Smaller isolated mitochondria and lipid membrane-based vesicles encapsulating isolated mitochondria

Abstract

According to the present invention, there are provided compositions comprising a population of smaller sized mitochondria and a population of lipid membrane-based vesicles encapsulating mitochondria in enclosed spaces, as well as methods for producing the compositions.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to Japanese Application No. 2019-239479, filed December 27, 2019, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to isolated mitochondria that are smaller in size and to lipid membrane-based vesicles encapsulating isolated mitochondria. [Background technology]

[0003] Mitochondrial dysfunction, such as respiratory chain complex dysfunction, is a major cause of mitochondrial diseases and aging. Deterioration of mitochondrial function affects cells in many organs, which is primarily involved in mitochondrial diseases and age-related diseases. To overcome this, attempts have been made to introduce mitochondria into cells (U.S. Patent No. 9,603,872). In U.S. Patent No. 9,603,872, mitochondria are mixed with Lipofectamine 2000 reagent to obtain lipoplexes with mitochondrial lipofectamine (i.e., the assembly of Lipofectamine particles and free mitochondrial particles). U.S. Patent No. 9,603,872 confirmed that the resulting lipoplexes were introduced into cells. However, U.S. Patent No. 9,603,872 did not confirm the physiological effects of the introduced lipoplexes.

[0004] A technology called a microflow channel device has been developed to form liposomes using channels with widths of 100 to 200 μm (Kimura N. et al., ACS Omega, Vol. 3: 5044-5051, 2018). This device, based on lipid membranes, allows for the production of vesicles with diameters of approximately 10 nm to approximately 100 nm. Summary of the Invention

[0005] The present invention provides lipid membrane-based vesicles encapsulating isolated mitochondria and methods for producing the vesicles.

[0006] The present inventors have obtained a composition containing a population of lipid membrane-based vesicles encapsulating mitochondria. Each vesicle is thought to have a sac-like membrane structure (closed space) formed by a lipid membrane to contain mitochondria.

[0007] In this specification, for example, the following inventions are provided. (1) A composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria. (2) The composition according to item (1), wherein the population of lipid membrane-based vesicles has a particle size distribution with a peak at less than 1 μm, as determined by dynamic light scattering. (3) The composition according to item (2), wherein the population of lipid membrane-based vesicles has a particle size distribution with a peak at less than 500 nm, as determined by dynamic light scattering. (4) The composition according to any one of items (1) to (3), wherein the population of lipid membrane-based vesicles has a PDI of less than 0.5. (5) The composition according to any one of items (1) to (4), wherein the encapsulated mitochondria may be integrated into the cytoplasm of a cell in contact therewith, or the mitochondria may be fused with endogenous mitochondria in the cytoplasm. (6) A composition according to any one of items (1) to (6) for use in delivering mitochondria to a cell. (7) The composition according to item (6) for use in improving mitochondrial respiratory activity in a cell. (8) A method for producing a composition according to item (1), comprising: A method comprising contacting an aqueous solution containing isolated mitochondria with an ethanol solution containing lipids capable of forming a lipid membrane in a confluent channel of a microflow channel device to mix the solutions. (9) The method according to item (8), wherein the microflow channel device comprises a flow channel for facilitating mixing of solutions brought into contact with each other in the confluence channel, and the flow channel has a baffle structure.

[0008] Furthermore, the following inventions are provided: [1] A composition comprising a population of mitochondria, the population having a particle size distribution with a peak at less than 1 μm as determined by dynamic light scattering. [2] The composition according to item [1], wherein the population has a particle size distribution with a peak at less than 500 nm, as determined by dynamic light scattering. [3] A composition according to item [1] or [2], wherein the population has a PDI of less than 0.5. [4] A composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria, wherein the population of lipid membrane-based vesicles has a particle size distribution with a peak at less than 1 μm as determined by dynamic light scattering. [4A] The composition according to item [1], wherein 50% or more of the mitochondria are each encapsulated in a lipid membrane-based vesicle. [5] The composition according to item [4] or [4A], wherein the population of lipid membrane-based vesicles has a particle size distribution with a peak at less than 500 nm, as determined by dynamic light scattering. [6] A composition according to any one of items [4] to [5] (i.e., items [4], [4A] and [5]), wherein the population of lipid membrane-based vesicles has a PDI of less than 0.5. [7] The composition according to any one of items [4] to [6], wherein the encapsulated mitochondria may be integrated into the cytoplasm of a cell in contact with the encapsulated mitochondria, or the mitochondria may be fused with endogenous mitochondria in the cytoplasm. [8] A composition according to any one of items [4] to [7] for use in delivering mitochondria to cells. [9] The composition according to item [8] for use in improving mitochondrial respiratory activity in a cell.

[10] A method for producing a composition according to item [4] or [4A], comprising contacting an aqueous solution containing isolated mitochondria with an ethanol solution containing lipids capable of forming a lipid membrane in a confluent channel of a microflow channel device to mix the solutions.

[11] The method according to item

[10] , wherein the microflow channel device includes a flow channel for facilitating mixing of solutions that are brought into contact with each other in the confluence channel, and the flow channel has a baffle structure.

[0009] Mitochondria can be encapsulated in the vesicles of the present invention and are suitable for pharmaceutical formulations. A population of mitochondria-containing lipid membrane-based nanovesicles with a monodisperse size distribution (i.e., PDI≦0.5) and / or a peak in the size distribution below 1 μm is more suitable for pharmaceutical formulations.

[12] A method for measuring mitochondrial DNA levels in isolated or packaged mitochondria, comprising amplifying at least a portion of the mitochondrial DNA in a sample containing isolated or packaged mitochondria to obtain amplified DNA amplicons, and counting the amplicons to obtain the mitochondrial DNA level.

[13] The method according to item

[12] , further comprising comparing the measured mitochondrial DNA level with a standard value. [Brief explanation of the drawings]

[0010] [Figure 1] Figure 1 shows particle size distribution and PDI as well as zeta potential (ζ) determined by dynamic light scattering (DLS) for suspensions of mitochondria isolated from cells. a) Data for isolated mitochondria before freezing (prepared product) and b) data for isolated mitochondria after a freeze-thaw process (frozen product). [Figure 2]1 shows the results of mitochondrial polarization in the ready-to-use product and the frozen product, detected with a fluorescent dye (TMRE). Fluorescence was observed in both the ready-to-use product and the frozen product. [Figure 3] Panel a) shows a scheme for preparing nanovesicles using a microflow channel device and subsequent dialysis. Panel b) shows a scheme for preparing nanoparticles by contacting a lipid-containing organic phase and an aqueous buffer (aqueous phase) in a confluent channel of a microflow channel device and mixing them; the composition of the organic and aqueous phases and their flow rates, the particle size distribution of the resulting nanoparticles by DLS, the PDI, and their zeta potential (ζ) are shown. [Figure 4] This figure shows a scheme for preparing lipid membrane-based vesicles encapsulating mitochondria by contacting and mixing a lipid-containing organic phase and an aqueous buffer (aqueous phase) containing isolated mitochondria in a confluent channel of a microflow channel device; the composition of the organic and aqueous phases and their flow rates, the particle size distribution and PDI of the resulting vesicles measured by DLS, and their zeta potential (ζ). [Figure 5-1] Panel a) shows the particle size distribution and PDI by DLS of the resulting particles, as well as their zeta potential (ζ), when the organic phase is a lipid-free organic phase (50% ethanol solution) and the aqueous phase is an aqueous buffer containing isolated mitochondria; Panel b) shows the particle size distribution and PDI by DLS of the resulting particles, as well as their zeta potential (ζ), when the organic phase is an organic phase containing stearylated octaarginine (STR-R8) and the aqueous phase is an aqueous buffer containing isolated mitochondria. [Figure 5-2] Panel c) shows the size distribution and PDI by DLS of the resulting particles, as well as their zeta potential (ζ), when an aqueous buffer is used instead of the organic phase and the aqueous phase is the aqueous buffer containing isolated mitochondria. [Figure 6]Figure 4 shows fluorescence microscopy images of nanoparticles prepared by a microflow channel device according to the scheme in Figure 4 (where lipids are fluorescently stained with DOPE-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-DOPE); mitochondria are stained with MitoTracker™ Deep Red). Panel a) shows the fluorescence signal from isolated mitochondria; panel b) shows the fluorescence signal from lipids; and panel c) shows the merged signals, demonstrating their almost perfect colocalization. [Figure 7] FIG. 1 shows electron microscopy images of isolated mitochondria fixed by chemical fixation. [Figure 8] This is an electron microscope image of nanocapsules prepared according to the scheme in Figure 3, panel b) and stained by negative staining. Figure 8 shows that the interior of the obtained nanocapsules is unexpectedly filled with lipids (lipid layer). [Figure 9] This figure shows an electron microscope image of nanoparticles prepared according to the scheme in Figure 4 and stained by negative staining. With negative staining, mitochondria do not produce contrast, and as a result, they cannot be detected. [Figure 10] FIG. 5 shows an electron microscope image of isolated mitochondria obtained according to the scheme in FIG. 5-1, panel b), treated with STR-R8, and fixed by chemical fixation. [Figure 11] This figure shows the DLS particle size distribution and PDI, as well as the zeta potential (ζ), of lipid membrane-based vesicles encapsulating mitochondria, obtained according to the same scheme as in Figure 4, except that the type of lipid was changed. Panels a) and b) show data from a negative control using an aqueous solution containing no mitochondria. Panels c) and d) show results obtained under the same conditions as in panels a) and b), respectively, except that the aqueous solution was an aqueous buffer containing isolated mitochondria. [Figure 12]Figure 1 shows the flow rate of the solution introduced into the microflow channel device; the particle size distribution and PDI of the resulting lipid membrane-based vesicles encapsulating mitochondria measured by DLS; and the zeta potential (ζ). [Figure 13] Figure 13 shows confocal laser scanning microscopy images of human cultured cells obtained by contacting the lipid membrane-based vesicles containing the resulting mitochondria with the cells and incubating them for 3 hours. In Figure 13, mitochondria present in the human cells are stained with MitoTracker™ Green; mitochondria in the lipid membrane-based vesicles containing the mitochondria are stained with MitoTracker™ Deep Red. In the incubated cells, the images formed by the fluorescence emitted from these mitochondria are almost perfectly aligned (see Hela mito green, isolated mito red, and merged). [Figure 14] Figure 14 shows confocal laser scanning microscope images of cells obtained by contacting isolated mitochondria not packaged in vesicles with cultured human cells and incubating them for 3 hours. In Figure 14, mitochondria present in the human cells were stained with MitoTracker™ Green; isolated mitochondria were stained with MitoTracker™ Deep Red. Figure 14 shows that virtually no signal from isolated mitochondria was observed in the cells. [Figure 15]Figure 15 shows confocal laser scanning microscopy images of cells obtained by contacting STR-R8-treated isolated mitochondria, which are not packaged in vesicles, with cultured human cells and incubating them for 3 hours. In Figure 15, mitochondria present in the human cells were stained with MitoTracker™ Green; isolated mitochondria were stained with MitoTracker™ Deep Red. Figure 15 shows that virtually no signal from isolated mitochondria was observed in the cells. In Figure 15, the lower left panel shows a light microscopy image of the incubated cultured human cells, suggesting that cell death was induced. [Figure 16] Figure 16 shows confocal laser scanning microscopy images of cells obtained by contacting isolated mitochondria not packaged in vesicles with cultured human cells (human cardiac progenitor cells) and incubating them for 3 hours. In Figure 16, mitochondria present in the human cells were stained with MitoTracker™ Green; isolated mitochondria were stained with MitoTracker™ Deep Red. Figure 16 shows that virtually no signal from isolated mitochondria was observed in the cells. [Figure 17] Figure 17 shows confocal laser scanning microscopy images of cells obtained by contacting isolated STR-R8-treated mitochondria, not packaged in vesicles, with cultured human cells (human cardiac progenitor cells) and incubating them for 3 hours. In Figure 17, mitochondria present in the human cells were stained with MitoTracker™ Green; isolated mitochondria were stained with MitoTracker™ Deep Red. Figure 17 shows that virtually no signal from isolated mitochondria was observed in the cells. [Figure 18] FIG. 1 shows a scheme for obtaining lipid membrane-based vesicles encapsulating mitochondria derived from human cardiac stem cells (hCDCs) by obtaining them from cardiac muscle and isolating mitochondria from the hCDCs. [Figure 19]This figure shows the results of measuring the mitochondrial respiratory activity of cells obtained by contacting lipid membrane-based vesicles encapsulating hCDC-derived mitochondria with skin fibroblasts obtained from a MELAS patient and incubating them for 3 hours or 24 hours. [Figure 20] This figure shows the results of measuring the mitochondrial respiratory activity of cells obtained by contacting lipid membrane-based vesicles encapsulating hCDC-derived mitochondria with skin fibroblasts obtained from LHON patients and incubating them for 3 hours or 24 hours. [Figure 21] This figure shows the results of measuring the mitochondrial respiratory activity of normal fibroblasts by contacting them with lipid membrane-based vesicles encapsulating hCDC-derived mitochondria or lipofectamine and mitochondrial lipid complexes (LFN-isolated Mitochondria) and incubating them for 24 hours. LFN-isolated Mitochondria reduced the mitochondrial respiratory activity of the cells. This suggests that LFN-isolated Mitochondria enters the cells and causes toxicity to mitochondria. [Figure 22] This figure shows the results of measuring the mitochondrial respiratory activity of cells obtained by contacting lipid membrane-based vesicles encapsulating hCDC-derived mitochondria or lipofectamine and mitochondrial lipid complexes (LFN-isolated Mt) with skin fibroblasts obtained from a patient with Leigh encephalopathy and incubating them for 24 hours. [Figure 23] This figure shows the results of measuring the mitochondrial respiratory activity of cells obtained by contacting lipid membrane-based vesicles encapsulating hCDC-derived mitochondria or lipofectamine and mitochondrial lipid complexes (LFN-isolated Mt) with skin fibroblasts obtained from LHON patients and incubating them for 24 hours. [Figure 24] FIG. 1 shows the ability of lipid membrane-based vesicles encapsulating mitochondria and LFN-isolated Mt to be incorporated into cells. [Figure 25]This figure shows the particle size distribution and PDI measured by DLS, as well as the zeta potential (ζ) of LFN-isolated mitochondria. The zeta potential of LFN-isolated mitochondria is close to 0. This suggests that a complex consisting of negatively charged mitochondria and positively charged LFN is obtained, in which the mitochondria are electrically neutralized. [Figure 26] FIG. 1 shows electron micrographs of negatively stained (Panels B and A, respectively) Lipofectamine 2000 (LFN) and a mixture of LFN and isolated mitochondria (LFN+Mt). [Figure 27] Figure 27 shows electron micrographs of isolated mitochondria stained by chemical fixation (Panel A) and a mixture of Lipofectamine 2000 (LFN) and isolated mitochondria (LFN+Mt) (Panel B). Figure 27, Panel C shows the survival ratios of cells treated with MITO-Q and LFN+Mt, respectively. [Figure 28] FIG. 1 shows a schematic diagram of the complex of Lipofectamine and isolated mitochondria (panel a) and a schematic diagram of lipid membrane-based vesicles encapsulating mitochondria (panel b) based on the results obtained. [Figure 29A] Figure 1 shows the results of assaying for membrane potential of mitochondria isolated by the various methods indicated in the figure, where MitoTracker Deep Red was used as a mitochondrial membrane potential indicator. [Figure 29B] Figure 1 shows the results of assaying for membrane potential of mitochondria isolated by the various methods indicated in the figure. Tetramethylrhodamine methyl ester (TMRM) was used as a mitochondrial membrane potential indicator. [Figure 30A] FIG. 1 shows an outline of the assay for mitochondrial respiratory activity in cells treated with encapsulated mitochondria or mitochondria treated with LFN. [Figure 30B] FIG. 1 shows the results of an assay for mitochondrial respiratory activity. [Figure 30C] FIG. 1 shows the results of an assay for mitochondrial respiratory activity in bar graph format. [Figure 31A] FIG. 1 shows fitted calibration curves for DNA concentration by quantitative PCR and protein concentration measured by the Bradford method. [Figure 31B] FIG. 1 shows calibration curves fitted for PCR amplicon copy number and template concentration. [Figure 31C]

[0023] Figure 1 shows the copy number of mtDNA in mitochondria isolated by the method shown in the figure. Copy number was normalized using total mitochondrial protein content. [Figure 31D]

[0023] Figure 1 shows the copy number of mtDNA in enclosed mitochondria isolated by the method shown in the figure. Copy number was normalized using total mitochondrial protein content. [Figure 31E] FIG. 1 shows the basal respiratory activity of cells treated with encapsulated mitochondria isolated by the method shown in the figure. [Figure 31F] FIG. 1 shows the maximum respiratory activity of cells treated with encapsulated mitochondria isolated by the method shown in the figure. [Figure 31G] Figure 1 shows the amount of TFAM in mitochondria isolated by the method shown in the figure. The amount was normalized using the total mitochondrial protein amount. [Figure 31H] Figure 1 shows the concentration of total protein in mitochondria isolated by the various methods shown in the figure. D-Mt was isolated by the conventional detergent method, Q (pH 7.4) was isolated by iMIT using pH 7.4 buffer, and Q (pH 8.9) was isolated by iMIT using pH 8.9 buffer. [Figure 32A] FIG. 1 shows the size distribution of hCPC-MITO-Q, which was prepared by encapsulating mitochondria isolated from human cardiac progenitor cells (hCPCs) by the iMIT method. [Figure 32B] FIG. 1 shows the results of staining of mitochondria in cells treated with TMRM with or without hCPC-MITO-Q. [Figure 33]1 shows the results of mitochondrial respiratory activity in cells treated with the samples shown in the figure. The term "Res-hCPC-MITO-Q" refers to MITO-Q prepared from hCPCs treated with MITO-Porter containing resveratrol. [Figure 34A] FIG. 1 shows the time course of the assay protocol. [Figure 34B] FIG. 1 shows the maximum respiratory activity of cells treated with MITO-Q after the incubation times indicated in the figure. [Figure 35] FIG. 1 shows the maximum respiratory activity of cells treated with MITO-Q stored at 4° C. for various periods indicated in the figure. [Figure 36] 1 is a reference drawing showing the structure of a microflow channel device, in which large arrows indicate the direction of liquid flow in the channels. DETAILED DESCRIPTION OF THE INVENTION

[0011] As used herein, "mitochondria" are intracellular organelles present in the cytoplasm of eukaryotic cells. Mitochondria likely play a role in generating ATP (by oxidative phosphorylation) within cells through the electron transport chain. Mitochondria have their own DNA (mitochondrial DNA), which encodes mitochondrial components (e.g., proteins of the respiratory chain complexes in the electron transport chain) that are independent of the DNA of the cell nucleus. Mutations in mitochondrial DNA sometimes impair mitochondrial function. Mitochondrial dysfunction can cause diseases called mitochondrial diseases. To overcome this, attempts to provide exogenous mitochondria have been implemented as a therapy.

[0012] As used herein, a "vesicle" refers to a particulate object having a closed space surrounded by a membrane, in which the physical, chemical, and / or physiological movement of substances into and out of the space is restricted. As used herein, "lipid membrane-based vesicles" refer to vesicles, such as liposomes, formed from a membrane containing lipids as the main component. Amphipathic lipids or cationic or anionic lipids placed in aqueous solution can form vesicles composed of a lipid bilayer (particularly a single lipid bilayer) with a closed space containing the aqueous solution therein.

[0013] As used herein, "encapsulated" refers to a state in which a given substance is encapsulated in a closed space in which the movement of the substance inside / outside is restricted. Therefore, the encapsulated mitochondria are isolated in a closed space within a hollow sac or vesicle formed by a membrane structure. Lipid membrane-based vesicles with a lipid bilayer membrane are commonly called liposomes. Lipid membrane structures usually have little permeability to water, blood, or other aqueous solutions. Therefore, the encapsulated mitochondria can be protected in body fluids by a membrane structure that completely packages the entire mitochondria during delivery to cells. The membrane structure can facilitate the entry of the encapsulated mitochondria into cells when the membrane composition is similar to that of the cell membrane or the surface of the membrane structure has a positive charge. Whether mitochondria are encapsulated in lipid membrane-based vesicles can be confirmed, for example, by staining mitochondria and lipids separately, forming vesicles encapsulating mitochondria, and observing the coexistence of the respective dyes of mitochondria and lipids under an optical microscope (e.g., a fluorescence microscope if a fluorescent dye is used), and by observing the morphology under an electron microscope to find the presence of a hollow lipid membrane (or its cross-section) by negative staining. Whether mitochondria are encapsulated in lipid membrane-based vesicles can be confirmed, for example, by contacting the vesicles with cells and observing whether mitochondria can be introduced into the cytoplasm; or, if the lipid membrane of the vesicle is positively charged, by examining whether a positive zeta potential of, for example, 10 mV or more can be obtained. In this specification, "free" mitochondria refers to isolated mitochondria, and is used to specifically describe mitochondria that are not encapsulated in vesicles. In this specification, unless otherwise specified, "isolated mitochondria" or "mitochondria" refers to free mitochondria.

[0014] As used herein, the term "population" refers to a group of multiple identical or different substances. As used herein, a "population of lipid membrane-based vesicles encapsulating mitochondria" refers to a group of lipid membrane-based vesicles encapsulating at least multiple identical or different mitochondria. A population does not necessarily have to be homogeneous, and can have physical, chemical, and / or physiological distributions. Examples of physical distributions include particle size and polydispersity index. Examples of chemical distributions include zeta potential distribution and lipid composition distribution. Examples of physiological distributions include differences in physiological function (e.g., respiratory activity).

[0015] As used herein, "dynamic light scattering" (DLS) refers to a technique for determining the size of nanometer-order particles in solution. Methods for measuring particle size and polydispersity are specified, for example, in ISO 22412:2017. Dynamic light scattering can obtain particle size distributions. Specifically, the average particle size can be obtained from the autocorrelation function of scattered light intensity using the cumulant analysis method (ISO 22412). As used herein, the term "peak" refers to the maximum frequency in a histogram showing the measured particle size distribution. When mitochondria are isolated intact, they likely exhibit a particle size distribution with a peak typically around 1 μm.

[0016] As used herein, the "polydispersity index" (PDI) (also referred to as polydispersity) is an index used to evaluate the width of particle size distribution obtained by DLS. PDI can be obtained from the autocorrelation function of scattered light intensity using the cumulant analysis method (ISO 22412). PDI = 0 means that a group of particles in a solution consists of particles of exactly the same size, and the maximum PDI is 1. If the PDI is 0.5 or greater, the group of particles is considered to have polydispersity. The particle size distribution of mitochondria (intracellular organelles) has polydispersity, and isolated mitochondria are usually considered to have a PDI of 0.5 or greater.

[0017] As used herein, "zeta potential" (ζ potential) refers to the potential that can be calculated by electrophoretic light scattering using the Helmholtz-Smoluchowski equation. Zeta potential is defined as follows: When a particle moves relative to a solution, a layer of solution with a certain thickness moves along with the particle. The potential difference between the surface (sliding plane) of the layer and the bulk of the solution sufficiently far away from the surface is defined as the zeta potential. Zeta potential can be measured using the electrophoretic light scattering method and can be obtained using the Helmholtz-Smoluchowski equation based on the dielectric constant of the solution, the viscosity of the solution, the particle's migration speed, and the electric field. Mitochondria release protons from their inner membranes to the outside during respiration. As a result, mitochondria are negatively polarized and have a negative zeta potential. When mitochondria are encapsulated in lipid membrane-based vesicles, the zeta potential is determined based on the composition of the lipid membrane. Therefore, mitochondria have no or limited influence on the zeta potential.

[0018] As used herein, "respiration" refers to the activity of mitochondria to generate ATP by consuming oxygen using the concentration gradient of protons released by mitochondria from within the electron transport chain. Mitochondrial respiratory activity refers to the respiratory capacity of mitochondria, which can be determined, for example, by the mitochondrial oxygen consumption rate (OCR). The oxygen consumption rate can be determined, for example, by an extracellular flux analyzer. More specifically, a substrate for a respiratory chain complex, such as malate, is added to mitochondria, and the OCR of the mitochondrial solution (designated "OCR1") is then measured. An ATP synthase inhibitor (e.g., oligomycin) is then added to mitochondria, and the OCR of the mitochondrial solution (designated "OCR2") can then be measured. An uncoupler (e.g., FCCP) is then added to mitochondria, and the OCR of the mitochondrial solution (designated "OCR3") can then be measured. Respiratory chain complex inhibitors (e.g., complex I inhibitors such as rotenone and complex III inhibitors such as antimycin A) are then added, and the OCR of the mitochondrial solution (designated "OCR4") can then be measured. The basal mitochondrial respiration rate, ATP-generating respiration rate, and maximum respiration rate can be obtained by the following equations: Basal mitochondrial respiration rate = OCR1-OCR4; Mitochondrial ATP-producing respiration rate = OCR1-OCR2; Maximum mitochondrial respiration rate = OCR3 - OCR4.

[0019] In one embodiment, the mitochondrial respiratory activity analyzed may be the maximum mitochondrial respiratory activity.

[0020] As used herein, the terms "microflow channel device" and "microfluidic device" are used interchangeably and refer to a device containing channels with diameters or widths and heights on the order of μm. In the channels of a microflow channel device, two different compositions introduced from two different inlets can be combined in a confluence channel. A confluence channel is a section where two flow paths, each connected to two different inlets, join. In addition to the confluence channel, a microflow channel device can have a channel (mixing channel) for mixing the combined solutions. The mixing channel can have a structure (e.g., bends) to facilitate mixing and stirring. The mixing channel may or may not have concave and convex surfaces on its internal surface.

[0021] As used herein, "ethanol solution" refers to an aqueous solution containing ethanol. As used herein, "organic phase" refers to a phase containing an organic solvent, and may be a phase containing an organic solvent capable of dissolving lipids capable of forming a lipid membrane. The organic solvent in the organic phase may be, for example, a water-soluble organic solvent. A water-soluble organic solvent is advantageous because it can be easily removed by a method such as dialysis after the formation of vesicles. Ethanol can be mentioned as an example of a water-soluble organic solvent. In an embodiment, the organic phase may be, for example, an aqueous ethanol solution.

[0022] The present inventors have found that lipid membrane-based vesicles and populations of vesicles encapsulating mitochondria can be obtained by contacting an aqueous solution containing isolated mitochondria with an organic phase (e.g., an ethanol solution) containing lipids capable of forming a lipid membrane in a confluent channel of a microflow channel device and mixing them. The present invention provides a method for producing lipid membrane-based vesicles encapsulating mitochondria, or a population thereof, or a composition containing vesicles, comprising contacting an aqueous solution containing isolated mitochondria with an organic phase (e.g., an ethanol solution) containing lipids capable of forming a lipid membrane in a confluent channel of a microflow channel device and mixing them.

[0023] As used herein, the term "mitochondrial activator" refers to a substance capable of activating mitochondrial respiratory chain complexes (electron transport system), particularly a substance capable of polarizing mitochondria with respect to membrane potential, and it is particularly preferable to use a substance capable of hyperpolarizing mitochondria. Examples of mitochondrial activators include antioxidants such as resveratrol (3,5,4'-trihydroxy-trans-stilbene), coenzyme Q10, vitamin C, vitamin E, N-acetylcysteine, 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO), superoxide dismutase (SOD), and glutathione, and resveratrol is particularly preferred (see WO2018 / 092839). Resveratrol preferably used in the present invention can be extracted from plants by known methods, or chemically synthesized by known methods, such as the method of Andrus et al. (Tetrahedron Lett. 2003, 44, 4819-4822). Other examples of mitochondrial activators include mitochondrial DNA and mitochondrial RNA, such as 12S rRNA and 16S rRNA (see WO2020 / 230601, which is incorporated herein by reference in its entirety), as well as any other components of mitochondria.

[0024] The microflow channel device includes a confluence channel where flow channels extending from at least two inlets join. The microflow channel device may further include a mixing channel to promote mixing of the solutions that join in the confluence channel. The mixing channel may be a linearly extending path, or may have at least one bend (e.g., a baffle structure or multiple bends arranged in series) to further promote mixing. The mixing channel may or may not have concave and convex surfaces on its internal surface.

[0025] The channels of a microflow channel device can have thicknesses on the order of μm. When a channel has a circular cross section, its thickness is represented by its diameter. When the cross section is elliptical, the thickness can be represented by one or both of the major and minor axes. When the cross section is rectangular, one or both of the width and height can be used. The width and height of a channel can each independently be, for example, 100 μm to 400 μm. Referring to FIG. 36, a microflow channel device suitable for use in the present invention is described. As shown in FIG. 36, the channel (10) of the microflow channel device has two liquid sample inlets (11a and 12a), channels (11 and 12) connecting the liquid sample inlets (11a and 12a) to the confluence channel (13), and a mixing channel (14). The confluence channel 13 is where the channels (11 and 12) extending from the two liquid sample inlets join. The mixing channel 14 is a channel for mixing the combined liquid samples. The mixing channel 14 may be a linear channel or a channel with bends. As shown in Figure 36, in the mixing channel 14, the solution moves along the flow direction indicated by the large arrow and is directed through the bend toward the outlet 14c. The mixing channel 14 may have a single or multiple sets (e.g., 10-30 sets, 15-25 sets, 20 sets) of bends represented by 14a and 14b. In Figure 36, 14a represents the region where the channel narrows, and 14b represents the region where the narrow channel widens.

[0026] Examples of lipids that can form the lipid membrane in lipid membrane-based vesicles include phospholipids, glycolipids, sterols, and saturated or unsaturated fatty acids. The lipid can include multiple lipids.

[0027] Phospholipids refer to lipids having a phosphate ester in their structure. Phospholipids may be phospholipids of a type that can constitute cell membranes. Examples of phospholipids include phosphatidylcholine (e.g., dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine), phosphatidylglycerol (e.g., dioleoylphosphatidylglycerol, dilauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol), phosphatidylethanolamine (e.g., dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine), and phospholipids (e.g., dioleoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine). phosphatidylcholine, phosphatidylinositol, phosphatidic acid, cardiolipin, sphingomyelin, ceramide phosphorylethanolamine, ceramide phosphorylglycerol, ceramide phosphorylglycerol phosphate, 1,2-dimyristoyl-1,2-deoxyphosphatidylcholine, dioleoylphosphatidylethanolamine, soy phosphatidylcholine, plasmalogen, egg yolk lecithin, soy lecithin, their hydrogenated products, 3β-[N-(N'-,N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-chol), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), and 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA).

[0028] Glycolipids refer to lipids to which sugars are attached. In glycolipids, sugars can be attached to the end of the lipid. Examples of glycolipids include glyceroglycolipids (e.g., sulfoxyribosylglyceride, diglycosyldiglyceride, digalactosyldiglyceride, galactosyldiglyceride, glycosyldiglyceride) and glycosphingolipids (e.g., galactosylcerebroside, lactosylcerebroside, ganglioside). Examples of sterols include animal-derived sterols (e.g., cholesterol, cholesterol succinate, cholestanol, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol), plant-derived sterols (phytosterols) (e.g., stigmasterol, sitosterol, campesterol, brassicasterol) and microbial-derived sterols (e.g., zymosterol, ergosterol).

[0029] Sterol refers to the steroid alcohols that exist in the animal and plant kingdoms.Examples of sterols include animal-derived sterols (such as cholesterol, cholesterol succinate, cholestanol, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol) and plant-derived sterols (such as stigmasterol, sitosterol, campesterol, brassicasterol).Sterols also include sterols that are derived from microorganisms, such as zymosterol and ergosterol.

[0030] In some embodiments, a mixture of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM), 1,2-dimyristoyl-sn-glycerol, and methoxypolyethylene glycol can be used to prepare lipid membrane-based vesicles. In one embodiment, alkylated polyarginine or S2 peptide, such as stearylated octaarginine (STR-R8) or S2 peptide, can be further included in the vesicles.

[0031] The ethanol solution can have an ethanol concentration of, for example, 10 V / V% to 50 V / V% or 10 V / V% to 20 V / V%, as long as the solution can solubilize the lipid components.

[0032] As mitochondria, isolated mitochondria can be used. Isolation refers to taking something out of cells. As mitochondria, purified mitochondria can be used. Purification refers to completely or partially separating a component from at least one other component after isolation. Purified mitochondria can be isolated mitochondria because they have already been isolated. In this specification, isolated mitochondria and purified mitochondria referred to as isolated mitochondria are sometimes referred to as purified mitochondria.

[0033] Mitochondria can be isolated from cells by the shear stress of water, for example, by homogenization. Mitochondria can also be isolated from cells by disrupting the cell membrane through repeated freeze-thaw processes. Mitochondria can also be isolated from cells by disrupting the cell membrane with a detergent (at a concentration equal to or greater than the critical micelle concentration). Mitochondria can also be isolated from cells by contacting cells with a detergent (at a concentration less than the critical micelle concentration), then incubating the treated cells on ice, and optionally subjecting the treated cells to the shear stress of water. Shear stress can be applied to the treated cells, for example, by pipetting the solution containing the treated cells, preferably without bubbling. Since the damage to the mitochondria removed by the detergent can be minimized, detergents with a concentration less than the critical micelle concentration can be advantageously used for isolation.

[0034] The isolated mitochondria can be collected by centrifugation. Mitochondria can be separated from cellular components such as nuclei (e.g., high-density cellular components) by a centrifugation process performed at 500 × g for several minutes (e.g., 4 minutes). Therefore, mitochondria can be collected by collecting the supernatant after the centrifugation process. Furthermore, mitochondria can be further centrifuged to precipitate. In this way, mitochondria can be separated from other cellular components (e.g., low-density cellular components) that have not been precipitated.

[0035] The aqueous solution containing mitochondria can be maintained in a buffer (e.g., a mitochondrial storage buffer). Maintenance can be performed at 4°C. Examples of buffers that can be used include physiological saline, Tris buffer, Hepes buffer, and phosphate buffer. The buffer can contain a tonicity agent. Examples of tonicity agents include sugars such as sucrose. The buffer can contain a divalent ion chelator, such as ethylenediaminetetraacetic acid (EDTA) and glycoletherdiaminetetraacetic acid. The buffer can contain a physiologically acceptable salt (e.g., sodium chloride, magnesium chloride).

[0036] Lipid membrane-based vesicles encapsulating mitochondria can be obtained by contacting an aqueous solution containing mitochondria with an organic phase (e.g., an ethanol solution) containing lipids capable of forming lipid membranes in the confluent channel of a microflow channel device and mixing them. The inventors discovered that providing isolated mitochondria having a particle size of approximately 1 μm to a microflow channel device can produce lipid membrane-based vesicles encapsulating mitochondria with a particle size substantially smaller than 1 μm; and that introducing the vesicles into cells can improve the mitochondrial activity of the cells. The resulting lipid membrane-based vesicles encapsulating mitochondria have a smaller PDI than isolated mitochondria and exhibit relatively high monodispersity as organelles. Therefore, the present invention provides a method for producing a composition (or preparation) comprising a population of lipid membrane-based vesicles encapsulating mitochondria. The present invention also provides a composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria or a mitochondrial preparation comprising the population. A composition comprising a group of mitochondria not encapsulated in vesicles can be obtained by contacting a lipid-free organic phase with an aqueous solution containing mitochondria in the contacting step. Such compositions containing unencapsulated mitochondria can be further subjected to an encapsulation procedure to form lipid membrane-based vesicles encapsulating the mitochondria.

[0037] The flow rates and flow rate ratio of the aqueous solution containing mitochondria and the organic phase (e.g., an ethanol solution) containing lipids capable of forming a lipid membrane, which are introduced into the confluent channel of the microflow channel device, can be appropriately determined by those skilled in the art.

[0038] In one embodiment, the present invention provides a population of mitochondria that is not encapsulated in vesicles. In one embodiment, the present invention provides a composition or pharmaceutical composition comprising a population of mitochondria that is not encapsulated in vesicles. In a preferred embodiment, the population of mitochondria has a particle size distribution with a peak at less than 1 μm, 900 nm or less, 800 nm or less, 700 nm or less, 600 nm or less, 500 nm or less, 400 nm or less, or 300 nm or less, as determined by dynamic light scattering. The PDI of the population of mitochondria may be 0.5 or less, 0.4 or less, or 0.3 or less. The population may be monodisperse. Such a population can be obtained by fractionating mitochondria isolated by iMIT using a microfluidic device into smaller ones. Small mitochondria are believed to be beneficial in preparing lipid membrane-based vesicles encapsulating the fractionated isolated mitochondria, and also in parenteral administration to avoid any blockage in the vascular system. Therefore, pharmaceutical compositions can be formulated for parenteral administration (e.g., intravenous, intramuscular, intraventricular, intracerebroventricular), for example, by encapsulating fractionated isolated mitochondria in lipid membrane-based vesicles. In one embodiment, a divided Q may have cristae equivalent in number and / or density to an undivided Q.

[0039] In a preferred embodiment, the mitochondria encapsulated in lipid membrane-based vesicles may be segmented, capable of being segmented after being encapsulated in lipid membrane structures (i.e., vesicles or sacs). Mitochondrial segmentation can be achieved using a microfluidic device in the presence or absence of lipids. Thus, lipid membrane-based vesicles can encapsulate segmented mitochondria, which can have a population size distribution with a peak of less than 1000 nm, 900 nm or less, 800 nm or less, 700 nm or less, 600 nm or less, 500 nm or less, 400 nm or less, or 300 nm or less. In one embodiment, the segmented mitochondria have cristae and a matrix within the mitochondrion. In a specific embodiment, the segmented mitochondria have cristae filled within the inner membrane. According to the present invention, such segmented mitochondria do not necessarily have a membrane potential, but introducing the vesicles into cells can improve mitochondrial respiration in cells. In one embodiment, the segmented, isolated mitochondria within the vesicles cannot have a detectable membrane potential. The mitochondrial membrane potential can be detected using a mitochondrial membrane potential indicator.

[0040] In one embodiment, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the population of encapsulated mitochondria consists of fractionated isolated mitochondria.

[0041] In one embodiment, the mitochondria of the population can be encapsulated in lipid membrane-based vesicles. In one embodiment, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the mitochondria in the population are encapsulated in lipid membrane-based vesicles, and the encapsulated mitochondria can be formulated as a pharmaceutical composition.

[0042] In one embodiment of the present invention, a composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria of the present invention, or a mitochondrial preparation comprising this population, can have a particle size distribution with a peak at less than 1 μm, 900 nm or less, 800 nm or less, 700 nm or less, 600 nm or less, 500 nm or less, 400 nm or less, or 300 nm or less, as determined by dynamic light scattering. A composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria of the present invention, or a mitochondrial preparation comprising this population, can have a particle size distribution with a peak, for example, at 200 nm or more and less than 500 nm, as determined by dynamic light scattering.

[0043] In one embodiment of the present invention, a composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria of the present invention, or a mitochondrial preparation comprising this population, can have a particle size distribution with a peak at 50 nm to 200 nm, 50 nm to 150 nm, 100 nm to 500 nm, 200 nm to 400 nm, 300 nm to 500 nm, 500 nm to 1500 nm, 600 nm to 1400 nm, 700 nm to 1300 nm, 800 nm to 1200 nm, or about 1000 nm (or about 1 μm), as determined by dynamic light scattering.

[0044] In another embodiment, mitochondria or divided mitochondria encapsulated in lipid membrane-based vesicles may not themselves possess a substantial membrane potential within the vesicles, and may not retain substantial respiratory capacity. Even if the encapsulated or divided mitochondria do not possess a substantial membrane potential or substantial respiratory capacity within the vesicles, the encapsulated or divided mitochondria may potentially deliver their components, such as mitochondrial DNA, coenzyme Q10, and other components, to cellular mitochondria, potentially benefiting the cells that receive the components. Mitochondrial membrane potential or respiratory capacity may be reduced under higher pH conditions, such as pH 8-10 or 8-9. Such encapsulated or divided mitochondria may optionally exhibit or recover their physiological functions, such as membrane potential or respiration, after reintroduction into cells.

[0045] Mitochondrial DNA leaking from mitochondria can have some negative effects on cellular function. Furthermore, mitochondrial DNA in isolated mitochondria improves mitochondrial function, particularly in cells with mitochondrial dysfunction. Therefore, in a preferred embodiment, the divided mitochondria can maintain mitochondrial DNA within the mitochondria. Non-nucleic acid components within the isolated mitochondria improve intracellular mitochondrial function. Therefore, in a preferred embodiment, the divided mitochondria can maintain non-nucleic acid components within or on the mitochondria.

[0046] The size distribution or upper peak limit of the population of lipid membrane-based vesicles encapsulating mitochondria of the present invention can vary depending on the cells that serve as the source and the method of isolating mitochondria from the cells.

[0047] A composition of the present invention comprising a population of lipid membrane-based vesicles encapsulating mitochondria, or a mitochondrial preparation comprising this population, can have a polydispersity index (PDI) of 0.5 or less, 0.4 or less, or 0.3 or less, as determined by dynamic light scattering. A composition of the present invention comprising a population of lipid membrane-based vesicles encapsulating mitochondria, or a mitochondrial preparation comprising this population, can have a PDI of 0.2 to 0.4.

[0048] A composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria of the present invention, or a mitochondrial preparation comprising this population, can have a positive, zero, or negative zeta potential. The zeta potential can be positive or negative to improve the dispersibility of the vesicles in solution (to prevent aggregation in solution). The vesicles can have a positive zeta potential. The positive zeta potential of the vesicles can improve cellular uptake. A composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria of the present invention, or a mitochondrial preparation comprising this population, can have a zeta potential of, for example, -10 mV or less, -11 mV or less, -12 mV or less, -13 mV or less, -14 mV or less, -15 mV or less, -16 mV or less, -17 mV or less, -18 mV or less, -19 mV or less, or -20 mV or less. Compositions comprising a population of lipid membrane-based vesicles encapsulating mitochondria of the present invention, or mitochondrial preparations comprising this population, can have a zeta potential of, for example, 10 mV or more, 11 mV or more, 12 mV or more, 13 mV or more, 14 mV or more, 15 mV or more, 16 mV or more, 17 mV or more, 18 mV or more, 19 mV or more, or 20 mV or more. To allow the vesicle population to have a positive zeta potential, the lipid membrane can be formed from a material that can impart a positive zeta potential (e.g., a lipid membrane formed from electrically neutral lipids, including cationic lipids and lipids with cationic moieties (e.g., lipids with cationic moieties such as stearylated octaarginine and S2 peptide)). To allow the vesicle population to have a negative zeta potential, the lipid membrane can be formed from a material with a negative zeta potential (e.g., a lipid membrane formed from anionic lipids).

[0049] A composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria of the present invention, or a mitochondrial preparation comprising this population, can have a particle size distribution with a peak at less than 1 μm, a PDI of 0.5 or less, and a positive zeta potential, as determined by dynamic light scattering. A composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria of the present invention, or a mitochondrial preparation comprising this population, can have a particle size distribution with a peak at less than 500 nm, a PD1 of 0.5 or less, and a positive zeta potential, as determined by dynamic light scattering. A composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria of the present invention, or a mitochondrial preparation comprising this population, can have a particle size distribution with a peak at less than 500 nm, a PDI of 0.5 or less, and a zeta potential of 10 mV or more, as determined by dynamic light scattering.

[0050] A composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria of the present invention, or a mitochondrial preparation comprising this population, can have a particle size distribution with a peak at less than 1 μm, a PDI of 0.5 or less, and a negative zeta potential, as determined by dynamic light scattering.A composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria of the present invention, or a mitochondrial preparation comprising this population, can have a particle size distribution with a peak at less than 500 nm, a PDI of 0.5 or less, and a negative zeta potential, as determined by dynamic light scattering.A composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria of the present invention, or a mitochondrial preparation comprising this population, can have a particle size distribution with a peak at less than 500 nm, a PDI of 0.5 or less, and a zeta potential of -10 mV or less, as determined by dynamic light scattering.

[0051] A composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria of the present invention, or a mitochondrial preparation comprising this population, can have a particle size distribution with a peak at 500 nm to 1500 nm (or about 1 μm) as determined by dynamic light scattering, and can have a positive zeta potential. A composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria of the present invention, or a mitochondrial preparation comprising this population, can have a particle size distribution with a peak at 500 nm to 1500 nm (or about 1 μm) as determined by dynamic light scattering, a PDI of 0.5 or less, and can have a positive zeta potential. A composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria of the present invention, or a mitochondrial preparation comprising this population, can have a particle size distribution with a peak at 500 nm to 1500 nm (or about 1 μm) as determined by dynamic light scattering, a PDI of 0.5 or less, and can have a zeta potential of 10 mV or more.

[0052] A composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria of the present invention, or a mitochondrial preparation comprising this population, can have a particle size distribution with a peak at 500 nm to 1500 nm (or about 1 μm) as determined by dynamic light scattering, and can have a negative zeta potential. A composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria of the present invention, or a mitochondrial preparation comprising this population, can have a particle size distribution with a peak at 500 nm to 1500 nm (or about 1 μm) as determined by dynamic light scattering, a PDI of 0.5 or less, and can have a negative zeta potential. A composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria of the present invention, or a mitochondrial preparation comprising this population, can have a particle size distribution with a peak at 500 nm to 1500 nm (or about 1 μm), a PDI of 0.5 or less, and can have a zeta potential of -10 mV or less as determined by dynamic light scattering.

[0053] The composition of the present invention, which comprises a population of lipid membrane-based vesicles encapsulating mitochondria, or the mitochondrial preparation comprising this population, when introduced into cells, releases mitochondria into the cytoplasm, and improves mitochondrial function (e.g., respiratory activity) in cells.The improvement of mitochondrial function in cells can be determined, for example, by measuring the oxygen consumption rate of mitochondria.

[0054] When the composition of the present invention, comprising a population of lipid membrane-based vesicles encapsulating mitochondria, or a mitochondrial preparation comprising this population, is introduced into cells, it releases mitochondria into the cytoplasm, and the released mitochondria fuse with endogenous mitochondria.More specifically, the composition of the present invention, comprising a population of lipid membrane-based vesicles encapsulating mitochondria, or a mitochondrial preparation comprising this population, encapsulates mitochondria in lipid membrane-based vesicles.When the composition or preparation is incorporated into cells, it is sequestered in endosomes.Mitochondria are protected by lipid membrane even in the endosomal environment, and can then be released from endosomes; more specifically, endosomes fuse with the lipid membrane of vesicles to release mitochondria from the lipid membrane.In this way, mitochondria that are separated from all or part of their lipid membranes can be released into the cytoplasm.Mitochondria that are separated from all or part of their lipid membranes can be brought into contact with other mitochondria (for example, endogenous mitochondria) and fuse with them. Mitochondrial fusion in cells can be confirmed by separately labeling mitochondria in cells and mitochondria contained in vesicles with different fluorescent dyes (distinguishable by wavelength) and examining whether they coexist in cells.

[0055] In the composition of the present invention that comprises a group of lipid membrane-based vesicles that contain mitochondria, or in the mitochondria preparation that comprises this group, the internal and external environments of the vesicles are physically, biochemically, and / or physiologically separated by lipid membrane.More specifically, the vesicles have a closed space formed by lipid membrane, which acts as a barrier that prevents the free transport of substances between the internal and external regions.The encapsulation of mitochondria in lipid membrane-based vesicles can be confirmed by observing the image of the hollow space in lipid membrane-based vesicles using negative staining and electron microscope; and by separately labeling mitochondria and lipid membrane with different fluorescent dyes (distinguishable by wavelength) and examining whether they coexist when observed under a microscope.

[0056] The composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria of the present invention, or a mitochondrial preparation comprising this population, encapsulates mitochondria. The mitochondria can have respiratory activity in cells or in the presence of substrates for respiratory chain complexes. The composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria of the present invention, or a mitochondrial preparation comprising this population, can be used to improve impaired mitochondrial function in cells. The composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria of the present invention, or a mitochondrial preparation comprising this population, can be administered to, for example, tissues with impaired mitochondrial function. The composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria of the present invention, or a mitochondrial preparation comprising this population, can be administered to, for example, tissues damaged by myocardial infarction. The composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria of the present invention, or a mitochondrial preparation comprising this population, can be administered to, for example, subjects with mitochondrial dysfunction. Examples of mitochondrial dysfunction include neurodegenerative disorders and neuropsychiatric disorders.

[0057] The composition of the present invention comprising a population of lipid membrane-based vesicles encapsulating mitochondria, or a mitochondrial preparation comprising this population, can contain an effective amount of the vesicles, which refers to an amount sufficient to improve mitochondrial function in a cell when the vesicles are introduced into the cell.

[0058] The composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria of the present invention, or a mitochondrial preparation comprising this population, can be provided in a frozen state.The composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria of the present invention, or a mitochondrial preparation comprising this population, in a frozen state, can further comprise a cryoprotectant (e.g., glycerol).

[0059] A composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria of the present invention, or a mitochondrial preparation comprising this population, can be stored at 4° C. for 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 1 week or more. The stored composition can be used to enhance intracellular mitochondria in cells contacted with the composition.

[0060] According to the present invention, there is provided a method for producing a composition comprising a population of lipid membrane-based vesicles encapsulating mitochondria or a mitochondrial preparation comprising said population, the method comprising: A method is provided that includes contacting and mixing an aqueous buffer containing isolated mitochondria and an organic phase containing lipids capable of forming a lipid membrane in a converging channel of a microflow channel device.

[0061] The organic phase is not particularly limited, as long as it can be removed by subsequent dialysis; for example, an ethanol solution can be mentioned.

[0062] In the method of the present invention, the microflow channel device can have a channel with at least one bend to facilitate mixing of solutions contacting each other in the confluence channel. In the method of the present invention, the microflow channel device includes a flow channel to facilitate mixing of solutions contacting each other in the confluence channel, the flow channel having a baffle structure.

[0063] The method of the present invention can further include removing ethanol from the resulting mixture (a solution containing lipid membrane-based vesicles encapsulating mitochondria). The removal of ethanol can be carried out by subjecting the resulting mixture to dialysis using a buffer that preserves mitochondria as the external solution.

[0064] The method of the present invention can further include adding pharmaceutically acceptable excipients (e.g., buffers, tonicity agents, stabilizers, dispersing agents, salts, and cryoprotectants) to the solution containing the lipid membrane-based vesicles encapsulating mitochondria (buffer solution).

[0065] In the methods of the present invention, isolated mitochondria can be stained with a potential-dependent dye (e.g., a mitochondrial membrane potential indicator). Examples of mitochondrial membrane potential indicators that can be used in the present invention include tetramethylrhodamine methyl ester (TMRM), tetramethylrhodamine ethyl ester (TMRE), 3,3'-dihexyloxacarbocyanine iodide (DiOC6), 6-amino-9-(2-methoxycarbonylphenyl)xanthen-3-ylidene)azanium chloride (rhodamine 123), and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). JC-1 accumulates in mitochondria in a membrane potential-dependent manner, forms associations at high concentrations, and changes color from green to red. TMRM and TMRE each accumulate in mitochondria in a membrane potential-dependent manner and exhibit high fluorescence intensity at high concentrations. TMRE, for example, can be used in the present invention. Therefore, when a mitochondrial membrane potential indicator is used, its fluorescence intensity reflects the magnitude of the mitochondrial membrane potential. The relationship between fluorescence intensity and membrane potential can be determined based on a pre-prepared calibration curve. This method allows easy evaluation of whether lipid membrane-based vesicles encapsulating mitochondria encapsulate functional mitochondria.

[0066] In one embodiment, the present disclosure provides mitochondria isolated by a novel method, referred to herein as the "detergent and homogenization-free (DHF)" method, or alternatively, the "iMIT" method. As described herein, mitochondria isolated by the iMIT method are undamaged (e.g., retain the integrity of their inner and outer membranes) and maintain functional capacity (e.g., membrane potential). Mitochondria obtained by the iMIT method are referred to herein as "Q" mitochondria. These mitochondria are suitable for use in the treatment of various diseases and disorders, including those described herein, e.g., by mitochondrial transplantation. Mitochondrial transplantation is a treatment expected to be useful in various diseases and disorders. To restore and / or enhance mitochondrial function, exogenous mitochondria (e.g., Q mitochondria) are internalized into cells with severely dysfunctional mitochondria and / or cells that would benefit from the influx of highly functional mitochondria.

[0067] In one embodiment, the present disclosure provides a method for recovering or isolating mitochondria from cells by treating cells in solution with a detergent at a concentration below the critical micelle concentration (CMC), removing the detergent from the solution containing the treated cells, and then incubating the detergent-treated cells to recover the mitochondria in solution, thereby recovering the mitochondria from the cells. This method is referred to herein as "iMIT." Accordingly, provided herein is iMIT, a method for obtaining mitochondria from cells, comprising: (A) treating cells in a first solution with a surfactant at a concentration below the critical micelle concentration (CMC); (B) removing the surfactant from the first solution to form a second solution; and (C) Incubating the detergent-treated cells in a second solution to recover mitochondria in the second solution. (A) through (C) above and additional components of the method are described below.

[0068] According to the disclosed method, cells having mitochondria in their cytoplasm are treated with a detergent in a solution at a concentration below the critical micelle concentration. Thus, in some embodiments, the structural strength of the cell membrane is weakened, but the low concentration of detergent does not cause permeabilization, leaving the mitochondrial membrane largely unexposed to the detergent and intact. In some embodiments, the cell membrane may be partially permeabilized, but the low concentration of detergent causes the mitochondrial membrane to be largely unexposed to the detergent and remain intact.

[0069] In some embodiments, the solution (A) can contain a buffer. Exemplary buffers for use in the methods provided herein include, for example, Tris buffer, HEPES buffer, and phosphate buffer. The buffer can have a pH of, for example, 6.7 to 7.6 (e.g., pH 6.8 to 7.4, pH 7.0 to 7.4, e.g., pH 7.2 to 7.4, e.g., pH 7.4). In some embodiments, the buffer can contain a tonicity agent and an osmolality adjuster. Exemplary tonicity and osmolality adjusting agents include monosaccharides (e.g., glucose, galactose, mannose, fructose, inositol, ribose, xylose, etc.), disaccharides (e.g., lactose, sucrose, cellobiose, trehalose, maltose, etc.), trisaccharides (e.g., raffinose, melesinose, etc.), polysaccharides (e.g., cyclodextrin, etc.), sugar alcohols (e.g., erythritol, xylitol, sorbitol, mannitol, maltitol, etc.), glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, etc. The buffer may also contain a chelating agent, particularly a chelating agent for divalent metals, e.g., a chelating agent for calcium ions. Chelating agents include, for example, glycol ether diamine tetraacetic acid (EGTA) and ethylenediamine tetraacetic acid (EDTA).

[0070] In some embodiments, the buffer may be a Tris buffer containing sucrose and a chelator, and has a pH of 6.7 to 7.6 (e.g., pH 6.8 to 7.4, pH 7.0 to 7.4, e.g., pH 7.2 to 7.4, e.g., pH 7.4). In some embodiments, the Tris buffer may contain digitonin or saponin, or another detergent provided herein. In some embodiments, the digitonin or saponin or other detergent may have a concentration of 20% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, or 10% or less of the critical micelle concentration. In some embodiments, digitonin can be used at a concentration of 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 or less, or 30 μM or less (e.g., at a concentration of 30 μM). In some embodiments, saponin can be used at a concentration of 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 or less, or 30 μM or less (e.g., at a concentration of 30 μM).

[0071] In some embodiments, the surfactant used in the methods provided herein may be an ionic or nonionic surfactant. Nonionic surfactants used in the present invention may include, for example, ester, ether, and alkyl glycoside types. Nonionic surfactants include, for example, alkyl polyethylene glycols, polyoxyethylene alkyl phenyl ethers, and alkyl glycosides. Nonionic surfactants may include Triton-X 100, Triton-X 114, Nonidet P-40, n-dodecyl-D-maltoside, Tween-20, Tween-80, saponin, and / or digitonin. In the treatment step (A), at least one surfactant selected from the group consisting of Triton-X 100, saponin, and digitonin is used. In some embodiments, the surfactant is saponin or digitonin.

[0072] In one embodiment, the treatment step (A) comprises treating cells with a surfactant at a concentration below the critical micelle concentration. The treatment time for cells in step (A) may be, for example, 1 to 30 minutes, for example, 1 to 10 minutes, or for example, 1 to 5 minutes, for example, 2 to 4 minutes, for example, 3 minutes. Treatment of cells in (A) may be carried out on ice, at 4°C, or at room temperature, or at any temperature therebetween.

[0073] In one embodiment, the concentration of the surfactant in treatment step (A) may be a concentration less than the critical micelle concentration, for example, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less of the critical micelle concentration, for example, 5 to 15%, for example, 8 to 12%, for example, 10%.

[0074] In one embodiment, treatment step (A) is a pretreatment of cells. Without wishing to be bound by theory, it is believed that treatment of cells with detergents below the critical micelle concentration can reduce the strength of cell membranes and / or partially or completely eliminate the effects of detergent treatment on intracellular mitochondria.

[0075] Therefore, in consideration of minimizing the effects of surfactants on mitochondria, the concentration of surfactant in the solution with which mitochondria come into contact in at least any of the steps (e.g., each of steps (B) to (E)) during and after the recovery of mitochondria from cells may be less than the critical micelle concentration, for example, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the critical micelle concentration; or may be below the detection limit. In consideration of minimizing the effects of surfactants on mitochondria, preferably, no surfactant should be added to the solution with which mitochondria come into contact during and after the recovery of mitochondria from cells.

[0076] In some embodiments, the cells may be in the form of cells present in a tissue, or they may be isolated from the tissue (e.g., single cells) or populations thereof. Cells isolated from a tissue may be cultured cells, or may be single cells or populations thereof obtained by treating the tissue or cultured cells with an enzyme, such as collagenase, used to reduce them to single cells. The tissue may optionally be minced prior to treatment with an enzyme, such as collagenase.

[0077] In one embodiment, to reduce the concentration of detergent in contact with the mitochondria or to sufficiently reduce the detergent in contact with the mitochondria, the detergent can be removed from the solution before the mitochondria are recovered from the detergent-treated cells in (A).

[0078] In the removal step (B), the detergent can be removed by, for example, replacing the buffer with a solution containing a lower or reduced concentration of detergent (preferably a detergent-free solution) (e.g., a buffer) or adding the solution to the buffer. When the detergent-treated cells are adherent cells, the detergent-containing buffer can be removed by aspirating the solution, washing the cells with a solution containing a lower or reduced concentration of detergent (preferably a detergent-free solution) (e.g., a buffer) if necessary, and adding a solution containing a lower or reduced concentration of detergent (preferably a detergent-free solution) (e.g., a buffer). When the detergent-treated cells are suspension cells, the detergent can be removed by centrifuging the cells, removing the supernatant, washing the cells with a solution containing a lower or reduced concentration of detergent (preferably a detergent-free solution) (e.g., a buffer) if necessary, and adding a solution containing a lower or reduced concentration of detergent (preferably a detergent-free solution) (e.g., a buffer).

[0079] By removed is meant at least reducing the concentration of detergent in the solution contacting the mitochondria, including, for example, to less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the detergent concentration; or below the detection limit in the solution contacting the mitochondria. To ensure removal of detergent from the solution, (B) can include washing the cells with a solution (preferably a detergent-free solution) (e.g., a buffer) containing a lower or reduced concentration of detergent.

[0080] In (B), the solution that is added to or exchanged with the solution to remove detergent from the solution is preferably a buffer, which may be a buffer as described in (A) above (but containing a lower concentration of detergent, preferably a solution that is detergent-free or contains undetectable levels of detergent).

[0081] Cells treated with (A) have reduced plasma membrane strength, allowing mitochondria to be released from the cell interior to the extracellular space simply by incubating them in the solution. However, in the previous step (C), the amount of detergent contacting the mitochondria is small, and the effect of the detergent on the mitochondria is limited, and therefore, the reduction in mitochondrial membrane strength is also limited, and / or the mitochondrial membrane remains intact.

[0082] In one embodiment, the method comprises obtaining mitochondria released into the second solution by simply allowing the cells to rest in the second solution.

[0083] Therefore, in the present invention, detergent-treated cells can be incubated in a solution to release mitochondria from the inside of the cell to the extracellular space. The term "release" in (C) means that mitochondria escape from the inside of the cell to the outside of the area surrounded by the plasma membrane (e.g., to the solution side or outside the cell).

[0084] The solution used for incubation in (C) ("second solution") may be a solution containing a lower concentration of detergent. In a preferred embodiment, the second solution is a detergent-free solution or a solution containing negligible and / or undetectable amounts of detergent. The solution used for incubation in (C) may be, for example, a buffer as described in (A) above, or a buffer (containing a lower concentration of detergent than that described in (A) above) (preferably a detergent-free solution). The solution used in (C) may be, for example, a solution containing a buffer, an osmolality adjusting agent, and a divalent metal chelator, but is substantially free of detergent. As used herein, "substantially free" does not exclude the presence of contaminating amounts of "substantially free components" that cannot be removed or detected.

[0085] In (C), the incubation may be, for example, 1 to 30 minutes, for example, 5 to 25 minutes, or for example, 5 to 20 minutes, for example, 5 to 15 minutes, for example, 10 minutes. Treatment of the cells in (C) may be carried out on ice, at room temperature, or at a temperature therebetween.

[0086] In (C), to enhance recovery of mitochondria from cells, a physical stimulus can be applied so as not to cause mechanical disruption of the mitochondrial lipid bilayer. Thus, in (C), for example, incubation can be performed under shaking or non-shaking conditions. In (C), for example, incubation can be performed under stirring or non-stirring conditions. In (C), detergent treatment makes it easier for cells to detach from the adhesive surface, and therefore, detachment of cells from the adhesive surface by a gentle water stream, as described above, does not appear to negatively affect the polarization ratio. Alternatively, in (C), incubation can be performed to a degree that does not cause cells to detach.

[0087] In (C), the mitochondria recovered in solution can be used in various applications as a population of isolated mitochondria. In some embodiments, the present disclosure provides a population of mitochondria, referred to herein as "Q" mitochondria, produced by the methods provided herein. In some embodiments, the present disclosure provides individual mitochondria (i.e., individual Q) produced by the methods provided herein.

[0088] In some embodiments, the methods provided herein further include purifying the mitochondria recovered in the solution (D). Mitochondria can be separated from one or more other cellular components by centrifugation. For example, mitochondria can be purified as a supernatant by centrifugation of the mitochondrial population recovered in (C) at 1500 g or less, 1000 g or less, or 500 g or less to precipitate contaminants, such as detached cells, contained in the mitochondrial population. Mitochondria can be purified as a supernatant, preferably by centrifugation at 500 g, for example. Mitochondria can also be recovered as a precipitate by subjecting the resulting supernatant to further centrifugation (e.g., 8000 g to 12000 g) for enrichment or the like. As used herein, the term "purified" means that mitochondria are separated from at least one other component in the solution by a process.

[0089] The mitochondrial population obtained in (C) and / or (D) above can be used as an isolated mitochondrial population in various applications.

[0090] The methods of the present invention can further include (E) freezing the mitochondria. Freezing can be carried out by gently suspending the mitochondria in a freezing buffer. The freezing buffer can be the buffer described in (A), but without detergent and can further contain a cryoprotectant. Exemplary cryoprotectants are known in the art and include glycerol, sucrose, trehalose, dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, diethyl glycol, triethylene glycol, glycerol-3-phosphate, proline, sorbitol, formamide, and polymers. Thus, the mitochondria provided herein can be preserved by freezing. In the methods of the present disclosure, mitochondria are not frozen if cryopreservation is not required; for example, mitochondria can be used as freshly isolated. In other embodiments, mitochondria can be stored at about 4°C ± 3°C or on ice. In certain embodiments, the mitochondria provided herein produced by the methods provided herein can be stored in liquid nitrogen at about -80°C ± 3°C or below, about -20°C ± 3°C or below, or about 4°C ± 3°C. In some embodiments, mitochondria can be stored for days, weeks, or months or longer and retain the ability to function after thawing.

[0091] In some embodiments, the methods provided herein further include thawing mitochondria that have been isolated and subsequently frozen as defined herein. The methods of thawing mitochondria provided herein include thawing mitochondria at a temperature of about 20° C.±3° C. or less, and thawing the mitochondria rapidly, e.g., within about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, or about 1 minute. In some embodiments, rapid thawing of mitochondria results in the mitochondria retaining their functional capabilities as described herein.

[0092] In some embodiments, the methods provided herein do not involve disrupting cell membranes throughout the entire process of recovering mitochondria from cells in a manner that disrupts the mitochondrial membrane. For example, in the methods provided herein, cells are not disrupted by homogenization during the process of recovering mitochondria from cells. That is, in some embodiments, the methods provided herein do not involve homogenization; in some embodiments, the methods include homogenization, but the homogenization is performed to the extent that it does not cause any bubbles or does not cause bubbles in the solution relative to the cells or tissue. In some embodiments, the methods do not involve freezing and thawing cells. Repeated freezing and thawing of cells is suitable for disrupting the plasma membrane and recovering its contents and can be used to recover mitochondria from cells, but the resulting mitochondrial membrane potential is not maintained (in contrast to the methods disclosed herein, in which the mitochondrial membrane potential is maintained), and it is believed that freezing and thawing also disrupts the mitochondrial lipid bilayer.

[0093] In some embodiments, the method of the present disclosure does not involve other methods that disrupt cell membranes (e.g., ultrasonication, treatment with a strong water stream that causes bubbles or foaming in the solution) throughout the entire process of recovering mitochondria from cells. In some embodiments, the method of the present disclosure is carried out without any process that may substantially cause physical, chemical, or physiological damage to mitochondria, although freeze-thaw cycles may be applied to mitochondria for preservation. Therefore, the method of the present disclosure makes it possible to obtain mitochondria with minimal damage.

[0094] The methods of the present invention do not require one or more filtration steps to purify mitochondria recovered from cells.

[0095] In some embodiments, the methods provided herein gently separate mitochondria from the microtubule system while they are still in the cell, without damaging them. During the incubation period, mitochondria, which have become non-filamentous due to the detachment of microtubules from the mitochondrial surface, can escape from the cell through detergent-treated cell membranes. Thus, mitochondria obtained from cells through the disclosed methods are obtained without rupturing or tearing the mitochondrial membrane or otherwise damaging the mitochondrial structure. Thus, the isolated mitochondria and populations thereof provided herein are able to maintain function after isolation, making them significantly more suitable for use in treating disease states than any previously described isolated mitochondria.

[0096] Thus, the methods provided herein differ in important respects from conventional methods for isolating mitochondria, and provide isolated or obtained mitochondria with unexpected and advantageous functionality compared to mitochondria isolated by conventional methods or any other previously disclosed methods.

[0097] In a preferred embodiment, mitochondria can be isolated from cell lines such as cardiac cells, myocytes, cardiomyocytes, cardiac progenitor cells, cardiac stem cells, HUVEC cells, and HeLa cells.

[0098] 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 a population of isolated mitochondria, wherein 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 about 95% of the mitochondria in the population have intact inner and outer membranes; and / or 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 about 95% of the mitochondria in the population are polarized as measured by a fluorescent indicator. In some embodiments, the fluorescent indicator is selected from the group consisting of positively charged dyes, such as JC-1, tetramethylrhodamine methyl ester (TMRM), and tetramethylrhodamine ethyl ester (TMRE).

[0099] In some embodiments, 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 about 95% or more of the isolated or obtained mitochondrial population are polarized as measured by a fluorescent indicator. In some embodiments, the fluorescent indicator may be any fluorescent indicator known to those of skill in the art to be suitable for measuring mitochondrial membrane potential. In some embodiments, the fluorescent indicator is selected from the group consisting of JC-1, TMRM, and TMRE. In some embodiments, the extracellular environment may comprise a total calcium concentration of about 4 mg / dL to about 12 mg / dL or about 1 mmol / L (1000 μM) to about 3 mmol / L (3000 μM). For example, in some embodiments, the extracellular environment may comprise a total calcium concentration of about 8 mg / dL to about 12 mg / dL or about 2 mmol / L (2000 μM) to about 3 mmol / L (3000 μM). In some embodiments, the extracellular environment comprises a free or active calcium concentration of about 4 mg / dL to about 6 mg / dL or about 1 mmol / L (1000 μM) to about 1.5 mmol / L (1500 μM). In some embodiments, the mitochondrial population maintains functional capacity in an environment having a higher calcium concentration compared to the calcium environment in the cell.

[0100] In certain embodiments, provided herein is a population of isolated mitochondria, wherein 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 about 95% of the mitochondria in the population have not undergone dynamin-related protein 1 (drp1)-dependent division. In certain embodiments, provided is a population of isolated mitochondria having an inner membrane and an outer membrane, wherein the inner mitochondrial membrane comprises densely folded cristae.

[0101] In some embodiments, a population of isolated mitochondria is provided, in which 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 about 95% of the mitochondria in the population have a substantially non-filamentous, non-branched structure or shape. For example, in some embodiments, the mitochondria provided herein appear round, punctate, spherical, irregularly shaped, and / or slightly elongated, or any mixture thereof, when viewed under a microscope. In some embodiments, 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 about 95% of the mitochondria in the population have a ratio of major to minor diameters of 4:1 or less, 3.5:1 or less, or 3:1 or less. In some embodiments, 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 about 95% of the isolated mitochondria in a population of mitochondria provided herein have a length that is less than two or three times the hydrodynamic diameter of the mitochondria. Thus, the isolated mitochondria provided herein have a significantly different shape (non-filamentous) compared to the shape of most mitochondria present in cells (filamentous). Accordingly, in some embodiments, the population of mitochondria provided herein has a shape that differs from unisolated mitochondria present in cells in that 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 about 95% of the mitochondria in the population are non-filamentous in shape. In some embodiments, the population of isolated mitochondria provided herein exhibits reduced association with the mitochondria-associated membrane (MAM). In one embodiment, association with MAM is measured by expression of glucose-regulated protein 75 (GRP75).In certain embodiments, the populations of isolated mitochondria provided herein exhibit about 60%, at least about 65%, at least about 70%, about 60%, about 50%, about 40%, about 30% or less association with MAM when compared to mitochondria in cells and / or mitochondria obtained by conventional isolation methods, e.g., methods involving homogenization and / or high levels of detergent, as further described in certain embodiments. In certain embodiments, the populations of isolated mitochondria provided herein exhibit a decrease in association with MAM, the decrease being at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or more compared to the association with MAM of mitochondria in cells or mitochondria isolated by conventional isolation methods.

[0102] In some embodiments, a population of isolated mitochondria provided herein is between about 500 nm and about 3500 nm in size, and in some embodiments, 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% of the mitochondria in the population are between about 500 nm and about 3500 nm in size. In some embodiments, the average size of mitochondria in the population is about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, about 1000 nm, about 1100 nm, about 1200 nm, about 1300 nm, about 1400 nm, about 1500 nm, about 1600 nm, about 1700 nm, about 1800 nm, about 1900 nm, about 2000 nm, about 2100 nm, about 2200 nm, about 2300 nm, about 2400 nm, about 2500 nm, about 2600 nm, about 2700 nm, about 2800 nm, about 2900 nm, about 3000 nm, about 3100 nm, about 3200 nm, about 3300 nm, about 3400 nm, or about 3500 nm. In some embodiments, the polydispersity index (PDI) of the isolated mitochondrial population is about 0.2 to about 0.8. In some embodiments, the PDI of the isolated mitochondrial population is about 0.2 to about 0.5. In some embodiments, the PDI of the isolated mitochondrial population is about 0.25 to about 0.35. In some embodiments, the zeta potential of the mitochondrial population is about -15 mV to about -40 mV. In some embodiments, the zeta potential of the mitochondrial population is about -20 mV, about -25 mV, about -30 mV, about -35 mV, or about -40 mV.

[0103] In certain embodiments, the population of isolated mitochondria provided herein is capable of incorporating into cells and / or co-localizing with endogenous mitochondria in cells when the population of isolated mitochondria is contacted with a population of cells. For example, in certain embodiments, the present disclosure provides a method of obtaining mitochondria from cells and then contacting a population of cells (e.g., ex vivo or in vivo cells) with the population of isolated mitochondria. In certain such embodiments, the mitochondria provided herein isolated through the iMIT method described herein are capable of co-localizing with endogenous mitochondria present in cells. In certain embodiments, the mitochondria provided herein are further capable of fusing with mitochondria present in cells with which they are contacted. In certain embodiments, a significant proportion of the population of isolated mitochondria are capable of co-localizing and / or fusing with endogenous mitochondria in cells. For example, in certain embodiments, 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 at least about 95% of the mitochondria in the population are capable of co-localizing and / or fusing with endogenous mitochondria in the cell. Thus, the mitochondria provided herein differ significantly from mitochondria isolated through conventional methods in that they are capable of co-localizing and / or fusing with endogenous mitochondria in the cell.

[0104] In certain embodiments, the isolated mitochondria provided herein are stable and / or polarized and / or maintain a membrane potential and / or maintain intact inner and outer membranes and / or maintain the ability to function after exposure to an extracellular environment (e.g., after exposure to a total calcium concentration of about 4 mg / dL to about 12 mg / dL) after storage at about 4° C. For example, in certain embodiments, 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 about 95% of the mitochondria in the population are stable and / or polarized and / or maintain a membrane potential and / or maintain intact inner and outer membranes and / or maintain the ability to function after exposure to an extracellular environment (e.g., after exposure to a total calcium concentration of about 4 mg / dL to about 12 mg / dL) after storage at about 4° C. In certain embodiments, the isolated mitochondria provided herein are stable and / or polarized and / or maintain a membrane potential and / or maintain intact inner and outer membranes and / or maintain the ability to function after exposure to an extracellular environment (e.g., after exposure to a total calcium concentration of about 4 mg / dL to about 12 mg / dL) after storage at or below about −20° C. For example, in certain embodiments, 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 about 95% of the mitochondria in the population are stable and / or polarized and / or maintain a membrane potential and / or maintain intact inner and outer membranes and / or maintain the ability to function after exposure to an extracellular environment (e.g., after exposure to a total calcium concentration of about 4 mg / dL to about 12 mg / dL) after storage at about −20° C. In some embodiments, the isolated mitochondria provided herein are stable and / or polarized and / or maintain a membrane potential and / or maintain intact inner and outer membranes and / or maintain the ability to function after exposure to the extracellular environment (e.g., after exposure to a total calcium concentration of about 4 mg / dL to about 12 mg / dL) after storage at or below about -80°C.For example, in certain embodiments, 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 about 95% of the mitochondria in a population are stable, and / or polarized, and / or maintain a membrane potential, and / or maintain intact inner and outer membranes, and / or maintain the ability to function after exposure to an extracellular environment (e.g., after exposure to a total calcium concentration of about 4 mg / dL to about 12 mg / dL) after storage at about −80° C. In certain embodiments, the isolated mitochondria provided herein are stable, and / or polarized, and / or maintain a membrane potential, and / or maintain intact inner and outer membranes, and / or maintain the ability to function after exposure to an extracellular environment (e.g., after exposure to a total calcium concentration of about 4 mg / dL to about 12 mg / dL) after storage in liquid nitrogen. For example, in certain embodiments, 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 about 95% of the mitochondria in the population are stable after storage in liquid nitrogen, and / or are polarized, and / or maintain a membrane potential, and / or maintain intact inner and outer membranes, and / or maintain the ability to function after exposure to the extracellular environment (e.g., after exposure to a total calcium concentration of about 4 mg / dL to about 12 mg / dL).

[0105] In some embodiments, storage is for at least about 2 hours, at least about 6 hours, at least about 12 hours, at least about 24 hours, 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 differ significantly from mitochondria isolated through traditional methods in that they maintain functional capacity, at least when freshly isolated and even after storage.

[0106] In certain embodiments, the isolated mitochondria provided herein are stable and / or polarized and / or maintain a membrane potential and / or maintain intact inner and outer membranes and / or maintain the ability to function after exposure to the extracellular environment (e.g., after exposure to a total calcium concentration of about 4 mg / dL to about 12 mg / dL) after a population of mitochondria has been frozen for storage and subsequently thawed. In certain embodiments, after freezing and subsequent thawing, the membrane potential is maintained at about 90% of the membrane potential of the mitochondria before freezing. For example, in certain embodiments, the polarization ratio of a frozen and thawed population of mitochondria is about 90% of the polarization ratio of the population before freezing. In certain embodiments, 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 about 95% of the mitochondria in a population are stable and / or polarized and / or maintain a membrane potential and / or maintain intact inner and outer membranes and / or maintain the ability to function after exposure to the extracellular environment (e.g., after exposure to a total calcium concentration of about 4 mg / dL to about 12 mg / dL) after being frozen for storage and subsequently thawed, e.g., after being frozen for storage and subsequently thawed one, two, three, or more times. Thus, in certain embodiments, the isolated mitochondria provided herein differ significantly from mitochondria isolated through conventional methods in that they maintain functional capacity, at least even when frozen for storage and subsequently thawed.

[0107] In certain embodiments, the populations of isolated mitochondria provided herein are capable of incorporating into cells and / or co-localizing and / or fusing with endogenous mitochondria in cells following storage of the mitochondria at any of the temperatures provided herein (e.g., at 4°C ± 3°C, -20°C ± 3°C, -80°C ± 3°C, or in liquid nitrogen). For example, in certain embodiments, 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 about 95% of the mitochondria in the population are capable of incorporating into cells and / or co-localizing and / or fusing with endogenous mitochondria in cells following storage of the mitochondria and / or following one or more freeze-thaw cycles. In certain embodiments, the method for storing and thawing a population of isolated mitochondria provided herein comprises storing the population at about -20°C ± 3°C, about -80°C ± 3°C, or lower (e.g., in liquid nitrogen) and then thawing the mitochondria at about 20°C ± 3°C or lower, wherein the mitochondria thaw within about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, or about 1 minute. In certain embodiments, the population of mitochondria thaws within about 1 minute. Thus, in certain embodiments, the mitochondria provided herein differ significantly from mitochondria isolated through conventional methods in that they are at least capable of incorporating into cells and / or colocalizing and / or fusing with endogenous mitochondria in cells, whereas mitochondria isolated by conventional methods are unable or have a greatly reduced ability to incorporate into cells and / or colocalize and / or fuse with endogenous mitochondria in cells. In certain embodiments, the colocalized isolated mitochondria are capable of forming filamentous, network, and / or reticular structures.

[0108] In some embodiments, the present disclosure provides compositions comprising the isolated mitochondria provided herein, which in some embodiments further comprise one or more pharmaceutically acceptable carriers.

[0109] In some embodiments, the present disclosure provides a method for isolating mitochondria from cells, which, unlike previously known methods, provides mitochondria with superior functionality and other characteristics as provided herein. In some embodiments, the method for isolating mitochondria from cells includes treating cells in a first solution with a detergent at a concentration below the detergent's critical micelle concentration (CMC), removing the detergent to form a second solution, incubating the cells in the second solution, and recovering mitochondria from the second solution. In some embodiments, the concentration of the detergent in the first solution is about 50% or less of the detergent's CMC. For example, in some embodiments, the concentration of the detergent in the first solution is about 40% or less, about 30% or less, about 20% or less, or about 10% or less of the detergent's CMC.

[0110] In some embodiments, the detergent is a non-ionic detergent. In some embodiments, the detergent is selected from the group consisting of Triton-X 100, Triton-X 114, Nonidet P-40, n-dodecyl-D-maltoside, Tween-20, Tween-80, saponin, and digitonin. In some embodiments, the detergent is saponin or digitonin. In some embodiments, the concentration of the detergent is less than about 400 μM. For example, in some embodiments, the concentration of the detergent in the first solution is less than about 300 μM, less than about 200 μM, less than about 100 μM, or less than about 50 μM. In some embodiments, the concentration of the detergent in the first solution is about 100 μM, about 75 μM, about 60 μM, about 50 μM, about 40 μM, about 30 μM, or about 20 μM. In one embodiment, the concentration of the surfactant in the first solution is from about 20 μM to about 50 μM or from about 30 μM to about 40 μM.

[0111] In some embodiments, the first solution further comprises a buffer containing one or more of a tonicity agent, an osmolality adjusting agent, or a chelating agent. In some embodiments, the first solution comprises a Tris buffer, sucrose, and a chelator.

[0112] In some embodiments, treating the cells in a first solution containing a low concentration of detergent (e.g., below the detergent's CMC) involves incubating the cells in the first solution at room temperature for about 2 to about 30 minutes. For example, in some embodiments, treating the cells in the first solution involves incubating the cells in the first solution for about 2, about 5, about 10, about 15, about 20, about 25, or about 30 minutes. Incubation can be carried out at a temperature of about 4°C to about 37°C.

[0113] In some embodiments, the step of removing the detergent comprises reducing the detergent in the solution to less than 10% of the detergent concentration in the first solution, or to less than 1% of the detergent concentration in the first solution, hi some embodiments, the step of removing the detergent comprises washing the cells with a buffer.

[0114] In some embodiments, incubating the second solution comprises incubating the cells in the second solution for about 5 minutes to about 30 minutes. For example, in some embodiments, incubating the cells in the second solution comprises incubating the cells in the second solution for about 5, about 10, about 15, about 20, about 25, or about 30 minutes. In some embodiments, incubating the cells in the second solution is performed at a temperature of about 4°C ± 3°C or on ice.

[0115] In some embodiments, recovering the mitochondria from the second solution comprises collecting the supernatant to recover the isolated mitochondria, hi some embodiments, recovering the mitochondria from the second solution comprises centrifuging the second solution and collecting the supernatant after centrifugation to recover the isolated mitochondria.

[0116] In some embodiments, iMIT can be performed on cells attached to a culture surface. In some embodiments, iMIT can be performed on cells attached to a culture surface without detaching the cells from the surface. In some embodiments, recovering mitochondria from the second solution can include recovering the supernatant to recover the isolated mitochondria, after which the remaining cells on the culture surface can be optionally washed with the second solution or a separate second solution and combined with the supernatant.

[0117] In some embodiments, the methods provided herein further comprise freezing the isolated mitochondria. In some embodiments, the methods comprise freezing the mitochondria in a buffer containing a cryoprotectant (e.g., glycerol). In some embodiments, the methods comprise freezing the mitochondria in a buffer in liquid nitrogen. In some embodiments, the methods further comprise thawing the mitochondria after freezing. In some embodiments, the method of thawing mitochondria comprises thawing the mitochondria rapidly, for example, within about 5 minutes, or within about 1 minute. In some embodiments, the mitochondria are thawed in a water bath having a temperature of about 20°C ± 3°C to about 37°C ± 3°C. In some embodiments, the mitochondria are thawed at a temperature of about 20°C ± 3°C or lower.

[0118] In some embodiments, the present disclosure provides a population of isolated mitochondria obtained by the methods provided herein. In some embodiments, the methods provided herein are "iMIT" methods, and mitochondria obtained by these methods are referred to herein as "Q" mitochondria. In some embodiments, the present disclosure provides compositions and / or formulations comprising a population of isolated mitochondria obtained by the methods provided herein.

[0119] In some embodiments, the present disclosure provides a method for treating or preventing a disease or disorder associated with mitochondrial dysfunction, comprising contacting cells of a subject with a population of isolated mitochondria, e.g., Q mitochondria, provided herein. In some embodiments, the disease or disorder is an ischemia-related disease or disorder. For example, in some embodiments, the ischemia-related disease or disorder is selected from the group consisting of cerebral ischemia-reperfusion, hypoxic-ischemic encephalopathy, acute coronary syndrome, myocardial infarction, hepatic ischemia-reperfusion injury, ischemic injury-compartment syndrome, vascular blockage, 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 some embodiments, the disease or disorder is cancer, cardiovascular disease, eye disorder, ear disorder, autoimmune disease, inflammatory disease, or fibrotic disorder. In some embodiments, the disease is acute respiratory distress syndrome (ARDS). In some embodiments, the disease or disorder is an age-related disease or disorder or an age-related condition. In some embodiments, the disease or disorder is pre-eclampsia or intrauterine growth restriction (IUGR).

[0120] In some embodiments, the present disclosure provides a method of treating or preventing a disease or disorder defined herein, comprising administering to a subject in need thereof a population or composition of isolated mitochondria. In some embodiments, the route of administration of the isolated mitochondria is intravenous, intraarterial, intratracheal, subcutaneous, intramuscular, inhalation, or intrapulmonary. In some embodiments, the subject is a mammal, e.g., a human.

[0121] In some embodiments, the present disclosure provides isolated mitochondria having intact inner and outer membranes, wherein the inner membrane comprises folded cristae, the mitochondria are isolated from cells, the mitochondria are polarized as measured by a fluorescent indicator (e.g., JC-1, TMRM, or TMRE), and the mitochondria are capable of maintaining polarization in an extracellular environment. In some embodiments, the folded cristae are densely folded cristae. In some embodiments, the mitochondria have a substantially non-filamentous shape. In some embodiments, the mitochondria comprise voltage-dependent anion channels (VDACs) associated with tubulin on their surface. For example, in some embodiments, the isolated mitochondria comprise dimeric tubulin associated with VDACs on their surface. In some embodiments, the tubulin comprises at least α-tubulin. In some embodiments, the tubulin is a heterodimer comprising α-tubulin and β-tubulin. In some embodiments, the tubulin is a homodimer. In some embodiments, the isolated mitochondria exhibit reduced association with MAM as measured by GRP75 expression. For example, in certain embodiments, isolated mitochondria exhibit about 70%, about 60%, about 50%, about 40%, about 30% or less association with MAM when compared to mitochondria present in cells (i.e., not isolated) and / or mitochondria obtained by conventional isolation methods, e.g., methods involving homogenization and / or high levels of detergent, as further described herein. In certain embodiments, the isolated mitochondria provided herein exhibit a reduction in association with MAM, wherein the reduction is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or more, compared to the association with MAM of mitochondria present in cells (i.e., not isolated) and mitochondria isolated by conventional isolation methods.

[0122] In some embodiments, the isolated mitochondria provided herein have a membrane potential of about -30 mV to about -220 mV. In some embodiments, the isolated mitochondria are non-filamentous in shape. In some embodiments, the isolated mitochondria have not undergone drp1-dependent fission. In some embodiments, the isolated mitochondria are about 500 nm to 3500 nm in size. For example, in some embodiments, the isolated mitochondria are about 500, about 600, about 700, about 800 nm, about 900 nm, about 1000 nm, about 1100 nm, about 1200 nm, about 1500 nm, about 2000 nm, about 2500 nm, about 3000 nm, or about 3500 nm in size.

[0123] In some embodiments, the present disclosure provides isolated mitochondria obtained by the methods provided herein. In some embodiments, the present disclosure provides compositions and formulations comprising the isolated mitochondria provided herein.

[0124] In a preferred embodiment, mitochondria can be isolated from MITO-cells. MITO-cells are activated cells that are contacted with MITO-Porter (see WO2018 / 092839, which is incorporated herein by reference in its entirety). MITO-Porter contains a mitochondrial activator in a liposome, which optionally provides a mitochondrial targeting signal molecule, such as an alkylated physiologically acceptable polycation, for example, polyarginine or S2 peptide, or a lipid conjugated to a polycation such as polyarginine or S2 peptide (see WO2017 / 090763 and WO2018 / 092839, which are incorporated herein by reference in their entirety). Examples of mitochondrial activators include antioxidants such as resveratrol (3,5,4'-trihydroxy-trans-stilbene), coenzyme Q10, vitamin C, vitamin E, N-acetylcysteine, 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO), superoxide dismutase (SOD), and glutathione, with resveratrol being particularly preferred (see WO2018 / 092839). Resveratrol promotes NAD + It has been reported that resveratrol can activate SIRT1, a member of the sirtuin family, a family of enzymes with NAD-dependent histone deacetylase activity. Resveratrol can promote the transcription of FOXO1 by combing the sirtuin-AMPK-PPAR-PGC-1α complex, resulting in mitochondrial biogenesis. MITO-cells, which are cells treated with MITO-Porter containing mitochondrial activators, have activated respiratory activity and respiratory complex activity due to mitochondrial activators such as antioxidants. Sirtuin-AMPK-PPAR-PGC-1α axis signal-induced mitochondrial biogenesis is thought to be due to the NAD + e from -It has also been reported that MITO-Porter activates cristae fusion and fission to accommodate increased oxidative phosphorylation due to the influx of mitochondrial proteins. Cristae fusion leads to cristae density, resulting in a significant increase in the fluorescence of mitochondrial indicators within the mitochondria. Therefore, theoretically, Q isolated from MITO-cells activated by MITO-Porter (also known as "Super Q") may have a higher cristae density, antioxidants contained in MITO-Porter, such as resveratrol, more nuclear inclusions, and more activated SIRT3-AMPK-PPAR-PGC1α complexes or activated SIRT1-AMPK-PPAR-PGC-1α complexes, as well as more transcripts than Q from untreated cells due to complex activation. Therefore, it is believed that internalized Super Q may exhibit more potent effects than internalized Q after intracellular mitochondrial activation.

[0125] In one embodiment, the present invention provides mitochondria isolated from cells treated with Super Q or MITO-Porter. In one embodiment, Super Q can be isolated by the iMIT method. In one embodiment, Super Q and encapsulated Super Q can contain mitochondrial activators incorporated using MITO-Porter, such as resveratrol. In one embodiment, Super Q and encapsulated Super Q can contain more mitochondrial activators than Q and encapsulated Super Q isolated from untreated cells.

[0126] In one aspect, the present disclosure provides a group of mitochondria that are isolated from cells using the method provided herein, and thus are highly functional.As mentioned above, the novel isolation method provided herein is interchangeably referred to as " DHF " method or " iMIT " method; the mitochondria obtained by DHF or iMIT method are herein referred to as " Q " mitochondria.Q mitochondria avoid the destruction and membrane disruption that occurs when mitochondria are isolated through traditional methods, and therefore are structurally and functionally superior to the mitochondria that are isolated through traditional methods.

[0127] In some embodiments, the present disclosure provides a population of isolated or obtained mitochondria, wherein the population contains a high percentage of polarized mitochondria (i.e., the population has a high polarization ratio). Thus, the population of mitochondria provided herein includes a high percentage of mitochondria with membrane potential. In some embodiments, the present disclosure provides a population of mitochondria, wherein a high percentage of mitochondria in the population have intact inner and outer membranes. In some embodiments, the presence of intact inner and outer membranes can be determined by mitochondrial functional activity, such as membrane potential and polarization.

[0128] The mitochondrial populations provided herein are therefore superior to mitochondrial populations obtained from cells using conventional methods, such as homogenization and / or cell freeze-thawing and / or high-concentration detergent or detergent-containing methods, as described above. For example, mitochondria isolated from cells through conventional methods are inevitably damaged by the isolation process and lose their functional capacity. Thus, the present disclosure provides isolated mitochondrial populations having a higher polarization ratio and / or a higher percentage of polarization, and / or a higher percentage of mitochondria with intact inner and outer membranes than mitochondrial populations obtained by conventional methods.

[0129] In certain embodiments, the polarization ratio of the isolated or obtained population of mitochondria can be, for example, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, or 85% or more.

[0130] In some embodiments, 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 of the isolated or obtained population of mitochondria are polarized as measured by a fluorescent indicator. In some embodiments, the fluorescent indicator may be any fluorescent indicator known to those skilled in the art to be suitable for measuring mitochondrial membrane potential. In some embodiments, the fluorescent indicator is selected from the group consisting of JC-1, TMRM, and TMRE.

[0131] In some embodiments, 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 of a population of isolated or obtained mitochondria have intact inner and outer membranes. In some embodiments, 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 of a population of isolated or obtained mitochondria have densely packed cristae in the inner membrane. For example, in some embodiments, the cristae structure of Q mitochondria resembles that of mitochondria in cells, i.e., not isolated from cells. As used herein, the term "densely packed cristae" means that mitochondria contain cristae present at a high density, i.e., highly folded cristae. Crista density can be examined using microscopy (e.g., transmission electron or light microscopy, including confocal microscopy). In certain embodiments, cristae density in mitochondria can be measured by the number of cristae folds per square micrometer, which can be determined manually by counting the number of folds and / or by automated software programs. In certain embodiments, "dense cristae," "densely folded cristae," and the like refer to 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 cristae (i.e., cristae folds) per square micrometer. Alternatively, or in addition, cristae density in mitochondria can be measured by cristae surface area per mitochondrial volume. Thus, in certain embodiments, "dense cristae," "densely folded cristae," and the like refer to cristae surface area per mitochondrial volume (μm 2 μm -3), means at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, or more. Methods for determining cristae density are known in the art (see, e.g., Segawa et al., "Quantification of cristae architecture reveals time-dependent characteristics of individual mitochondria," Life Science Alliance, Vol. 3, No. 7, June 2020; and Nielsen et al., The Journal of Physiology 595.9 (2017) pp. 2839-47). In certain embodiments, 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 of the mitochondria in a population of mitochondria provided herein have 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 cristae per square micrometer; and / or a cristae surface area (μm) of at least about 20 per mitochondrial volume. 2 μm -3 ), at least about 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 -3or greater. In certain embodiments, the isolated mitochondria provided herein have an average or representative cristae density that is equivalent to and / or not significantly less than the cristae density of mitochondria in the cell type from which the isolated mitochondria were obtained. In certain embodiments, 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 of the mitochondria in a population of mitochondria provided herein exhibit a cristae density that is equivalent to and / or not significantly less than the average or representative cristae density of mitochondria in the cell type from which the isolated mitochondria were obtained.

[0132] In some embodiments, the isolated populations of mitochondria provided herein are enriched in high calcium (Ca 2+ ) environment, maintaining functional competence. In some embodiments, the isolated mitochondrial populations provided herein maintain functional competence in the extracellular environment due to the isolation methods provided herein. In some embodiments, 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 of the isolated or obtained mitochondrial population maintain functional competence in the extracellular environment. In some embodiments, the extracellular environment comprises a total calcium concentration of about 6 mg / dL to about 14 mg / dL, or about 8 mg / dL to about 12 mg / dL. In some embodiments, the extracellular environment comprises a free / active calcium concentration of about 3 mg / dL to about 8 mg / dL, or about 4 mg / dL to about 6 mg / dL. Thus, in one embodiment, the Q mitochondria provided herein have the notable feature of being isolated from the cellular environment with minimal or negligible damage, and retain the ability to function even when exposed to an extracellular environment, e.g., a calcium-rich environment that would otherwise be expected to cause damage to the mitochondria and / or significantly inhibit their functional capacity.

[0133] Without wishing to be bound by theory, in some embodiments, the ability of the isolated or derived mitochondria provided herein to maintain functional capacity in an extracellular environment is due, in part or in whole, to the association of tubulin with voltage-dependent anion channels (VDACs) on the mitochondrial surface. For example, in some embodiments, during the iMIT isolation process provided herein, tubulin can associate with all or a significant number of VDACs on the surface of mitochondria, thereby enabling mitochondria to maintain function even in calcium-rich environments (e.g., extracellular environments containing about 3 mg / dL to about 14 mg / dL of calcium or more). In some embodiments, the association of tubulin with VDACs on the surface of isolated mitochondria can be determined by detecting the presence of tubulin on the surface of mitochondria, e.g., by staining.

[0134] Without wishing to be bound by theory, in some embodiments, the isolated Q mitochondria provided herein are able to maintain functional capacity in an extracellular environment due, in whole or in part, to the loss of cholesterol, ergosterol, and / or related molecules in the Q mitochondrial outer membrane during iMIT isolation. That is, cholesterol (which stabilizes VDAC structure) may be somewhat lost due to small amounts of detergent contacting the mitochondrial membrane during the isolation procedure, resulting in isolated mitochondria with VDAC on their surface that has lost some or all function, thereby rendering the mitochondria resistant to extracellular calcium concentrations (e.g., extracellular environments containing about 3 mg / dL to about 14 mg / dL of calcium or higher). Accordingly, in some embodiments, the isolated mitochondria provided herein contain very low levels of sterol in the mitochondrial membrane.

[0135] In some embodiments, the isolated or obtained population of mitochondria further exhibits reduced association with mitochondria-associated membranes (MAMs) compared to mitochondria in cells and / or mitochondria isolated or obtained using conventional methods, such as methods involving cell homogenization and / or cell freeze-thawing. In some embodiments, reduced MAM association is measured by expression of the glucose-regulated protein GRP75 on the surface of mitochondria.

[0136] In some embodiments, the isolated mitochondria are substantially non-filamentous in shape. "Non-filamentous" can be used interchangeably with "non-network-like," etc., and means that the mitochondria do not exhibit the branched and reticulated network of mitochondria present in cells. In some embodiments, rather than having a filamentous, networked, or branched structure, the mitochondria provided herein appear round, spherical, irregularly shaped, and / or slightly elongated, or any mixture thereof, when viewed under a microscope. At lower magnifications, the isolated mitochondria appear as punctate structures. In contrast, at lower magnifications, the highly elongated, network-like, or branched structure of mitochondria in cells is visible. In some embodiments, 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 of the isolated or obtained population of mitochondria have a long-to-short diameter ratio of 4:1 or less, 3.5:1 or less, or 3:1 or less. Without wishing to be bound by theory, the shape of mitochondria isolated through the methods provided herein results from the gentle removal of motor protein attachment to microtubules while the mitochondria are still in the cell prior to isolation. That is, when mitochondria are no longer attached to cellular microtubules, they lose the highly elongated, branched / networked shape they had in the cell to instead form the non-filamentous shape described herein.

[0137] In some embodiments, 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 about 95% of the isolated mitochondria in the mitochondrial populations provided herein have a length that is less than twice the hydrodynamic diameter of the mitochondria. In some embodiments, the hydrodynamic diameter is about 1 μm, and 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 about 95% of the isolated mitochondria in the mitochondrial populations provided herein have a major axis length of 2 μm or less, 1.9 μm or less, 1.8 μm or less, 1.7 μm or less, 1.6 μm or less, 1.5 μm or less, 1.4 μm or less, or 1.3 μm or less. In some embodiments, the hydrodynamic diameter is measured by dynamic light scattering (DLS). In some embodiments, the hydrodynamic diameter is measured by a median diameter D 50 is.

[0138] In cells, mitochondria are generally highly elongated or filamentous branched structures, as described above. Non-filamentous, non-elongated mitochondria are generally only present in cells undergoing Drp1-mediated division or fission. During this process, interactions with the endoplasmic reticulum cause the initial constriction of the mitochondrion. Drp1 protein is recruited to the mitochondrion and aggregates on its surface, causing further constriction. DYN2 is then recruited to carry out the final step of membrane scission. The resulting mitochondria may be generally spherical in shape. In cells, such spherical mitochondria can retain their spherical shape for a limited period before becoming elongated or forming more typical branch-like structures. In contrast, mitochondria isolated using the iMIT method are non-filamentous in shape without undergoing Drp1-mediated fission. Furthermore, mitochondria isolated by conventional methods, such as those involving cell homogenization, yield mitochondria that are non-filamentous in shape and are largely round or spherical because they are damaged and torn from the microtubules in the cell that otherwise cause them to maintain an elongated shape. In contrast to mitochondria isolated by such methods, mitochondria isolated by the iMIT method provided herein have not undergone deleterious removal from microtubules and have not undergone drp1-mediated fission. Thus, the mitochondria of the present disclosure differ from native mitochondria in cells and from mitochondria isolated by more traditional methods. For example, in certain embodiments, mitochondria provided herein obtained through the iMIT method are substantially non-filamentous in shape, have not undergone drp1 fission, and simultaneously exhibit a highly functional state (e.g., polarized), intact inner and outer membrane structures, including densely folded cristae.

[0139] In some embodiments, the Q mitochondria provided herein, upon contact with a cell or a population of cells, exhibit the unexpected ability to colocalize with endogenous mitochondria in the cell. The Q mitochondria colocalize to a much greater extent with endogenous mitochondria than mitochondria isolated through conventional methods. In some embodiments, the Q mitochondria provided herein, upon contact with a cell or a population of cells, fuse with endogenous mitochondria in the cell. The fusion of isolated Q mitochondria is distinctly different from and more advantageous than mitochondria isolated through conventional methods. In some embodiments, the mitochondria retain this ability even after storage. Thus, in some embodiments, the Q mitochondria provided herein are superior to conventionally isolated mitochondria, at least in that they colocalize and / or fuse more efficiently with endogenous mitochondria in the cell, and therefore exhibit superior clinical efficacy when used to treat any disease or disorder, such as those described herein. This may suggest that the Q mitochondria provided herein have a more robust and largely intact outer membrane than conventionally isolated mitochondria.

[0140] In some embodiments, the present disclosure provides a population of mitochondria isolated or obtained by the methods provided herein. For example, the present disclosure provides a population of mitochondria isolated or obtained by a method comprising steps (A) through (C) of the iMIT method described hereinabove. In some embodiments, the present disclosure provides a population of mitochondria isolated or obtained by a method comprising steps (A) through (E) described hereinabove.

[0141] The present disclosure provides a composition comprising the isolated mitochondrial population of the present invention. The present disclosure provides a mitochondrial preparation comprising the isolated mitochondrial population of the present invention. The composition comprising the isolated mitochondrial population of the present invention may further comprise a buffer. The mitochondrial preparation comprising the isolated mitochondrial population of the present invention is pharmaceutically acceptable and may further comprise additional pharmaceutically acceptable components, such as excipients. The isolated mitochondrial population of the present disclosure, or a composition or mitochondrial preparation containing the same, can be obtained during the separation process without using cell sorting by a flow cytometer, such as fluorescence-activated cell sorting (FACS). Therefore, the isolated mitochondrial population of the present disclosure, or a composition or mitochondrial preparation containing the same, does not contain fluorescent dyes and fluorescent probes (as well as non-fluorescent mitochondrial stains and probes). In some embodiments, the composition is a pharmaceutical composition. All of these functions of Q can be performed by preparing Super Q.

[0142] In one embodiment, detergent treatment can be carried out at temperatures below 10° C., 9° C., 8° C., 7° C., 6° C., 5° C., or 4° C., preferably at about 0° C. to about 4° C., or on ice (as long as the sample is not frozen). In one embodiment, the entire isolation procedure can be carried out at temperatures between about 0° C. and room temperature, preferably below 10° C., 9° C., 8° C., 7° C., 6° C., 5° C., or 4° C., preferably at about 4° C., or on ice.

[0143] The present disclosure provides a population of mitochondria isolated or obtained from cells whose mitochondria have been activated by the methods provided herein. Mitochondrial activation can be achieved by various methods, such as contacting mitochondria with a mitochondrial activator. Such activation of mitochondria can be achieved by various methods, including the MITO-Porter technique. The MITO-Porter technique can use a complex of a mitochondrial-targeted carrier and a mitochondrial activator. In the complex, the mitochondrial-targeted carrier can be covalently or non-covalently linked to the mitochondrial activator, optionally via a linker. In the complex, the mitochondrial-targeted carrier, such as an MTS peptide, polycation, octaarginine, or S2 peptide, can be covalently bound to a lipid or hydrophobic moiety (e.g., a hydrocarbon) displayed on the surface of a lipid membrane-based vesicle, such as a liposome, through hydrophobic interactions. In one embodiment, the mitochondrial-targeted carrier in the form of a vesicle can encapsulate or contain a mitochondrial activator. In one embodiment, activated mitochondria have increased membrane potential and / or increased respiratory activity, which can be assessed by oxygen consumption rate (OCR), compared to untreated mitochondria or mitochondria prior to the mitochondrial activation treatment.

[0144] The present disclosure also provides a population of mitochondria isolated or obtained by the methods provided herein from cells treated with MITO-Porter containing a mitochondrial activator, such as resveratrol. In some embodiments, the present disclosure provides a population of mitochondria containing a mitochondrial activator, such as resveratrol, isolated or obtained from cells by the methods provided herein. The present invention includes a step of introducing a complex of a mitochondrial-targeted carrier and a mitochondrial activator into cells, such as CPC or non-CPC cells. In some embodiments, the cells may not be cardiac cells.

[0145] The present invention involves introducing a complex of a mitochondrial-targeted carrier and a mitochondrial activator into cells, such as CPC or non-CPC cells. In a preferred embodiment, the complex of the mitochondrial-targeted carrier and the mitochondrial activator is a lipid membrane-based vesicle (i.e., a mitochondrial-targeted liposome) that encapsulates or contains the mitochondrial activator.

[0146] Mitochondria-targeted carriers have the function of selectively reaching mitochondria as one of the intracellular organelles when introduced into cells. Examples of mitochondria-targeted carriers include lipid-soluble cationic substances, such as lipophilic triphenylphosphonium cation (TPP) and rhodamine 123; polypeptides, such as mitochondrial targeting sequence (MTS) peptides (Kong, BW. et al., Biochimica et Biophysica Acta 2003, 1625, 98-108) and S2 peptides (Szeto, HH. et al., Pharm. Res. 2011, 28, 2669-2679); and mitochondria-targeted liposomes, such as DQAsomes (Weissig, V. et al., J. Control. Release 2001, 75, 401-408), MITO-Porter (Yamada, Y. et al., Biochim. Biophys. Examples of such carriers include DF-MITO-Porter (Yamada, Y. et al., Mol. Ther. 2011, vol. 19, pp. 1449-1456), and modified DF-MITO-Porter modified with S2 peptide (Kawamura, E. et al., Mitochondrion 2013, vol. 13, pp. 610-614). These documents are incorporated herein by reference with respect to the production and use of carriers in the present invention.

[0147] The preferred mitochondria-targeted carrier in the present invention is a mitochondria-targeted liposome, and in particular, MITO-Porter, DF-MITO-Porter or modified DF-MITO-Porter is preferred.

[0148] A complex of a mitochondrial-targeted carrier and a mitochondrial activator is a substance in which the mitochondrial-targeted carrier and the mitochondrial activator act in a unified manner, regardless of whether chemical bonding, physical encapsulation, etc. are used to form the complex. For example, when a lipophilic cationic lipid or polypeptide is the mitochondrial-targeted carrier, the complex of the mitochondrial-targeted carrier and the mitochondrial activator can be formed by binding the mitochondrial-targeted carrier to the mitochondrial activator using a chemical method such as a covalent bond or an ionic bond, for example, the method of Murphy et al. (GF Kelso et al., J. Biol. Chem., 2001, Vol. 276, pp. 4588-4596) for lipophilic cationic substances or the method for the Szeto peptide described in Japanese Patent Application Laid-Open No. 2007-503461.

[0149] Furthermore, when the mitochondrial-targeted carrier is a liposome, a complex of the mitochondrial-targeted carrier and the mitochondrial activator can be formed by chemically binding the mitochondrial activator to the lipid membrane surface of the liposome, or by physically encapsulating the mitochondrial activator in the liposome, i.e., the internal space blocked by the lipid membrane.

[0150] The complex can be introduced into cells, such as CPC or non-CPC cells, by a method known for introducing a complex into cells for a mitochondrial-targeted carrier. The complex can be introduced into cells, for example, by culturing cells, such as CPC or non-CPC cells, in an appropriate medium containing the complex, or by incubating the complex and cells, such as CPC or non-CPC cells, in the presence of a known substance capable of promoting the uptake of a substance into cells, such as lipofectamine or polyethylene glycol.

[0151] In the first aspect of the present invention, a preferred example of the step of introducing a complex of a mitochondrial-targeted carrier and a mitochondrial activator into cells such as CPC or non-CPC cells is a step of introducing the complex into cells such as CPC or non-CPC cells by incubating cells such as CPC or non-CPC cells with a complex that is a mitochondrial-targeted liposome encapsulating a mitochondrial activator, particularly a complex that is a MITO-Porter or DF-MITO-Porter having a surface modified with an MTS peptide or an S2 peptide and encapsulating a mitochondrial activator.

[0152] The mitochondrial activator is a substance that can activate mitochondrial respiratory chain complexes (electron transport system), particularly a substance that can polarize mitochondria with respect to membrane potential, and particularly preferably a substance that can hyperpolarize mitochondria. Examples of mitochondrial activators include antioxidants such as resveratrol (3,5,4'-trihydroxy-trans-stilbene), coenzyme Q10 (see WO2020 / 203961A, which is fully incorporated herein by reference), vitamin C, vitamin E, N-acetylcysteine, TEMPO, SOD, and glutathione, and particularly preferred is resveratrol.

[0153] Resveratrol preferably used in the present invention may be extracted from plants by known methods, or may be chemically synthesized by known methods, such as the method of Andrus et al. (Tetrahedron Lett. 2003, 44, 4819-4822).

[0154] Cells such as CPC or non-CPC cells produced by the methods according to the invention are a further aspect of the invention and are capable of significantly improving the survival of mice receiving doxorubicin, as shown in the Examples below.

[0155] Administration of doxorubicin, a type of anthracycline-based drug, is clinically known to cause severe myocardial damage, and mice receiving doxorubicin are used as heart failure model mice. Therefore, cells such as CPC or non-CPC cells produced by the method of the present invention can be used for treating and / or preventing myocardial damage, particularly severe myocardial damage, restoring, protecting against, or suppressing deterioration of cardiac function, treating and / or preventing heart failure, etc.

[0156] Another aspect of the present invention relates to a cell population containing cardiac stem cells, in which when the cell population is stained with the fluorescent dye JC-1, the average ratio of the fluorescence intensity of the JC-1 dimer to the fluorescence intensity of the JC-1 monomer (fluorescence intensity of the JC-1 dimer / fluorescence intensity of the JC-1 monomer) is 1 to 4.

[0157] Under the action of the respiratory chain complexes present in mitochondria, mitochondria generate a proton concentration gradient across the membrane, resulting in a polarized membrane potential. When mitochondria are subjected to apoptosis, metabolic stress, etc., the polarized mitochondria change to a depolarized state in which the membrane potential is reduced. Thus, the polarized state of mitochondria is a parameter that indicates the metabolic activity of mitochondria, and cells with a large number of polarized mitochondria are considered to have active mitochondria.

[0158] The fluorescent dye JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide), a mitochondrial membrane potential probe, is known to form a monomer that emits green fluorescence in depolarized mitochondria, but a dimer that emits red fluorescence in polarized mitochondria. Therefore, the ratio of the fluorescence intensities between the JC-1 monomer and the JC-1 dimer is an index of the mitochondrial polarization state. The fluorescence intensity ratio can be measured using commercially available JC-1 from Thermo Fisher Scientific or Cosmo Bio Co., Ltd. by detecting the fluorescence ratio according to the manufacturer's protocol.

[0159] The cell population according to this embodiment is a cell population containing cells such as CPC or non-CPC cells having activated mitochondria, and the degree of mitochondrial activation of the cells such as CPC or non-CPC cells contained in the population can be represented by the average ratio of the fluorescence intensity of the JC-1 dimer to the fluorescence intensity of the JC-1 monomer (fluorescence intensity of the JC-1 dimer / fluorescence intensity of the JC-1 monomer) when the cell population is stained with JC-1.

[0160] The average fluorescence intensity ratio can be determined by measuring the ratio of the fluorescence intensity of the JC-1 dimer to the fluorescence intensity of the JC-1 monomer (fluorescence intensity of JC-1 dimer / fluorescence intensity of JC-1 monomer) for any number of cells, such as CPCs or non-CPCs, contained in a cell population, preferably more than 10 but less than 100 CPCs or non-CPCs, and calculating the average of the measured ratios. The average ratio of the fluorescence intensity of the JC-1 dimer to the fluorescence intensity of the JC-1 monomer in a cell population containing CPCs with activated mitochondria exceeds 1, preferably 1 to 4.

[0161] The cell population according to this aspect is a cell population that consists primarily of cells such as CPC or non-CPC cells. The cell population may generally be produced by the above-described method according to the first aspect of the invention.

[0162] The mitochondria isolated from cells are treated with a mitochondrial activator. Therefore, the mitochondria are preferably isolated from cells treated with MITO-Porter (or lipid membrane-based vesicles or liposomes) that encapsulate or contain the mitochondrial activator.

[0163] In one embodiment, the mitochondrial DNA concentration in isolated or divided mitochondria is 10 per μg of protein. 5 ~10 7The copy number of mitochondrial DNA can be calculated by quantitative PCR using a primer set comprising a forward primer having the sequence of SEQ ID NO: 1 and a reverse primer having the sequence of SEQ ID NO: 2. In one embodiment, isolated or divided mitochondria may contain mitochondrial transcription factor A (TFAM) at a concentration of between about 50 ng / mg of total protein and about 300 ng / mg of total protein (e.g., about 100 ng / mg of total protein and 250 ng / mg of total protein), which can be calculated by ELISA using an anti-TFAM antibody. This calculation can be performed by comparing a standard value or sample of TFAM.

[0164] In one aspect, the present invention provides a method for measuring mitochondrial DNA levels in packaged mitochondria, the method comprising providing isolated mitochondria and measuring the mitochondrial DNA level in the isolated mitochondria. In one embodiment, the method can further comprise measuring the amount of protein in the isolated mitochondria. In one embodiment, the method can further comprise measuring the amount of protein in the isolated mitochondria and calculating the ratio of the mitochondrial DNA level to the amount of protein. In one embodiment, the mitochondrial DNA level can be expressed as the copy number or concentration of mitochondrial DNA. In a preferred embodiment, the isolated mitochondria can be packaged in vesicles, such as lipid membrane-based vesicles. In one embodiment, the amount of protein can be measured by the Bradford method. In one embodiment, the amount of DNA can be measured by quantitative PCR. In a preferred embodiment, the step of measuring the mitochondrial DNA level can comprise amplifying mitochondrial DNA using a primer set comprising a first primer having the nucleotide sequence set forth in SEQ ID NO: 1 and a second primer having the nucleotide sequence set forth in SEQ ID NO: 2. The method can further comprise comparing the measured mitochondrial DNA level to a standard value. The standard value can be a value for functional isolated mitochondria obtained by the iMIT method. The level of mitochondrial DNA in isolated or packaged mitochondria can be an indicator of damage to mitochondria during the isolation process or storage. In one embodiment, if the level is a predetermined value that is 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the standard value, the method can further include predicting that the sample having a level at or above the predetermined value is a pharmaceutically active ingredient for a pharmaceutical composition. In other words, the level can be an indicator of the efficacy, function, or usefulness of the sample as a pharmaceutically active ingredient.In one embodiment, the method can further include selecting samples with levels equal to or greater than a predetermined value and / or discarding samples with levels below the predetermined value. The method can be a method for testing mitochondrial function in a sample containing isolated mitochondria or packaged mitochondria. Thus, the method according to the present invention is useful for predicting damage to mitochondria in a sample, such as an isolated mitochondrial sample, a preserved mitochondrial sample, a packaged mitochondrial sample, a pharmaceutically acceptable mitochondrial preparation, or a pharmaceutically acceptable mitochondrial preparation. The predicting step can include comparing the DNA level or ratio with a predetermined value. The predetermined value is 2 x 10 per μg of protein. 5 Copies to 5 x 10 per μg of protein 6 The lower limit of the given value is 2 x 10 per μg of protein. 5 Copies, 3 × 10 per μg of protein 5 Copies, 4 × 10 per μg of protein 5 Copies, 5 × 10 per μg of protein 5 Copies, 6 × 10 per μg of protein 5 Copies, 7 × 10 per μg of protein 5 Copies, 8 × 10 per μg of protein 5 Copies, 9 × 10 per μg of protein 5 Copies, 1 × 10 per μg of protein 6 Copies, 2 × 10 per μg of protein 6 Copies, 3 × 10 per μg of protein 6 Copies, 4 × 10 per μg of protein 6 copies, or 5 × 10 per μg of protein 6 The upper limit of the specified value is 5 × 10 per μg of protein. 6 Copies, 4 × 10 per μg of protein 6 Copies, 3 × 10 per μg of protein 6 Copies, 2 × 10 per μg of protein 6 copies, or 1 × 10 per μg of protein 6 It may be a copy.

[0165] The present disclosure also provides a population of isolated, obtained, or processed mitochondria that are artificially activated, wherein the mitochondria in the population exhibit superior functional capacity. For example, in one aspect, the present disclosure provides a population of isolated mitochondria, wherein 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 about 95% of the mitochondria in the population have intact inner and outer membranes; and / or 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 about 95% of the mitochondria in the population are polarized as measured by a fluorescent indicator. In some embodiments, the fluorescent indicator is selected from the group consisting of positively charged dyes, such as JC-1, tetramethylrhodamine methyl ester (TMRM), and tetramethylrhodamine ethyl ester (TMRE).

[0166] In some embodiments, the present disclosure provides a population of isolated mitochondria, wherein 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 about 95% of the mitochondria in the population maintain functional capacity (e.g., are polarized) in an extracellular environment. In some embodiments, functional capacity in the extracellular environment is measured by a fluorescent indicator of membrane potential. In some embodiments, the fluorescent indicator is selected from the group consisting of positively charged dyes, such as JC-1, TMRM, and TMRE. In some embodiments, the extracellular environment can comprise a total calcium concentration of about 4 mg / dL to about 12 mg / dL or about 1 mmol / L (1000 μM) to about 3 mmol / L (3000 μM). For example, in some embodiments, the extracellular environment comprises a total calcium concentration of about 8 mg / dL to about 12 mg / dL or about 2 mmol / L (2000 μM) to about 3 mmol / L (3000 μM). In some embodiments, the extracellular environment comprises a free or active calcium concentration of about 4 mg / dL to about 6 mg / dL or about 1 mmol / L (1000 μM) to about 1.5 mmol / L (1500 μM). In some embodiments, the mitochondrial population maintains functional capacity in an environment having a higher calcium concentration compared to the calcium environment in the cell.

[0167] In certain embodiments, provided herein is a population of isolated mitochondria, wherein 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 about 95% of the mitochondria in the population have not undergone dynamin-related protein 1 (drp1)-dependent fission. In certain embodiments, provided herein is a population of isolated mitochondria having an inner membrane and an outer membrane, wherein the inner membrane of the mitochondria comprises densely folded cristae.

[0168] In certain embodiments, populations of isolated mitochondria are provided herein, wherein 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 about 95% of the mitochondria in the population have a substantially non-filamentous, non-branched structure or shape. For example, in certain embodiments, the mitochondria provided herein appear round, punctate, spherical, irregularly shaped, and / or slightly elongated, or any mixture thereof, when viewed under a microscope. In certain embodiments, 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 about 95% of the mitochondria in the population have a ratio of major to minor diameters of 4:1 or less, 3.5:1 or less, or 3:1 or less. In some embodiments, 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 about 95% of the isolated mitochondria in a population of mitochondria provided herein have a length that is shorter than two or three times the hydrodynamic diameter of the mitochondria. Thus, the isolated mitochondria provided herein have a significantly different shape (non-filamentous) compared to the shape of most mitochondria present in cells (filamentous). Accordingly, in some embodiments, the population of mitochondria provided herein has a shape that differs from unisolated mitochondria present in cells in that 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 about 95% of the mitochondria in the population are non-filamentous in shape. In some embodiments, the population of isolated mitochondria provided herein exhibits reduced association with the mitochondria-associated membrane (MAM). In one embodiment, association with MAM is measured by expression of glucose-regulated protein 75 (GRP75).In certain embodiments, the populations of isolated mitochondria provided herein exhibit about 60%, at least about 65%, at least about 70%, about 60%, about 50%, about 40%, about 30% or less association with MAM when compared to mitochondria in cells and / or mitochondria obtained by conventional isolation methods, e.g., methods involving homogenization and / or high levels of detergent, as further described herein. In certain embodiments, the populations of isolated mitochondria provided herein exhibit a decrease in association with MAM, which decrease is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or more when compared to the association with MAM of mitochondria in cells or mitochondria isolated by conventional isolation methods.

[0169] In some embodiments, a population of isolated mitochondria provided herein is between about 500 nm and about 3500 nm in size, hi some embodiments, 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% of the mitochondria in the population are between about 500 nm and about 3500 nm in size. In some embodiments, the average size of mitochondria in the population is about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, about 1000 nm, about 1100 nm, about 1200 nm, about 1300 nm, about 1400 nm, about 1500 nm, about 1600 nm, about 1700 nm, about 1800 nm, about 1900 nm, about 2000 nm, about 2100 nm, about 2200 nm, about 2300 nm, about 2400 nm, about 2500 nm, about 2600 nm, about 2700 nm, about 2800 nm, about 2900 nm, about 3000 nm, about 3100 nm, about 3200 nm, about 3300 nm, about 3400 nm, or about 3500 nm. In some embodiments, the polydispersity index (PDI) of the isolated mitochondrial population is about 0.2 to about 0.8. In some embodiments, the PDI of the isolated mitochondrial population is about 0.2 to about 0.5. In some embodiments, the PDI of the isolated mitochondrial population is about 0.25 to about 0.35. In some embodiments, the PDI is about 0 to 0.8, preferably about 0 to 0.5, and more preferably about 0 to 0.35. In some embodiments, the zeta potential of the mitochondrial population is about -15 mV to about -40 mV. In some embodiments, the zeta potential of the mitochondrial population is about -20 mV, about -25 mV, about -30 mV, about -35 mV, or about -40 mV.

[0170] In certain embodiments, the population of isolated mitochondria provided herein is capable of incorporating into cells and / or coexisting with endogenous mitochondria in cells when the population of isolated mitochondria is contacted with a population of cells. For example, in certain embodiments, the present disclosure provides a method of obtaining mitochondria from cells and then contacting a population of cells (e.g., ex vivo or in vivo cells) with the population of isolated mitochondria. In such embodiments, the mitochondria provided herein isolated via the iMIT method described herein are capable of coexisting with endogenous mitochondria present in the cells. In certain embodiments, the mitochondria provided herein are further capable of fusing with mitochondria present in the cells with which they are contacted. In certain embodiments, a significant proportion of the population of isolated mitochondria are capable of coexisting and / or fusing with endogenous mitochondria in the cells. For example, in certain embodiments, 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 at least about 95% of the mitochondria in the population are capable of coexisting and / or fusing with endogenous mitochondria in the cells. Thus, the mitochondria provided herein differ significantly from mitochondria isolated through conventional methods in that they are capable of coexisting with and / or fusing with endogenous mitochondria in cells.

[0171] In certain embodiments, the isolated mitochondria provided herein are stable and / or polarized and / or maintain a membrane potential and / or maintain intact inner and outer membranes after storage at about 4° C., and / or maintain the ability to function after exposure to an extracellular environment (e.g., after exposure to a total calcium concentration of about 4 mg / dL to about 12 mg / dL). For example, in certain embodiments, 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 about 95% of the mitochondria in the population are stable and / or polarized and / or maintain a membrane potential and / or maintain intact inner and outer membranes after storage at about 4° C., and / or maintain the ability to function after exposure to an extracellular environment (e.g., after exposure to a total calcium concentration of about 4 mg / dL to about 12 mg / dL). In certain embodiments, the isolated mitochondria provided herein are stable and / or polarized and / or maintain a membrane potential and / or maintain intact inner and outer membranes after storage at or below about −20° C., and / or maintain the ability to function after exposure to an extracellular environment (e.g., after exposure to a total calcium concentration of about 4 mg / dL to about 12 mg / dL). For example, in certain embodiments, 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 about 95% of the mitochondria in the population are stable and / or polarized and / or maintain a membrane potential and / or maintain intact inner and outer membranes after storage at about −20° C., and / or maintain the ability to function after exposure to an extracellular environment (e.g., after exposure to a total calcium concentration of about 4 mg / dL to about 12 mg / dL). In some embodiments, the isolated mitochondria provided herein are stable and / or polarized after storage at or below about -80°C, and / or maintain a membrane potential, and / or maintain intact inner and outer membranes, and / or maintain the ability to function after exposure to the extracellular environment (e.g., after exposure to a total calcium concentration of about 4 mg / dL to about 12 mg / dL).For example, in certain embodiments, 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 about 95% of the mitochondria in a population are stable and / or polarized and / or maintain a membrane potential and / or maintain intact inner and outer membranes and / or maintain the ability to function after exposure to an extracellular environment (e.g., after exposure to a total calcium concentration of about 4 mg / dL to about 12 mg / dL) after storage at about −80° C. In certain embodiments, the isolated mitochondria provided herein are stable and / or polarized and / or maintain a membrane potential and / or maintain intact inner and outer membranes and / or maintain the ability to function after exposure to an extracellular environment (e.g., after exposure to a total calcium concentration of about 4 mg / dL to about 12 mg / dL) after storage in liquid nitrogen. For example, in some embodiments, 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 about 95% of the mitochondria in the population are stable and / or polarized and / or maintain a membrane potential and / or maintain intact inner and outer membranes and / or maintain the ability to function after exposure to the extracellular environment (e.g., after exposure to a total calcium concentration of about 4 mg / dL to about 12 mg / dL) after storage in liquid nitrogen. In some embodiments, storage is for at least about 2 hours, at least about 6 hours, at least about 12 hours, at least about 24 hours, 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 certain embodiments, the isolated mitochondria provided herein differ significantly from mitochondria isolated through conventional methods in that they retain at least some functional capacity when freshly isolated and even after storage.

[0172] In certain embodiments, the isolated mitochondria provided herein are stable and / or polarized and / or maintain a membrane potential and / or maintain intact inner and outer membranes and / or maintain the ability to function after exposure to the extracellular environment (e.g., after exposure to a total calcium concentration of about 4 mg / dL to about 12 mg / dL) after the population of mitochondria has been frozen for storage and subsequently thawed. In certain embodiments, after freezing and subsequent thawing, the membrane potential is maintained at about 90% of the membrane potential of the mitochondria before freezing. For example, in certain embodiments, the polarization ratio of the frozen and thawed population of mitochondria is about 90% of the polarization ratio of the population before freezing. In certain embodiments, 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 about 95% of the mitochondria in the population are stable and / or polarized and / or maintain a membrane potential and / or maintain intact inner and outer membranes and / or maintain the ability to function after exposure to the extracellular environment (e.g., after exposure to a total calcium concentration of about 4 mg / dL to about 12 mg / dL) after being frozen for storage and subsequently thawed, e.g., after being frozen for storage and subsequently thawed one, two, three, or more times. Thus, in certain embodiments, the isolated mitochondria provided herein differ significantly from mitochondria isolated through conventional methods in that they maintain functional capacity, at least even when frozen for storage and subsequently thawed.

[0173] In certain embodiments, the populations of isolated mitochondria provided herein are capable of incorporating into cells and / or coexisting and / or fusing with endogenous mitochondria in cells following storage of the mitochondria at any of the temperatures provided herein (e.g., 4°C ± 3°C, -20°C ± 3°C, -80°C ± 3°C, or in liquid nitrogen). For example, in certain embodiments, 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 about 95% of the mitochondria in the population are capable of incorporating into cells and / or coexisting and / or fusing with endogenous mitochondria in cells following storage of the mitochondria and / or following one or more freeze-thaw cycles. In certain embodiments, the method for storing and thawing a population of isolated mitochondria provided herein comprises storing the population at about -20°C ± 3°C, about -80°C ± 3°C, or lower (e.g., in liquid nitrogen) and then thawing the mitochondria at about 20°C ± 3°C or lower, wherein the mitochondria thaw within about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, or about 1 minute. In certain embodiments, the population of mitochondria thaws within about 1 minute. Thus, in certain embodiments, the mitochondria provided herein differ significantly from mitochondria isolated through conventional methods in that they are at least capable of incorporating into cells and / or coexisting and / or fusing with endogenous mitochondria in cells, whereas mitochondria isolated by conventional methods are unable or have a greatly reduced ability to incorporate into cells and / or coexisting and / or fusing with endogenous mitochondria in cells. In certain embodiments, the coexisting isolated mitochondria can form thread-like, network, and / or reticular structures.

[0174] In some embodiments, the present disclosure provides compositions comprising the isolated mitochondria provided herein, which in some embodiments further comprise one or more pharmaceutically acceptable carriers.

[0175] In some embodiments, the present disclosure provides a method for isolating mitochondria from cells that differs from previously known methods and provides mitochondria with superior functionality and other characteristics. In some embodiments, the method for isolating mitochondria from cells includes treating cells in a first solution with a detergent at a concentration below the detergent's critical micelle concentration (CMC), removing the detergent to form a second solution, incubating the cells in the second solution, and recovering mitochondria from the second solution. In some embodiments, the concentration of the detergent in the first solution is about 50% or less of the detergent's CMC. For example, in some embodiments, the concentration of the detergent in the first solution is about 40% or less, about 30% or less, about 20% or less, or about 10% or less of the detergent's CMC.

[0176] In some embodiments, the detergent is a non-ionic detergent, hi some embodiments, the detergent is selected from the group consisting of Triton-X 100, Triton-X 114, Nonidet P-40, n-dodecyl-D-maltoside, Tween-20, Tween-80, saponin, and digitonin.

[0177] In some embodiments, the surfactant is saponin or digitonin. In some embodiments, the concentration of the surfactant is less than about 400 μM. For example, in some embodiments, the concentration of the surfactant in the first solution is less than about 300 μM, less than about 200 μM, less than about 100 μM, or less than about 50 μM. In some embodiments, the concentration of the surfactant in the first solution is about 100 μM, about 75 μM, about 60 μM, about 50 μM, about 40 μM, about 30 μM, or about 20 μM. In some embodiments, the concentration of the surfactant in the first solution is about 20 μM to about 50 μM, or about 30 μM to about 40 μM.

[0178] In some embodiments, the first solution further comprises a buffer containing one or more of a tonicity agent, an osmolality adjusting agent, or a chelating agent. In some embodiments, the first solution comprises a Tris buffer, sucrose, and a chelator.

[0179] In some embodiments, treating the cells in a first solution containing a low concentration of detergent (e.g., below the detergent's CMC) comprises incubating the cells in the first solution at room temperature for about 2 to about 30 minutes. For example, in some embodiments, treating the cells in the first solution comprises incubating the cells in the first solution for about 2, about 5, about 10, about 15, about 20, about 25, or about 30 minutes. Incubation can be carried out at a temperature of about 4°C to about 37°C.

[0180] In some embodiments, the step of removing the detergent comprises reducing the detergent in the solution to less than 10% of the detergent concentration in the first solution, or to less than 1% of the detergent concentration in the first solution, hi some embodiments, the step of removing the detergent comprises washing the cells with a buffer.

[0181] In some embodiments, incubating the cells in the second solution comprises incubating the cells in the second solution for about 5 minutes to about 30 minutes. For example, in some embodiments, incubating the cells in the second solution comprises incubating the cells in the second solution for about 5, about 10, about 15, about 20, about 25, or about 30 minutes. In some embodiments, incubating the cells in the second solution is performed at a temperature of about 4°C ± 3°C or on ice.

[0182] In some embodiments, recovering the mitochondria from the second solution comprises collecting the supernatant to recover the isolated mitochondria, hi some embodiments, recovering the mitochondria from the second solution comprises centrifuging the second solution and collecting the supernatant after centrifugation to recover the isolated mitochondria.

[0183] In some embodiments, iMIT can be performed on cells attached to a culture surface. In some embodiments, iMIT can be performed on cells attached to a culture surface without detaching the cells from the surface. In some embodiments, recovering mitochondria from the second solution includes collecting the supernatant to recover the isolated mitochondria, after which the remaining cells on the culture surface can be optionally washed with the second solution or a separate second solution and combined with the supernatant.

[0184] In some embodiments, the methods provided herein further comprise freezing the isolated mitochondria. In some embodiments, the methods comprise freezing the mitochondria in a buffer containing a cryoprotectant (e.g., glycerol). In some embodiments, the methods comprise freezing the mitochondria in a buffer in liquid nitrogen. In some embodiments, the methods further comprise thawing the mitochondria after freezing. In some embodiments, the method of thawing mitochondria comprises thawing the mitochondria rapidly, e.g., within about 5 minutes, or within about 1 minute. In some embodiments, the mitochondria are thawed in a water bath having a temperature of about 20°C ± 3°C to about 37°C ± 3°C. In some embodiments, the mitochondria are thawed at a temperature of about 20°C ± 3°C or lower.

[0185] In some embodiments, the present disclosure provides a population of isolated mitochondria obtained by the methods provided herein. In some embodiments, the methods provided herein are "iMIT" methods, and mitochondria obtained by these methods are referred to herein as "Q" mitochondria. In some embodiments, the present disclosure provides compositions and / or formulations comprising a population of isolated mitochondria obtained by the methods provided herein.

[0186] In some embodiments, the present disclosure provides a method for treating or preventing a disease or disorder associated with mitochondrial dysfunction, comprising contacting a subject's cells with a population of isolated mitochondria, e.g., Q mitochondria, provided herein. In some embodiments, the disease or disorder is an ischemia-related disease or disorder. For example, in some embodiments, the ischemia-related disease or disorder is selected from the group consisting of cerebral ischemia-reperfusion, hypoxic-ischemic encephalopathy, acute coronary syndrome, myocardial infarction, hepatic ischemia-reperfusion injury, ischemic injury-compartment syndrome, vascular blockage, 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 some embodiments, the disease or disorder is cancer, cardiovascular disease, eye disorder, ear disorder, autoimmune disease, inflammatory disease, or fibrotic disorder. In some embodiments, the disease is acute respiratory distress syndrome (ARDS). In some embodiments, the disease or disorder is an age-related disease or disorder or an age-related condition. In some embodiments, the disease or disorder is pre-eclampsia or intrauterine growth restriction (IUGR).

[0187] In some embodiments, the present disclosure provides a method of treating or preventing a disease or disorder defined herein, comprising administering to a subject in need thereof a population or composition of isolated mitochondria. In some embodiments, the route of administration of the isolated mitochondria is intravenous, intraarterial, intratracheal, subcutaneous, intramuscular, inhalation, or intrapulmonary. In some embodiments, the subject is a mammal, e.g., a human.

[0188] In some embodiments, the present disclosure provides isolated mitochondria having intact inner and outer membranes, wherein the inner membrane comprises folded cristae, the mitochondria are isolated from cells, the mitochondria are polarized as measured by a fluorescent indicator (e.g., JC-1, TMRM, or TMRE), and the mitochondria are capable of maintaining polarization in an extracellular environment. In some embodiments, the folded cristae are densely folded cristae. In some embodiments, the mitochondria have a substantially non-filamentous shape. In some embodiments, the mitochondria comprise voltage-dependent anion channels (VDACs) on their surface associated with tubulin. For example, in some embodiments, the isolated mitochondria comprise dimeric tubulin associated with VDACs on their surface. In some embodiments, the tubulin comprises at least α-tubulin.

[0189] In some embodiments, the tubulin is a heterodimer comprising α-tubulin and β-tubulin. In some embodiments, the tubulin is a homodimer. In some embodiments, the isolated mitochondria exhibit reduced association with MAM as measured by GRP75 expression. For example, in some embodiments, the isolated mitochondria exhibit about 70%, about 60%, about 50%, about 40%, about 30% or less association with MAM when compared to mitochondria present in cells (i.e., not isolated) and / or mitochondria obtained by conventional isolation methods, e.g., methods involving homogenization and / or high levels of detergent, as further described herein. In some embodiments, the isolated mitochondria provided herein exhibit reduced association with MAM, which reduction is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or more when compared to the association with MAM of mitochondria present in cells (i.e., not isolated) and mitochondria isolated by conventional isolation methods.

[0190] In some embodiments, the isolated mitochondria provided herein have a membrane potential of about -30 mV to about -220 mV. In some embodiments, the isolated mitochondria are non-filamentous in shape. In some embodiments, the isolated mitochondria have not undergone drp1-dependent division. In some embodiments, the isolated mitochondria are about 500 nm to 3500 nm in size. For example, in some embodiments, the isolated mitochondria are about 500, about 600, about 700, about 800 nm, about 900 nm, about 1000 nm, about 1100 nm, about 1200 nm, about 1500 nm, about 2000 nm, about 2500 nm, about 3000 nm, or about 3500 nm in size.

[0191] In some embodiments, the present disclosure provides isolated mitochondria obtained by the methods provided herein. In some embodiments, the present disclosure provides compositions and formulations comprising the isolated mitochondria provided herein.

[0192] The present disclosure may also provide inventions as described below. Item 1. A population of isolated mitochondria: (i) at least 80% of the mitochondria in the population have intact inner and outer membranes; (ii) at least 80% of the mitochondria in the population are polarized as measured by a fluorescent indicator; and / or (iii) At least 80% of the mitochondria in the population maintain functional capacity in the extracellular environment. In a preferred embodiment, the mitochondria are activated. In a more preferred embodiment, the mitochondria are activated with a mitochondrial activator, such as resveratrol. In a more preferred embodiment, such mitochondria can be obtained from cells treated with a lipid membrane-based vesicle (e.g., liposome) containing or encapsulating a mitochondrial activator, such as resveratrol. Item 2. 3. The population of isolated mitochondria according to item 1, wherein (iii) the functional capacity in the extracellular environment is measured by a fluorescent indicator of membrane potential. Item 3. (iii) The isolated population of mitochondria according to item 1, wherein the extracellular environment comprises a total calcium concentration of about 8 to about 12 mg / dL. Item 4. (iii) The isolated population of mitochondria according to item 1, wherein the extracellular environment comprises a free / active calcium concentration of about 4 to about 6 mg / dL. Item 5. 2. The isolated population of mitochondria of item 1, wherein at least 80% of the mitochondria in the population have not undergone dynamin-related protein 1 (drp1)-dependent division. Item 6. 2. The isolated population of mitochondria of item 1, wherein the inner membrane of the mitochondria comprises densely folded cristae. Item 7. 7. The population of isolated mitochondria according to any one of items 1 to 6, wherein at least 80% of the mitochondria in the population have a non-filamentous shape. Item 8. 8. The population of isolated mitochondria of item 7, wherein at least 85% of the mitochondria have a non-filamentous shape. Item 9. 9. The population of isolated mitochondria of item 8, wherein at least 90% of the mitochondria have a non-filamentous shape. Item 10. 10. The population of isolated mitochondria of any one of paragraphs 1 to 9, wherein the mitochondria exhibit reduced association with mitochondrial-associated membranes (MAMs) as measured by expression of glucose-regulated protein 75 (GRP75). Item 11. 11. The population of isolated mitochondria of item 10, wherein the reduction in association is at least about 30% compared to the association with MAM of mitochondria in cells or isolated mitochondria obtained by a method comprising cell homogenization. Item 12. 12. The population of isolated mitochondria of item 11, wherein the reduction in association is at least about a 50% reduction. Item 13. (i) at least 85% of the mitochondria in the population have intact inner and outer membranes; (ii) at least 85% of the mitochondria in the population are polarized as measured by a fluorescent indicator; and / or (iii) at least 85% of the mitochondria in said population maintain functional capacity in the extracellular environment; 13. The population of isolated mitochondria according to any one of items 1 to 12. Item 14. (i) at least 90% of the mitochondria in the population have intact inner and outer membranes; (ii) at least 90% of the mitochondria in the population are polarized as measured by a fluorescent indicator; and / or (iii) at least 90% of the mitochondria in said population maintain functional capacity in the extracellular environment; 13. The population of isolated mitochondria according to any one of items 1 to 12. Item 15. 15. The population of isolated mitochondria according to any one of items 1 to 14, wherein the fluorescent indicator is selected from the group consisting of JC-1, tetramethylrhodamine methyl ester (TMRM), and tetramethylrhodamine ethyl ester (TMRE). Item 16. 16. The population of isolated mitochondria according to any one of items 1 to 15, wherein at least 80% of the mitochondria in the population are between about 500 nm and about 3500 nm in size. Item 17. 17. The population of isolated mitochondria according to any one of items 1 to 16, wherein the polydispersity index (PDI) of the population is from about 0.2 to about 0.8. Item 18. 17. The population of isolated mitochondria according to any one of items 1 to 16, wherein the polydispersity index (PDI) of the population is about 0.2 to about 0.3. Item 19. 19. The isolated population of mitochondria according to any one of items 1 to 18, wherein the zeta potential of the population of mitochondria is about −15 mV to about −40 mV. Item 20. 20. The population of isolated mitochondria of any one of items 1 to 19, wherein when the population of isolated mitochondria is contacted with a population of cells, the isolated mitochondria are capable of co-localizing with endogenous mitochondria in the cells. Item 21. 20. The population of isolated mitochondria of any one of items 1 to 19, wherein when the population of isolated mitochondria is contacted with a population of cells, the mitochondria are capable of fusing with endogenous mitochondria in the cells. Item 22. 21. The population of isolated mitochondria of item 20, wherein the mitochondria are capable of co-localizing with the endogenous mitochondria after storage at 4° C. for at least 12 hours. Item 23. 22. The isolated population of mitochondria of item 21, wherein the mitochondria are capable of fusing with the endogenous mitochondria after storage at 4°C for at least 12 hours. Item 24. 24. The population of isolated mitochondria of any one of items 1 to 23, wherein at least 70% of the isolated mitochondria in the population are polarized as measured by a fluorescent indicator after the population has been subjected to one or more freeze-thaw cycles. Item 25. 25. The population of isolated mitochondria of any one of paragraphs 1 to 24, wherein the mitochondria are capable of co-localizing with endogenous mitochondria after the population is subjected to one or more freeze-thaw cycles. Item 26. 26. The population of isolated mitochondria of item 24 or 25, wherein the population is frozen at -80°C or below for at least 2 weeks and then thawed at 20°C or below within about 5 minutes. Item 27. 27. The population of isolated mitochondria of item 26, wherein the population is thawed within about 1 minute. Item 28. 28. The population of isolated mitochondria of item 26 or 27, wherein the population is frozen in liquid nitrogen for at least two weeks. Item 29. 29. The population of isolated mitochondria of item 28, wherein the population is frozen in liquid nitrogen for at least two months. Item 30. 30. The population of isolated mitochondria of any one of items 24 to 29, wherein when the population of thawed mitochondria is contacted with a population of cells, isolated mitochondria in the population are capable of fusing with endogenous mitochondria in the cells. Item 31. A composition comprising the population of isolated mitochondria according to any one of items 1 to 30. Item 32. 32. A formulation comprising the composition of item 31 and a pharmaceutically acceptable carrier. Item 33. 1. A method for isolating mitochondria from cells, comprising: (i) treating the cells in the first solution with a detergent at a concentration below the critical micelle concentration for the detergent; (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. Including, Here, the method comprises contacting the cells with lipid membrane-based vesicles containing or encapsulating a mitochondrial activator, for example, to provide the cells with activated mitochondria. Item 34. 34. The method of claim 33, wherein the concentration of the surfactant in the first solution is less than or equal to about 50% of the critical micelle concentration of the surfactant. Item 35. 35. The method of claim 33 or 34, wherein the concentration of the surfactant in the first solution is less than or equal to about 10% of the critical micelle concentration of the surfactant. Item 36. 36. The method according to any one of items 33 to 35, wherein the surfactant is a nonionic surfactant. Item 37. 37. The method according to any one of items 33 to 36, wherein the surfactant is selected from the group consisting of Triton-X 100, Triton-X 114, Nonidet P-40, n-dodecyl-D-maltoside, Tween-20, Tween-80, saponin and digitonin. Item 38. 38. The method of claim 37, wherein the surfactant is saponin or digitonin, and the concentration of the surfactant in the first solution is less than about 400 μM. Item 39. 38. The method of claim 37, wherein the surfactant is saponin or digitonin, and the concentration of the surfactant in the first solution is less than about 50 μM. Item 40. Item 38. The method according to Item 37, wherein the surfactant is saponin or digitonin, and the concentration of saponin or digitonin in the first solution is about 30 μM to about 40 μM. Item 41. 41. The method of any one of items 33 to 40, wherein the first solution further comprises a buffer containing one or more of a tonicity agent, an osmolality adjusting agent, or a chelating agent. Item 42. 42. The method of claim 41, wherein the first solution comprises a Tris buffer, sucrose, and a chelator. Item 43. 43. The method according to any one of items 33 to 42, wherein treating the cells in the first solution comprises incubating the cells in the first solution at room temperature for about 2 minutes to about 30 minutes. Item 44. 44. The method of any one of items 33 to 43, wherein removing the surfactant comprises reducing the concentration of the surfactant in the solution to less than 10% of the concentration of the surfactant in the first solution. Item 45. 45. The method of any one of items 33 to 44, wherein removing the surfactant comprises reducing the concentration of the surfactant in the solution to less than 1% of the concentration of the surfactant in the first solution. Item 46. 46. ​​The method of any one of items 33 to 45, wherein removing the detergent comprises washing the cells with a buffer. Item 47. 47. The method of any one of items 33 to 46, wherein incubating the second solution comprises incubating the cells in the second solution at about 4°C for about 5 minutes to about 30 minutes. Item 48. 48. The method of any one of items 33 to 47, wherein recovering the mitochondria from the second solution comprises recovering the supernatant to recover isolated mitochondria. Item 49. 49. The method of any one of items 33 to 48, wherein recovering the mitochondria from the second solution comprises centrifuging the second solution and recovering the supernatant after centrifugation to recover isolated mitochondria. Item 50. 50. The method of any one of items 33 to 49, further comprising freezing the isolated mitochondria. Item 51. 51. The method of claim 50, comprising freezing the isolated mitochondria in a buffer containing a cryoprotectant. Item 52. A population of isolated mitochondria obtained by the method according to any one of items 33 to 51. Item 53. A method of treating a disease or disorder, comprising contacting cells of a subject in need thereof with the isolated population of mitochondria described in any one of items 1 to 30, or the composition of item 31, or the formulation of item 32, wherein the disease or disorder is selected from the group consisting of diabetes (types I and II), metabolic diseases, eye disorders associated with mitochondrial dysfunction, hearing loss, mitochondrial toxicity associated with therapeutic agents, cardiotoxicity associated with chemotherapy or other therapeutic agents, mitochondrial dysfunction disorders, and migraine. Item 54. 32. A method for treating a disease or disorder associated with mitochondrial dysfunction, comprising contacting cells of a subject in need thereof with the population of isolated mitochondria of any one of items 1 to 30, or the composition of item 31, or the formulation of item 32. Item 55. 55. The method of item 54, wherein the disease or disorder is selected from the group consisting of mitochondrial myopathy, diabetes and deafness (DAD) syndrome, Barth syndrome, Leber's hereditary ophthalmo-neuropathy (LHON), Leigh syndrome, NARP (neuropathy, ataxia, retinitis pigmentosa and ptosis syndrome), myoneurologic gastrointestinal encephalopathy (MNGIE), MELAS (mitochondrial encephalopathy, lactic acidosis and stroke-like episodes) syndrome, myoclonic epilepsy with rough red fibers (MERRF) syndrome, Kearns-Sayre syndrome and mitochondrial DNA wasting syndrome. Item 56. 55. The method of item 54, wherein the disease or disorder is an ischemia-related disease or disorder. Item 57. 57. The method of item 56, wherein the ischemia-related disease or disorder is selected from the group consisting of cerebral ischemia-reperfusion, hypoxic-ischemic encephalopathy, acute coronary syndrome, myocardial infarction, hepatic ischemia-reperfusion injury, ischemic injury-compartment syndrome, vascular blockage, wound healing, spinal cord injury, sickle cell disease, and reperfusion injury of transplanted organs. Item 58. 55. The method of item 54, wherein the disease or disorder is a genetic disorder. Item 59. 55. The method of item 54, wherein the disease or disorder is an age-related disease or disorder. Item 60. 55. The method of item 54, wherein the disease or disorder is a neurodegenerative or cardiovascular condition. Item 61. 61. The method of item 60, wherein the neurodegenerative condition is selected from the group consisting of dementia, Friedreich's ataxia, amyotrophic lateral sclerosis, mitochondrial myopathy, encephalopathy, lactic acidosis, stroke (MELAS), myoclonic epilepsy with ragged-red fibers (MERFF), epilepsy, Parkinson's disease, Alzheimer's disease, or Huntington's disease. Exemplary neuropsychiatric disorders include bipolar disorder, schizophrenia, depression, addictive disorders, anxiety disorders, attention deficit disorder, personality disorders, autism, and Asperger's disease. Item 62. 61. The method of item 60, wherein the cardiovascular condition is selected from the group consisting of coronary heart disease, myocardial infarction, atherosclerosis, hypertension, cardiac arrest, cerebrovascular disease, peripheral artery disease, rheumatic heart disease, congenital heart disease, congestive heart failure, arrhythmia, stroke, deep vein thrombosis, and pulmonary embolism. Item 63. 55. The method of item 54, wherein the disease or disorder is cancer, an autoimmune disease, an inflammatory disease, or a fibrotic disorder. Item 64. 55. The method of item 54, wherein the disease is acute respiratory distress syndrome (ARDS). Item 65. 55. The method of item 54, wherein the disease or disorder is preeclampsia or intrauterine growth restriction (IUGR). Item 66. 66. The method of any one of items 54 to 65, comprising administering the population of isolated mitochondria or the composition to the subject via an intravenous, intra-arterial, intratracheal, subcutaneous, intramuscular, inhalation or intrapulmonary administration route. Item 67. 1. An isolated mitochondrion having an intact inner and outer membrane, the inner membrane comprising folded cristae, the mitochondrion being isolated from a cell, the mitochondrion being polarized as measured by a fluorescent indicator, and the mitochondrion being capable of maintaining polarization in an extracellular environment. Item 68. 68. The isolated mitochondria according to item 67, which have a non-filamentous shape. Item 69. 69. The isolated mitochondrion of item 67 or 68, wherein the voltage-dependent anion channel (VDAC) on the surface of the mitochondrion is associated with tubulin on its surface. Item 70. 68. The isolated mitochondria of item 67, wherein the tubulin is a dimeric tubulin. Item 71. 71. The isolated mitochondrion of item 70, wherein the tubulin is a heterodimer comprising α-tubulin and β-tubulin. Item 72. 72. The isolated mitochondria according to any one of items 67 to 71, wherein the fluorescent indicator is selected from the group consisting of JC-1, tetramethylrhodamine methyl ester (TMRM), and tetramethylrhodamine ethyl ester (TMRE). Item 73. 73. The isolated mitochondria of any one of paragraphs 67 to 72, which exhibits reduced association with mitochondria-associated membranes (MAMs) as measured by expression of glucose-regulated protein 75 (GRP75). Item 74. 74. The isolated mitochondria of item 73, wherein the reduction in association is at least about 30% compared to the association with MAM of mitochondria in cells or isolated mitochondria obtained by a method comprising cell homogenization. Item 75. 75. The isolated mitochondria of item 74, wherein the reduction in association is at least about a 50% reduction. Item 76. 76. The isolated mitochondrion according to any one of items 67 to 75, having a membrane potential of about −30 mV to about −220 mV. Item 77. 77. The isolated mitochondrion of any one of items 67 to 76, which has not undergone drp1-dependent division. Item 78. 78. The isolated mitochondria of any one of items 67 to 77, having a size of about 500 nm to 3500 nm. Item 79. 79. A composition comprising the isolated mitochondria according to any one of items 67 to 78. Item 80. 31. The population of isolated mitochondria of any one of items 1 to 30, wherein the isolated mitochondria are derived from or isolated from cells that have been treated with a mitochondrial activator. Item 81. 53. The method of any one of items 33 to 52, wherein mitochondria in the cells are treated with a mitochondrial activator prior to (i) to (iv). Item 82. 79. The composition of item 30 or 79 or the formulation of item 31, wherein the isolated mitochondria are derived from or isolated from cells whose mitochondria have been treated with a mitochondrial activator. Item 83. Isolated mitochondria, which are derived from or isolated from cells that have been treated with a mitochondrial activator. Item 84. 79. The isolated mitochondria of any one of items 67 to 78, wherein the mitochondria are derived from or isolated from cells treated with a mitochondrial activator. Item 85. The method of any one of items 53 to 66, wherein the administered population of isolated mitochondria is the population of isolated mitochondria of item 80, the composition of item 82, or the formulation of item 82. Item 86. The population of isolated mitochondria of item 80, the composition or formulation of item 82, or the isolated mitochondria of item 83 or 84, further comprising a mitochondrial activator. Item 87. The isolated population of mitochondria of item 80, the composition or formulation of item 82, or the isolated mitochondria of item 83 or 84, wherein the mitochondrial activator is resveratrol. Item 88. 87. The population, composition, formulation, or isolated mitochondria of item 86, wherein the mitochondrial activator is resveratrol. Item 89. Isolated mitochondria derived from or isolated from cells treated with lipid membrane-based vesicles encapsulating or containing a mitochondrial activator. Item 90. 89. The isolated mitochondria of item 89, wherein the mitochondrial activator is resveratrol. Item 91. 91. The isolated mitochondria of item 89 or 90, further comprising a mitochondrial activator. Item 92. A population of isolated mitochondria from any one of items 88-90. Item 93. A composition comprising the population of isolated mitochondria of any one of items 88 to 90. Item 94. A pharmaceutical composition comprising the population of isolated mitochondria of any one of items 88 to 90. Item 95. 67. The method of any one of items 33 to 66, wherein the cells are treated with lipid membrane-based vesicles encapsulating or containing a mitochondrial activator. Item 96. Item 96. The method of item 95, wherein the mitochondrial activator is resveratrol.

[0193] In one embodiment, encapsulated mitochondria can be prepared from isolated mitochondria as described above. [Example]

[0194] Example 1 Isolation of mitochondria Mitochondria were isolated from HeLa cells as follows. Human HeLa cells (RCB3680) purchased from the Riken Cell Bank were cultured in MEM + 10% FBS medium, and subcultured once or twice a week. 1) The cells were cultured in a 100 mm diameter dish and confirmed to be 80% confluent. 2) The medium was discarded, and the dish was washed twice with 3 mL of isolation buffer (10 mM Tris-HCl, 250 mM sucrose, 0.5 mM EGTA, pH 7.4). 3) 3 mL of isolation buffer containing 30 μM digitonin was added to the dish, and the dish was allowed to stand at room temperature for 3 minutes. 30 μM is approximately 1 / 10 of the critical micelle concentration (cmc) of digitonin. 4) The inside of the dish was washed twice with 3 mL of isolation buffer. 5) 3 mL of isolation buffer was added to the dish, and the dish was allowed to stand at 4°C for 10 minutes. 6) The cells were detached by gentle pipetting using a micropipette. 7) The suspension containing the mitochondria and detached cells was then transferred to a 15 mL centrifuge tube and centrifuged at 500 × g for 10 minutes at 4 °C. The supernatant (2 mL) was collected to obtain the isolated mitochondrial population (hereafter referred to as the "prepared product"). 8) For freezing, glycerol was added to the freezing buffer (10 mM Tris-HCl, 225 mM mannitol, 75 mM sucrose, 0.5 mM EGTA, pH 7.4) to a concentration of 10% and suspended in the buffer. The suspension was frozen in liquid nitrogen to obtain frozen isolated mitochondria (hereafter referred to as the "frozen product").

[0195] The product prepared immediately for use was stained with 250 nM tetramethylrhodamine methyl ester (TMRM) in the presence of malic acid and glutamine (5 mM each) to assess the activity of isolated mitochondria. As a result, mitochondrial polarization was confirmed. The purity of isolated mitochondria was examined by staining with 100 nM MitoTracker Deepred. Bright green fluorescence was emitted from almost the entire solution containing isolated mitochondria. This confirmed that the isolated mitochondria were present in almost the entire solution. In other words, the isolated mitochondria comprised more than 98% of the mitochondrial solution, and 90% of the mitochondria showed polarization.

[0196] Frozen mitochondria were thawed by exposure to tap water and centrifuged at 500 × g and 4°C for 10 minutes. The supernatant was collected and subsequently centrifuged to precipitate the mitochondria. The supernatant was discarded, and Tris buffer was added instead to obtain the samples. The particle size (measured by dynamic light scattering) and zeta potential (measured by electrophoretic light scattering) of the mitochondria contained in these samples were measured using a Zetasizer NanoZS (Malvern Instruments, Ltd., Worcestershire, UK). More specifically, the mean mitochondrial particle size (mean hydrodynamic diameter) and polydispersity index (PDI) were obtained from the autocorrelation function of the scattered light intensity by cumulant analysis (ISO 22412). A histogram was then created based on the particle size. The results are shown in Figure 1. In Figure 1, panel a) shows the results for the product obtained in step 7 above, which is prepared for use before freezing; panel b) shows the results for the sample obtained by thawing the frozen product obtained in step 8 above. As shown in Figure 1, the particle size of mitochondria in the isolated mitochondrial population was distributed around 1,000 nm (see Figure 1, panel a). The same distribution was obtained in the frozen and thawed isolated mitochondrial population (see Figure 1, panel b). Furthermore, after the freeze-thaw process, the zeta potential remained negative (see Figure 1, zeta potential).

[0197] Subsequently, confocal laser scanning microscopy was performed to assess the surface potential of mitochondria contained in the isolated mitochondrial population. More specifically, to assess membrane potential, a mitochondrial potential-dependent reagent, tetramethylrhodamine ethyl ether (TMRE) (excitation wavelength: 549 nm, emission wavelength: 574 nm) (Thermo Fisher Scientific, Waltham, MA), was used. TMRE emits red fluorescence when mitochondrial membrane potential is maintained. The isolated mitochondrial solution (300 μL) was added to a 3.5 cm glass-based Petri dish (AGC TECHNO GLASS Co., Ltd. (IWAKI), Shizuoka, Japan) and centrifuged at 10 × g and 4 °C for 10 minutes. The supernatant was discarded. A staining solution was added to obtain final concentrations of 10 nM TMRE, 0.33 mg / mL bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO), 5 mM malic acid (Wako Pure Chemical Industries, Ltd., Osaka, Japan), and 5 mM glutamic acid (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Incubation was performed at room temperature for 10 minutes. Observation was performed using an FV10i-LVI (Olympus Corporation, Tokyo, Japan). The results are shown in Figure 2. In Figure 2, the results for the isolated mitochondrial population obtained in step 7) above (i.e., the isolated mitochondrial population before the freeze-thaw process, hereafter also referred to as the "prepared product") are shown in panel a); whereas, the results for the sample obtained by thawing the frozen material obtained in step 8) above (i.e., the isolated mitochondrial population after the freeze-thaw process, hereafter also referred to as the "frozen product") are shown in panel b). As shown in Figure 2, red fluorescence is also satisfactorily detected in the frozen product, demonstrating that the membrane potential is maintained even after the freeze-thaw process.

[0198] Example 2 Lipid film coating and characterization of the resulting particlesThe mitochondria contained in the isolated mitochondrial population were each coated with a lipid membrane having a lamellar structure that served as a boundary to topologically separate the inside and outside (to obtain mitochondria encapsulated in a lipid membrane). We investigated whether a microflow channel could be used for lipid membrane coating. Specifically, the process was as follows. As shown in Figure 36, the iLiNP™ (Lilac Pharma Inc.) with a baffle structure was used as the microflow channel device. The iLiNP device has two solution inlets (11a and 12a), channels (11 and 12) that connect the solution inlets to a confluence channel (13) and a mixing channel (14), respectively. The confluence channel 13 is the site where the channels (11 and 12) extending from the two solution inlets join. The mixing channel 14 is the channel where the combined solutions are mixed. As shown in Figure 36, in the mixing channel 14, the solution moves along the flow direction indicated by the large arrow and is guided through a bend toward the outlet 14c. The mixing channel 14 had single or multiple sets (20 sets) of bends represented by 14a and 14b. In Figure 36, 14a represents the region where the channel narrows (50 μm wide), and 14b (200 μm wide) represents the region where the narrowed region returns to its original width. The channel height was 100 μm. The microflow channel device allows for rapid mixing of organic and aqueous solvents (see Figure 3, panel a). In this example, the organic phase used was a 7.7 mM lipid solution dissolved in ethanol (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) / sphingomyelin (SM) / 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol 2000 (DMG-PEG2000) / stearylated octaarginine (STR-R8) = 9 / 2 / 0.33 / 1.1 (molar ratio)); the aqueous phase used was a solution of isolated mitochondria. These two solutions were poured into the two inlets of the microflow channel device, respectively, and mixed in the converging channel of the microflow channel device (see Figure 4).The mixing conditions were as follows: overall flow rate: 500 μL / min (organic phase: 100 μL / min, aqueous phase: 400 μL / min). The syringe pump used here was a PUMP 11 ELITE (Harvard Apparatus, Holliston, MA). As controls, a sample without isolated mitochondria in the aqueous phase (see Figure 3, panel b), a sample using only ethanol in the organic phase (see Figure 5, panel a), and a sample using only STR-R8 in the organic phase with ethanol (see Figure 5, panel b) were evaluated. Additionally, a sample using a mixture of isolated mitochondrial solution instead of the aqueous solvent and organic phase was also evaluated (see Figure 5, panel c). The particle size and zeta potential of each sample were measured. As shown in Figure 4, the particles obtained from the microflow channel device showed a monodisperse particle size distribution with a peak around 100 nm. Active mitochondria are polarized, so they have a negative zeta potential. In contrast, particles obtained by the microflow channel device exhibited a positive zeta potential (see Figure 4), suggesting that mitochondria were encapsulated by a lipid membrane exposing cationic STR-R8 on the surface and that the zeta potential was positively altered due to the presence of said R8.

[0199] Furthermore, by using a mitochondrial membrane potential indicator, it was shown that after partitioning in the microflow channel, mitochondria split into smaller ones (size: 262 nm; PDI: 0.301; ζ potential: −19.7 mV) with no detectable negative membrane polarization.

[0200] Example 3 Observation of lipid membrane-based vesicles encapsulating mitochondria by fluorescence microscopy Next, to determine whether mitochondria were successfully coated with lipid membranes, mitochondria were stained red (MitoTracker™ Deep Red (excitation wavelength: 644 nm, emission wavelength: 665 nm) (Thermo Fisher, Waltham, MA)), while lipids were stained green (DOPE-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-DOPE) (excitation wavelength: 465 nm, emission wavelength: 535 nm) (Avanti Polar lipids, Alabaster, AL). The isolated mitochondria were then subjected to the coating process using the microflow channel device as described above and observed by fluorescence imaging. If mitochondria were successfully coated, red and green overlapping areas and yellow fluorescence should be produced. The particles obtained by the microflow channel device were added to a glass slide and then imaged with a Nikon A1 (Nikon The particles were observed using a microscope (Microscopy, Fluorescence Imaging, Fluorescence Spectro ...

[0201] Example 4 Observation of lipid membrane-based vesicles encapsulating mitochondria by electron microscopy The structures of lipid membrane-based vesicles encapsulating mitochondria (see Figure 4) obtained using the microflow channel device described above were observed by electron microscopy. Isolated mitochondria (prepared at the time of use) and isolated mitochondria with STR-R8 modification (see Figure 5, panel b)) were used as controls. More specifically, isolated mitochondria and isolated mitochondria with STR-R8 modification were stained using a chemical fixation method commonly used for observing biological samples. Simple lipid membrane-based vesicles (see Figure 3, panel b)) and lipid membrane-based vesicles encapsulating mitochondria (see Figure 4) were stained using a negative staining method, which is suitable for observing nanoparticles. The chemical fixation method was performed as follows. First, the samples were fixed in an equal volume of 0.1 M cacodylate buffer (pH 7.4) containing 4% paraformaldehyde and 2% glutaraldehyde, cooled, and then further fixed overnight at 4°C in 0.1 M cacodylate buffer (pH 7.4) containing 2% glutaraldehyde. The fixed samples were washed with cacodylate buffer and then further fixed in 0.1 M cacodylate buffer (pH 7.4) containing 2% osmium tetraoxide. To remove water, the samples were immersed in graded ethanol-containing solutions. The samples were treated twice with propylene oxide and then incubated for 1 hour in a mixture containing propylene oxide and resin (Quetol-812; Nisshin EM Co., Tokyo, Japan) at a 70:30 ratio. The tube caps were then removed and the samples were left overnight to allow the propylene oxide to evaporate. The samples were then embedded in 100% resin and polymerized at 60°C for 48 hours. The samples were sliced ​​into 70-nm-thick sections. The sections were observed under an electron microscope. The nanoparticles were observed using a negative staining method because the lipid membrane was decomposed by the chemical fixation method, making the resulting samples unsuitable for observing membrane structure. The electron microscope used here was a JEM-1400 Plus (JEOL Ltd., Tokyo, Japan). Analysis of the observations was outsourced to Tokai Electron Microscopy, Inc.The results are shown in Figures 7-10. Using chemical fixation, as shown in Figure 7, isolated mitochondria were observed to have the characteristic cristae structure of mitochondria. As shown in Figure 8, lipid membrane-based vesicles that did not encapsulate mitochondria were observed to have a particle shape; however, the interior of the particles was filled with lipid membranes. In contrast, lipid membrane-based vesicles encapsulating mitochondria (see Figure 4) were observed to have hollow lipid membrane structures, as shown in Figure 9. When no substance is encapsulated within the lipid membrane, the interior of the particle is filled with lipid membranes, as shown in Figure 8. In the particle shown in Figure 9, the lipid membrane was unable to enter the interior, so it was assumed that mitochondria were encapsulated. Note that negative staining is not an appropriate method for observing mitochondrial structure, as shown in Figure 7. For this reason, mitochondria were not shown in Figure 9. However, in isolated mitochondria modified with STR-R8, the mitochondrial structure was observed to be disrupted, as shown in Figure 10. It was found that mitochondria were favorably modified with STR-R8 after being encapsulated by lipid membranes.

[0202] Example 5 Lipid membrane-based vesicles encapsulating mitochondria using various lipids Instead of the lipid membrane composition (nanocapsule material: DOPE / SM / STR-R8) of the mitochondria-encapsulating lipid membrane-based vesicles prepared in Figure 4, we investigated whether various lipid membrane compositions could be used to prepare mitochondria-encapsulating lipid membrane-based vesicles from these compositions. The lipid membrane material compositions used here were neutral lipid membrane compositions: hydrogenated soybean phosphatidylcholine (HSPC) / cholesterol (Chol) / 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) = 3 / 2 / 0.25 (molar ratio), which is the same composition as that of the clinically used nanocapsule Doxil; and DOPE / cholesterol hemisuccinate (CHEMS) = 9 / 2 (molar ratio), which is a component of a negatively charged lipid membrane composition. Mitochondria-encapsulating lipid membrane-based vesicles were prepared using the microflow channel device described above. As negative controls, particles prepared from a solution without mitochondria and organic solvent containing lipids were used. The results are shown in Figure 11. As shown in Figure 11, all particle groups were observed to exhibit a particle size distribution without aggregation. It was found that the particle size of lipid membrane-based vesicles encapsulating mitochondria (referred to as "mitochondrial packaging" in the figure) tended to be larger than that of particles without mitochondria encapsulated therein (referred to as "nanoparticles" in the figure). It was also found that lipid membrane-based vesicles encapsulating mitochondria could be obtained not only with the positively charged composition shown in Figure 4 but also with both neutral lipids (see Figure 11, panel c)) and negatively charged lipids (see Figure 11, panel d)). Based on a comparison between particle sizes, it was suggested that the particle size distribution may vary depending on the lipid membrane composition (see Figure 11).

[0203] Example 6 Lipid membrane-based vesicles encapsulating packaged mitochondria at various flow rates Lipid membrane-based vesicles encapsulating mitochondria were prepared using the same microflow channel device (see Figure 4) as above, except that only the flow rate and ethanol concentration of the organic phase were changed. The overall flow rate was changed to 50 μL / min, 100 μL / min, 250 μL / min, or 500 μL / min. The ethanol concentration of the organic phase was changed to 10%, 20%, or 40%. The particle size and zeta potential of the resulting lipid membrane-based vesicles encapsulating mitochondria were measured in the same manner as above. The results are shown in Figure 12. As shown in Figure 12, the particle size was approximately 100 to 150 nm under all conditions.

[0204] Example 7 Integration of the resulting lipid membrane-based vesicles encapsulating mitochondria into cells We observed the integration of the mitochondria-encapsulating lipid membrane-based vesicles (Figure 4) into cells and their intracellular dynamics after integration. Prior to packaging of isolated mitochondria, the mitochondria in the lipid membrane-based vesicles were stained red with MitoTracker™ Deep Red (by incubating them at 4°C for 15 minutes at a concentration of 100 nM to stain the mitochondria). HeLa cells were prepared, and the mitochondria in the HeLa cells were stained green with MitoTracker™ Green (by incubating them at 37.0°C for 15 minutes at a concentration of 100 nM in 5% CO2). The resulting HeLa cells were then added with the mitochondria-encapsulating lipid membrane-based vesicles. The mixture was incubated for 3 hours. After incubation, the cells were observed using a confocal laser scanning microscope (CLSM) using an Olympus FV10i-LIV instrument with an UPlanSApo 60x objective (NA = 1.2) and laser diodes (LD) of 473 nm and 635 nm. The results are shown in Figure 13. As shown in Figure 13, in HeLa cells, red signals of mitochondria derived from lipid membrane-based vesicles encapsulating mitochondria and green signals of mitochondria derived from HeLa cells were observed. Almost all mitochondria showed colocalization. This demonstrated that mitochondria derived from lipid membrane-based vesicles encapsulating mitochondria were incorporated into cells, that mitochondria incorporated into cells were fused with mitochondria in the cells, and that fusion was uniformly carried out.

[0205] In contrast, when the same amount of isolated mitochondria before packaging was added to HeLa cells, virtually no red signal was observed in the cells (see Figure 14). When STR-R8-modified isolated mitochondria (not lipid-coated) were added to HeLa cells, many cells were observed to have died (see Figure 15).

[0206] Mitochondria were isolated from human cardiac stem cells (hCDCs) and contacted with HeLa cells as described above. After 30 minutes, the isolated mitochondria were observed to be integrated into the cells. The isolated mitochondria that were not packaged in lipids were not observed to be integrated into the cells (see Figures 16 and 17).

[0207] Example 8 Rescue experiments of mitochondrial disease model cells In this experiment, mitochondria were isolated from human cardiac stem cells (hCDCs), packaged in lipids, and transplanted into cells with mitochondrial mutations. This method rescued the mitochondrial mutations. A population of isolated mitochondria was collected from hCDCs in the same manner as above, except that the cells were changed to hCDCs (see Figure 18). To obtain lipid membrane-based vesicles encapsulating hCDC-derived mitochondria, the collected population of isolated mitochondria was packaged in lipid membranes as shown in Figure 4. The resulting lipid membrane-based vesicles encapsulating hCDC-derived mitochondria were then added to dermal fibroblasts isolated and cultured from MELAS patients (MELA cells) and LHON patients (LHON cells). After 3 and 24 hours, mitochondrial respiratory activity was assessed using an extracellular flux analyzer (Extracellular Flux Analyzer XFp, Agilent Technologies, California, USA). Cells were seeded into wells of an assay plate at a rate of 15,000 cells / well. Lipid membrane-based vesicles containing hCDC-derived mitochondria were added 3 and 24 h before the assay. The basal medium for respiratory activity measurements contained glucose (5.5 mM), pyruvate (1.25 mM), and glutamine (4.0 mM). After measuring basal respiration using an extracellular flux analyzer, oligomycin (final concentration 1 μM), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (final concentration 1.5 μM), and rotenone and antimycin A (final concentrations of 0.5 μM each) were added sequentially to measure mitochondrial oxygen consumption rates.

[0208] The results are shown in Figures 19 and 20. As shown in Figures 19 and 20, the addition of lipid membrane-based vesicles encapsulating hCDC-derived mitochondria significantly improved mitochondrial respiratory activity after the addition of FCCP. The improvement in respiratory activity was observed even after 3 hours, with an even greater improvement observed after 24 hours. As described, transplantation of lipid membrane-based vesicles encapsulating mitochondria into cells was found to improve mitochondrial function in treated cells. The improvement was observed within only 3 hours, suggesting that mitochondrial genomic DNA is not necessarily required for improvement, and that some of the other components in the encapsulated mitochondria may help support intracellular mitochondrial function.

[0209] Example 9 Delivery of mitochondria into cells by lipid membrane-based vesicles encapsulating mitochondria compared with delivery of mitochondria into cells by lipofection Lipid membrane-based vesicles encapsulating mitochondria from hCDCs, prepared as described above, were used. For lipofection, we used the ready-to-use product prepared as described above and a lipid complex (lipoplex, hereafter referred to as "LFN-isolated mitochondria") prepared by mixing 0.32 μg of mitochondria (protein equivalent) with 1 μL of Lipofectamine® 2000 (Invitrogen, California, USA). Normal skin fibroblasts (normal fibroblasts), skin fibroblasts isolated and cultured from patients with Leigh encephalopathy (Leigh encephalopathy cells), and skin fibroblasts isolated and cultured from patients with LHON (LHON cells) were used. Cells were seeded into wells of an assay plate at a rate of 15,000 cells / well. Lipid membrane-based vesicles encapsulating hCDC-derived mitochondria and LFN-isolated mitochondria were added 24 hours before the assay. The amount of mitochondria introduced was the same. The basal medium for measuring respiratory activity was supplemented with glucose (5.5 mM), pyruvate (1.25 mM), and glutamine (4.0 mM). After measuring basal respiration using an extracellular flux analyzer, oligomycin (final concentration 1 μM), FCCP (final concentration 1.5 μM), and rotenone and antimycin A (final concentrations of 0.5 μM each) were added sequentially to measure mitochondrial oxygen consumption rates.

[0210] The results are shown in Figures 21 to 23. As shown in Figures 21 to 23, lipid membrane-based vesicles encapsulating mitochondria increased mitochondrial respiratory activity in all cells, including normal cells, Leigh syndrome fibroblasts, and LHON fibroblasts. In contrast, no or limited increase in mitochondrial respiratory activity was observed in the group using lipofectamine (LFN-isolated Mitochondria). As shown in Figure 21, the group using lipofectamine (LFN-isolated Mitochondria) had a negative effect on mitochondrial respiratory activity in normal cells. This suggests that the group using lipofectamine (LFN-isolated Mitochondria) is likely cytotoxic.

[0211] The potential for cellular uptake of lipid membrane-based vesicles encapsulating mitochondria was then compared with that of LFN-isolated mitochondria. Test samples were prepared in the same manner as above, except that mitochondria were obtained from normal skin fibroblasts, similar to the product prepared at the time of use. The potential for cellular uptake of the test samples was assessed using a flow cytometer (CytoFlex, Beckman Coulter, Inc., Tokyo, Japan). HeLa cells were cultured at 2.0 × 10 in a 6-well plate. 5 The cells were seeded at a ratio of 1:1, 1:2, 1:3, 1:4, 1:5 ...

[0212] The results are shown in Figure 24. As shown in Figure 24, it was confirmed that the group using Lipofectamine (LFN-isolated Mitochondria) had a lower probability of entering cells than isolated mitochondria. It was confirmed that lipid membrane-based vesicles encapsulating mitochondria were efficiently incorporated into cells, similar to the observation images shown in Figure 13.

[0213] Example 10 Comparison between lipid membrane-based vesicles encapsulating mitochondria and lipofectamine-mitochondria complexes Isolated mitochondria (prepared product) and a mixture of the preparative product and Lipofectamine 2000 (LFN-isolated Mt) were prepared by combining 1 μL of Lipofectamine solution (OptiMEM) with mitochondria (0.32 μg protein equivalent). Their particle size distributions and zeta potentials were measured using a Zetasizer. The results are shown in Figure 25. As shown in Figure 25, the lipofectamine particles had a particle size of approximately 2670 nm, and the lipofectamine-mitochondria complex had a particle size of approximately 3500 nm. The lipofectamine-mitochondria complex had a small negative zeta potential. This means that the complex was electrically neutral; in other words, the mitochondria were not completely encapsulated by lipofectamine (positive charge), suggesting that lipofectamine and free mitochondria could form a complex to neutralize the charge.

[0214] For the mixture of the ready-to-use product and Lipofectamine 2000, Lipofectamine-mitochondria associations formed in the mixture were observed by electron microscopy. The mixture of the ready-to-use product and Lipofectamine 2000 was routinely stained using chemical fixation and negative staining, respectively, and then the associations were observed. The results of negative staining are shown in Figure 26, and the results of chemical fixation are shown in Figure 28. As shown in Figure 26, Lipofectamine 2000 (LFN only) formed particle associations in solution. In contrast, in the mixture of Lipofectamine 2000 and mitochondria (LFN+Mt), associations between LFN particles and debris-like mitochondria (enclosed by the white dashed line) were observed. As shown in Figure 27, compared to the ready-to-use product (Panel A), aggregates of Lipofectamine particles were observed associated with some sides of mitochondria in the Lipofectamine 2000-mitochondria mixture (LFN+Mt; Panel B). Therefore, the complex of LFN and mitochondria is clearly not a lipid membrane-based vesicle encapsulating mitochondria.

[0215] Furthermore, the cytotoxicity of MITO-Q was measured and compared with that of LFN+Mt. HeLa cells were cultured at 1.0 × 10 4Cells / well were plated and cultured for 24 hours. HeLa cells were then contacted with MITO-Q or Q treated with LFN at different concentrations. After 1 hour of incubation, the medium was removed, and 500 μL of fresh medium (FBS(-)) was added to each well, and the cells were further incubated. 24 hours after mitochondrial treatment, the cells were washed with 500 μL of PBS. To measure the viability (%) of the treated cells, the incubated cells were then subjected to a WST-1 assay (Premix WST-1 Cell Proliferation Assay System, Takara Bio, Japan). The results are shown in panel C of Figure 27. As shown in panel C of Figure 27, HeLa cells showed dose-dependent cytotoxicity upon treatment with LFN+Mt, whereas HeLa cells showed virtually no cytotoxicity upon treatment with MITO-Q. This suggests that fully encapsulated mitochondria can reduce the cytotoxicity of particles encapsulating mitochondria. This suggests that smaller mitochondria may be beneficial in the preparation of lipid membrane-based nanovesicles without exhibiting substantial cytotoxicity.

[0216] The results are summarized in Figure 28. As shown in Figure 28, particles are formed when Lipofectamine and isolated mitochondria are mixed; however, mitochondria are not encapsulated within the particles. It is believed that mitochondria (particles) and Lipofectamine particles form a complex. In contrast, the present invention yielded lipid membrane-based vesicles encapsulating mitochondria in a closed space. Lipid membrane-based vesicles encapsulating mitochondria have mitochondrial activity. Introducing vesicles into cells can enhance mitochondrial activity in cells. Furthermore, because a microflow channel device was used to mix isolated mitochondria with a lipid solution, the isolated mitochondria were divided into small populations while retaining their activity, and were encapsulated in the lipid membrane-based vesicles in this divided small population state. The present invention not only provides lipid membrane-based vesicles encapsulating mitochondria, but also allows for miniaturization of lipid membrane-based vesicles encapsulating mitochondria. Lipid membrane-based vesicles encapsulating miniaturized mitochondria can also be provided.

[0217] Example 11 The effect of loss of membrane potential on improving intracellular mitochondrial function As shown in Figures 29A and 29B, isolated mitochondria were prepared as described in Example 1, except that solutions with a pH of 7.4 to 8.9 were used throughout the isolation process. Next, mitochondria isolated in solutions with different pHs were stained with 100 nM MitoTracher Deep Red or 250 nM tetramethylrhodamine methyl ester (TMRM) in the presence of malate and glutamine (5 mM each) to assess the activity of the isolated mitochondria. The results are shown in Figures 29A and 29B. As shown in Figures 29A and 29B, mitochondria isolated in solutions with a pH of 8.0 to 8.9 exhibit a reduced membrane potential, as evidenced by the decreased fluorescence intensity in these samples.

[0218] To evaluate the effect of mitochondria import with reduced membrane potential on improving intracellular mitochondrial function, mitochondria isolated in a solution with reduced membrane potential (pH 8.9) were encapsulated in lipid membrane-based vesicles and then introduced into HeLa cells using the procedure described in Example 9. Encapsulated mitochondria isolated in a solution with pH 7.4 were used as a positive control, and Tris buffer was used as a negative control. The experiment was performed as shown in Figure 30A. Briefly, HeLa cells were plated 24 hours before the experiment. After 24 hours, the cells were subcultured, and then the prepared encapsulated mitochondria were added to the culture. After 1 hour of incubation, FBS was added to the culture at a final concentration of 20%. After 24 hours, cellular respiratory function was measured as described in Example 9.

[0219] As shown in Figures 30B and 30C, mitochondrial respiration in treated cells was improved in the group treated with encapsulated mitochondria prepared in pH 8.9 buffer. This improvement in respiratory function was comparable to that in the group treated with encapsulated mitochondria prepared in pH 7.4 buffer and was significantly greater than that in the negative control group. These results suggest that mitochondrial membrane potential is not necessarily required to improve mitochondrial function in mitochondria-implanted cells.

[0220] Example 12 Abundance of mtDNA in MITO-Q and its effect on MITO-Q activity MITO-Q was prepared from Q isolated in pH 7.4 buffer, Q isolated in pH 8.9 buffer, and mitochondria isolated by the conventional method (hereafter also referred to as "method D") using detergent at a concentration higher than the critical micelle concentration.

[0221] Mitochondrial DNA (also called "mtDNA") was measured by quantitative PCR. Prior to the measurement, a primer set was selected to linearly detect mtDNA due to its wide dynamic range. After selecting a vast number of primer pairs, a forward primer having the nucleic acid shown in SEQ ID NO: 1 and a reverse primer having the nucleic acid sequence shown in SEQ ID NO: 2 were identified to achieve linear amplification of mtDNA by PCR. In Figure 31A, isolated mitochondria were diluted with Tris buffer to obtain a dilution series. Protein concentration was measured by the conventional Bradford method, and mtDNA concentration was calculated by PCR amplification using the above primer pair. The relationship between protein concentration and mtDNA concentration is shown in Figure 31A. As shown in Figure 31A, the concentrations were linearly and highly correlated with each other. Furthermore, tRNA Leu pT7-tRNA (3230-3304) was inserted into the EcoRI and EcoRV restriction enzyme sites of the pUC57-Amp plasmid vector. Leu The plasmid was diluted in Tris buffer to obtain a dilution series. Next, the plasmid was amplified at different concentrations to measure the copy number of the amplicon. Figure 31B shows that the measured copy number (or amplicon concentration) (ng / μL) was highly correlated with the plasmid concentration, suggesting that the selected primer pair can linearly amplify mtDNA and is useful for quantifying the template mtDNA contained in the sample.

[0222] If the membranes of isolated mitochondria are disrupted, mtDNA leaks out of the mitochondria, resulting in a decrease in the amount of mtDNA in the resulting mitochondria. The number of mtDNA fragments in mitochondria isolated by various methods was calculated. Mitochondria isolated by iMIT in a pH 7.4 solution contained 9.3 x 10 fragments per μg of protein. 6 Mitochondria isolated by iMIT in a pH 8.9 solution containing 5.6 × 10 copies of mtDNA per μg of protein 6Mitochondria isolated by the D method had 8.7 x 10 copies of mtDNA per μg of protein at concentrations above the CMC. 5 copies of mtDNA, however, 1 ng of mtDNA contains 1.0 x 10 6 Therefore, it was thought that mitochondria isolated by iMIT retained their mtDNA inside the mitochondria, whereas mitochondria isolated by the D method lost most of their mtDNA from the mitochondria during the isolation process.

[0223] Furthermore, these isolated mitochondria were packaged to obtain packaged mitochondria, and then the number of mtDNA copies in the packaged mitochondria was calculated in a similar manner. The results are shown in Table 1 and Figure 3 ID.

[0224] [Table 1]

[0225] The resulting entrapped mitochondria were then contacted with cells to introduce the mitochondria into the cells. Basal and maximum mitochondrial respiration were measured. The results are shown in Figures 31E and 31F. As shown in Figures 31E and 31F, MITO-Q isolated at pH 7.4 and MITO-Q isolated at pH 8.9 exhibited dramatic increases in respiratory activity, whereas mitochondria isolated by the D method exhibited only a moderate increase in respiratory activity. These results suggest that mitochondria may lose some mitochondrial components during the isolation process, but that MITO-Q isolated by iMIT maintains them.

[0226] The amount of transcription factor A, mitochondrial (TFAM) contained in each of the encapsulated mitochondria was measured using an enzyme-linked immunosorbent assay kit for transcription factor A, mitochondrial (TFAM) organism species: Homo sapiens (human) (#MBS2706301) according to the manufacturer's instructions. Mitochondria isolated by the iMIT method using pH 7.4 or pH 8.9 buffer or by method D were used. These mitochondria were encapsulated in lipid membrane-based nanovesicles according to the example shown above. The results from the WST-1 assay are shown in Figure 31G. As shown in Figure 31G, the levels of TFAM contained in mitochondria isolated by various methods were comparable to each other. The amounts of total protein in mitochondria isolated by these methods were also comparable to each other (Figure 31H).

[0227] Divided mitochondria or split Q lose their respiratory activity. Therefore, some of the mitochondrial components that are reduced during the isolation process by conventional methods may play an important role in activating mitochondrial function. It has also been shown that the concentration of mtDNA in vesicles is an important indicator of MITO-Q function.

[0228] Example 13 Preparation of lipid membrane-based vesicles containing mitochondria from human cardiac progenitor cells Mitochondria were isolated from human cardiac progenitor cells (hCPCs) according to Example 1 and then encapsulated in lipid membrane-based vesicles according to Example 2. The size distribution of isolated hCPC mitochondria and encapsulated mitochondria is shown in Figure 32A and Table 2 below.

[0229] [Table 2]

[0230] As shown in Table 2 and Figure 32A, isolated hCPC mitochondria (i.e., isolated hCPC Mt) have a size distribution peak at approximately 800 nm, a PDI of 0.7 (greater than 0.5), and a negative zeta potential. After encapsulation in lipid membrane-based vesicles presenting cationic peptides, the vesicles (i.e., hCPC-MITO-Q) have a size distribution peak at 85 nm, a PDI of less than 0.5, and a positive zeta potential. These results indicate that hCPC mitochondria were successfully encapsulated in vesicles presenting cationic peptides.

[0231] Next, the prepared encapsulated mitochondria (hCPC-MITO-Q) were contacted with hCPCs, and the degree of membrane polarization of the resulting hCPCs was measured by staining the mitochondria with TMRM. As shown in Figure 32B, hCPC-MITO-Q induces stronger fluorescence in treated cells than in untreated control cells.

[0232] Example 14 Effect of importing isolated mitochondria from cells whose mitochondrial function was activated by MITO-Porter In this example, hCPCs were treated with the mitochondrial activator resveratrol using MITO-Porter, a drug delivery system for mitochondria, in the same manner as described in Example 1 of WO2018 / 092839. Mitochondria were then isolated from the resveratrol-treated hCPCs and encapsulated in lipid membrane-based vesicles displaying S2 peptide by the method described in Example 12, using stearylated S2 peptide instead of stearylated octaarginine (Szeto, HH et al., Pharm. Res., 2011, 28, 2669-2679). The resulting vesicles were designated Res-hCPC-MITO-Q.

[0233] The basal and maximum respiration of hCPCs treated with Tris buffer, hCPC-MITO-Q prepared in Example 12, and Res-hCPC-MITO-Q prepared in Example 13 are shown in Figure 33. As shown in Figure 33, Res-hCPC-MITO-Q induces a greater improvement in the respiratory capacity of treated cells than hCPC-MITO-Q. These results indicate that activated mitochondria can further improve the respiratory capacity of treated cells.

[0234] Example 15 MITO-Q prepared as described in Examples 1 and 2 was contacted with normal fibroblasts, and the treated cells were then incubated for different periods of time as shown in Figure 34A. Maximum respiratory activity was measured for each sample. The results are shown in Figure 34B. As shown in Figure 34B, all treated cells exhibited increased maximum respiratory activity compared to the untreated group (NT). Because MITO-Q contains mitochondrial DNA and other components of mitochondria, the improvement observed in samples incubated for shorter periods (e.g., 3 to 48 hours) likely results from the introduction of non-DNA components, including proteins and metabolites, whereas the improvement observed in samples incubated for longer periods (e.g., 72 hours) likely results from the introduction of mitochondrial DNA into the cells, given the mitochondrial turnover period (approximately 2 to 3 days).

[0235] Example 16 Preservation of lipid membrane-based vesicles encapsulating mitochondria Mitochondria were isolated from hCPCs and encapsulated in lipid membrane-based vesicles displaying the S2 peptide / aptamer by the method described in Example 12. The resulting hCPC-MITO-Q was stored at 4°C for one week. The stored hCPC-MITO-Q was then contacted with hCPCs, and the maximum respiratory capacity was measured. Pre-storage hCPC-MITO-Q was used as a positive control. As shown in Figure 35, the maximum respiration of cells treated with stored hCPC-MITO-Q was greater than that of untreated cells (NT) and cells treated with pre-storage hCPC-MITO-Q. Therefore, these results indicate that formulations containing lipid membrane-based vesicles can be stably stored for at least one week.

[0236] Reference Number List for Figure 36 11: Channel 1 11a: Liquid sample inlet for channel 1 12: Channel 2 12a: Liquid sample inlet for channel 2 13: Channel 1 and Channel 2 merge channel 14: Mixing channel for mixing liquid samples that are combined in the confluence channel 14a: Channel narrowing region 14b: A narrowed area widens 14c: outlet for discharging the liquid sample mixture

Claims

1. A composition comprising a population of lipid membrane-based nanovesicles encapsulating mitochondria, wherein the population of lipid membrane-based vesicles has a particle size distribution with a peak at 50 nm to 200 nm as determined by dynamic light scattering, the mitochondria are contained in a sac-like membrane structure having a closed space formed by a lipid membrane in the lipid membrane-based vesicles, and the lipid membrane comprises an amphipathic lipid or a cationic lipid or an anionic lipid.

2. 10. The composition of claim 1, wherein the population of lipid membrane-based nanovesicles has a PDI of less than 0.

5.

3. 3. The composition of claim 1 or 2, wherein the encapsulated mitochondria may be integrated into the cytoplasm of a cell in contact therewith, or the mitochondria may be fused with endogenous mitochondria in the cytoplasm.

4. A composition according to any one of claims 1 to 3 for use in delivering mitochondria into cells.

5. The composition of claim 4 for use in improving mitochondrial respiratory activity in a cell.

6. 1. A method of producing a composition, comprising: The method comprises bringing an aqueous solution containing isolated mitochondria into contact with an ethanol solution containing amphipathic lipids or cationic lipids or anionic lipids in a confluent channel of a microflow channel device, thereby mixing the solutions; The lipid membrane-based nanovesicle has a morphology in which mitochondria are contained in a sac-like membrane structure having a closed space formed by a lipid membrane, the lipid membrane containing an amphipathic lipid, a cationic lipid, or an anionic lipid; The composition comprises a population of lipid membrane-based nanovesicles encapsulating mitochondria; The method, wherein the population of lipid membrane-based nanovesicles has a particle size distribution that peaks at less than 1 μm as determined by dynamic light scattering.

7. The method of claim 6, wherein the microflow channel device comprises a flow channel for facilitating mixing of the solutions that contact each other in the confluence channel, the flow channel having a baffle construction.

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