comprising isolated mitochondria and a biocompatible polymer. [Means for solving the problem]

[0013] One embodiment is a composition containing HEPES (Hydroxyethyl piperazine ethanol sulfonic acid), a chelator, and magnesium ions (Mg 2+ The present invention provides a composition for storing isolated mitochondria, comprising one or more selected from the group consisting of:

[0014] As used herein, "mitochondria" refers to double-membrane-bound organelles found in most eukaryotic organisms that produce most of the intracellular adenosine triphosphate (ATP).

[0015] As used herein, the term "isolated mitochondria" refers to mitochondria obtained from autologous, allogeneic, or xenogeneic sources.

[0016] As used herein, the term "autologous mitochondria" refers to mitochondria obtained from the plasma, tissue, bone marrow, or cells of the same individual. The term "allogeneic mitochondria" refers to mitochondria obtained from the plasma, tissue, bone marrow, or cells of an individual that belongs to the same species as the individual but has a different genotype for the allele. The term "heterogeneous mitochondria" refers to mitochondria obtained from the plasma, tissue, bone marrow, or cells of an individual that belongs to a different species than the individual.

[0017] In this case, the individual may be a mammal, preferably a human.

[0018] The mitochondria may be isolated from cells, bone marrow, or plasma of an individual. The mitochondria may be obtained from autologous or allogeneic cells cultured in vitro. In this case, the cells, bone marrow, or plasma may have normal biological activity.

[0019] As used herein, the term "cell" refers to a structural or functional unit of an organism, consisting of a cytoplasm surrounded by a cell membrane and containing biomolecules such as proteins and nucleic acids. The cell refers to a cell containing mitochondria within the cell membrane.

[0020] The mitochondria may be obtained by concentrating tissue, plasma, bone marrow, cerebrospinal fluid, umbilical cord blood, peripheral blood, cells and / or organelles, followed by fragmentation and isolation, or may be obtained by storing frozen and then thawing tissue, plasma, bone marrow or cell samples, followed by fragmentation and isolation.

[0021] In one embodiment, the cell or organelle is any one selected from the group consisting of a stem cell, a somatic cell, a germ cell, and a platelet.

[0022] As used herein, the term "stem cells" refers to undifferentiated cells capable of differentiating into various types of tissue cells. The stem cells may be any one selected from the group consisting of mesenchymal stem cells, adult stem cells, dedifferentiated stem cells, embryonic stem cells, bone marrow stem cells, neural stem cells, limbal stem cells, and tissue-derived stem cells.

[0023] In this case, the mesenchymal stem cells may be any one selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta, and may preferably be derived from human umbilical cord.

[0024] As used herein, the term "somatic cells" refers to cells that constitute an individual, excluding germ cells. The somatic cells are selected from the group consisting of muscle cells, hepatocytes, fibroblasts, epithelial cells, nerve cells, adipocytes, osteocytes, periosteal cells, leukocytes, lymphocytes, and mucosal cells. Preferably, the somatic cells may be obtained from muscle cells or hepatocytes with excellent mitochondrial activity. Alternatively, the somatic cells may be obtained from autologous or allogeneic blood PBMC cells.

[0025] As used herein, the term "cerebrospinal fluid" refers to the fluid that fills the subarachnoid space, the space between the pia mater and the arachnoid membrane that surrounds the brain and spinal cord, and the ventricles. The cerebrospinal fluid contains extracellular mitochondria.

[0026] As used herein, the term "germ cell" refers to a cell that forms a zygote during reproduction in a sexually reproducing individual. The mitochondria may be derived from autologous or allogeneic germ cells. The germ cell may be a sperm or an egg.

[0027] As used herein, the term "platelet" refers to the solid components of blood that play an important role in blood clotting by binding fibrin together to form clots. The mitochondria may be obtained from autologous or allogeneic platelets.

[0028] As used herein, the term "bone marrow" refers to the semi-solid tissue found in the spongy portion of bone. Human bone marrow produces approximately 500 billion blood cells per day. In particular, bone marrow contains active mitochondria.

[0029] As used herein, the term "plasma" refers to the liquid component of blood, excluding blood cells, that is the extracellular fluid within the blood vessels. Plasma contains up to 95% water and 6-8% dissolved proteins or electrolytes. In particular, plasma contains active mitochondria.

[0030] The plasma can be obtained by separation from blood. Specifically, it can be obtained by spinning blood containing an anticoagulant in a centrifuge and separating the supernatant from the blood. Alternatively, plasma can be extracted from blood through filtration or coagulation. The plasma can also be classified according to the blood source. In one specific example, the plasma can be separated from umbilical cord blood or peripheral blood. Preferably, it can be separated from umbilical cord blood.

[0031] In one embodiment, the plasma or bone marrow may be obtained from an individual and stored. Specifically, the plasma or bone marrow may be frozen.

[0032] The isolated mitochondria may also have normal biological activity. Specifically, the mitochondria with normal biological activity may have one or more properties selected from the group consisting of (i) having a membrane potential, (ii) producing ATP within the mitochondria, and (iii) removing ROS or reducing ROS activity within the mitochondria.

[0033] The mitochondria can be separated from specific cells by various known methods, for example, using a specific buffer solution, or using a potential difference and a magnetic field, etc. The mitochondria separation may also include centrifuging and filtering the plasma to remove all cellular components, and centrifuging the filtered plasma.

[0034] The mitochondrial isolation is achieved by disrupting the cells and centrifuging them while maintaining mitochondrial activity, and the centrifugation may be performed in the first to third stages.

[0035] In one embodiment, the method may include culturing cells, subjecting a pharmaceutical composition containing the cells to a first centrifugation to produce a pellet, resuspending and homogenizing the pellet in a buffer solution, subjecting the homogenized solution to a second centrifugation to produce a supernatant, and subjecting the supernatant to a third centrifugation to purify mitochondria. In this case, it is desirable to adjust the time for the second centrifugation to be shorter than the times for the first and third centrifugations in terms of maintaining cell activity, and the speed may be increased from the first to the third centrifugation.

[0036] The mitochondria can be separated from plasma by various known methods, such as using a specific buffer solution, ultrasound, a concentration gradient, and a magnetic field.

[0037] The mitochondrial isolation includes removing cells or organelles from plasma and purifying mitochondria. The mitochondrial isolation may also include physically separating endoplasmic reticulum, mitochondrial-associated membrane debris, and mitochondria.

[0038] In one embodiment, the separation may be performed by centrifugation. Specifically, the separation may be performed by first centrifuging the plasma at low speed to remove cells from the plasma, then filtering the plasma to remove cellular debris, and then centrifuging the plasma supernatant for a second time.

[0039] In one embodiment, the separation may be performed using a discontinuous concentration gradient and centrifugation. The discontinuous concentration gradient may be a sucrose or Percoll concentration gradient. Specifically, the separation may be performed through the steps of: lysing cells using ultrasound; subjecting the plasma to a low-speed first centrifugation to remove plasma cells; subjecting the plasma to a second centrifugation to remove endoplasmic reticulum; loading the plasma supernatant onto a discontinuous concentration gradient; and subjecting the separated product to a third centrifugation.

[0040] The first to third centrifugation steps may be performed at a temperature of 0 to 10°C, preferably 3 to 5°C. The duration of the centrifugation may be 1 to 50 minutes, and may be appropriately adjusted depending on the number of centrifugation steps, the sample content, etc. The first centrifugation step may be performed at a speed of 100 to 1,000 xg, 200 to 700 xg, or 300 to 450 xg. The second or third centrifugation step may be performed at a speed of 1 to 2,000 xg, 25 to 1,800 xg, 500 to 1,600 xg, 100 to 20,000 xg, 500 to 18,000 xg, or 800 to 15,000 xg.

[0041] The storage is refrigerated or frozen. The temperature range for the refrigeration is not particularly limited, and may be, for example, above 0°C and 1 to 10°C or lower, 1 to 8°C or lower, 1 to 6°C or lower, 1 to 4°C or lower, 1 to 3°C or lower, or 1 to 2°C or lower. The temperature range for the cooling is 0°C or lower, -35°C or lower, or -196°C or lower. The cooling may be performed using liquid nitrogen.

[0042] The chelating agent may include one or more selected from the group consisting of EGTA (Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid) and EDTA (Ethylenediaminetetraacetic acid), and specifically may include EGTA.

[0043] The magnesium ions may be contained in the form of one or more selected from the group consisting of MgCl 2 , MgSO 4 , Mg(OAc) 2 and Mg(NO 3 ) 2 , and specifically, may be contained in the form of MgCl 2 .

[0044] The composition is for preserving the activity of isolated mitochondria, and may specifically be for preserving the activity of mitochondria that have been stored in a refrigerator or freezer.

[0045] The maintenance of mitochondrial activity means that one or more selected from the group consisting of electron transport complex function, ATP synthesis ability, and membrane potential are maintained.

[0046] In one embodiment, the activity of mitochondria preserved using the composition is 50% to 100% of the activity of mitochondria immediately after isolation, specifically 50% to 100%, 50% to 95%, 50% to 90%, 50% to 85%, 50% to 80%, 50% to 75%, 60% to 100%, 60% to 95%, 60% to 90%, 60% to 85%, 60% to 80%, 60% to 75%, 70% to 100%, 70% to 95%, 70% to 90%, 70% to 85%, 70% to 80%, or 70% to 75% of the activity of mitochondria immediately after isolation.

[0047] The composition may be free of one or more selected from the group consisting of sugar, bicarbonate, KCl, and albumin, and may specifically be free of sugar, bicarbonate, KCl, and albumin.

[0048] The sugars include, but are not limited to, one or more selected from the group consisting of sucrose, dextrose, lactose, trehalose, maltose, galactose, and fructose.

[0049] The bicarbonate salts include, but are not limited to, one or more selected from the group consisting of sodium bicarbonate (NaHCO), sodium carbonate (NaCO), potassium bicarbonate (KHCO), calcium bicarbonate (Ca(HCO)), and calcium carbonate (CaCO).

[0050] The albumin may be animal albumin or plant albumin. The animal albumin may be ovalbumin, serum albumin, lactalbumin, or miogen. The plant albumin may be leucosin, legumelin, or lysine. The albumin may be albumin derived from genetically modified rice. The serum albumin may be albumin derived from human or animal serum, for example, human serum albumin or bovine serum albumin.

[0051] The composition may be one in which the components contained therein are dissolved in an aqueous medium, and therefore the composition may further comprise an aqueous medium.

[0052] Mitochondria stored using the composition may retain 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, or 90% or more of mitochondrial activity compared to membrane-isolated mitochondria (fresh MTs) after 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 weeks of storage under refrigerated conditions.

[0053] In the composition, HEPES is contained at a concentration of 1 to 500 mM, specifically, 1 to 500 mM, 1 to 300 mM, 1 to 100 mM, 1 to 80 mM, 1 to 60 mM, 1 to 50 mM, 1 to 40 mM, 1 to 30 mM, 1 to 25 mM, 1 to 20 mM, 5 to 500 mM, 5 to 300 mM, 5 to 100 mM, 5 to 80 mM, 5 to 60 mM, 5 to 50 mM, 5 to 40 mM, 5 to 30 mM, 5 to 25 mM, 5 to 20 mM, 10 to 500 mM, 10 to 300 mM, 10 to 100 mM, 10 to 80 mM, 1 It may be contained at a concentration of 0 to 60 mM, 10 to 50 mM, 10 to 40 mM, 10 to 30 mM, 10 to 25 mM, 10 to 20 mM, 15 to 500 mM, 15 to 300 mM, 15 to 100 mM, 15 to 80 mM, 15 to 60 mM, 15 to 50 mM, 15 to 40 mM, 15 to 30 mM, 15 to 25 mM, 15 to 20 mM, 20 to 500 mM, 20 to 300 mM, 20 to 100 mM, 20 to 80 mM, 20 to 60 mM, 20 to 50 mM, 20 to 40 mM, 20 to 30 mM, or 20 to 25 mM.

[0054] In the composition, the chelating agent is contained at a concentration of 0.1 to 50 mM, specifically, 0.1 to 50 mM, 0.1 to 30 mM, 0.1 to 10 mM, 0.1 to 8 mM, 0.1 to 6 mM, 0.1 to 5 mM, 0.1 to 4 mM, 0.1 to 3 mM, 0.1 to 2.5 mM, 0.1 to 2 mM, 0.5 to 50 mM, 0.5 to 30 mM, 0.5 to 10 mM, 0.5 to 8 mM, 0.5 to 6 mM, 0.5 to 5 mM, 0.5 to 4 mM, 0.5 to 3 mM, 0.5 to 2.5 mM, 0.5 to 2 mM, 1 to 50 mM, It may be contained at a concentration of 1 to 30 mM, 1 to 10 mM, 1 to 8 mM, 1 to 6 mM, 1 to 5 mM, 1 to 4 mM, 1 to 3 mM, 1 to 2.5 mM, 1 to 2 mM, 1.5 to 50 mM, 1.5 to 30 mM, 1.5 to 10 mM, 1.5 to 8 mM, 1.5 to 6 mM, 1.5 to 5 mM, 1.5 to 4 mM, 1.5 to 3 mM, 1.5 to 2.5 mM, 1.5 to 2 mM, 2 to 50 mM, 2 to 30 mM, 2 to 10 mM, 2 to 8 mM, 2 to 6 mM, 2 to 5 mM, 2 to 4 mM, 2 to 3 mM, or 2 to 2.5 mM.

[0055] In the composition, magnesium ions are contained at a concentration of 0.1 to 50 mM, specifically, 0.1 to 50 mM, 0.1 to 30 mM, 0.1 to 10 mM, 0.1 to 8 mM, 0.1 to 6 mM, 0.1 to 5 mM, 0.1 to 4 mM, 0.1 to 3 mM, 0.1 to 2.5 mM, 0.1 to 2 mM, 0.1 to 1.5 mM, 0.5 to 50 mM, 0.5 to 30 mM, 0.5 to 10 mM, 0.5 to 8 mM, 0.5 to 6 mM, 0.5 to 5 mM, 0.5 to 4 mM, 0.5 to 3 mM 1.5 to 2.5 mM, 0.5 to 2 mM, 0.5 to 1.5 mM, 1 to 50 mM, 1 to 30 mM, 1 to 10 mM, 1 to 8 mM, 1 to 6 mM, 1 to 5 mM, 1 to 4 mM, 1 to 3 mM, 1 to 2.5 mM, 1 to 2 mM, 1 to 1.5 mM, 1.5 to 50 mM, 1.5 to 30 mM, 1.5 to 10 mM, 1.5 to 8 mM, 1.5 to 6 mM, 1.5 to 5 mM, 1.5 to 4 mM, 1.5 to 3 mM, 1.5 to 2.5 mM, or 1.5 to 2 mM.

[0056] In the composition, HEPES and the chelating agent are contained in a concentration ratio of 2:1 to 20:1 (HEPES:chelating agent), and specifically, 2:1 to 20:1, 2:1 to 18:1, 2:1 to 15:1, 2:1 to 13:1, 2:1 to 11:1, 2:1 to 10:1, 5:1 to 20:1, 5:1 to 18:1, 5:1 to 15:1, 5:1 to 13:1, 5:1 to 11:1, 5:1 to 10:1, 7:1 The concentration ratio may be 1:1 to 20:1, 7:1 to 18:1, 7:1 to 15:1, 7:1 to 13:1, 7:1 to 11:1, 7:1 to 10:1, 9:1 to 20:1, 9:1 to 18:1, 9:1 to 15:1, 9:1 to 13:1, 9:1 to 11:1, 9:1 to 10:1, 10:1 to 20:1, 10:1 to 18:1, 10:1 to 15:1, 10:1 to 13:1, or 10:1 to 11:1.

[0057] In the composition, HEPES and magnesium ions may be contained in a concentration ratio of 2:1 to 30:1 (HEPES:magnesium ions), and specifically, 2:1 to 30:1, 2:1 to 25:1, 2:1 to 20:1, 2:1 to 17:1, 2:1 to 15:1, 2:1 to 14:1, 2:1 to 13.5:1, 5:1 to 30:1, 5:1 to 25:1, 5:1 to 20:1, 5:1 to 17:1, 5:1 to 15:1, 5:1 to 14:1, 5:1 to 13.5:1, 7:1 to 30:1, 7:1 to 25:1, 7:1 to 20:1, 7:1 to 17:1, 7:1 to 15:1, 7:1 1 to 14:1, 7:1 to 13.5:1, 10:1 to 30:1, 10:1 to 25:1, 10:1 to 20:1, 10:1 to 17:1, 10:1 to 15:1, 10:1 to 14:1, 10:1 to 13.5:1, 12:1 to 30:1, 12:1 to 25:1, 12:1 to 20:1, 12:1 to 17:1, 12:1 to 15:1, 12:1 to 14:1, 12:1 to 13.5:1, 13:1 to 30:1, 13:1 to 25:1, 13:1 to 20:1, 13:1 to 17:1, 13:1 to 15:1, 13:1 to 14:1, or 13:1 to 13.5:1.

[0058] In the composition, the chelating agent and magnesium ions are contained at a concentration ratio of 1:1 to 3:1 (chelating agent:magnesium ions), and specifically, may be contained at a concentration ratio of 1:1 to 3:1, 1:1 to 2.5:1, 1:1 to 2:1, 1:1 to 1.5:1, 1:1 to 1.34:1, 1.2:1 to 3:1, 1.2:1 to 2.5:1, 1.2:1 to 2:1, 1.2:1 to 1.5:1, 1.2:1 to 1.34:1, 1.3:1 to 3:1, 1.3:1 to 2.5:1, 1.3:1 to 2:1, 1.3:1 to 1.5:1, or 1.3:1 to 1.34:1.

[0059] Another embodiment provides a method for storing isolated mitochondria, comprising: 1) preparing isolated mitochondria from a mitochondria-containing sample; 2) mixing the isolated mitochondria with the storage composition; and 3) storing the mixture in a refrigerator or freezer. The same provisions as those described above also apply to this method.

[0060] In the above method, the mixing step may further include mixing a biocompatible polymer.

[0061] Yet another embodiment provides a pharmaceutical composition for treating or preventing a disease caused by a functional defect of mitochondria, comprising isolated mitochondria. The same provisions as those described above also apply to the composition.

[0062] The isolated mitochondria may be stored using the storage composition or the storage method. The pharmaceutical composition may contain the storage composition or a component thereof. The pharmaceutical composition has the long-term mitochondrial preservation and activity-maintaining effects of the mitochondrial preservation composition described above, and is therefore convenient for storage.

[0063] As used herein, the term "diseases caused by mitochondrial functional defects" may include both primary mitochondrial diseases (PMDs) and secondary mitochondrial dysfunction (SMDs). Primary mitochondrial diseases may be caused by congenital damage to mitochondrial DNA (mtDNA) or the nuclear DNA encoding ETC proteins. Secondary mitochondrial dysfunction refers to diseases caused by abnormalities in mitochondrial function other than primary mitochondrial diseases, and may be caused by acquired damage to mtDNA due to environmental factors such as aging, inflammation, or drugs. The mitochondrial functional defects may be caused by loss or reduced function of mtDNA.

[0064] Diseases caused by functional defects of mitochondria include, for example, diabetes and deafness,DAD, multiple sclerosis, encephalomyelitis, peripheral neuropathy, Leigh syndrome, MELAS syndrome, migraine, psychosis, Alzheimer's disease, Parkinson's disease, optic atrophy, optic neuropathy, retinitis pigmentosa, cataract, hyperaldosteronism, hypoparathyroidism, myopathy, myoatrophy, myoglobinuria, hypotonia, myalgia, renal failure, hepatic failure failure, hepatomegaly, sideroblastic anemia, neutropenia, thrombocytopenia, villous atrophy, dysphagia, sensorineural hearing loss, epilepsy, mental retardation, Huntington's disease, Leber's hereditary optic neuropathy (LHON), ptosis, ataxia, retinitis pigmentosa, myoneurogenic gastrointestinal encephalopathy, MERRF syndrome, rheumatism, arthritis, alopecia, ischemic The cause may be, but is not limited to, tendon disease or tendinopathy.

[0065] When the pharmaceutical composition is administered to an individual suffering from a disease caused by a functional defect of mitochondria, mitochondria can be transferred or transplanted into the target cells, thereby restoring mitochondrial function.

[0066] The target cells may be cells with defective mitochondrial function, and the type of target cells may be, for example, muscle cells, endothelial cells, nerve cells, epithelial cells, leukocytes, or lymphocytes, but is not limited thereto.

[0067] The pharmaceutical composition may contain a surfactant, such as poloxamer, a nonionic surfactant, to enhance the efficiency of mitochondria delivery to target cells. Specific types of surfactants that can enhance the efficiency of mitochondria delivery may be found in Korean Patent Registration No. 10-1846460.

[0068] The mitochondria in the pharmaceutical composition are contained at a concentration of 0.1 μg / ml to 1,000 μg / ml, 1 μg / ml to 750 μg / ml, 25 μg / ml to 500 μg / ml, 25 μg / ml to 150 μg / ml, or 25 μg / ml to 100 μg / ml, specifically 1 to 100 μg / ml, 1 to 80 μg / ml, 1 to 60 μg / ml, 1 to 50 μg / ml, 1 to 40 μg / ml, 1 to 30 μg / ml, 1 to 25 μg / ml, 1 to 20 μg / ml, 5 to 100 μg / ml, 5 to 80 μg / ml, 5 to 60 μg / ml, 5 to 50 μg / ml, 5 to 40 μg / ml, 5 to 30 μg / ml, 5 to 25 μg / ml, 5 to 20μg / ml, 10~100μg / ml, 10~80μg / ml, 10~60μg / ml, 10~50μg / ml, 10~40μg / ml, 10~3 0μg / ml, 10~25μg / ml, 10~20μg / ml, 15~100μg / ml, 15~80μg / ml, 15~60μg / ml, 15~50μ g / ml, 15-40 μg / ml, 15-30 μg / ml, 15-25 μg / ml, 15-20 μg / ml, 20-100 μg / ml, 20-80 μg / ml, 20-60 μg / ml, 20-50 μg / ml, 20-40 μg / ml, 20-30 μg / ml or 20-25 μg / ml.

[0069] The number of isolated mitochondria can be measured using a particle counter (Multisizer 4e, Beckman Coulter).

[0070] The mitochondria in the pharmaceutical composition are 1×10 5 Mitochondria / ml ~ 9x10 9 The amount of mitochondria in the pharmaceutical composition may be 1×10 5 / ml~5X10 9 / ml, 2X10 5 / ml~2X10 9 / ml, 5X10 5 / ml~1X10 9 / ml, 1X10 6 / ml~5X10 8 / ml, 2X10 6 / ml~2X10 8 / ml, 5X10 6 / ml~1X10 8 / ml, or 1X10 7 / ml~5X10 7 / ml.

[0071] The term "treatment" as used herein means any action in which the symptoms of the disease are improved or alleviated by administering the composition of the present invention.

[0072] The term "prevention" as used herein means any action in which the disease or the likelihood of developing the disease is inhibited or delayed by administering the composition of the present invention.

[0073] The pharmaceutical composition may further contain an ingredient known to be effective in preventing, treating, or ameliorating diseases caused by mitochondrial dysfunction, which may be in the form of, but is not limited to, a compound, a protein, and / or a nucleic acid.

[0074] The pharmaceutical composition may contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the compound to be injected. Here, "pharmaceutically acceptable" means that it does not inhibit the activity of the active ingredient and does not have toxicity beyond the range acceptable for the subject to which it is applied (prescribed). Any type of carrier that can be used in the pharmaceutical composition is commonly used in the art and is pharmaceutically acceptable. Non-limiting examples of the carrier include lactose, dextrose, maltodextrin, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, glycerol, ethanol, starch, acacia gum, alkynate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. These may be used alone or in combination. The pharmaceutical composition may be prepared as an oral or parenteral dosage form depending on the route of administration, by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. The pharmaceutical compositions can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions by conventional methods.

[0075] When the pharmaceutical composition is formulated, it is prepared using a commonly used diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, or surfactant, but is not limited thereto.

[0076] When the pharmaceutical composition is prepared into an oral dosage form, it can be prepared with a suitable carrier into dosage forms such as powder, granules, tablets, pills, sugar-coated tablets, capsules, liquids, gels, suspensions, wafers, etc., by methods known in the art. Examples of suitable pharmaceutically acceptable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol; starches such as corn starch, potato starch, and wheat starch; celluloses such as methylcellulose, ethylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyols, and vegetable oils. When formulated, diluents and / or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants can be added as needed.

[0077] When the pharmaceutical composition is prepared as a parenteral dosage form, it can be formulated with a suitable carrier into the form of an injection, transdermal agent, nasal inhalant, or suppository by methods known in the art. When formulated as an injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof. Preferably, an infusion solution, phosphate buffered saline (PBS) containing triethanolamine, sterile water for injection, or an isotonic solution such as 5% dextrose can be used. When formulated as a transdermal administration, it can be formulated into the form of an ointment, cream, lotion, gel, external solution, paste, liniment, aerosol, etc. In the case of nasal inhalants, they are formulated into an aerosol spray form using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc., and when formulated as a suppository, the base may be witepsol, Tween 61, polyethylene glycols, cocoa butter, laurin butter, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, etc.

[0078] The pharmaceutical composition may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" refers to an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention. The effective dose level may be determined based on factors including the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, and sensitivity to the drug, the administration time, route of administration, and excretion rate of the composition of the present invention used, the duration of treatment, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered alone or in combination with an ingredient known to have a therapeutic effect against a known disease. Taking all of the above factors into consideration, it is important to administer an amount that will achieve maximum efficacy at the minimum dose without side effects.

[0079] The dosage of the pharmaceutical composition can be determined by one skilled in the art, taking into consideration the purpose of use, the degree of toxicity of the disease, the patient's age, weight, sex, and medical history, and the type of substance used as the active ingredient. For example, the pharmaceutical composition of the present invention is administered at approximately 0.1 ng to 1,000 mg / kg, preferably 1 ng to 100 mg / kg, per adult. The administration frequency of the composition of the present application is not particularly limited, but it can be administered once a day or in divided doses and administered several times. The dosage or frequency of administration does not in any way limit the scope of the present application.

[0080] Yet another embodiment provides a method for treating or preventing a disease caused by mitochondrial dysfunction, comprising administering to an individual the pharmaceutical composition for treating or preventing a disease caused by mitochondrial dysfunction. The same parts as those described above also apply to the method.

[0081] As used herein, the term "individual" includes, without limitation, mammals, birds, reptiles, amphibians, etc., including dogs, cats, rats, livestock, and humans, which may experience a functional defect in mitochondria, develop a disease caused by such a defect, or are at risk of developing a disease. The individual may also be a non-human.

[0082] The pharmaceutical composition may be administered in a single or multiple doses in a pharmaceutically effective amount. The composition may be administered in the form of a liquid, powder, aerosol, injection, infusion (Ringer's solution), capsule, pill, tablet, suppository, or patch. The pharmaceutical composition for preventing or treating diseases caused by mitochondrial dysfunction may be administered via any common route as long as it can reach the target tissue.

[0083] The pharmaceutical composition may be administered via routes such as intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, transdermal patch, oral, intranasal, pulmonary, and rectal administration, depending on the intended purpose, without any particular limitation. However, when administered orally, it may be administered in an unformulated form, and since the active ingredient of the pharmaceutical composition may be denatured or decomposed by gastric acid, oral compositions may be administered in a formulated form or oral patch form so that the active agent is coated or protected from degradation in the stomach. In addition, the composition may be administered by any device that can deliver the active agent to target cells.

[0084] Yet another embodiment provides a mitochondrial transplant composition comprising isolated mitochondria. The same applies to the composition as described above.

[0085] The isolated mitochondria may be stored using the storage composition or the storage method. The composition may contain the storage composition or a component thereof. The composition has the long-term mitochondrial preservation and activity-maintaining effects of the mitochondrial preservation composition described above, and is therefore convenient for storage.

[0086] The mitochondrial transplant composition can deliver mitochondria to target cells to increase mitochondrial activity in the target cells or produce cells with increased mitochondrial activity.

[0087] The mitochondrial transplantation can be carried out by contacting the composition with cells cultured in vitro to transfer mitochondria, or by administering the composition to an individual to transfer mitochondria to cells in vivo.

[0088] The mitochondrial transplant composition may comprise the components of the pharmaceutical composition described above.

[0089] Yet another embodiment provides a hydrogel comprising isolated mitochondria and a biocompatible polymer, and the same applies as described above.

[0090] The isolated mitochondria may be stored using the storage composition or the storage method. The hydrogel may contain the storage composition or a component thereof. The hydrogel has the long-term mitochondrial preservation and activity-maintaining effects of the mitochondrial preservation composition, and is therefore convenient for storage.

[0091] The hydrogel prevents mitochondria from dispersing to other tissues, and the mitochondrial delivery effect can be concentrated at the administration site, thereby increasing the efficiency of mitochondrial delivery to the target site or target cells.

[0092] The hydrogel is prepared by mixing isolated mitochondria and a biocompatible polymer, and may further contain the storage composition or other components.

[0093] The biocompatible polymer can be selected appropriately depending on the site of administration.

[0094] The biocompatible polymer may be a synthetic polymer or a natural polymer. The synthetic polymers include polyethylene (PEA), polyethylene glycol (PEG), polycaprolactone (PCL), polyalkylcarbonate, polyamino acid, polyhydroxybutyric acid, polyorthoester, polyanhydride, Pluronic (registered trademark) (Poly(ethyleneoxide) poly(propyleneoxide) poly(ethyleneoxide)), polylactide (PLA), polyglycolide (PGA), polybutylenesuccinate (PBS), poly(3 The polymer may be any one or more selected from the group consisting of poly(3-hydroxybutyrate-co-3-hydroxyvalerate, PHBV), polybutyleneterephthalate (PBT), polytrimethyleneterephthalate (PTT), polyethylenenaphthalate (PEN), poly(lactic-co-glycolicacid, PLGA), (polylactic-co-glycolide)-glucose (PLGA-glucose), and methoxypolyethylene glycol-(polycaprolactone-co-polylactic acid) (MPEG-(PCL-co-PLLA).

[0095] The natural polymer may be one or more selected from the group consisting of carboxymethylcellulose (CMC), algin, alginic acid, alginate, hyaluronic acid, polypeptide, protein, gelatin, collagen, albumin, dextran, starch, casein, chitin derivatives, chitosan, and small intestinal submucosa (SIS).

[0096] The natural polymer may be an extracellular matrix component, such as hyaluronic acid, collagen, gelatin, albumin, or glycosamidoglycan, and the glycosamidoglycan may be chondroitin sulfate, heparan sulfate, or keratan sulfate.

[0097] The biocompatible polymer may be crosslinked.

[0098] The biocompatible polymer may have an average molecular weight of 2,000,000 to 4,000,000 Da, 2,300,000 to 4,000,000 Da, 2,000,000 to 3,700,000 Da, 2,200,000 to 3,700,000 Da, or 2,500,000 to 3,500,000 Da.

[0099] The content of the biocompatible polymer may be appropriately adjusted as needed, for example, 1 to 10 wt %, 1 to 5 wt %, or 1 to 3 wt %.

[0100] The hydrogel may be used to treat a disease caused by a functional defect of mitochondria, and may further contain a drug used in the treatment of the disease.

[0101] The mitochondrial content in the hydrogel is, for example, 0.1 μg / ml to 10 mg / ml, 0.1 μg / ml to 5 mg / ml, 0.1 μg / ml to 1 mg / ml, 1 μg / ml to 10 mg / ml, 1 μg / ml to 5 mg / ml, 1 μg / ml to 1 mg / ml, 5 μg / ml to 10 mg / ml, 5 μg / ml to 5 mg / ml, 5 μg / ml to 1 mg / ml, 10 μg / ml to 10 mg / ml, 10 μg / ml to 5 mg / ml, or 10 μg / ml to 1 mg / ml based on mitochondrial protein.

[0102] The hydrogel may be in an injectable dosage form. When the hydrogel is in an injectable dosage form, it is advantageous that the storage modulus (G') is high. For example, the storage modulus of an easily injectable hydrogel is 2.00X10 as measured using a rheometer in the frequency range of 0.1 Hz to 10 Hz. 2 Pa~2.00Х10 3 Pa, or 2.00Х10 2 Pa~4.00Х10 2 It may be Pa, but is not limited to it.

[0103] The complex viscosity of this easy-to-inject hydrogel at 25°C is 3.00X10 1 Pa·s~3.0X10 2 Pa·s, or 3.00X10 1 It may be, but is not limited to, Pa·s to 6.0×101 Pa·s.

[0104] The hydrogel can be prepared as an injectable support (filler), which can be a hyaluronic acid filler, for example, a monophasic filler or a biphasic filler.

[0105] Yet another embodiment provides a container filled with the hydrogel. The container may be a vial or a pre-filled syringe. The same as described above also applies to the container. [Effects of the Invention]

[0106] The storage composition according to one embodiment can maintain the activity of mitochondria isolated from cells for a long period of time.

[0107] The pharmaceutical composition according to one embodiment is administered to a subject to restore mitochondrial function, thereby treating or ameliorating a mitochondrial-related disease, and has excellent storage stability.

[0108] A mitochondrial transplant composition according to one embodiment delivers mitochondria to cells to create cells with restored or increased mitochondrial activity, and has excellent storage stability.

[0109] The hydrogel according to one embodiment may prevent mitochondria from dispersing to other tissues, concentrating the mitochondrial delivery effect at the local administration site, thereby increasing the efficiency of mitochondrial delivery to the target site or target cells. [Brief explanation of the drawings]

[0110] [Figure 1] 1 is a diagram showing a method for isolating mitochondria from various cells or plasma. [Figure 2] 1 is a diagram showing a method for isolating mitochondria from various cells or plasma. [Figure 3] 1 is a diagram showing a method for isolating mitochondria from various cells or plasma. [Figure 4] 1 shows the results of evaluating the mitochondrial refrigeration efficiency of compositions containing various ingredient combinations. [Figure 5] 1 shows the results of evaluating the mitochondrial refrigeration efficiency of compositions containing various ingredient combinations. [Figure 6] 1 shows the results of evaluating the mitochondrial refrigeration efficiency of compositions containing various ingredient combinations. [Figure 7] 1 shows the results of evaluating the activity level of electron transport complexes in mitochondria stored in a refrigerator for 4 weeks using HEM buffer. [Figure 8] 1 shows the results of evaluating the level of restoration of cellular metabolic function of mitochondria stored in a refrigerator for 4 weeks using HEM buffer. [Figure 9] 1 shows the results of evaluating the membrane potential level of mitochondria stored in a refrigerator for 4 weeks using HEM buffer. [Figure 10] 1 shows the results of evaluating the activity level of electron transport complexes in mitochondria stored in a refrigerator for 12 weeks using HEM buffer. [Figure 11] 1 shows the results of evaluating the activity levels of electron transport complexes in mitochondria of various origins that had been refrigerated and stored for 4 weeks using HEM buffer. [Figure 12] 1 shows the results of evaluating the activity level of electron transport complexes in mitochondria stored in a refrigerator for 4 weeks using HEM buffer, compared with various control groups. [Figure 13] 1 shows the results of evaluating the activity level of electron transport complexes in mitochondria stored in a refrigerator for 4 weeks using HEM buffer, compared with various control groups. [Figure 14] 1 is a diagram showing the results of confirming the transfer efficiency by mixing mitochondria and hydrogel. DETAILED DESCRIPTION OF THE INVENTION

[0111] The present invention will be described in more detail with reference to the following examples, but these examples are merely illustrative and should not be construed as limiting the scope of the present invention.

[0112] Example 1: Harvesting mitochondria from umbilical cord-derived mesenchymal stem cells 1-1: Cultivation of umbilical cord-derived stem cells Umbilical cord-derived mesenchymal stem cells (IRB number: No. 201411-BR-022-02 or No. 201806-BR-029-03) were obtained from Wharton's jelly of umbilical cords and used for the experiments. Isolated umbilical cord-derived mesenchymal stem cells were cultured in T-175 culture flasks using Minimum Essential Medium Alpha Modification (MEM Alpha Modification, Hyclone) medium containing 10% fetal bovine serum (FBS; Gibco, Waltham, USA) and 1% penicillin / streptomycin antibiotics (P / S, Hyclone, Logan, USA). Cells were incubated at 37°C and 5% CO. 2 The cells were maintained under these conditions until the cell density reached approximately 80% to 90%, at which point the next subculture was carried out.

[0113] 1-2: Isolation of mitochondria from umbilical cord-derived mesenchymal stem cells Mitochondria were isolated from the umbilical cord-derived mesenchymal stem cells cultured in Example 1-1. 7Cells were suspended in 200 μl of SHE buffer [0.25 M sucrose, 20 mM HEPES (pH 7.4), 2 mM EGTA, 10 mM KCl, 1.5 mM MgCl2, 0.1% defatted bovine serum albumin (BSA), pH 7.4] per 100 cells, and then incubated at 4°C for 5 minutes. The cell membrane was disrupted using a 1 ml syringe (Koreavaccine, Seoul, South Korea). To remove undisrupted cells and nuclei, the cells were centrifuged at 1,500 × g for 5 minutes at 4°C. The supernatant was collected and centrifuged at 20,000 × g for 10 minutes at 4°C. To wash the isolated mitochondria, 2 ml of SHE buffer without BSA was added and centrifuged at 20,000 × g for 10 minutes at 4°C. After removing the supernatant, the mitochondrial pellet was washed twice with Dulbecco's phosphate-buffered saline (DPBS; Welgene, South Korea). 200 μL of DPBS was added and the pellet was resuspended. The pellet was then stored at 4°C to obtain umbilical cord-derived mesenchymal stem cell mitochondria. The isolated mitochondria were confirmed to have the appearance of a pale white suspension (Figure 1).

[0114] Example 2: Harvesting mitochondria from hepatocytes 2-1: Human hepatocyte culture Hepatocytes obtained from ATCC (American Type Culture Collection) and constructed in the laboratory were used for the experiments. Hepatocytes were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Wellgene) containing 10% FBS and 1% P / S in T-175 culture flasks. The cells were maintained at 37°C and 5% CO 2 While maintaining the conditions, the next subculture was carried out when the cell density reached approximately 80% to 90%.

[0115] 2-2: Isolation of mitochondria from human hepatocytes Mitochondria were isolated from the cultured hepatocytes in the same manner as in Example 1-2 (FIG. 2).

[0116] Example 3: Harvesting of plasma-derived mitochondria 3-1: Platelet separation Donor blood was transported in heparin tubes and used for the experiment. Donor blood was placed in a 50 ml tube and centrifuged at 3,000 rpm for 5 minutes. After centrifugation, the plasma was separated and collected in a new 50 ml tube and centrifuged at 2,000 x g for 10 minutes. After centrifugation, platelets were collected from the resulting pellet. The platelet-separated plasma was then separated and collected in a new 50 ml tube.

[0117] 3-2: Isolation of plasma-derived mitochondria The platelet-separated plasma obtained in Example 3-1 was centrifuged at 20,000 x g for 20 minutes at 4°C to precipitate mitochondria present in the plasma, and the supernatant was removed. The mitochondria were then washed in the same manner as in Example 1-2 using the SHE buffer (Figure 3).

[0118] Example 4: Discovery of optimal ingredient combinations for mitochondrial cold storage compositions In the prior art, mitochondrial storage solutions were used without distinction between a mitochondrial isolation composition (isolation buffer) and a storage composition (storage buffer). In this regard, in the present invention, the following experiment was carried out to identify essential components of existing mitochondrial isolation / storage solutions in order to develop a storage composition for refrigerated storage of mitochondria.

[0119] Specifically, isolated mitochondria were stored in a refrigerator for one week using candidate buffers in which each component of a known mitochondrial isolation / storage solution, SHEMK buffer, containing sucrose, HEPES, EGTA, MgCl2, and KCl, had been removed.

[0120] As a result, it was confirmed that mitochondrial activity was significantly reduced when HEPES, EGTA, or MgCl2 was omitted from the six components, and that HEPES, EGTA, and MgCl2 had a particularly large effect on mitochondrial activity (Figures 4 and 5). Based on these results, it can be seen that HEPES, EGTA, and MgCl2 are essential components of existing mitochondrial isolation / storage solutions.

[0121] Next, to confirm whether sucrose or HEPES could be replaced with dextrose or NaHCO3, respectively, the isolated mitochondria in the replaced buffer were stored in a refrigerator for 2 weeks.

[0122] As a result, it was confirmed that there was not much difference in the storage effect when sucrose was replaced with dextrose, but when HEPES was replaced with NaHCO3, the storage effect was significantly reduced (Figure 6).

[0123] Based on the above results, it can be seen that a composition for refrigerated storage of mitochondria must contain HEPES, EGTA, and MgCl2 as essential components.

[0124] Example 5: Comparative evaluation of the refrigerated storage efficacy of novel mitochondrial storage compositions (1) In order to evaluate the refrigerated storage efficacy of the mitochondria storage composition prepared based on the optimal ingredient combination identified in Example 4, the following experiment was carried out.

[0125] First, a storage composition containing HEPES, EGTA, and MgCl2 (hereinafter referred to as HEM buffer) was prepared as confirmed in Example 4. As comparison groups, a composition containing sucrose, HEPES, EGTA, MgCl2, and KCl (hereinafter referred to as SHEMK buffer) and a composition containing dextrose, HEPES, EGTA, and MgCl2 (hereinafter referred to as DHEM buffer) were prepared. The composition and content of the buffers are shown in the table below. Furthermore, DW, DPBS, and saline were used as negative control storage compositions.

[0126] [Table 1]

[0127] 5-1: Evaluation of the efficacy of maintaining mitochondrial activity after refrigeration for 4 weeks Mitochondria isolated using the above-prepared composition and a control were stored in a refrigerator for 4 weeks (100 μl of buffer per 5 μg of isolated mitochondria, 4°C). To analyze the activity of the refrigerated mitochondria, the function of the electron transport complex was analyzed. Meanwhile, membrane-isolated mitochondria (fresh MTs) and damaged mitochondria (damaged MTs) that were not stored in a refrigerator / freezer were used as controls for activity comparison.

[0128] As a result, it was confirmed that the activity of refrigerated mitochondria maintained significantly better efficiency when HEM buffer was used than when other buffers and the control group were used (Figure 7).

[0129] 5-2: Evaluation of the efficacy of refrigerated stored mitochondria in restoring cellular metabolic function The mitochondria isolated using the above-prepared compositions and the control group were stored in a refrigerator for 4 weeks (100 μl of buffer per 5 μg of isolated mitochondria, 4° C.).

[0130] To confirm whether the refrigerated mitochondria could restore the metabolic function of cells, a cell-based assay was performed. Specifically, tenocytes were treated with TNF-α (20 ng / mL) for 24 hours to induce inflammation, and then the refrigerated mitochondria were treated and the function of electron transport complexes was compared.

[0131] As a result, we confirmed that HEM buffer can more effectively restore electron transport complex function in refrigerated mitochondria than other buffers or the control group (Figure 8).

[0132] 5-3: Evaluation of the membrane potential maintenance ability of refrigerated stored mitochondria Mitochondria isolated using the above-prepared composition and a control were stored in a refrigerator for 4 weeks (100 μl of buffer per 5 μg of isolated mitochondria, 4°C). To analyze the activity of the refrigerated mitochondria, the ability to maintain mitochondrial membrane potential was compared. Meanwhile, membrane-isolated mitochondria (fresh MTs) that were not stored in a refrigerator / freezer were used as a control for activity comparison.

[0133] To measure the membrane potential of the refrigerated mitochondria, analysis was performed using the mitochondrial-specific marker MitoTracker CMXRos Red dye (CMXRos; Thermofisher, USA) and Tetramethylrhodamine, Methyl Ester, Perchlorate (TMRM; Thermofisher). After 4 weeks of storage, the mitochondrial pellet was obtained by centrifugation at 20,000 x g for 10 minutes at 4°C. The supernatant was removed, and the mitochondrial pellet was washed twice with DPBS. The mitochondrial membrane potential was then measured by staining with the mitochondrial-specific markers CMXRos (200 nM) and TMRM (200 nM).

[0134] As a result, it was confirmed that mitochondria stored in a refrigerator using HEM buffer had significantly better membrane potential maintenance ability than mitochondria stored in other buffers (Figure 9).

[0135] 5-4: Evaluation of the efficacy of maintaining mitochondrial activity after long-term refrigeration Mitochondria isolated using the above-prepared composition and a control group were refrigerated for 12 weeks (100 μl of buffer per 5 μg of isolated mitochondria, 4°C), and the function of electron transport complexes was compared to analyze the activity of mitochondria stored for a long period of time.

[0136] As a result, it was confirmed that the activity of refrigerated stored mitochondria was maintained with significantly greater efficiency using HEM buffer than using other buffers and the control group (Figure 10).

[0137] 5-5: Evaluation of refrigerated storage efficacy according to mitochondrial origin To confirm whether the HEM buffer exhibits excellent refrigerated storage efficacy for mitochondria derived from various cells, the following experiment was carried out.

[0138] Specifically, umbilical cord-derived stem cell mitochondria (uMT), hepatocyte-derived mitochondria (wMT), and plasma-derived mitochondria (pMT) isolated and harvested in Examples 1 to 3 using the HEM buffer were refrigerated (100 μl of buffer per 5 μg of isolated mitochondria, 4°C), and their ATP synthesis capacities were compared and analyzed at 0, 1, and 4 weeks after refrigerated storage.

[0139] As a result, we confirmed that all mitochondria, regardless of their origin, retained their ability to synthesize ATP under refrigerated storage conditions using HEM storage solution (Figure 11).

[0140] Example 6: Comparative evaluation of the refrigerated storage efficacy of novel mitochondrial storage compositions (2) The following experiment was carried out to comparatively evaluate the refrigerated storage efficacy of the mitochondria storage compositions prepared based on the optimal ingredient combinations identified in Example 4.

[0141] First, a storage composition containing HEPES, EGTA, and MgCl2 was prepared as described in Example 4 (hereinafter referred to as HEM buffer). For comparison, a composition containing Tris instead of HEPES as a buffer (hereinafter referred to as TEM buffer), a composition containing EDTA instead of EGTA as a chelating agent (hereinafter referred to as HDM buffer), and a composition not containing magnesium ions (MgCl2) (hereinafter referred to as HE buffer) were prepared. The compositions and contents of the buffers are listed in the table below.

[0142] [Table 2]

[0143] The mitochondria isolated using the above-prepared composition and the control group were stored in a refrigerator for 4 weeks (100 μl of buffer per 5 μg of isolated mitochondria, 4°C), and the function of the electron transport complex was comparatively analyzed to analyze the activity of the refrigerated mitochondria.

[0144] As a result, it was confirmed that the activity of refrigerated mitochondrial samples was maintained with significantly greater efficiency when HEM buffer was used than when other buffers and the control group were used (Figure 12).

[0145] Based on the above results, it can be seen that the mitochondria storage composition of the present invention can exhibit significantly excellent storage effects by using HEPES as a buffering agent and EGTA as a chelating agent, and that magnesium ions are also a major component.

[0146] Example 7: Comparative evaluation of the refrigerated storage efficacy of novel mitochondrial storage compositions (3) The following experiment was carried out to comparatively evaluate the refrigerated storage efficacy of the mitochondria storage compositions prepared based on the optimal ingredient combinations identified in Example 4.

[0147] First, a storage composition containing HEPES, EGTA, and MgCl2 was prepared (hereinafter referred to as HEM buffer) as confirmed in Example 4. For comparison, compositions based on the components of previously known mitochondrial storage or loading solutions were prepared (D1, D2, D3, D4, and THEKB). The composition and contents of the buffers are listed in the table below.

[0148] [Table 3]

[0149] The mitochondria isolated using the above-prepared composition and the control group were stored in a refrigerator for 4 weeks (100 μl of buffer per 5 μg of isolated mitochondria, 4°C), and the function of the electron transport complex was comparatively analyzed to analyze the activity of the refrigerated mitochondria.

[0150] As a result, it was confirmed that the activity of refrigerated mitochondria was maintained significantly more efficiently using HEM buffer than using other buffers or the control group (Figure 13).

[0151] Example 8: Evaluation of transfer efficiency by mixing isolated mitochondria with hydrogel The following experiment was carried out to determine the mixing ratio of the isolated mitochondria stored under refrigeration in Example 4 and hydrogel, and to increase the efficiency of mitochondrial transfer by transplantation into target cells or tissues using the mixture.

[0152] First, the mitochondria stored in a refrigerator in Example 4 were mixed with the same volume of mitochondrial mixture and hyaluronic acid gel using a 3-way stopcock and a 1 ml syringe. The mixing conditions varied depending on the concentration of hyaluronic acid, and the results were determined by swelling and viscosity.

[0153] Next, to facilitate mitochondrial transfer into cells or tissues, the following experiment was carried out. Specifically, isolated mitochondria were stained with the specific marker MitoTracker dye green and transferred to target cells using centrifugation. 24 hours later, the intracellular mitochondrial transfer rate was measured using a flow cytometer. The results confirmed that the amount of intracellular mitochondrial transfer was increased when mitochondria were mixed with hyaluronic acid compared to the mitochondria alone treatment group.

[0154] Next, the mitochondria and hydrogel mixture was subcutaneously implanted into animals, and the expression of mitochondrial-specific mtDNA in the tissue was compared. As a result, it was confirmed that the mitochondria in the target tissue were transferred, and remained in the tissue for up to 14 days after administration.

[0155] The above description of the present invention is merely an example, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. Therefore, it should be understood that the above-described embodiment is illustrative in all respects and is not limiting.

Claims

1. HEPES (Hydroxyethyl piperazine Ethane Sulfonic acid), chelating agent and magnesium ion (Mg ２＋ ) A composition for storing isolated mitochondria, comprising one or more selected from the group consisting of:

2. 2. The composition of claim 1, wherein the mitochondria are isolated from one or more selected from the group consisting of tissue, plasma, bone marrow, cerebrospinal fluid, umbilical cord blood, peripheral blood, cells, and organelles.

3. 3. The composition of claim 2, wherein the cells and organelles are derived from one or more selected from the group consisting of stem cells, somatic cells, germ cells, and platelets.

4. The composition of claim 1 , wherein the storage is refrigerated or frozen.

5. The composition of claim 1 , wherein the chelating agent comprises EGTA (Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid).

6. The magnesium ions are MgCl ２ , MgSO ４ , Mg(OAc) ２ and Mg(NO ３ ) ２ The composition of claim 1, wherein the composition is contained in one or more forms selected from the group consisting of:

7. The composition of claim 1, wherein the composition is for preserving the activity of isolated mitochondria.

8. 10. The composition of claim 1, wherein the composition does not contain one or more selected from the group consisting of sugar, bicarbonate, KCl, and albumin.

9. 1) providing mitochondria isolated from a mitochondria-containing sample; 2) mixing the isolated mitochondria with the storage composition of claim 1; 3) storing the mixture in a refrigerator or freezer.

10. A pharmaceutical composition for treating or preventing a disease caused by a mitochondrial functional defect, comprising isolated mitochondria, A composition, wherein the isolated mitochondria have been stored using the storage composition of claim 1.

11. A mitochondrial transplant composition comprising isolated mitochondria, A composition, wherein the isolated mitochondria have been stored using the storage composition of claim 1.

12. A hydrogel comprising isolated mitochondria and a biocompatible polymer, A hydrogel, wherein the isolated mitochondria have been stored using the storage composition of claim 1.

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