Organelle complex

Abstract

This specification discloses a population of organelle complexes. The organelle complex can include mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. In some embodiments, the organelle complex is isolated or derived from floating cells and / or frozen cells. In some embodiments, the organelle complex is isolated or derived from cells that have been contacted with a surfactant at a concentration above the critical micelle concentration (CMC) of the surfactant. At least about 80% of the mitochondria of the organelle complex can maintain structural integrity in the extracellular environment. This specification also provides a method for generating a first population of organelle complexes.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of 35 U.S.C.§119(e) of U.S. Provisional Patent Application No. 63 / 359,110, filed on July 7, 2022, and the content of this related application is hereby incorporated by reference in its entirety for all purposes.

Background Art

[0002] The present disclosure relates to a method for obtaining an organelle complex from cells, an organelle complex obtained by such a method, and uses of an organelle complex obtained by such a method.

[0003] Mitochondria are intracellular organelles that undertake multiple metabolic conversion and regulatory functions. They generate most of the ATP used by eukaryotic cells. Regarding mitochondrial function, the folded inner membrane and the surrounding outer membrane, as well as the electron transport system arranged within the inner membrane, play important roles. The inner membrane forms a highly folded structure called cristae that is thought to keep the proton concentration high by retaining supercomplexes of the electron transport system within the cristae membrane and trapping excited protons in the cristae space. The electrochemical proton gradient formed by the electron transport system enables the transport of anions, as well as ATP synthesis and cation transport.

[0004] Mitochondria are also highly dynamic organelles that move throughout the cell, undergo structural transitions, and change in length, morphology, shape, and size. Additionally, mitochondria are continuously removed and regenerated in a process known as mitochondrial biogenesis. Although most mitochondrial genes have been transferred to the nuclear genome, the mitochondrial genome still encodes rRNA, tRNA, and 13 subunits of the electron transport system (ETC). Thus, functional communication between the nuclear and mitochondrial genomes is essential for mitochondrial biogenesis, efficient oxidative phosphorylation, and normal health. Mitochondria are also a major source of free radicals and reactive oxygen species (ROS) that cause oxidative stress. Furthermore, mitochondria play an important role in intracellular signaling and the control of cell death, including apoptosis and necrosis.

[0005] Evidence is increasing that mitochondrial dysfunction is associated with a wide range of human diseases. Mitochondrial dysfunction, such as respiratory chain complex dysfunction, is a major cause involved in mitochondrial diseases and aging. When mitochondrial function declines, it affects the cells of many organs that are mainly involved in mitochondrial diseases or age-related diseases. Introducing exogenous mitochondria into target cells that require treatment is a promising approach for treating or preventing multiple diseases and disorders. However, there is a need for a scalable and surfactant-compatible method to isolate populations containing mitochondria from cells in a way that preserves mitochondrial function and structural integrity. SUMMARY OF THE INVENTION

[0006] This specification discloses a population of organelle complexes. In some embodiments, the organelle complex is isolated or derived from floating cells and / or frozen cells. In some embodiments, the organelle complex comprises mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. In some embodiments, the organelle complex is depleted of cytoplasmic macromolecules. In some embodiments, at least about 80% of the mitochondria of the organelle complex maintain structural integrity in the extracellular environment.

[0007] This specification discloses a population of organelle complexes. In some embodiments, the organelle complex is isolated or derived from cells contacted with the surfactant at a concentration above the critical micelle concentration (CMC) of the surfactant. In some embodiments, the organelle complex comprises mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. In some embodiments, the organelle complex is depleted of cytoplasmic macromolecules. In some embodiments, at least about 80% of the mitochondria of the organelle complex maintain structural integrity in the extracellular environment.

[0008] In some embodiments, the structural integrity includes the intramembrane structural integrity and / or the extramembrane structural integrity of the mitochondria. In some embodiments, at least two-fold or more of the mitochondria of the organellar complex maintain structural integrity in the extracellular environment as compared to a homogenized population of mitochondria in the extracellular environment. In some embodiments, the structural integrity is measured by citrate synthase (CS) activity and / or cytochrome c oxidase (COX) activity. In some embodiments, the extracellular environment includes a total calcium concentration of about 1 to about 20 mg / dL and / or a free / active calcium concentration of about 1 to about 6 mg / dL. In some embodiments, at least about 80% of the mitochondria of the organellar complex maintain functionality. In some embodiments, the mitochondria of the organellar complex are capable of generating ATP. In some embodiments, they include at least two-fold to six-fold the mitochondrial DNA (mtDNA) copy number as compared to a homogenized population of mitochondria. In some embodiments, the cytoplasmic macromolecules include cytoplasmic proteins, and the abundance of one or more cytoplasmic proteins (e.g., p70S6K and / or glyceraldehyde 3-phosphate dehydrogenase (GAPDH)) is depleted by at least about 90% as compared to the cells from which the organellar complex population is derived.

[0009] In some embodiments, the organelle complex comprises one or more mitochondrial matrix proteins (e.g., mitochondrial transcription factor A (TFAM), citrate synthase (CS)). In some embodiments, the organelle complex comprises one or more outer mitochondrial membrane proteins (e.g., translocase of outer mitochondrial membrane 20 (TOMM20)). In some embodiments, the organelle complex comprises one or more lysosomal proteins (e.g., lysosome-associated membrane protein 2 (LAMP2), mannose 6-phosphate receptor (M6PR), lysosome-associated membrane protein 1 (LAMP1)). In some embodiments, the organelle complex comprises one or more peroxisomal proteins (e.g., catalase, ATP-binding cassette transporter 1, subfamily D, type 3 (ABCD3)). In some embodiments, the organelle complex comprises one or more Golgi apparatus proteins (e.g., Golgin-97, Syntaxin-6, TGOLN2 / trans-Golgi network protein 2 (TGN46), Golgi matrix protein 130 (GM130), mannosidase alpha class 2A member 1 (MAN2A1)). In some embodiments, the organelle complex comprises one or more endoplasmic reticulum proteins (e.g., calreticulin, calnexin).

[0010] In some embodiments, the organelle complex population comprises a first organelle complex, or a combination of a first organelle complex and a second organelle complex. In some embodiments, the first organelle complex is derived from (i) frozen cells, (ii) floating cells, and / or (iii) cells contacted with the surfactant at a concentration equal to or higher than the critical micelle concentration (CMC) of the surfactant. In some embodiments, the second organelle complex is derived from (i) adherent cells, and / or (ii) cells contacted with the surfactant at a concentration lower than the critical micelle concentration (CMC) of the surfactant. In some embodiments, the first organelle complex comprises one or more lysosomal proteins (e.g., lysosome-associated membrane protein 2 (LAMP2), mannose-6-phosphate receptor (M6PR), and / or lysosome-associated membrane protein 1 (LAMP1)) that are at least about 1.1-fold or more compared to the second organelle complex. In some embodiments, the first organelle complex comprises one or more peroxisomal proteins (e.g., catalase, ATP-binding cassette transporter 1, subfamily D, type 3 (ABCD3)) that are at least about 1.1-fold compared to the second organelle complex. The first organelle complex comprises one or more Golgi apparatus proteins (e.g., Golgin-97, Syntaxin-6, trans-Golgi network protein 2 (TGN46), Golgi matrix protein 130 (GM130), mannosidase alpha class 2A member 1 (MAN2A1)) that are at least about 1.1-fold or more compared to the second organelle complex. In some embodiments, the first organelle complex comprises one or more endoplasmic reticulum proteins (e.g., calreticulin, calnexin) that are at least about 1.1-fold or more compared to the second organelle complex. In some embodiments, the second organelle complex comprises one or more cytoplasmic proteins (e.g., p70S6K, glyceraldehyde 3-phosphate dehydrogenase (GAPDH)) that are at least 1.1-fold or more compared to the first organelle complex.

[0011] In some embodiments, when the organelle complex contacts the cell population, the organelle complex can be taken up into the cell. In some embodiments, at least about two-fold or more organelle complexes can be taken up into the cell as compared to a population of homogenized mitochondria. In some embodiments, when the organelle complex contacts the host cell population, the organelle complex is superior in the ability to be taken up into the host cell as compared to a population of homogenized mitochondria. In some embodiments, when the first organelle complex contacts the host cell population, the first organelle complex is superior in the ability to be taken up into the host cell as compared to the second organelle complex. In some embodiments, when the organelle complex contacts the host cell population, the organelle complex is superior in the ability to be taken up into the host cell. In some embodiments, at least two-fold or more organelle complexes can be taken up into the host cell as compared to a population of homogenized mitochondria. In some embodiments, the mitochondria of the organelle complex can be incorporated into cells after the population has undergone one or more freeze-thaw cycles. In some embodiments, at least two-fold the mitochondria of the organelle complex can be incorporated into cells after the population has undergone one or more freeze-thaw cycles as compared to a population of homogenized mitochondria. In some embodiments, at least about 80% of the organelle complex is between about 500 nm and about 3500 nm in size, and optionally between about 200 nm and about 1000 nm in size. In some embodiments, it is derived from cells treated with a mitochondrial activator (e.g., resveratrol).

[0012] This specification discloses a composition comprising the organelle complex population provided herein. This specification discloses a formulation comprising the composition provided herein (e.g., a composition comprising an organelle complex population) and a pharmaceutically acceptable carrier.

[0013] This specification discloses a method for generating a first population of organelle complexes. In some embodiments, the method includes incubating cells in a first solution containing a surfactant at a first temperature, removing the surfactant to form a second solution, and recovering a first organelle complex from the second solution. In some embodiments, the first organelle complex includes mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. In some embodiments, the first population of organelle complexes is depleted of cytoplasmic macromolecules. In some embodiments, (i) the cells in the first solution are incubated with the surfactant at a concentration above the critical micelle concentration (CMC) of the surfactant, and / or (ii) the cells include or are derived from floating cells or frozen cells.

[0014] In some embodiments, the method includes incubating the second solution at a second temperature. In some embodiments, the first organelle complex includes mitochondria and two, three, or four of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. In some embodiments, the cells in the first solution are contacted with the surfactant at a concentration at least 5% higher than the critical micelle concentration (CMC) of the surfactant. In some embodiments, the surfactant is saponin. In some embodiments, the surfactant is present at a concentration of about 50 μg / mL. In some embodiments, the surfactant is a nonionic surfactant. In some embodiments, the surfactant is selected from the group consisting of Triton-X100, Triton-X114, Nonidet® P-40, n-dodecyl-D-maltoside, Tween-20, Tween-80, saponin, and digitonin. In some embodiments, the first solution further includes a buffer. In some embodiments, the buffer includes one or more of an isotonic agent, an osmolyte, and a chelating agent. In some embodiments, the first solution includes a Tris buffer, sucrose, and / or a chelating agent.

[0015] In some embodiments, incubating the cells in the first solution includes incubating the cells in the first solution for about 1 minute to about 120 minutes, optionally for about 30 minutes. In some embodiments, the first temperature and / or the second temperature is from about 0°C to about 50°C, optionally the first temperature is 25°C and the second temperature is from about 0°C to about 4°C. In some embodiments, removing the surfactant includes washing one or more times with a buffer, optionally a Tris buffer. In some embodiments, incubating the second solution includes incubating the second solution for about 1 minute to about 120 minutes, optionally for about 20 minutes. Recovering the first organellar complex from the second solution can include tangential flow filtration (TFF). (i) Optionally, a low-viscosity buffer that reduces shear rate, (ii) about 22°C to about 25°C, (iii) a shear rate of less than about 2000 sec -1 -1, (iv) below room temperature, optionally 4°C, and / or (v) performing TFF with a buffer containing human albumin or recombinant albumin (HA). In some embodiments, recovering the first organellar complex from the second solution includes performing TFF using a TFF membrane with a molecular weight cut-off of at least 100 kDa, preferably 750 kDa. In some embodiments, recovering the first organellar complex from the second solution includes one or more centrifugation steps. In some embodiments, recovering the first organellar complex from the second solution includes centrifuging the second solution at a first centrifugal force, collecting the supernatant, centrifuging the supernatant at a second centrifugal force, and collecting the pellet to recover the first organellar complex. In some embodiments, the first centrifugal force and / or the second centrifugal force is from about 100 to about 5000 g, optionally the first centrifugal force is about 500 g and the second centrifugal force is about 3000 g. The centrifugation can be performed for about 10 minutes to about 20 minutes. In some embodiments, centrifuging the supernatant at a second centrifugal force includes centrifuging at 8000 g for about 20 minutes.

[0016] In some embodiments, incubating the cells in the first solution and / or incubating the second solution includes applying physical stimuli, optionally shaking and / or stirring, to the first solution and / or the second solution. In some embodiments, applying physical stimuli to the first solution and / or the second solution includes the first solution and / or the second solution flowing through a flow device. In some embodiments, the flow device includes a flow path that includes two or more portions with different cross-sectional diameters. In some embodiments, the cross-sectional diameter ranges from about 0.8 mm to about 25.4 mm. In some embodiments, flowing through the flow device creates additional flow and / or shear. In some embodiments, the method further includes optionally freezing the first organelle complex in a buffer containing a cryoprotectant. In some embodiments, the cryoprotectant includes human albumin (HA) and / or glycerol. In some embodiments, the method further includes treating the cells with a mitochondrial activator (e.g., resveratrol) prior to the incubating step. This specification discloses a first organelle complex population obtained by a method for generating the first organelle complex population provided herein.

[0017] This specification discloses a method of treating a disease or disorder. In some embodiments, the method includes contacting cells of a subject in need of treatment with an effective amount of (i) the organelle complex population provided herein, (ii) a composition provided herein (e.g., a composition comprising the organelle complex population), and / or (iii) a formulation provided herein, thereby treating the disease or disorder.

[0018] This specification discloses methods for treating diseases or disorders associated with mitochondrial dysfunction. In some embodiments, the method comprises contacting cells of a subject in need of treatment with an effective amount of (i) a population of organelle complexes provided herein, (ii) a composition provided herein (e.g., a composition comprising a population of organelle complexes), and / or (iii) a formulation provided herein, thereby treating a disease or disorder associated with mitochondrial dysfunction.

[0019] In some embodiments, contacting the cells of the subject comprises an administration route selected from the group consisting of intravenous administration, intraarterial administration, intratracheal administration, subcutaneous administration, intramuscular administration, inhalation, intrapulmonary administration, and intraocular administration. In some embodiments, the disease or disorder is selected from the group consisting of diabetes (type I and type II), metabolic diseases, eye disorders associated with mitochondrial dysfunction, hearing loss, mitochondrial toxicity associated with therapeutic agents, mitochondrial dysfunction associated with space travel, cardiac toxicity associated with chemotherapy or other therapeutic agents, mitochondrial dysfunction, and migraine. In some embodiments, the disease or disorder is selected from the group consisting of mitochondrial diseases, diabetes and deafness (DAD) syndrome, bark syndrome, Leber hereditary optic neuropathy (LHON), Leigh syndrome, NARP (neurogenic, ataxia, pigmentary retinopathy and ptosis syndrome), myoneurogastrointestinal encephalopathy (MNGIE), MELAS (mitochondrial encephalopathy, lactic acidosis and stroke-like episodes) syndrome, myoclonic epilepsy with ragged red fibers (MERRF) syndrome, Kearns-Sayre syndrome, and mitochondrial DNA depletion syndrome.

[0020] In some embodiments, the disease or disorder is an ischemia-related disease or disorder, a genetic disorder, an aging disease or disorder, a neurodegenerative condition, a cardiovascular disease, cancer, an autoimmune disease, an inflammatory disease, a fibrotic disorder, or any combination thereof. In some embodiments, the ischemia-related disease or disorder is selected from the group consisting of cerebral ischemia reperfusion, hypoxic ischemic brain tissue, acute coronary syndrome, myocardial infarction, liver ischemia reperfusion injury, ischemic compartment syndrome, vascular occlusion, wound healing, spinal cord injury, disease, and reperfusion injury of transplanted organs.

[0021] In some embodiments, the neurodegenerative condition is selected from the group consisting of dementia, Friedreich's ataxia, amyotrophic lateral sclerosis, mitochondrial encephalopathy, lactic acidosis, and strokelike episodes (MELAS), myoclonic epilepsy with ragged red fibers (MERFF), epilepsy, Parkinson's disease, Alzheimer's disease, or Huntington's disease. Examples of neurite diseases include, for example, bipolar disorder, schizophrenia, depression, addiction disorder, anxiety disorder, attention deficit hyperactivity disorder, personality disorder, autism spectrum disorder, and Asperger's syndrome. In some embodiments, the cardiovascular disease is selected from the group consisting of coronary heart disease, myocardial infarction, atherosclerosis, hypertension, cardiac arrest, cerebrovascular disease, peripheral arterial disease, rheumatic heart disease, congenital heart disease, congestive heart failure, arrhythmia, stroke, deep vein thrombosis, and pulmonary embolism. In some embodiments, the disease or disorder is acute respiratory distress syndrome (ARDS) or intrauterine growth restriction (IUGR).

Brief Description of the Drawings

[0022]

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DETAILED DESCRIPTION OF THE INVENTION

[0023] In the following detailed description, reference is made to the accompanying drawings, which form a part of this specification. In the drawings, like symbols typically identify like components unless the context dictates otherwise. The illustrative embodiments described in the embodiments of the invention, the drawings, and the claims are not meant to be limiting. Other embodiments may be utilized and other changes may be made without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that all aspects of the disclosure generally described herein and illustrated in the figures are fully contemplated herein and can be arranged, substituted, combined, separated, and designed in a variety of different configurations that form a part of the disclosure herein.

[0024] All patents, published patent applications, other publications, and sequences from GenBank, as well as other databases, referred to in this specification are hereby incorporated by reference in their entirety for the relevant art.

[0025] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994), Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For the purposes of this disclosure, the following terms are defined below.

[0026] As used herein, "isolated" shall be given its ordinary meaning and shall refer to a substance or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (in nature or in an experimental setting), and / or (2) produced, prepared, and / or manufactured by the hand of man. In some embodiments, isolated mitochondria or a population of isolated organelle complexes are processed for obtaining from a cellular environment via the methods provided herein.

[0027] As used herein, the term "cell" is given its ordinary meaning and also refers to eukaryotic cells, i.e., cells containing mitochondria in the cytoplasm, such as animal cells, such as mammalian cells, preferably human cells. As used herein, the term "cell" is used in the sense of including cells present in tissues, cells isolated from tissues (e.g., single cells), and cells within a population of cells (e.g., a population of cells obtained from a subject's tissue and / or a population of cells obtained from a cell line).

[0028] As used herein, the term "mitochondria" is given its ordinary meaning and also refers to an organelle present in eukaryotic cells having a double lipid membrane, an inner membrane and an outer membrane, and a matrix surrounded by the cristae membrane and the inner membrane. Mitochondria (plural mitochondria) have enzymes such as respiratory chain complexes involved in oxidative phosphorylation on their inner membranes. The inner membrane has a membrane potential due to, for example, an internal and external proton gradient formed by the action of the respiratory chain complexes. Mitochondria are thought to be unable to maintain the membrane potential when the inner membrane collapses.

[0029] As used herein, the term "organelle complex" shall have its ordinary meaning and shall refer to a complex of mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. The organelle complex can deplete cytoplasmic macromolecules (e.g., cytoplasmic proteins). In some embodiments, the organelle complex is free of cytoplasmic macromolecules. In some embodiments, the organelle complex population comprises homogenized mitochondria. As used herein, the term "population" shall have its ordinary meaning and shall refer to a group of multiple same or different substances. For example, an "organelle complex population" is a group of at least multiple same or different organelle complexes. The population may not always be homogeneous and may have a physical, chemical, and / or physiological distribution. The physical distribution includes, for example, particle size and polydispersity index. The chemical distribution includes, for example, zeta potential distribution and lipid composition distribution. The physiological distribution includes, for example, differences in physiological functions (e.g., respiratory activity). The organelle complex population can include a first organelle complex, a second organelle complex, homogenized mitochondria, or any combination thereof. As used herein, the term "homogenized mitochondria" shall have its ordinary meaning and shall refer to mitochondria isolated via a method including one or more homogenization steps.

[0030] As used herein, the term "surfactant" shall have its ordinary meaning and shall refer to a molecule having a hydrophilic portion and a hydrophobic portion in one molecule. The surfactant serves to reduce the surface tension at the interface or to mix polar and nonpolar substances by forming micelles. Surfactants are broadly classified into nonionic surfactants and ionic surfactants. Nonionic surfactants are those in which the hydrophilic portion is not ionized, and ionic surfactants are those in which the hydrophilic portion contains a cation or an anion or both a cation and an anion.

[0031] As used herein, the term "critical micelle concentration" (CMC) is given its ordinary meaning and refers to the concentration at which surfactants form micelles and additional surfactant added to the system contributes to micelle formation, particularly the concentration in bulk. At concentrations above the critical micelle concentration, adding surfactant to the system ideally increases the amount of micelles, particularly the number of micelles.

[0032] As used herein, "subject" refers to an animal that is the object of treatment, observation, or experiment. "Animal" includes cold-blooded vertebrates, warm-blooded vertebrates, and invertebrates such as fish, shellfish, reptiles, particularly mammals. "Mammal" as used herein refers to an individual belonging to the class of mammals, including but not limited to humans, domestic animals and livestock, zoo animals, sports and pet animals. Examples of mammals include, but are not limited to, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, and primates such as monkeys, chimpanzees, and apes, particularly humans. In some embodiments, the mammal is a human. However, in some embodiments, the mammal is not a human. As used herein, the term "host cell" is given its ordinary meaning and refers to in vivo cells, in vitro cells, and / or ex vivo cells into which the uptake of exogenous mitochondria and / or organelle complexes is intended.

[0033] As used herein, the term "treatment" refers to an intervention performed in response to a disease, disorder or physiological condition manifested by a patient. The purposes of treatment include, but are not limited to, one or more of alleviation or prevention of symptoms, delay or arrest of the progression or worsening of a disease, disorder or condition, and remission of a disease, disorder or condition. The terms "treating" and "treatment" include, for example, therapeutic treatment, prophylactic treatment, and uses for reducing the risk that a subject will develop a disorder or other risk factor. Treatment does not require complete cure of the disorder and encompasses embodiments that reduce symptoms or underlying risk factors. In some embodiments, "treatment" refers to both therapeutic and prophylactic or prophylactic means. Subjects in need of treatment include subjects already affected by a disease or disorder or undesirable physiological condition, as well as subjects in which a disease or disorder or undesirable physiological condition is prevented. As used herein, the term "prevention" refers to any activity that reduces the burden on an individual in whom those symptoms will later develop. This can be done at primary, secondary, and / or tertiary prevention levels, where a) primary prevention avoids the onset of symptoms / disorders / conditions, b) secondary prevention activities target the early stages of condition / disorder / symptom treatment, thereby increasing the opportunity for intervention to prevent the progression of the condition / disorder / symptom and the emergence of symptoms, and c) tertiary prevention reduces the adverse effects of an already established condition / disorder / symptom, for example, by restoring function and / or reducing any condition / disorder / symptom or associated complications. The term "preventing" does not require the elimination of 100% of the likelihood of an event. Rather, it indicates that the likelihood of the occurrence of an event has been reduced in the presence of the compound or method.

[0034] As used herein, the term "oxidative stress" shall have its ordinary meaning and shall also refer to an imbalance between the production of reactive oxygen species, reactive nitrogen species, and / or free radicals and the antioxidant capacity of a biological system.

[0035] As used herein, the term "effective amount" refers to an amount sufficient to produce a beneficial or desired biological and / or clinical result.

[0036] The methods, compositions, systems, and kits provided herein can, in some embodiments, be used in combination with the methods, compositions, systems, and kits described in PCT Patent Application Publication Nos. WO2018 / 092839, WO2017 / 090763, WO2020 / 230601, WO2019 / 164003, WO2020 / 054824, WO2020 / 203961, WO2020 / 054829, WO2021 / 015298, and WO2021 / 132735, the entire contents of which are incorporated herein by reference.

[0037] Organellar complex This specification discloses a population of organellar complexes. The organellar complexes can be isolated or derived from floating cells and / or frozen cells. The organellar complexes can be isolated or derived from cells contacted with a surfactant at a concentration above the critical micelle concentration (CMC) of the surfactant. The organellar complexes can include mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. This specification discloses a composition comprising the population of organellar complexes provided herein. This specification also discloses a formulation comprising a composition provided herein (e.g., a composition comprising a population of organellar complexes) and a pharmaceutically acceptable carrier. This specification also discloses a first population of organellar complexes obtained by a method for generating the first population of organellar complexes provided herein. These first organellar complexes can be suitable for use in the treatment of various diseases and disorders including those described herein, such as mitochondrial transplantation. In some embodiments, the organellar complexes (e.g., the first organellar complexes) are introduced into cells with severely reduced mitochondrial function and / or cells with a predominance of influx of functional mitochondria to restore and / or enhance mitochondrial function.

[0038] The organelle complexes provided in this specification (e.g., the first organelle complex, the second organelle complex) can include mitochondria and one, two, three, or four of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. The organelle complexes (e.g., the first organelle complex, the second organelle complex) can include (i) mitochondria and endoplasmic reticulum, (ii) mitochondria and peroxisomes, (iii) mitochondria and lysosomes, (iv) mitochondria and Golgi apparatus, (v) mitochondria, endoplasmic reticulum, and peroxisomes, (vi) mitochondria, endoplasmic reticulum, and lysosomes, (vii) mitochondria, endoplasmic reticulum, and Golgi apparatus, (viii) mitochondria, endoplasmic reticulum, peroxisomes, and lysosomes, (ix) mitochondria, endoplasmic reticulum, peroxisomes, and Golgi apparatus, (x) mitochondria, endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus, (xi) mitochondria, endoplasmic reticulum, lysosomes, and Golgi apparatus, (xii) mitochondria, peroxisomes, and lysosomes, (xiii) mitochondria, peroxisomes, and Golgi apparatus, (xiv) mitochondria, peroxisomes, lysosomes, and Golgi apparatus, and / or (xv) mitochondria, lysosomes, and Golgi apparatus. The ratio of mitochondria to additional organelles (e.g., endoplasmic reticulum, peroxisomes, lysosomes, and / or Golgi apparatus) within the organelle complex population can vary.For example, in some embodiments, the molar ratio of mitochondrial proteins to proteins associated with additional organelles (e.g., endoplasmic reticulum proteins, peroxisome proteins, lysosome proteins, and / or Golgi apparatus proteins) in a mitochondrial complex population can be 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65, 1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77, 1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89, 1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000, or a number or range between any two of these values, or the degree of those values.In some embodiments, the molar ratio of mitochondrial proteins to proteins associated with additional organelles (e.g., endoplasmic reticulum proteins, peroxisomal proteins, lysosomal proteins, and / or Golgi apparatus proteins) in a mitochondrial complex population can be 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1, 90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1, 3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, 10000:1, or a numerical value or range between any two of these values, or the degree of those values.

[0039] The organelle complex can deplete cytoplasmic macromolecules. The cytoplasmic macromolecules can be absent from the population of organelle complexes provided herein. The population of organelle complexes provided herein can contain a negligible amount and / or an undetectable amount of cytoplasmic macromolecules. The population of organelle complexes can be a substantially pure population of organelle complexes. A substantially pure population of organelle complexes can contain less than about 20% (e.g., about 20%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, 1%, 0.1%, 0.01%, 0.001%, 0%, or a range between any two of these values) of cytoplasmic macromolecules. The cytoplasmic macromolecules can include cytoplasmic proteins, and the abundance of one or more cytoplasmic proteins (e.g., p70S6K and / or glyceraldehyde 3-phosphate dehydrogenase (GAPDH)) can be depleted by at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) compared to the cells from which the population of organelle complexes is derived. The organelle complex can include one or more mitochondrial matrix proteins (e.g., mitochondrial transcription factor A (TFAM), citrate synthase (CS)). The organelle complex can include one or more outer mitochondrial membrane proteins (e.g., translocase of outer mitochondrial membrane 20 (TOMM20)). The organelle complex can include one or more lysosomal proteins (e.g., lysosome-associated membrane protein 2 (LAMP2), mannose-6-phosphate receptor (M6PR), lysosome-associated membrane protein 1 (LAMP1)). The organelle complex can include one or more peroxisomal proteins (e.g., catalase, ATP-binding cassette transporter 1, subfamily D, type 3 (ABCD3)).The organelle complex can include one or more Golgi apparatus proteins (e.g., Golgin-97, Syntaxin-6, TGOLN2 / trans-Golgi network protein 2 (TGN46), Golgi matrix protein 130 (GM130), mannosidase alpha class 2A member 1 (MAN2A1)). The organelle complex can include one or more endoplasmic reticulum proteins (e.g., calreticulin, calnexin).

[0040] At least about 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) of the organelle complexes in the population can be between about 500 nm and about 3500 nm in size (e.g., about 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1250 nm, 1500 nm, 1750 nm, 2000 nm, 2250 nm, 2500 nm, 2750 nm, 3000 nm, 3250 nm, 3500 nm, or a number or range between any two of these values). The organelle complexes can exhibit a size distribution of about 200 nm to 1000 nm and, in some embodiments, can exhibit two peaks. In some embodiments, the first organelle complex population exhibits a (large) shift in size distribution relative to the second organelle complex population. In some embodiments, the production scalability (e.g., time, reagent cost, and / or labor cost) of the first organelle complex is at least about 1.1 times (e.g., 1.1 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, or a number or range between any of these values) compared to the second organelle complex. The organelle complex population can contain at least 2 times (e.g., 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, or a number or range between any of these values) more mitochondrial DNA (mtDNA) copies than a population of homogenized mitochondria.The first population of organellar complexes can include a mitochondrial DNA (mtDNA) copy number and / or an ability to incorporate mtDNA into recipient cells that is at least two-fold (e.g., two-fold, three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, twenty-fold, thirty-fold, forty-fold, fifty-fold, sixty-fold, seventy-fold, eighty-fold, ninety-fold, one hundred-fold, or any number or range between these values) greater compared to the second population of organellar complexes.

[0041] The population of organellar complexes can include the first organellar complex, or a combination of the first organellar complex and the second organellar complex. The first organellar complex can be derived from (i) frozen cells, (ii) floating cells, and / or (iii) cells contacted with a surfactant at a concentration above the critical micelle concentration (CMC) of the surfactant. The second organellar complex can be derived from (i) adherent cells and / or (ii) cells contacted with a surfactant at a concentration below the critical micelle concentration (CMC) of the surfactant. The population of organellar complexes can be derived from cells treated with a mitochondrial activator (e.g., resveratrol).

[0042] In some embodiments, a second organelle complex is provided. In some embodiments, a method for isolating a second organelle complex from a cell comprises treating the cells in a first solution having a surfactant at a concentration below the critical micelle concentration (CMC) of the surfactant, removing the surfactant to form a second solution, incubating the cells in the second solution, and recovering the second organelle complex from the second solution. The first organelle complex can comprise one or more lysosomal proteins (e.g., lysosome-associated membrane protein 2 (LAMP2), mannose-6-phosphate receptor (M6PR), lysosome-associated membrane protein 1 (LAMP1)) that are at least about 1.1-fold (e.g., 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or a number or range between any of these values) or more compared to the second organelle complex. The first organelle complex can comprise one or more peroxisomal proteins (e.g., catalase, ATP-binding cassette transporter 1, subfamily D, type 3 (ABCD3)) that are at least about 1.1-fold (e.g., 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or a number or range between these) compared to the second organelle complex. The first organelle complex can comprise Golgi apparatus proteins (e.g., Golgin-97, Syntaxin-6, TGOLN2 / trans-Golgi network protein 2 (TGN46), Golgi matrix protein 130 (GM130), mannosidase alpha class 2A member 1 (MAN2A1)) that are at least about 1.1-fold (e.g., 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or a number or range between any of these values) or more compared to the second organelle complex.The first organelle complex can contain one or more endoplasmic reticulum lumen proteins (e.g., calreticulin, calnexin) that are at least about 1.1-fold (e.g., 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or any number or range between these values) or more compared to the second organelle complex. The second organelle complex can contain one or more cytoplasmic proteins (e.g., p70S6K, glyceraldehyde 3-phosphate dehydrogenase (GAPDH)) that are at least about 1.1-fold (e.g., 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or any number or range between these numbers) or more compared to the first organelle complex.

[0043] In some embodiments, at least about 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) of the mitochondria of the organelle complex maintain structural integrity in the extracellular environment. The structural integrity can include the structural integrity inside and / or outside the membrane of the mitochondria. 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 mitochondria of the organelle complex have intact inner and outer membranes. In some embodiments, at least two-fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or a number or range between any of these values) or more of the mitochondria of the organelle complex maintain structural integrity in the extracellular environment compared to a population of homogenized mitochondria in the extracellular environment. The population of organelle complexes provided herein has a high calcium (Ca 2+) It has the surprising feature of maintaining structural integrity and / or functionality even when exposed to the environment. The extracellular environment can contain a total calcium concentration of about 1 to about 20 mg / dL and / or a free / active calcium concentration of about 1 to about 6 mg / dL. The extracellular environment may contain 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 contains 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 contains a concentration of free or active calcium 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 mitochondria of the organelle complex maintain functional capacity in an environment with a higher calcium concentration compared to the intracellular calcium environment. Thus, the mitochondria of the organelle complex provided herein are isolated with minimal or negligible damage from the cell environment (including frozen or floating cells) and maintain the ability to function even when exposed to an extracellular environment, such as a calcium-rich environment, that is expected to damage the mitochondria and / or significantly inhibit their functional capacity.

[0044] In some embodiments, the inner membrane structural integrity and / or outer membrane structural integrity (e.g., an intact inner membrane and / or outer membrane) can be determined by the functional activity of the mitochondria, such as membrane potential and polarization. The structural integrity can be measured by citrate synthase (CS) activity and / or cytochrome c oxidase (COX) activity. In some embodiments, at least about 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) of the mitochondria of the organelle complex maintain functionality. The mitochondria of the organelle complex can be capable of ATP production in some embodiments provided herein. The ATP production of the organelle complex population provided herein can exceed the ATP production of homogenized mitochondria by at least about 2-fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or a number or range between any of these values) in some embodiments. In some embodiments, the functional ability in the extracellular environment is measured by a fluorescent indicator of membrane potential. In some embodiments, the fluorescent indicator is selected from positively charged dyes such as JC-1, TMRM, and TMRE.

[0045] The organelle complex population may have various characteristics that facilitate the delivery of a payload, such as a desired transgene or an exogenous agent, to a target cell. The transgene can encode a therapeutic protein. The exogenous agent can be selected from the group consisting of nucleic acid molecules, proteins or polypeptides, small molecules, hormones, and any combination thereof. The exogenous agent can include a viral vector, a bacterial vector, a plasmid vector, or any combination thereof. In some embodiments, the exogenous agent comprises a nucleic acid molecule selected from the group consisting of ribonucleic acid, small RNA molecule, complementary RNA, non-coding RNA molecule, siRNA, pi-RNA molecule, microRNA molecule, sno-RNA molecule, long non-coding RNA molecule, messenger RNA molecule, ribosomal RNA molecule, antisense nucleic acid molecule, locked nucleic acid (LNA), antagomir, CRISPR / Cas gene editing RNA, trans-activating crRNA (tracrRNA), short synthetic RNA (gRNA) consisting of a "scaffold" sequence, small Cajal body-specific RNA (scaRNA), natural cis-antisense siRNA (cis-nat-siRNA), trans-activating siRNA (tasiRNA), repeat-associated small interfering RNA (rasiRNA), 7SK, transfer-messenger RNA (tmRNA), transfer RNA (tRNA), 7SL RNA, signal recognition particle RNA (SRP), or any combination thereof.

[0046] When an organelle complex contacts a cell population, the organelle complex provided herein can be taken up into the cell. In some embodiments, the intracellular incorporation includes co-localization and / or fusion with endogenous mitochondria within the cell. The cells can be in vivo, in vitro or ex vivo. In some embodiments, at least about 2-fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or any number or range between these values) or more of the organelle complex can be taken up into the cell as compared to a population of homogenized mitochondria. When the organelle complex contacts a host cell population, the organelle complex is superior in the ability to be taken up by the host cell as compared to a population of homogenized mitochondria. When a first organelle complex contacts a host cell population, the first organelle complex is superior in the ability to be taken up by the host cell as compared to a second organelle complex. In some embodiments, at least about 2-fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or any number or range between these values) or more of the organelle complex can be taken up into the cell as compared to the second organelle complex. When the organelle complex contacts a host cell population, the organelle complex is superior in the ability to be taken up by the host cell. In some embodiments, at least 2-fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or any number or range between these values) or more of the organelle complex can be taken up into the host cell as compared to a population of homogenized mitochondria. In some embodiments, without being particularly bound by theory, the superior uptake ability of the organelle complex provided herein is at least partly responsible for the superior clinical effect exerted by the organelle complex when used in the treatment of any disease or disorder as described herein.

[0047] In some embodiments, the population of organelle complexes provided herein can be incorporated into cells after storing mitochondria at any temperature provided herein (e.g., 4°C ± 3°C, -20°C ± 3°C, -80°C ± 3°C, or in liquid nitrogen). The mitochondria of the organelle complexes can be incorporated into cells after the population has undergone one or more freeze-thaw cycles. At least two-fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or any number or range between these values) or more mitochondria of the organelle complexes can be incorporated into cells after the population has undergone one or more freeze-thaw cycles as compared to a population of homogenized mitochondria.

[0048] Method for generating a first population of organelle complexes Disclosed herein is a method for generating a first population of organelle complexes. In some embodiments, the method includes incubating cells in a first solution containing a surfactant at a first temperature, removing the surfactant to form a second solution, and recovering the first organelle complexes from the second solution. The first organelle complexes can include mitochondria and one or more of endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. The first population of organelle complexes can be depleted of cytoplasmic macromolecules. In some embodiments, (i) the cells in the first solution are incubated with the surfactant at a concentration above the critical micelle concentration (CMC) of the surfactant, and / or (ii) the cells include or are derived from suspension cells or frozen cells.

[0049] In some embodiments, a method for generating a first organelle complex is provided. The generation of the first organelle complex includes (step A) providing adherent cells, floating cells, and / or frozen cells, thawing them, and placing them in a tube. The generation of the first organelle complex includes (step A) providing adherent cells, floating cells, and / or frozen cells, centrifuging them, and collecting the precipitate. The generation of the first organelle complex includes (step A) providing adherent cells, aspirating, adding a solution (e.g., PBS(-)), aspirating, adding TrypLE, incubating, adding a solution (e.g., PBS(-)), placing the cell suspension in a tube, centrifuging, and collecting the precipitate. The generation of the first organelle complex can include one or more of the steps of (step B) adding Tris buffer, centrifuging, and collecting the precipitate, (step C) adding Tris buffer and vortexing, (step D) adding a solution containing a surfactant and incubating, (step E) centrifuging to collect the precipitate, (step F) adding Tris buffer, centrifuging to collect the precipitate, (step G) adding Tris buffer and pipetting, (step H) transferring to another tube and collecting and washing away the buffer solution in the original tube, (step I) centrifuging to collect the supernatant, (step J) centrifuging to collect the precipitate, (step K) pipetting the above steps. One or more of the above steps can include an incubation period. One or more of the above steps can include a centrifugation step followed by collection of the supernatant and / or precipitate. One or more of the above steps can be omitted and one or more additional steps can be included. Depending on the embodiment, the time, volume, concentration, and centrifugal force can be varied.

[0050] In some embodiments, the cells used in the disclosed methods for generating a first population of organelle complexes can be adherent cells, floating cells, detached cells, suspension cells, frozen cells, or any combination thereof. The cells can be in the form of cells present in a tissue, or they can be isolated from a tissue (e.g., single cells) or a population thereof. Cells isolated from a tissue can be cultured cells, or they can be single cells or a population thereof obtained by treating a tissue or cultured cells with an enzyme (such as collagenase) used to make them into single cells. Also, before enzyme treatment such as collagenase, the tissue can be cut if desired. The method can further include treating the cells with a mitochondrial activator (e.g., resveratrol) before the step of incubating the cells in the first solution. The first organelle complex can be derived from the cells, and the mitochondria are activated. Activation of the mitochondria can be achieved by various methods, for example, by contacting the mitochondria with a mitochondrial activator. Such activation of the mitochondria can be achieved by various methods including MITO-Porter technology. In MITO-Porter technology, a complex of a mitochondrial targeting carrier and a mitochondrial activator may be used. As used herein, the term "mitochondrial activator" refers to a substance that can activate the mitochondrial respiratory chain complex (electron transport system), particularly a substance that can polarize mitochondria with a membrane potential, and particularly preferably a substance that can hyperpolarize mitochondria. Examples of mitochondrial activators may 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 particularly preferably resveratrol (see WO2018 / 092839).As mitochondrial activators, mitochondrial DNA, mitochondrial RNAs such as 12S rRNA and 16S rRNA (see W02020 / 230601, which is hereby incorporated by reference in its entirety), and any other components of mitochondria may also be mentioned.

[0051] Step A can include adding a solution of about 1 mL to about 10 mL (e.g., PBS(-)). Step A can include adding about 1 mL to about 5 mL of TrypLE. Step A can include incubating at about 30 °C to 40 °C for about 2 min to about 10 min. Step A can include thawing at a temperature of about 30 °C to 42 °C for about 1 min to about 7 min. Step A can include using adherent cells from a dish of about 5 cm to about 20 cm. Steps A, B, E, and / or I can include centrifuging at about 200 g to about 800 g, at about 0 °C to 10 °C for about 5 min to about 20 min. Steps A and / or H can include using a tube of about 10 mL to about 75 mL. Steps B, C, F, and / or G can include adding about 0.5 mL to about 6 mL of Tris buffer. Step C can include vortexing for about 5 sec to about 20 sec. Step D can include adding a solution containing a surfactant of about 0.5 mL to about 2 mL. The surfactant can be present at a concentration of about 50 μg / mL to about 200 μg / mL in the solution containing the surfactant. The final concentration of the surfactant after addition in Step D can be about 25 μg / mL to about 75 μg / mL. Step D can include incubating at about 18 °C to 28 °C for about 20 min to about 40 min. Step F can include centrifuging at about 500 g to about 1500 g, at about 0 °C to 10 °C for about 1 min to about 10 min. Step G can include incubating at about 0 °C to 5 °C for about 10 min to about 30 min. Steps G and / or K can include pipetting about 5 times to about 30 times. Step H can include collecting about 0.5 mL to about 2 mL of the buffer solution into the original tube and washing it away. Step J can include centrifuging at about 2000 g to about 4000 g, at about 0 °C to 10 °C for about 5 min to about 20 min. Step J can include centrifuging at about 6000 g to about 10000 g, at about 0 °C to 10 °C for about 10 min to about 30 min.The collection of the precipitate can include removing the supernatant (e.g., 0.5 mL to about 2.5 mL).

[0052] In some embodiments, a first generation method (e.g., a method based on centrifugation) is provided for generating a first organelle complex that can include some or all of the steps provided above (e.g., step A, step B, step C, step D, step E, step F, step G, step H, step I, step J, and / or step K). In some embodiments, a second generation method (e.g., the TFF method) is provided for generating a first organelle complex that can include one or more substitutions (and / or additions thereto) of step A, step B, step C, step D, step E, step F, step G, step H, step I, step J, and / or step K. For example, saponin treatment and / or extraction of the first organelle complex can include the use of a flow device (e.g., a reducing agent flow device). The flow device can include a fluid channel that includes two or more portions with different cross-sectional diameters. In some embodiments, without being particularly limited by theory, flowing through the reducing agent flow device can result in a change in flow velocity due to a change in the cross-sectional area of the flow path. The reducing agent flow device provided herein can have various configurations, such as, for example, a square reducer, a tapered reducer, a concentric reducer, and / or an eccentric reducer. The flow device can include tubes of various types and sizes to create additional flow and shear for the extraction of the first organelle complex. Lysate polishing can include prefiltration and / or Rotea. Tangential flow filtration (TFF) can be used for purification and / or buffer exchange in some embodiments of the second generation method (e.g., the TFF method) provided herein. Step B can include the use of Rotea. Step G can include the use of a flow device (e.g., a reducing agent flow device). Step I can include cell lysate polishing (prefilter, Rotea, etc.). Step J can include TFF. Step K can include the use of a cryoprotectant containing human albumin (HA) and / or glycerol.By using HA and / or glycerol as a storage buffer, the stability of the first organelle complex during storage at low temperatures (e.g., -80 °C) can be enhanced, and their quality after thawing can be improved.

[0053] Cells in the first solution can be contacted with a surfactant at a concentration that is at least about 5% (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50%, 75%, 100% or more, and overlapping ranges thereof) greater than the critical micelle concentration (CMC) of the surfactant. The surfactant can be saponin and / or digitonin. The surfactant can be present at a concentration of about 50 μg / mL. The surfactant can be a nonionic surfactant. The surfactant can be selected from the group consisting of Triton-X100, Triton-X114, Nonidet® P-40, n-dodecyl-D-maltoside, Tween-20, Tween-80, saponin, and digitonin.

[0054] In some embodiments, the surfactant used in the methods provided herein may be an ionic surfactant or a non-ionic surfactant. The non-ionic surfactants used in this disclosure may include, for example, in the form of esters, ethers, and alkyl glycosides. Non-ionic surfactants include, for example, alkyl polyethylene glycols, polyoxyethylene alkyl phenyl ethers, and alkyl glycosides. Non-ionic surfactants may include Triton-X100, Triton-X114, Nonidet® P-40, n-dodecyl-D-maltoside, Tween-20, Tween-80, saponin and / or digitonin.The concentration of the surfactant(s) present in the first solution can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 128, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000 μg / mL or μM, or a number or range between any two of these values, the degree of those numbers, at least those numbers, or at most those numbers.

[0055] In some embodiments, the first solution and / or the second solution can contain a buffer. The buffer can contain one or more of an isotonic agent, an osmotic regulator, and a chelating agent. The first solution and / or the second solution can contain a Tris buffer, sucrose, and / or a chelating agent. Exemplary buffers used in the methods provided herein include, for example, Tris buffer, HEPES buffer, and phosphate buffer. The buffer can be, for example, at pH 6.7 to 7.6 (for example, pH 6.8 to 7.4, pH 7.0 to 7.4, for example, pH 7.2 to 7.4, for example, pH 7.4). In some embodiments, the buffer can contain an isotonic agent and an osmotic regulator. Examples of isotonic agents and osmotic regulators include monosaccharides (such as glucose, galactose, mannose, fructose, inositol, ribose, xylose, etc.), disaccharides (such as lactose, sucrose, cellobiose, trehalose, maltose, etc.), trisaccharides (such as raffinose, melibiose, etc.), polysaccharides (such as cyclodextrin, etc.), sugar alcohols (such as erythritol, xylitol, sorbitol, mannitol, maltitol, etc.), glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, and polyethylene glycol, etc. The buffer can also contain a chelating agent, particularly a chelating agent for divalent metals such as a chelating agent for calcium ions. Chelating agents include, for example, glycol ether diamine tetraacetic acid (EGTA) and ethylenediamine tetraacetic acid (EDTA).

[0056] The method can include incubating the second solution at a second temperature. The first temperature and / or the second temperature can be from about 0°C to about 50°C. The first temperature can be 25°C, and the second temperature can be from about 0 to about 4°C. The first temperature and / or the second temperature can be 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, or a number or range between any two of these values, to the extent of those numbers, at least those numbers, or at most those numbers. Removing the surfactant can include washing one or more times with a buffer (e.g., Tris buffer). One or more steps of the methods provided herein can be performed on ice or at room temperature.

[0057] Incubating the cells in the first solution can include incubating the cells in the first solution for about 1 minute to about 120 minutes, optionally for about 30 minutes. Incubating the second solution can include incubating the second solution for about 1 minute to about 120 minutes, optionally for about 20 minutes. The step of incubating the cells in the first solution and / or the step of incubating the second solution can include a period of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 128, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500 minutes, or a number or range of periods between any two of these values. Incubating the cells in the first solution and / or incubating the second solution can include applying physical stimuli, such as pipetting, shaking, and / or stirring, to the first solution and / or the second solution, respectively. Applying physical stimuli to the first solution and / or the second solution can include the first solution and / or the second solution flowing through a flow device (e.g., a reducing agent flow device). The above flow device can include a flow path including two or more portions with different cross-sectional diameters. The above cross-sectional diameter can be about 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 21 mm, 22 mm, 23 mm, 24 mm, 25 mm, or a number or range thereof, the degree of those numbers, at least those numbers, or at most those numbers.In some embodiments, additional rolling and / or shearing occurs by flowing through the flow device.

[0058] The step of removing the surfactant to form the second solution can include reducing the concentration of the surfactant in the solution contacted by the first organelle complex to, for example, less than 10%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% of the concentration of the surfactant, or below the detection limit in the solution contacted by the first organelle complex. To ensure removal of the surfactant from the solution, removing the surfactant to form the second solution may include washing the cells with a solution containing a lower or reduced concentration of surfactant (preferably, a surfactant-free solution). To remove the surfactant from the first solution, the solution added to or exchanged with the first solution can be a buffer, and in some embodiments, is the buffer used for the first solution (however, a solution containing a lower concentration of surfactant, preferably, a surfactant-free solution or a solution having a non-detectable level of surfactant). The second solution can be a solution containing a lower concentration of surfactant. In some embodiments, the second solution is a surfactant-free solution or a solution having a negligible amount and / or non-detectable amount of surfactant. The solution used in the recovery step (e.g., the second solution) can be, for example, a solution containing a buffer, an osmotic adjuster, and a divalent metal chelator that is substantially free of surfactant. As used herein, "substantially free of" is used in the sense of not precluding the presence of contaminants in an amount of the "substantially free of component" that cannot be removed or detected.

[0059] In some embodiments, recovering the first organelle complex comprises subjecting the second solution to one or more physical stimuli. The recovery step can include incubating the second solution at a second temperature. Thus, the recovery step can be performed under shaking conditions or non-shaking conditions. The incubation of the recovery step can be performed under stirring conditions or non-stirring conditions. The first organelle complex can be collected as a precipitate by performing one or more centrifugation steps on the second solution. Recovering the first organelle complex from the second solution can include one or more centrifugation steps. Recovering the first organelle complex from the second solution can include centrifuging the second solution at a first centrifugal force, collecting the supernatant, centrifuging the supernatant at a second centrifugal force, and collecting the pellet to recover the first organelle complex. The first centrifugal force and / or the second centrifugal force can be from about 100 g to about 5000 g. The first centrifugal force can be about 500 g and the second centrifugal force can be about 3000 g.The first centrifugal force and / or the second centrifugal force can be 100 g, 110 g, 120 g, 128 g, 130 g, 140 g, 150 g, 160 g, 170 g, 180 g, 190 g, 200 g, 210 g, 220 g, 230 g, 240 g, 250 g, 260 g, 270 g, 280 g, 290 g, 300 g, 310 g, 320 g, 330 g, 340 g, 350 g, 360 g, 370 g, 380 g, 390 g, 400 g, 410 g, 420 g, 430 g, 440 g, 450 g, 460 g, 470 g, 480 g, 490 g, 500 g, 510 g, 520 g, 530 g, 540 g, 550 g, 560 g, 570 g, 580 g, 590 g, 600 g, 610 g, 620 g, 630 g, 640 g, 650 g, 660 g, 670 g, 680 g, 690 g, 700 g, 710 g, 720 g, 730 g, 740 g, 750 g, 760 g, 770 g, 780 g, 790 g, 800 g, 810 g, 820 g, 830 g, 840 g, 850 g, 860 g, 870 g, 880 g, 890 g, 900 g, 910 g, 920 g, 930 g, 940 g, 950 g, 960 g, 970 g, 980 g, 990 g, 1000 g, 1100 g, 1200 g, 1300 g, 1400 g, 1500 g, 1600 g, 1700 g, 1800 g, 1900 g, 2000 g, 2100 g, 2200 g, 2300 g, 2400 g, 2500 g, 2600 g, 2700 g, 2800 g, 2900 g, 3000 g, 3250 g, 3500 g, 3750 g, 4000 g, 4250 g, 4500 g, 4750 g, 5000 g, 5500 g, 6000 g, 6500 g, 7000 g, 7500 g, 8000 g, 8500 g, 9000 g, 9500 g, 10000 g, or a number or range between any two of these numbers, or to the extent of these numbers, or at least these numbers, or at most these numbers.The centrifugation step can include a period of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 128, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500 minutes, or a number or range of periods between any two of these values. Centrifugation can be performed for about 10 minutes to about 20 minutes. Centrifuging the supernatant at a second centrifugal force can include centrifuging at 8000g for about 20 minutes. Recovering the first organelle complex from the second solution can include tangential flow filtration (TFF). TFF can be performed with a low-viscosity buffer, and in some embodiments, the low-viscosity buffer reduces the shear rate.The viscosity of the TFF buffer can be 1 centipoise (cP), 2 cP, 3 cP, 4 cP, 5 cP, 6 cP, 7 cP, 8 cP, 9 cP, 10 cP, 11 cP, 12 cP, 13 cP, 14 cP, 15 cP, 16 cP, 17 cP, 18 cP, 19 cP, 20 cP, 21 cP, 22 cP, 23 cP, 24 cP, 25 cP, 26 cP, 27 cP, 28 cP, 29 cP, 30 cP, 31 cP, 32 cP, 33 cP, 34 cP, 35 cP, 36 cP, 37 cP, 38 cP, 39 cP, 40 cP, 41 cP, 42 cP, 43 cP, 44 cP, 45 cP, 46 cP, 47 cP, 48 cP, 49 cP, 50 cP, 51 cP, 52 cP, 53 cP, 54 cP, 55 cP, 56 cP, 57 cP, 58 cP, 59 cP, 60 cP, 61 cP, 62 cP, 63 cP, 64 cP, 65 cP, 66 cP, 67 cP, 68 cP, 69 cP, 70 cP, 71 cP, 72 cP, 73 cP, 74 cP, 75 cP, 76 cP, 77 cP, 78 cP, 79 cP, 80 cP, 81 cP, 82 cP, 83 cP, 84 cP, 85 cP, 86 cP, 87 cP, 88 cP, 89 cP, 90 cP, 91 cP, 92 cP, 93 cP, 94 cP, 95 cP, 96 cP, 97 cP, 98 cP, 99 cP, 100 cP, 200 cP, 300 cP, 400 cP, 500 cP, 600 cP, 700 cP, 800 cP, 900 cP, 1000 cP, 2500 cP, 5000 cP, 7500 cP, 10000 cP, or a number or range between any two of these values, the degree of these numbers, at least these numbers, or at most these numbers. The temperature at which TFF is performed can be 0 °C, 1 °C, 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 11 °C, 12 °C, 13 °C, 14 °C, 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45 °C, 46 °C, 47 °C, 48 °C, 49 °C, 50 °C, or a number or range between any two of these values, the degree of those numbers, at least those numbers, or at most those numbers.The shear rate of the TFF procedure and / or the flow device (e.g., the reducing agent flow device) is 1 sec. -1 , 2 sec -1 , 3 sec -1 , 4 sec -1 , 5 sec -1 , 6 sec -1 , 7 sec -1 , 8 sec -1 , 9 sec -1 , 10 sec -1 , 11 sec -1 , 12 sec -1 , 13 sec -1 , 14 sec -1 , 15 sec -1 , 16 sec -1 , 17 sec -1 , 18 sec -1 , 19 sec -1 , 20 sec -1 , 25 sec -1 , 30 sec -1 , 35 sec -1 , 40 sec -1 , 45 sec -1 , 50 sec -1 , 60 sec -1 , 70 sec -1 , 80 sec -1 , 90 sec -1 , 100 sec -1 , 110 sec -1 , 120 sec -1 , 128 sec -1 , 130 sec -1 , 140 sec -1 , 150 sec -1 , 160 sec -1 , 170 sec -1 , 180 sec -1 , 190 sec -1 , 200 sec -1 , 210 sec -1 , 220 sec -1 , 230 sec -1 , 240 sec -1 , 250 sec -1 , 260 sec -1 , 270 sec -1 , 280 sec -1, 290 sec -1 , 300 sec -1 , 310 sec -1 , 320 sec -1 , 330 sec -1 , 340 sec -1 , 350 sec -1 , 360 sec -1 , 370 sec -1 , 380 sec -1 , 390 sec -1 , 400 sec -1 , 410 sec -1 , 420 sec -1 , 430 sec -1 , 440 sec -1 , 450 sec -1 , 460 sec -1 , 470 sec -1 , 480 sec -1 , 490 sec -1 , 500 sec -1 , 510 sec -1 , 520 sec -1 , 530 sec -1 , 540 sec -1 , 550 sec -1 , 560 sec -1 , 570 sec -1 , 580 sec -1 , 590 sec -1 , 600 sec -1 , 610 sec -1 , 620 sec -1 , 630 sec -1 , 640 sec -1 , 650 sec -1 , 660 sec -1 , 670 sec -1 , 680 sec -1 , 690 sec -1 , 700 sec -1 , 710 sec -1 , 720 sec -1 , 730 sec -1 , 740 sec -1 , 750 sec -1 , 760 sec -1 , 770 sec -1 , 780 sec -1, 790 sec -1 , 800 sec -1 , 810 sec -1 , 820 sec -1 , 830 sec -1 , 840 sec -1 , 850 sec -1 , 860 sec -1 , 870 sec -1 , 880 sec -1 , 890 sec -1 , 900 sec -1 , 910 sec -1 , 920 sec -1 , 930 sec -1 , 940 sec -1 , 950 sec -1 , 960 sec -1 , 970 sec -1 , 980 sec -1 , 990 sec -1 , 1000 sec -1 , 1100 sec -1 , 1200 sec -1 , 1300 sec -1 , 1400 sec -1 , 1500 sec -1 , 1600 sec -1 , 1700 sec -1 , 1800 sec -1 , 1900 sec -1 , 2000 sec -1 , 2100 sec -1 , 2200 sec -1 , 2300 sec -1 , 2400 sec -1 , 2500 sec -1 , 2600 sec -1 , 2700 sec -1 , 2800 sec -1 , 2900 sec -1 , 3000 sec -1 , 3250 sec -1 , 3500 sec -1 , 3750 sec -1 , 4000 sec -1 , 4250 sec -1 , 4500 sec -1 , 4750 sec-1 、5000 seconds -1 、5500 seconds -1 、6000 seconds -1 、6500 seconds -1 、7000 seconds -1 、7500 seconds -1 、8000 seconds -1 、8500 seconds -1 、9000 seconds -1 、9500 seconds -1 、10000 seconds -1 or can be a number or range between any two of these values, can be the degree of those numbers, can be at least those numbers, or can be at most those numbers. TFF can be performed with a buffer containing human albumin (HA). Recovering the first organelle complex from the second solution can include TFF using a TFF membrane. The molecular weight cut-off of the TFF membrane is 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, 15 kDa, 16 kDa, 17 kDa, 18 kDa, 19 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 128 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, 200 kDa, 210 kDa, 220 kDa, 230 kDa, 240 kDa, 250 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, 300 kDa, 310 kDa, 320 kDa, 330 kDa, 340 kDa, 350 kDa, 360 kDa, 370 kDa, 380 kDa, 390 kDa, 400 kDa, 410 kDa, 420 kDa, 430 kDa, 440 kDa, 450 kDa, 460 kDa, 470 kDa, 480 kDa, 490 kDa, 500 kDa, 510 kDa, 520 kDa, 530 kDa, 540 kDa, 550 kDa, 560 kDa, 570 kDa, 580 kDa, 590 kDa, 600 kDa, 610 kDa, 620 kDa, 630 kDa, 640 kDa, 650 kDa, 660 kDa, 670 kDa, 680 kDa, 690 kDa, 700 kDa, 710 kDa, 720 kDa, 730 kDa, 740 kDa, 750 kDa, 760 kDa, 770 kDa, 780 kDa, 790 kDa, 800 kDa, 810 kDa, 820 kDa, 830 kDa, 840 kDa, 850 kDa, 860 kDa, 870 kDa, 880 kDa, 890 kDa, 900 kDa, 910 kDa, 920 kDa, 930 kDa, 940 kDa, 950 kDa, 960 kDa, 970 kDa, 980 kDa, 990 kDa, 1000 kDa, 1100 kDa, 1200 kDa, 1300 kDa, 1400 kDa,1500 kDa, 1600 kDa, 1700 kDa, 1800 kDa, 1900 kDa, 2000 kDa, 2100 kDa, 2200 kDa, 2300 kDa, 2400 kDa, 2500 kDa, 2600 kDa, 2700 kDa, 2800 kDa, 2900 kDa, 3000 kDa, 3250 kDa, 3500 kDa, 3750 kDa, 4000 kDa, 4250 kDa, 4500 kDa, 4750 kDa, 5000 kDa, 5500 kDa, 6000 kDa, 6500 kDa, 7000 kDa, 7500 kDa, 8000 kDa, 8500 kDa, 9000 kDa, 9500 kDa, 10000 kDa, or a number or range between any two of these values, the degree of those numbers, at least those numbers, or at most those numbers.

[0060] The method can further include freezing the organelle complex. Freezing can be performed by gently suspending the organelle complex in a freezing buffer (e.g., a storage buffer). The freezing buffer may be the buffer used in the first solution, may not contain a surfactant, and may further contain a cryoprotectant. The cryoprotectant can include human albumin (HA) and / or glycerol. The proportion of glycerol can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or a number or range between any two of these values, can be to the extent of those numbers, can be at least those numbers, or can be at most those numbers.The proportion of HA can be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.20%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.30%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.40%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.50%, 0.51%, 0.52%, 0.53%, 0.54%, 0.55%, 0.56%, 0.57%, 0.58%, 0.59%, 0.60%, 0.61%, 0.62%, 0.63%, 0.64%, 0.65%, 0.66%, 0.67%, 0.68%, 0.69%, 0.70%, 0.71%, 0.72%, 0.73%, 0.74%, 0.75%, 0.76%, 0.77%, 0.78%, 0.79%, 0.80%, 0.81%, 0.82%, 0.83%, 0.84%, 0.85%, 0.86%, 0.87%, 0.88%, 0.89%, 0.90%, 0.91%, 0.92%, 0.93%, 0.94%, 0.95%, 0.96%, 0.97%, 0.98%, 0.99%, 1.00%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or a number or range between any two of these values, the degree of those numbers, at least those numbers, or at most those numbers. Exemplary cryoprotectants are known in the art and include, for example, glycerin, sucrose, trehalose, dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, diethyl glycol, triethylene glycol, glycerol-3-phosphate, proline, sorbitol, formamide, and polymers.Accordingly, the organelle complexes provided herein can be stored by freezing. In the methods of the present disclosure, the organelle complexes need not be frozen if freezing storage is not required; for example, the organelle complexes may be used when newly isolated. In other embodiments, the organelle complexes may be stored at 4°C ± 3°C or on ice. In some embodiments, the organelle complexes provided herein produced by the methods provided herein may 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, the method further includes thawing the organelle complexes after freezing. In some embodiments, the method of thawing the organelle complexes includes thawing the organelle complexes rapidly, for example, within about 5 minutes or within about 1 minute. In some embodiments, the organelle complexes are thawed in a warm bath at a temperature of about 20°C ± 3°C to about 37°C ± 3°C. In some embodiments, the organelle complexes are thawed at a temperature of about 20°C ± 3°C or below. In some embodiments, the organelle complexes may be stored for days, weeks, months or more and retain the ability to function after thawing.

[0061] In some embodiments, the methods provided herein do not include homogenization. In some embodiments, the method includes a homogenization treatment, but the homogenization is performed only to the extent that it does not create bubbles in the solution with respect to any bubbles or cells or tissues. In some embodiments, the method also does not include freeze-thawing of cells. In some embodiments, the methods of this disclosure do not require one or more filtration steps when purifying the organelle complex recovered from cells. In some embodiments, the method does not include the application of shear forces (e.g., down-sizing, needle passage) and / or the addition of proteases to the cells, the first solution, and / or the second solution. In some embodiments, the methods of this disclosure do not include other methods of disrupting the cell membrane (e.g., sonication, treatment with a strong water stream that causes the solution to form bubbles, or treatment with a strong water stream that causes the solution to foam) throughout the step of recovering the first organelle complex from the cells. In some embodiments, the methods of this disclosure can perform freeze-thaw cycles on the organelle complex for storage, but are performed without performing any treatment that may substantially cause physical, chemical, or physiological damage to the organelle complex. Thus, the methods of this disclosure can obtain the organelle complex with minimal damage.

[0062] In some embodiments, the methods provided herein include encapsulating the first organelle complex in a lipid membrane-based vesicle. In some embodiments, provided is a method of generating a composition comprising a lipid membrane-based vesicle or vesicles encapsulating an organelle complex or a population thereof, the method comprising contacting and mixing an aqueous solution comprising the organelle complex with an organic phase (e.g., an ethanol solution) comprising a lipid capable of forming a lipid membrane in a confluence channel within a microfluidic device. The method of encapsulating in lipid membrane-based vesicles is disclosed in PCT Patent Application Publication No. WO2021 / 132735, the entire contents of which are incorporated herein by reference.

[0063] Treatment method In some embodiments, methods of treating or preventing a disease or disorder are provided. In some embodiments, the method comprises contacting cells of a subject in need of treatment with an effective amount of (i) the population of organelle complexes provided herein, (ii) a composition provided herein (e.g., a composition comprising a population of organelle complexes), and / or (iii) a formulation provided herein, thereby treating or preventing the disease or disorder.

[0064] In some embodiments, methods of treating or preventing a disease or disorder associated with mitochondrial dysfunction are provided. In some embodiments, the method comprises contacting cells of a subject in need of treatment with an effective amount of (i) the population of organelle complexes provided herein, (ii) a composition provided herein (e.g., a composition comprising a population of organelle complexes), and / or (iii) a formulation provided herein, thereby treating or preventing the disease or disorder associated with mitochondrial dysfunction.

[0065] The present disclosure also provides the use of a population of organelle complexes in the manufacture of a medicament for treating the diseases and disorders provided herein. In some embodiments, the population of organelle complexes is administered to a subject in combination with one or more additional medicaments and / or additional therapies designed to treat the disease or disorder. In some embodiments, the present disclosure provides methods for treating diseases and disorders associated with mitochondrial insufficiency or diseases or disorders that would benefit from replenishment of healthy, functional mitochondria.

[0066] Contacting the cells of the subject can include an administration route selected from the group including intravenous administration, intraarterial administration, intratracheal administration, subcutaneous administration, intramuscular administration, inhalation, intrapulmonary administration, and intraocular administration. The population of organelle complexes can be administered locally or systemically.

[0067] As used herein, the term "local administration" or "topical administration" refers to any route of administration by which an organelle complex population obtained in the body contacts the body of an individual such that the location of the organelle complex population obtained in the body is topical (i.e., limited to a particular tissue, organ, or other body part where imaging is desired). Exemplary local administration routes include injection into a particular tissue using a needle, enteral nutrition into the gastrointestinal tract, and spreading a solution containing the organelle complex population onto the skin surface.

[0068] As used herein, the term "systemic administration" refers to any route of administration by which an organelle complex population obtained in the body contacts the body of an individual such that the location of the organelle complex population obtained in the body is systemic (i.e., not limited to a particular tissue, organ, or other body part where imaging is desired). Systemic administration includes enteral and parenteral administrations. Enteral administration is a route of systemic administration by which a substance is administered via the gastrointestinal tract and includes, but is not limited to, oral administration, administration by gastrostomy tube, administration by duodenal tube, gastric fistula, enteral nutrition, and rectal administration. Parenteral administration is a route of systemic administration by which a substance is administered via a route other than the gastrointestinal tract and includes, but is not limited to, intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, intradermal administration, intraperitoneal administration, and intravesical instillation.

[0069] In another aspect, this disclosure provides a pharmaceutically acceptable composition comprising a therapeutically effective amount of an organelle complex population disclosed herein. As described in detail below, the pharmaceutical compositions of this disclosure can be administered (1) orally, for example, as a drip (aqueous or non-aqueous solution or suspension), tablet, bolus, powder, granule, paste, (2) parenterally, for example, as a sterile solution or suspension, for example, by subcutaneous, intramuscular or intravenous injection, (3) topically, for example, as a cream, ointment or spray applied to the skin, (4) intravaginally or rectally, for example, as a pessary, cream or foam, or (5) by aerosol, for example, an aqueous aerosol, a liposomal preparation or an aerosol as solid particles containing an organelle complex population, and may be specially formulated for administration in solid or liquid form, including dosage forms suitable for. The pharmaceutical composition can comprise one or more pharmaceutically acceptable carriers. As used herein, the phrase "therapeutically effective amount" can refer to an amount of the organelle complex population disclosed herein that is effective, at a reasonable benefit / risk ratio, to produce some desired therapeutic effect, for example, cancer treatment.

[0070] As used herein, the phrase "pharmaceutically acceptable" refers to those agents, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, in contact with human and animal tissue, are suitable for use without excessive toxicity, irritation, allergic response, or other problems or complications and commensurate with a reasonable benefit / risk ratio.

[0071] As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material involved in carrying or transporting a subject chemical substance from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials that can act as pharmaceutically acceptable carriers are: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0072] Formulations useful in the methods of this disclosure include formulations suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and / or parenteral administration. The formulations may, for convenience, be in unit dosage form and may be prepared by any method well known in the pharmaceutical arts. The amount of active ingredient (e.g., the organelle complex population) that can be combined with the carrier materials to produce a single dosage form will vary depending on the host being treated and the particular mode of administration. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form is generally the amount of the organelle complex population that achieves a therapeutic effect. Generally, out of 100 percent, this amount ranges from about 1% to about 99% of the active ingredient, preferably from about 5% to about 70%, most preferably from about 10% to about 30%.

[0073] Suspensions may contain, in addition to the active agent, suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and mixtures thereof.

[0074] Dosage forms for topical or transdermal administration of the organelle complex population include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active ingredient may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants as may be required.

[0075] Ointments, pastes, creams and gels may contain excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0076] Ophthalmic formulations, eye ointments, powders, solutions, etc. are also considered to be within the scope of this disclosure.

[0077] Suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions of this disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Suitable fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0078] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenolsorbic acid, etc. It may also be desirable to include in the composition isotonic agents such as sugars, sodium chloride, etc. Furthermore, delayed absorption of injectable pharmaceutical forms may be caused by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0079] The actual dosage level of the active ingredient in the pharmaceutical compositions of this disclosure can be determined by the methods of this disclosure in order to obtain the amount of the active ingredient and is non-toxic to the subject and effective in achieving the desired therapeutic response for a particular subject, composition, and mode of administration.

[0080] The disease or disorder can be selected from the group consisting of diabetes (type I and type II), metabolic diseases, eye disorders associated with mitochondrial dysfunction, hearing loss, mitochondrial toxicity associated with therapeutic agents, mitochondrial dysfunction associated with space travel, cardiac toxicity associated with chemotherapy or other therapeutic agents, mitochondrial dysfunction, and migraine.

[0081] The disease or disorder can be selected from the group consisting of mitochondrial disease, diabetes and deafness (DAD) syndrome, bark syndrome, Leber hereditary optic neuropathy (LHON), leisure syndrome, NARP (neurogenic, ataxia, pigmentary retinopathy and Leigh syndrome), myoneurogenic encephalopathy with lactic acidosis (MNGIE), MELAS (mitochondrial encephalopathy, lactic acidosis and strothosis, etc.) syndrome, myoclonic epilepsy with ragged red fibers (MERRF) syndrome, Caret-Sawry syndrome and mitochondrial DNA depletion syndrome.

[0082] The disease or disorder can be an ischemia-related disease or disorder, genetic disorder, aging disease or disorder, neurodegenerative condition, cardiovascular disease, cancer, autoimmune disease, inflammatory disease, fibrotic disorder, or any combination thereof. The ischemia-related disease or disorder can be selected from the group consisting of cerebral ischemia reperfusion, hypoxic ischemic brain tissue, acute coronary syndrome, myocardial infarction, hepatic ischemia reperfusion injury, ischemia injury compartment syndrome, vascular occlusion, wound healing, spinal cord injury, disease, and reperfusion injury of transplanted organs. The neurodegenerative condition can be selected from the group consisting of dementia, Friedreich's ataxia, amyotrophic lateral sclerosis, mitochondrial brain disorder, lactic acidosis, and stroke-like episode (MELAS), myoclonic epilepsy with ragged red fibers (MERFF), epilepsy, Parkinson's disease, Alzheimer's disease, or Huntington's disease. Examples of neurite diseases include, for example, bipolar disorder, schizophrenia, depression, addiction disorder, anxiety disorder, attention deficit hyperactivity disorder, personality disorder, autism spectrum disorder, Asperger's syndrome, etc. The cardiovascular disease can be selected from the group consisting of coronary heart disease, myocardial infarction, atherosclerosis, hypertension, cardiac arrest, cerebrovascular disease, peripheral arterial disease, rheumatic heart disease, congenital heart disease, congestive heart failure, arrhythmia, stroke, deep vein thrombosis and pulmonary embolism. The disease or disorder can be acute respiratory distress syndrome (ARDS) or intrauterine growth restriction (IUGR).

[0083] Ischemic-related diseases and disorders include, for example, cerebral ischemia-reperfusion, hypoxic-ischemic encephalopathy, acute coronary syndrome, myocardial infarction, hepatic ischemia-reperfusion injury, ischemic compartment syndrome, vascular occlusion, wound healing (e.g., acute or chronic wounds, incisions, lacerations, contusions, burns (e.g., chemical, heat or flame, wind, or sunburn), or wounds due to medical or surgical intervention), spinal cord injury, sickle cell disease, and reperfusion injury of transplanted organs. In some embodiments, the organelle complex population may treat, prevent, ameliorate, and / or improve clinical conditions due to ischemia-reperfusion injury. In some embodiments, the organelle complex population may improve ejection fraction (EF), inhibit cardiac hypertrophy, and / or treat, prevent, ameliorate, and / or improve fibrosis after ischemia-reperfusion injury.

[0084] This specification also provides a kit comprising one or more of the compositions described herein (e.g., a formulation comprising an organelle complex population) in suitable packaging, and may further include written materials that can include instructions for use, conclusions of clinical studies, lists of side effects, etc. Such kits may also include information such as references to scientific literature, package insert materials, clinical trial results, and / or summaries thereof, that indicate and / or establish the activity and / or advantages of the composition and / or describe dosage, administration, side effects, drug interactions, or other information useful to a healthcare provider. Such information may be based on the results of various studies, for example, studies using experimental animals including in vivo models and studies based on human clinical trials. The kit may include one or more unit doses described herein.

Examples

[0085] Some aspects of the above-described embodiments are disclosed in more detail in the following examples, which are in no way intended to limit the scope of the present disclosure.

[0086] Example 1 Characterization of the Organelle Complex Population The composition of the organellar complex population provided in this specification, as well as the functional differences between them (and the homogenized mitochondria obtained via currently available methods), were examined in a series of experiments.

[0087] Homogenized mitochondria, the first organellar complex, and the second organellar complex were isolated from HeLa cells (Pierce μg / mL: HeLa H-mito, 650.1; HeLa 2 nd OC, 238.3; HeLa 1 st OC, 660). The isolated homogenized mitochondria and organellar complex population were resuspended in H2O (Milli-Q) and Laemmli sample buffer. The final concentration was adjusted to approximately 3 mg per 15 ml, and then the samples were boiled at 96 °C for 5 min. The samples were run on a 4 - 20% gradient gel (100 V, 75 min). The antibodies used were AMPKa (D5A2) Rabbit mAb #5831, Phospho-AMPKα (Thr172) (40H9) Rabbit mAb #2535, TFAM (D5C8) Rabbit mAb #8076, Tom20 (F-10) Mouse mAb #sc-17764, p70 S6 Kinase (49D7) Rabbit mAb #2708, Golgin-97 (D8P2K) Rabbit mAb #13192, Catalase (D4P7B) XP® Rabbit mAb #12980, Citrate Synthase (D7V8B) Rabbit mAb #14309, Calreticulin (D3E6) XP® Rabbit mAb#12238;, Anti-Lamin A / C Antibody (E-1) #sc-376248, and OxPhos Human WB Antibody Cocktail, Rabbit mAb #45-8199. Figures 1A - 1B show non-limiting exemplary data related to the characterization of the organellar complex population provided in this specification. Figure 1A shows homogenized mitochondria (H-mito), the first organellar complex (1 st OC) and the second organellar complex (2 ndShows the intracellular structure / organelle Western blot protein analysis of (OC). Figure 1B shows homogenized mitochondria (H-mito), the first organelle complex (1 st (OC) and the second organelle complex (2 nd (OC) mitochondrial marker Western blot protein analysis. Figures 2A-2B show non-limiting exemplary data related to the characterization of the organelle complex population provided herein. The first organelle complex and the second organelle complex were isolated from HEK293T cells. Figure 2A shows the intracellular structure / organelle Western blot protein analysis of the first organelle complex (1 st (OC) and the second organelle complex (2nd OC). Figure 2B shows the mitochondrial marker Western blot protein analysis of the first organelle complex (1 st (OC) and the second organelle complex (2 nd (OC). These data show that organelle complex populations isolated from different types of cells show similar results with respect to the differences between the first organelle complex and the second organelle complex. Also, these data show that the differences in mitochondrial markers between the first organelle complex and the second organelle complex are greater compared to homogenized mitochondria.

[0088] Figures 3A - 3B show non - limiting exemplary data regarding the structural integrity of the first organelle complex and the second organelle complex, namely, outer membrane integrity (Figure 3A) and inner membrane integrity (Figure 3B). In some embodiments, membrane integrity (%) = ((amount of final product with detergent - amount of final product without detergent) / amount of final product with detergent). Figure 4 shows non - limiting exemplary data regarding ATP production by the first organelle complex population within the range of Pi concentration. Figures 5A - 5B show non - limiting exemplary data regarding the structural integrity of the first organelle complex. The citrate synthase activity (Figure 5A) and cytochrome c oxidase activity (Figure 5B) of the first organelle complex were measured within the range of Ca 2+ concentration. Figure 6A shows the experimental setup, and Figures 6B - 6D show data regarding the structural integrity of the first organelle complex and the second organelle complex. Figures 6B - 6D show ATP production of the second organelle complex (2 nd OC, Figure 6C) and the first organelle complex (1 st OC, Figure 6B) at the illustrated concentrations, and Figure 6D shows the ATP production rate of both organelle complexes. In short, these data show the structural and functional differences between the organelle complex populations provided herein and the homogenized mitochondria generated by currently available methods. The first organelle complex and the second organelle complex provided herein include various organelles (in addition to mitochondria), exhibit excellent functionality, and maintain structural integrity within the range of the extracellular environment.

[0089] Next, the effect of cellular uptake of the second organelle complex on ATP production was examined. Figures 7A - 7B show the experimental setup (Figure 7A) and data (Figure 7B) regarding the effect of cellular uptake of the second organelle complex (2 nd OC) on ATP production. It was found that cellular uptake of the second organelle complex increased ATP production in a dose - dependent manner.

[0090] Next, qPCR analysis of the first organelle complex and the second organelle complex was performed. Figure 8 shows the first organelle complex (1 st(OC) and the second organelle complex (2 nd nd shows qPCR data regarding (OC). The data shows that when quantified by qPCR, there is a large difference in the relative mitochondrial DNA copy number (mtDNA CN) between the first organelle complex and the second organelle complex, while the first organelle complex has about 2 to 6 times or more mtDNA CN in the isolated organelle complex.

[0091] Next, a comparison of the uptake of mtDNA into recipient C6 rho0 cells incubated with homogenized mitochondria, the first organelle complex, and the second organelle complex was performed. The recipient used was the C6 rho0 cell line, a derivative of rat C6 glioma cells, and their mitochondrial DNA (mtDNA) disappeared as a result of transient transfection with a plasmid. The mitochondrial donor was HeLa, an immortal cell line derived from human cervical cancer cells. Homogenized mitochondria (H-mito), the first organelle complex (1 st (OC) and the second organelle complex (2 nd (OC) were isolated from HeLa cells. C6 rho0 cells were cultured in a 12-well plate at a cell concentration of 2x10 5 cells / well. Homogenized mitochondria (H-mito), the first organelle complex (1 st (OC) and the second organelle complex (2 nd (OC) were co-incubated at 42 mg / well. Figure 9A shows the experimental setup, and Figure 9B shows data regarding the uptake of mtDNA into recipient C6 rho0 cells incubated with homogenized mitochondria (H-mito), the first organelle complex (1 st (OC) and the second organelle complex (2 nd (OC). Figure 9B shows the ratio of 1 st OC and 2 ndShows the ratio of mtDNA incorporated with OC. An increase in the incorporation of mtDNA in the first organelle complex treatment group into recipient C6 rho0 cells was observed. Specifically, a three-fold increase in the incorporated mtDNA was observed using the first organelle complex as the donor.

[0092] Example 2 Alternative final centrifugation conditions In some embodiments of the methods provided herein, the second solution is centrifuged at a first centrifugal force, the supernatant is collected and centrifuged at a second centrifugal force, and the pellet is collected to recover the first organelle complex. In this example, the results of increasing the second centrifugal force from 3000 g to 8000 g (angled, soft brake) and increasing the second centrifugation time from 10 min to 20 min were examined. Figures 10A - 10G show data regarding the protein concentration (Figure 10A), total ATP production (Figure 10B), ATP production in the presence of oligomycin (Figure 10C), inner membrane integrity (Figure 10D), outer membrane integrity (Figure 10E), COX activity (Figure 10F), and citrate synthase (CS) activity (Figure 10G) of the first organelle complex recovered using final centrifugation conditions of 3000 g (10 min) or 8000 g (20 min). Figures 11A - 11G show data regarding the protein levels of LAMP2 (lysosome, Figure 11B), golgin 97 (Golgi, Figure 11C), catalase (peroxisome, Figure 11D), calreticulin (ER, Figure 11E), VDAC (mitochondria (outer), Figure 11F), TOMM20 (mitochondria (outer), Figure 11G), and TFAM (mitochondria (matrix), Figure 11H) of the first organelle complex recovered using final centrifugation conditions of 3000 g (10 min) or 8000 g (20 min) on a stained SDS-PAGE gel (17 μg of protein / lane) (Figure 11A).

[0093] Example 3 Bioactivities of homogenized mitochondria, the first organelle complex, and the second organelle complex In this example, the biological activities of homogenized mitochondria, the first organelle complex, and the second organelle complex were compared. Fibroblasts (5000 cells / well, 96-well plate) were incubated for about 24 hours, and then FBS(-) vehicle (Tris-sucrose buffer), homogenized mitochondria, the first organelle complex, or the second organelle complex (each derived from HeLa cells) was added at concentrations of 5 μg / ml, 15 μg / ml, or 50 μg / ml. After 24 hours of incubation, ATP production measurement (in triplicate) was performed using Cell Titer-Glo. FIG. 12 shows data on ATP production of fibroblasts incubated with homogenized mitochondria (H-mito), the first organelle complex, or the second organelle complex. These data indicate that a high dose of homogenized mitochondria may induce cell death (a trend also seen in microscopic observations at medium doses). Both the second organelle complex and the first organelle complex showed a dose-dependent increase in intracellular ATP production, while the first organelle complex was shown to be more effective at low doses.

[0094] Example 4 Reductant flow device As described herein, the first generation method for generating the first organelle complex can be modified, and in some embodiments, can include using a flow device (e.g., a reductant flow device) instead of (or in addition to) pipetting (e.g., step G) as a means of applying a physical stimulus to the first solution and / or the second solution. FIG. 14 shows data on the first organelle complex obtained by the first generation method (using pipetting as the physical stimulus) and a modified method employing a reducer flow device as the physical stimulus. It was found that a higher protein recovery rate was obtained using the reductant flow device compared to the first generation method (including pipetting).

[0095] Example 5 Tangential flow filtration In this example, the use of a TFF system during the isolation of the first organellar complex was investigated, including various TFF parameters such as shear rate and buffer composition. First, the use of a lower shear rate during TFF purification was investigated. The shear rate was about 1000 - 2000 sec -1 -1. It was found that by using a lower shear rate, the outer membrane integrity in TFF purification could be improved (Table 2). [Table 1]

[0096] Next, the effect of buffer viscosity during TFF purification was investigated. It was found that by using a lower viscosity buffer, the outer membrane integrity of the first organellar complex in TFF purification could be improved (Table 3). Thus, in some of the embodiments provided herein, a lower viscosity buffer (e.g., a mannitol-based buffer) is used for the separation of the first organellar complex during TFF. [Table 2]

[0097] Next, the effect of HA and glycerol in the storage buffer on the outer membrane integrity of mitochondria was investigated (Table 4). [Table 3]

[0098] In at least some of the foregoing embodiments, one or more elements used in the embodiments can also be used interchangeably with other embodiments, provided that such substitution is technically feasible. Those skilled in the art will understand that various other omissions, additions, and modifications can be made to the above methods and structures without departing from the scope of the claimed subject matter. All such modifications and changes are intended to be within the scope of the subject matter defined by the appended claims.

[0099] Regarding the use of substantially any plural and / or singular terms in this specification, one of ordinary skill in the art can convert from plural to singular and / or from singular to plural as appropriate for the context and / or application. Various singular / plural substitutions may be explicitly described in this specification for clarity. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Any reference to "or" in this specification shall be inclusive of "and / or" unless specifically stated otherwise.

[0100] One of ordinary skill in the art will generally understand that terms used herein, and particularly in the appended claims (e.g., the body of the appended claims), are generally intended to be open terms (e.g., the term "including" should be construed as "including but not limited to", the term "having" should be construed as "having at least", the term "includes" should be construed as "including but not limited to", etc.). One of ordinary skill in the art will further understand that if a specific number is intended in the recitation of an introduced claim, such intent is explicitly recited in the claim, and if there is no such recitation, there is no such intent. For example, for purposes of illustration, the following appended claims may include the use of introductory phrases "at least one" and "one or more" to introduce the recitation of the claim. However, the use of such phrases should not be construed to limit a particular claim that includes such a recited claim to embodiments that include only one such recitation, even if the same claim includes an introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be construed to mean "at least one" or "one or more"). Also, even if a specific number of recited claims is explicitly recited, one of ordinary skill in the art will recognize that such recitation should be construed to mean at least the recited number (e.g., a mere recitation of "two recitations" without other modifiers means at least two recitations, or two or more recitations). Further, when a convention similar to "at least one of A, B, and C, etc." is used, generally such construction is intended in the sense that one of ordinary skill in the art understands the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, a system having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together).When a convention similar to "at least one of A, B, or C, etc." is used, generally, such an interpretation is intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, or C" includes, but is not limited to, a system having only A, only B, only C, both A and B together, both A and C together, both B and C together, and / or all of A, B, and C together). One of ordinary skill in the art can further understand that, in any of the specification, claims, and drawings, substantially any disjunctive and / or clause presenting two or more alternative terms should be understood to contemplate the possibility of including one of the terms, any of the terms, or both terms.

[0101] Furthermore, when a feature or aspect of the present disclosure is described with respect to a Markush group, one of ordinary skill in the art will recognize that the present disclosure is also described with respect to any individual member or subgroup of members of the Markush group.

[0102] As will be understood by one of ordinary skill in the art, for any and all purposes, such as for the purpose of providing a written description, all ranges disclosed herein also include any and all possible subranges and combinations of those subranges. Any recited range can be readily recognized and described as being divisible into at least half, one third, one fourth, one fifth, one tenth, etc. of the same range. By way of non-limiting example, each range described herein can be readily subdivided into a lower third, a middle third, an upper third, etc. Also, as will be understood by one of ordinary skill in the art, all words such as "maximum," "at least," "greater than," "less than," etc. include the recited numbers and refer to ranges that can be later subdivided into subranges as described above. Finally, as will be understood by one of ordinary skill in the art, ranges include each individual member. Thus, for example, a group having 1 to 3 articles refers to a group having 1, 2, or 3 articles. Similarly, a group having 1 to 5 articles refers to a group having 1, 2, 3, 4, or 5 articles, etc.

[0103] Although various aspects and embodiments are disclosed herein, other aspects and embodiments will be apparent to those of ordinary skill in the art. The various aspects and embodiments disclosed herein are presented by way of example only and not by way of limitation, while the true scope and spirit are indicated by the following claims.

Claims

1. A population of organelle complexes, wherein the organelle complex includes mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus, the organelle complex is isolated or derived from floating cells and / or frozen cells, the organelle complex has depleted cytoplasmic macromolecules, and at least about 80% of the mitochondria of the organelle complex maintain structural integrity in the extracellular environment. A population of organelle complexes.

2. A population of organelle complexes, wherein the organelle complex includes mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus, the organelle complex is isolated or derived from cells contacted with the surfactant at a concentration equal to or higher than the critical micelle concentration (CMC) of the surfactant, the organelle complex has depleted cytoplasmic macromolecules, and at least about 80% of the mitochondria of the organelle complex maintain structural integrity in the extracellular environment. A population of organelle complexes.

3. The population of organelle complexes according to any one of claims 1 to 2, wherein the structural integrity includes the structural integrity inside and / or outside the membrane of the mitochondria.

4. Compared with the population of homogenized mitochondria in the extracellular environment, at least twice as many mitochondria of the organelle complex maintain structural integrity in the extracellular environment. The population of organelle complexes according to any one of claims 1 to 3.

5. The population of organelle complexes according to any one of claims 1 to 4, wherein the structural integrity is measured by citrate synthase (CS) activity and / or cytochrome c oxidase (COX) activity.

6. The population of organelle complexes according to any one of claims 1 to 5, wherein the extracellular environment includes a total calcium concentration of about 1 to about 20 mg / dL and / or a free / active calcium concentration of about 1 to about 6 mg / dL.

7. At least about 80% of the mitochondria of the organelle complex maintain functionality. The population of organelle complexes according to any one of claims 1 to 6.

8. The mitochondria of the organelle complex are capable of generating ATP. The population of organelle complexes according to any one of claims 1 to 7.

9. The organelle complex population according to any one of claims 1 to 8, comprising at least 2 to 6 times the mitochondrial DNA (mtDNA) copy number as compared to a population of homogenized mitochondria.

10. The cytoplasmic macromolecule includes cytoplasmic proteins, and the abundance of one or more cytoplasmic proteins is at least about 90% depleted as compared to the cells from which the organelle complex population is derived. Optionally, the cytoplasmic protein is p70S6K and / or glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The organelle complex population according to any one of claims 1 to 9.

11. One or more mitochondrial matrix proteins, optionally mitochondrial transcription factor A (TFAM) and / or citrate synthase (CS), and One or more outer mitochondrial membrane proteins, optionally outer mitochondrial membrane complex subunit 20 (TOMM20), and One or more lysosomal proteins, optionally lysosome-associated membrane protein 2 (LAMP2), mannose-6-phosphate receptor (M6PR), and / or lysosome-associated membrane protein 1 (LAMP1), and One or more peroxisomal proteins, optionally catalase and / or ATP-binding cassette transporter 1, subfamily D, type 3 (ABCD3), and One or more Golgi apparatus proteins, optionally Golgin-97, Syntaxin-6, TGOLN2 / trans-Golgi network protein 2 (TGN46), Golgi matrix protein 130 (GM130), and / or mannosidase alpha class 2A member 1 (MAN2A1), and One or more endoplasmic reticulum proteins, optionally calreticulin and / or calnexin. The organelle complex population according to any one of claims 1 to 10.

12. Comprising a first organelle complex, or a combination of a first organelle complex and a second organelle complex, The first organelle complex is derived from (i) frozen cells, (ii) floating cells, and / or (iii) cells contacted with the surfactant at a concentration equal to or higher than the critical micelle concentration (CMC) of the surfactant, and The second organelle complex is a population of organelle complexes according to any one of claims 1 to 11, which is derived from (i) adherent cells and / or (ii) cells contacted with the surfactant at a concentration below the critical micelle concentration (CMC) of the surfactant.

13. The first organelle complex contains one or more lysosomal proteins at least about 1.1-fold or more compared to the second organelle complex, optionally lysosome-associated membrane protein 2 (LAMP2), mannose-6-phosphate receptor (M6PR), and / or lysosome-associated membrane protein 1 (LAMP1). The first organelle complex contains one or more peroxisomal proteins at least about 1.1-fold or more compared to the second organelle complex, optionally catalase and / or ATP-binding cassette transporter 1, subfamily D, type 3 (ABCD3). The first organelle complex contains one or more Golgi apparatus proteins at least about 1.1-fold or more compared to the second organelle complex, optionally Golgin-97, Syntaxin-6, trans-Golgi network protein 2 (TGN46), Golgi matrix protein 130 (GM130), and / or mannosidase alpha class 2A member 1 (MAN2A1). The first organelle complex contains one or more endoplasmic reticulum proteins at least about 1.1-fold or more compared to the second organelle complex, optionally calreticulin and / or calnexin, and / or The second organelle complex contains one or more cytoplasmic proteins at least about 1.1-fold or more compared to the first organelle complex, optionally p70S6K and / or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in a population of organelle complexes according to any one of claims 1 to 12.

14. When the organelle complex is contacted with a cell population, the organelle complex can be taken up into the cell, and optionally, at least about 2-fold or more of the organelle complex can be taken up into the cell compared to a population of homogenized mitochondria, in a population of organelle complexes according to any one of claims 1 to 13.

15. When the organelle complex is brought into contact with the host cell population, the organelle complex has an excellent ability to be taken up by the host cell as compared with a population of homogenized mitochondria. The organelle complex population according to any one of claims 1 to 14.

16. When the first organelle complex is brought into contact with the host cell population, the first organelle complex has an excellent ability to be taken up by the host cell as compared with the second organelle complex. The organelle complex population according to any one of claims 1 to 15.

17. When the organelle complex is brought into contact with the host cell population, the organelle complex has an excellent ability to be taken up by the host cell as compared with a population of homogenized mitochondria. Optionally, at least twice or more of the organelle complex can be taken up into the host cell as compared with a population of homogenized mitochondria. The organelle complex population according to any one of claims 1 to 16.

18. The mitochondria of the organelle complex can be incorporated into cells after the population has undergone one or more freeze-thaw cycles. Optionally, at least twice or more of the mitochondria of the organelle complex can be incorporated into cells after the population has undergone one or more freeze-thaw cycles as compared with a population of homogenized mitochondria. The organelle complex population according to any one of claims 1 to 17.

19. At least about 80% of the organelle complex has a size between about 500 nm and about 3500 nm. Optionally, the size is between about 200 nm and about 1000 nm. The organelle complex population according to any one of claims 1 to 18.

20. A mitochondrial activator, optionally derived from cells treated with resveratrol. The organelle complex population according to any one of claims 1 to 19.

21. A composition comprising the organelle complex population according to any one of claims 1 to 20.

22. A formulation comprising the composition according to claim 21 and a pharmaceutically acceptable carrier.

23. A method for generating a first organelle complex population, incubating cells in a first solution containing a surfactant at a first temperature, removing the surfactant to form a second solution, and recovering a first organelle complex from the second solution. The first organelle complex includes mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus, the first organelle complex population has depleted cytoplasmic macromolecules, (i) incubating the cells in the first solution with the surfactant at a concentration equal to or higher than the critical micelle concentration (CMC) of the surfactant, and / or (ii) the cells include or are derived from floating cells or frozen cells, a method.

24. The method according to claim 23, comprising incubating the second solution at a second temperature.

25. The method according to any one of claims 23 to 24, wherein the first organelle complex includes mitochondria and two, three, or four of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus.

26. The method according to any one of claims 23 to 25, wherein the cells in the first solution are contacted with the surfactant at a concentration at least 5% higher than the critical micelle concentration (CMC) of the surfactant.

27. The method according to any one of claims 23 to 26, wherein the surfactant is saponin, and optionally, the surfactant is present at a concentration of about 50 μg / mL.

28. The method according to any one of claims 23 to 27, wherein the surfactant is a nonionic surfactant.

29. The method according to any one of claims 23 to 28, wherein the surfactant is selected from the group consisting of Triton-X100, Triton-X114, Nonidet® P-40, n-dodecyl-D-maltoside, Tween-20, Tween-80, saponin, and digitonin.

30. The method according to any one of claims 23 to 29, wherein the first solution further comprises a buffer containing one or more of an isotonic agent, an osmotic regulator, and a chelating agent, optionally.

31. The method according to any one of claims 23 to 30, wherein the first solution contains Tris buffer, sucrose, and / or a chelating agent.

32. The method according to any one of claims 23 to 31, wherein incubating the cells in the first solution includes incubating the cells in the first solution for about 1 minute to about 120 minutes, optionally for about 30 minutes.

33. The first temperature and / or the second temperature is from about 0°C to about 50°C, and optionally, the first temperature is 25°C and the second temperature is from about 0°C to about 4°C, the method according to any one of claims 23 to 32.

34. Removing the surfactant includes washing one or more times with a buffer, optionally a Tris buffer, the method according to any one of claims 23 to 33.

35. Incubating the second solution includes incubating the second solution for from about 1 minute to about 120 minutes, optionally for about 20 minutes, the method according to any one of claims 24 to 34.

36. Recovering the first organelle complex from the second solution includes tangential flow filtration (TFF), and optionally, (i) optionally, a low-viscosity buffer that reduces the shear rate, (ii) from about 22°C to about 25°C, (iii) a shear rate of less than about 2000 sec －１ The method according to any one of claims 23 to 35, wherein TFF is performed with a buffer containing less than (iv) room temperature, optionally 4°C, and / or (v) human albumin (HA) or recombinant albumin ().

37. Recovering the first organelle complex from the second solution includes one or more centrifugation steps, the method according to any one of claims 23 to 36.

38. Recovering the first organelle complex from the second solution includes centrifuging the second solution at a first centrifugal force, collecting the supernatant, centrifuging the supernatant at a second centrifugal force, collecting the pellet to recover the first organelle complex, the method according to any one of claims 23 to 37.

39. The first centrifugal force and / or the second centrifugal force is from about 100 to about 5000 g, and optionally, the first centrifugal force is about 500 g and the second centrifugal force is about 3000 g, and optionally, the centrifugation is carried out for from about 10 minutes to about 20 minutes, the method according to any one of claims 23 to 38.

40. Centrifuging the supernatant at a second centrifugal force includes centrifuging at 8000 g for about 20 minutes, the method according to any one of claims 23 to 39.

41. Incubating the cells in the first solution and / or incubating the second solution includes subjecting the first solution and / or the second solution to physical stimulation, optionally shaking and / or stirring, the method according to any one of claims 23 to 40.

42. Applying a physical stimulus to the first solution and / or the second solution includes the first solution and / or the second solution flowing through a flow device, the flow device including a flow path having two or more portions with different cross-sectional diameters, optionally, the cross-sectional diameter ranges from about 0.8 mm to about 25.4 mm, and further optionally, the cross-sectional diameter ranges from about 1.5 mm to 6.5 mm, and optionally, flowing through the flow device creates additional flow and / or shear. The method according to claim 41.

43. The method according to any one of claims 23 to 42, further comprising freezing the first organelle complex, optionally, in a buffer containing a cryoprotectant.

44. The method according to claim 43, wherein the cryoprotectant includes human albumin (HA) and / or glycerol.

45. The method according to any one of claims 23 to 44, further comprising treating the cells with a mitochondrial activator, optionally, resveratrol, prior to the incubating step.

46. A population of organelle complexes obtained by the method according to any one of claims 23 to 45.

47. A method for treating a disease or disorder, comprising contacting cells of a subject in need of treatment with an effective amount of (i) a population of organelle complexes according to any one of claims 1 to 20 or 46, (ii) a composition according to claim 21, and / or (iii) a formulation according to claim 22, thereby treating the disease or disorder.

48. A method for treating a disease or disorder associated with mitochondrial dysfunction, comprising contacting cells of a subject in need of treatment with an effective amount of (i) a population of organelle complexes according to any one of claims 1 to 20 or 46, (ii) a composition according to claim 21, and / or (iii) a formulation according to claim 22, thereby treating the disease or disorder associated with mitochondrial dysfunction.

49. The method according to any one of claims 47 to 48, wherein contacting the cells of the subject includes an administration route selected from the group consisting of intravenous administration, intra-arterial administration, intratracheal administration, subcutaneous administration, intramuscular administration, inhalation, intrapulmonary administration, and intraocular administration.

50. The disease or disorder is selected from the group consisting of diabetes (type I and type II), metabolic diseases, eye disorders associated with mitochondrial dysfunction, hearing loss, mitochondrial toxicity associated with therapeutic agents, mitochondrial dysfunction associated with space travel, cardiac toxicity associated with chemotherapy or other therapeutic agents, mitochondrial dysfunction, and migraine, according to any one of claims 47 to 49.

51. The disease or disorder is selected from the group consisting of mitochondrial diseases, diabetes and deafness (DAD) syndrome, bark syndrome, Leber hereditary optic neuropathy (LHON), leisure syndrome, NARP (neurogenic, ataxia, pigmentary retinopathy and ptosis syndrome), mitochondrial neurogastrointestinal encephalopathy (MNGIE), MELAS (mitochondrial encephalopathy, lactic acidosis and strothosis, etc.) syndrome, myoclonic epilepsy with ragged red fibers (MERRF) syndrome, Caret-Sawry syndrome, and mitochondrial DNA depletion syndrome, according to any one of claims 47 to 50.

52. The disease or disorder is an ischemia-related disease or disorder, genetic disorder, aging disease or disorder, neurodegenerative condition, cardiovascular disease, cancer, autoimmune disease, inflammatory disease, fibrosis disorder, or any combination thereof, according to any one of claims 47 to 51.

53. The ischemia-related disease or disorder is selected from the group consisting of cerebral ischemia reperfusion, hypoxic ischemic brain tissue, acute coronary syndrome, myocardial infarction, hepatic ischemia reperfusion injury, ischemic compartment syndrome, vascular occlusion, wound healing, spinal cord injury, disease, and reperfusion injury of transplanted organs, according to any one of claims 47 to 52.

54. The neurodegenerative condition is selected from the group consisting of dementia, Friedreich's ataxia, amyotrophic lateral sclerosis, mitochondrial brain disorder, lactic acidosis, and stroke-like episodes (MELAS), myoclonic epilepsy with ragged red fibers (MERRFF), epilepsy, Parkinson's disease, Alzheimer's disease, or Huntington's disease. Examples of neurite diseases include bipolar disorder, schizophrenia, depression, addiction disorder, anxiety disorder, attention deficit hyperactivity disorder, personality disorder, autism spectrum disorder, Asperger's syndrome, etc., according to any one of claims 47 to 53.

55. The cardiovascular disease is selected from the group consisting of coronary heart disease, myocardial infarction, atherosclerosis, hypertension, cardiac arrest, cerebrovascular disease, peripheral arterial disease, rheumatic heart disease, congenital heart disease, congestive heart failure, arrhythmia, stroke, deep vein thrombosis and pulmonary embolism, according to any one of claims 47 to 54.

56. The disease or disorder is acute respiratory distress syndrome (ARDS) or intrauterine growth restriction (IUGR), according to any one of claims 47 to 55.

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