Organic organelle complex

Abstract

The contents disclosed herein include populations of organelle complexes. The organelle complex can include mitochondria and one or more of endoplasmic reticulum, peroxisome, lysosome, and Golgi. In some embodiments, the organelle complexes are isolated from or derived from floating cells and / or frozen cells. In some embodiments, the organelle complexes are isolated from or derived from cells contacted with a surfactant at a concentration equal to or higher than the critical micelle concentration (CMC) of the surfactant. At least about 80% of the mitochondria of the organelle complex is capable of maintaining structural integrity in an extracellular environment. Also provided herein are methods for generating a first population of organelle complexes.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 359,110 filed on July 7, 2022 under 35 U.S.C. §119(e), the contents of this related application being incorporated herein by reference in its entirety for all purposes. Background Art Technical Field

[0003] The present disclosure generally relates to methods of obtaining organelle complexes from cells, organelle complexes obtained by such methods, and uses of organelle complexes obtained by such methods.

[0004] Description of Related Technology

[0005] Mitochondria are intracellular organelles responsible for many metabolic transformations and regulatory functions. They produce most of the ATP used by eukaryotic cells. For mitochondrial function, the folded inner membrane and the surrounding outer membrane and the electron transport system located in the inner membrane play a vital role. The inner membrane forms a highly folded structure (called cristae), which is believed to hold the supercomplex of the electron transport system in the cristae membrane and maintain a high proton concentration by trapping pumped protons in the cristae space. The electrochemical proton gradient formed by the electron transport system enables the transport of anions and ATP synthesis and cation transport.

[0006] Mitochondria are also highly dynamic organelles, which move throughout the cell and undergo structural transitions, changing length, morphology, shape and size. In addition, mitochondria are constantly eliminated and regenerated in a process called mitochondrial biogenesis. Although most mitochondrial genes have been transferred to the nuclear genome, the mitochondrial genome still encodes 13 subunits of rRNA, tRNA and electron transport chain (ETC). Therefore, functional communication between the nuclear genome and the mitochondrial genome is crucial for mitochondrial biogenesis, effective oxidative phosphorylation and normal health. Mitochondria are also the main source of free radicals and reactive oxygen species (ROS) that cause oxidative stress. In addition, mitochondria play a key role in the control of intracellular signal transduction and cell death (including apoptosis and necrosis).

[0007] There is increasing evidence that mitochondrial dysfunction is associated with a variety of human diseases. Mitochondrial dysfunction (e.g., respiratory chain complex dysfunction) is the main cause of mitochondrial disease and aging. Decreased mitochondrial function can affect cells in many organs, primarily involving mitochondrial diseases and age-related diseases. Introducing exogenous mitochondria into cells of subjects in need is a promising method for treating or preventing many diseases and disorders. However, a scalable and highly surfactant-compatible method is needed to separate a population containing mitochondria from cells in a manner that retains mitochondrial function and structural integrity. Summary of the invention

[0008] Disclosed herein are populations of organelle complexes. In some embodiments, these organelle complexes are isolated or derived from floating cells and / or frozen cells. In some embodiments, these organelle complexes comprise mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi bodies. In some embodiments, these organelle complexes are depleted of cytoplasmic macromolecules. In some embodiments, at least about 80% of the mitochondria of these organelle complexes maintain structural integrity in the extracellular environment.

[0009] Disclosed herein are cell organelle complex populations. In some embodiments, these cell organelle complexes are separated from or derived from cells contacted with a surfactant at a concentration equal to or higher than the critical micelle concentration (CMC) of the surfactant. In some embodiments, these cell organelle complexes comprise mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi bodies. In some embodiments, these cell organelle complexes are depleted of cytoplasmic macromolecules. In some embodiments, at least about 80% of the mitochondria of these cell organelle complexes maintain structural integrity in the extracellular environment.

[0010] In some embodiments, structural integrity includes the structural integrity of the inner membrane and / or the structural integrity of the outer membrane of the mitochondria. In some embodiments, at least 2 times of the mitochondria of the organelle complexes maintain structural integrity in the extracellular environment compared to the homogenized mitochondrial population in the extracellular environment. In some embodiments, structural integrity is measured by citrate synthase (CS) activity and / or cytochrome c oxidase (COX) activity. In some embodiments, the extracellular environment comprises 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 organelle complexes maintain functional capacity. In some embodiments, the mitochondria of the organelle complexes can achieve ATP production. In some embodiments, compared to the homogenized mitochondrial population, the organelle complex populations comprise at least 2 to 6 times the number of mitochondrial DNA (mtDNA) copies. 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% compared to the cells from which the organelle complex populations are derived.

[0011] In some embodiments, the organelle complexes comprise one or more mitochondrial matrix proteins (e.g., mitochondrial transcription factor A (TFAM), citrate synthase (CS). In some embodiments, the organelle complexes comprise one or more mitochondrial outer membrane proteins (e.g., mitochondrial outer membrane complex subunit 20 (TOMM20)). In some embodiments, the organelle complexes comprise one or more lysosomal proteins (e.g., lysosomal associated membrane protein 2 (LAMP2), mannose-6-phosphate receptor (M6PR), lysosomal associated membrane protein 1 (LAMP1)). In some embodiments, the organelle complexes The organelle complexes include: one or more peroxisomal proteins (e.g., catalase, ATP-binding cassette transporter 1, subfamily D, type 3 (ABCD3)). In some embodiments, the organelle complexes include one or more Golgi proteins (e.g., Golgi protein-97, syntaxin-6, TGOLN2 / trans-Golgi network protein 2 (TGN46), Golgi matrix protein 130 (GM130), mannosidase alpha 2A class member 1 (MAN2A1)). In some embodiments, the organelle complexes include one or more endoplasmic reticulum proteins (e.g., calreticulin, calnexin).

[0012] In some embodiments, these organelle complex populations include 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 a 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 a surfactant at a concentration lower than the critical micelle concentration (CMC) of the surfactant. In some embodiments, the first organelle complex comprises at least about 1.1 times more one or more lysosomal proteins (e.g., lysosomal associated membrane protein 2 (LAMP2), mannose-6-phosphate receptor (M6PR), lysosomal associated membrane protein 1 (LAMP1)) than the second organelle complex. In some embodiments, the first organelle complex comprises at least about 1.1 times more one or more peroxisomal proteins (e.g., catalase, ATP binding cassette transporter 1, subfamily D, type 3 (ABCD3)) than the second organelle complex. In some embodiments, the first organelle complex comprises at least about 1.1 times more of one or more Golgi proteins (e.g., Golgi protein-97, syntaxin-6, TGOLN2 / trans-Golgi network protein 2 (TGN46), Golgi matrix protein 130 (GM130), mannosidase alpha 2A class member 1 (MAN2A1)) than the second organelle complex. In some embodiments, the first organelle complex comprises at least about 1.1 times more of one or more endoplasmic reticulum proteins (e.g., calreticulin, calnexin) than the second organelle complex. In some embodiments, the second organelle complex comprises at least about 1.1 times more of one or more cytoplasmic proteins (e.g., p70S6K, glyceraldehyde 3-phosphate dehydrogenase (GAPDH)) than the first organelle complex.

[0013] In some embodiments, when these organelle complexes are in contact with a cell population, these organelle complexes can be incorporated into these cells. In some embodiments, compared to a homogenized mitochondrial population, at least about 2 times of organelle complexes can be incorporated into these cells. In some embodiments, when these organelle complexes are in contact with a host cell population, compared to a homogenized mitochondrial population, these organelle complexes have excellent ability to be incorporated into host cells. In some embodiments, when a first organelle complex is in contact with a host cell population, compared to a second organelle complex, these first organelle complexes have excellent ability to be incorporated into host cells. In some embodiments, when these organelle complexes are in contact with a host cell population, these organelle complexes have excellent ability to be incorporated into host cells. In some embodiments, compared to a homogenized mitochondrial population, at least 2 times of organelle complexes can be incorporated into host cells. In some embodiments, the mitochondria of the organelle complex can be incorporated into cells after the population undergoes one or more freeze-thaw cycles. In some embodiments, compared to a homogenized mitochondrial population, at least 2 times of mitochondria of these organelle complexes can be incorporated into cells after the population undergoes one or more freeze-thaw cycles. In some embodiments, at least about 80% of the organelle complexes in the population are between about 500 nm and about 3500 nm in size, optionally between about 200 nm and about 1000 nm in size. In some embodiments, the organelle complex populations are derived from cells treated with a mitochondrial activator (e.g., resveratrol).

[0014] Disclosed herein include compositions comprising a population of organelle complexes provided herein.Disclosed herein include formulations comprising a composition provided herein (eg, a composition comprising a population of organelle complexes) and a pharmaceutically acceptable carrier.

[0015] Disclosed herein include methods for producing a first population of organelle complexes. In some embodiments, the method includes: incubating cells in a first solution comprising a surfactant at a first temperature; removing the surfactant to form a second solution; and recovering the first organelle complex from the second solution. In some embodiments, the first organelle complexes include mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi bodies. In some embodiments, the first organelle complex populations are depleted of cytoplasmic macromolecules. In some embodiments, (i) the cells in the first solution are incubated with the surfactant at a concentration equal to or greater than the critical micelle concentration (CMC) of the surfactant; and / or (ii) the cells include or are derived from floating cells or frozen cells.

[0016] In some embodiments, the method includes incubating the second solution at a second temperature. In some embodiments, the first organelle complexes include mitochondria and two, three or four of the endoplasmic reticulum, peroxisomes, lysosomes and Golgi bodies. In some embodiments, the cells in the first solution are contacted with the surfactant at a concentration at least about 5% higher than the critical micelle concentration (CMC) of the surfactant. In some embodiments, the surfactant is a 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 the following items: Triton-X100, Triton-X 114, 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 a tension agent, an osmotic regulator and a chelating agent. In some embodiments, the first solution includes Tris buffer, sucrose and / or a chelating agent.

[0017] In some embodiments, incubating cells in the first solution comprises 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 about 0°C to about 50°C, optionally the first temperature is 25°C and the second temperature is about 0°C to about 4°C. In some embodiments, removing the surfactant comprises washing one or more times with a buffer, optionally a Tris buffer. In some embodiments, incubating the second solution comprises incubating the second solution for about 1 minute to about 120 minutes, optionally for about 20 minutes. Recovering the first organelle complexes from the second solution may comprise tangential flow filtration (TFF). TFF may be performed as follows: (i) using a low viscosity buffer, optionally the low viscosity buffer reduces the shear rate; (ii) at about 22°C to about 25°C; (iii) at less than about 2000sec -1(iv) below room temperature, optionally at 4°C; and / or (v) using a buffer comprising human albumin or recombinant albumin (HA). In some embodiments, recovering the first organelle complexes from the second solution comprises performing TFF using a TFF membrane having a molecular weight cutoff of at least 100 kDa, preferably 750 kDa. In some embodiments, recovering the first organelle complexes from the second solution comprises one or more centrifugation steps. In some embodiments, recovering the first organelle complexes from the second solution comprises: centrifuging the second solution at a first centrifugal force; collecting the supernatant; centrifuging the supernatant at a second centrifugal force; and collecting the precipitate to recover the first organelle complexes. In some embodiments, the first centrifugal force and / or the second centrifugal force is from about 100 g to about 5000 g, optionally, the first centrifugal force is about 500 g, and the second centrifugal force is about 3000 g. Centrifugation can be performed for about 10 minutes to about 20 minutes. In some embodiments, centrifuging the supernatant at a second centrifugal force comprises centrifuging at 8000 g for about 20 minutes.

[0018] In some embodiments, incubating cells in the first solution and / or incubating the second solution includes applying physical stimulation to the first solution and / or the second solution, respectively, optionally oscillating and / or stirring. In some embodiments, applying physical stimulation to the first solution and / or the second solution includes causing the first solution and / or the second solution to flow through a flow device. In some embodiments, the flow device includes a fluid channel, which includes two or more sections 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, the flow through the flow device produces additional flow and / or shear. In some embodiments, the method further includes optionally freezing the first organelle complex in a buffer comprising 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) before the incubation step. Disclosed herein include a first organelle complex population, which is obtained by a method for producing a first organelle complex population provided herein.

[0019] Disclosed herein include methods for treating a disease or disorder. In some embodiments, the method includes: contacting a cell of a subject in need thereof with an effective amount of: (i) a cell organelle complex population provided herein; (ii) a composition provided herein (e.g., a composition comprising a cell organelle complex population); and / or (iii) a formulation provided herein, thereby treating the disease or disorder.

[0020] Disclosed herein include methods for treating diseases or conditions associated with mitochondrial dysfunction. In some embodiments, the method includes: contacting a cell of a subject in need thereof with an effective amount of: (i) a cell organelle complex population provided herein; (ii) a composition provided herein (e.g., a composition comprising a cell organelle complex population); and / or (iii) a formulation provided herein, thereby treating a disease or condition associated with mitochondrial dysfunction.

[0021] In some embodiments, contacting the subject's cells comprises a route of administration 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 II), metabolic diseases, eye disorders associated with mitochondrial dysfunction, hearing loss, mitochondrial toxicity associated with therapeutic agents, mitochondrial dysfunction associated with space travel, cardiotoxicity associated with chemotherapy or other therapeutic agents, mitochondrial dysfunction disorders, and migraine. In some embodiments, the disease or disorder is selected from the group consisting of mitochondrial myopathy, diabetes and deafness (DAD) syndrome, Barth syndrome, Leber's hereditary optic neuropathy (LHON), Reye's syndrome, NARP (neuropathy, ataxia with retinitis pigmentosa and ptosis syndrome), myoneurogastrointestinal encephalopathy (MNGIE), MELAS (mitochondrial encephalopathy with lactic acidosis and stroke-like episodes) syndrome, myoclonic epilepsy with red ragged fibers (MERRF) syndrome, Kearns-Sell syndrome, and mitochondrial DNA depletion syndrome.

[0022] In some embodiments, the disease or disorder is an ischemia-related disease or disorder, a genetic disorder, a disease or disorder of aging, a neurodegenerative disorder, a cardiovascular disorder, 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 encephalopathy, acute coronary syndrome, myocardial infarction, liver ischemia-reperfusion injury, ischemic injury-compartment syndrome, vascular occlusion, wound healing, spinal cord injury, sickle cell disease, and reperfusion injury of a transplanted organ.

[0023] In some embodiments, the neurodegenerative disorder is selected from the group consisting of: dementia, Friedreich's ataxia, amyotrophic lateral sclerosis, mitochondrial encephalopathy with hyperlactosis and stroke-like episodes (MELAS), myoclonic epilepsy with red ragged fibers (MERFF), epilepsy, Parkinson's disease, Alzheimer's disease or Huntington's disease. Exemplary neuropsychiatric disorders include bipolar disorder, schizophrenia, depression, addiction disorders, anxiety disorders, attention deficit disorder, personality disorders, autism and Asperger's disease. In some embodiments, the cardiovascular disorder 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 illness is acute respiratory distress syndrome (ARDS) or preeclampsia or intrauterine growth restriction (IUGR). BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figures 1A to 1B Depicted are non-limiting exemplary data related to the characterization of organelle complex populations provided herein. Figure 1A Depicted are intracellular structure / organelle western blot protein analysis of homogenized mitochondria (H-mito), first organelle complex (1st OC), and second organelle complex (2nd OC) prepared from HeLa cells using the methods disclosed herein. Figure 1B Depicted are Western blot protein analysis of mitochondrial markers of homogenized mitochondria (H-mito), first organelle complex (1st OC), and second organelle complex (2nd OC) prepared from HeLa cells using the methods disclosed herein.

[0025] Figures 2A to 2B Depicted are non-limiting exemplary data related to the characterization of organelle complex populations provided herein. Figure 2A Depicted are Western blot protein analysis of the intracellular structure / organelle of the first organelle complex (1st OC) and the second organelle complex (2nd OC) prepared from HEK293T cells using the methods disclosed herein. Figure 2B Depicted are Western blot protein analysis of mitochondrial markers of the first organelle complex (1st OC) and the second organelle complex (2nd OC) prepared from HEK293T cells using the methods disclosed herein.

[0026] Figures 3A to 3B Depicted are non-limiting exemplary data related to the structural integrity of the first organelle complex (1st OC) and the second organelle complex (2nd OC): Outer membrane integrity ( Figure 3A ) and endometrial integrity ( Figure 3B ).

[0027] Figure 4 Depicted are non-limiting exemplary data relating to ATP production by a population of the first organelle complex over a range of Pi concentrations.

[0028] Figures 5A to 5B Depicted are non-limiting exemplary data related to the structural integrity of the first organelle complex. 2+ The citrate synthase activity of the first organelle complex was measured at Figure 5A ) and cytochrome c oxidase activity ( Figure 5B ).

[0029] Figures 6A to 6D Data related to the structural integrity of the first organelle complex (1st OC) and the second organelle complex (2nd OC) are depicted. Fig. 6A The experimental setup is depicted, and Figures 6B to 6C Depicted are the second organelle complex (2OC; Figure 6C ) and the first organelle complex (1st OC; Figure 6B ) of ATP production, and Fig.6D The ratio of ATP production by the two organelle complexes is depicted.

[0030] Figures 7A to 7B Depicts the experimental setup related to the effect of the second organelle complex (2OC) on ATP production ( Fig. 7A ) and data( Figure 7B ).

[0031] Figure 8 Depicted are qPCR data associated with the first organelle complex (1st OC) and the second organelle complex (2nd OC) isolated from HeLa cells. Depicted are the relative ratios of mitochondrial DNA (mtDNA) copy number / ug protein between the 1st OC and the 2nd OC.

[0032] Figures 9A to 9B Depicted is the experimental setup related to mtDNA incorporation in recipient C6 rho0 cells incubated with homogenized mitochondria (H-mito), the first organelle complex (1st OC), and the second organelle complex (2nd OC) ( Fig.9A ) and data( Fig. 9B ). The incorporated mtDNA ( Fig. 9B ).

[0033] Figures 10A to 10GThe protein concentration of the first organelle complex recovered using final centrifugation conditions of 3000 g (10 min) or 8000 g (20 min) is depicted. Fig. 10A ), total ATP production ( Fig. 10B ), ATP production in the presence of oligomycin ( Fig. 10C ), endometrial integrity ( Fig. 10D ), outer membrane integrity ( Fig.10E )、COX activity( Fig.10F ) and citrate synthase (CS) activity ( Figure 10G ) related data.

[0034] Figures 11A to 11G Depicted is a stained SDS-PAGE gel (17 μg protein / lane) of the first organelle complex recovered using final centrifugation conditions of 3000 g (10 min) or 8000 g (20 min) ( Fig.11A ) and LAMP2 (lysosomal; Fig. 11B ), Golgi protein 97 (Golgi; Fig. 11C ), catalase (peroxisome; Fig.11D ), calreticulin (ER; Fig.11E ), VDAC (mitochondria (outer membrane); Fig.11F ), TOMM20 (mitochondria (outer membrane); Fig.11G ) and TFAM (mitochondria (matrix); Fig.11H ) protein level data.

[0035] Fig.12 Depicted are data relating to ATP production by fibroblasts incubated with homogenized mitochondria (H-mito), the first organelle complex, or the second organelle complex.

[0036] Fig.13 Depicted are data associated with a first organelle complex obtained via a first generation method (employing pipetting as a physical stimulus) compared to an improved method employing a flow reduction device as a physical stimulus. DETAILED DESCRIPTION

[0037] In the following detailed description, the accompanying drawings are mentioned, which form a part of this paper. Unless the context indicates otherwise, in the accompanying drawings, similar symbols generally represent similar components. The illustrative embodiments described in the detailed description, the accompanying drawings and the claims are not intended to be restrictive. Other embodiments may be used and other changes may be made without departing from the spirit or scope of the subject matter described herein. It will be easily understood that aspects of the present disclosure (as generally described herein and shown in the accompanying drawings) may be arranged, substituted, combined, separated and designed in various different configurations, all of which are clearly considered herein and become part of the disclosure herein.

[0038] With respect to the related art, all patents, published patent applications, other publications, and sequences from GenBank and other databases mentioned herein are incorporated by reference in their entirety.

[0039] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd Edition, 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 as follows.

[0040] As used herein, "isolated" shall be given its ordinary meaning and shall also refer to a substance or entity that has been (1) separated from at least some of the components with which it was originally produced (whether in nature or in an experimental setting), and / or (2) produced, prepared and / or manufactured by the hand of man. In some embodiments, the isolated mitochondria or isolated population of organelle complexes have been processed to be obtained from a cellular environment via the methods provided herein.

[0041] As used herein, the term "cell" shall be given its ordinary meaning and shall also refer 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 to include cells present in tissues, cells separated from tissues (e.g., single cells), and cells within cell populations (e.g., cell populations obtained from tissues of subjects and / or cell populations obtained from cell lines).

[0042] As used herein, the term "mitochondria" should be given its ordinary meaning and should also refer to an organelle present in a eukaryotic cell having a double lipid membrane, an inner membrane and an outer membrane, and a matrix surrounded by cristae and the inner membrane. Mitochondria (more than one mitochondria) have enzymes on their inner membrane, such as respiratory chain complexes involved in oxidative phosphorylation. The inner membrane has a membrane potential due to the internal-external proton gradient formed by the action of respiratory chain complexes, etc. When the inner membrane is destroyed, mitochondria are considered to be unable to maintain membrane potential.

[0043] As used herein, the term "organelle complex" should be given its usual meaning, and should also refer to a complex of mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi bodies. Organelle complexes can be depleted of cytoplasmic macromolecules (e.g., cytoplasmic proteins). In some embodiments, organelle complexes do not contain cytoplasmic macromolecules. In some embodiments, organelle complex populations include homogenized mitochondria. As used herein, the term "population" should be given its usual meaning, and should also refer to a group with multiple identical or different substances. For example, a "organelle complex population" is a group with at least multiple identical or different organelle complexes. A population may not always be homogenous, and may have a physical distribution, a chemical distribution, and / or a physiological distribution. Physical distribution includes, for example, particle size and polydispersity index. Chemical distribution includes, for example, zeta potential distribution and lipid composition distribution. Physiological distribution includes, for example, differences in physiological functions (e.g., respiratory activity). Organelle complex populations can include a first organelle complex, a second organelle complex, homogenized mitochondria, or any combination thereof. As used herein, the term "homogenized mitochondria" shall be given its ordinary meaning, and shall also refer to mitochondria isolated via a method comprising one or more homogenization steps.

[0044] As used herein, the term "surfactant" shall be given its ordinary meaning and shall also refer to a molecule having a hydrophilic portion and a hydrophobic portion in one molecule. Surfactants have the effect of reducing interfacial surface tension or mixing polar and non-polar substances by forming micelles. Surfactants are roughly classified into nonionic surfactants and ionic surfactants. Nonionic surfactants are those in which the hydrophilic portion is not ionized, while ionic surfactants are those in which the hydrophilic portion contains cations or anions or contains both cations and anions.

[0045] As used herein, the term "critical micelle concentration" (CMC) shall be given its ordinary meaning and shall also refer to the concentration at which the surfactant forms micelles and further addition of surfactant to the system contributes to micelle formation, particularly bulk concentrations. When the concentration is above the critical micelle concentration, adding surfactant to the system may desirably increase the amount of micelles, particularly the number of micelles.

[0046] As used herein, "subject" refers to an animal that is the subject of treatment, observation or experiment. "Animal" includes cold-blooded vertebrates and warm-blooded vertebrates and invertebrates, such as fish, shellfish, reptiles, and particularly mammals. As used herein, "mammal" refers to an individual belonging to the class mammalia, and includes, but is not limited to, humans, domestic animals and farm animals, zoo animals, sports animals, and pet animals. Non-limiting examples of mammals include mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats; cattle; horses; primates, such as monkeys, chimpanzees, and apes, and 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" should be given its ordinary meaning, and should also refer to cells in vivo, in vitro, and / or ex vivo cells intended to incorporate exogenous mitochondria and / or organelle complexes.

[0047] As used herein, the term "treatment" refers to an intervention in response to a disease, illness or physiological condition exhibited by a patient. The purpose of treatment may include, but is not limited to, one or more of the following: relieving or preventing symptoms, slowing or stopping the progression or deterioration of a disease, illness or condition, and the alleviation of a disease, illness or condition. The terms "treat" and "treatment" include, for example, therapeutic treatments, preventive treatments, and applications in which the risk of a subject suffering from a disease or other risk factors is reduced. Treatment does not require a complete cure of the disease and encompasses embodiments in which symptoms or potential risk factors are alleviated. In some embodiments, "treatment" refers to both therapeutic treatments and preventive measures or preventive measures. Those that require treatment include those that have been affected by a disease or illness or an undesirable physiological condition and those in which a disease or illness or an undesirable physiological condition is to be prevented. As used herein, the term "prevention" refers to any activity that reduces the burden on individuals who subsequently exhibit these symptoms. This can occur at the primary, secondary and / or tertiary prevention levels, where: a) primary prevention avoids the progression of a symptom / disorder / condition; b) secondary prevention activities target the early stages of treatment of a symptom / disorder / symptom, thereby increasing the chances of intervention to prevent the progression of the symptom / disorder / symptom and the onset of symptoms; and c) tertiary prevention reduces the negative impact of an established symptom / disorder / symptom by, for example, restoring function and / or reducing any symptom / disorder / symptom or associated complications. The term "prevention" does not require 100% elimination of the likelihood of an event. Rather, it indicates that the likelihood of an event occurring has been reduced in the presence of a compound or method.

[0048] As used herein, the term "oxidative stress" shall be given its ordinary meaning, and shall also refer to an imbalance between the generation of reactive oxygen species, reactive nitrogen species and / or free radicals and the antioxidant capacity of a biological system.

[0049] As used herein, the term "effective amount" refers to an amount sufficient to achieve beneficial or desired biological and / or clinical results.

[0050] In some embodiments, the methods, compositions, systems and kits provided herein can be used in conjunction 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 contents of which are incorporated herein by reference in their entirety).

[0051] Organelle complex

[0052] Disclosed herein include organelle complex populations. Organelle complexes can be separated from or derived from floating cells and / or frozen cells. Organelle complexes can be separated from or derived from cells contacted with a surfactant at a concentration equal to or higher than the critical micelle concentration (CMC) of the surfactant. These organelle complexes can contain mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi bodies. Disclosed herein include compositions comprising organelle complex populations provided herein. Also disclosed herein include formulations comprising compositions provided herein (e.g., compositions comprising organelle complex populations) and pharmaceutically acceptable carriers. Also disclosed herein include a first organelle complex population, which is obtained by a method provided herein for producing a first organelle complex population. These first organelle complexes can be suitable for, for example, treating various diseases and disorders, including those described herein, by mitochondrial transplantation. In some embodiments, an organelle complex (e.g., a first organelle complex) is internalized into cells in which mitochondria have severe dysfunction and / or cells in which the influx of highly functional mitochondria is beneficial to restore and / or enhance mitochondrial function.

[0053] The organelle complexes provided herein (e.g., the first organelle complex, the second organelle complex) can include mitochondria and one, two, three or four of the endoplasmic reticulum, peroxisome, lysosome 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 peroxisome; (iii) mitochondria and lysosome; (iv) mitochondria and Golgi apparatus; (v) mitochondria, endoplasmic reticulum and peroxisome; (vi) mitochondria, endoplasmic reticulum and lysosome; (vii) mitochondria, endoplasmic reticulum and Golgi apparatus; (viii) mitochondria, endoplasmic reticulum, peroxisome and lysosome; The ratio of mitochondria to other organelles (e.g., endoplasmic reticulum, peroxisome, lysosome, and Golgi) may vary in the population of organelle complexes.For example, in some embodiments, the molar ratio of mitochondrial proteins to other organelle-associated proteins (e.g., endoplasmic reticulum proteins, peroxisomal proteins, lysosomal proteins, and / or Golgi proteins) in a population of organelle complexes can be or is about 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, 1:10 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 range between any two of these values.In some embodiments, the molar ratio of mitochondrial proteins to other organelle-associated proteins (e.g., endoplasmic reticulum proteins, peroxisomal proteins, lysosomal proteins, and / or Golgi proteins) in a population of organelle complexes can be or is about 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 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、5 1:1、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 range between any two of these values.

[0054] Organelle complexes can be depleted of cytoplasmic macromolecules. Cytoplasmic macromolecules may not be present in the organelle complex populations provided herein. The organelle complex populations provided herein may include negligible and / or undetectable amounts of cytoplasmic macromolecules. The organelle complex populations provided herein may be substantially pure organelle complex populations. Substantially pure organelle complex populations may include less than about 20% (e.g., less than about 20%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, 1%, 0.1%, 0.01%, 0.001%, 0% or a number or range between any two of these values) cytoplasmic macromolecules. The cytoplasmic macromolecules may comprise cytoplasmic proteins, and the abundance of one or more cytoplasmic proteins (e.g., p70S6K and / or glyceraldehyde 3-phosphate dehydrogenase (GAPDH)) may 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 cell from which the organelle complex population is derived. The organelle complex may comprise one or more mitochondrial matrix proteins (e.g., mitochondrial transcription factor A (TFAM), citrate synthase (CS). The organelle complex may comprise one or more mitochondrial outer membrane proteins (e.g., mitochondrial outer membrane complex subunit 20 (TOMM20)). The organelle complex may comprise one or more lysosomal proteins (e.g., lysosomal associated membrane protein 2 (LAMP2), mannose-6-phosphate receptor (M6PR), lysosomal associated membrane protein 1 (LAMP1)). The organelle complex may comprise one or more Peroxisomal proteins (e.g., catalase, ATP-binding cassette transporter 1, subfamily D, type 3 (ABCD3)). The organelle complex may include one or more Golgi proteins (e.g., Golgi protein-97, syntaxin-6, TGOLN2 / trans-Golgi network protein 2 (TGN46), Golgi matrix protein 130 (GM130), mannosidase alpha 2A class member 1 (MAN2A1)). The organelle complex may include one or more endoplasmic reticulum proteins (e.g., calreticulin, calnexin).

[0055] 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 a population can have a size between about 500 nm and about 3500 nm (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 complex 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 shift (larger) relative to the size distribution of the second organelle complex population. In some embodiments, the production scalability of the first organelle complex (e.g., in terms of time, reagent cost and / or labor cost) 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 any number or range between these values) compared to the second organelle complex. The organelle complex population can comprise 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) of mitochondrial DNA (mtDNA) copies compared to the homogenized mitochondrial population. The first organelle complex population can comprise 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) of mitochondrial DNA (mtDNA) copies and / or mtDNA incorporated into recipient cells compared to the second organelle complex population.

[0056] The organelle complex population may include a first organelle complex or a combination of a first organelle complex and a second organelle complex. The first organelle complex may be derived from (i) frozen cells; (ii) floating cells; and / or (iii) cells contacted with a surfactant at a concentration equal to or higher than the critical micelle concentration (CMC) of the surfactant. The second organelle complex may be derived from (i) adherent cells; and / or (ii) cells contacted with a surfactant at a concentration lower than the critical micelle concentration (CMC) of the surfactant. The organelle complex population may be derived from cells treated with a mitochondrial activator (e.g., resveratrol).

[0057] In some embodiments, a second organelle complex is provided. In some embodiments, the method for separating the second organelle complex from the cell includes treating the cells in the first solution with a surfactant at a concentration lower than 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 include 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 any number or range between these values) of one or more lysosomal proteins (e.g., lysosomal associated membrane protein 2 (LAMP2), mannose-6-phosphate receptor (M6PR), lysosomal associated membrane protein 1 (LAMP1)) compared to the second organelle complex. The first organelle complex can comprise 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) of one or more peroxisomal proteins (e.g., catalase, ATP-binding cassette transporter 1, subfamily D, type 3 (ABCD3)) as compared to the second organelle complex. The first organelle complex can comprise 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) of one or more Golgi proteins (e.g., Golgi protein-97, syntaxin-6, TGOLN2 / trans-Golgi network protein 2 (TGN46), Golgi matrix protein 130 (GM130), mannosidase alpha 2A class member 1 (MAN2A1)) as compared to the second organelle complex. The first organelle complex can comprise 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 any number or range between these values) of one or more endoplasmic reticulum proteins (e.g., calreticulin, calnexin) as compared to the second organelle complex.The second organelle complex can comprise 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 any number or range between these values) of one or more cytoplasmic proteins (e.g., p70S6K, glyceraldehyde 3-phosphate dehydrogenase (GAPDH)) as compared to the first organelle complex.

[0058] 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 of the inner membrane and / or the structural integrity of the outer 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 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) of the mitochondria of the organelle complex maintain structural integrity in the extracellular environment compared to a homogenized population of mitochondria in the extracellular environment. In some embodiments, even when exposed to high calcium (Ca 2+) environment, the organelle complex population provided herein also has the unexpected characteristics of maintaining structural integrity and / or functional performance. The extracellular environment can include 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 can include 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 includes 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 includes a free or active calcium concentration of about 4 mg / dL to about 6 mg / dL, or about 1 mmol / L (1000 μM) to about 1.5 mmol / L (1500 μM). In some embodiments, the mitochondria of the organelle complex maintain functional capacity in an environment with a higher calcium concentration than the calcium environment in the cell. Therefore, in some embodiments, the mitochondria of the organelle complex provided herein have the significant feature of being isolated from the cell environment (including from frozen cells or floating cells) with minimal or negligible damage, and retain functional capacity even when exposed to an extracellular environment (e.g., an environment rich in calcium, which would have been expected to cause damage to the mitochondria and / or significantly inhibit their functional capacity).

[0059] In some embodiments, the structural integrity of the inner membrane and / or the structural integrity of the outer membrane (e.g., the presence of a complete inner membrane and / or outer membrane) can be determined by the functional activity of the mitochondria (e.g., membrane potential and polarization). 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 any two of these values) of the mitochondria of the organelle complex maintain functional capacity. In some embodiments provided herein, the mitochondria of the organelle complex can achieve ATP production. In some embodiments, the ATP production of the organelle complex population provided herein exceeds the ATP production of homogenized mitochondria by at least about 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). In some embodiments, the functional capacity in the extracellular environment is measured by a fluorescent indicator of membrane potential. In some embodiments, the fluorescent indicator is selected from a positively charged dye, such as JC-1, TMRM and TMRE.

[0060] The organelle complex population may have various properties that facilitate the delivery of a payload, such as a desired transgene or exogenous agent, to a target cell. The transgene may encode a therapeutic protein. The exogenous agent may be selected from the group consisting of a nucleic acid molecule, a protein or polypeptide, a small molecule, a hormone, and any combination thereof. The exogenous agent may comprise or be 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, a small RNA molecule, a complementary RNA, a non-coding RNA molecule, a siRNA, a pi-RNA molecule, a microRNA molecule, a sno-RNA molecule, a long non-coding RNA molecule, a messenger RNA molecule, a ribosomal RNA molecule, an antisense nucleic acid molecule, a locked nucleic acid (LNA), an antagomir, a CRISPR / Cas gene editing RNA, a trans-activating crRNA (tracrRNA), a short synthetic RNA (gRNA) composed of a "scaffold" sequence, a small Cajal body-specific RNA (scaRNA), a natural cis-antisense siRNA (cis-nat-siRNA), a trans-acting siRNA (tasiRNA), a repeat-associated small interfering RNA (rasiRNA), 7SK, a transfer-messenger RNA (tmRNA), a transfer RNA (tRNA), a 7SL RNA, a signal recognition particle RNA (SRP), or any combination thereof.

[0061] When the organelle complex is contacted with the cell colony, the organelle complex provided herein can be incorporated into the cell. In certain embodiments, incorporation into the cell includes co-location and / or fusion with the endogenous mitochondria in the cell. The cell can be in vivo, in vitro or in vitro. In certain embodiments, compared with the homogenized mitochondrial colony, at least about 2 times (for example, 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 any number or range between these values) of the organelle complex can be incorporated into the cell. When the organelle complex is in contact with the host cell colony, compared with the homogenized mitochondrial colony, the organelle complex can have excellent ability to be incorporated into the host cell. When the first organelle complex is in contact with the host cell colony, compared with the second organelle complex, the first organelle complex can have excellent ability to be incorporated into the host cell. In some embodiments, at least about 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 any number or range between these values) of the first organelle complex can be incorporated into the cell compared to the second organelle complex. When the organelle complex is contacted with a host cell population, the organelle complex can have an excellent ability to be incorporated into the host cell. In some embodiments, 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 any number or range between these values) of the organelle complex can be incorporated into the host cell compared to a homogenized mitochondrial population. In some embodiments, and without being bound by any particular theory, the superior incorporation capabilities of the organelle complexes provided herein are at least partially responsible for the superior clinical effects exhibited by the organelle complexes when used to treat any disease or condition, such as those described herein.

[0062] In some embodiments, the organelle complex population provided herein can be incorporated into the cell after storing mitochondria at any temperature provided herein (e.g., 4°C ± 3°C, -20°C ± 3°C, -80°C ± 3°C or in a liquid nitrogen environment). The mitochondria of the organelle complex can be incorporated into the cell after the population undergoes one or more freeze-thaw cycles. Compared to the homogenized mitochondrial population, 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 any number or range between these values) of the organelle complex can be incorporated into the cell after the population undergoes one or more freeze-thaw cycles.

[0063] Method for producing a first organelle complex population

[0064] Disclosed herein are methods for producing a first population of organelle complexes. In some embodiments, the method comprises: incubating cells in a first solution comprising a surfactant at a first temperature; removing the surfactant to form a second solution; and recovering the first organelle complex from the second solution. The first organelle complex may comprise mitochondria and one or more of an endoplasmic reticulum, a peroxisome, a lysosome, and a Golgi apparatus. The first population of organelle complexes may be depleted of cytoplasmic macromolecules. In some embodiments, (i) the cells in the first solution are incubated with the surfactant at a concentration equal to or greater than the critical micelle concentration (CMC) of the surfactant; and / or (ii) the cells comprise or are derived from floating cells or frozen cells.

[0065] In some embodiments, a method for producing a first organelle complex is provided. The production of the first organelle complex may include: (step A) providing adherent, floating and / or frozen cells, thawing and placing in a tube. The production of the first organelle complex may include: (step A) providing adherent, floating and / or frozen cells, centrifuging and collecting the precipitate. The production of the first organelle complex may include: (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 production of the first organelle complex may include one or more of the following steps: (step B) adding Tris buffer, centrifuging and collecting precipitate; (step C) adding Tris buffer and vortexing; (step D) adding a solution containing a surfactant and incubating; (step E) centrifuging and collecting precipitate; (step F) adding Tris buffer, centrifuging and collecting precipitate; (step G) adding Tris buffer and pipetting; (step H) transferring to another tube and collecting the buffer solution in the original tube and rinsing; (step I) centrifuging and collecting supernatant; (step J) centrifuging and collecting precipitate; and (step K) pipetting, one or more of the above steps may include an incubation period. One or more of the above steps may include a centrifugation step, followed by collecting supernatant and / or precipitate. One or more of the above steps may be omitted and one or more additional steps may be included. The time, volume, concentration and centrifugal force may vary according to the embodiment.

[0066] In some embodiments, the cells used in the disclosed method for producing the first cell organelle complex colony can be adherent cells, floating cells, separated cells, suspended cells, frozen cells or any combination thereof. Cells can be in the form of cells present in tissues, or they can be separated from tissues (e.g., single cells) or their colonies. Cells separated from tissues can be cultured cells, or single cells or their colonies, obtained by treating tissues or cultured cells with enzymes (such as collagenase) for making them single cells. If necessary, tissues can be chopped before enzyme (such as collagenase) treatment. The method can further include treating cells with mitochondrial activators (e.g., resveratrol) before the step of incubating cells in the first solution. The first cell organelle complex can be derived from cells in which mitochondria are activated. The activation of mitochondria can be achieved by a variety of methods (e.g., by contacting mitochondria with mitochondrial activators). The activation of this mitochondria can be achieved by a variety of methods (including MITO-Porter technology). The MITO-Porter technology can use a complex of mitochondrial targeting vectors and mitochondrial activators. As used herein, the term "mitochondrial activator" may refer to a substance capable of activating the mitochondrial respiratory chain complex (electron transport system), particularly in terms of membrane potential, a substance capable of causing mitochondria to enter a polarized state, and in particular, preferably using a substance capable of causing mitochondria to enter a hyperpolarized state. The example of a mitochondrial activator 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 in particular, resveratrol is preferred (see WO2018 / 092839). Other examples of mitochondrial activators include mitochondrial DNA and mitochondrial RNA, such as 12S rRNA and 16S rRNA (see WO2020 / 230601, which is incorporated herein by reference in its entirety), and any other component of mitochondria.

[0067] Step A may include adding about 1 mL to about 10 mL of a solution (e.g., PBS(-)). Step A may include adding about 1 mL to about 5 mL of TrypLE. Step A may include incubating at about 30°C to 40°C for about 2 min to about 10 min. Step A may include thawing at a temperature of about 30°C to 42°C for about 1 min to about 7 min. Step A may include using adherent cells from a culture dish of about 5 cm to about 20 cm. Step A, step B, step E, and / or step I may include centrifuging at about 200 g to about 800 g at 0°C to 10°C for about 5 min to about 20 min. Step A and / or step H may include using a tube of about 10 mL to about 75 mL. Step B, step C, step F, and / or step G may include adding about 0.5 mL to about 6 mL of Tris buffer. Step C may include vortexing for about 5 sec to about 20 sec. Step D may include adding about 0.5 mL to about 2 mL of a solution containing a surfactant. The surfactant can be present in a solution containing the surfactant at a concentration of about 50 μg / mL to about 200 μg / mL. 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 500g to about 1500g for about 1 min to about 10 min at about 0°C to 10°C. Step G can include incubating at about 0°C to 5°C for about 10 min to about 30 min. Step G and / or step 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 in the original tube and rinsing. Step J can include centrifuging at about 2000g to about 4000g at about 0°C to 10°C for about 5 min to about 20 min. Step J can include centrifuging at about 6000g to about 10000g at about 0°C to 10°C for about 10 min to about 30 min. Collecting the precipitate can include removing the supernatant (eg, 0.5 mL to about 2.5 mL).

[0068] In some embodiments, a first generation method (e.g., a centrifugation-based method) for producing a first organelle complex is provided, which may 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., a TFF method) for producing a first organelle complex is provided, which may include replacing (and / or adding) one or more of the following steps: 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, in some embodiments, cell washing / preparation may include the use of a countercurrent centrifugation system (e.g., Rotea). For example, saponin treatment and / or first organelle complex extraction may include the use of a flow device (e.g., a flow reduction device). The flow device may include a fluid channel including two or more sections having different cross-sectional diameters, and in some embodiments, and without being bound by any particular theory, the flow through the flow reduction device may cause a change in flow rate due to a change in the flow cross-sectional area. The flow reduction device provided herein can have various configurations, such as square reducers, conical reducers, concentric reducers and / or eccentric reducers. The flow device can include tubes of various types and sizes to generate additional flow and shear forces for extraction of the first organelle complex. Lysate fine purification can include pre-filtration and / or Rotea. In some embodiments of the second generation method (e.g., TFF method) provided herein, tangential flow filtration (TFF) can be used for purification and / or buffer exchange. Step B can include the use of Rotea. Step G can include the use of a flow device (e.g., a flow reduction device). Step I can include cell lysate fine purification (e.g., pre-filter or Rotea or combination). Step J can include TFF. Step K can include the use of a cryoprotectant comprising human albumin (HA) and / or glycerol. Using HA and / or glycerol in a preservation buffer can enhance the stability of the first organelle complex during storage at low temperatures (e.g., -80°C), and can improve its quality after thawing.

[0069] The 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 therein) higher 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 50ug / mL. The surfactant can be a nonionic surfactant. The surfactant can be selected from the group consisting of Triton-X100, Triton-X 114, Nonidet P-40, n-dodecyl-D-maltoside, Tween-20, Tween-80, saponin and digitonin.

[0070] In certain embodiments, the surfactant used in the method provided herein can be an ionic surfactant or a nonionic surfactant. The nonionic surfactant used in the present disclosure can include, for example, esters, ethers, and alkyl glycoside forms. The nonionic surfactant includes, for example, alkyl polyethylene glycols, polyoxyethylene alkylphenyl ethers, and alkyl glycosides. The nonionic surfactant can include Triton-X 100, Triton-X 114, Nonidet P-40, n-dodecyl-D-maltoside, Tween-20, Tween-80, saponin, and / or digitonin. The concentration of the one or more surfactants present in the first solution may be, may be about, may be at least, or may be at most 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 20, 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 0, 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, 180 0, 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, or a number or range in μg / mL or μM between any two of these values.

[0071] In some embodiments, the first solution and / or the second solution may include a buffer. The buffer may include one or more of a tonicity agent, an osmotic regulator, and a chelating agent. The first solution and / or the second solution may include a Tris buffer, sucrose, and / or a chelating agent. Exemplary buffers for use in the methods provided herein include, for example, Tris buffer, HEPES buffer, and phosphate buffer. The buffer may be, for example, pH 6.7-7.6 (e.g., pH 6.8-7.4, pH 7.0-7.4, such as pH 7.2-7.4, such as pH 7.4). In some embodiments, the buffer may include a tonicity agent and an osmotic regulator. Exemplary tonicity agents and osmotic regulators include monosaccharides (e.g., glucose, galactose, mannose, fructose, inositol, ribose, xylose, etc.), disaccharides (e.g., lactose, sucrose, cellobiose, trehalose, maltose, etc.), trisaccharides (e.g., raffinose, melesinose, etc.), polysaccharides (e.g., cyclodextrins, etc.), sugar alcohols (e.g., erythritol, xylitol, sorbitol, mannitol, maltitol, etc.), glycerol, diglycerol, polyglycerol, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, etc. The buffer may also contain a chelating agent, particularly a chelating agent for divalent metals, such as a chelating agent for calcium ions. Chelating agents include, for example, ethylene glycol ether diaminetetraacetic acid (EGTA) and ethylenediaminetetraacetic acid (EDTA).

[0072] The method may include incubating the second solution at a second temperature. The first temperature and / or the second temperature may be from about 0°C to about 50°C. The first temperature may be 25°C, and the second temperature may be from about 0°C to about 4°C. The first temperature and / or the second temperature can be, can be about, can be at least or can be at most 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. Removing the surfactant can include performing one or more washes using a buffer (e.g., a Tris buffer). One or more steps of the methods provided herein can be performed on ice or at room temperature.

[0073] Incubating the cells in the first solution may 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 may include incubating the second solution for about 1 minute to about 120 minutes, optionally for about 20 minutes. The steps of incubating the cells in the first solution and / or the step of incubating the second solution may include 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 or a time period of a number or range of minutes between any two of these values. Incubating cells in the first solution and / or incubating the second solution can include applying physical stimulation to the first solution and / or the second solution respectively, such as pipetting, vibration and / or stirring. Applying physical stimulation to the first solution and / or the second solution can include making the first solution and / or the second solution flow through a flow device (e.g., a flow reduction device). The flow device can include a fluid channel, which includes two or more sections with different cross-sectional diameters. The cross-sectional diameter can be, can be about, can be at least or can be at most 0.1mm, 0.2mm, 0.3mm, 0.4mm, 0.5mm, 0.6mm, 0.7mm, 0.8mm, 0.9mm, 1mm, 2mm, 3mm, 4mm, 5mm, 6mm, 7mm, 8mm, 9mm, 10mm, 11mm, 12mm, 13mm, 14mm, 15mm, 16mm, 17mm, 18mm, 19mm, 20mm, 21mm, 22mm, 23mm, 24mm, 25mm or a number or range between any two of these values. In some embodiments, the flow through the flow device produces additional flow and / or shear.

[0074] Removing the surfactant to form the second solution may include reducing the concentration of the surfactant in the solution contacted by the first organelle complex, including, for example, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% or less of the surfactant concentration; or below the detection limit in the solution contacted by the first organelle complex. To ensure that the surfactant is removed 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 solution without surfactant) (e.g., a buffer). In order to remove the surfactant from the first solution, the solution added to the first solution or exchanged with the first solution may be a buffer, and in some embodiments, it is the buffer used in the first solution (but containing a solution with a lower concentration of surfactant, preferably a solution without surfactant or containing an undetectable level of surfactant). The second solution may be a solution containing a lower concentration of surfactant. In some embodiments, the second solution is a solution without surfactant or a solution with a negligible and / or undetectable amount of surfactant. The solution used in the recovery step (e.g., the second solution) may be a solution containing, for example, a buffer, an osmotic regulator, and a divalent metal chelator, substantially free of surfactant. As used herein, "substantially free" is used in a sense that does not exclude the presence of contamination by a certain amount of the "substantially free component" that cannot be removed or detected.

[0075] In some embodiments, recovering the first organelle complex comprises applying one or more physical stimuli to the second solution. The recovery step may comprise incubating the second solution at a second temperature. Thus, the recovery step may be performed under oscillating or non-oscillating conditions. The incubation of the recovery step may be performed under stirring or non-stirring conditions. The first organelle complex may be collected as a precipitate by subjecting the second solution to one or more centrifugation steps. Recovering the first organelle complex from the second solution may comprise one or more centrifugation steps. Recovering the first organelle complex from the second solution may comprise: centrifuging the second solution at a first centrifugal force; collecting the supernatant; centrifuging the supernatant at a second centrifugal force; and collecting the precipitate to recover the first organelle complexes. The first centrifugal force and / or the second centrifugal force may be about 100 g to about 5000 g. The first centrifugal force may be about 500 g, and the second centrifugal force may be about 3000 g.The first centrifugal force and / or the second centrifugal force may be, may be about, may be at least or may be at most 100g, 110g, 120g, 128g, 130g, 140g, 150g, 160g, 170g, 180g, 190g, 200g, 210g, 220g, 230g, 240g, 250g, 260g, 270g, 280g, 290g, 300g, 310g, 320g, 330g, 340g, 350g, 360g, 370g, 380g, 390g, 400g, 410g, 420g, 430g, 440g, 450g, 460g, 470g, 480g, 490g, 500g, 510g, 520g, 530g, 540g, 550g, 560g, 570g, 580g, 590g, 600g, 610g, 620g, 630g, 640g 90g, 400g, 410g, 420g, 430g, 440g, 450g, 460g, 470g, 480g, 490g, 500g, 510g, 520g, 530g, 540g, 550g, 560g, 570 g, 580g, 590g, 600g, 610g, 620g, 630g, 640g, 650g, 660g, 670g, 680g, 690g, 700g, 710g, 720g, 730g, 740g, 750g, 7 60g, 770g, 780g, 790g, 800g, 810g, 820g, 830g, 840g, 850g, 860g, 870g, 880g, 890g, 900g, 910g, 920g, 930g, 940 g, 950g, 960g, 970g, 980g, 990g, 1000g, 1100g, 1200g, 1300g, 1400g, 1500g, 1600g, 1700g, 1800g, 1900g, 2000g, 2100g, 2200g, 2300g, 2400g, 2500g, 2600g, 2700g, 2800g, 2900g, 3000g, 3250g, 3500g, 3750g, 4000g, 4250g, 4500g, 4750g, 5000g, 5500g, 6000g, 6500g, 7000g, 7500g, 8000g, 8500g, 9000g, 9500g, 10000g, or a number or range between any two of these values.The centrifugation step may include 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, 21 In some embodiments, the first cell organelle complexes can be recovered from the second solution by centrifugation for about 10 minutes to about 20 minutes. The first cell organelle complexes can be recovered from the second solution by centrifugation for about 20 minutes. The first cell organelle complexes can be recovered from the second solution by 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 may be, may be about, may be at least, or may be at most 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, cP, 28cP, 29cP, 30cP, 31cP, 32cP, 33cP, 34cP, 35cP, 36cP, 37cP, 38cP, 39cP, 40cP, 41cP, 42cP, 43cP, 44cP, 45cP, 46cP, 47cP, 48cP, 49cP, 50cP, 51cP, 52cP, 53cP, 54cP, 55cP, 56cP, 57cP, 58c P、59cP、60cP、61cP、62cP、63cP、64cP、65cP、66cP、67cP、68cP、69cP、70cP、71cP、72cP、73cP、7 4cP, 75cP, 76cP, 77cP, 78cP, 79cP, 80cP, 81cP, 82cP, 83cP, 84cP, 85cP, 86cP, 87cP, 88cP, 89cP , 90cP, 91cP, 92cP, 93cP, 94cP, 95cP, 96cP, 97cP, 98cP, 99cP, 100cP, 200cP, 300cP, 400cP, 500cP, 600cP, 700cP, 800cP, 900cP, 1000cP, 2500cP, 5000cP, 7500cP, 10000cP or a number or range between any two of these values. The temperature at which TFF is performed can be, can be about, can be at least or can be at most 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 shear rate of the TFF procedure and / or the flow device (eg, flow reduction device) may be, may be about, may be at least, or may be at most 1 sec. -1 , 2sec -1 、3sec -1 、4sec -1 , 5sec -1、6sec -1 、7sec -1 、8sec -1 、9sec -1 、10sec -1 、11sec -1 、12sec -1 、13sec -1 、14sec -1 、15sec -1 、16sec -1 、17sec -1 、18sec -1 、19sec -1 、20sec -1 、25sec -1 、30sec -1 、35sec -1 、40sec -1 、45sec -1 、50sec -1 、60sec -1 、70sec -1 、80sec -1 、90sec -1 、100sec -1 、110sec -1 、120sec -1 、128sec -1 、130sec -1 、140sec -1 、150sec -1 、160sec -1 、170sec -1 、180sec -1 、190sec -1 、200sec -1 、210sec -1 、220sec -1 、230sec -1 、240sec -1 、250sec -1 、260sec -1 、270sec -1 、280sec -1 、290sec -1 、300sec -1 、310sec -1 、320sec -1 、330sec -1 、340sec -1 、350sec-1 、360sec -1 、370sec -1 、380sec -1 、390sec -1 、400sec -1 、410sec -1 、420sec -1 、430sec -1 、440sec -1 、450sec -1 、460sec -1 、470sec -1 、480sec -1 、490sec -1 、500sec -1 、510sec -1 、520sec -1 、530sec -1 、540sec -1 、550sec -1 、560sec -1 、570sec -1 、580sec -1 、590sec -1 、600sec -1 、610sec -1 、620sec -1 、630sec -1 、640sec -1 、650sec -1 、660sec -1 、670sec -1 、680sec -1 、690sec -1 、700sec -1 、710sec -1 、720sec -1 、730sec -1 、740sec -1 、750sec -1 、760sec -1 、770sec -1 、780sec -1 、790sec -1 、800sec -1 、810sec -1 、820sec -1 、830sec -1 、840sec -1 、850sec-1 、860sec -1 、870sec -1 、880sec -1 、890sec -1 、900sec -1 、910sec -1 、920sec -1 、930sec -1 、940sec -1 、950sec -1 、960sec -1 、970sec -1 、980sec -1 、990sec -1 、1000sec -1 、1100sec -1 、1200sec -1 、1300sec -1 、1400sec -1 、1500sec -1 、1600sec -1 、1700sec -1 、1800sec -1 、1900sec -1 、2000sec -1 、2100sec -1 、2200sec -1 、2300sec -1 、2400sec -1 、2500sec -1 、2600sec -1 、2700sec -1 、2800sec -1 、2900sec -1 、3000sec -1 、3250sec -1 、3500sec -1 、3750sec -1 、4000sec -1 、4250sec -1 、4500sec -1 、4750sec -1 、5000sec -1 、5500sec -1 、6000sec -1 、6500sec -1 、7000sec -1 、7500sec-1 、8000sec -1 、8500sec -1 、9000sec -1 、9500sec -1 、10000sec -1or a number or range between any two of these values. TFF can be performed using a buffer comprising human albumin (HA). Recovering the first organelle complex from the second solution can include performing TFF using a TFF membrane. The molecular weight cutoff of the TFF membrane can be, can be about, can be at least, or can be at least 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, kDa, 200kDa, 210kDa, 220kDa, 230kDa, 240kDa, 250kDa, 260kDa, 270kDa, 280kDa, 290kDa, 300kDa, 310kDa, 320kDa, 330kDa, 340kDa, 350kDa , 360kDa, 370kDa, 380kDa, 390kDa, 400kDa, 410kDa, 420kDa, 430kDa, 440kDa, 450kDa, 460kDa, 470kDa, 480kDa, 490kDa, 500kDa, 510kDa, 520 kDa, 530kDa, 540kDa, 550kDa, 560kDa, 570kDa, 580kDa, 590kDa, 600kDa, 610kDa, 620kDa, 630kDa, 640kDa, 650kDa, 660kDa, 670kDa, 680kDa , 690kDa, 700kDa, 710kDa, 720kDa, 730kDa, 740kDa, 750kDa, 760kDa, 770kDa, 780kDa, 790kDa, 800kDa, 810kDa, 820kDa, 830kDa, 840kDa, 85 0kDa, 860kDa, 870kDa, 880kDa, 890kDa, 900kDa, 910kDa, 920kDa, 930kDa, 940kDa, 950kDa, 960kDa, 970kDa, 980kDa, 990kDa, 1000kDa, 1100 kDa, 1200kDa, 1300kDa, 1400kDa, 1500kDa, 1600kDa, 1700kDa, 1800kDa, 1900kDa, 2000kDa, 2100kDa, 2200kDa, 2300kDa, 2400kDa, 2500kDa,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.

[0076] The method may further include freezing the organelle complex. Freezing can be performed by gently suspending the organelle complex in a buffer (e.g., a storage buffer) for freezing. The buffer for freezing can be a buffer used in the first solution but does not contain a surfactant, and may further include a cryoprotectant. The cryoprotectant may include human albumin (HA) and / or glycerol. The percentage of glycerol may be, may be about, may be at least or may be at most 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.The percentage of HA may be, may be about, may be at least or may be at most 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%, 0.8 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%, 0.5 9%, 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%, %, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or a number or range between any two of these values. Exemplary cryoprotectants are known in the art and include, for example, glycerol, sucrose, trehalose, dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, diethylene glycol, triethylene glycol, 3-phosphoglycerol, proline, sorbitol, formamide, and polymers. Therefore, the organelle complexes provided herein can be stored by freezing. In the methods disclosed herein, if cryopreservation is not required, the organelle complexes may not be frozen, for example, the organelle complexes may be used when freshly isolated. In other embodiments, the organelle complexes may be stored at 4°C ± 3°C or on ice.In some embodiments, the organelle complex provided herein produced by the methods provided herein can be stored in liquid nitrogen at about -80°C ± 3°C or lower, about -20°C ± 3°C or lower, or about 4°C ± 3°C. In some embodiments, the method further includes thawing the organelle complex after freezing. In some embodiments, the method for thawing the organelle complex includes, for example, rapidly thawing the organelle complex within about 5 minutes or within about 1 minute. In some embodiments, the organelle complex is thawed in a warm bath having a temperature of about 20°C ± 3°C to about 37°C ± 3°C. In some embodiments, the organelle complex is thawed at a temperature of about 20°C ± 3°C or lower. In some embodiments, the organelle complex can be stored for days, weeks, or months or longer, and retains the ability to function after thawing.

[0077] In some embodiments, the method provided herein does not include homogenization; in some embodiments, the method includes homogenization, but the homogenization is only performed to the extent that no bubbles or bubbles are generated in the solution relative to the cell or tissue. In some embodiments, the method also does not include freezing and thawing of cells. In some embodiments, the method of the present disclosure does not require one or more filtration steps when purifying the organelle complex recovered from the cell. In some embodiments, the method does not include applying shear force (e.g., passing through a needle) and / or adding proteases to the cell, the first solution, and / or the second solution. In some embodiments, the method of the present disclosure does not include other methods of destroying the cell membrane (e.g., ultrasonic treatment, treatment with a strong water flow to the extent that the solution produces bubbles, or to the extent that the solution foams) during the entire process of collecting the first organelle complex from the cell. In some embodiments, the method of the present disclosure is performed without any process that may substantially cause physical, chemical or physiological damage to the organelle complex, although freeze-thaw cycles may be applied to the organelle complex for storage. Therefore, the method of the present disclosure is able to obtain organelle complexes with minimal damage.

[0078] In some embodiments, the method provided herein includes encapsulating the first organelle complex in a lipid membrane-based vesicle. In some embodiments, a method for producing a lipid membrane-based vesicle or a composition comprising a vesicle that encapsulates an organelle complex or a population thereof is provided, the method comprising contacting an aqueous solution comprising an organelle complex and an organic phase (e.g., an ethanol solution) comprising a lipid that can form a lipid membrane in a microfluidic channel device with each other in a confluent channel to mix them. The method encapsulated in a lipid membrane-based vesicle is disclosed in PCT Patent Application Publication No. WO2021 / 132735, the contents of which are incorporated herein by reference in their entirety.

[0079] Treatment

[0080] In some embodiments, a method for treating or preventing a disease or illness is provided. In some embodiments, the method comprises: contacting a cell of a subject in need thereof with an effective amount of: (i) a cell organelle complex population provided herein; (ii) a composition provided herein (e.g., a composition comprising a cell organelle complex population); and / or (iii) a formulation provided herein, thereby treating or preventing a disease or illness.

[0081] In some embodiments, a method for treating or preventing a disease or disorder associated with mitochondrial dysfunction is provided. In some embodiments, the method comprises: contacting a cell of a subject in need thereof with an effective amount of: (i) a cell organelle complex population provided herein; (ii) a composition provided herein (e.g., a composition comprising a cell organelle complex population); and / or (iii) a formulation provided herein, thereby treating or preventing a disease or disorder associated with mitochondrial dysfunction.

[0082] The present disclosure also provides the use of organelle complex populations in the manufacture of medicaments for treating diseases and disorders provided herein. In some embodiments, the organelle complex population is administered to a subject in combination with one or more additional agents and / or additional therapies designed to treat a disease or disorder. In some embodiments, the present disclosure provides methods for treating diseases and disorders associated with mitochondrial dysfunction or diseases or disorders that otherwise benefit from supplementing healthy functional mitochondria.

[0083] Contacting the subject's cells may include a route of administration selected from the group consisting of intravenous administration, intraarterial administration, intratracheal administration, subcutaneous administration, intramuscular administration, inhalation, intrapulmonary administration, and intraocular administration. The organelle complex population may be administered orally or systemically.

[0084] As used herein, the term "local administration" or "topic administration" refers to any route of administration that brings a population of organelle complexes into contact with the body of an individual such that the location of the resulting population of organelle complexes within the body is local (limited to the specific tissue, organ, or other body part to be imaged). Exemplary routes of local administration include injection into a specific tissue by needle, tube feeding into the gastrointestinal tract, and application of a solution containing a population of organelle complexes onto the surface of the skin.

[0085] As used herein, the term "systemic administration" refers to any route of administration that contacts a population of organelle complexes with the body of an individual such that the location of the resulting population of organelle complexes in vivo is systemic (i.e., not limited to a specific tissue, organ, or other body part that requires imaging). Systemic administration includes enteral administration and parenteral administration. Enteral administration is a systemic route of administration in which a substance is administered via the digestive tract, and includes, but is not limited to, oral administration, administration through a gastric feeding tube, administration through a duodenal feeding tube, gastrostomy, enteral nutrition, and rectal administration. Parenteral administration is a systemic route of administration in which a substance is administered by a route other than the digestive tract, and includes, but is not limited to, intravenous administration, intra-arterial administration, intramuscular administration, subcutaneous administration, intradermal administration, intraperitoneal administration, and intravesical infusion.

[0086] In another aspect, the present disclosure provides a pharmaceutically acceptable composition comprising a therapeutically effective amount of a cell organelle complex population disclosed herein. As described in detail below, the pharmaceutical compositions of the present disclosure can be specifically formulated for administration in solid or liquid form, including those suitable for the following: (1) oral administration, such as an immersion (aqueous or non-aqueous solution or suspension), tablet, pill, powder, granule, paste; (2) parenteral administration, such as by subcutaneous, intramuscular or intravenous injection, such as as a sterile solution or suspension: (3) topical administration, such as as a cream, ointment or spray applied to the skin; (4) intravaginal or intrarectal, such as as a pessary, cream or foam; or (5) an aerosol containing a cell organelle complex population, such as an aqueous aerosol, a liposomal preparation or solid particles. The pharmaceutical composition may include one or more pharmaceutically acceptable carriers. As used herein, the phrase "therapeutically effective amount" may refer to an amount of a population of organelle complexes disclosed herein that is effective to produce some desired therapeutic effect, such as cancer treatment, at a reasonable benefit / risk ratio.

[0087] The phrase "pharmaceutically acceptable / pharmaceutically acceptable" as used herein refers to those agents, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0088] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, that is involved in carrying or transporting the subject chemical 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 causing harm to the subject. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (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, cellulose acetate; (4) astragalus powder; (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 tuna oil. soybean oil; (10) glycols such as propylene glycol; (1) polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer solution; and (21) other nontoxic compatible substances used in pharmaceutical preparations.

[0089] Preparations that can be used for the methods of the present disclosure include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, spray and / or parenteral administration. The preparation can be conveniently present in unit dosage form and can be prepared by any method well known in the pharmaceutical field. The amount of active ingredient (e.g., organelle complex population) that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the specific mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of organelle complex population that produces a therapeutic effect. In general, in one hundred percent, this amount will be in the range of from about 1% to about 99% of the active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%.

[0090] Suspensions, in addition to the active agents, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0091] Dosage forms for topical or transdermal administration of a population of organelle complexes 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 that may be required.

[0092] Ointments, pastes, creams and gels may contain excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicone, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0093] Ophthalmic formulations, eye ointments, powders, solutions, etc. are also contemplated as being within the scope of this disclosure.

[0094] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present 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). Appropriate fluidity can be maintained, for example, by the use of coating materials (such as lecithin), by maintaining the desired particle size in the case of dispersions, and by the use of surfactants.

[0095] These compositions can also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. Can ensure that prevention of microbial action is achieved by including various antibacterial and antifungal agents (e.g. parabens, chlorobutanol, phenol sorbic acid, etc.). It may also be desirable to include isotonic agents (such as sugar, sodium chloride, etc.) in the composition. In addition, the absorption of injectable drug forms can be extended by including agents that delay absorption (such as aluminum monostearate and gelatin).

[0096] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure can be determined by the methods of the present disclosure so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration without causing toxicity to the subject.

[0097] The disease or condition may be selected from the group consisting of diabetes (type I and II), metabolic diseases, eye disorders associated with mitochondrial dysfunction, hearing loss, mitochondrial toxicity associated with therapeutic agents, mitochondrial dysfunction associated with space travel, cardiotoxicity associated with chemotherapy or other therapeutic agents, mitochondrial dysfunction disorders, and migraine.

[0098] The disease or condition may be selected from the group consisting of mitochondrial myopathy, diabetes and deafness (DAD) syndrome, Barth syndrome, Leber's hereditary optic neuropathy (LHON), Reye's syndrome, NARP (neuropathy, ataxia with retinitis pigmentosa and ptosis syndrome), myoneurogastrointestinal encephalopathy (MNGIE), MELAS (mitochondrial encephalopathy with lactic acidosis and stroke-like episodes) syndrome, myoclonic epilepsy with red ragged fibers (MERRF) syndrome, Kearns-Sell syndrome and mitochondrial DNA depletion syndrome.

[0099] The disease or disorder can be an ischemia-related disease or disorder, a genetic disorder, an aging disease or disorder, a neurodegenerative disorder, a cardiovascular disorder, cancer, an autoimmune disease, an inflammatory disease, a 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 encephalopathy, acute coronary syndrome, myocardial infarction, liver ischemia-reperfusion injury, ischemic injury-compartment syndrome, vascular occlusion, wound healing, spinal cord injury, sickle cell disease, and reperfusion injury of transplanted organs. The neurodegenerative disorder can be selected from the group consisting of dementia, Friedreich's ataxia, amyotrophic lateral sclerosis, mitochondrial encephalopathy with hyperlactatemia and stroke-like episodes (MELAS), myoclonic epilepsy with red ragged fibers (MERFF), epilepsy, Parkinson's disease, Alzheimer's disease, or Huntington's disease. Exemplary neuropsychiatric disorders include bipolar disorder, schizophrenia, depression, addiction disorders, anxiety disorders, attention deficit disorder, personality disorder, autism and Asperger's disease. Cardiovascular disorders 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 illness can be acute respiratory distress syndrome (ARDS) or preeclampsia or intrauterine growth restriction (IUGR).

[0100] Exemplary ischemia-related diseases and disorders include cerebral ischemia-reperfusion, hypoxic-ischemic encephalopathy, acute coronary syndrome, myocardial infarction, hepatic ischemia-reperfusion injury, ischemic injury-compartment syndrome, vascular occlusion, wound healing (e.g., acute wounds or chronic wounds; cuts, lacerations, compression injuries, burns (e.g., chemical burns, thermal burns or flame burns, windburns or sunburns), or wounds caused by medical or surgical interventions), spinal cord injury, sickle cell disease, and reperfusion injury of transplanted organs. In some embodiments, the organelle complex population can treat, prevent, alleviate and / or improve clinical conditions caused by ischemia-reperfusion injury. In some embodiments, the organelle complex population can improve ejection fraction (EF), inhibit cardiac hypertrophy, and / or treat, prevent, alleviate and / or improve fibrosis after ischemia-reperfusion injury.

[0101] Also provided herein is a kit, which comprises one or more compositions described herein (e.g., a preparation comprising a cell organelle complex population) in a suitable package, and may further comprise written materials, which may include instructions for use, a discussion of clinical studies, a list of side effects, etc. Such kits may also include information, such as scientific literature references, package insert materials, clinical trial results, and / or summaries of these, etc., which indicate or establish the activity and / or advantages of the composition, and / or describe administration, application, side effects, drug interactions, or other information useful to healthcare providers. Such information may be based on the results of various studies, such as studies using experimental animals involving in vivo models and studies based on human clinical trials. The kit may comprise one or more unit doses as described herein.

[0102] Examples

[0103] Some aspects of the above-described embodiments are disclosed in further detail in the following examples, which are not intended to limit the scope of the present disclosure in any way.

[0104] Example 1

[0105] Characterization of organelle complex populations

[0106] The composition of the organelle complex populations provided herein and their functional differences from homogenized mitochondria obtained via currently available methods were examined in a series of experiments.

[0107] Homogenized mitochondria, first organelle complex, and second organelle complex were isolated from HeLa cells (Pierce μg / mL: HeLa H-mito, 650.1; HeLa 2OC, 238.3; HeLa 1OC, 660). The isolated homogenized mitochondrial and organelle complex populations were resuspended in H2O (Milli-Q) and Laemmli sample buffer. The final concentration was adjusted to about 3 mg in 15 ml, and the samples were then 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 as follows: 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; Golgi protein-97 (D8P2K) rabbit mAb #13192; catalase (D4P7B) Rabbit mAb #12980; Citrate synthase (D7V8B) Rabbit mAb #14309; Calreticulin (D3E6) Rabbit mAb #12238; anti-laminin A / C antibody (E-1) #sc-376248; and OxPhos human WB antibody cocktail, rabbit mAb #45-8199. Figures 1A to 1B Depicted are non-limiting exemplary data related to the characterization of organelle complex populations provided herein. Figure 1A Depicted are intracellular structure / organelle western blot protein analysis of homogenized mitochondria (H-mito), first organelle complex (1st OC), and second organelle complex (2nd OC) prepared from HeLa cells using the methods disclosed herein. Figure 1B Depicted are Western blot protein analysis of mitochondrial markers of homogenized mitochondria (H-mito), first organelle complex (1st OC), and second organelle complex (2nd OC) prepared from HeLa cells using the methods disclosed herein. Figures 2A to 2B Depicted are non-limiting exemplary data related to the characteristics of the organelle complex populations provided herein.The first organelle complex and the second organelle complex were isolated from HEK293T cells. Figure 2A Depicted are Western blot protein analysis of the intracellular structure / organelle of the first organelle complex (1st OC) and the second organelle complex (2nd OC) prepared from HEK293T cells using the methods disclosed herein. Figure 2B Depicted are Western blot protein analysis of mitochondrial markers of the first organelle complex (1st OC) and the second organelle complex (2nd OC) prepared from HEK293T cells using the methods disclosed herein. These data show that organelle complex populations isolated from different types of cells show similar results in terms of differences between the first organelle complex and the second organelle complex. In addition, these data show that mitochondrial markers between the first organelle complex and the second organelle complex are greatly different compared to homogenized mitochondria.

[0108] Figures 3A to 3B Depicted are non-limiting exemplary data related to the structural integrity of the first organelle complex and the second organelle complex: Outer membrane integrity ( Figure 3A ), endometrial integrity ( Figure 3B In some embodiments, membrane integrity (%) = ((amount of final product containing detergent - amount of final product without detergent) / amount of final product containing detergent). Figure 4 Depicted are non-limiting exemplary data relating to ATP production by a population of the first organelle complex over a range of Pi concentrations. Figures 5A to 5BDepicted are non-limiting exemplary data related to the structural integrity of the first organelle complex. 2+ The citrate synthase activity of the first organelle complex was measured at Figure 5A ) and cytochrome c oxidase activity ( Figure 5B ). Fig. 6A The experimental setup is depicted, and Figures 6B to 6D Data related to the structural integrity of the first organelle complex and the second organelle complex are depicted. Figures 6B to 6D Depicted are the second organelle complex (2OC; Figure 6C ) and the first organelle complex (1st OC; Figure 6B ) of ATP production, and Fig.6D Describe the ATP production ratio of two kinds of organelle complexes.In general, these data show that the structure and functional differences between the organelle complex population provided herein and the homogenized mitochondria produced by the current available methods.The first organelle complex and the second organelle complex provided herein comprise various organelles (except mitochondria), show favorable functional properties, and maintain structural integrity in a series of extracellular environments.

[0109] We next investigated the effect of cellular uptake of the second organelle complex on ATP production. Figures 7A to 7B Depicts the experimental setup related to the effect of the second organelle complex (2OC) on ATP production ( Fig. 7A ) and data( Figure 7B ). It was found that cellular uptake of the second organelle complex increased ATP production in a dose-dependent manner.

[0110] Then qPCR analysis of the first and second organelle complexes was performed. Figure 8 Depicted are qPCR data associated with the first organelle complex (1st OC) and the second organelle complex (2nd OC). The data demonstrate that when quantified by qPCR, there is a dramatic difference in relative mitochondrial DNA copy number (mtDNACN) between the first organelle complex and the second organelle complex, with the first organelle complex having between about 2-6 times the mtDNA CN in the isolated organelle complexes.

[0111] Next, a comparison of mtDNA incorporation in 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, which is a derivative of rat C6 glioma cells and has lost its mitochondrial DNA (mtDNA) due to transient transfection with a plasmid. The mitochondrial donor was HeLa, an immortalized cell line derived from human cervical cancer cells. Homogenized mitochondria (H-mito), the first organelle complex (1st OC), and the second organelle complex (2nd OC) were isolated from HeLa cells. C6 rho0 cells were incubated at 2x10 5 The cells were cultured in 12-well plates at a concentration of 10 cells / well. Homogenized mitochondria (H-mito), the first organelle complex (1st OC) and the second organelle complex (2nd OC) were co-incubated at 42 μg / well. Fig.9A Depicted is the experimental setup related to mtDNA incorporation in recipient C6 rho0 cells incubated with homogenized mitochondria (H-mito), the first organelle complex (1st OC), and the second organelle complex (2nd OC), and Fig. 9B The data associated therewith are depicted. Fig. 9B Depicted are the ratios of incorporated mtDNA of the 1st OC and 2nd OC relative to homogenized mitochondria after 24 hours of co-incubation with homogenized mitochondria, the first organelle complex, and the second organelle complex. Increased incorporation of mtDNA in the first organelle complex treatment group in recipient C6 rho0 cells was observed. Specifically, a three-fold increase in incorporated mtDNA was observed using the first organelle complex as a donor.

[0112] Example 2

[0113] Alternative Final Centrifugation Conditions

[0114] 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 precipitate is collected to recover the first organelle complex. In this example, the consequences of increasing the second centrifugal force from 3000 g to 8000 g (with angle, soft brake) and increasing the second centrifugation time from 10 min to 20 min were examined. Figures 10A to 10G The protein concentration of the first organelle complex recovered using final centrifugation conditions of 3000 g (10 min) or 8000 g (20 min) is depicted. Fig. 10A ), total ATP production ( Fig. 10B ), ATP production in the presence of oligomycin ( Fig. 10C ), endometrial integrity ( Fig. 10D ), outer membrane integrity ( Fig.10E)、COX activity( Fig.10F ) and citrate synthase (CS) activity ( Figure 10G ) related data. Figures 11A to 11G Depicted is a stained SDS-PAGE gel (17 μg protein / lane) of the first organelle complex recovered using final centrifugation conditions of 3000 g (10 min) or 8000 g (20 min) ( Fig.11A ) and with LAMP2 (lysosome; Fig. 11B ), Golgi protein 97 (Golgi; Fig. 11C ), catalase (peroxisome; Fig.11D ), calreticulin (ER; Fig.11E ), VDAC (mitochondria (outer membrane); Fig.11F ), TOMM20 (mitochondria (outer membrane); Fig.11G ) and TFAM (mitochondria (matrix); Fig.11H ) protein level data.

[0115] Example 3

[0116] Biological activities of homogenized mitochondria, organelle complex I, and organelle complex II

[0117] 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 plates) 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) were added at a concentration of 5ug / ml, 15ug / ml or 50ug / ml. After 24 hours of incubation, ATP generation was measured using Cell Titer-Glo (triplicate). Fig.12 Data related to ATP production of fibroblasts incubated with homogenized mitochondria (H-mito), the first organelle complex, or the second organelle complex are depicted. These data suggest that high doses of homogenized mitochondria may induce cell death (even at moderate doses this trend can be observed in microscopic observations). Both the second organelle complex and the first organelle complex show a dose-dependent increase in intracellular ATP production, with the first organelle complex appearing to be more effective at lower doses.

[0118] Example 4

[0119] Flow reducer

[0120] As described herein, the first method of producing a first organelle complex can be modified and can include using a flow device (e.g., a flow reduction device) instead of using pipetting (e.g., step G) as a means of applying a physical stimulus to the first solution and / or the second solution in some embodiments (or in addition to using pipetting, using a flow device as a means of applying a physical stimulus to the first solution and / or the second solution). Figure 14 depicts data related to a first organelle complex obtained via a first generation method (using pipetting as a physical stimulus) compared to an improved method using a flow reduction device as a physical stimulus. It was found that using a flow reduction device can obtain a higher protein recovery rate than the first generation method (including pipetting).

[0121] Example 5

[0122] Tangential Flow Filtration

[0123] In this example, the use of a TFF system (including various TFF parameters, such as shear rate and buffer composition) during the isolation of the first organelle complex was investigated. The use of lower shear rates during TFF purification was first examined. The AT shear rate was about 1000-2000 sec -1 It was found that the outer membrane integrity in TFF purification can be improved by using lower shear rates (Table 2).

[0124] Table 2: TFF shear rate test

[0125]

[0126] Next, the effect of buffer viscosity during TFF purification was examined. It was found that the use of a buffer with a lower viscosity can improve the outer membrane integrity of the first organelle complex in TFF purification (Table 3). Therefore, in some embodiments provided herein, a buffer with a lower viscosity (e.g., a buffer based on mannitol) is used for the separation of the first organelle complex during TFF.

[0127] Table 3: TFF buffer viscosity

[0128] TFF batch Buffer Run time Outer membrane integrity % 1 Tris-sucrose-buffer 2.5hr 60-70 2 Tris-mannitol-sucrose buffer 2.5hr 80-90

[0129] Next, the effects of HA and glycerol in the storage buffer on the outer membrane integrity of mitochondria were examined (Table 4).

[0130] Table 4: Storage buffer test results

[0131]

[0132] Unless such substitution is technically infeasible, in at least some of the previously described embodiments, one or more elements used in an embodiment may be interchangeably used in another embodiment. It should be understood by those skilled in the art that various other omissions, additions, and modifications may 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 fall within the scope of the subject matter defined by the appended claims.

[0133] Regarding the use of any plural and / or singular terms herein, those skilled in the art can translate plural into singular and / or singular into plural according to context and / or application. For the sake of clarity, various singular / plural arrangements can be explicitly set forth herein. Unless the context clearly indicates otherwise, as used in this specification and the appended claims, the singular forms "one / a kind (a)", "one / a kind (an)" and "the / said (the)" include plural references. Unless otherwise indicated, any reference to "or" herein is intended to encompass "and / or".

[0134] Those skilled in the art will understand that, in general, the terms used herein and particularly in the appended claims (e.g., the bodies of the appended claims) are generally intended to be "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "comprising" should be interpreted as "including but not limited to," etc.). Those skilled in the art will also understand that if a specific number of claim recitations is intended to be introduced, such intent will be explicitly recited in the claim, and in the absence of such a recitation, no such intent is present. For example, to aid understanding, the following appended claims may contain the use of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed as implying that the introduction of claim recitations by the indefinite article "a" or "an" limits any particular claim containing such introduced claim recitations to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "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"); the same is true for the use of definite articles to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, one skilled in the art will recognize that such recitation should be construed to mean at least the recited number (e.g., reciting "two recitations" alone without mentioning other modifiers means at least two recitations, or two or more recitations). Furthermore, in those cases where a convention similar to “at least one of A, B, and C, etc.” is used, generally speaking, such construction is intended to be in the sense that one skilled in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include, but is not limited to, systems having: A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those cases where a convention similar to “at least one of A, B, or C, etc.” is used, generally speaking, such construction is intended to be in the sense that one skilled in the art would understand the convention (e.g., “a system having at least one of A, B, or C” would include, but is not limited to, systems having: A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.).Those skilled in the art will also appreciate that virtually any disjunctive word and / or phrase presenting two or more alternative terms (whether in the specification, claims or drawings) should be understood to contemplate the possibility of including one of the terms, any one of the terms, or both of the terms.

[0135] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0136] As will be understood by those skilled in the art, for any and all purposes, such as in providing written specifications, all scopes disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof.Any listed scope can be easily considered to fully describe and enable the same scope to be decomposed into at least equal half, one-third, one-quarter, one-fifth, one-tenth, etc. As a non-limiting example, each scope discussed herein can be lower third, middle third, and upper third, etc. It can also be understood by those skilled in the art that all languages ​​(such as "until", "at least", "greater than", "less than", etc.) include quoted numbers and refer to the scope of the sub-ranges that can be decomposed into as described above subsequently.Finally, as will be understood by those skilled in the art, scope includes each individual member.Therefore, for example, a group with 1 to 3 articles refers to a group with 1, 2 or 3 articles.Similarly, a group with 1 to 5 articles refers to a group with 1, 2, 3, 4 or 5 articles, etc.

[0137] Although various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for illustrative purposes and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

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

2. A population of organelle complexes, wherein the organelle complex comprises mitochondria and one or more of endoplasmic reticulum, peroxisome, lysosome and Golgi apparatus, wherein the organelle complex is isolated or derived from a cell contacted with a surfactant at a concentration equal to or greater than the critical micelle concentration (CMC) of the surfactant, wherein the organelle complex is depleted of cytoplasmic macromolecules, and wherein at least about 80% of said mitochondria of said organelle complex maintain structural integrity in an extracellular environment.

3. The organelle complex population according to any one of claims 1 to 2, wherein the structural integrity comprises the structural integrity of the inner membrane and / or the structural integrity of the outer membrane of the mitochondria.

4. The population of organelle complexes according to any one of claims 1 to 3, wherein at least 2 times more of the mitochondria of the organelle complexes maintain structural integrity in the extracellular environment compared to a homogenized mitochondrial population in the extracellular environment.

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 organelle complex population according to any one of claims 1 to 5, wherein the extracellular environment comprises 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. The population of organelle complexes according to any one of claims 1 to 6, wherein at least about 80% of the mitochondria of the organelle complexes retain functional capacity.

8. The organelle complex population according to any one of claims 1 to 7, wherein the mitochondria of the organelle complex are capable of ATP production.

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

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

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

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

13. The organelle complex population according to any one of claims 1 to 12, wherein: The first organelle complex comprises at least about 1.1 times more of one or more lysosomal proteins, optionally lysosomal associated membrane protein 2 (LAMP2), mannose-6-phosphate receptor (M6PR), and / or lysosomal associated membrane protein 1 (LAMP1), than the second organelle complex; The first organelle complex comprises at least about 1.1 times more of one or more peroxisomal proteins, optionally catalase and / or ATP-binding cassette transporter 1, subfamily D, type 3 (ABCD3), than the second organelle complex; The first organelle complex comprises at least about 1.1 times more of one or more Golgi proteins, optionally Golgi protein-97, syntaxin-6, TGOLN2 / trans-Golgi net protein 2 (TGN46), Golgi matrix protein 130 (GM130), and / or mannosidase alpha 2A class member 1 (MAN2A1), than the second organelle complex; The first organelle complex comprises at least about 1.1 times more of one or more endoplasmic reticulum proteins, optionally calreticulin and / or calnexin, than the second organelle complex; and / or The second organelle complex comprises at least about 1.1 times more of one or more cytoplasmic proteins, optionally p70S6K and / or glyceraldehyde 3-phosphate dehydrogenase (GAPDH), than the first organelle complex.

14. A population of organelle complexes according to any one of claims 1 to 13, wherein when the organelle complexes are contacted with a cell population, the organelle complexes can be incorporated into the cells, and optionally, at least about 2 times more organelle complexes can be incorporated into the cells compared to a homogenized mitochondrial population.

15. The organelle complex population according to any one of claims 1 to 14, wherein when the organelle complex is contacted with a host cell population, the organelle complex has a superior ability to be incorporated into host cells compared to a homogenized mitochondrial population.

16. The organelle complex population according to any one of claims 1 to 15, wherein when the first organelle complex is contacted with a host cell population, the first organelle complex has a superior ability to be incorporated into host cells compared to the second organelle complex.

17. A population of organelle complexes according to any one of claims 1 to 16, wherein when the organelle complexes are contacted with a host cell population, the organelle complexes have excellent ability to be incorporated into host cells, optionally, at least 2 times more organelle complexes can be incorporated into host cells compared to a homogenized mitochondrial population.

18. A population of organelle complexes according to any one of claims 1 to 17, wherein the mitochondria of the organelle complexes can be incorporated into cells after the population undergoes one or more freeze-thaw cycles, optionally compared to a homogenized mitochondrial population, at least twice as many mitochondria of the organelle complexes can be incorporated into cells after the population undergoes one or more freeze-thaw cycles.

19. A population of organelle complexes according to any one of claims 1 to 18, wherein at least about 80% of the organelle complexes in the population have a size between about 500 nm and about 3500 nm, optionally between about 200 nm and about 1000 nm.

20. The population of organelle complexes according to any one of claims 1 to 19, wherein the population of organelle complexes is derived from cells treated with a mitochondrial activator, optionally resveratrol.

21. A composition comprising a population of organelle complexes according to any one of claims 1 to 20.

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

23. A method for producing a first population of organelle complexes, the method comprising: incubating the cells in a first solution comprising a surfactant at a first temperature; removing the surfactant to form a second solution; as well as recovering the first organelle complex from the second solution, wherein the first organelle complex comprises mitochondria and one or more of endoplasmic reticulum, peroxisome, lysosome and Golgi apparatus, wherein said first organelle complex population is depleted of cytoplasmic macromolecules, and wherein: (i) incubating the cells in the first solution with the surfactant at a concentration equal to or greater than the critical micelle concentration (CMC) of the surfactant; and / or (ii) the cells comprise or are derived from floating cells or frozen cells.

24. The method of claim 23, wherein the method comprises incubating the second solution at a second temperature.

25. The method of any one of claims 23 to 24, wherein the first organelle complex comprises mitochondria and two, three or four of the endoplasmic reticulum, peroxisome, lysosome and Golgi apparatus.

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

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

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

29. The method according to any one of claims 23 to 28, wherein the surfactant is selected from the group consisting of Triton-X 100, Triton-X 114, Nonidet P-40, n-dodecyl-D-maltoside, Tween-20, Tween-80, saponin and digitonin.

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

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

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

33. The method of any one of claims 23 to 32, wherein 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.

34. The method of any one of claims 23 to 33, wherein removing the surfactant comprises one or more washes with a buffer, optionally Tris buffer.

35. The method of any one of claims 24 to 34, wherein incubating the second solution comprises incubating the second solution for about 1 minute to about 120 minutes, optionally for about 20 minutes.

36. The method of any one of claims 23 to 35, wherein recovering the first organelle complex from the second solution comprises tangential flow filtration (TFF), optionally performed by: (i) using a low viscosity buffer, optionally the low viscosity buffer reduces shear rate; (ii) at about 22°C to about 25°C; (iii) at less than about 2000 sec -1 (iv) below room temperature, optionally at 4°C; and / or (v) using a buffer comprising human albumin (HA) or recombinant albumin ().

37. The method of any one of claims 23 to 36, wherein recovering the first organelle complex from the second solution comprises one or more centrifugation steps.

38. The method of any one of claims 23 to 37, wherein recovering the first organelle complex from the second solution comprises: centrifuging the second solution at a first centrifugal force; Collect the supernatant; centrifuging the supernatant at a second centrifugal force; as well as The precipitate is collected to recover the first organelle complex.

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

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

41. The method of any one of claims 23 to 40, wherein incubating cells in the first solution and / or incubating the second solution comprises applying a physical stimulus, optionally shaking and / or stirring, to the first solution and / or the second solution, respectively.

42. A method according to claim 41, wherein applying a physical stimulus to the first solution and / or the second solution comprises flowing the first solution and / or the second solution through a flow device, wherein the flow device comprises a fluid channel, the fluid channel comprising two or more segments having different cross-sectional diameters, optionally the cross-sectional diameters range from about 0.8 mm to about 25.4 mm, further optionally the cross-sectional diameters range from about 1.5 mm to 6.5 mm, optionally the flowing through the flow device generates additional flow and / or shear.

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

44. The method of claim 43, wherein the cryoprotectant comprises human albumin (HA) and / or glycerol.

45. The method of any one of claims 23 to 44, wherein the method further comprises treating the cells with a mitochondrial activator, optionally resveratrol, prior to the incubation 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, the method comprising: Contacting cells of a subject in need thereof 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 condition associated with mitochondrial dysfunction, the method comprising: Contacting cells of a subject in need thereof 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 a disease or disorder associated with mitochondrial dysfunction.

49. The method of any one of claims 47 to 48, wherein contacting the subject's cells comprises a route of administration 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 method of any one of claims 47 to 49, wherein the disease or condition is selected from the group consisting of diabetes (type I and II), metabolic diseases, eye disorders associated with mitochondrial dysfunction, hearing loss, mitochondrial toxicity associated with therapeutic agents, mitochondrial dysfunction associated with space travel, cardiotoxicity associated with chemotherapy or other therapeutic agents, mitochondrial dysfunction disorders, and migraine.

51. The method of any one of claims 47 to 50, wherein the disease or condition is selected from the group consisting of mitochondrial myopathy, diabetes and deafness (DAD) syndrome, Barth syndrome, Leber's hereditary optic neuropathy (LHON), Reye's syndrome, NARP (neuropathy, ataxia with retinitis pigmentosa and ptosis syndrome), myoneurogastrointestinal encephalopathy (MNGIE), MELAS (mitochondrial encephalopathy with lactic acidosis and stroke-like episodes) syndrome, myoclonic epilepsy with red ragged fibers (MERRF) syndrome, Kearns-Sell syndrome, and mitochondrial DNA depletion syndrome.

52. The method of any one of claims 47 to 51, wherein the disease or disorder is an ischemia-related disease or disorder, a genetic disorder, a disease or disorder of aging, a neurodegenerative disorder, a cardiovascular disorder, cancer, an autoimmune disease, an inflammatory disease, a fibrotic disorder, or any combination thereof.

53. The method of any one of claims 47 to 52, wherein the ischemia-related disease or condition is selected from the group consisting of cerebral ischemia-reperfusion, hypoxic-ischemic encephalopathy, acute coronary syndrome, myocardial infarction, liver ischemia-reperfusion injury, ischemic injury-compartment syndrome, vascular occlusion, wound healing, spinal cord injury, sickle cell disease, and reperfusion injury of a transplanted organ.

54. The method of any one of claims 47 to 53, wherein the neurodegenerative disorder is selected from the group consisting of dementia, Friedreich's ataxia, amyotrophic lateral sclerosis, mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS), myoclonic epilepsy with red ragged fibers (MERFF), epilepsy, Parkinson's disease, Alzheimer's disease, or Huntington's disease. Exemplary neuropsychiatric disorders include bipolar disorder, schizophrenia, depression, addiction disorders, anxiety disorders, attention deficit disorder, personality disorders, autism, and Asperger's disease.

55. The method of any one of claims 47 to 54, wherein the cardiovascular disorder 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.

56. The method of any one of claims 47 to 55, wherein the disease or condition is acute respiratory distress syndrome (ARDS) or pre-eclampsia or intrauterine growth restriction (IUGR).

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