Method of obtaining mitochondria from cells and obtained mitochondria

Abstract

To provide methods of obtaining mitochondria from cells, mitochondria obtained by such methods, and methods of use of mitochondria obtained by such methods.SOLUTION: A method of isolating mitochondria from cells, the method comprising: (i) subjecting cells in a first solution to treatment with a surfactant at a concentration lower than the critical micelle concentration of the surfactant, (ii) removing the surfactant to form a second solution substantially not containing the surfactant in order to prevent contact between the mitochondria in the cells and the surfactant, (iii) incubating the cells in the second solution, and (iv) recovering mitochondria released into the second solution.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is filed on July 24, 2019, under Japanese Patent Application No. 2019-136283. No. 60 / 699,999, filed on May 1, 2003, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Mitochondria are a type of organelle that play three important roles: 1) A 2) metabolism, such as TP synthesis; 3) intracellular signaling, such as Ca2+ and reactive oxygen species; and 3) Regulation of cell death, such as apoptosis and necrosis. In this sense, mitochondria are a valuable resource for disease prevention. It has been studied by many researchers from the perspective of health and its relationship to the environment.

[0003] For mitochondrial function, the folded inner membrane and surrounding outer membrane, and inner membrane The electron transport chain located in the inner membrane plays an important role. The cristae membranes contain the electron transport chain supercomplexes, forming a complex structure. By confining the pumped protons within the cristae space, It is believed that the concentration of the electrochemical protons formed by the electron transport chain is kept high. The anion gradient allows for ATP synthesis and cation transport in addition to anion transport.

[0004] Decreased mitochondrial function can lead to various diseases. To isolate mitochondria from cells in a manner that preserves their structural integrity, the present technology There are currently no known methods in the field. This disclosure addresses this and other needs. Summary of the Invention

[0005] The present disclosure provides a population of isolated, obtained or processed mitochondria, wherein The mitochondria in the population exhibit superior functional capacity. For example, in one aspect, the present disclosure provides isolated a population of mitochondria, wherein at least about 60% of the mitochondria in the population are %, at least about 65%, at least about 70%, at least about 75%, at least about 80 %, at least about 85%, at least about 90% or at least about 95% intact intima and and / or at least about 60% of the mitochondria in the population have an outer membrane; at least about 65%, at least about 70%, at least about 75%, at least about 80%, At least about 85%, at least about 90%, or at least about 95% is detected by the fluorescent indicator. In one embodiment, the method provides a population of isolated mitochondria that is polarized as measured by ATP. In this case, the fluorescent indicator is a positively charged dye, e.g., JC-1, tetramethylrhodamine methyl ester. The group consisting of tetramethylrhodamine ethyl ester (TMRM) and tetramethylrhodamine ethyl ester (TMRE) is selected from.

[0006] In one embodiment, the present disclosure provides a method for detecting at least about 60% of mitochondria in a population, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% function in the extracellular environment Provides a population of isolated mitochondria that maintains bioactivity (e.g., is polarized) In one embodiment, functional capacity in the extracellular environment is measured by a fluorescent indicator of membrane potential. In one embodiment, the fluorescent indicator is a positively charged dye, such as JC-1, TMRM, and and TMRE. In one embodiment, the extracellular environment is about 4 mg / dL ~ about 12 mg / dL or about 1 mmol / L (1000 μM) ~ about 3 mmol / L (30 For example, in one embodiment, the total calcium concentration may be 0.000 μM. The environment is about 8 mg / dL to about 12 mg / dL or about 2 mmol / L (2000 μM) to about In one embodiment, the total calcium concentration is 3 mmol / L (3000 μM). The environment is about 4 mg / dL to about 6 mg / dL or about 1 mmol / L (1000 μM) to about 1 Contains a free or active calcium concentration of 0.5 mmol / L (1500 μM). In embodiments, the mitochondrial population exhibits a higher calcium content compared to the calcium environment in the cell. Maintain functional ability in an environment with high sodium concentrations.

[0007] In one embodiment, at least about 60%, at least about 10%, or at least about 60% of the mitochondria in the population 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% are dynamin-related protein 1 A population of isolated mitochondria that are not undergoing (drp1)-dependent division is In one embodiment, an isolated mitochondrial cell line comprising an inner membrane and an outer membrane is provided. The inner membrane of mitochondria contains densely folded cristae. In one embodiment, an isolated population of mitochondria is provided.

[0008] In one embodiment, at least about 60%, at least about 10%, or at least about 60% of the mitochondria in the population 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% substantially non-filamentous, non-branched In one embodiment, a population of isolated mitochondria having a structure or shape of For example, in some embodiments, the mitochondria provided herein can be visualized under a microscope. When viewed, they may be circular, dotted, spherical, irregular and / or slightly elongated, or any of the above. In one embodiment, at least one of the mitochondria in the population at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least At least about 80%, at least about 85%, at least about 90%, or at least about 95% of the In some embodiments, the major axis to minor axis ratio is 3:1 or less, 3.5:1 or less, or 3:1 or less. Among the isolated mitochondria in the mitochondrial populations provided herein, fewer At least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least At least about 80%, at least about 85%, at least about 90%, or at least about 95% They have a length that is less than two or three times the hydrodynamic diameter of the mitochondria. The isolated mitochondria provided here represent the majority of mitochondria found in cells. It has a significantly different shape (non-filamentous) compared to the shape (filamentous) of A. Therefore, In some embodiments, the populations of mitochondria provided herein comprise a population of mitochondria. At least about 60%, at least about 65%, at least about 70%, at least about 7 5%, at least about 80%, at least about 85%, at least about 90% or at least Approximately 95% of mitochondria are non-filamentous in shape, meaning they are not isolated from cells. In one embodiment, the isolated mitochondria provided herein have a different shape than the mitochondrial The population of mitochondrial membranes is called the mitochondria-associated membrane (MAM). In one embodiment, the MAM and The association is measured by the expression of glucose-regulated protein 75 (GRP75). In some embodiments, the isolated populations of mitochondria provided herein are isolated from mitochondria in cells. and / or in certain embodiments, by conventional isolation methods as further described. methods, e.g., methods involving homogenization and / or high levels of detergent methods Approximately 60%, at least 65%, and at least At most about 70%, about 60%, about 50%, about 40%, about 30% or less of MAM In one embodiment, the population of isolated mitochondria provided herein exhibits: This indicates a decrease in the association with MAM, a decrease that is consistent with the presence of mitochondria in cells or with conventional isolation methods. At least 30% of the mitochondria isolated by , at least about 40%, at least about 50%, at least about 60%, at least about 70% Or even more.

[0009] In one embodiment, the population of isolated mitochondria provided herein is approximately 100% mitochondrial in size. In one embodiment, the mitochondria in the population are At least about 60%, at least about 65%, at least about 70%, at least about 75%, At least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99% are between about 500 nm and about 3500 nm in size. In these cases, the average size of mitochondria in the population ranged from about 500 nm, to about 600 nm, to about 70 nm, 0nm, approx. 800nm, approx. 900nm, approx. 1000nm, approx. 1100nm, approx. 1200n m, approx. 1300nm, approx. 1400nm, approx. 1500nm, approx. 1600nm, approx. 1700n m, approx. 1800nm, approx. 1900nm, approx. 2000nm, approx. 2100nm, approx. 2200n m, approx. 2300nm, approx. 2400nm, approx. 2500nm, approx. 2600nm, approx. 2700n m, approx. 2800nm, approx. 2900nm, approx. 3000nm, approx. 3100nm, approx. 3200n m, about 3300 nm, about 3400 nm, or about 3500 nm. The polydispersity index (PDI) of the isolated mitochondrial population ranges from about 0.2 to about 0.8. In one embodiment, the population of isolated mitochondria has a PDI of about 0.2 to about 0.5. In one embodiment, the PDI of the isolated mitochondrial population is about 0.25 to about 0. 35. In one embodiment, the zeta potential of a population of mitochondria is between about −15 mV and about In one embodiment, the zeta potential of the mitochondrial population is about -20 mV. V is about −25 mV, about −30 mV, about −35 mV, or about −40 mV.

[0010] In some embodiments, the population of isolated mitochondria provided herein comprises an isolated When a population of mitochondria comes into contact with a population of cells, they can integrate into the cells and / or For example, in one embodiment, the present disclosure provides a method for the co-localization of endogenous mitochondria in the mitochondrial matrix. obtain mitochondria from cells and then transfect the cells (e.g., ex vivo or in vivo) The present invention provides a method for contacting a population of cells (e.g., mitochondrial cells) with a population of isolated mitochondria. In one such embodiment, the presently disclosed subject matter is isolated through the iMIT method described herein. The mitochondria provided can co-localize with endogenous mitochondria present in the cell. In some embodiments, the mitochondria provided herein are capable of inhibiting the growth of cells with which they are contacted. It is further capable of fusing with mitochondria present in cells. A significant proportion of the isolated mitochondrial population coexists with endogenous mitochondria in the cell. For example, in one embodiment, mitochondria in a population can be localized and / or fused. At least about 30%, at least about 40%, at least about 50%, at least About 60%, at least about 70%, at least about 80%, at least about 90% or less Approximately 95% of all mitochondria are able to colocalize and / or fuse with endogenous mitochondria in cells. Therefore, the mitochondria provided herein are endogenous mitochondria in cells. It is not possible to simply identify and / or fuse with endogenous endothelial cells through conventional methods. This is significantly different from the mitochondria that are isolated.

[0011] In one embodiment, the isolated mitochondria provided herein are suitable for storage at about 4°C. maintain a stable and / or polarized and / or membrane potential after or maintain intact inner and outer membranes and / or after exposure to the extracellular environment (e.g. (e.g., after exposure to a total calcium concentration of about 4 mg / dL to about 12 mg / dL) For example, in one embodiment, at least about 10% of the mitochondria in the population are maintained at a constant concentration. 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% are at about 4°C. maintain a stable and / or polarized and / or membrane potential after storage at and / or maintain intact inner and outer membranes and / or exposure to the extracellular environment (e.g., after exposure to a total calcium concentration of about 4 mg / dL to about 12 mg / dL) In one embodiment, the isolated mitochondria provided herein maintain their ability to function. The rear is stable and / or polarized after storage at about -20°C or below, and and / or maintains the membrane potential, and / or maintains intact inner and outer membranes, and / or after exposure to the extracellular environment (e.g., about 4 mg / dL to about 12 mg / dL total calcium). maintain their ability to function (after exposure to high concentrations of methionine). For example, in one embodiment, At least about 60%, at least about 65%, at least about 70% of mitochondria At least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% are stable and / or polarized after storage at about -20°C. and / or maintain a membrane potential and / or maintain intact inner and outer membranes. and / or after exposure to the extracellular environment (e.g., about 4 mg / dL to about 12 mg In one embodiment, the patient maintains the ability to function (after exposure to a total calcium concentration of 1 / dL). The isolated mitochondria provided here are stable after storage at approximately -80°C or below. and / or polarized and / or maintains a membrane potential and / or is intact maintain the inner and outer membranes of the cells and / or after exposure to the extracellular environment (e.g., approximately 4 m maintain the ability to function (after exposure to total calcium concentrations of 1000 to 12000 mg / dL) For example, in one embodiment, at least about 60% of the mitochondria in the population at least about 65%, at least about 70%, at least about 75%, at least about 80%, At least about 85%, at least about 90%, or at least about 95% are stored at about -80°C. and / or maintain a stable and / or polarized and / or membrane potential after and / or maintain intact inner and outer membranes and / or after exposure to the extracellular environment (e.g., after exposure to total calcium concentrations of about 4 mg / dL to about 12 mg / dL) In one embodiment, the isolated mitochondria provided herein maintain the ability to , stable after storage in liquid nitrogen, and / or polarized, and / or membrane potential maintain the position and / or maintain intact inner and outer membranes and / or the extracellular environment after exposure to a total calcium concentration of about 4 mg / dL to about 12 mg / dL For example, in one embodiment, mitochondria in a population maintain their ability to function (after exposure). At least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least Approximately 95% of the samples are stable and / or polarized after storage in liquid nitrogen, and and / or maintain the membrane potential, and / or maintain intact inner and outer membranes, and / or or after exposure to the extracellular environment (e.g., about 4 mg / dL to about 12 mg / dL total cal In some embodiments, preservation is less than In either case, the temperature is maintained for at least about 2 hours, at least about 6 hours, at least about 12 hours, at least about 24 hours, or At least about 48 hours, at least about 1 week, at least about 2 weeks, at least about 3 weeks, At least about 1 month, at least about 2 months, at least about 3 months, or longer. Thus, in some embodiments, the isolated mitochondria provided herein contain at least Both of these proteins maintain their functional capacity when freshly isolated and even after storage. This is significantly different from mitochondria isolated through conventional methods.

[0012] In some embodiments, the isolated mitochondria provided herein are isolated from mitochondria. The population is stable after being frozen for storage and subsequently thawed, and / or Polarized and / or maintains a membrane potential and / or has intact inner and outer membranes and / or after exposure to the extracellular environment (e.g., about 4 mg / dL to about 12 In one embodiment, the patient maintains the ability to function (after exposure to a total calcium concentration of 100 mg / dL). So, after freezing and then thawing, the maintenance rate of the membrane potential is the same as the membrane potential of the mitochondria before freezing. For example, in one embodiment, frozen and thawed mitochondria The polarization ratio of the population is about 90% of the polarization ratio of the population before freezing. At least about 60%, at least about 65%, at least about 70% of the mitochondria in %, at least about 75%, at least about 80%, at least about 85%, at least about 90 % or at least about 95% after freezing for storage and subsequent thawing, e.g., storage is stable after being frozen for 1, 2, 3 or more times and subsequently thawed, and / or polarized and / or maintains membrane potential and / or is intact Maintaining the inner and outer membranes and / or after exposure to the extracellular environment (e.g., approximately 4 mg maintain the ability to function after exposure to total calcium concentrations between 10 mg / dL and approximately 12 mg / dL Thus, in some embodiments, the isolated mitochondria provided herein contain at least They maintain their functional capacity even when frozen for storage and then thawed. This is significantly different from mitochondria isolated through conventional methods.

[0013] In one embodiment, the population of isolated mitochondria provided herein comprises the mitochondrial at any temperature (e.g., 4°C ± 3°C, -20°C ± 3°C, -80°C ± 3°C, or Preservation of mitochondria (in vitro or in liquid nitrogen) followed by incorporation into cells and / or or can co-localize and / or fuse with endogenous mitochondria in cells. For example, in one embodiment, at least about 60% of the mitochondria in the population About 65%, at least about 70%, at least about 75%, at least about 80%, at least About 85%, at least about 90%, or at least about 95% of the mitochondria are preserved. Incorporated into cells after incubation and / or after undergoing one or more freeze-thaw cycles and / or co-localization and / or fusion with endogenous mitochondria in the cell. In one embodiment, the populations of isolated mitochondria provided herein can be The method of storage and thawing is to keep the population at approximately -20°C ± 3°C, approximately -80°C ± 3°C, or below this temperature. Store at low temperatures (e.g., in liquid nitrogen) and then allow the mitochondria to cool to approximately 20°C ± 3°C. This involves thawing at or below that temperature, and mitochondria within about 5 minutes, and within about 4 minutes. In certain embodiments, the microcapsules are thawed within about 3 minutes, within about 2 minutes, or within about 1 minute. The population of mitochondrial cells thaws within about 1 minute. The mitochondria provided are at least capable of being incorporated into cells and / or are capable of co-localizing and / or fusing with endogenous mitochondria in cells, while Mitochondria isolated by conventional methods can be incorporated into cells, and / or Are they unable to co-localize and / or fuse with endogenous mitochondria in cells? or mitochondria isolated through conventional methods in that their ability is greatly reduced. In one embodiment, the co-localized isolated mitochondria are filamentous. Structures, networks and / or reticulated structures can be formed.

[0014] In some embodiments, the present disclosure provides a composition comprising isolated mitochondria as provided herein. The compositions may, in some embodiments, be administered in one or more pharmaceutically acceptable carriers. Further includes:

[0015] In one embodiment, the present disclosure provides a method for isolating mitochondria from cells, the method comprising: Unlike known methods, the present invention provides a method for producing mitochondrial cells with superior functionality and other features. In one embodiment, the method comprises isolating mitochondria from cells. The method comprises: placing cells in a first solution in a surfactant solution at a concentration below the critical micelle concentration (CMC) of the surfactant; Treating with a surfactant, removing the surfactant to form a second solution, incubating the cells in the second solution and recovering the mitochondria from the second solution. In one embodiment, the concentration of the surfactant in the first solution is greater than or equal to the CMC of the surfactant. For example, in one embodiment, the concentration of surfactant in the first solution is about 50% or less. , about 40% or less, about 30% or less, about 20% or less, or about 10% or less of the CMC of the surfactant Below.

[0016] In some embodiments, the surfactant is a non-ionic surfactant. Detergents: Triton-X 100, Triton-X 114, Nonidet P -40, n-dodecyl-D-maltoside, Tween-20, Tween-80, Saponin In one embodiment, the surfactant is selected from the group consisting of saponin and digitonin. In one embodiment, the detergent concentration is less than about 400 μM. For example, in one embodiment, the concentration of the surfactant in the first solution is less than about 300 μM. In certain embodiments, the ATP concentration is less than about 200 μM, less than about 100 μM, or less than about 50 μM. The concentration of the surfactant in the first solution is about 100 μM, about 75 μM, about 60 μM, about 5 μM, In some embodiments, the first solution is about 0 μM, about 40 μM, about 30 μM, or about 20 μM. The concentration of the surfactant in the solution is about 20 μM to about 50 μM or about 30 μM to about 40 μM. do.

[0017] In one embodiment, the first solution contains one or more of a tonicity agent, an osmolality adjusting agent, or a chelating agent. In one embodiment, the first solution further comprises a buffer containing a plurality of Tris buffers. Contains sucrose and a chelator.

[0018] In one embodiment, a first surfactant containing a low concentration of surfactant (e.g., below the CMC of the surfactant) is used. The step of treating the cells in the first solution comprises treating the cells in the first solution at room temperature for about 2 minutes to about 30 minutes. For example, in one embodiment, the cells in the first solution are treated with The step of treating the cells in the first solution may include treating the cells for about 2, about 5, about 10, about 15, about 20, about 25 minutes. Incubation may be performed at about 4°C to about 30 minutes. It can be carried out at a temperature of 7°C.

[0019] In one embodiment, the step of removing the detergent comprises removing the detergent from the solution by dissolving it in a first solvent. to less than 10% of the surfactant concentration in the first solution, or to less than 1% of the surfactant concentration in the second solution. In one embodiment, the step of removing the detergent comprises reducing the cells to This includes washing with buffer.

[0020] In one embodiment, the step of incubating the second solution comprises incubating the cells in the second solution. For example, in one embodiment, the second the step of incubating the cells in the second solution for about 5, about 10, In some embodiments, the incubation is for about 15, about 20, about 25, or about 30 minutes. In the method, the step of incubating the cells in the second solution is performed at a temperature of about 4°C ± 3°C. is performed on ice.

[0021] In one embodiment, the step of recovering mitochondria from the second solution comprises recovering the supernatant. In one embodiment, the method further comprises recovering the isolated mitochondria from the second solution. The step of recovering mitochondria from the second solution is performed by centrifuging the second solution and then centrifugation. and recovering the isolated mitochondria.

[0022] In one embodiment, iMIT can be performed on cells attached to a culture surface. In one embodiment, iMITs allow cells to attach to a culture surface without detaching them from the surface. In one embodiment, the method can be performed on cells containing mitochondria from the second solution. The step of recovering the mitochondrion comprises recovering the supernatant and recovering the isolated mitochondria. and then, if necessary, wash the remaining cells on the culture surface with the second solution or a different second solution. The supernatant can be combined with the purified water.

[0023] In some embodiments, the methods provided herein involve freezing isolated mitochondria. In one embodiment, the method further comprises: precipitating the mitochondria in a cryoprotectant (e.g., In one embodiment, the method comprises freezing the cells in a buffer containing the desired product (e.g., glycerol). The method involves freezing mitochondria in a buffer in liquid nitrogen. In some embodiments, the method further comprises thawing the mitochondria after freezing. In this case, the method of thawing mitochondria is to thaw the mitochondria quickly, for example, within about 5 minutes. In one embodiment, the mitochondria are thawed within about 1 minute or within about 1 minute. The thawing is performed in a warm bath having a temperature of 20°C ± 3°C to about 37°C ± 3°C. Mitochondria thaw at temperatures of about 20°C ± 3°C or below.

[0024] In some embodiments, the present disclosure provides isolated mitochondria obtained by the methods provided herein. In one embodiment, the methods provided herein are "iMIT" methods. The mitochondria obtained by this method are referred to herein as "Q" mitochondria. In one embodiment, the present disclosure provides a method for producing a hydroxyapatite-containing ... The present invention provides compositions and / or formulations comprising a population of isolated mitochondria comprising:

[0025] In some embodiments, the present disclosure provides a method for treating a disease or disorder associated with mitochondrial dysfunction. The present invention also provides a method for treating or preventing a subject's cells with an isolated mitochondrial protein (IMP) provided herein. In one embodiment, the method includes contacting a population of mitochondria, e.g., Q mitochondria, with the mitochondria. In an aspect, the disease or disorder is an ischemia-related disease or disorder. For example, in one embodiment In this paper, ischemia-related diseases or disorders include cerebral ischemia-reperfusion, hypoxic-ischemic encephalopathy, acute coronary artery disease, and syndrome, myocardial infarction, hepatic ischemia-reperfusion injury, ischemic injury-compartment syndrome, hematopoietic A group consisting of vascular obstruction, wound healing, spinal cord injury, sickle cell disease, and reperfusion injury of transplanted organs In some embodiments, the disease or disorder is a genetic disorder. In this case, the disease or disorder is cancer, cardiovascular disease, eye disorders, ear disorders, autoimmune diseases, inflammation, etc. In one embodiment, the disease is acute respiratory distress syndrome (ARS). In one embodiment, the disease or disorder is an age-related disease or disorder, or an age-related disease or disorder. In some embodiments, the disease or disorder is pre-eclampsia or intrauterine fetal morbidity. Growth retardation (IUGR).

[0026] In some embodiments, the present disclosure provides a method for treating or preventing a disease or disorder as defined herein. A method comprising administering an isolated mitochondrial population or composition to a subject in need thereof. In one embodiment, the method comprises administering isolated mitochondria. The route of administration may be intravenous, intraarterial, intratracheal, subcutaneous, intramuscular, inhalation, or intrapulmonary. In some embodiments, the subject is a mammal, such as a human.

[0027] In one embodiment, the present disclosure provides an isolated mitochondrion having intact inner and outer membranes. The inner membrane contains folded cristae and mitochondria are isolated from cells. , mitochondria are visualized by fluorescent indicators (e.g., JC-1, TMRM, or TMRE). Measurements revealed that mitochondria were polarized, and mitochondria were able to maintain polarization in the extracellular environment. In one embodiment, the present invention provides an isolated mitochondrion. In some embodiments, mitochondria are densely packed cristae. In some embodiments, mitochondria have tubular structures on their surface. For example, in one embodiment, the ATP-dependent anion channel (VDAC) is associated with a ATP-dependent anion channel (ATP). In mitochondria, isolated mitochondria contain dimeric tubules associated with VDAC on their surface. In some embodiments, the tubulin comprises at least α-tubulin. In an embodiment, the tubulin is a heterodimer comprising α-tubulin and β-tubulin. In some embodiments, the tubulin is a homodimer. In this study, isolated mitochondria exhibited association with MAM as measured by GRP75 expression. For example, in one embodiment, the isolated mitochondria exhibit a reduction in the amount of mitochondria present in the cell. (i.e., not isolated) mitochondria and / or Conventional isolation methods, such as homogenization and / or high-resolution filtration, as described in Compared with mitochondria obtained by methods including Bell's detergent method, approximately 70 %, about 60%, about 50%, about 40%, about 30% or less of the MAM. In some embodiments, the isolated mitochondria provided herein have reduced association with MAM. where the decrease is due to the presence of mitochondria present in cells (i.e., not isolated). Comparison of the association of MAM with mitochondria isolated by conventional and conventional isolation methods. and at least about 30%, at least about 40%, at least about 50%, at least about 6 0%, at least about 70% or more.

[0028] In one embodiment, the isolated mitochondria provided herein have a pH of about -30 mV to about In one embodiment, the isolated mitochondria have a membrane potential of -220 mV. In one embodiment, the isolated mitochondria undergo drp1-dependent division. In one embodiment, the isolated mitochondria are between about 500 nm and 350 nm in size. For example, in one embodiment, the isolated mitochondria are approximately 100 nm in size. 500, approximately 600, approximately 700, approximately 800 nm, approximately 900 nm, approximately 1000 nm, approximately 110 0nm, approx. 1200nm, approx. 1500nm, approx. 2000nm, approx. 2500nm, approx. 300 0 nm or approximately 3500 nm.

[0029] In some embodiments, the present disclosure provides isolated mitochondria obtained by the methods provided herein. In one embodiment, the present disclosure provides an isolated mitochondrial cell line provided herein. Compositions and formulations comprising Andria are provided. [Brief explanation of the drawings]

[0030] [Figure 1A] This figure shows the distribution of the fluorescence intensity ratio (ratio of mitochondrial fluorescence intensity to background fluorescence intensity) of a fluorescent indicator for mitochondria depolarized with a depolarizing agent. In Figure 1A, 97.5% of the depolarized mitochondria showed a fluorescence intensity of less than 1.2. Therefore, in this experiment, mitochondria were considered to have a membrane potential if the fluorescence intensity ratio exceeded 1.2. [Figure 1B] 1 shows microscopic images of mitochondria obtained by the method of the present invention using transmitted light and a fluorescent image using a fluorescent indicator (TMRE) in the same region as the transmitted light image, with the scale bar representing 10 μm. [Figure 2A] TMRE fluorescence images of mitochondria obtained by the DHF method compared with those obtained by the homogenization method. The scale bar represents 25 μm. [Figure 2B] The upper panel shows the protein content (μg) of mitochondrial fractions isolated by DHF versus homogenization. The lower panel of Figure 2B shows the TMRE-positive mitochondria (%) of mitochondrial fractions isolated by DHF versus homogenization. The DHF method resulted in a statistically significantly higher % TMRE-positive fraction (p<0.05). [Figure 2C] Microscopic images (upper panel) and TMRE-stained fluorescent images (lower left panel) of isolated mitochondria are shown. The images are merged in the lower right panel, demonstrating that almost all mitochondria in the isolated population are polarized. [Figure 2D] Figure 1 shows an electron micrograph of mitochondria isolated by the method provided herein. The scale bar is 1 µm. The image shows that the isolated mitochondria have intact inner and outer membranes and densely folded cristae. [Figure 2E] Figure 1 shows STED microscopy images of Q mitochondria, H mitochondria (mitochondria isolated by conventional homogenization), and D mitochondria (mitochondria isolated by conventional detergent). The outer membrane of mitochondria was stained green (Tom20 immunofluorescence), and the inner membrane was stained red with Mitotracker Red. Quantification of the ratio and size of intact outer membranes of isolated mitochondria is provided in Table 3. [Figure 3A]FIG. 1 shows the size distribution, polydispersity (PDI) and measured zeta potential by dynamic light scattering of mitochondrial populations before freeze-thawing obtained by mitochondrial extraction at a concentration above the critical micelle concentration (DHF method) without homogenization or without detergent in the examples. [Figure 3B] FIG. 1 shows the size distribution, polydispersity (PDI) and measured zeta potential by dynamic light scattering of mitochondrial populations after freeze-thawing obtained by the DHF method. [Figure 3C] This figure shows the size distribution, polydispersity (PDI) and measured zeta potential of mitochondrial populations after freeze-thawing, obtained by the same method as the DHF method, except that the centrifugation method (section 7 of 2.2.1) was performed at 1000 × g. [Figure 3D] FIG. 1 shows a superimposed image of a transmission microscope image and a TMRE-stained image of a group of mitochondria before and after freezing and thawing obtained by the DHF method. [Figure 4] FIG. 1 shows the isolation of mitochondrial populations from each of the mouse tissues by the DHF method, as well as the size distribution, polydispersity (PDI) and measured zeta potential of the isolated mitochondrial populations by dynamic light scattering. [Figure 5A] FIG. 1 shows TMRE fluorescence intensity over time (min) in single mitochondria isolated by the iMIT method and treated with 1 mM malate and 1 μM oligomycin at the indicated time points. [Figure 5B] Figure 5A shows TMRE fluorescence images of single mitochondria isolated by the iMIT method over a typical time course with malate added at the times indicated (1 mM malate was added between 0 and 1 min, and 1 μM oligomycin was added between 5 and 6 min). [Figure 6A] Figure 1 shows isolated mitochondria (middle panel) co-localizing with mitochondria in recipient fibroblasts (left panel), with a merged image shown in the right panel. [Figure 6B]Figure 1 shows the co-localization of Q mitochondria with mitochondria in recipient cells (bottom panel). In contrast, mitochondria isolated through homogenization appear to be inside recipient cells but do not co-localize with endogenous mitochondria. [Figure 7A] Fixed cells stained to show mitochondrial shape in the absence (left panel) or presence (right panel) of 10 μM of the Drp1 inhibitor Mdivi1. Mitochondria in cells with or without 10 μM Mdivi1 maintained a networked, branched shape. [Figure 7B] After the addition of 30 μM digitonin and subsequent washing steps, mitochondria in cells with or without 10 μM Mdivi1 are shown to be non-filamentous in shape. [Figure 7C] FIG. 1 shows that GFP+ mitochondria isolated by iMIT from cells treated with 0 or 10 μM Mdivi1 retain a non-filamentous shape. [Figure 8] This figure shows a comparison of polarization between Q mitochondria (top panel) and mitochondria isolated by conventional detergent (middle panel) or conventional homogenization (bottom panel). The left column of panels shows microscopic images of mitochondria isolated by each method. The right panel shows TMRE fluorescence, demonstrating that only Q mitochondria have a high proportion of polarized mitochondria. [Figure 9A] FIG. 10 Calcein fluorescent staining shows that Q mitochondria have an intact mitochondrial membrane after isolation (top panel) and maintain an intact membrane even after the addition of 0.96 mM calcium (bottom panel). [Figure 9B] FIG. 10 shows TMRE fluorescent staining demonstrating that Q mitochondria are polarized before (top panel) and after (bottom panel) exposure to calcium. [Figure 10A]FIG. 1 shows that physical disruption of a population of mitochondria disrupts the mitochondrial membrane, and that the addition of 0.96 mM calcium further disrupts the membrane, such that calcein fluorescence can no longer be detected after the combination of agitation with a high calcium environment. [Figure 10B] FIG. 1 shows that membrane potential (measured by TMRE staining) is maintained after physical disruption of mitochondria by agitation, but is lost after addition of 0.96 mM calcium. [Figure 11] Figure 1 shows GRP75 protein content by Western blot assay (left panel) of iMIT mitochondria (Q mitochondria; right lane) compared with mitochondria isolated via conventional detergent method (D, left lane) or conventional homogenization method (H, center lane). Cytochrome oxidase was used as a protein content control (right panel). [Figure 12] FIG. 1 is a schematic diagram providing the study design for a 4-week myocardial infarction model study. [Figure 13] Figure 1 shows left ventricular systolic function in rats in an ischemia-reperfusion model. EF% is shown for animals in the sham, PBS, homogenized mitochondrial, and high and low Q mitochondrial groups. **(p<0.01). [Figure 14] FIG. 1 is a schematic diagram providing the study design for a 7-day myocardial infarction model study. [Figure 15] FIG. 1 shows the ejection fraction (EF%) after 1, 3 and 7 days of dosing with PBS or mitochondria (Q) in the test group (IR Q), negative control (IR PBS) or sham infarction (Sham Q) groups. DETAILED DESCRIPTION OF THE INVENTION

[0031] The present disclosure provides highly functional mitochondria that are beneficial in treating a variety of diseases, disorders, and conditions. In one embodiment, the method provides a population of chondria and highly functional mitochondria. The mitochondria are isolated from cells and retain their ability to function. For example, in one embodiment, The mitochondria provided herein are isolated from cells and are highly polarized and / or The present disclosure provides a method for the production of mitochondrial cells that retain other aspects of mitochondrial function as described herein. Further provided is a method for obtaining mitochondria such that the mitochondria are highly functional. The methods and isolated mitochondria provided herein involve extracting mitochondria from cells. known methods for isolating and the isolated mitochondria resulting from those known methods. There's a big improvement from the rear.

[0032] As used herein, the terms "isolate" and "isolating" refer to the removal of a substance from within a cell. The term "isolating" refers to the collection of extracellularly recovered mitochondria. and removing at least one of the other components in the solution from the solution containing the compound. Therefore, the term "isolated" as used herein means that mitochondria are The term "processed" or "obtained" means that the substance is no longer in the cell. "isolated" can be used interchangeably with "isolated." In some embodiments, isolated mitochondria Isolated populations of adrenaline or mitochondria can be isolated from the cell cycle through the methods provided here. The methods provided herein involve treating mitochondria with mitochondrial tissue. These results suggest that the membrane integrity and membrane potential are maintained even after isolation, causing minimal structural damage to the membrane. The goal is to obtain mitochondria from cells in a way that allows them to be maintained.

[0033] As used herein, the term "cell" refers to a eukaryotic cell, i.e., a cell containing mitochondria in the cytoplasm. The cell contains the antibody, for example an animal cell, for example a mammalian cell, preferably a human cell. As used herein, the term "cell" refers to cells present in a tissue as well as cells separated from a tissue. Cells obtained from a tissue of interest (e.g., single cells) and populations of cells (e.g., cells obtained from a tissue of interest) This term is used to include cells in a population of cells and / or a population of cells derived from a cell line. will be done.

[0034] As used herein, the term "mitochondria" refers to a mitochondrion that includes a bilayer lipid membrane, an inner membrane, and an outer membrane. Present in eukaryotic cells that have a matrix surrounded by cristae and an inner membrane Mitochondria (plural mitochondria) are organelles involved in oxidative phosphorylation These cells have enzymes on their inner membranes, such as the respiratory chain complexes, which act to Because of the internal-external proton gradient, the inner membrane has a membrane potential. It is believed that the membrane potential cannot be maintained if the inner membrane is disrupted. Mitochondria have their own genome (mitochondrial genome) that is different from the genome in the cell nucleus. As used herein, a "population of mitochondria" refers to a group of mitochondria. This is a group that includes Tochondria.

[0035] As used herein, the term "polarized" means that mitochondria exhibit a membrane potential. As used herein, the term "polarization ratio" refers to the ratio of polarized mitochondria to total mitochondria. Mitochondrial polarization can be measured, for example, by using commercially available fluorescent indicators. Those skilled in the art can conveniently detect the fluorescent indicator. Examples of the fluorescent indicator include, but are not limited to, JC-1 , tetramethylrhodamine methyl ester (TMRM) and tetramethylrhodamine ester Examples include methyl ester (TMRE).

[0036] As used herein, the term "surfactant" refers to a surfactant that contains a hydrophilic and a hydrophobic moiety in one molecule. Surfactants are molecules that reduce surface tension at interfaces or form micelles. The surfactant acts to mix polar and non-polar substances by forming a They are broadly classified into ionic surfactants and nonionic surfactants. surfactants whose hydrophilic portion is not ionized, and ionic surfactants whose hydrophilic portion is cationic. It may be an ion, an anion, or both a cation and an anion.

[0037] As used herein, the term "critical micelle concentration" (CMC) refers to the concentration at which The concentration at which surfactants form micelles is referred to as the concentration at which surfactants form micelles. Contributes to cell formation, especially in bulk concentrations. At concentrations above the critical micelle concentration, the interface to the system The addition of an active agent ideally increases the amount of micelles, particularly the number of micelles.

[0038] The traditional method for isolating mitochondria involves mechanically grinding (homogenizing) whole cells. or by solubilizing the cell membrane using detergents or detergent methods to remove the The latter method involves the use of detergents or surfactants to recover mitochondria. The reactive agent method involves the use of a compound at a concentration high enough to disrupt the cell membrane and any membranes within the cell (i.e., In some cases, these methods (homogenization) are used to administer the compound to cells. lysis and the use of high concentrations of detergents or detergent methods) Other methods have been used in combination to increase yield. These methods include freeze-thawing and / or ultrasonic treatment for the purpose of The resulting mitochondria can exhibit some functions, but these methods Considering the low polarization ratios required, (1) homogenizing the cells to disrupt the cell membrane; and / or freeze-thawing the cells and / or sonicating the cells, Mitochondria, which form a network structure within the membrane, are vulnerable to membrane-damaging shear stress and / or or physical injury by ice crystal formation and / or membrane-damaging ultrasound, respectively. and (2) in detergent-based methods, the mitochondrial membrane is In addition to the above, the cells are exposed to detergents that can solubilize the cell membrane, or The surfactant binds to mitochondrial membrane proteins, and then the isolated mitochondria are depleted of the surfactant. It is believed that the animals are chemically injured by sexual agents.

[0039] Researchers used proteins instead of detergents to form pores in the plasma membrane They also tried to recover mitochondria. (1) This method also yields some high-quality mitochondria. However, the yield was low and most of the mitochondria were damaged. The cells were repeatedly pipetted, so the mitochondria outside the cells appeared to be damaged. It was.

[0040] In one aspect, the present disclosure provides a "detergent-based and homogenization-free (DHF) The invention relates to a novel method for isolating mitochondria from mitochondria isolated by a novel method, referred to herein as the "iMIT" method or alternatively as the "iMIT" method. As described herein, the present invention provides mitochondrial cells isolated by the iMIT method. Mitochondria remain intact (e.g., retain the integrity of the inner and outer membranes) and remain functionally intact. The mitochondria obtained by the iMIT method maintain the membrane potential. These mitochondria are referred to herein as "Q" mitochondria. For a variety of diseases and disorders, including those described herein, mitochondrial transplantation, e.g., Mitochondrial transplantation is effective in treating a variety of diseases and disorders. It is a treatment that is expected to be useful for restoring and / or enhancing mitochondrial function. Therefore, cells with severely dysfunctional mitochondria and / or highly functional mitochondria Add exogenous mitochondria (e.g., Q mitochondria) to cells that benefit from the influx of mitochondria. ) is internalized.

[0041] Another application contemplated herein is the investigation of mitochondrial machinery, particularly mitochondria in response to stimulation. The purpose of isolated mitochondria is to study their response to the environment. Because of this potential for control, we are currently studying how mitochondria respond to intracellular signals. (e.g., how the proton motive force drives ATP synthesis) The role of protons is to transport electrons through the electron transport chain to the mitochondria. Peter Mitchell's chemiosmotic theory that oxygen is pumped across the inner membrane (This can be performed on isolated mitochondria). Considering the polarization ratio of mitochondria, isolated mitochondria obtained by conventional methods It is thought that both the adventitia and intima are severely damaged. By harvesting a large number of mitochondria, only a small fraction of their remaining functions can be measured. In contrast, the mitochondria obtained by the method of the present disclosure are thought to be damaged. This allows the measurement of many phenomena that are not measurable in irradiated mitochondria.

[0042] Contamination with damaged mitochondria can have deleterious effects on living organisms and cells. The mitochondria provided herein may be isolated by conventional methods. are superior to those used in other drugs, in part because they are associated with cytotoxicity. compared to mitochondria isolated from and / or through traditional methods, e.g. Furthermore, the present invention relates to the use of hydroxybenzoates in the treatment of cancer, as these are associated with much less cytotoxicity when administered intravenously. The mitochondria provided in the present invention are superior in their functional capacity as described herein. Therefore, they are superior to those isolated by conventional methods. The effects of physical or chemical destruction by surfactants on the endosperm during their recovery process and there is no contact with the surfactant or it cannot be removed from the first solution, i Much lower than CMC, which cannot damage mitochondria during the recovery process of MIT The degree of injury can be reduced by contacting the tissue with a surfactant at a high concentration. They are not in contact with detergents and therefore suffer damage to organisms and cells. If it can be expected to reduce the harmful effects of mitochondria, the extent of the damage can be minimized. It is expected that this will be possible.

[0043] How to get mitochondria In one embodiment, the present disclosure provides a method for treating cells in a solution at a concentration below the critical micelle concentration (CMC). Treating with a detergent, removing the detergent from the solution containing the treated cells, and The drug-treated cells are then incubated to recover the mitochondria into solution, thereby Harvesting or isolating mitochondria from cells by harvesting mitochondria from vacuoles This method is referred to herein as "iMIT." The specification provides iMIT, a method for obtaining mitochondria from cells, which includes: Served:

[0044] (A) Cells in the first solution were treated with a surfactant at a concentration below the critical micelle concentration (CMC). To do, (B) removing the surfactant from the first solution to form a second solution; and (C) Incubation of detergent-treated cells in the second solution to induce mitochondrial proliferation in the second solution. (A) through (C) above and additional aspects of the method are described below. can be.

[0045] By the methods of the present disclosure, cells having mitochondria in their cytoplasm are critically endowed with The cells are treated with a surfactant at a concentration below the micelle concentration. The membrane is weakened in structural strength but not permeabilized due to the low concentration of detergent, The dodder membrane is exposed to little surfactant and remains intact. In this case, the plasma membrane may be partially permeabilized, but the mitochondrial membrane is permeabilized by low detergent concentrations. Because of the low concentration, they are exposed to very little surfactant and remain intact.

[0046] In some embodiments, the solution of (A) can contain a buffer. Exemplary buffers for use in the method include, for example, Tris buffer, HEP Examples of the buffer include ES buffer and phosphate buffer. The buffer has a pH of, for example, 6.7 to 7. .6 (e.g. pH 6.8-7.4, pH 7.0-7.4, e.g. pH 7.2-7.4, e.g. In one embodiment, the buffer may contain a tonicity agent and an osmolality adjuster. Exemplary tonicity and osmolality adjusting agents include simple sugars (e.g., Glucose, galactose, mannose, fructose, inositol, ribose, xylose sugars (e.g., lactose, sucrose, cellobiose, trehalose, wheat saccharides (e.g., raffinose, melesinose, etc.), trisaccharides (e.g., raffinose, melesinose, etc.), Polysaccharides (e.g., cyclodextrin, etc.), sugar alcohols (e.g., erythritol, xylitol, thitol, sorbitol, mannitol, maltitol, etc.), glycerin, diglycerin , polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol Examples include diethylene glycol, triethylene glycol, and polyethylene glycol. The buffer may contain chelating agents, especially chelating agents for divalent metals, e.g., calcium. It may also contain a chelating agent for ammonium ions. Examples of the chelating agent include: Glycol ether diamine tetraacetic acid (EGTA) and ethylenediamine tetraacetic acid (EDT) A) is one example.

[0047] In one embodiment, the buffer is a Tris buffer containing sucrose and a chelator. The pH may be 6.7 to 7.6 (e.g., pH 6.8 to 7.4, pH 7.0 to 7.4 , for example, pH 7.2 to 7.4, for example, pH 7.4. The buffer may contain digitonin or saponin, or another detergent provided herein. In one embodiment, digitonin or saponin or other detergents can be used for clinical purposes. Boundary micelle concentration: 20% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% In one embodiment, digitonin can have a concentration of 10% or less. , 400 μM or less, 350 μM or less, 200 μM or less, 150 μM or less, 100 μM or less , 90 μM or less, 80 μM or less, 70 μM or less, 60 μM or less, 50 μM or less, 40 μM It can be used at a concentration of 0.1 or less or 30 μM or less (eg, at a concentration of 30 μM). In some embodiments, the saponin is at 400 μM or less, 350 μM or less, 200 μM or less, 1 50μM or less, 100μM or less, 90μM or less, 80μM or less, 70μM or less, 60μM At concentrations of 50 μM or less, 40 μM or less, or 30 μM or less (e.g., 30 μM can be used in various concentrations.

[0048] In some embodiments, the surfactants used in the methods provided herein are ionic or The nonionic surfactant used in the present invention may be: For example, ester, ether and alkyl glycoside forms may be included. Examples of the surfactant include alkyl polyethylene glycol, polyoxyethylene Nonionic surfactants include alkylphenyl ethers and alkylglycosides. The inhibitors include Triton-X 100, Triton-X 114, and Nonidet P. -40, n-dodecyl-D-maltoside, Tween-20, Tween-80, Saponin In the treatment step (A), Triton and / or digitonin may be included. -X 100, saponin, and digitonin. In one embodiment, the surfactant is saponin or digitonin. do.

[0049] In one embodiment, the treating step (A) comprises treating the cells with a detergent at a concentration below the critical micelle concentration. The treatment time for the cells in step (A) is, for example, 1 to 30 minutes, for example, It may be 1 to 10 minutes, or for example 1 to 5 minutes, for example 2 to 4 minutes, for example 3 minutes. (A) Cell processing can be performed on ice, at 4°C, or at room temperature, or at any temperature in between. can be done.

[0050] In one embodiment, the concentration of the surfactant in the treatment step (A) is less than the critical micelle concentration. Concentration, for example, 90% or less, 80% or less, 70% or less, 60% or less of the critical micelle concentration, 50% or less, 40% or less, 30% or less, 20% or less, 15% or less, 14% or less, 13% or less Below, 12% or less, 11% or less, 10% or less, for example, 5-15%, for example, 8-12%, for example For example, it may be 10%.

[0051] In one embodiment, the treatment step (A) is a pretreatment of the cells. Treatment of cells with detergents below their critical micelle concentration, although undesirable, reduces the strength of the cell membrane. and / or to investigate the effects of detergent methods on intracellular mitochondria. It is believed to partially or completely eliminate

[0052] Therefore, minimizing the effects of detergents on mitochondria is important. During and after the collection of mitochondria from the cells, at least one step (e.g., The concentration of surfactant in the solution that comes into contact with mitochondria in each of steps (B) to (E) is less than the critical micelle concentration, e.g., less than 10%, less than 5%, less than 4%, less than 3% of the critical micelle concentration It may be less than 2%, or less than 1% or less; or it may be below the limit of detection. In order to minimize the effect of surfactants on mitochondria, it is preferable to use surfactants in the cells. During and after recovery of mitochondria from the No additives should be added.

[0053] In some embodiments, the cells may be in the form of cells present in a tissue, or they may be It may be isolated from tissue (e.g., a single cell) or a population thereof. The cells to be isolated may be cultured cells or may be cultured cells used to make them single cells. A single protein obtained by treating tissue or cultured cells with enzymes such as collagenase The tissue may be a cell or a population thereof. The tissue may be optionally treated with an enzyme such as collagenase. may be shredded before use.

[0054] In one embodiment, the concentration of detergent in contact with the mitochondria is reduced or In order to sufficiently reduce the surfactant in contact with mitochondria, (A) Detergent must be removed from the solution before being recovered from the detergent-treated cells. can.

[0055] In the removal step (B), the detergent is removed, for example, by lowering or reducing the buffer. a solution containing a surfactant at a given concentration (preferably a surfactant-free solution) (e.g., a buffer solution) This can be done by replacing the solution with HCl or by adding the solution to the buffer. If the detergent-treated cells are adherent cells, the buffer containing the detergent Aspirate the solution and, if necessary, transfer the cells to a solution containing a lower or reduced concentration of detergent. Wash with a solution (preferably a detergent-free solution) (e.g., a buffer) and or a solution containing a reduced concentration of surfactant (preferably a surfactant-free solution) (e.g. The detergent-treated cells can be removed by adding a buffer. If cells are present, centrifuge the cells, remove the supernatant, and if necessary, reduce or eliminate the cells. A solution containing a surfactant at a given concentration (preferably a surfactant-free solution) (e.g., a buffer solution) Wash with water and a solution containing a lower or reduced concentration of surfactant (preferably Remove detergent by adding a detergent-free solution (e.g., buffer). It is possible to remove it.

[0056] Removal means, for example, reducing the surfactant concentration to less than 10%, less than 5%, less than 4%, less than 3%, Less than 2%, or less than 1%; or below the detection limit in solutions that come into contact with mitochondria At least reduce the concentration of detergent in the solution that the mitochondria contact, including To ensure the removal of detergent from the solution, (B) is used to better immerse the cells. a solution containing a lower or reduced concentration of surfactant (preferably a surfactant-free solution) This may include washing with a buffer (e.g., a buffer).

[0057] In (B), a surfactant is added to or mixed with the solution to remove it from the solution. The exchanged solution may preferably be a buffer, such as the buffer described in (A) above. (However, solutions containing lower concentrations of surfactants, preferably surfactant-free or undetectable levels of surfactant).

[0058] (A) Cells treated with ATP have reduced plasma membrane strength, and simply immobilizing them in solution Incubation releases mitochondria from the inside of the cell to the extracellular space. However, in the previous step (C), the boundary that contacts the mitochondria is The amount of detergent was small, and the effect of detergent on mitochondria was limited; therefore, Therefore, the decrease in mitochondrial membrane strength is limited and / or the mitochondrial membrane Remains intact.

[0059] In one embodiment, the method comprises simply placing the cells in the second solution. This involves obtaining mitochondria that are released into a solution of 2.

[0060] Therefore, in the present invention, in order to release mitochondria from inside the cell to the extracellular space, Then, detergent-treated cells can be incubated in solution. (C) "Release" The term "mitochondrion" refers to the area outside the plasma membrane that surrounds the mitochondria from the inside of the cell. (e.g., to the solution side or outside the cell)

[0061] (C) The solution used for incubating (the "second solution") is lower In a preferred embodiment, the second solution may be a solution containing a surfactant at a concentration of is a surfactant-free solution or a negligible and / or undetectable amount of surfactant The solution for use in incubating in (C) is, for example, the solution (A) and the buffer (described in (A) above) (preferably a surfactant-free solution) The solution used in (C) may contain, for example, a buffer, an osmotic pressure adjusting agent, and a divalent metal. It may be a solution that contains a chelator and is substantially free of surfactants. "Substantially free" means "substantially contained" that cannot be removed or detected. The term "not excluding the presence of contaminating ingredients" is used to mean that the presence of contaminating ingredients in small amounts is not excluded.

[0062] In (C), the incubation may be, for example, 1 to 30 minutes, for example, 5 to 25 minutes, or, for example, 1 to 30 minutes. For example, the treatment may be for 5 to 20 minutes, for example, for 5 to 15 minutes, for example, for 10 minutes. can be performed on ice or at room temperature, or at any temperature in between.

[0063] In (C), mitochondrial lipids were removed to enhance recovery of mitochondria from cells. A physical stimulus can be applied to prevent mechanical breakdown of the lipid bilayer. In (C), for example, incubation is performed under shaking or non-shaking conditions. In (C), for example, the incubation can be carried out under stirring or non-stirring conditions. In (C), detergent treatment prevents cells from detaching from the adhesive surface. This makes it easier to remove the cells from the adhesive surface by the gentle water flow described above. The desorption of ions does not appear to have a negative effect on the polarization ratio. The incubation can be carried out for a period of time that does not cause the cells to detach.

[0064] In (C), the mitochondria recovered in solution are compared with the isolated mitochondrial population. In some embodiments, the present disclosure provides The mitochondrial population resulting from the method, referred to herein as "Q" mitochondria, In some embodiments, the present disclosure provides a method for producing a compound comprising: , providing individual mitochondria (i.e., individual Q).

[0065] In some embodiments, the methods provided herein further comprise (D) recovering mitochondria in solution. The method further comprises purifying the mitochondria by centrifugation. It can be separated from other cellular components. For example, mitochondria can be collected in (C). Contaminants in the mitochondrial population, such as detached cells, contained in the mitochondrial population Centrifugation at ≤1500g, ≤1000g or ≤500g to precipitate Therefore, the mitochondria can be purified as a supernatant. The resulting supernatant can be purified by centrifugation. Further centrifugation (e.g., 8000g to 12000g) for enrichment etc. Therefore, it can be recovered as a precipitate. As used herein, the term "purified" means , mitochondria are separated from at least one other component in the solution by a procedure This means that...

[0066] The mitochondrial populations obtained in (C) and / or (D) above can be isolated for various applications. The mitochondrial population can be used as a purified mitochondrial population.

[0067] The method of the present invention may further comprise (E) freezing the mitochondria. Freezing is performed by gently suspending mitochondria in freezing buffer. The buffer for freezing may be the buffer described in (A), but It may be surfactant-free and may further comprise a cryoprotectant. Exemplary cryoprotectants include These are known in the art and examples include glycerol, sucrose, trehalose, dimethicone, Dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, diethyl glycol Licorice, Triethylene Glycol, Glycerol-3-Phosphate, Proline, Sorbitol Examples of the methyl group include methyl methyl acrylate, methyl meth ... Chondria can be preserved by freezing. The disclosed methods do not require cryopreservation. Otherwise, the mitochondria were not frozen, e.g., they were freshly isolated. In some other embodiments, mitochondria are cultured at about 4°C ± 3°C. Alternatively, the solution can be stored on ice. In some embodiments, the methods provided herein The mitochondria provided herein are produced in liquid nitrogen at approximately -80°C ± 3°C or below, approximately It can be stored at -20°C ± 3°C or below, or at about 4°C ± 3°C. Mitochondria can be stored for days, weeks, or even months, and remain functional after thawing. maintain the ability to

[0068] In some embodiments, the methods provided herein involve isolating and The present invention further includes a method for thawing frozen mitochondria. The method for thawing mitochondria is to thaw mitochondria at a temperature of approximately 20°C ± 3°C or less. and mitochondria rapidly, e.g., within about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, or about 1 minute. In one embodiment, rapid thawing of mitochondria includes rapidly thawing mitochondria. This results in the retention of functional abilities as described herein by the lear.

[0069] In some embodiments, the methods provided herein involve disrupting mitochondrial membranes. The entire process of extracting mitochondria from cells using this method does not involve disrupting the cell membrane. For example, in the methods provided herein, the cells are homogenized during the process of recovering mitochondria from the cells. That is, in some embodiments, the compounds provided herein are not destroyed by genization. The method does not involve homogenization; in some embodiments, the method does not involve homogenization. Homogenization involves the lysis of cells, but the homogenization does not cause any bubbles or Or it is only performed to the extent that it does not cause bubbles in the solution relative to the tissue. In embodiments, the method also does not involve freezing and thawing the cells. Repeated freezing and thawing of the cells can damage the plasma membrane. Suitable for disrupting and recovering its contents, for recovering mitochondria from cells However, the resulting mitochondrial membrane potential is not maintained (mitochondrial (In contrast to the disclosed method, where mitochondrial membrane potential is maintained), freezing and thawing It is also thought to disrupt the lipid bilayer.

[0070] In one embodiment, the disclosed method comprises the steps of: Other methods of disrupting the vesicle membrane (e.g., sonication, soaking the solution to the extent that it causes bubbles or dissolving the solution) In one embodiment, the method of the present disclosure does not include treatment with a strong water stream that causes the liquid to foam. may cause substantial physical, chemical, or physiological damage to mitochondria. It is carried out without any process, but it is possible to perform freeze-thaw cycles for storage. Therefore, the method of the present invention can be applied to chondria. Chondria can be obtained.

[0071] The method of the present invention involves purifying mitochondria recovered from cells using one or more No filtration step is required.

[0072] In some embodiments, the methods provided herein involve removing mitochondria while they are still in the cell. This gently separates mitochondria from the microtubule system without damaging the mitochondria. During incubation, the mitochondrial structure becomes nonfilamentous due to the detachment of microtubules from the mitochondrial surface. The mitochondria can escape from the cell through the detergent-treated cell membrane. Thus, mitochondria obtained from cells through the disclosed methods may contain mitochondrial membranes. without tearing and tearing or otherwise damaging the mitochondrial structure Thus, the isolated mitochondria and populations thereof provided herein are It is possible to maintain function after isolation and has been previously described for use in treating disease states. This is significantly more preferable than any isolated mitochondria that has been isolated.

[0073] Therefore, the method provided here is different from conventional methods for isolating mitochondria. and which differ in important respects from those isolated by conventional methods or any other method previously disclosed. The isolated or obtained mitochondria have unexpected and advantageous functions compared to the mitochondria Offer Andria.

[0074] Mitochondrial population In one aspect, the present disclosure provides a method for the production of a medicament for the treatment of a medicament comprising isolating a medicament from a cell using the methods provided herein, and As described above, the present invention provides a population of mitochondria that are highly functional. The novel isolation method is referred to interchangeably as the "DHF" method or the "iMIT" method; Mitochondria obtained by the iMIT method are referred to herein as "Q" mitochondria. Q Mitochondria are called mitochondria when they are isolated through traditional methods. They avoid the disruption and membrane destruction that occurs in mitochondria and therefore are less susceptible to being isolated through traditional methods. It is structurally and functionally superior to the Doria.

[0075] In some embodiments, the present disclosure provides a population of isolated or obtained mitochondria, The population contains a high proportion of polarized mitochondria (i.e., the population has a high polarization ratio Therefore, the population of mitochondria presented here is a population of mitochondria with a membrane potential. In one embodiment, the present disclosure provides a population of mitochondria, A high percentage of mitochondria in the population have intact inner and outer membranes. The presence of intact inner and outer membranes ensures the functional activity of mitochondria, e.g., membrane potential and differentiation. The poles can be determined by the

[0076] The populations of mitochondria provided herein can therefore be isolated by conventional methods, e.g., As mentioned above, homogenization and / or cell freeze-thawing and / or high concentration Mitochondria obtained from cells using the detergent method or a method including a detergent For example, mitochondria isolated from cells through traditional methods are superior to conventional populations. are inevitably damaged by the isolation process and lose their functional capacity. The mitochondrial populations obtained by conventional methods have higher polarization ratios and / or better Mitochondria with a higher % polarization and / or a higher % intact inner and outer membranes The present invention provides a population of isolated mitochondria having a

[0077] In some embodiments, the polarization ratio of an isolated or obtained population of mitochondria can be determined, e.g., by: 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more or more, 75% or more, 80% or more, or 85% or more.

[0078] In one embodiment, at least about 60% of the isolated or obtained mitochondrial population , at least about 65%, at least about 70%, at least about 75%, at least about 80% , at least about 85%, at least about 90%, at least about 95% or more of the fluorescent indicator In one embodiment, the fluorescent indicator is a mitochondrial membrane It can be any fluorescent indicator known to those skilled in the art to be suitable for measuring electrical potential. In one embodiment, the fluorescent indicator is selected from the group consisting of JC-1, TMRM, and TMRE. It is selected.

[0079] In one embodiment, at least about 60% of the isolated or obtained mitochondrial population , at least about 65%, at least about 70%, at least about 75%, at least about 80% , at least about 85%, at least about 90%, at least about 95% or more In one embodiment, the isolated or obtained mitochondria have an inner and outer membrane. At least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or more have densely packed cristae in the inner membrane. In one embodiment, the cristae of Q mitochondria are isolated from the cell, i.e., from the cell. It resembles the cristae structure of unisolated mitochondria. The term "densely packed cristae" refers to cristae with a high density of mitochondria. The density of the cristae is using microscopy (e.g., transmission electron or light microscopy, including confocal microscopy); In one embodiment, the cristae density in mitochondria can be measured by the square root of the It can be measured by the number of cristae per micrometer, which is the Manually by counting and / or by automated software programs In one embodiment, "dense cristae," "densely folded" The "contained cristae" etc. are at least about 3, at least about 4, At least about 5, at least about 6, at least about 7, at least about 8 or more Listeria (i.e., cristae) or, alternatively, mitochondrial Cristae density in mitochondria is measured by the cristae surface area per mitochondrial volume. Thus, in one embodiment, "dense cristae," "densely folded "Cristae surface area" is the area of the cristae per mitochondrial volume (μm 2 μm -3 ) is at least about 20, at least about 25, at least about 30, at least about 35, This means that the cristae density is at least about 40 or greater. It is well known in the art (see, for example, Segawa et al., "Quantification of cristae architecture reveals time dep endent characteristics of individual mit ochondria,” Life Science Alliance, Vol. 3, No. 7, 202 June 2006; and Nielsen et al., The Journal of Physiology (See, Phys. 595.9 (2017) 2839-47). In one embodiment, At least about 60% of the mitochondria in the population of mitochondria provided by at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least At least about 85%, at least about 90%, at least about 95%, or more, square microphone at least about 3, at least about 4, at least about 5, at least about 6 per meter, having at least about 7, at least about 8, or more cristae; and / or At least about 20 cristae surface area (μm 2 μm -3 ), less Both are approximately 25 μm 2 μm -3 , at least about 30 μm 2 μm -3 , at least about 35 μm 2 μm -3 , at least about 40 μm 2 μm -3 In some embodiments, the nucleotides provided herein have a molecular weight of 100 or more. The isolated mitochondria provided are those derived from the cell type from which they were obtained. Average cristae density equal to and / or not significantly lower than that of mitochondria or have a typical cristae density. At least about 60%, at least about 65%, at least about 60% of the mitochondria in the rear population at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least Approximately 90%, at least 95% or more of the isolated mitochondria were obtained. and / or equivalent to the average or representative cristae density of mitochondria in a given cell type. The cristae density is not significantly lower than that of the nuclei.

[0080] In some embodiments, the isolated populations of mitochondria provided herein are high calcium mitochondrial Mu (Ca 2+ ) has the surprising ability to maintain functional capacity even when exposed to environmental In embodiments, the population of isolated mitochondria provided herein comprises the mitochondrial fragments provided herein. Due to the isolation method, the cells maintain their functional capacity in the extracellular environment. At least about 60%, at least about 65%, or at least about 60% of the mitochondrial population obtained or At least about 70%, at least about 75%, at least about 80%, at least about 85%, At least about 90%, at least about 95% or more of the cells retain their functional capacity in the extracellular environment. In one embodiment, the extracellular environment is maintained at about 6 mg / dL to about 14 mg / dL, or about In one embodiment, the total calcium concentration is between about 8 mg / dL and about 12 mg / dL. The environment is free from about 3 mg / dL to about 8 mg / dL, or about 4 mg / dL to about 6 mg / dL. Thus, in one embodiment, the QM provided herein comprises a concentration of 0.1% or more of the active calcium. Remarkably, mitochondrial cells are isolated from their cellular environment with minimal or negligible damage. and have desirable characteristics that would otherwise cause damage to the extracellular environment, e.g., mitochondria. calcium-rich foods that are expected to significantly impair the function and / or function of the retains the ability to function even when exposed to a toxic environment.

[0081] Without wishing to be bound by theory, in some embodiments, functional ability in the extracellular environment is The ability of the isolated or derived mitochondria provided herein to maintain potency is due in part to Overall, the mitochondrial surface voltage-dependent anion channels (VDA) and tubulin are involved in the C). For example, in some embodiments, the iMIT provided herein During the isolation process, tubulin binds to all or a significant number of V It can associate with DAC, and therefore, it is necessary to maintain a calcium-rich environment (e.g., about 3 mg / dL Even in an extracellular environment containing approximately 14 mg / dL of calcium or more, In one embodiment, the ribosome is isolated from tubulin and is capable of maintaining its function. The association of VDAC with the mitochondrial surface is related to the presence of tubulin at the mitochondrial surface. The presence of the protein can be determined by detecting it, for example, by staining.

[0082] While not wishing to be bound by theory, in some embodiments, the isolated The isolated Q mitochondria were either totally or partially iMIT-extramitochondrial during isolation. Due to the loss of cholesterol, ergosterol and / or related molecules in membranes, It is possible to maintain functional capacity in the extracellular environment. During the isolation procedure, small amounts of detergent are introduced into the mitochondrial membrane (which stabilizes the VDAC structure). may be impaired to some extent due to contact with the VDAC, and may lose some or all of its function. This results in isolated mitochondria with extracellular cAMPs. Calcium concentration (e.g., about 3 mg / dL to about 14 mg / dL of calcium or more) Thus, in one embodiment, the present invention provides a method for the preparation of a medicament for the treatment of dengue fever, which is characterized by the presence of dengue fever and flu-like symptoms. Isolated mitochondria express very low levels of sterols in the mitochondrial membrane. Included.

[0083] In some embodiments, the isolated or obtained population of mitochondria is a population of mitochondria in a cell. Doria and / or conventional methods, e.g., homogenization of cells and / or Compared to mitochondria isolated or obtained using methods involving freezing and thawing cells, Meeting with MAM (mitochondria-associated membrane) In one embodiment, the decreased association with MAM further indicates a decrease in mitochondrial This is measured by the expression of the glucose-regulated protein GRP75 on the surface of the skin.

[0084] In some embodiments, the isolated mitochondria are substantially non-filamentous in shape. "Network-like" can be used interchangeably with "non-network-like" and refers to the way mitochondria function in cells. This means that the branched and reticular network of mitochondria present in the (See, for example, Figure 7A, which shows the typical filamentous shape of mitochondria in a cell.) In embodiments, rather than having a thread-like, networked, or branched structure, The mitochondria provided herein may be round, spherical, irregular, or globular when viewed under a microscope. and / or slightly elongated, or any mixture thereof. In contrast, at lower magnifications, isolated mitochondria appear as punctate structures. The highly elongated network or branched structure of mitochondria in cells is visible. In one embodiment, at least about 6% of the isolated or obtained population of mitochondria 0%, at least about 65%, at least about 70%, at least about 75%, at least about 8 0%, at least about 85%, at least about 90%, at least about 95% or more , having a major axis to minor axis ratio of 4:1 or less, 3.5:1 or less, or 3:1 or less. Although we do not wish to be identified as such, we do wish to identify mitochondria isolated through the methods provided herein. The shape is due to the motor proteins attached to the microtubules while the mitochondria were still in the cell before isolation. This results from the gentle removal of the connections made by mitochondria to the cell's microtubules. When no longer linked, they instead form the non-filamentous shapes described herein. The very elongated, branched / networked shape that the cells had in order to form lose.

[0085] In some embodiments, the isolated mitochondria in the mitochondrial populations provided herein At least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or less Approximately 95% of all mitochondrial proteins have a length less than twice the hydrodynamic diameter of the mitochondria. In an embodiment, the hydrodynamic diameter is about 1 μm, and in the mitochondrial populations provided herein At least about 60%, at least about 65%, at least about 60% of the isolated mitochondria at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least At least about 90%, or at least about 95%, have a major axis length of 2 μm or less, 1.9 μm or less, 1.8μm or less, 1.7μm or less, 1.6μm or less, 1.5μm or less, 1.4μm or less, or a length of 1.3 μm or less. In some embodiments, the hydrodynamic diameter is measured by dynamic light scattering. In one embodiment, the hydrodynamic diameter is measured by DLS. 50 Yes do.

[0086] Generally, in cells, mitochondria are very elongated or filamentous in shape, as mentioned above. Non-filamentous, non-elongated mitochondria are generally drp1-dependent. It is only present in cells when they are undergoing division or drp1-dependent division. During this process of mitochondrial division, interaction with the endoplasmic reticulum induces the initial constriction of mitochondria. Drp1 protein is recruited to mitochondria and assembled on their surface, causing further DYN2 is recruited to carry out the final step of membrane scission. Mitochondria may be generally spherical in shape. In cells, such spherical mitochondria The endothelial cells remain for a limited period before becoming elongated or forming more common branch-like structures. In contrast, mitochondria isolated using the iMIT method are able to retain their spherical shape. The endothelial cells are non-filamentous in shape without undergoing Drp1-mediated division. Mitochondria isolated by conventional methods, such as those involving homogenization, They produce mitochondria that are non-filamentous in shape and mostly round or spherical, The reason is that they are injured and the cells that would otherwise cause them to maintain their elongated shape This is because the mitochondria isolated in this way are torn from their microtubules. In contrast, mitochondria isolated by the iMIT method provided herein are microscopic. They have not undergone deleterious removal from the tube and have not undergone drp1-mediated division. The disclosed mitochondria are naturally occurring mitochondria in cells and those derived by more traditional methods. For example, in one embodiment, mitochondria isolated through the iMIT method The mitochondria provided herein are substantially non-filamentous in shape, but contain drp Without undergoing division, the cells are in a highly functional state (e.g., polarized), densely folded, The intact inner and outer membrane structures containing the staphylococcus aureus are shown together.

[0087] In one embodiment, the Q mitochondria provided herein are attached to a cell or population of cells. When exposed to these proteins, they exhibit an unexpected function: co-localization with endogenous mitochondria in cells. Chondria are significantly more abundant than mitochondria isolated through conventional methods. In one embodiment, the Q-micoproteins provided herein co-localize to a small extent with endogenous mitochondria. When contacted with a cell or group of cells, mitochondria act to stimulate the endogenous mitochondria in the cell. Fusion of isolated Q mitochondria is more efficient than that of conventionally isolated mitochondria. In one embodiment, mitochondria are It retains this ability even after storage. Thus, in one embodiment, the antibodies provided herein Q Mitochondria are at least partially responsible for their co-localization with endogenous mitochondria in cells. mitochondria are more efficient at integrating and / or fusion than conventionally isolated mitochondria. and therefore, are superior in treating any disease or disorder, such as those described herein. This is because the Q mitochondrial globulin provided herein shows excellent clinical efficacy when used to treat Mitochondria have a more robust and nearly intact outer membrane compared to conventionally isolated mitochondria. It may suggest that it has.

[0088] In some embodiments, the present disclosure provides a method for the production of a mitochondrial protein comprising administering to a subject a subject therapies, including but not limited to, a mitochondria, a mitochondrial protein ... For example, the present disclosure provides a population of chondria, including the process of the iMIT method described hereinabove. A population of mitochondria isolated or obtained by a method comprising steps (A) to (C) is provided. In one embodiment, the present disclosure includes steps (A) to (E) described herein above. A population of mitochondria isolated or obtained by the method is provided.

[0089] The present disclosure provides compositions comprising a population of isolated mitochondria of the present invention. The present disclosure provides mitochondrial preparations comprising a population of isolated mitochondria of the present invention. The composition comprising a population of isolated mitochondria of the present invention further comprises a buffer. The mitochondrial product containing the isolated mitochondrial population of the present invention can be included in the The formulation is pharmaceutically acceptable and further comprises additional pharmaceutically acceptable components, e.g., excipients. The isolated mitochondrial population of the present disclosure or a composition containing the same can be Alternatively, mitochondrial preparations can be analyzed using flow cytometers such as fluorescence activated cell sorting (FACS). can be obtained during the isolation process without the use of cell sorting by The isolated mitochondrial population of the present invention, or a composition or mitochondrial composition containing the same Mitochondrial preparations contain fluorescent dyes and fluorescent probes (as well as non-fluorescent mitochondrial stains) and probe). In some embodiments, the composition is a pharmaceutical composition.

[0090] Detection of mitochondrial membrane potential Whether mitochondria have a membrane potential (whether they are polarized or not) depends on the The mitochondrial membrane potential can be determined by detecting the mitochondrial membrane potential. The detection can be performed using an indicator, such as a fluorescent indicator. The fluorescent indicators used are JC-1 and tetramethylrhodamine methyl ester (TMRM). ) and tetramethylrhodamine ethyl ester (TMRE). JC-1 is It accumulates in mitochondria and changes color from green to red upon sensing the mitochondrial membrane potential. M and TMRE accumulate in mitochondria and sense the mitochondrial membrane potential to induce red light. Generate.

[0091] Depolarized mitochondria were used as a negative control after detecting the mitochondrial membrane potential. Mitochondria can be depolarized by a mitochondrial depolarizing agent. Examples of mitochondrial depolarizing agents include carbonyl cyanide-m- For example, in the presence of 5 μM CCCP, Mitochondrial membrane potential after depolarization by incubation at room temperature for 1 hour under (or the fluorescence intensity by the fluorescent indicator) can be used as a negative control, Mitochondria with a membrane potential (or fluorescence intensity measured by a fluorescent indicator) higher than , it can be determined that the mitochondria have a membrane potential. The fluorescence intensity of the sample is determined by eliminating the effect of background fluorescence. can be calculated (for example, as a ratio to or difference from background fluorescence intensity). When the mitochondrial membrane potential of the negative control fluctuates, it is 90% or more, 91% or more of the negative control. 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more Mitochondria with membrane potentials greater than or equal to 99% of the mitochondria have membrane potentials In this way, it can be determined that the mitochondria have the same position. In the disclosed method, mitochondrial membrane potential can be detected in a population of mitochondria. An additional step leading to loss of mitochondrial membrane potential is performed in the detection of mitochondrial membrane potential. I can't.

[0092] The mitochondrial polarization ratio is the ratio of the number of mitochondria with membrane potential to the total number of mitochondria. The mitochondrial polarization ratio is the ratio (%) of the mitochondrial polarization ratio to the mitochondrial polarization ratio. A specific area (e.g., 100 μm) of a substrate board such as glass 2 ~10,000μm 2 ) The number of mitochondria contained in the region and the mitochondria with membrane potential within that region Mitochondria can be counted using a light microscope, for example. It is possible.

[0093] Treatment method In one aspect, the present disclosure provides a method for treating a disease or disorder associated with mitochondrial dysfunction, or Treating a disease or disorder where supplementation of otherwise healthy, functional mitochondria is beneficial This provides a way to

[0094] In one embodiment, the Q mitochondria provided herein are suitable for treating a disease or condition. The disorder may be a genetic disease or disorder, an ischemia-related disease or disorder, a neurodegenerative disease or disorder, or disorders, cancer, cardiovascular diseases or disorders, autoimmune diseases, inflammatory diseases, fibrotic diseases, It is an age-related disease or disorder, or a disease associated with complications of childbirth.

[0095] Exemplary ischemia-related diseases and disorders include cerebral ischemia-reperfusion, hypoxic-ischemic encephalopathy, Acute coronary syndrome, myocardial infarction, hepatic ischemia-reperfusion injury, ischemic injury-compartment syndrome, vascular blockage, wound healing (e.g., acute or chronic wounds; cuts, lacerations, pressure wounds) wounds, burns (e.g., chemical, heat or flame, wind or sunburn) or medical or wounds resulting from surgical interventions, spinal cord injuries, sickle cell disease and recurrence of transplanted organs In one embodiment, Q mitochondria are involved in ischemia-reperfusion injury. In one embodiment, the present invention can treat, prevent, ameliorate, and / or improve the clinical condition of a patient. Q: Mitochondria improve ejection fraction (EF), suppress cardiac hypertrophy, and and / or to treat, prevent, ameliorate, and / or improve fibrosis following ischemia-reperfusion injury. It is possible.

[0096] Exemplary autoimmune and / or inflammatory and / or fibrotic diseases and disorders include: These include acute respiratory distress syndrome (ARDS), abdominal disease, vasculitis, lupus, and chronic obstructive pulmonary disease. COPD, irritable bowel disease, inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease), These include multiple sclerosis, atherosclerosis, arthritis and psoriasis.

[0097] Exemplary cancers include, for example, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, Adenocarcinoma, testicular cancer, lung cancer, hepatocellular carcinoma, kidney cancer, bladder cancer, stomach cancer, colorectal cancer, Pancreatic cancer, esophageal cancer, melanoma, lymphoma, leukemia, and blastoma (e.g., neuroblastoma) Examples include:

[0098] Additional diseases and disorders that may be treated by administration of Q mitochondria provided herein Harms include diabetes (types I and II), metabolic disorders (e.g., hyperglycemia, hypoglycemia, glutathione-deficiency syndrome ... Course intolerance, insulin resistance, hyperinsulinemia, metabolic syndrome, syndrome, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, dyslipidemia Diabetes, hypertriglyceridemia, kidney disease, ketoacidosis, thrombotic disorders, nephropathy, diabetic neuropathy disorder, fatty liver, nonalcoholic fatty liver disease and steatohepatitis), mitochondrial dysfunction eye disorders associated with diabetes (e.g., glaucoma, diabetic retinopathy, or age-related macular degeneration), Hearing, mitochondrial toxicity associated with therapeutic drugs, cardiotoxicity associated with chemotherapy or other therapeutic drugs Mitochondrial dysfunction disorders (e.g., mitochondrial myopathy, diabetes, and hearing loss) Disorders of the optic nerve (DAD) syndrome, Barth syndrome, Leber's hereditary optic neuropathy (LHON), Leigh syndrome Leigh) syndrome, NARP (Neuropathy, Ataxia, Retinitis Pigmentosa and Ptosis Syndrome) , myoneuropathic gastrointestinal encephalopathy (MNGIE), MELAS (mitochondrial encephalopathy, lactic acidosis) Myoclonic epilepsy with ragged-red fibers (MERRF) syndrome ) syndrome, Kearns-Sayre syndrome and mitochondrial DNA depletion syndrome), or In some embodiments, the disease or disorder is pre-eclampsia or intrauterine fetal morbidity. Growth retardation (IUGR).

[0099] In one embodiment, the present disclosure provides a method for administering to a subject in need thereof the Q mitochondrial endothelial cell line provided herein. and methods for treating aging and age-related conditions by administering levothyroxine to the subject. Normal aging and age-related conditions can be treated with the compositions and methods provided herein. Age-related conditions include neurodegenerative conditions, cardiovascular conditions, hypertension, obesity, bone These include porosis, cancer and type II diabetes.

[0100] Exemplary neurodegenerative diseases and disorders include, for example, dementia, Friedreich's ataxia, Symptoms of dyslipidemia, amyotrophic lateral sclerosis, mitochondrial myopathy, MELAS (encephalopathy, lactic acidosis) sis, stroke), myoclonic epilepsy with ragged red fibers (MERFF), epilepsy, Exemplary neuropsychiatric disorders include Parkinson's disease, Alzheimer's disease, and Huntington's disease. Psychological disorders include bipolar disorder, schizophrenia, depression, addictive disorders, anxiety disorders, and attention These include deficit disorders, personality disorders, autism and Asperger's disease.

[0101] Exemplary cardiovascular diseases include coronary heart disease, myocardial infarction, atherosclerosis, hypertension, and Blood pressure, cardiac arrest, cerebrovascular disease, peripheral arterial disease, rheumatic heart disease, congenital heart disease, congestive heart failure These include heart failure, arrhythmia, stroke, deep vein thrombosis, and pulmonary embolism.

[0102] In one aspect, the present disclosure provides methods for the production of mitochondrial cells in cells, tissues of a subject, organs, egg cells, or embryos. In one embodiment, the organ is a heart, a lung, a kidney, or a brain, skeletal muscle, skin tissue, facial muscle, bone marrow tissue, or white adipose tissue. In some embodiments, the organ is a transplant organ. In some embodiments, the cells are transplanted cells. In embodiments, the tissue is transplanted tissue, for example, transplanted bone marrow tissue.

[0103] In one aspect, the present disclosure provides a method for detecting mitochondrial dysfunction. In embodiments, the methods include detecting a biomarker of mitochondrial dysfunction. In some embodiments, the present disclosure provides a method for treating mitochondrial dysfunction in a subject, comprising: Detect mitochondrial dysfunction in subjects with Q mitochondria provided in In an exemplary embodiment, a method for detecting a biomarker of mitochondrial dysfunction is provided. - heteroplasmy, peripheral mitochondrial number, mitochondrial DNA deletion or replication, and / or DNA methylation levels. Markers include growth differentiation factor 15 (GDF15), apelin, humanin, and / or fibrinogen. It may be blood levels of blast growth factor 21 (FGF21).

[0104] In some embodiments, the Q mitochondria provided herein are administered systemically (e.g., intranasally, intramuscularly, subcutaneously, intra-arterially, intratracheally, by inhalation, intrapulmonary, or intravenously) or topically. In one embodiment, the mitochondria are administered to a subject in a pharmaceutically acceptable carrier. In some embodiments, the mitochondria are engineered to treat a disease or disorder. administered to a subject in combination with one or more additional agents and / or therapies In some embodiments, the mitochondria are syngeneic, allogeneic, or xenogeneic. It is mitochondria.

[0105] The present disclosure relates to methods for the manufacture of medicaments for treating the diseases and disorders defined herein. The present disclosure also provides for the use of Q mitochondria in any of the methods provided herein. Q mitochondria are also provided for use.

[0106] The present disclosure also provides kits for use in treating the diseases and disorders defined herein. In one embodiment, the kit comprises a population of Q mitochondria provided herein. In some embodiments, the kit includes instructions for administering the Q mitochondria to a subject. In one embodiment, the present disclosure further includes a kit for isolating Q mitochondria, Kits are also provided, e.g., for carrying out the iMIT isolation method. In one embodiment, the kit The present invention provides a novel method for isolating mitochondria from cells via the iMIT method. Includes detergent, buffer and instructions for use.

[0107] Including, but not limited to, patents, patent applications, articles, books, papers and internet web pages. All literature and similar materials cited in this application, including, but not limited to, the following, are hereby incorporated by reference for all purposes. Unless otherwise specified, all references made herein to the Technical and scientific terms are used to refer to those skilled in the art to which various embodiments described herein belong. have the same meaning as commonly understood by one skilled in the art. When a definition appears to differ from the definitions set forth in the present teachings, the definitions set forth in the present teachings shall control. Let's say. [Example]

[0108] Example 1: Surfactant method and homogenization-free (DH) compared with conventional methods F) Method ("iMIT") Conventional isolation methods involving homogenization and / or high concentrations of detergents are For comparison with the surfactant and homogenization-free methods provided in the specification The mitochondria isolated by the iMIT method were used in the present study. These are called "Q" mitochondria. The isolated mitochondria are herein referred to as H-mitochondria or H- Mito, and D-mitochondria or D-mito.

[0109] The cells used in this study were human-derived HeL cells purchased from the RIKEN cell bank. The culture medium used was MEM + 10% FB. The cells were passaged once or twice a week in S. For the DHF method, the following steps were carried out. 1) Culture the cells in a 100 mm diameter dish until they are 80% confluent. Confirmed. 2) Discard the medium and place the cells in isolation buffer (10 mM Tris-HCl, 250 mM Sodium Chloride). The cells were washed twice with 3 mL of cellulose, 0.5 mM EGTA, pH 7.4. 3) Add 3 mL of isolation buffer containing 30 μM digitonin and incubate the dish at room temperature. It was allowed to stand for 3 minutes. 30 μM is approximately 1 / 10 of the critical micelle concentration (cmc) of digitonin. (2,3) . 4) The inside of the dish was washed twice with 3 mL of isolation buffer. 5) 3 mL of isolation buffer was added, and the dish was allowed to stand at 4°C for 10 minutes. 6) Detach the cells by gently pipetting them with a micropipette. Ta. 7) Then, transfer the suspension containing the detached cells and mitochondria to a 15 mL centrifuge tube. Transfer the cells to the wells, centrifuge at 500 x g for 10 min at 4 °C, and collect 2 mL of the supernatant to obtain isolated mitochondria. The isolated mitochondrial population was obtained at this stage by the following method. It may or may not be frozen throughout. 8) When freezing, do not use isolation buffer, but freezing buffer (10 mM Tris s-HCl, 225 mM mannitol, 75 mM sucrose, 0.5 mM EGTA, p Glycerol was added to H7.4) to make the glycerol concentration 10%. Frozen material from the rear (a population of isolated mitochondria in a frozen state or a composition containing the same) The product was obtained by freezing in liquid nitrogen.

[0110] Isolation of mitochondria using higher concentrations of detergent (above the critical micelle concentration) A second isolation method was performed to test the following steps: 1) Culture the cells in a 100 mm diameter dish until they are 80% confluent. Confirmed. 2) The medium was discarded, and the cells were washed twice with 3 mL of isolation buffer. 3) Digitonin dissolved in 3 mL of isolation buffer was added to a concentration of 400 μM (critical micelle concentration The dish was then allowed to stand at room temperature for 3 minutes. 4) Detach the cells by gently pipetting them with a micropipette. Ta. 5) Transfer 3 mL of the suspension to a 15 mL centrifuge tube and centrifuge at 500 × g for 10 minutes at 4 °C. 2 mL of the supernatant was collected to obtain the isolated mitochondrial population. 6) When freezing, add glycerol to the freezing buffer to suspend it, and The concentration of the solution was adjusted to 10% and frozen in liquid nitrogen to obtain isolated mitochondria. The material was obtained.

[0111] Carry out the third isolation method using conventional homogenization methods and the following steps: Ta. 1) Culture the cells in a 100 mm diameter dish until they are 80% confluent. Confirmed. 2) The medium was discarded, and the cells were washed twice with 2 mL of isolation buffer. 3) 3 mL of isolation buffer was added, and the cells were detached using a cell scraper. 4) The suspension of detached cells was homogenized using a Potter-type glass Teflon (registered trademark) homogenizer. The suspension was homogenized in a centrifuge while being cooled in ice. The robot was moved up and down. 5) Transfer the homogenate to a 15 mL centrifuge tube and centrifuge at 500 × g for 10 minutes at 4 °C. 2 mL of the supernatant was collected to obtain the isolated mitochondrial population. 6) When freezing, add glycerol to the freezing buffer to suspend it, and The concentration of the solution was adjusted to 10% and frozen in liquid nitrogen to obtain isolated mitochondria. The material was obtained.

[0112] Mitochondria isolated by each of the above methods adhere to glass-based Petri dishes. The membrane potential of individual mitochondria was observed using a fluorescence microscope. Ta: 1) The suspension (300 μL) containing isolated mitochondria is placed on a glass-based sieve. The cells were spread on a glass surface of a glass tray and left to stand on ice for 1 hour to form isolated mitochondria on the glass surface. Then, 2 mL of 1 M KOH was added to a glass-based Petri dish (35 mm). In addition, the glass surface was washed. 2) The dish was washed twice with 2 mL of Milli-Q water. 3) Washed with 2 mL of ethanol. 4) The dish was washed twice with 2 mL of Milli-Q water. 5) The dish was washed twice with 2 mL of isolation buffer.

[0113] Using a zetasizer (Nanosize Nano-ZS, Malvern) Particle size analysis, zeta potential analysis, and polydispersity index (PDI) analysis of isolated mitochondria The procedure was performed according to the manufacturer's manual. Mitochondrial depolarization and membrane potential sensitivity Staining of mitochondria with dyes was performed as follows. 1) Mitochondria adsorbed on a glass-based Petri dish were added to 2 mL of isolation buffer. Washed with. 2) Add 2 mL of isolation buffer containing 5 μM CCCP and incubate the dish at room temperature for 1 It was left to stand for an hour. 3) 2700 μL of TMRE staining buffer containing 5 μM CCCP - (10 mM Tris-HCl, 250 mM sucrose, 10 nM TMRE, 0.3 The plate was then replaced with 3 mg / mL BSA, and the dish was left to stand at room temperature in the dark for 10 minutes. Ta. 4) A total of 56 μL of malic acid and glutamic acid was added to bring the concentration of each to 5 mM. Fluorescence observation was performed at room temperature within 5 minutes.

[0114] Isolated mitochondria were stained with membrane potential-sensitive dyes as follows. 1) Mitochondria adsorbed on a glass-based Petri dish were added to 2 mL of isolation buffer. Washed with. 2) Buffer: 2700 μL of TMRE staining buffer (10 mM TrisHCl , 250 mM sucrose, 10 nM TMRE, 0.33 mg / mL BSA) The dish was then left to stand at room temperature in the dark for 10 minutes. 3) A total of 56 μL of malic acid and glutamic acid was added to bring the concentration of each to 5 mM. Fluorescence observation was performed at room temperature within 5 minutes.

[0115] For observation, an Olympus IX70 fluorescence microscope and a cooled CCD camera (Sensica m QE, PCO AG; Kelheim, Germany) (6.45μm / pixel ) was used under the following conditions: Objective lens: ×40, NA0.9 Light source: Halogen lamp Absorption filter: Center wavelength 546nm and bandpass 10nm CCD camera: Binning: 2 x 2 Exposure time: 1 second

[0116] The same field of view was observed with the same equipment as in the case of transmitted light. The different conditions from the case of transmitted light were as follows: Light source: Xenon lamp Excitation filter: Bandpass filter that passes 520 to 550 nm Fluorescence filter: Sharp-cut filter that passes light above 580 nm

[0117] For analysis of the fluorescence images, 0.94 μm of the transmitted light images of individual mitochondria were used. 2 Take the area of The average fluorescence intensity in the region was calculated. The ratio of this value to the background fluorescence intensity is The fluorescence intensity ratio was calculated by multiplying the fluorescence intensity of each mitochondrion by 2000 to the second decimal place. Calculations were made by rounding down.

[0118] The distribution of the ratio of fluorescence intensities was determined as described in section 2.4.7. The threshold for the fluorescence intensity ratio, which is obtained from the ratio of the fluorescence intensities of polarized mitochondria and represents depolarized mitochondria, is The results are shown in Figure 1A. As shown in Figure 1A, over 97% of mitochondria were The rear existed with a fluorescence intensity ratio of 1.2 or less in the presence of 5 μM CCCP. We used a fluorescence intensity ratio of 1.2 as a threshold to determine whether mitochondria are polarized. It was decided that:

[0119] The percentage of polarized mitochondria in isolated mitochondria was determined by the DHF method and homogenization. The results were determined by the lysis method and the surfactant method (n = 130 for each). 150) The results are shown in Table 1. The polarization ratio can be calculated by, for example, the transmitted light image in the left panel of Figure 1B. The percentage of black spots that appear to be 0.5-1.5 μm in diameter is shown in the right panel of Figure 1B. The fluorescence intensity ratio of TMRE in the panel is greater than the threshold of 1.2. The mitochondria examined were pre-frozen.

[0120] [Table 1]

[0121] Figure 2A shows Q mitochondria (iMIT method) versus homogenized mitochondria. Tables 1 and 2 and Figure 2B show the TMRE fluorescence of the same number of cells at the time of isolation and and the resulting mitochondrial fractions isolated by the iMIT method with comparable protein content. The percentage of TMRE-positive mitochondria compared to conventional methods It is confirmed that there is a statistically significant increase in (*p<0.05, Figure 2B, lower panel). Figure 2C shows that almost all mitochondria obtained through the iMIT method were TMRE+. A random selection of 10 black dots from the bright field image compared with TMRE positive staining was Figure 2D shows that 90% of mitochondria were TMRE-positive. The isolated mitochondria retain their double membranes (inner and outer membranes) and cristae structure. This indicates that

[0122] [Table 2]

[0123] Through conventional homogenization method (H-Mito) or conventional surfactant method (D-Mito) Compared with mitochondria obtained through the iMIT method, Q mitochondria were obtained through the iMIT method. To visualize the outer membrane, stimulated emission depletion (STED) microscopy was used (Figure 2E). stained for Tom20 (immunofluorescence), and for the inner membrane with Mitotracker Red. As seen in Figure 2E, Q mitochondria had intact inner membranes and H-mites had an outer membrane, whereas H-mites had much less detectable inner membrane or an intact outer membrane. However, D mitos had even fewer detectable inner or outer membranes. It was shown that a portion of the inner membrane protrudes from the outer membrane, suggesting that the outer membrane was physically damaged during the isolation process. In D-mitochondrial cells, many small fragments from the outer membrane were detected, suggesting that some of the outer membrane Some do not have an inner membrane inside the outer membrane, which is due to the detergent used during the isolation process. The isolated mitochondria will be chemically disrupted to solubilize the outer and inner membranes. The ratio of mitochondria with membrane potential obtained from the test to mitochondria with intact outer membranes was Quantitative analysis of mitochondrial ratio, mitochondrial length along the major axis, and the ratio of major to minor axis The results are provided in Table 3 below. Protein content was also measured (mitochondrial DNA from HeLa cells). Indria-1 dish 150Φ, 12,000,000 cells). Measured in mitochondria isolated from vesicles with intact outer membrane, diameter, and long:short diameter ratio was measured in mitochondria isolated from HeLa cells.

[0124] [Table 3]

[0125] The results of these tests demonstrate that the iMIT method of the present disclosure maintains structural integrity and exhibits polarization. The results demonstrated that the method is suitable for preparing mitochondria that can be cultured. The mitochondria obtained by the iMIT method shown in Fig. 1 were prepared by conventional mitochondrial preparation methods ( Higher concentrations of cellulose can be used to show polarization than homogenization and high surfactant methods. The study also showed that mitochondria had a high polarization ratio. The isolated mitochondria had a non-filamentous shape, compared with H and D mitochondria. Tests also demonstrated that the particles were generally less round or spherical. Pretreatment of cells with detergents is sufficient for the recovery of mitochondria from inside the cells. Furthermore, the study revealed that mitochondria isolated by the iMIT method , which are fundamentally different and functionally superior compared to mitochondria isolated by conventional methods. This shows that

[0126] The size distribution and zeta potential of mitochondria isolated by the iMIT method were measured. Pre-frozen samples (i.e., freshly isolated Q mitochondria via the iMIT method) The size distribution and zeta potential of the iMIT method are shown in Figure 3A. The mitochondria isolated by this method showed a monodisperse shape with a size (particle size) of 1034 nm. This result indicates that there is less contamination with nuclear DNA and debris from cells, and This suggests that the majority of the recovered material is mitochondria.

[0127] The size distribution and zeta potential of samples obtained through the iMIT method after freezing and subsequent thawing The potential of the mitochondria is shown in Figure 3B. This was performed by freezing the sample in liquid nitrogen afterwards. The samples were transported on dry ice. Thawing was performed within 3 minutes. The mitochondria were isolated by holding the vial under running tap water while swirling the vial. As shown in Figure 3B, mitochondria isolated by the iMIT method were frozen and then resuspended in 100 ml of water. Even after sintering, the particle size was 1171 nm.

[0128] Additionally, iMI was performed except that centrifugation was performed at 1000 × g instead of 500 × g. The size distribution and zeta potential of mitochondria isolated by the T method were determined, and the results were The isolated mitochondria were frozen and thawed, as shown in Figure 3C. The size distribution and zeta potential were 858.5 nm.

[0129] The zeta (ζ) potentials were excellent for all of the above samples (-22.4 mV to 31.0 mV). V).

[0130] The specimens in Figure 3A and Figure 3B were subjected to TMRE staining, and the staining (Margaret's staining) was superimposed on the bright-field image. The results are shown in Figure 3D. The isolated mitochondria showed a high polarization ratio before and after freeze-thawing. It was shown that

[0131] Studies were performed to investigate the use of detergents other than digitonin. Instead, saponin was used (concentration: approximately 40 μM, the concentration is approximately 1 / 15 of the CMC). ), and mitochondria were isolated from cells in a similar manner as described above. The CMC of saponin was 538 ~646 μM (Komatsu et al., J. Oleo. Sci. 54 :265-270 (2002). A superior population of mitochondria with characteristics similar to those of the mitochondrial endothelial cells was obtained by this method.

[0132] Approximately 0.1 g, 1.2 g, and 1.5 g of heart, liver, and skeletal muscle obtained from a mouse, respectively 1.2 g of the tissue was cut into small pieces and treated with collagenase (concentration: 0.2% by weight) at 37°C for 30 minutes. This method uses the iMIT method described above with digitonin below the CMC. In this study, mitochondria were isolated from single-celled cells. The size, polydispersity and zeta potential of the ria were measured, and the results are shown in Figure 4. As shown in Figure 4, mitochondria isolated from liver and heart have excellent zeta potentials. Mitochondria isolated from skeletal muscle also showed excellent zeta potential (i.e., approximately -1 5mV) and has a size distribution of about 100nm to about 2,000nm. We have demonstrated that mitochondria can be isolated directly from tissue samples using the iMIT method. In particular, mitochondria obtained from liver exhibited the lowest zeta potential, and these was considered preferable among the examples.

[0133] The activity of mitochondria isolated by the iMIT method was significantly improved as shown in Figures 5A and 5B. After isolation, the cells were incubated in 20 nM TMRE, 1 mM KH2PO4, and 0.5 mM ADP. -K, 0.33 mg / ml BSA, 0.5 M EGTA, 10 mM Tris, 110 Incubate mitochondria in 70 mM sucrose and 70 mM KCl for 10 min at room temperature. TMRE fluorescence was averaged over 10 mitochondria and normalized to 1 at t=0. Between 0 and 1 minute, malic acid was added at 1 mM. Between 5 and 6 minutes, oligomycin was added at 1 mM. Figure 5A shows that TMRE fluorescence changes in single mitochondria. Figure 5B shows a typical time course of TMRE fluorescence imaging in a single mitochondrion. The time interval between images was 1 min.

[0134] Example 2: Co-localization of Q mitochondria with endogenous mitochondria in recipient cells Mitochondria isolated through the iMIT method (Q) were cloned into mitochondria in recipient cells. Tests were performed to determine whether co-localization with mitochondria was possible. were isolated from cardiac progenitor cells using the iMIT method and visualized with Mito Tracker (red). Endogenous mitochondria in recipient LHON fibroblasts were labeled green. Exogenous Mitotracker mitochondria were contacted with recipient cells and co-transfected. Focusing microscopy images were taken. A representative image is provided in Figure 6A, showing that isolated mitochondria Figure 6B shows that the ATP-dependent ATP transporter migrated into cells overnight and colocalized with recipient mitochondria. Mitochondria isolated using conventional homogenization methods and the method provided herein We provide a comparison of colocalization between mitochondria isolated using the iMIT method and those isolated using the iMIT method. The upper panel of Figure 6B shows that a few mitochondria isolated by conventional methods were transferred to cells. However, the vast majority of the proteins isolated through the iMIT method The mitochondria are transferred to the cells, and only the mitochondria from the iMIT method are transferred to the recipient cells. They colocalized with mitochondrial cells and formed filamentous, network-like, and / or reticulate structures (Fig. 6B, lower panel).

[0135] Example 3: Shape of functional isolated mitochondria Mitochondria isolated by the iMIT method provided herein express drp1-mediated Tests were performed to determine whether cells were undergoing division. Generally, most cell types Mitochondria in the brain are long and filamentous, forming a network-like or reticulated structure. Any mitochondria in cells that do not have long filamentous and reticular shapes are mediated by drp1. They are generally non-filamentous because they undergo fission.

[0136] In the study, the drp1 inhibitor Mdivi1 was added to the cells. The iMIT method exhibits a networked and filamentous shape (Figure 7A). Mitochondria in cells treated with α-diaminobenzyl methyltransferase (MDM) were expressed in the absence and presence of Mdivi1 (0 or 1). 10 μM or 10 μM Mdivi1 (Fig. 7B) were non-filamentous. The non-filamentous form of iMIT is initiated while the ribosome is still in the cell, and is involved in drp1-mediated division. Figure 7C shows mitochondria after iMIT isolation, showing that they have a non-filamentous shape. Therefore, this study demonstrates that the iMIT method provided herein This indicates that the non-filamentous shape of isolated mitochondria is independent of drp1 fission. That is, Q mitochondria, at least when they are nonfilamentous, are drp1-dependent. They differ from mitochondria present in cells in that they do not undergo division.

[0137] Example 4: Polarization under Ca2+ conditions Studies were performed to investigate the function of Q mitochondria under high calcium conditions. Mitochondria are isolated from cells through the iMIT method, detergent method, or homogenization method. Each of these three mitochondrial populations was separated into two subpopulations. The 1 subpopulation was treated with BSA as a control and 10 nM TMRE and TrisHCl-sucrose. The second subpopulation was incubated in BSA and 100% EGTA buffer. in DMEM containing 0 nM TMRE and 200 mg / mL CaCl2 for 10 min Incubated.

[0138] The results of the test showed that mitochondria isolated through the iMIT method (Q) were The polarity was observed by the surfactant method (D-Mito) or the homogenization method (H-Mito). showed that mitochondria isolated in the presence of Ca2+ did not show polarizability. In the center row of Figure 8, a magnified image of the D-mitochondrial micrograph shows that there are many mitochondria in the sample. Although there are mitochondrial structures, the TMRE image does not show any TMRE+ mitochondria. In contrast, in the top row of Figure 8, many TMRE+Q mitochondria are observed. Therefore, this study demonstrates that Q mitochondria remain functional even when exposed to a high Ca2+ environment. demonstrated that it retains its capabilities.

[0139] Further testing was performed using calcein fluorescence to detect holes in the mitochondrial membrane. Q Mitochondria were isolated from HUVEC cells using the iMIT method and placed in glass vessels. The TMRE and calcein in individual mitochondria were absorbed into a petri dish. Fluorescence was observed by fluorescence microscopy. Changes in fluorescence after the indicated treatments were observed under the same microscope. Observations were made sequentially in the field.

[0140] Q Mitochondria were isolated in an isolation buffer containing 5 mM malate and 5 mM glutamate. Incubate with 1 μM calcein-AM in isolation buffer for 10 min at room temperature. The upper panel of Figure 9A shows the isolation of calcein Q with 1 mL of isolation buffer. The fluorescence is shown in Figure 9A. The lower panel of Figure 9A shows the results of the experiment using 4 mL of HBS (10 mM HEPES , 120mM NaCl, 4mM KCl, 0.5mM MgSO4, 1mM NaH2 PO4, 4 mM NaHCO3, 25 mM glucose, 1.2 mM CaCl2, 0.1 Calcein fluorescence of Q after addition of 100% bovine serum albumin (HBS, pH 7.4) is shown. The calcium was added gently towards the edge of the dish. Without altering the mitochondrial function, Q mitochondria isolated through the iMIT method are membrane-integrated in a Ca2+-rich environment. It was confirmed that the efficacy was maintained.

[0141] This study demonstrated that Q mitochondria in a Ca2+-rich environment, as measured by TMRE fluorescence, We further confirmed that 5 mM malate and 5 mM glutamate maintained the membrane potential (Fig. 9B). Q was incubated with 10 nM TMRE for 10 min in isolation buffer containing thiamin mononitrate. The upper panel of Figure 9B shows the results of the 1 mL of isolation buffer. The TMRE fluorescence of Q is shown. The bottom panel of Figure 9B shows the TM with 4 mL of HBS added. HBS was gently added towards the edge of the dish. TMRE fluorescence was observed when HBS was added. It was maintained even after the addition of

[0142] Interestingly, the presence of a Ca2+ environment and the physical stimulus of pipetting (agitation) When mitochondria are physically destroyed by heating, the mitochondria react with calcein. The fluorescence was lost (Figure 10A). Q contains 5 mM malate and 5 mM glutamate. Incubate with 1 μM calcein-AM in isolation buffer for 10 min at room temperature and isolate The cells were gently washed with buffer. As shown in the upper right panel of Figure 10A, the cells were washed with pipetting (1 Agitation of Q (0–15 times) reduced calcein fluorescence. Addition of 0.96 mM Ca2+ Calcein fluorescence after addition is shown in the center row of the right panel. Gently add 4 mL of HBS containing 5 mM malate and 5 mM glutamate to the edge of the dish. In the presence of Ca2+, the Q was stirred again by pipetting 10-15 times, and Ca2 Calcein fluorescence after addition of + and stirring is shown in the center column of the left panel. Sein fluorescence is shown in the lower panel of Figure 10A. Furthermore, Figure 10B shows the presence of mitochondrial membrane Despite the effect of agitation (10-5 times pipetting), the membrane potential (as determined by TMRE staining) TMRE staining showed that the Ca2+ After exposure of stirred mitochondria to the environment (0.96 mM Ca2+), mitochondria Loss of membrane potential. Isolation buffer containing 5 mM malate and 5 mM glutamate Q was incubated with 20 nM TMRE in PBS for 10 min at room temperature. Fluorescence values were obtained from the mitochondria. The ratio of TMRE fluorescence in chondria to background (upper left panel, Figure 10B TMRE fluorescence after 10–15 pipetting cycles is shown in the upper right panel of Figure 10B. TMRE fluorescence in Q after addition of Ca2+ is shown in the lower right panel of Figure 10B. Add 4 mL of HBS containing 5 mM malic acid and 5 mM glutamic acid to the edge of the dish. After 10-15 pipetting cycles, the Q value in the presence of Ca2+ was The TMRE fluorescence in the mitochondria is shown in the lower left panel of Figure 10B. Although resistant to the Ca2+ environment, mitochondria undergo C Therefore, without wishing to be bound by theory, However, the results are not consistent with physical disruption of mitochondria (shaking, stirring, etc.) and / or enrichment of Ca2 + The combination of environmental and physical disruption, e.g., shaking or agitation, can cause mitochondria to lose membrane integrity. and / or may cause loss of membrane potential. Although we do not wish to be bound by this, the results show that mitochondria isolated through the iMIT method are C When in contact with or stored in a2+ environment, e.g., before use in a therapeutic procedure suggested that the materials should be handled with minimal disruption.

[0143] Example 5: GRP27 content Glucose Regulation of Mitochondria Isolated via the iMIT Method Provided Herein Protein 75 (GRP75) content measured by detergent or homogenization method Tests were carried out to compare the results with those of isolated mitochondria. GRP75 protein was isolated from HeLa cells by each of the three methods and analyzed using a lysate assay. A protein blot was performed using cytochrome oxidase as a protein content control. The results of the study showed that mitochondria isolated through the iMIT method provided herein compared to mitochondria isolated through detergent or homogenization methods. Compared to the control, the sucrose-derived cereals had much lower GRP75 content (Figure 11 and Table 4). .

[0144] [Table 4]

[0145] Example 6: In vivo effects in a myocardial infarction model with a 4-week follow-up The effects on cardiac function improvement and prevention of cardiac remodeling, as well as Q mitochondria ( The safety of local injection of mitochondrial DNA (isolated through the iMIT method provided in this paper) was evaluated in rat myocardial infarction. The evaluation was carried out using a coronary artery ischemia-reperfusion model.

[0146] The test article, "Q" mitochondrial population, was derived from HUVEC (an immortalized human umbilical vein endothelial cell line, HUEhT-1) was prepared by the iMIT method provided herein. The mitochondrial population was then cryopreserved in liquid nitrogen for 1-2 weeks before use. The collected mass was thawed and formulated at the study site according to the internal procedures in use. A 30-minute surgical occlusion of the left anterior descending (LAD) artery was used to study the effects of thrombosis on the skeletal muscle of 11- to 12-week-old male rats. Slc:Wister rats were used in the study. The mean body weights were approximately equal among the groups. The animals were then allocated using the Stratified Random Allocation Method. Grouped by.

[0147] One minute before reperfusion, needles (26-3 mm) were inserted into three sites near the infarct area on the left ventricular myocardial tissue. 0.23 μg / body (low dose) or 11.5 μg / body (low dose) in 30 μL per site using Q mitochondria were injected locally at the same dose (high dose). PBS (-) was used as a negative control. A conventional mitochondrial isolation kit (89874, Thermo Scientific) was used. Mitochondria isolated using Fiscial were administered in the same manner as Q mitochondria. A higher dose (11.5 μg / body) was used as a comparator. The rest were untreated. Ten animals were assigned to each group. After that, body weight was measured weekly, and echocardiography was performed before dosing, at week 2, and at week 4. For the purpose of this study, blood sampling and isolation of the heart and lungs were performed. The heart, left and right atria and ventricles were then isolated. The left ventricle was isolated and the lungs were weighed. Histopathological examination was performed using the isolated left ventricle. The size of the myocardial infarct area and the relative area of cardiac fibrosis were determined. All animals were sacrificed 4 weeks after administration, and organ weights and heart and lung tissues were examined. Histopathology was performed. Figure 12 provides a schematic of the study design.

[0148] The ischemia-reperfusion (IR) model animal prepared in this study was used to investigate the effects of left ventricular tissue remodeling (hypertrophy). abnormal left ventricular contractility (LVADs and LVADs, and decreased LVAWd) The results showed that the EF and %FS decreased, the relative organ weight increased significantly, and the shape of the myocardial infarction lesions increased. Additionally, histopathological examination revealed cardiac regenerative necrosis, inflammatory cell infiltration, and cardiac fibrosis. In the control group, 1 out of 10 animals showed signs of myocardial infarction. The patient died 8 days after the preparation of the drug (8 days after administration). This event was not due to a myocardial infarction-related pathology. The death was considered to be a clinical death.

[0149] There was no significant difference in body weight in the Q group compared to the PBS control and conventional mitochondrial control groups. It was.

[0150] Echocardiography data are shown in Table 5. Compared to the PBS control or conventional mitochondrial group, In comparison, LVIDd (left ventricular internal dimension during diastole: internal dimension during cardiac expansion) and LVAWd (left ventricular internal dimension during diastole) Left ventricular wall thickness during diastole (LVWPd) or left ventricular posterior wall thickness during diastole (LVWPd) There was no significant difference in the Q group regarding the thickness of the posterior wall during vasodilation.

[0151] Regarding LVIDs (left ventricular internal dimensions during systole), high dose Q is demonstrated a significant reduction compared to the BS and conventional mitochondrial control groups (PBS control p<0.05 vs. conventional mitochondrial control, p<0.01 vs. conventional mitochondrial control) (Table 5).

[0152] When examining the ejection fraction (EF; an index of the total blood volume ejected by a single cardiac contraction), both Group Q was compared with PBS control and conventional mitochondrial control at weeks 2 and 4. A significant increase (p<0.01) was observed (Table 5). The data are also shown in Figure 13. .

[0153] Fractional shortening (FS; index of left ventricular contractility) was measured at week 2 for both Q groups. and a significant increase compared to PBS control and conventional mitochondria at week 4 (p<0. 01) was also observed (Table 5).

[0154] [Table 5] JPEG2025118621000006.jpg229164

[0155] Organ weights are shown in Table 6. Overall heart weights between groups or overall relative heart weights between groups (total There were no significant differences in body weight or organ weight. However, compared with the conventional mitochondrial control group, A significant inhibitory effect (p<0.01) on left atrial weight was observed in the high-dose Q group. (p<0.05) and conventional mitochondrial control (p<0.01) groups. The relative weight of the left atrium was also significantly suppressed at the high dose of Q compared with the conventional mitochondrial control group. When the high-dose Q was administered, the relative weight of the right ventricle was significantly reduced (p<0.05). at high dose Q (p<0.01) and low dose Q (p<0.05) compared with the control group. Lung weights were significantly lower than those of the PBS control (p<0.05) and the mitochondrial control (p<0. Compared with the 01) group, the relative lung weight was significantly suppressed in the high-dose Q group, and Compared with the control group, the low dose of Q significantly suppressed the activity (p<0.05).

[0156] [Table 6]

[0157] [Table 7]

[0158] There was no significant difference in myocardial infarct size compared to conventional mitochondrial controls, but myocardial fibrillation The relative area of fibrosis was significantly smaller at the high dose of Q (p<0.05) (Table 7).

[0159] [Table 8]

[0160] Histopathological studies revealed no abnormalities in specimens obtained from the 10 animals in the sham group. In all other groups, mild to moderate myocardial regenerative necrosis, mild to moderate interstitial edema, and mild Severe to moderate fibrosis was reported.

[0161] In summary, the high-dose (11.5 μg) Q group was compared with the negative control and comparator (conventional There was a statistically significant difference (p<0.01) between the left and right mitochondria groups. Ventricular tissue remodeling (suppression of LVIDs hypertrophy) and left ventricular systolic function (EF and %F The results demonstrated a statistically significant improvement in relative organ weights (heart and lungs) (Figure 13). A significant reduction in the area of cardiac fibrosis and fibrosis was also observed in the Q group. The degree of change in interstitial edema was compared with that observed in the negative control and comparator groups. The statistical significance (p<0.01) was observed in the negative control and comparison group. Compared with the control group, the low-dose (0.23 μg) group showed significantly improved left ventricular systolic function (EF, %FS) ) and the degree of interstitial edema (Figure 13).

[0162] In summary, based on the studies provided herein, the 0.23% IR in rat models Local injection of Q mitochondria at a dose of 11.5 μg / body or 11.5 μg / body was performed using echocardiography. Based on laboratory tests, it prevented left ventricular hypertrophy and improved left ventricular systolic function. Additionally, histopathology The results showed that Q mitochondria significantly reduced the relative organ weight of the heart and lungs and cardiac fibrosis formation. The effect of Q mitochondria was higher at the 11.5 μg dose.

[0163] Example 7: Myocardial infarction model with 7-day analysis To further explore the ability of Q mitochondria to protect against ischemia-reperfusion injury A second study was performed in a rat model of myocardial infarction (coronary ischemia-reperfusion). Test product "Q" is GFP-HUVEC (green fluorescent protein-labeled immortalized human umbilical vein endothelial cells). The cells were prepared by the iMIT method using a human epithelial cell line (Human epithelial cell line) and stored in liquid nitrogen for approximately 4 weeks before use in the test. In this example, Q mitochondria are designated "QN-01."

[0164] The animals were kept in a stratified ranch so that the average body weights were approximately equal among the groups. Grouping was performed using the m Allocation Method. g-labeled QN-01, sham procedure (open surgery, no IR preparation) and control group (PBS) Two animals were assigned to each of the observations on days 1, 3, and 7. Three animals were assigned to each of the same observations in the 0.23 μg labeled QN-01 test group.

[0165] Rats were prepared by occlusion of the left anterior descending aorta (LAD) for 30 minutes followed by reperfusion. As in the above study in Example 6, 1 min before reperfusion (after 29 min of occlusion), the LV proximal QN-01 was administered at a dose of 0.23 μg to the myocardial tissue at three sites.

[0166] After 1, 3 or 7 days of administration, body weights were measured and echocardiography was performed. After 1, 3 or 7 days, blood sampling is performed, the heart and lungs are isolated, and the heart, left and right The atria and ventricles and lungs of each animal were weighed. All animals were examined for organ weights and cardiac and ventricular function. Lung histopathology was performed on specimens obtained from test animals to detect the presence of GFP. Examine by microscopy and use anti-human mitochondrial antibody to investigate the cellular uptake of labeled QN-01 The antibodies were tested by immunohistochemical staining using the primary antibody. , provided in Figure 14 .

[0167] No deaths were reported in this study and all animals were monitored until scheduled necropsy. In cardiac examination, the negative control group showed a decreased EF ( showed decreased %FS (fractional shortening) and hypertrophied LVIDs. IDs were observed 3 and 7 days after administration. Compared with the negative control, the Q group showed a significant improvement in left ventricular function. demonstrated a significant effect in improving contractile function (EF and %FS), but this study was limited No statistical analysis was planned due to the number of animals studied (Figure 15 and Table 8). In addition, the hypertrophy of LVIDd and LVIDs was also suppressed (Table 9).

[0168] [Table 9]

[0169] [Table 10]

[0170] Histopathology showed that fluorescein staining intensity did not differ among dose groups, including the negative control group. , Q was not detected in any of the specimens collected on days 1, 3, or 7. Immunohistochemical staining showed that none of the specimens tested positive for Q. Clinical studies showed that at 7 days, there was a significant reduction in myocardial degeneration and necrosis as well as inflammatory processes compared to the negative control. The degree of cellular infiltration was slightly reduced in group Q (Table 10). Plasma collected from all of the animals The samples were analyzed using a Multiplex Assay system (Luminex 17 Plex* Assay, R&D System) for cytokines and chemokines. and analyzed.

[0171] [Table 11]

[0172] In summary, in the QN-01 group, echocardiography showed significant improvement in EF at 1, 3, and 7 days after administration. The decrease in LV and %FS was suppressed compared to the PBS control group. The hypertrophy of IDd and LVIDs was also suppressed. Histopathological examination after 7 days of administration showed that the control group Myocardial regenerative necrosis and inflammatory cell infiltration were slightly increased compared to the QN-01 group. Therefore, this study focused on a myocardial infarction model prepared by 30-minute infarction. Local injection of QN-01 in rats prevents left ventricular hypertrophy and improves left ventricular systolic function Additionally, QN-01 inhibits myocardial regeneration, necrosis, and inflammation in the heart. The degree of sexual cell infiltration was altered.

[0173] References 1. Shibata et al., Biochem. Biophys. Res. Commun. (2015) 463, 563-8. doi: 10.101 6 / j.brc.2015.05.095. 2. Baker, et al. Eur. J. Transl. Myol. 2015, 25, 35-48. 3. Stetsenko and Gustov. Crystal. 2017, 7:197. https: / / doi.org / 10.3390 / cryst 707 0197 4. Shin B, Cowan DB, Emani SM, Del Nido PJ, McCully JD. Mitochondrial Transplant ation in Myocardial Ischemia and Reperfusion Injury. Adv. Exp. Med. Biol. 2017; 982:595-619 5. Emani SM, Piekarski BL, Harrild D, Del Nido PJ, McCully JD. Autologous mitoch ondrial transfer for dysfunction after ischemia-reperfusion injection.2) J. Thor ac. Cardiovascular. Surg. 2017; 154(1):286-289 6. Bertero E, Mack C, O'Rourke B. Mitochondrial Transplantation in Humans: "Magi cal" cure or cause for concern? J. Clin. Invest. 2018 Dec 3; 128(12):5191-5194 7. Segawa et al., "Quantification of cristae architecture reveals time dependent characteristics of individual mitochondria" Life Science Alliance vol. 3 no. 7, June 2020 8. Nielsen et al., The Journal of Physiology 595.9 (2017) pp. 2839-47

Claims

1. 1. A population of isolated mitochondria, comprising: (i) at least 80% of the mitochondria in said population have intact inner and outer membranes; 、 (ii) at least 80% of the mitochondria in the population are measured by a fluorescent indicator; It is polarized, and / or (iii) at least 80% of the mitochondria in the population are functionally capable in the extracellular environment; maintain, Isolated mitochondrial populations.

2. (iii) wherein said functional capacity in the extracellular environment is measured by a fluorescent indicator of membrane potential.

2. The isolated population of mitochondria of claim 1.

3. (iii) The extracellular environment of claim 1, wherein the extracellular environment comprises a total calcium concentration of about 8 to about 12 mg / dL.

2. A population of isolated mitochondria according to claim 1.

4. (iii) the extracellular environment comprises a free / active calcium concentration of about 4 to about 6 mg / dL; 2. The isolated population of mitochondria of claim 1.

5. At least 80% of the mitochondria in the population express dynamin-related protein 1 (dr 10. The isolated population of mitochondria of claim 1, which has not undergone p1)-dependent division. 。

6. 2. The method of claim 1, wherein the inner membrane of the mitochondria comprises densely folded cristae. Populations of isolated mitochondria.

7. 1 to 3, wherein at least 80% of the mitochondria in the population have a non-filamentous shape.

7. A population of isolated mitochondria according to any one of claims 6 to 6.

8. 8. The method of claim 7, wherein at least 85% of the mitochondria have a non-filamentous shape. Isolated mitochondrial populations.

9. 9. The method of claim 8, wherein at least 90% of the mitochondria have a non-filamentous shape. Isolated mitochondrial populations.

10. The mitochondria are stimulated by the expression of glucose-regulated protein 75 (GRP75). Any of claims 1 to 9, which, when measured, shows reduced association with the mitochondrial associated membrane (MAM). A population of isolated mitochondria described in any one of claims 1 to 4.

11. The reduction in association involves homogenization of mitochondria in cells or cells. The isolated mitochondria obtained by the method have at least a low MAM association compared to the MAM association.

11. The population of isolated mitochondria of claim 10, wherein the reduction in the number of mitochondria is about 30%.

12. The isolated mitochondrial cell of claim 11, wherein the reduction in association is at least about a 50% reduction. Andria group.

13. (i) at least 85% of the mitochondria in said population have intact inner and outer membranes; 、 (ii) at least 85% of the mitochondria in the population are detected by a fluorescent indicator; It is polarized, and / or (iii) at least 85% of the mitochondria in said population are functionally capable in the extracellular environment; maintain, A population of isolated mitochondria according to any one of claims 1 to 12.

14. (i) at least 90% of the mitochondria in said population have intact inner and outer membranes; 、 (ii) at least 90% of the mitochondria in the population are measured by a fluorescent indicator; It is polarized, and / or (iii) at least 90% of the mitochondria in the population are functionally capable in the extracellular environment; maintain, A population of isolated mitochondria according to any one of claims 1 to 12.

15. The fluorescent indicator is JC-1, tetramethylrhodamine methyl ester (TMRM), and and tetramethylrhodamine ethyl ester (TMRE), Item 15. The isolated population of mitochondria according to any one of Items 1 to 14.

16. At least 80% of the mitochondria in the population are between about 500 nm and about 3500 nm in size.

16. The population of isolated mitochondria according to any one of claims 1 to 15, wherein the population is nm.

17. 17. The method of claim 1, wherein the polydispersity index (PDI) of the population is from about 0.2 to about 0.

8.

2. A population of isolated mitochondria according to any one of claims 1 to 11.

18. 17. The method of claim 1, wherein the polydispersity index (PDI) of the population is from about 0.2 to about 0.

3.

2. A population of isolated mitochondria according to any one of claims 1 to 11.

19. The zeta potential of the population of mitochondria is from about -15 mV to about -40 mV.

19. A population of isolated mitochondria according to any one of 1 to 18.

20. When the population of isolated mitochondria is contacted with a population of cells, the isolated mitochondria 1 to 3, wherein the chondria are capable of co-localizing with endogenous mitochondria in the cell.

10. A population of isolated mitochondria according to any one of claims 9 to 9.

21. When the population of isolated mitochondria is contacted with a population of cells, the mitochondria is capable of fusing with endogenous mitochondria in said cells. A population of isolated mitochondria described in any one of claims 1 to 4.

22. The mitochondria were stored at 4°C for at least 12 hours before the endogenous mitochondria were detected.

21. The population of isolated mitochondria of claim 20, which is capable of co-localizing with ribosomes.

23. The mitochondria were stored at 4°C for at least 12 hours before the endogenous mitochondria were detected.

22. The isolated population of mitochondria of claim 21, which is capable of fusing with a leukocyte. 。

24. At least 70% of the isolated mitochondria in the population are isolated from one or more Polarized as measured by fluorescent indicators after undergoing multiple freeze-thaw cycles.

24. The isolated population of mitochondria of any one of claims 1 to 23.

25. After the population has been subjected to one or more freeze-thaw cycles, the mitochondria are The monoclonal antibody of any one of claims 1 to 24, which is capable of co-localizing with localized mitochondria. Isolated mitochondrial populations.

26. The population is frozen at -80°C or below for at least two weeks, and then frozen at 20°C or below for approximately five minutes.

26. The population of isolated mitochondria of claim 24 or 25, which is thawed to

27. 27. The method of claim 26, wherein the population is thawed within about 1 minute. Group.

28. 28. The method of claim 26 or 27, wherein the population is frozen in liquid nitrogen for at least two weeks. Isolated mitochondrial populations.

29. 29. The isolated population of claim 28, wherein the population is frozen in liquid nitrogen for at least two months. A group of mitochondria.

30. When the population of thawed mitochondria is contacted with a population of cells, the isolated mitochondria in the population wherein the mitochondria are capable of fusing with endogenous mitochondria in the cell.

30. The isolated population of mitochondria according to any one of claims 24 to 29.

31. A composition comprising a population of isolated mitochondria according to any one of claims 1 to 30. 。

32. 32. A formulation comprising the composition of claim 31 and a pharmaceutically acceptable carrier.

33. 1. A method for isolating mitochondria from cells, comprising: (i) soaking the cells in the first solution in a surfactant solution at a concentration less than the critical micelle concentration for the surfactant; Treating with an agent, (ii) removing the surfactant to form a second solution; (iii) incubating the cells in the second solution; and (iv) recovering mitochondria from the second solution. A method comprising:

34. The concentration of the surfactant in the first solution is about 50% of the critical micelle concentration of the surfactant. % or less.

35. The concentration of the surfactant in the first solution is about 10 times the critical micelle concentration of the surfactant.

35. The method of claim 33 or 34, wherein the amount of the hydroxyl group is 0.05 or less.

36. The method according to any one of claims 33 to 35, wherein the surfactant is a nonionic surfactant. How to post.

37. The surfactant is Triton-X 100, Triton-X 114, Nonid et P-40, n-dodecyl-D-maltoside, Tween-20, Tween-80 37. Any one of claims 33 to 36, wherein the hydroxybenzoate is selected from the group consisting of saponin and digitonin. The method described in paragraph .

38. the surfactant is saponin or digitonin, and 38. The method of claim 37, wherein the concentration of the agent is less than about 400 μM.

39. the surfactant is saponin or digitonin, and 38. The method of claim 37, wherein the concentration of the agent is less than about 50 μM.

40. the surfactant is saponin or digitonin, and the saponin or digitonin in the first solution 38. The method of claim 37, wherein the concentration of digitonin is about 30 μM to about 40 μM.

41. The first solution may comprise one or more of a tonicity agent, an osmolality adjusting agent, or a chelating agent. The method of any one of claims 33 to 40, further comprising a buffer.

42. 5. The method of claim 4, wherein the first solution comprises Tris buffer, sucrose, and a chelator.

1. The method according to claim 1.

43. Treating the cells in the first solution includes treating the cells in the first solution at room temperature.

43. The method of any one of claims 33 to 42, comprising incubating for about 2 minutes to about 30 minutes. The method described.

44. Removing the surfactant from the solution may be performed by removing the surfactant from the first solution.

44. The method of claim 33, further comprising reducing the concentration of the surfactant to less than 10%.

1. The method according to claim 1.

45. Removing the surfactant from the solution may be performed by removing the surfactant from the first solution. Any of claims 33 to 44, including reducing the concentration of the surfactant to less than 1%.

10. The method according to claim 1.

46. 10. The method of claim 9, wherein removing the detergent comprises washing the cells with a buffer. 33 to 45. A method according to any one of claims 33 to 45.

47. Incubating the second solution includes incubating the cells in the second solution at about 4° C.

47. The method of any one of claims 33 to 46, comprising incubating for about 5 minutes to about 30 minutes. The method described.

48. Recovering the mitochondria from the second solution includes recovering the isolated supernatant.

48. The method of any one of claims 33 to 47, comprising recovering mitochondria.

49. Recovering the mitochondria from the second solution may include centrifuging the second solution. and recovering the supernatant after centrifugation to recover the isolated mitochondria. Item 49. The method according to any one of Items 33 to 48.

50. 50. The method of any one of claims 33 to 49, further comprising freezing the isolated mitochondria. The method according to any one of claims 1 to 5.

51. freezing the isolated mitochondria in a buffer containing a cryoprotectant.

51. The method of claim 50, comprising:

52. Isolated mitochondria obtained by the method according to any one of claims 33 to 51. Doria's group.

53. A method for treating a disease or disorder, comprising injecting cells of a subject in need thereof into a cell line comprising ...

30. The isolated population of mitochondria of claim 31.

33. The method of claim 32, wherein the disease or disorder is: Diabetes (types I and II), metabolic diseases, and eye disorders associated with mitochondrial dysfunction , hearing loss, mitochondrial toxicity associated with therapeutic drugs, chemotherapy or other therapeutic drugs The method is selected from the group consisting of cardiotoxicity, mitochondrial dysfunction and migraine.

54. A method for treating a disease or disorder associated with mitochondrial dysfunction, comprising: The cells of interest are cultured in the presence of an isolated mitochondrial cell line according to any one of claims 1 to 30. or a population of mice or a composition of claim 31 or a formulation of claim 32. and a method comprising:

55. The disease or disorder is mitochondrial myopathy, diabetes, and deafness (DAD). syndrome, Barth syndrome, Leber's hereditary optic neuropathy (LHON), Leigh syndrome, NARP ( Neuropathy, ataxia, retinitis pigmentosa, and ptosis syndrome), myoneuronal gastrointestinal encephalopathy (MNGIE) ), MELAS (Mitochondrial Encephalopathy, Lactic Acidosis and Stroke-like Episodes) Syndrome, Myoclonic epilepsy with ragged-red fibers (MERRF) syndrome, Kearns-Sayre syndrome 55. The method of claim 54, wherein the mitochondrial DNA is selected from the group consisting of mitochondrial DNA depletion syndrome and mitochondrial DNA wasting syndrome. How to post.

56. 55. The method of claim 54, wherein the disease or disorder is an ischemia-related disease or disorder.

57. The ischemia-related disease or disorder is cerebral ischemia-reperfusion, hypoxic-ischemic encephalopathy, acute coronary artery disease, syndrome, myocardial infarction, hepatic ischemia-reperfusion injury, ischemic injury-compartment syndrome, hematopoietic A group consisting of vascular obstruction, wound healing, spinal cord injury, sickle cell disease, and reperfusion injury of transplanted organs 57. The method of claim 56, wherein the

58. 55. The method of claim 54, wherein the disease or disorder is a genetic disorder.

59. 55. The method of claim 54, wherein the disease or disorder is an age-related disease or disorder.

60. 55. The method of claim 54, wherein the disease or disorder is a neurodegenerative or cardiovascular condition. 。

61. The neurodegenerative condition is dementia, Friedreich's ataxia, amyotrophic lateral sclerosis, mitochondrial infarction, Endorhiomyopathies, encephalopathy, lactic acidosis, stroke (MELAS), ragged red fibers Myoclonic epilepsy (MERFF), epilepsy, Parkinson's disease, Alzheimer's 61. The method of claim 60, wherein the disease is selected from the group consisting of: rheumatoid arthritis, ... and Huntington's disease. Exemplary neuropsychiatric disorders include bipolar disorder, schizophrenia, depression, addictive disorders, These include anxiety disorders, attention deficit disorder, personality disorders, autism and Asperger's disease.

62. The cardiovascular condition is coronary heart disease, myocardial infarction, atherosclerosis, hypertension, cardiac arrest, , cerebrovascular disease, peripheral arterial disease, rheumatic heart disease, congenital heart disease, congestive heart failure, arrhythmia 61. The method of claim 60, wherein the vasoconstrictor is selected from the group consisting of: stroke, deep vein thrombosis, and pulmonary embolism. How to do it.

63. the disease or disorder is cancer, an autoimmune disease, an inflammatory disease or a fibrotic disorder; 55. The method of claim 54.

64. 55. The method of claim 54, wherein the disease is acute respiratory distress syndrome (ARDS).

65. The disease or disorder is preeclampsia or intrauterine growth restriction (IUGR). Item 55. The method according to item 54.

66. The population or composition of isolated mitochondria is administered intravenously, intraarterially, intratracheally, Administering to the subject via a subcutaneous, intramuscular, inhalation, or pulmonary route of administration. 54-65. A method according to any one of claims 54 to 65.

67. Isolated mitochondria with intact inner and outer membranes, said inner membrane being folded. the mitochondria are isolated from the cells and contain packed cristae; The mitochondria are polarized as measured by a fluorescent indicator, and the mitochondria are separated into different parts of the extracellular environment. Isolated mitochondria capable of maintaining their poles.

68. 68. The isolated mitochondrion of claim 67, having a non-filamentous shape.

69. The voltage-dependent anion channel (VDAC) on the surface of the mitochondria 69. The isolated mitochondrion of claim 67 or 68, which is associated with tubulin.

70. 68. The isolated mitochondrial protein of claim 67, wherein the tubulin is a dimeric tubulin. Andria.

71. The tubulin is a heterodimer containing α-tubulin and β-tubulin.

71. The isolated mitochondrion of claim 70, wherein

72. The fluorescent indicator is JC-1, tetramethylrhodamine methyl ester (TMRM), and and tetramethylrhodamine ethyl ester (TMRE), 72. The isolated mitochondrion of any one of paragraphs 67 to 71.

73. Mitochondrial function, as measured by glucose-regulated protein 75 (GRP75) expression Mitochondria-associated membrane (MAM) 73. The isolated mitochondria of any one of claims 67 to 72, which exhibits reduced association with Chondria.

74. The reduction in association involves homogenization of mitochondria in cells or cells. The isolated mitochondria obtained by the method have at least a low MAM association compared to the MAM association.

74. The isolated mitochondrion of claim 73, wherein the reduction in mitochondrial activity is about 30%.

75. 75. The isolated mitochondrial protein of claim 74, wherein the reduction in association is at least about a 50% reduction. Andria.

76. 76. The method according to any one of claims 67 to 75, wherein the membrane potential is from about -30 mV to about -220 mV. Isolated mitochondria as described above.

77. The isolated vector according to any one of claims 67 to 76, which is not undergoing drp1-dependent division. mitochondria.

78. 78. The method according to any one of claims 67 to 77, wherein the size is about 500 nm to 3500 nm. Isolated mitochondria of.

79. A composition comprising the isolated mitochondria of any one of claims 67 to 78.

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