Frozen and freeze-dried mitochondria and uses thereof

Abstract

The present invention relates to a method for freezing and / or lyophilizing mitochondria isolated from cells, and the use of frozen and / or lyophilized mitochondria. The frozen mitochondria according to the present invention showed similarity to unfrozen mitochondria in terms of anti-inflammatory activity and energy metabolism-related enzyme activity. The lyophilized mitochondria also showed similar levels of anti-inflammatory activity, energy metabolism-related enzyme activity, and long-term storage effect after recovery to mitochondria before lyophilization. Therefore, it is expected that the frozen and / or lyophilized mitochondria according to the present invention will greatly enhance the commercial availability of therapeutic agents containing mitochondria, by enabling isolated mitochondria to find a wide range of applications in the treatment of various diseases caused by mitochondrial dysfunction or disorder.

Description

[Technical field]

[0001] The present invention relates to a method for freezing and / or lyophilizing mitochondria isolated from cells, and uses of the frozen and / or lyophilized mitochondria. More specifically, the present invention relates to a method for freezing and / or lyophilizing mitochondria, and to pharmaceutical compositions and long-term storage solution compositions containing, as active ingredients, mitochondria frozen and / or lyophilized by this method. [Background technology]

[0002] Freeze-drying is a drying method in which a sample containing water is frozen and placed under reduced pressure to sublimate and remove the water from the sample. It is used for long-term preservation of water-containing substances such as plant or animal cells or tissues, or food.

[0003] However, when freezing a substance containing water, water molecules form ice crystals during freezing, and the solute or impurity medium in the substance containing water diffuses unevenly, resulting in freeze concentration, so cryoprotectants are used to improve the stability of the tissue. However, in the case of eukaryotic cells, even if cryoprotectants are used, it is difficult to stably preserve the cells without damaging the cell structure such as the cell membrane during freeze-drying, and therefore there is a problem that the function of the active ingredient in the cells cannot be maintained.

[0004] Mitochondria play an important role in various physiological processes such as the synthesis of ATP as an energy source, the production of reactive oxygen species, and apoptosis. Therefore, mitochondrial damage can cause various diseases, and most mitochondrial disorders are caused by inherited or acquired mutations in mitochondrial DNA. For example, mitochondrial function can be altered by swelling caused by abnormal mitochondrial membrane potential, oxidative stress caused by reactive oxygen species or free radicals, and defective oxidative phosphorylation function for energy production in mitochondria. These mitochondrial dysfunctions have been reported to cause mitochondrial genetic diseases, inflammatory diseases such as chronic rheumatoid arthritis, ischemic diseases, infectious diseases, heart diseases, muscle diseases, degenerative diseases such as Parkinson's disease and Alzheimer's disease, and the development and metastasis of various cancers.

[0005] In order to treat these diseases associated with mitochondrial dysfunction, there have been increasing attempts recently to isolate mitochondria from cells or tissues and reinject them into the body for treatment (Korean Patent No. 10-2126199, Korean Patent No. 10-2019277).

[0006] Although various studies have been conducted to show the possibility of using mitochondria to treat certain diseases, the development of a method for stably maintaining and storing mitochondria and application technology for a stable dosage form is insufficient. Therefore, it is necessary to develop a new method for stably maintaining and storing mitochondria and the bioactive factors contained therein. Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, the present inventors have studied a method for efficiently preserving mitochondria for transplantation therapy as part of the treatment of mitochondrial-related diseases. As a result, the present inventors have found that mitochondria freeze-dried by the method of the present invention can be preserved at room temperature and at low temperatures, and that frozen and / or freeze-dried mitochondria maintain the activity of mitochondria before freezing. Based on the above, the present inventors have completed the present invention. [Means for solving the problem]

[0008] In order to solve the above problems, one aspect of the present invention provides frozen or lyophilized mitochondria, a pharmaceutical composition containing the same, and a long-term storage solution composition kit.

[0009] In another aspect of the invention, methods for stably freezing or lyophilizing isolated mitochondria and a cryoprotectant, as well as frozen compositions comprising the cryoprotectant and mitochondria, are provided.

[0010] In another aspect of the invention there is provided a use of isolated frozen or lyophilized mitochondria for the prevention or treatment of a mitochondrial associated disease.

[0011] Another aspect of the invention provides a method for preventing or treating a mitochondrial associated disease comprising administering isolated frozen or lyophilized mitochondria. Effect of the Invention

[0012] It was confirmed that the frozen and / or lyophilized mitochondria according to the present invention show similarity to non-frozen mitochondria. Moreover, the frozen and / or lyophilized mitochondria showed similarity to non-frozen mitochondria in terms of anti-inflammatory activity and activity of energy metabolism-related enzymes. Furthermore, the lyophilized mitochondria showed a similar level of long-term storage efficacy to non-freeze-dried mitochondria. Therefore, it is expected that the frozen and / or lyophilized mitochondria according to the present invention will greatly enhance the commercial availability of therapeutic agents containing mitochondria by enabling isolated mitochondria to find a wide range of applications in the treatment of various diseases caused by mitochondrial dysfunction or disorder. [Brief description of the drawings]

[0013] [Figure 1] 1 is a graph showing the results of confirming the anti-inflammatory efficacy of frozen platelet-derived mitochondria. [Diagram 2] FIG. 1 is a graph showing the results of confirming the anti-inflammatory efficacy of umbilical cord-derived mesenchymal stem cells or mitochondria derived from platelets isolated from human blood, with or without freezing. [Diagram 3] 1 is a graph showing the results of measuring the mitochondrial protein concentration before freeze-drying and the mitochondrial protein concentration after volume recovery after freeze-drying. [Figure 4] FIG. 1 shows the results of confirming the location of mitochondria after applying mitochondria isolated from HEK293 cells and freeze-dried to cultured cells. [Diagram 5] 1 is a graph showing the results of measuring the activity of mitochondrial energy synthesis-related enzymes in mitochondria isolated from platelets, with or without freeze-drying. [Figure 6] This is a graph showing the results of confirming the anti-inflammatory effect of mitochondria isolated from platelets with and without freeze-drying. [Figure 7] 1 is a graph showing the results of confirming the anti-inflammatory effect of mitochondria isolated from platelets or umbilical cord-derived mesenchymal stem cells as a function of concentration with or without lyophilization. [Figure 8] FIG. 1 shows a diagram (top) and a graph (bottom) comparing the cardiac protective efficacy depending on whether mitochondria contained in a long-term storage solution were freeze-dried or not. [Figure 9] FIG. 1 shows the results of Western blotting to confirm the concentration of cytochrome C in the supernatant of each mixed preservation solution after freezing and thawing mitochondria isolated from umbilical cord-derived mesenchymal stem cells mixed with a TTG mixed preservation solution or an SHE mixed preservation solution. [Figure 10] Graph showing the results of measuring the activity of each mitochondrial energy synthesis-related enzyme after freezing and thawing mitochondria isolated from umbilical cord-derived mesenchymal stem cells mixed with TT mixed preservation solution, TTG mixed preservation solution, TT mixed preservation solution containing DMSO (TT+DMSO), TTG mixed preservation solution containing DMSO (TTG+DMSO), or SHE mixed preservation solution. *p<0.05, **p<0.01, ***p<0.001 vs. CTR, #p<0.05, ##p<0.05, ###p<0.001 vs. TTG. [Figure 11] 1 is a graph showing the results of confirming the anti-inflammatory efficacy of each mitochondrion after freezing and thawing mitochondria isolated from umbilical cord-derived mesenchymal stem cells mixed with TTG mixed preservation solution or SHE mixed preservation solution. *p<0.05, **p<0.01, ***p<0.001 vs. CTR, #p<0.05, ##p<0.05, ###p<0.001 vs. TTG. [Figure 12] FIG. 1 shows the results of confirming the platelet aggregation inhibitory effect of each mitochondrion after freezing and thawing mitochondria isolated from umbilical cord-derived mesenchymal stem cells mixed with a TTG mixed preservation solution or an SHE mixed preservation solution. [Figure 13] This figure shows the results of confirming changes in total mitochondrial protein amount, ATP synthesis rate, staining rate of a mitochondrial-specific staining reagent, and Tom20 expression level after freezing and thawing mitochondria isolated from umbilical cord-derived mesenchymal stem cells mixed with a TTG mixed preservation solution one month later. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Frozen and / or freeze-dried mitochondria In one aspect of the invention, frozen and / or lyophilized (lyophilized) mitochondria are provided.

[0015] As used herein, the term "mitochondria" refers to the organelles of eukaryotic cells that are involved in the synthesis and regulation of adenosine triphosphate (ATP), the intracellular energy source. Mitochondria are involved in the control of various metabolic pathways in vivo, such as cell signaling, cell differentiation, cell death, and cell cycle and cell proliferation. Thus, mitochondrial dysfunction or disorder due to genetic, environmental, or unknown causes has been reported to be related to the development of various diseases, such as mitochondrial-related genetic diseases, inflammatory diseases such as rheumatoid arthritis, ischemic diseases, infectious diseases, heart diseases, muscle diseases, degenerative diseases such as Parkinson's disease or Alzheimer's disease, and the development and metastasis of various cancers.

[0016] The mitochondria may be obtained from a mammal or from a human. Specifically, the mitochondria may be isolated from cells or tissues, or from blood cells or platelets. Furthermore, the mitochondria may be normal mitochondria obtained from cells with normal mitochondrial biological activity. In this case, the mitochondria may be isolated and used after enrichment and disruption of tissue or cells, or may be isolated and disrupted from a tissue or cell sample that has been frozen and then thawed. Furthermore, the mitochondria may be isolated from a sample that has been frozen and then thawed after culturing cells or tissues in vitro. For example, the mitochondria may be obtained from somatic cells, germ cells, or stem cells.

[0017] In particular, the somatic cell may be a muscle cell, a liver cell, a nerve cell, a fibroblast, an epithelial cell, an adipocyte, a bone cell, a leukocyte, a lymphocyte, a platelet, or a mucosal cell.

[0018] Furthermore, the stem cells are undifferentiated cells capable of differentiating into various types of tissue cells, and 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, corneal epithelial stem cells, and tissue-derived stem cells, but are not limited thereto. In this case, the mesenchymal stem cells may be obtained from any one selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, synovial fluid, testis, amniotic membrane, and placenta.

[0019] Furthermore, mitochondria may be obtained from autologous, allogeneic, or xenogeneic subjects. Specifically, autologous mitochondria refers to mitochondria obtained from tissues or cells of the same subject. Furthermore, allogeneic mitochondria refers to mitochondria obtained from a subject belonging to the same species as the subject, but with a different allelic genotype. Furthermore, heterologous mitochondria refers to mitochondria obtained from a subject belonging to a different species than the subject. Furthermore, heterologous mitochondria refers to mitochondria obtained from a subject belonging to a different species than the subject.

[0020] Furthermore, the mitochondria may be mitochondria isolated from a cell. Furthermore, the mitochondria may be intact and may have mitochondrial activity.

[0021] The frozen mitochondria of the present invention can be thawed in a refrigerator. Specifically, the mitochondria can be thawed at about 0°C to about 10°C. Furthermore, the thawed mitochondria can be stored in a refrigerator. Specifically, the mitochondria can be stored at about 0°C to about 10°C.

[0022] The freeze-dried mitochondria of the present invention can be stored at room temperature or in a refrigerator. Specifically, the mitochondria can be stored at about 0° C. to about 37° C. The mitochondria can be stored at about 0° C. to about 8° C., about 9° C. to about 15° C., about 16° C. to about 22° C., about 23° C. to about 29° C., or about 30° C. to about 37° C.

[0023] Furthermore, freeze-dried mitochondria can exhibit mitochondrial activity when recovered and used.

[0024] As used herein, the term "restoration" refers to the restoration of lyophilized mitochondria to their original state, which is accomplished by adding an aqueous solution to the lyophilized mitochondria. In this case, the aqueous solution may be water for injection, saline, phosphate buffer, purified water, or deionized water. Deionized water may be preferred.

[0025] In this case, the above-mentioned restoration process can be carried out at room temperature or in a refrigerator. Specifically, it can be carried out at about 0°C to about 37°C. Mitochondria can be heated at about 0°C to about 8°C, about 9°C to about 15°C, about 16°C to about 22°C, about 23°C to about 29°C, or about 30°C to about 37°C. Furthermore, the above-mentioned restoration process can be carried out for about 1 minute to about 1 hour, about 1 minute to about 50 minutes, about 1 minute to about 40 minutes, about 1 minute to about 30 minutes, about 1 minute to about 20 minutes, about 1 minute to about 10 minutes, or about 1 minute to about 5 minutes. The above-mentioned restoration process is not particularly limited as long as it restores freeze-dried mitochondria and dries them.

[0026] In one embodiment of the present invention, it was confirmed that when freeze-dried mitochondria were restored and used, the activity of energy synthesis-related enzymes present in the mitochondria was equivalent to that of non-freeze-dried mitochondria (Figure 5).

[0027] Furthermore, frozen and / or lyophilized mitochondria can exhibit anti-inflammatory and protective effects on isolated organs. In one embodiment of the present invention, it was confirmed that when macrophages were treated with frozen and / or lyophilized mitochondria derived from platelets or umbilical cord-derived mesenchymal stem cells to induce an inflammatory response, the induction of an inflammatory response was suppressed (FIGS. 1, 2, 6, and 7). Furthermore, when isolated hearts were stored in a long-term storage solution containing lyophilized mitochondria, cardioprotective effects were observed (FIG. 8).

[0028] Freezing and freeze-drying methods In another aspect of the invention, a method is provided for stably freezing and / or lyophilizing isolated mitochondria.

[0029] The freezing method may include isolating mitochondria and freezing the isolated mitochondria by treating them with a mitochondrial cryoprotectant comprising glycine, where the cryoprotectant may further comprise any one selected from the group consisting of trehalose, Tris, and combinations thereof.

[0030] Furthermore, the freeze-drying method can include isolating mitochondria, treating the isolated mitochondria with a mitochondrial cryoprotectant containing glycine to freeze them, and freeze-drying the frozen mitochondria, where the mitochondria are as described above. In this case, the cryoprotectant can further include any one selected from the group consisting of trehalose, Tris, and combinations thereof.

[0031] When isolating mitochondria from particular cells, the mitochondria can be isolated by a variety of modified methods, including various known methods such as, for example, using specific buffers or using potential differences and magnetic fields.

[0032] From the viewpoint of maintaining mitochondrial activity, the isolation of mitochondria can be performed by disrupting tissues or cells and centrifuging them. In one embodiment, the isolation of mitochondria can be performed by culturing cells, performing a first centrifugation on a composition containing the cells to produce a pellet, resuspending the pellet in a buffer solution, homogenizing it, performing a second centrifugation on the homogenized solution to produce a supernatant, and performing a third centrifugation on the supernatant to purify mitochondria. In this case, from the viewpoint of maintaining cellular activity, the time for performing the second centrifugation is preferably adjusted to be shorter than the time for performing the first and third centrifugations, and the speed may be increased between the first and third centrifugations.

[0033] Specifically, the first to third centrifugations can be carried out at a temperature of about 0° C. to about 10° C., preferably about 3° C. to about 5° C. Furthermore, the time for carrying out the centrifugation may be about 1 minute to 50 minutes, and can be appropriately adjusted depending on the number of centrifugations and the contents of the sample.

[0034] Furthermore, the first centrifugation can be performed at a speed of about 100×g to 1,000×g, about 200×g to 700×g, or about 300×g to 450×g. Furthermore, the second centrifugation can be performed at a speed of about 1×g to 2,000×g, about 25×g to 1,800×g, or about 500×g to 1,600×g. Furthermore, the third centrifugation can be performed at a speed of about 100×g to 20,000×g, about 500×g to 18,000×g, or about 800×g to 15,000×g.

[0035] Furthermore, pharmaceutically acceptable sugars can be used to stabilize the obtained mitochondria.Specifically, the sugars can be, but are not limited to, sucrose, mannitol, trehalose, etc. Furthermore, the pH buffer can be, but is not limited to, pharmaceutically acceptable Tris, HEPES (hydroxyethylpiperazineethanesulfonic acid), phosphate, etc.

[0036] On the other hand, after obtaining mitochondria, additives such as chelating agents and antioxidants can be used to remove damage caused by ion outflow and suppress oxidative stress, and these can be used without limitation, including reagents known in the art, such as, but not limited to, EDTA, EGTA, citric acid, glycine, taurine, ATP, etc.

[0037] Furthermore, from the viewpoint of maintaining mitochondrial activity, the isolation of mitochondria can be carried out by thawing and disrupting frozen cells or tissues, followed by centrifugation. The method for obtaining mitochondria can be carried out by freezing cells or tissues, thawing the cells or tissues, and disrupting the thawed cells or tissues.

[0038] Specifically, in the present invention, the isolated mitochondria can be mixed with a mitochondrial cryoprotectant containing glycine and frozen. In this case, the cryoprotectant can further contain any one selected from the group consisting of trehalose, tris, and combinations thereof. In the present invention, the cryoprotectant can be used in the form of an aqueous solution, which can be water for injection, physiological saline, phosphate buffer, purified water, or deionized water.

[0039] When mitochondria are frozen, the cryoprotectant can maintain and / or improve the stability of the mitochondria and can also stably maintain the activity of the mitochondria.

[0040] The cryoprotectant of the present invention may contain glycine. Glycine may be contained in the cryoprotectant at a concentration of about 15 mM or more, but is not limited thereto. Specifically, glycine may be contained at a concentration of about 15 mM to about 150 mM, about 17 mM to about 130 mM, about 20 mM to about 120 mM, about 22 mM to about 110 mM, or about 25 mM to about 100 mM. Furthermore, glycine may be used together with at least one amino acid selected from the group consisting of, but not limited to, histidine, isoleucine, leucine, lysine acetate, methionine, phenylalanine, threonine, tryptophan, valine, alanine, arginine, aspartic acid, cysteine, glutamic acid, proline, serine, and tyrosine.

[0041] The cryoprotectant may further contain a sugar. In this case, the sugar may be at least one sugar selected from the group consisting of sucrose, trehalose, mannitol, sorbitol, glucose, fructose, mannose, maltose, lactose, isomaltose, dextran, and dextrin. Preferably, the sugar may be trehalose. Specifically, trehalose in the cryoprotectant may be contained at a concentration of about 40 mM to about 400 mM, about 50 mM to about 350 mM, about 60 mM to about 300 mM, about 70 mM to about 250 mM, or about 80 mM to about 200 mM.

[0042] The cryoprotectant may further comprise a buffer. The buffer contained in the injectable liquid composition may be selected from the group consisting of Tris buffer, HEPES buffer, MOPS (3-(N-morpholino)propanesulfonic acid) buffer, and acetate or phosphate-containing buffer, but is not limited thereto. In this case, the pH of the buffer may be in the range of about 7.0 to about 7.8, about 7.2 to about 7.6, or about 7.3 to about 7.5, but is not limited thereto. Preferably, in the present invention, the buffer may be a Tris buffer. Specifically, the cryoprotectant may be contained at a concentration of about 5 mM to about 50 mM, about 8 mM to about 40 mM, about 10 mM to about 35 mM, about 13 mM to about 30 mM, or about 15 mM to about 25 mM.

[0043] More specifically, the cryoprotectant of the present invention may include glycine and may further include any one selected from the group consisting of trehalose, tris, and combinations thereof. In one embodiment, the cryoprotectant may include glycine and trehalose. The cryoprotectant may include glycine and tris. In one embodiment, the cryoprotectant may include glycine, trehalose, and tris. In one embodiment of the present invention, a mixed preservation solution containing glycine, trehalose, and tris (TTG) reduced damage to mitochondria during the freezing and thawing steps compared to a mixed preservation solution containing trehalose and tris (TT) (Figures 9 to 12).

[0044] Additionally, the cryoprotectant may further comprise a chelating agent.

[0045] The chelating agent may be, but is not limited to, at least one chelating agent selected from the group consisting of injectable grade EGTA (ethylene glycol tetraacetic acid), EDTA (ethylenediaminetetraacetic acid) and BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). The chelating agent can suppress damage caused by ion flux after obtaining mitochondria contained in the injectable liquid composition.

[0046] Additionally, the cryoprotectant may contain additives such as antioxidants, ATP, magnesium, etc., which are effective in maintaining mitochondrial function and activity.

[0047] Freezing can be performed at a temperature range of, but not limited to, −1° C. or lower. Specifically, freezing can be performed at a temperature range of about −5° C. to about −200° C., about −15° C. to about −180° C., about −25° C. to about −160° C., about −40° C. to about −140° C., about −55° C. to about −120° C., about −60° C. to about −100° C., or about −70° C. to about −90° C.

[0048] Freezing can be performed using liquid nitrogen (LN2) or a freezing device. Specifically, it can be performed by using LN2 for quick freezing during the refrigeration or freezing process, or by using a freezing device such as a deep freezer or a freezer. Freezing is not particularly limited as long as it can refrigerate or freeze mitochondria. When freezing mitochondria using a freezing device, a freezing container can be used. The freezing container can freeze cells by lowering the temperature by about 1°C per minute. Preferably, when using a deep freezer, a freezing container can be used. Furthermore, when freezing mitochondria using a freezing device, it is not necessary to use a freezing container.

[0049] Freezing can be carried out for about 24 hours or more, about 48 hours or more, or about 96 hours or more, but is not particularly limited as long as the frozen mitochondria have normal activity.

[0050] Freeze-drying can be performed by dividing the process into freezing and drying mitochondria, but is not limited thereto. In this case, freeze-drying can be performed under vacuum. Freeze-drying of mitochondria can be performed by gradually increasing the temperature after freezing mitochondria under vacuum.

[0051] When lyophilizing mitochondria, the freezing step can be carried out at about -40°C or lower. Specifically, the freezing can be carried out at about -80°C to about -40°C, about -70°C to about -40°C, about -60°C to about -40°C, or about -50°C to about -40°C. In this case, the freezing can be carried out for about 5 minutes to about 120 minutes, about 10 minutes to about 100 minutes, about 15 minutes to about 80 minutes, about 20 minutes to about 60 minutes, or about 25 minutes to about 40 minutes. In one embodiment of the present invention, the freezing was carried out at about -40°C for 30 minutes.

[0052] When freeze-drying mitochondria, the drying step can be carried out in three stages. The first stage of drying can be carried out at about -30°C to about 0°C. More specifically, it can be carried out at about -40°C to about 0°C, about -30°C to about -5°C, or about -20°C to about -10°C. In this case, the first stage of drying can be carried out for about 1 hour to about 50 hours. Specifically, it can be carried out for about 1 hour to about 50 hours, about 5 hours to about 50 hours, about 10 hours to about 50 hours, about 20 hours to about 50 hours, or about 30 hours to about 50 hours. In one embodiment of the present invention, the first stage of drying is carried out at about -20°C for 25 hours, and further carried out at about -20°C for 8.3 hours depending on the state of the mitochondria.

[0053] The second stage drying can be carried out at about 0°C to about 10°C or lower. More specifically, it can be carried out at about 0°C to about 10°C, about 2°C to about 9°C, about 4°C to about 8°C, or about 5°C to about 7°C. In this case, the second stage drying can be carried out for about 1 hour to about 24 hours. Specifically, the second stage drying can be carried out for about 1 hour to about 16 hours, about 2 hours to about 16 hours, about 4 hours to about 16 hours, about 6 hours to about 16 hours, or about 8 hours to about 16 hours. In one embodiment of the present invention, the second stage drying was carried out at about 6°C for 8.3 hours.

[0054] The third stage drying can be carried out at about 11° C. to about 30° C. or lower. More specifically, it can be carried out at about 11° C. to about 30° C., about 15° C. to about 30° C., about 20° C. to about 30° C., or about 25° C. to about 30° C. In this case, the third stage drying can be carried out for about 1 hour to about 18 hours. Specifically, the third stage drying can be carried out for about 1 hour to about 18 hours, about 3 hours to about 18 hours, about 6 hours to about 18 hours, or about 9 hours to about 18 hours. In one embodiment of the present invention, the third stage drying was carried out at about 25° C. for 9 hours.

[0055] The concentration of mitochondria mixed with the cryoprotectant may be about 0.1 mg to about 10 mg, about 0.2 mg to about 9 mg, about 0.3 mg to about 8 mg, about 0.4 mg to about 7 mg, about 0.5 mg to about 6 mg, about 0.6 mg to about 5 mg, about 0.7 mg to about 4 mg, about 0.8 mg to about 3 mg, or about 0.9 mg to about 2 mg per mL.

[0056] Additionally, the frozen mitochondria can be lyophilized.

[0057] Cryoprotectants and cryocompositions In another aspect of the present invention, there is provided a mitochondrial cryoprotectant comprising glycine, and a freezing composition comprising the cryoprotectant and mitochondria, wherein the cryoprotectant may further comprise any one selected from the group consisting of trehalose, tris, and combinations thereof.

[0058] Mitochondria and cryoprotectants were as previously described.

[0059] Pharmaceutical Compositions In another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating a disease, comprising frozen and / or lyophilized mitochondria as an active ingredient.

[0060] In this case, the use of the pharmaceutical composition may be for preventing or treating a mitochondrial-related disease.

[0061] As used herein, the term "mitochondrial-related disease" refers to diseases related to mitochondrial dysfunction or disorder due to genetic, environmental, or unknown causes.For example, diseases include, but are not limited to, genetic diseases such as Leber's hereditary optic neuropathy (LHON), Leigh syndrome, myoclonus epilepsy with ragged-red fibers (MERRF), mitochondrial myopathy, encephalopathy, lactacidosis, and stroke (MELAS), degenerative diseases such as Parkinson's disease and Alzheimer's disease, metabolic diseases such as diabetes and obesity, inflammatory diseases such as sepsis, rheumatoid arthritis, osteoarthritis, and systemic inflammatory response syndrome (SIRS), hearing loss, cancer, ischemic disease, neurological disease, heart disease, muscle disease, degenerative disease, fibrotic disease, eye disease, hair loss, autoimmune diseases of the nervous system, etc.

[0062] The cancer may be any one selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, glioblastoma, gastric cancer, liver cancer, colon cancer, prostate cancer, ovarian cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma, but is not limited thereto.

[0063] Further, the ischemic disease includes, but is not limited to, severe limb ischemia, ischemic stroke, ischemic heart disease, ischemic colitis, etc. In this case, the ischemic disease may be a disease caused by mitochondrial abnormality, and includes all ischemic cell damage caused by mitochondrial disorder.

[0064] Neurological disorders include, but are not limited to, epilepsy, drug-resistant epilepsy, bipolar disorder or bipolar manic depression, headache, migraine, anxiety disorder, attention deficit disorder, addictive disorders, personality disorders, autism, traumatic brain injury, Friedreich's ataxia, optic atrophy, and the like.

[0065] Further, infectious diseases include, but are not limited to, hepatitis B, hepatitis C, human papillomavirus (HPV) infection, cytomegalovirus infection, viral respiratory diseases, influenza virus infection, and the like.

[0066] Further, muscle diseases include, but are not limited to, MELAS syndrome, MERRF syndrome, Kearns-Sayre syndrome, myopathy, encephalomyopathy, myasthenia, myasthenia gravis, amyotrophic lateral sclerosis, muscular dystrophy, muscle atrophy, hypotonia, muscle weakness, myotonia, etc. In this case, muscle diseases can include all muscle cell disorders caused by mitochondrial disorders.

[0067] Degenerative diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease (amyotrophic lateral sclerosis), Huntington's disease, multiple sclerosis, brain dysfunction due to immune system disorders, progressive neurodegenerative diseases, metabolic brain diseases, Niemann-Pick disease, and dementia due to cerebral ischemia and cerebral hemorrhage.

[0068] Eye diseases include, but are not limited to, glaucoma, diabetic retinopathy, age-related macular degeneration, and the like.

[0069] Autoimmune diseases of the nervous system include, but are not limited to, multiple sclerosis, neuromyelitis optica, acute disseminated encephalomyelitis, ascending myelitis, central myelitis, descending myelitis, transverse myelitis, myasthenia gravis, Guillain-Barre syndrome, autoimmune uveitis, autoimmune encephalopathy, and chronic inflammatory demyelinating polyneuropathy.

[0070] In the pharmaceutical composition of the present invention containing frozen and / or lyophilized mitochondria as an active ingredient, mitochondria may be contained at a concentration of about 0.1 μg / mL to about 500 μg / mL, about 0.2 μg / mL to about 450 μg / mL, or about 0.5 μg / mL to about 400 μg / mL, but is not limited thereto. By containing mitochondria in the above range, it is possible to easily adjust the dose of mitochondria at the time of administration, and it is possible to further improve the degree of symptom improvement of the patient's mitochondria-related disease, specifically inflammatory disease. In this case, the dose of mitochondria can be determined through mitochondrial quantification by quantifying membrane proteins after thawing frozen mitochondria or recovering lyophilized mitochondria. Specifically, isolated mitochondria can be quantified through Bradford protein analysis [reference by James D. McCully (J Vis Exp. 2014; (91): 51682)].

[0071] In particular, the pharmaceutical composition according to the present invention can be administered in an amount of about 0.01 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 4 mg / kg, or about 0.25 mg / kg to about 2.5 mg / kg per dose based on the body weight of the subject to be administered, but is not limited thereto. That is, it is most preferable in terms of cell activity to administer the pharmaceutical composition together with the amount of mitochondria in the above range based on the body weight of the subject suffering from the mitochondrial-related disease.

[0072] Furthermore, the pharmaceutical composition can be administered 1 to 10 times, 3 to 8 times, or 5 to 6 times, preferably 5 times. In this case, the administration interval may be 1 to 7 days or 2 to 5 days, preferably 3 days.

[0073] As used herein, the term "prevention" refers to any action of administering a pharmaceutical composition to inhibit or delay the onset of cancer. Additionally, "treatment" refers to any action of administering a pharmaceutical composition to improve or beneficially alter the symptoms of cancer.

[0074] As used herein, the term "efficacy" can be determined by one or more parameters, such as survival or disease-free survival over a period of 1 year, 5 years, 10 years, etc. Additionally, the parameters can include inhibition of the size of at least one tumor in a subject.

[0075] The preferred dosage of the pharmaceutical composition varies depending on the patient's condition and body weight, the severity of the disease, the form of the drug, the route of administration and the duration, but can be appropriately selected by those skilled in the art. In the pharmaceutical composition for preventing or treating cancer of the present invention, the active ingredient may be contained in any amount (effective amount) depending on the use, dosage form, purpose of combination, etc., as long as it can exhibit anti-cancer activity. Here, the "effective amount" refers to the amount of active ingredient that can induce anti-cancer effect. Such an effective amount can be determined experimentally within the scope of the ordinary ability of a person skilled in the art.

[0076] The pharmaceutical composition of the present invention may further comprise a pharma- ceutically acceptable carrier. The pharma- ceutically acceptable carrier may be any carrier as long as it is a non-toxic material suitable for delivery to a patient. Distilled water, alcohol, fat, wax and inert solids may be included as carriers. In addition, pharma- ceutically acceptable auxiliary agents (buffers, dispersants) may be included in the pharmaceutical composition.

[0077] Specifically, the pharmaceutical composition contains a pharma- ceutical acceptable carrier in addition to the active ingredient, and can be prepared as a parenteral preparation according to the administration route by a conventional method known in the art. Here, "pharma- ceutical acceptable" means that it does not inhibit the activity of the active ingredient and does not exceed the toxicity that can be applied to the application (formulation) target.

[0078] When the pharmaceutical composition of the present invention is prepared as a parenteral preparation, it can be formulated in the form of an injection, a transdermal preparation and a nasal inhalant with a suitable carrier by a method known in the art.When it is formulated as an injection, the suitable carrier can be sterilized water, ethanol, polyol such as glycerol or propylene glycol, or a mixture thereof, and preferably, Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine, or isotonic solution such as sterilized water for injection, 5% dextrose, etc.

[0079] The pharmaceutical composition of the present invention may be an injection preparation. Therefore, the pharmaceutical composition of the present invention can be prepared as a physically or chemically very stable injection by adjusting pH using an acidic aqueous solution or a buffer such as phosphoric acid, which can be used as an injection, to ensure the stability of the product according to the distribution of the injection to be formulated.

[0080] Specifically, the pharmaceutical composition of the present invention may comprise water for injection.

[0081] Water for injection refers to distilled water prepared for dissolving a solid injection or diluting a water-soluble injection, and may be, for example, glucose injection, xylitol injection, D-mannitol injection, fructose injection, physiological saline, dextran 40 injection, dextran 70 injection, amino acid injection, Ringer's solution, lactate-Ringer's solution, or phosphate buffer or sodium dihydrogen phosphate-citrate buffer with a pH of about 3.5 to about 7.5.

[0082] The pharmaceutical composition of the present invention can further comprise a stabilizer or solubilizer.For example, the stabilizer can be pyrosulfite or ethylenediaminetetraacetic acid, and the solubilizer can be hydrochloric acid, acetic acid, potassium phosphate hydroxide, potassium bicarbonate, potassium carbonate, or Tris.In one embodiment, the pharmaceutical composition can comprise a mixed preservative solution, such as TTG (trehalose-tris-glycine) solution, which can be commonly used in pharma-ceutically acceptable drug formulations.

[0083] Specifically, the pharmaceutical composition of the present invention may contain a liquid composition for injection in addition to frozen and / or lyophilized mitochondria. In this case, the frozen and / or lyophilized mitochondria are as described above. The pharmaceutical composition of the present invention contains a liquid composition for injection, thereby forming a composition for preventing or treating diseases related to mitochondrial function, and when mitochondria are administered by injection, it can suppress thrombosis that may occur due to mitochondrial aggregation, thrombocytopenia and aggregation, and can maintain and / or improve mitochondrial stability and stably maintain mitochondrial activity.

[0084] Here, the liquid composition may contain glycine, and may further contain a sugar and / or a buffer.

[0085] Glycine may be present in the injectable liquid composition at a concentration of about 15 mM or more, but is not limited thereto, specifically, at a concentration of about 15 mM to about 150 mM, about 17 mM to about 130 mM, about 20 mM to about 120 mM, about 22 mM to about 110 mM, or about 25 mM to about 100 mM. Furthermore, glycine may be used together with at least one amino acid selected from the group consisting of, but not limited to, histidine, isoleucine, leucine, lysine acetate, methionine, phenylalanine, threonine, tryptophan, valine, alanine, arginine, aspartic acid, cysteine, glutamic acid, proline, serine, and tyrosine.

[0086] Furthermore, the sugar contained in the injectable liquid composition may be one or more selected from the group consisting of sucrose, trehalose, mannitol, sorbitol, glucose, fructose, mannose, maltose, lactose, isomaltose, dextran and dextrin, but is not limited thereto. In particular, the sugar may be trehalose, mannitol or sucrose. Preferably, the sugar may be trehalose.

[0087] The buffer solution contained in the injectable liquid composition may be selected from the group consisting of, but is not limited to, Tris buffer, HEPES buffer, MOPS buffer, and acetate or phosphate-containing buffer.Preferably, the buffer solution may be an injectable Tris buffer.

[0088] Preferably, the liquid composition may include glycine, trehalose, and Tris.

[0089] In this case, the pH of the buffer solution may be, but is not limited to, in the range of about 7.0 to about 7.8, in the range of about 7.2 to about 7.6, or in the range of about 7.3 to about 7.5.

[0090] Furthermore, the buffer may be present in the injectable liquid composition at a concentration of, but not limited to, about 5 mM to about 50 mM, about 8 mM to about 40 mM, about 10 mM to about 35 mM, about 13 mM to about 30 mM, or about 15 mM to about 25 mM.

[0091] The injectable liquid composition may have an osmolality in the range of about 200 to about 400 mOsm, about 230 to about 380 mOsm, about 250 to about 350 mOsm, about 260 to about 320 mOsm, about 270 to about 330 mOsm, or about 280 to about 300 mOsm. In this case, the osmolality in this range facilitates long-term storage at temperatures of 2° C. to 8° C. or higher, and can produce a composition suitable for parenteral administration, such as intravenous, intramuscular, or subcutaneous injection, without causing side effects in the subject.

[0092] As used herein, the term "osmolality" refers to the number of moles of solute contributing to the osmotic pressure of a solution per kilogram of solvent, and osmolality is determined by measuring the freezing point depression of a sample using an osmometer.

[0093] Additionally, the injectable liquid composition may further comprise a chelating agent.

[0094] The chelating agent may be at least one selected from the group consisting of, but not limited to, injection grade EGTA, EDTA, and BAPTA. The chelating agent can eliminate damage caused by ion flux after obtaining mitochondria contained in the injectable liquid composition.

[0095] Furthermore, the pharmaceutical composition may contain additives such as antioxidants, ATP, magnesium, etc., which are effective in maintaining mitochondrial function and activity.

[0096] The pharmaceutical composition of the present invention can be stored in a container selected from the group consisting of a vial, a cartridge, a syringe, and an auto-injector. Furthermore, the container in which the pharmaceutical composition is stored can be stored at room temperature, a refrigerated temperature of about 2°C to about 8°C, or about 25°C to about 40°C until it is administered to a subject in need of treatment.

[0097] The subject may be a mammal, such as, but not limited to, a human, dog, cow, horse, pig, sheep, goat, cat, mouse, rabbit, rat, and preferably a human.

[0098] On the other hand, the pharmaceutical composition of the present invention is administered in a pharmacologic effective amount. The term "therapeutically effective amount" or "pharmaceutical effective amount" refers to an amount of a compound or composition effective to prevent or treat a target disease, which is an amount sufficient to treat the disease with a reasonable benefit / risk ratio applicable to medical treatment and without causing side effects. The level of the effective amount can be determined according to factors including the patient's health condition, the type and severity of the disease, the activity of the drug, sensitivity to the drug, the method of administration, the time of administration, the route of administration and the excretion rate, the duration of treatment, the drugs used in combination or simultaneously, and other factors known in the medical field. In one embodiment, the therapeutically effective amount refers to the amount of a drug effective to treat a disease.

[0099] As used herein, the term "administration" refers to introducing a predetermined substance into a subject in an appropriate manner, and the route of administration of the composition can be through any common route as long as it can reach the target tissue. Administration can be, but is not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, topical administration, intranasal administration, and intrarectal administration.

[0100] The preferred dose of the pharmaceutical composition of the present invention may be, but is not limited to, about 0.01 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 4 mg / kg, or about 0.25 mg / kg to about 2.5 mg / kg of mitochondria per dose based on the body weight of the subject to be administered. That is, it is most preferable from the viewpoint of cell activity that the pharmaceutical composition is administered together with frozen and / or lyophilized mitochondria in the above-mentioned range based on the body weight of the subject suffering from the disease. Furthermore, the pharmaceutical composition can be administered 1 to 10 times, 3 to 8 times, or 5 to 6 times, preferably 5 times. In this case, the administration interval may be 1 to 7 days or 2 to 5 days, preferably 3 days. Such dosages should not be construed as limiting the scope of the present invention in any aspect.

[0101] In this case, the pharmaceutical composition may be parenterally administered using an 18G to 32G needle in a volume of about 5 ml or less, about 3 ml or less, or about 2 ml or less, but is not limited thereto.

[0102] The term "subject" refers to a subject to which the composition of the present invention can be applied (prescribed), and may be a subject suffering from a disease. Furthermore, the subject may be a mammal such as a rat, mouse, or livestock, including a human, but preferably a human. In addition to the frozen and / or lyophilized mitochondria, the composition of the present invention may further contain any compound or natural extract whose safety has been verified and which is known to have a preventive or therapeutic effect against each disease. In this case, the pharmaceutical composition and the active compound or natural extract may be administered simultaneously or sequentially.

[0103] In another aspect of the present invention, there is provided a use of frozen and / or lyophilized mitochondria for producing a pharmaceutical composition for preventing or treating a disease. Furthermore, the present invention can provide a method for preventing or treating a disease, comprising administering the pharmaceutical composition to a subject. Here, the subject may be a mammal, preferably a human. The frozen mitochondria, the lyophilized mitochondria, the prevention and the treatment are as described above.

[0104] The disease is a mitochondrial-associated disease, and specific diseases are as described above. In this case, administration can be performed by injection into the affected area, subcutaneous injection, or intravenous injection. Through this, the pharmaceutical composition according to the present invention can directly supply mitochondria having normal activity to the affected area where the disease has occurred, thereby increasing the activity of cells with reduced mitochondrial function or regenerating cells with abnormal mitochondrial function, and can be used to prevent or treat mitochondrial-associated diseases.

[0105] Long-term storage solution composition kit In another aspect of the present invention, there is provided a long-term preservation solution composition kit for preserving isolated cells, tissues or organs, comprising frozen and / or lyophilized mitochondria and a long-term preservation solution. The frozen and lyophilized mitochondria are as described above.

[0106] The long-term storage solution may include one or more selected from the group consisting of histidine, tryptophan, and ketoglutaric acid. The ketoglutaric acid may be alpha-ketoglutaric acid.

[0107] The frozen and / or lyophilized mitochondria of the kit can be mixed with a long-term storage solution containing one or more selected from the group consisting of histidine, tryptophan and ketoglutarate prior to use.

[0108] When the frozen and / or lyophilized mitochondria of the kit are mixed with a storage solution for use, the concentration of the frozen and / or lyophilized mitochondria may be about 0.01 μg / mL to about 1 mg / mL, about 0.01 μg / mL to about 100 μg / mL, about 0.01 μg / mL to about 90 μg / mL, about 0.01 μg / mL to about 80 μg / mL, about 0.01 μg / mL to about 70 μg / mL, about 0.01 μg / mL to about 60 μg / mL, about 0.01 μg / mL to about 50 μg / mL, about 0.01 μg / mL to about 40 μg / mL, about 0.01 μg / mL to about 30 μg / mL, or about 0.01 μg / mL to about 50 μg / mL.

[0123] The amount of the glycerol in the solution may be about 0.01 μg / mL, about 0.01 μg / mL to about 20 μg / mL, about 0.01 μg / mL to about 10 μg / mL, about 0.01 μg / mL to about 9 μg / mL, about 0.01 μg / mL to about 8 μg / mL, about 0.01 μg / mL to about 7 μg / mL, about 0.01 μg / mL to about 6 μg / mL, about 0.01 μg / mL to about 5 μg / mL, about 0.1 μg / mL to about 10 μg / mL, about 0.5 μg / mL to about 10 μg / mL, about 1 μg / mL to about 10 μg / mL, about 2.5 μg / mL to about 10 μg / mL, or about 5 μg / mL to about 10 μg / mL.

[0109] The concentration of histidine in the long-term storage solution may be about 1 mM to about 500 mM, about 5 mM to about 500 mM, about 10 mM to about 500 mM, about 25 mM to about 500 mM, about 50 mM to about 500 mM, about 75 mM to about 500 mM, about 100 mM to about 500 mM, about 125 mM to about 500 mM, about 150 mM to about 500 mM, about 175 mM to about 500 mM, about 198 mM to about 500 mM, about 100 mM to about 400 mM, about 100 mM to about 300 mM, about 100 mM to about 200 mM, or about 100 mM to about 198 mM.

[0110] The concentration of tryptophan in the long-term storage solution may be about 1 μM to about 100 mM, about 1 μM to about 90 mM, about 1 μM to about 80 mM, about 1 μM to about 70 mM, about 1 μM to about 60 mM, about 1 μM to about 50 mM, about 1 μM to about 40 mM, about 1 μM to about 30 mM, about 1 μM to about 20 mM, about 1 μM to about 10 mM, about 1 μM to about 5 mM, about 10 μM to about 10 mM, about 100 μM to about 10 mM, about 0.5 mM to about 10 mM, about 1 mM to about 10 mM, about 1.5 mM to about 10 mM, or about 2 mM to about 10 mM.

[0111] The concentration of ketoglutaric acid in the long-term storage solution may be about 1 μM to about 100 mM, about 1 μM to about 90 mM, about 1 μM to about 80 mM, about 1 μM to about 70 mM, about 1 μM to about 60 mM, about 1 μM to about 50 mM, about 1 μM to about 40 mM, about 1 μM to about 30 mM, about 1 μM to about 20 mM, about 1 μM to about 10 mM, about 1 μM to about 5 mM, about 10 μM to about 10 mM, about 100 μM to about 10 mM, about 250 μM to about 10 mM, about 500 μM to about 10 mM, about 750 μM to about 10 mM, about 1 mM to about 10 mM, about 1 mM to about 5 mM, or about 1 mM to about 2 mM.

[0112] The cells, tissues or organs may be from a mammal, which may be a human, cow, horse, pig, dog, sheep, goat or cat.

[0113] The cells, tissues or organs may be for transplant into a living organism.

[0114] The cell may be a muscle cell, a hepatocyte, a fibroblast, an epithelial cell, a neuron, an adipocyte, a bone cell, a leukocyte, a lymphocyte, a stem cell or a mucosal cell.

[0115] The tissue may be selected from skin, muscle, cornea, cartilage, and bone.

[0116] The organ may be selected from the heart, liver, kidney, small intestine, stomach, pancreas, lung, gallbladder, nerve, skin, blood vessel, tooth, eye, and cornea.

[0117] The long-term preservation solution composition kit may be for perfusion or reperfusion. Perfusion may be the replacement of plasma with an artificial fluid by injecting or continuously flowing a perfusion fluid into an isolated cell, tissue, or organ. Reperfusion may be the temporary cessation of blood supply to an isolated cell, tissue, or organ, followed by restoration of blood flow.

[0118] The long-term storage solution composition kit may be for parenteral administration. The composition may be liquid. In addition, the composition may include one or more additives. The additives may include excipients, diluents, anti-adhesives, preservatives, vehicles, surfactants, or combinations thereof. In addition, the composition may further include a salt, such as MgCl2, CaCl2, NaCl, KCl, or combinations thereof.

[0119] The long-term preservation solution composition may include an additive that can enhance the long-term preservation efficiency of isolated cells, tissues, or organs. The additive may be water, sugar (e.g., sucrose, raffinose, starch), pH buffer (e.g., sodium phosphate, potassium phosphate), ATP, a buffer, albumin, or an antioxidant (e.g., vitamin E).

[0120] In another aspect of the present invention, there is provided a use of frozen and / or lyophilized mitochondria for manufacturing a long-term storage solution composition kit.Furthermore, in another aspect of the present invention, there is provided a method for preserving an isolated cell, tissue or organ, comprising preserving the isolated cell, tissue or organ in the presence of a long-term storage solution composition comprising frozen and / or lyophilized mitochondria.

[0121] The mitochondria, frozen and / or lyophilized mitochondria, cells, tissues, organs, long-term storage solution compositions, and storage are as described above.

[0122] The storage can be performed under refrigerated conditions. The refrigerated conditions may be about -4°C to about 25°C, about -4°C to about 20°C, about -4°C to about 15°C, about -4°C to about 10°C, about -2°C to about 10°C, about -2°C to about 8°C, about 0°C to about 15°C, about 0°C to about 10°C, about 0°C to about 8°C, about 0°C to about 6°C, about 0°C to about 5°C, about 0°C to about 4°C, about 0°C to about 3°C, or about 2°C to about 5°C.

[0123] Storage can be performed for about 0 hours to about 2 days, about 0 hours to about 42 hours, about 0 hours to about 36 hours, about 0 hours to about 30 hours, about 0 hours to about 24 hours, about 0 hours to about 18 hours, about 0 hours to about 12 hours, about 0 hours to about 9 hours, about 0 hours to about 6 hours, or about 0 hours to about 3 hours.

[0124] In this case, when frozen mitochondria are used, the method may further include isolating mitochondria, and mixing the isolated mitochondria with a cryoprotectant and freezing them. Furthermore, when freeze-dried mitochondria are used, the method may further include isolating mitochondria, mixing the isolated mitochondria with a cryoprotectant and freezing them, and freeze-drying the frozen mitochondria. The method for isolating mitochondria, the method for freezing the isolated mitochondria, the method for freeze-drying the frozen mitochondria, and the cryoprotectant are as described above. EXAMPLES

[0125] The present invention will now be described in more detail with reference to the following examples. However, these examples are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these examples.

[0126] Preparation Example 1. Preparation of mitochondria After physical disruption of enriched platelets, umbilical cord-derived mesenchymal stem cells, or HEK293 cells, precipitated mitochondria were obtained using differential centrifugation.

[0127] Example 1. Freezing and freeze-drying of mitochondria The obtained mitochondria were resuspended in a TTG mixed storage solution (0.195M trehalose, 20mM Tris, 65mM glycine) at a concentration of 1mg / mL. Then, they were quickly frozen using LN2 or frozen at -80°C. When the mitochondria were frozen at -80°C, they were frozen with or without a freezing container (FC). The freeze-drying process was carried out using a freeze-dryer in a vacuum state, while changing the temperature to -40°C, -20°C, 6°C, and 25°C in sequence. Specifically, the freeze-drying process was carried out at -40°C for 30 minutes, and then the reaction was carried out at -20°C for 25 hours. The reaction was carried out at -20°C for another 8.3 hours while checking the state of the mitochondria. Next, the reaction was carried out at 6°C for 8.3 hours and at 25°C for 9 hours, respectively, to finally obtain freeze-dried mitochondria.

[0128] Example 2. Confirmation of the anti-inflammatory efficacy of frozen mitochondria 4×10 5 THP-1 human monocytic cells were cultured in RPMI1640 medium containing 1% FBS (fetal bovine serum). After 16-24 h of culture, THP-1 cells were treated with frozen platelet-derived mitochondria (40 μg) for 24-96 h.

[0129] After 1 hour of treatment, the cells were treated with LPS (lipopolysaccharide) at a concentration of 2 μg / mL to construct an inflammatory cell model. After 4 hours of treatment with LPS, the culture medium of the THP-1 cells was collected and centrifuged at 20,000 × g for 10 minutes, and the concentration of TNF-α, an inflammatory cytokine, in the supernatant was measured by ELISA (Figure 1). In addition, the anti-inflammatory efficacy of stem cell-derived mitochondria and platelet-derived mitochondria (isolated from the blood of three individuals) was also confirmed using the same method. At this time, mitochondria were treated at a concentration of 40 μg.

[0130] As a result, as shown in Figures 1 and 2, the frozen platelet-derived mitochondria or stem cell-derived mitochondria were confirmed to have an anti-inflammatory effect compared to the group treated with vehicle alone (control group).

[0131] Example 3. Recovery of freeze-dried mitochondria The freeze-dried mitochondria were stored at room temperature or in a refrigerator, and immediately before use, ultrapure distilled water was added to restore the volume to that before freeze-drying.

[0132] The mitochondrial protein concentration before lyophilization and after recovery was measured by the BCA (bicinchoninic acid) method (Figure 3).

[0133] Example 4. Confirmation of intracellular transport of freeze-dried mitochondria Cells with red fluorescent protein-labeled mitochondria (HEK293-MT dsRED ) and lyophilized and then recovered by the method of Example 3. Cultured HEK293-MTS-GFP cells (mitochondria were labeled with green fluorescent protein) were treated with 10 μg of recovered mitochondria for 1 day, and the cells were observed using a confocal microscope.

[0134] As a result, as shown in FIG. 4, mitochondria recovered after freeze-drying were transferred into the transplanted cells.

[0135] Example 5. Confirmation of energy synthesis-related enzyme activity in frozen and freeze-dried mitochondria To confirm changes in mitochondrial activity due to freezing or lyophilization, the protein concentrations of freeze-dried and non-lyophilized platelet-derived mitochondria were measured using the BCA method, and the activities of energy synthesis-related enzymes per equivalent protein amount were compared.

[0136] Specifically, the activity of complex I and complex III (oxidative phosphorylation complex) present in the mitochondrial inner membrane was evaluated by measuring the change in absorbance intensity during reduction of oxidized cytochrome c (Sigma aldrich, catalog number C2037) at a wavelength of 550 nm at 1-minute intervals for 20 minutes. Furthermore, the activity of complex IV was evaluated by reducing cytochrome c using sodium bisulfite (Sigma aldrich, catalog number 157953), and then measuring the change in absorbance intensity during oxidation at a wavelength of 550 nm at 1-minute intervals for 20 minutes. After adding ADP, ATP synthesis ability was evaluated by measuring luminescence at 1-minute intervals for 20 minutes using a CellTiter-Glo luminescence kit (Promega). Acetyl-CoA (Sigma aldrich, catalog number A2181) and oxaloacetate (Sigma aldrich, catalog number O4126) are converted to coenzyme A (CoA-SH) via mitochondrial citrate synthase, and coenzyme A reacts with DTNB (5,5'-dithiobis(2-nitrobenzoic acid), Sigma aldrich, catalog number D218200) to produce color at a wavelength of 450 nm. Mitochondrial citrate synthase activity was measured at 1-minute intervals for 20 minutes (Figure 5).

[0137] As a result, as shown in FIG. 5, it was confirmed that the activity of enzymes present in mitochondria was maintained even after freezing or lyophilization.

[0138] Example 6. Confirmation of the anti-inflammatory efficacy of freeze-dried mitochondria 4×10 5 THP-1 human monocytic cells were cultured in RPMI1640 medium containing 1% FBS (fetal bovine serum). After 16 to 24 hours of culture, the THP-1 cells were treated with non-lyophilized or lyophilized mitochondria (40 μg) isolated from platelets or umbilical cord-derived mesenchymal stem cells and prepared by the method of Example 3.

[0139] After 1 hour of treatment, the cells were treated with 2 μg / mL LPS to induce intracellular inflammatory responses. After 4 hours of LPS treatment, the media from the THP-1 cells were collected and centrifuged at 20,000 × g for 10 minutes, and the concentration of the inflammatory cytokine TNF-α in the supernatant was measured by ELISA. In addition, the anti-inflammatory efficacy of freeze-dried or non-freeze-dried mitochondria at different concentrations was also confirmed using the same method.

[0140] As a result, as shown in Figures 6 and 7, compared to the group treated with vehicle only (control group), the groups treated with non-lyophilized mitochondria or freeze-dried mitochondria exhibited an anti-inflammatory effect dependent on the mitochondrial concentration.

[0141] Example 7. Measurement of the cardioprotective effect of freeze-dried mitochondria Example 7.1. Heart removal and stabilization To confirm cardiac function, the hearts were first excised from the rats and stabilized.

[0142] Specifically, Sprague-Dawley rats (SD, 12 weeks old, male) were anesthetized with ketamine and then sacrificed. The rat hearts were excised and placed in a Langendorff tube via an aortic catheter. The excised hearts were then perfused with Krebs-Henseleit solution for about 20 minutes. The hearts were stabilized by injecting a refrigerated histidine-tryptophan-ketoglutaric acid (HTK) solution through the aorta of the heart and perfusing for about 10 minutes. At this time, the HTK solution contained 198 mM histidine, 2 mM tryptophan, 1 mM ketoglutaric acid, 30 mM mannitol, 4 mM MgCl2, 15 μM CaCl2, 15 mM NaCl, and 9 mM KCl.

[0143] Example 7.2. Maintenance of perfusate with mitochondria-containing solution in isolated hearts Lyophilized mitochondria or mitochondria were added to HTK solution to prepare 5 μg / mL lyophilized mitochondria-HTK and mitochondria-HTK solution.

[0144] The stabilized hearts prepared in Example 7.1 were injected with 5 μg / mL mitochondria-HTK solution (MT), 5 μg / mL freeze-dried mitochondria-HTK solution (freeze-dried MT), or mitochondria-free HTK solution (control) for about 2 minutes. The hearts were then immersed in 150 μg / 30 mL mitochondria-HTK solution (MT), freeze-dried mitochondria-HTK solution (freeze-dried MT), or mitochondria-free HTK solution, respectively, and stored at about 4° C. After 9 hours, the preserved hearts were placed in a Langendorff via the aortic catheter and passed through the Krebs-Henseleit solution. The amount of perfusate flowing out of the heart was measured ( FIG. 8 ).

[0145] As a result, as shown in Figure 8, compared with when the hearts were stored in HTK solution alone (control), the volume of perfusate in the hearts increased by approximately 1.97-fold when stored in mitochondria-HTK solution and by approximately 1.48-fold when stored in freeze-dried mitochondria-HTK solution.

[0146] Example 8. Confirmation of the effect of reducing mitochondrial damage by the mixed preservation solution during cryopreservation Example 8.1. Comparison of mitochondrial protection effects of mixed preservation solutions upon freezing and thawing Mitochondria isolated from umbilical cord-derived mesenchymal stem cells were mixed with SHE (250 mM sucrose, 20 mM HEPES, 2 mM EGTA) mixed storage solution, TTG (20 mM Tris, 195 mM trehalose, 66.6 mM glycine) mixed storage solution, or TT (20 mM Tris, 195 mM trehalose) mixed storage solution, frozen, thawed, and centrifuged at 12,000 g for 10 minutes. After centrifugation, the supernatant was obtained, and the amount of cytochrome C released when mitochondrial damage was induced was confirmed using Western blot. As a control group, mitochondria disrupted by ultrasonication were centrifuged under the same conditions as above, and the supernatant was obtained and used.

[0147] As a result, as shown in Figure 9, it was confirmed that cytochrome c efflux was increased in the mitochondrial supernatant mixed with SHE or TT mixed preservation solution and frozen, compared to the mitochondrial supernatant mixed with TTG mixed preservation solution and frozen and then thawed. Through the above results, it was confirmed that the TTG mixed preservation solution can more stably protect mitochondria from mitochondrial damage during the freezing and thawing process.

[0148] Example 8.2. Comparison of activity of mitochondria frozen and then thawed in mixed preservation solutions In order to confirm the mitochondrial protection effect against stress occurring during the freezing and thawing process of the TTG mixed preservation solution, mitochondria isolated from umbilical cord-derived mesenchymal stem cells were mixed with SHE mixed preservation solution alone, TTG mixed preservation solution alone, TT mixed preservation solution alone, TTG mixed preservation solution containing DMSO (TTG + DMSO), or TT mixed preservation solution containing DMSO (TT + DMSO), respectively, and then frozen and thawed to compare the mitochondrial activity. At this time, DMSO was used at a concentration of 10% as a commonly used freezing and preservation solution. Mitochondrial activity was confirmed by measuring the activity of citrate synthase and ATP synthase, which are mitochondrial-specific enzymes, in the same manner as in Example 5.

[0149] As a result, as shown in FIG. 10, mitochondria mixed with the TTG mixed preservation solution and frozen had higher citrate synthase and ATP synthase activities than mitochondria mixed with the SHE mixed preservation solution and frozen.

[0150] Example 8.3. Comparison of the anti-inflammatory efficacy of mitochondria frozen and then thawed with a mixed preservation solution Mitochondria isolated from umbilical cord-derived mesenchymal stem cells were mixed with SHE mixed preservation solution, TTG mixed preservation solution alone, TT mixed preservation solution alone, TTG mixed preservation solution containing DMSO (TTG+DMSO), or TT mixed preservation solution containing DMSO (TT+DMSO), and then frozen and thawed to confirm the anti-inflammatory effect of mitochondria. At this time, the anti-inflammatory effect of mitochondria was confirmed by the same method as in Example 2.

[0151] As a result, as shown in Figure 11, mitochondria mixed and frozen in the TTG mixed preservation solution showed higher anti-inflammatory efficacy than mitochondria mixed and frozen in the SHE mixed preservation solution, TT mixed preservation solution, or TT + DMSO mixed preservation solution. The results showed that the activity of mitochondria mixed and frozen in the TTG mixed preservation solution was superior to the activity of mitochondria mixed and frozen in other mixed preservation solutions, suggesting that the TTG mixed preservation solution can more stably protect mitochondria from mitochondrial damage that occurs during the freezing and thawing process.

[0152] Example 8.4. Comparison of platelet aggregation inhibitory effects of frozen-thawed mitochondria according to mixed storage solution Mitochondria isolated from umbilical cord-derived mesenchymal stem cells were mixed with a SHE mixed preservation solution or a TTG mixed preservation solution, frozen and thawed, and the platelet aggregation inhibitory effects of mitochondria were compared.

[0153] Specifically, frozen and thawed mitochondria were stained with 0.1 μM MitoTracker Orange for 10 min and washed twice with mitochondrial isolation solution. Mitochondria (5 μg) were mixed with platelets (1 × 10 7 ) and incubated for 1 day under shaking. The shaking culture medium (5 μl) was applied to a glass slide and observed using a confocal laser microscope.

[0154] As a result, as shown in Figure 12, platelet aggregation was not induced when mitochondria were mixed with the TTG mixed preservation solution and frozen, whereas platelet aggregation was induced when mitochondria were mixed with the SHE mixed preservation solution and frozen.

[0155] Example 9. Confirmation of damage to mitochondria mixed with TTG mixed preservation solution, frozen, and thawed Mitochondria isolated from umbilical cord-derived mesenchymal stem cells were mixed with a TTG preservation solution, frozen, and thawed one month later to confirm mitochondrial damage.

[0156] Specifically, the total mitochondrial protein amount, ATP synthesis rate, Mitotracker staining rate, and expression level of Tom20, a specific protein of the outer membrane, were measured, and then the same mitochondria were mixed with a TTG mixed storage solution and frozen. After one month of freezing, the mitochondria were thawed and the above items were measured again. At this time, the total mitochondrial protein amount was measured using a BCA assay kit, and the ATP synthesis rate was measured using the method of Example 5. The Mitotracker staining rate and Tom20 expression level were analyzed using a flow cytometer after staining with 0.5 μM Mitotracker green or Mitotracker Orange, or after staining with a fluorescent protein-conjugated Tom20 antibody (Santacruz, sc-17764-FITC).

[0157] As a result, as shown in Figure 13, when mitochondria were frozen and stored in the TTG mixed storage solution, it was confirmed that there were no significant changes in the total mitochondrial protein amount, ATP synthesis rate, Mitotracker staining rate, or Tom20 expression level before and after freezing.

Claims

1. Frozen and / or lyophilized mitochondria.

2. 2. The frozen and / or lyophilized mitochondria of claim 1, wherein the lyophilized mitochondria are stored at 37° C. or below.

3. A method for stably freezing isolated mitochondria.

4. Isolating mitochondria; freezing the isolated mitochondria by treatment with a mitochondrial cryoprotectant comprising glycine; 4. A method for freezing mitochondria according to claim 3.

5. 5. The method of freezing mitochondria according to claim 4, wherein the cryoprotectant further comprises any one selected from the group consisting of trehalose, Tris, and combinations thereof.

6. 5. The method for freezing mitochondria according to claim 4, wherein the freezing is carried out at a temperature of -1°C or lower.

7. 5. The method of freezing mitochondria according to claim 4, wherein the mitochondria are obtained from platelets, muscle cells, hepatocytes, fibroblasts, epithelial cells, nerve cells, adipocytes, bone cells, leukocytes, lymphocytes, stem cells, or mucosal cells.

8. A method for stably lyophilizing isolated mitochondria.

9. Isolating mitochondria; freezing the isolated mitochondria by treatment with a mitochondrial cryoprotectant comprising glycine; and freeze-drying the frozen mitochondria. A method for freeze-drying mitochondria according to claim 8.

10. 10. The method for freeze-drying mitochondria according to claim 9, wherein the cryoprotectant further comprises any one selected from the group consisting of trehalose, Tris, and combinations thereof.

11. 10. The method for freeze-drying mitochondria according to claim 9, wherein the freezing is carried out at a temperature of -1°C or lower.

12. 10. The method of freeze-drying mitochondria according to claim 9, wherein the mitochondria are obtained from platelets, muscle cells, hepatocytes, fibroblasts, epithelial cells, neuronal cells, adipocytes, bone cells, leukocytes, lymphocytes, stem cells, or mucosal cells.

13. A glycine-containing cryoprotectant for mitochondria.

14. The mitochondrial cryoprotectant of claim 13, further comprising any one selected from the group consisting of trehalose, tris, and combinations thereof.

15. A composition for freezing mitochondria, comprising a mitochondrial cryoprotectant comprising glycine, and mitochondria.

16. 16. The composition for freezing mitochondria of claim 15, wherein the cryoprotectant further comprises any one selected from the group consisting of trehalose, Tris, and combinations thereof.

17. A pharmaceutical composition for preventing or treating a disease, comprising frozen and / or lyophilized mitochondria as an active ingredient.

18. 18. The pharmaceutical composition for preventing or treating a disease according to claim 17, wherein the disease is a mitochondrial-related disease including chronic inflammatory diseases, acute inflammatory diseases, ischemic diseases, neurological diseases, cardiac diseases, muscular diseases, degenerative diseases, metabolic diseases, fibrosis, joint diseases, eye diseases, hair loss, cancer, hearing loss, and immune-related diseases.

19. A long-term preservation solution composition kit for preserving isolated cells, tissues, or organs, comprising frozen and / or lyophilized mitochondria and a long-term preservation solution.

20. 20. The long-term storage solution composition kit of claim 19, wherein the frozen and / or lyophilized mitochondria are mixed with a long-term storage solution prior to use.

21. Use of frozen and / or lyophilized mitochondria for preventing or treating a mitochondrial associated disease.

22. 1. A method for preventing or treating a mitochondrial associated disease comprising administering frozen and / or lyophilized mitochondria.

Images