System and method for growing and coating mitochondria

Abstract

A method for growing isolated mitochondria includes the steps of selecting a source of mitochondria and stem cells, extracting the stem cells and mitochondria from the source, separating the stem cells and mitochondria into a first pool and a second pool respectively, incubating the extracted mitochondria / transferring them to the extracted stem cells to produce packed stem cells, expanding the packed stem cells in a bioreactor, adjusting the environmental conditions of the bioreactor to be favorable for the growth of the mitochondria of the packed stem cells, converting the packed stem cells into megakaryocytes, isolating the mitochondria from the megakaryocytes, and applying a coating to the mitochondria after isolation from the megakaryocytes.

Description

Technical Field

[0001] Incorporation by reference to priority applications Any application identified in a PCT application for which foreign or domestic priority is claimed and that is filed with the present application is incorporated herein by reference.

[0002] The invention disclosed and claimed herein generally relates to systems and methods for growing and coating mitochondria. More particularly, it relates to systems and methods for obtaining coated bioreactors that grow mitochondria, which are for the treatment of various pathologies or for maintaining / increasing cellular energy for the purpose of promoting longevity.

Background Art

[0003] Researchers have conducted tests on exogenous mitochondrial transplantation, but due to the lack of donor mitochondria, this has been limited mainly to rare pediatric diseases and surgeries rather than a wide range of areas such as adult diseases and longevity. Similar to how organ transplants such as those of the liver or kidney are severely restricted by the availability of donor organs, "organelle transplantation" of mitochondria is also restricted by the lack of donor mitochondria. Mitochondria are the "powerhouses of the cell," small organelles that produce the energy necessary for cells to replicate and function. Research has shown that mitochondria are highly mobile and can migrate directly from cell to cell and also via the bloodstream, and that mitochondria can cause diseases due to injury, age, or dysfunction due to mutations. Therefore, the lack of donor mitochondria available for the treatment of adult diseases has long hindered the development of the field of mitochondrial-based therapies and has thus become one of the main obstacles to expanding such treatments beyond their current limited scope. Therefore, there has long been a felt need to develop systems and methods for growing mitochondria and enhancing the effectiveness of the uptake of such mitochondria into cells.

Prior Art Documents

Patent Document

[0004]

Patent Document 1

Non-Patent Document

[0005]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Means for Solving the Problems

[0006] The methods disclosed herein each have several aspects, none of which alone relate to a desirable property. Without limiting the claims, some prominent features are briefly considered here. Numerous other embodiments are also contemplated, including embodiments having fewer, additional, and / or different components, steps, features, objectives, benefits, and advantages. The components, aspects, and steps can also be in different arrangements and orders. After considering this discussion, particularly after reading the section entitled "Detailed Description of the Invention," one will understand what advantages the features of the devices and methods disclosed herein provide compared to other known devices and methods.

[0007] In some embodiments, a method of growing isolated mitochondria is provided, the method comprising selecting a source comprising mitochondria and stem cells; extracting the stem cells and mitochondria from the source, and separating the stem cells and mitochondria into a first pool and a second pool, respectively; incubating the extracted mitochondria / transferring them to the extracted stem cells to produce packed stem cells; expanding the packed stem cells in a bioreactor; adjusting the conditions of the bioreactor environment to be favorable for the growth of the mitochondria of the packed stem cells; converting the packed stem cells into megakaryocytes; isolating the mitochondria from the megakaryocytes; and applying a coating to the mitochondria after isolation from the megakaryocytes.

[0008] In some embodiments, the source is placental tissue comprising mitochondria and stem cells. In some embodiments, the source is bone marrow comprising mitochondria and stem cells. In some embodiments, the source is adipose tissue comprising mitochondria and stem cells. In some embodiments, the source is peripheral blood comprising platelet-derived extracellular vesicles (PEVs). In some embodiments, the PEVs comprise mitochondria.

[0009] In some embodiments, the coating comprises Asialorosomucoid (AsOR). In some embodiments, the coating further comprises poly-L-lysine. In some embodiments, the coating further comprises listeriolysin O (LLO).

[0010] In some embodiments, the method further comprises the step of preserving the coated mitochondria. In some embodiments, the preserving step comprises suspending the mitochondria in a cryoprotectant. In some embodiments, the cryoprotectant comprises trehalose. In some embodiments, the cryoprotectant comprises phosphate buffered saline (PBS). In some embodiments, the coating is applied at a ratio of 1 μg of coating per 128 μg of bioreactor-grown mitochondria. In some embodiments, the coating is applied at a ratio that is at least 2-fold the ratio of 1 μg of coating per 128 μg of bioreactor-grown mitochondria.

[0011] In some embodiments, the method further comprises the step of administering a therapeutic amount of the coated mitochondria to a subject.

[0012] In some embodiments, the method comprises obtaining blood from one or more donors; adding an anticoagulant and a buffer to the blood to form a mixture; separating the mixture into a supernatant and platelet-rich plasma (PRP); collecting the PRP; releasing extracellular vesicles from platelets in the PRP by stimulating the collected PRP; and collecting the extracellular vesicles as PEV. In some embodiments, the collected PRP is stimulated by immune complexes in the presence of Ca 2+ . In some embodiments, the immune complexes comprise heat-aggregated IgG. In some embodiments, the concentration of heat-aggregated IgG is from 0.1 mg / mL to 2.5 mg / mL and Ca 2+The concentration is from 1 mM to 25 mM. In some embodiments, the anticoagulant is anticoagulant citrate dextrose (ACD). In some embodiments, the buffer is a Tyrode's buffer with a pH from 6 to 7.

[0013] In some embodiments, a method for growing isolated mitochondria is provided, the method comprising: selecting a source comprising mitochondria and stem cells; extracting the stem cells and mitochondria from the source, and separating the stem cells and mitochondria into a first pool and a second pool, respectively; incubating the extracted mitochondria and transferring them to the extracted stem cells to produce packed stem cells; expanding the packed stem cells in a bioreactor; adjusting the conditions of the bioreactor environment to be favorable for the growth of the mitochondria of the packed stem cells; converting the packed stem cells into megakaryocytes; and isolating the mitochondria from the megakaryocytes. In some embodiments, the method further comprises applying a coating to the mitochondria after isolation from the megakaryocytes.

[0014] In some embodiments, the source is placental tissue comprising mitochondria and stem cells. In some embodiments, the source is bone marrow comprising mitochondria and stem cells. In some embodiments, the source is adipose tissue comprising mitochondria and stem cells. In some embodiments, the source is peripheral blood comprising platelet-derived extracellular vesicles (PEV). In some embodiments, the PEV comprises mitochondria.

[0015] In some embodiments, the coating comprises asialoorosomucoid (AsOR). In some embodiments, the coating further comprises poly-L-lysine. In some embodiments, the coating further comprises listeriolysin O (LLO).

[0016] In some embodiments, the method further includes the step of preserving the coated mitochondria. In some embodiments, the step of preserving includes the step of suspending the mitochondria in a cryoprotectant. In some embodiments, the cryoprotectant includes trehalose. In some embodiments, the cryoprotectant includes phosphate buffered saline (PBS). In some embodiments, the coating is applied at a ratio of 1 μg of coating per 128 μg of bioreactor-grown mitochondria. In some embodiments, the coating is applied at a ratio that is at least twice the ratio of 1 μg of coating per 128 μg of bioreactor-grown mitochondria.

[0017] In some embodiments, the method further includes the step of administering a therapeutic amount of the coated mitochondria to a subject.

[0018] In some embodiments, the method further includes the steps of obtaining blood from one or more donors; adding an anticoagulant and a buffer to the blood to form a mixture; separating the mixture into a supernatant and platelet-rich plasma (PRP); collecting the PRP; releasing extracellular vesicles from platelets in the PRP by stimulating the collected PRP; and collecting the extracellular vesicles as PEVs. In some embodiments, the collected PRP is stimulated by immune complexes in the presence of Ca 2+ . In some embodiments, the immune complexes include heat-aggregated IgG. In some embodiments, the concentration of heat-aggregated IgG is from 0.1 mg / mL to 2.5 mg / mL, and the concentration of Ca 2+ is from 1 mM to 25 mM. In some embodiments, the anticoagulant is anticoagulant citrate dextrose (ACD). In some embodiments, the buffer is a Tyrode's buffer with a pH from 6 to 7.

[0019] The features of the examples of the present disclosure will become apparent by referring to the following detailed description and drawings (like reference numerals may correspond to similar components although not necessarily the same). For the sake of brevity, features having reference numerals or previously described functions may or may not be described in connection with other drawings in which they appear.

Brief Description of the Drawings

[0020]

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Mode for Carrying Out the Invention

[0021] In the above "Summary of the Invention" section and "Detailed Description of the Invention" section, as well as in the following claims, specific features of the present invention are referred to. It should be understood that the disclosure of the present invention in this specification includes all possible combinations of such specific features. For example, if a particular feature is disclosed in connection with a particular aspect or embodiment of the present invention, or a particular claim, that feature can also be used, to the extent possible, in combination with and / or in connection with other particular aspects and embodiments of the present invention, and also throughout the present invention.

[0022] Definitions The section headings used in this specification are for organization purposes only and are not to be construed as limiting the subject matter described.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The use of the term "including" is not limiting in the same manner as other forms such as "include", "includes", and "included". The use of the term "having" is not limiting in the same manner as other forms such as "have", "has", and "had". The use of the term "containing" is not limiting in the same manner as other forms such as "contain", "contains", and "contained". As used herein, whether in a transitional phrase or the body of a claim, the terms "comprise(s)" and "comprising" are to be interpreted as having a non-limiting meaning. That is, the above terms are to be interpreted as synonymous with the phrase "at least having" or "at least including". For example, when used in connection with a process, the term "comprising" means that the process includes at least the recited steps, but may also include additional steps. When used in connection with a compound, composition, or device, the term "comprising" means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components.

[0024] As used herein, the singular forms "a", "an", and "the" include the plural referents unless the context clearly dictates otherwise.

[0025] As used herein, the term "mitochondria" includes membrane-bound organelles characterized by a unique double-membrane structure. This membrane structure includes an outer membrane that is relatively permeable to small molecules and an inner membrane (folded into structures known as cristae) that is highly impermeable. The space enclosed by the inner membrane is called the mitochondrial matrix. The functional aspects of mitochondria, particularly their role in cellular respiration and energy production, involve a complex series of biochemical reactions that include, but are not limited to, the citric acid cycle (Krebs cycle) and oxidative phosphorylation, by which mitochondria convert nutrients into adenosine triphosphate (ATP), the cell's main energy currency. Furthermore, the term "mitochondria" includes the contributions of these membrane-bound organelles to various cellular functions other than energy production. These roles include, but are not limited to, involvement in cell differentiation, cell death, regulation of the cell cycle and cell proliferation, and maintenance of intracellular calcium levels. Additionally, "mitochondria" is used to describe the organelle-specific genetic characteristics. Each mitochondrion contains its own DNA, known as mitochondrial DNA (mtDNA), which is independent of nuclear DNA and is primarily maternally inherited. As used herein, the term mtDNA includes small circular double-stranded DNA molecules that exist within mitochondria independent of the nuclear genome found in the cell nucleus.

[0026] As used herein, the terms "mito", "mitos", and "mitlet" include naked mitochondria and bioreactor-propagated mitochondria, unless otherwise specified. Naked mitochondria refer to mitochondria that have not been propagated or expanded in a bioreactor. In other words, naked mitochondria can be derived from a source after digestion and fractionation to obtain a small amount of mitochondria. Naked mitochondria have several drawbacks, such as low yield, aging of mtDNA (and thus low resistance to the accumulation of damage from reactive oxygen species), and susceptibility to the effects of the immune response.

[0027] As used herein, the term "bioreactor" includes a device or apparatus configured to support the growth and proliferation of biological entities under controlled and regulated environmental conditions. A bioreactor includes a containment chamber, which is constructed from biocompatible materials to minimize negative interactions with the biological entities contained therein. This chamber provides an isolated and controlled environment that is effective in preventing contamination from external sources, and this environment optimizes the growth conditions for the biological entities to grow or proliferate in the presence of an appropriate medium. The "bioreactor" may further include a series of sensors designed to continuously monitor and record important parameters including, but not limited to, temperature, pH, oxygen and carbon dioxide concentrations, and nutrient and waste levels. Feedback from these sensors is essential to inform adjustments to the internal environment of the bioreactor, thereby maintaining optimal growth conditions. The bioreactor may further include a control system operably connected to these sensors and the containment chamber. This control system interprets the data from the sensors and accordingly adjusts the environmental conditions within the containment chamber. By controlling and manipulating these parameters, it is possible to fine-tune the environment of the biological entities to enhance growth and productivity. The "bioreactor" may be configured as an input / output system constructed for the introduction of fresh medium necessary to provide nutrients and the removal of used medium essential for the removal of waste. This system ensures a dynamic environment within the containment chamber and supports the sustainable viability and optimal productivity of the biological entities.

[0028] As used herein, the term "stem cell" includes unspecialized living cells that are capable of self-renewal and differentiation into specialized cell types. Stem cells include both embryonic stem cells (ESCs) and adult stem cells. ESCs are derived from the inner cell mass of the blastocyst, an early-stage embryo. ESCs are essentially pluripotent, meaning they have the ability to differentiate into any cell type found in the adult body. Adult stem cells, also known as somatic stem cells or tissue-specific stem cells, have been found to be scattered throughout adult tissues such as bone marrow, blood, brain, and skin. These cells are typically multipotent and can differentiate into a limited number of cell types related to the tissue in which they are present. The term "stem cell" also includes induced pluripotent stem cells (iPSCs), which are adult cells that have been genetically reprogrammed into an ESC-like state. This reprogramming enables iPSCs to differentiate into virtually any cell type, potentially providing an unlimited source of any human cell type.

[0029] As used herein, the term "iPSC" includes adult somatic cells that have been genetically reprogrammed into an ESC-like state. This reprogramming confers pluripotency, the ability to differentiate into virtually any cell type found in the body, to these cells. iPSCs are generated by introducing specific transcription factors such as Oct4, Sox2, Klf4, and c-Myc into adult somatic cells. This reprogramming process resets the cell state, enabling the modified cells to proliferate indefinitely and differentiate into a wide range of cell types upon appropriate stimulation.

[0030] The present invention provides a method for overcoming the shortage of donor mitochondria available for treating adult diseases. In some embodiments described herein, the first step is to grow the mitochondria in a bioreactor. These mitochondria are then provided with a unique coating to protect them from the immune response, along with a receptor for targeting specific tissue types. These coated mitochondria are injected into the body, travel via the blood to the desired tissue, and become established within the cells.

[0031] In some embodiments, a method 100 for growing mitochondria is provided as shown in FIG. 1, and the method 100 includes a step of selecting a source in step 110. In some embodiments, the source includes mitochondria and stem cells. In some embodiments, the source includes naturally occurring vesicles derived from donor blood. In some embodiments, the source includes stem cells because the mitochondria age is generally younger, as demonstrated by the fact that the accumulation of damage by reactive oxygen species is lower in the mitochondrial DNA of mitochondria derived from stem cells compared to that of mitochondria derived from older cells. In some embodiments, the stem cells match the subject's haplotype as shown in FIG. 4. In some embodiments, the stem cells include pluripotent stem cells. In some embodiments, the pluripotent stem cells include induced pluripotent stem cells. In some embodiments, the stem cells are obtained from umbilical cord blood. In some embodiments, the stem cells are obtained from the placenta. In some embodiments, the stem cells are obtained from dental pulp. In some embodiments, the stem cells are obtained from the dermis. In some embodiments, the stem cells are obtained from amniotic fluid. In some embodiments, the stem cells are obtained from tumors. In some embodiments, the stem cells include hematopoietic stem cells obtained from bone marrow. In some embodiments, the stem cells are obtained from adipose tissue. In some embodiments, the method 100 further includes a step of extracting mitochondria from the stem cells in step 120. In some embodiments, the method 100 includes a step of selecting healthy mitochondria from the extracted mitochondria.

[0032] In some embodiments, the vesicles derived from donor blood are collected from platelets. In some embodiments, the step of obtaining mitochondria includes the step of extracting extracellular vesicles (PEVs) containing platelet-derived mitochondria. In some embodiments, the extracting step further includes: 1) obtaining blood from a donor; 2) adding an anticoagulant and a buffer to the blood to form a mixture; 3) separating the mixture into a supernatant and platelet-rich plasma (PRP); 4) collecting the platelet-rich plasma (PRP); 5) stimulating the collected platelets; and collecting the PEVs. Finding a source of mitochondria for therapeutic use (e.g., administration of mitochondrial formulations) has been a challenge. Similar to all organ donations, the supply of mitochondria from young and healthy donors is insufficient. Some diseases or injuries may be treatable with autologous mitochondria (in a non-limiting example, taken from the leg muscles of one's own body), however, in many other diseases, the "patient" has poor-quality mitochondria due to age or mutations in mtDNA. In these patients, the provision of mitochondria is a preferred solution. Furthermore, mitochondria immediately after being completely isolated rapidly die within minutes of isolation, and when introduced naked into the bloodstream, they can also cause an immune reaction, potentially reducing the effectiveness of treatment. Therefore, it would be convenient to find an easily accessible source of mitochondria that can be provided (simultaneously encapsulated in some coating, vesicle, or vehicle suspension (or any combination thereof) that protects them from the immune system). Platelets derived from human blood contain an average of 4-5 mitochondria, and when the platelets are activated, the mitochondria are released in extracellular vesicles. Extracellular vesicles containing platelet-derived mitochondria are referred to herein as PEVs. These PEVs are typically larger (greater than 400 nM) than other platelet extracts or lysates (30-100 nM), and although less well-known, other sizes may also be applicable. PEVs have been shown to provide mitochondria to nearby cells and increase the respiratory activity of the cells that absorb them.PEVs have several advantages that favor rapid commercialization and, thus, the potential for worldwide use of PEV products in the very near future: In particular, they can be derived from platelets that are provided but must be discarded as "expired"; they represent another medically appropriate and good alternative use for platelets that would otherwise be wasted; and they can be collected in most blood banks that already have all of the required skilled personnel, clean handling facilities, and required equipment and that are already located near hospitals. Since PEVs are a type of platelet transfusion, they are likely to be accepted and tested by medical experts already familiar with platelet transfusion therapy. Further, PEVs can be prepared for local infusion into various internal anatomical regions using delivery devices already on the market to treat a variety of clinical disorders.

[0033] Following extraction step 120 of method 100, in step 130, the stem cells and various mitochondria are separated and isolated into two different material pools. In some embodiments, the first material pool contains stem cells. In some embodiments, the stem cells are isolated prior to the various mitochondria. In some embodiments, the second pool contains the various mitochondria extracted from the remainder of the tissue from which the stem cells were previously extracted. In some embodiments, the remainder of the tissue is ground prior to extracting the various mitochondria. In some embodiments, the tissue includes umbilical cord blood. In some embodiments, the tissue includes the umbilical cord. In some embodiments, the tissue includes bone marrow. In some embodiments, the tissue includes adipose tissue. In some embodiments, the tissue includes any tissue associated with stem cells. In some embodiments, the tissue includes any tissue containing stem cells.

[0034] In some embodiments, method 100 further includes the step of growing mitochondria in a bioreactor. In some embodiments, method 100 includes the step of incubating / transferring the extracted mitochondria to stem cells in step 140. In some embodiments, method 100 includes the step of expanding the packed stem cells in a bioreactor in step 150, as shown in FIGS. 1 and 4. In some embodiments, the extracted mitochondria not used in the stem cells can be used for treatment. In some embodiments, the step of growing mitochondria in a bioreactor further includes the step of expanding the mitochondria-packed stem cells at the highest rate allowed by the bioreactor. In some embodiments, method 100 further includes the step of adjusting the selection conditions in step 160 to be favorable for high-quality mitochondria. In some embodiments, the selection conditions include hypoxia, glucose starvation, the use of Treefrog treatment, or any combination thereof. In some embodiments, method 100 further includes the step of differentiating the remaining stem cells into megakaryocytes (MK) in step 170, thereby increasing the number of mitochondria generated by a hundredfold or even a thousandfold compared to conventional cell culture techniques.

[0035] As used herein, MK is a polyploid cell derived from hematopoietic stem cells found in the bone marrow. The role of MK includes the production of platelets involved in local blood clot formation to prevent bleeding. MK grown in a bioreactor can be obtained not only from hematopoietic stem cells found in the bone marrow. In some embodiments, MK can be produced / induced from stem cells derived from adipose tissue. In some embodiments, the adipose tissue includes subcutaneous adipose tissue. In some embodiments, the method further includes the step of obtaining a source. In some embodiments, the source includes adipose tissue, and the adipose tissue is obtained by any technique known in the art. In some embodiments, after obtaining the adipose tissue, the method further includes the step of digesting the adipose tissue with a digestive agent. In some embodiments, the digestive agent is type II collagenase. In some embodiments, the method further includes the step of centrifuging the digested adipose tissue to produce an adipose-derived mesenchymal stromal cell line (ASCL). In some embodiments, the method further includes the step of treating the ASCL with an MK lineage induction medium to produce MK. In some embodiments, the MK lineage induction medium contains in IMDM (Iscove's Modified Dulbecco's Medium), 2 mM L-glutamine; 100 U / mL penicillin-streptomycin solution; 0.5% bovine serum albumin (BSA); 4 μg / mL LDL cholesterol; 200 μg / mL iron-saturated transferrin; 10 μg / ml insulin; 50 μM 2-β-mercaptoethanol, nucleotides (each of ATP, UTP, GTP, and GTP at about 20 μM), and 50 ng / ml thrombopoietin (TPO). See also U.S. Patent No. 10,113,147.

[0036] In some embodiments, MK can be produced / derived from stem cells derived from pluripotent stem cells (PSCs). In some embodiments, the method further comprises the step of obtaining PSCs. In some embodiments, the method further comprises the step of transducing the expression of transcription factors into PSCs via a vector. In some embodiments, the transcription factors are cloned into a vector backbone. In some embodiments, the vector comprises a lentiviral vector. In some embodiments, the transcription factors include GATA binding protein 1 (GATA1); FLI1 (friend leukemia integration 1); and T cell acute lymphoblastic leukemia protein 1 (TAL1). See also Moreau T et al. "Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming." Nat Commun. 2016;7:11208. In some embodiments, the method further comprises the step of maintaining the transduced PSCs in PSC medium for about 2 days. In some embodiments, the PSC medium comprises fibroblast growth factor (FGF2) and activin-A. In some embodiments, the method further comprises the step of maintaining the transduced PSCs in MK medium. In some embodiments, after the step of maintaining in PSC medium, the MK medium comprises TPO and SCF for at least 5 days.

[0037] In some embodiments, method 100 further includes, in step 180, separating a percentage of the expanded MK and isolating the mitochondria contained in the MK. In some embodiments, the percentage is 75%, and the mitochondria isolated from the percentage are packed in the remaining 25% MK, and then, by isolating the mitochondria from the latter, the copy number of each mitochondrion in the 25% MK increases. In some embodiments, the percentage is 80%. In some embodiments, the percentage is 70%. In some embodiments, the percentage is from 70% to 80%. In some embodiments, the percentage is 65%. In some embodiments, the percentage is from 65% to 80%. In some embodiments, the method further includes incubating the MK with mitochondrial transcription factor A (TFAM) to increase the copy number of the mitochondria.

[0038] In some embodiments, method 100 further includes, at step 180, isolating MKs that have been grown in a bioreactor from mitochondria. In some embodiments, the isolating step 180 includes incubating the MKs in 500 μL of isolation buffer at 4° C. for at least 15 minutes. In some embodiments, the isolation buffer includes 2 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, and 1 mM dithiothreitol (DTT). In some embodiments, the isolation buffer has a pH of 7.5. In some embodiments, the isolating step 180 further includes lysing the MKs. In some embodiments, the lysing step includes using a plastic potter / pestle for 20 strokes and performing a short centrifugation spin between each stroke. In some embodiments, the centrifugation spin includes a first spin at 1,000 g for 10 min at 4° C. for supernatant recovery and pellet discard, and the pellet of the first spin includes debris and intact cells. In some embodiments, the centrifugation spin includes a second spin at 13,000 g for 15 min at 4° C. for pellet acquisition, and the pellet of the second spin includes mitochondria. As shown in FIG. 5, the yield of mitochondria was improved to 20.22%, which was significantly improved compared to previous methods.

[0039] In some embodiments, the step of lysing comprises treating the MK with an extraction buffer. In some embodiments, the step of lysing further comprises transferring the treated MK to a dissociation device to homogenize the treated MK. In some embodiments, the step of lysing further comprises filtering the homogenized MK and labeling the mitochondria with magnetic microbeads, and the labeled mitochondria remain in the homogenate. In some embodiments, the microbeads comprise anti-TOM22 microbeads. In some embodiments, the anti-TOM22 microbeads are of human or mouse origin. In some embodiments, the step of lysing further comprises applying a magnetic field to a column placed in a separation device and flowing the labeled mitochondria through the column, and the magnetically labeled mitochondria are retained in the column. In some embodiments, the step of lysing further comprises removing the column from the separation device and eluting the mitochondria from the column.

[0040] In some embodiments, as shown in Figure 3, the method includes the step of maintaining stocks of multiple cell types. These cell types include, but are not limited to, hepatocytes, endothelial cells, neurons, hematopoietic cells, or any combination thereof. The cell types included in the method start with a mitochondrial energy level measured by oxygen consumption rate (OCR) under a Seahorse assay at 100% of the baseline. In some embodiments, the method further includes the step of reducing mitochondria within the cell line from about 0% to about 50% of the baseline using 2',3'-dideoxycytidine (ddC), a drug that depletes mtDNA, which significantly reduces mitochondrial activity (thus reducing the oxidative respiration of the cells). In some embodiments, the health of the cell type is determined after the step of reducing the mitochondria. In some embodiments, the determination of the health of the cell type is performed at least once or multiple times (i.e., cycles) for each of the cell types. In some embodiments, the determination of the health of the cell type is performed for each of the cell types for at least multiple cycles. In some embodiments, the method further includes the step of growing multiple types of transplantable mitochondrial species. The mitochondrial species can be derived from any source and any step of the methods disclosed herein. In some embodiments, the method further includes the step of restoring cellular energy by pipetting multiple mitochondria from the step of growing multiple types of transplantable mitochondrial species into multiple assay cells. In some embodiments, the restoring step further includes determining the duration required for the mitochondria to be pipetted into the target assay cells; and the duration required for the cellular energy to reach the normal baseline. In some other embodiments, the method further includes the step of deriving characteristics for each cell type, and the characteristics include, but are not limited to: the tendency of the cell type to absorb the transplanted mitochondria. The rate of recovery of cell respiration (e.g., depleting mtDNA using ddC before transplantation of the obtained mitochondria); the rate of regeneration of cellular energy after reaching a threshold; and other variables of energy management.In some embodiments, the threshold value is 25%, 30%, 35%, 40%, 45%, 50%, 55%, or any integer between 25% and 55%.

[0041] In the non-limiting example shown in FIG. 6, the basal respiration of HepG2 cells (immortalized human liver cell line), determined by measuring cell respiration via OCR (pmol / min), shows a significant decrease from about 85% to about 24.4% of OCR when ddC is incubated in HepG2 cell culture. Administration of 0.1 ng of mito restores the OCR to about 36.7% ± 2.8% from about 24.4% ± 4.6% of OCR; administration of 10 ng of mito restores the OCR to about 37.7% ± 3.0%; administration of 100 ng of mito restores the OCR to 36.6% ± 3.5%; administration of 1000 ng of mito restores the OCR to about 37.3% ± 3.4%. Thus, HepG2 cells that exhibited a significant decrease in cell respiration after ddC treatment showed a significant increase in cell respiration at basal respiration (measured by OCR) with administration of at least 0.1 ng of mito.

[0042] In some embodiments, method 100 further includes applying a coating to the mitochondria in step 190. In some embodiments, the mitochondria include mitochondria extracted from MK. In some embodiments, the coating includes an Asian orthochromatic coid (AsOR). In some embodiments, the coating further includes poly-L-lysine added to the AsOR. As shown in FIG. 8, the mito Asian sugar protein receptor (AsGR) functions to internalize AsG in the endosome of the cell. The AsOR can be coated on the cell. Since the AsOR is a ligand of the AsGR, cell substances or organelles coated with the AsOR-based coating can be targeted for uptake into the endosomal compartment of the cell. Thus, mitochondria (derived from another cell) coated with the AsOR-based coating can internalize into another cell and be delivered to the endosome of that cell. To avoid targeting the degradation pathway leading to the lysosome (e.g., the endosome transfers its contents to the lysosome), the coating further includes listeriolysin O (LLO). LLO is an endosome destabilizing peptide. By adding this to a coating containing AsOR and PL, it creates a porin-like channel in the endosome, and the resulting osmotic pressure causes the endosome to rupture, thereby releasing the coated mitochondria into the cell. As shown in FIGS. 7 and 8, the AsOR-PL+LLO and AsOR-PL coatings readily associate with mito as determined by flow cytometry assays.

[0043] As shown in the non-limiting examples of FIGS. 9 and 10, the basal respiration of cells incorporated with mito (coated or uncoated) at a specific concentration, particularly mito treated with coating: AsOR-PL, AsOR-PL and LLO, or AsOR-LLO for 24 hours, showed stronger basal respiration compared to the control of dimethyl sulfoxide (DMSO)-treated cells. The importance of mito incorporation was clarified by another group of HepG2 cells incubated in a medium containing ddC. Further, the results of FIGS. 9 and 10 show a tendency that energy-deficient cells are rescued when coated mitochondria are administered to the culture at 0.01 pg and 10 pg of mito per cell. The example of FIG. 7 shows that HepG2 cells were cultured for one week in normal medium with either DMSO or 10 μM ddC. Mitochondria were extracted from HepG2 cells. 64 μg of mitochondria were left untreated, coated with AsOR-PL only, coated with AsOR-PL + AsOR-LLO, or coated with AsOR-LLO only. Mito (10 pg / cell or 0.01 pg / cell) was added to the cells in a medium with +1% serum (to limit cell growth). The cells were incubated with a dilution of AsOR-PL / AsOR-LLO corresponding to the 10 pg / cell condition. The cells were incubated at 37 °C overnight or for 72 hr before evaluating cell respiration. As shown in FIGS. 15 and 16, the cellular energy of mito in DMSO-treated HepG2 decreased at the 72-hour time point and appeared to return to baseline. However, naked mitochondria administered to the cells at 10 pg / cell appeared to have a significant decrease in the basal respiration of HepG2 cells, further clarifying how naked mitochondria can be harmful to their target compared to coated cells at a similar concentration. Regarding mtDNA-depleted ddC-treated HepG2 cells, mito administration seems to tend to be beneficial in increasing the basal respiration rate of these cells.

[0044] Regarding maximal respiration at 24 hours shown in FIGS. 11 and 12, coated mito, particularly mito having AsOR-PL-PL, showed significantly superior OCR compared to the control. There did not appear to be a dose-dependent response to the amount of coated mito administered to HepG2 cells, and administration of 0.01 pg of mito per cell showed the same results as administration of 10 pg of mito per cell. Regarding the rescue of mtDNA depletion generated by ddC, low concentrations (0.01 pg / cell) of coated mito (AsOR-PL, AsOR-PL + AsOR-LLO, AsOR-LLO) were shown to have more consistent rescue of OCR compared to administration at 10 pg / cell. FIGS. 13 and 14 show how much of a significant decrease in OCR was shown by mtDNA-depleted ddC-treated HepG2 cells (without mito) in all phases tested by the Seahorse assay: basal respiration (the energy demand of cells under baseline conditions), ATP production (ATP produced by the mitochondria of HepG2 cells, contributing to meeting the energy needs of the cells), proton leak (providing signs of potential mitochondrial damage), maximal respiration (simulating the physiological energy demand by respiratory chain stimulation for operation at maximum capacity by adding the uncoupler FCCP), spare capacity (the ability of HepG2 cells to respond to demand, an indicator of the fitness or flexibility of the cells), and non-mitochondrial oxygen consumption (the persistence of oxygen consumption from a subset of cellular enzymes that continue to consume oxygen even after addition of rotenone and antimycin A). As shown in FIGS. 17 and 18, after 72 hours, the naked mito administered at 10 pg / cell appeared to have a significant decrease in maximal respiration OCR.

[0045] FIG. 19 shows the effect of administration of naked mito at various concentrations to ddC-treated retinal pigment epithelial cells (RPEC). Here, the naked mito is not coated. FIGS. 20A and 20B show ATP production and total cell respiration, respectively, in ddC-treated RPEC administered naked mito. FIG. 21 shows data collected on fold change of ddC-treated RPEC treated with various concentrations of naked mito.

[0046] As shown in FIGS. 22A and 22B, the coating can be optimized. In a non-limiting example, various amounts of AsOR-PL were tested against fresh HepG2 mitochondria to determine whether the percentage of coated mitochondria could be increased. Here, the mitochondria were extracted from HepG2 cells. These fresh mito were stained with Mitotracker. The Mitotracker dye is a fluorescent compound that is cell permeable and mitochondria selective. These dyes bind to thiol-reactive chloromethyl groups in the mitochondrial membrane or to the free thiol groups of cysteine residues belonging to mitochondrial proteins. As shown in FIG. 22A, the AsOR-PL coating was easily applied to the mitochondria and resulted in 52% of the Mitotracker events shown in the flow cytometry assay. Typically, conventional methods show a mitochondrial coating yield of about 50%. To increase the yield of coated mitochondria, the amount was doubled (1 μg of AsOR-PL per 128 μg of mitochondria), and a significantly larger number of coated mitochondria (about 70-72%) were obtained, as shown in FIG. 22B. A typical yield of 51% is shown as 0.125 μg. Increases in yields of 72%, 71%, 67%, and 67% are shown as 0.25 μg, 0.375 μg, 0.5 μg, and 0.625 μg, respectively.

[0047] In some embodiments, the method further comprises administering a therapeutic amount of bioreactor-propagated mitochondria systemically (enterally or parenterally) to a subject. In some embodiments, the bioreactor-propagated mitochondria are administered intravitreally, intravenously, or intraarterially to a subject. In some embodiments, the bioreactor-propagated mitochondria comprise a coating. In some embodiments, the coating comprises AsOR-PL. In some embodiments, the coating further comprises LLO.

[0048] In some embodiments, the method further includes mitochondrial preservation, and results from non-limiting examples thereof are shown in FIGS. 23-28. Mitochondria were stored at 10 mg / ml (100 μg / tube). The conditions were as follows: stored as pellets; stored in PBS or in trehalose buffer (300 mM trehalose, 10 mM HEPES-KOH pH 7.7, 10 mM KCl, 0.1% BSA, 1 mM EDTA, 1 mM EGTA). Tubes were stored at room temperature (RT), 4° C., and -80° C. (instantly frozen prior to storage). After one week, mitochondria were extracted from fresh liver for controls. All samples were tested for: protein concentration (BCA); mitochondrial number (flow cytometry); mtDNA in supernatant and cells (qPCR) (in the case of pellets, first resuspended in PBS (same volume as PBS condition), then centrifuged to recover free mitochondrial DNA in the supernatant); ATP production assay (bioluminescence); and electron flow assay (Seahorse). As shown in FIG. 24, at 4° C., the protein concentration was similar to fresh mitochondria under all conditions. At -80° C., resuspension of the pellet was difficult. In FIG. 25A, in the supernatant, the trehalose buffer appears to prevent the release of mtDNA during freezing at -80° C. In the pellet, fresh cells also had less mtDNA, however, the mtDNA was still very similar to fresh mitochondria under multiple storage conditions. In FIGS. 25B and 25C, mtDNA is more concentrated in the pellet after a series of centrifugations. In FIG. 26, ATP levels were determined according to mitochondrial storage conditions. As shown, the luminescence signal measuring ATP levels showed at least a two-fold increase in mitochondria stored at room temperature in some type of solution. In some embodiments, the solution includes phosphate buffered saline (PBS). In some embodiments, the solution includes trehalose. The dry pellet appears to be well maintained at 4° C., but the dry pellet does not appear to be a suitable solution for storage at -80° C. compared to PBS and trehalose.

[0049] As shown in FIG. 27, the Seahorse assay reveals that PBS and trehalose solutions may not be physiologically suitable for storage at room temperature. However, at lower temperatures, PBS appears to be more advantageous than trehalose at 4°C, while trehalose appears to be more advantageous than PBS at -80°C. For long-term storage, trehalose appears to be a better option. From the flow cytometry data in FIG. 28, PBS appears to be a preferred option for storage at 4°C, and trehalose appears to be a preferred option for long-term freezing at -80°C.

[0050] FIG. 29 is a non-limiting example of the treatment by mitochondrial injection in an amyotrophic lateral sclerosis (ALS) animal model. As used herein, the term "amyotrophic lateral sclerosis" (ALS) refers to a progressive neurodegenerative disease that mainly affects motor neurons in the brain and spinal cord, causing muscle weakness and ultimately paralysis. ALS is characterized by the degeneration and eventual death of both upper motor neurons (UMNs) in the motor cortex of the brain and lower motor neurons (LMNs) in the brainstem and spinal cord. The specific cause of ALS is unknown, but it is thought to be caused by a combination of genetic and environmental factors. ALS also encompasses its clinical findings, which typically include muscle weakness, atrophy, fasciculation (muscle twitching), spasticity, and difficulties with speech, swallowing, and breathing. The symptoms generally progress from the site of onset to most voluntary muscles. Animal models of ALS include SOD1 transgenic mice (SOD1 mice), which are mouse models of ALS. In SOD1 mice, a mutant version of human superoxide dismutase 1 (SOD1) is inserted into the mouse genome and overexpressed.

[0051] Figure 30 shows several phenotypes due to overexpression of SOD1 in SOD1 mice, and these phenotypes include the onset of a progressive neurodegenerative disease that closely mimics human ALS with respect to both its pathological and clinical characteristics. Some of these characteristics include the presence of SOD1 aggregates, selective degeneration of motor neurons in the brain and spinal cord, muscle weakness and atrophy, neuroinflammation, oxidative stress, cognitive abnormalities, paralysis, and a progressive decline in motor function that often culminates in death due to respiratory failure. These symptoms typically begin to appear when the mice are several months old and progress rapidly, reflecting the aggressive nature of human ALS. As shown, the survival rate of SOD1 is approximately 50% at around 18 weeks.

[0052] Figure 31 shows a study design for evaluating the effectiveness of mitochondrial injection in an SOD1 mouse model of ALS. To ensure sufficient availability of mitoTEMPO, mitoTEMPO is obtained from any of the methods disclosed herein. SOD1 mice are divided into five treatment groups. As shown, group 1 functions as a negative control group containing wild-type (WT) mice administered a vehicle; group 2 functions as a positive control group containing SOD1 mice injected with a vehicle; group 3 contains SOD1 mice injected with 120 μL of mitoTEMPO; group 4 contains SOD1 mice injected with 120 μL of naked mitochondria derived from mouse liver; and group 5 contains SOD1 mice administered mitoTEMPO and naked mitochondria derived from mouse liver. These groups undergo various endpoint tests, including behavioral / clinical, survival, and SOD / TDP aggregation assessments. Behavioral / clinical assessments include, but are not limited to, rotarod tests and hanging wire tests, neuroinflammation assays, body weight, hSOD aggregation scores, and histological assays. Histological assays include assays targeting astrocytes, hippocampus, and cortex. Non-limiting examples include examining sections of the hippocampus to assay mitochondrial number and its variations. Behavioral tests can be scored using the Vercelli scoring system as a reference (Vercelli A et al., "Human mesenchymal stem cell transplantation extends survival, improves motor performance and decreases neuroinflammation in mouse model of amyotrophic lateral sclerosis." Neurobiol Dis. 2008;31(3):395-405).In the Vercelli scoring system, SOD1 mice were evaluated for the symptoms of motor impairment based on the following point scoring: 4 points for normal (no symptoms of motor dysfunction); 3 points if tremors of the hind limbs were evident when suspended by the tail; 2 points if there was abnormal gait; 1 point if at least one hind limb was dragged; 0 points if unable to stand independently within 30 s. The onset of motor impairment was retrospectively defined as the earliest time point when the mice showed symptoms (score < 4) continuously for more than two weeks. The results are shown in Figure 32.

[0053] Supplementary Note It should be understood that all combinations of the foregoing concepts and additional concepts discussed in more detail below are contemplated as part of the subject matter of the invention disclosed herein (provided such concepts are not mutually inconsistent). In particular, all combinations of the claimed subject matter listed at the end of this disclosure are contemplated as part of the subject matter of the invention disclosed herein. It should also be understood that terms explicitly used herein that may also appear in any disclosure incorporated by reference should be given the meaning that most closely matches the particular concepts disclosed herein.

[0054] Throughout this specification, references to "one example", "another example", and "an example", etc., mean that a particular element (e.g., a feature, structure, and / or property) described in connection with the example is included in at least one example described herein, and may or may not be present in other examples. Furthermore, it should be understood that elements described for any example can be combined in any suitable manner in various examples, unless the context clearly dictates otherwise. Although several examples have been described in detail, it should be understood that the disclosed examples can be modified. Accordingly, the foregoing description is to be regarded as non-limiting.

[0055] Features, materials, characteristics, or groups described in connection with a particular embodiment or example are understood to be applicable to any other embodiment or example described in this section or elsewhere in this specification, so long as they are not inconsistent therewith. All features disclosed in this specification (including the appended claims, abstract, and drawings), and / or all of the steps of any methods or processes so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The protection is not limited to the details of any of the foregoing examples. The protection extends to any novel one, or any novel combination, of the features disclosed in this specification (including the appended claims, abstract, and drawings), and / or to any novel one, or any novel combination, of the steps of any methods or processes so disclosed.

[0056] Furthermore, certain features described in this disclosure in connection with individual embodiments may also be implemented in combination in a single embodiment. Conversely, various features described in connection with a single embodiment may also be implemented separately in a plurality of embodiments, or in any suitable sub-combination. Additionally, although features may have been described above as functioning in a particular combination, one or more features of a claimed combination may, in some cases, be excised from the combination, and the combination may be claimed as a sub-combination or variation of a sub-combination.

[0057] Moreover, the operations may be depicted in the drawings or described herein in a particular order, but such operations need not be performed in the particular order shown or in a sequential order to achieve the desired result, nor is it necessary to perform all of the operations. Other operations not shown or described may also be incorporated into the exemplary methods and processes. For example, one or more additional operations may be performed before, after, simultaneously with, or between any of the operations described. Further, the operations may be rearranged in their arrangement or order in other embodiments. Those skilled in the art will understand that in some instances, the actual steps executed in the processes shown and / or disclosed may differ from those shown in the drawings. In some examples, certain ones of the steps described above may be omitted or others may be added. Additionally, the features and characteristics of the specific examples disclosed above may be combined in different manners to form additional examples, all of which are within the scope of the present disclosure.

[0058] For purposes of the present disclosure, certain aspects, advantages, and novel features are described herein. Not all of such advantages may be achieved in accordance with any particular example. Thus, for example, those skilled in the art will recognize that the present disclosure may be implemented or carried out so as to achieve one advantage or group of advantages taught herein without necessarily achieving other advantages taught or suggested herein.

[0059] Conditional language, such as "can", "could", "might", or "may", unless specifically stated otherwise or otherwise understood within the context in which it is used, generally intends to convey that a particular feature, element, and / or step is included in a particular instance but not in other instances. Thus, such conditional language generally does not intend to mean that a feature, element, and / or step is required in any way in one or more instances, or that one or more instances necessarily include logic for determining whether these features, elements, and / or steps are included in or implemented in any particular instance, regardless of the presence or absence of user input or prompting.

[0060] Conjunctive language, such as the phrase "at least one of X, Y, and Z", is understood as a context generally used to clearly convey that an item, term, etc. can be either X, Y, or Z, unless specifically stated otherwise. Thus, such conjunctive language generally does not intend to mean that a particular instance requires the presence of at least one X, at least one Y, and at least one Z.

[0061] Language used herein to indicate degree, such as the terms "about", "approximately", "generally", and "substantially", represents a value, quantity, or characteristic that is close to the stated value, quantity, or characteristic and still performs the desired function or achieves the desired result.

[0062] The following treatment methods may be derived from the methods disclosed in this specification: treatment methods that can reverse the photoaging process of the face and hands; treatment methods that improve the endpoints of neurodegenerative indications (e.g., Alzheimer's disease, Parkinson's disease, ALS) (as mitochondrial transplantation regenerates mitochondrial energy in the hippocampus of aged mice); treatment methods that can regenerate the retina, which is one of the most energy-consuming parts of the body, in AMD and glaucoma indications; treatment methods that assist the immune system in fighting sepsis and infectious diseases, such as Covid-19, and include the possibility of reversing immune system aging; potential anti-aging treatment methods that affect physical strength, cognitive ability, and vitality.

[0063] The scope of the present disclosure is not intended to be limited by the specific disclosure of preferred examples in this section or elsewhere in this specification, but may be defined by the claims presented in this section or elsewhere in this specification, or as presented hereinafter. The terms of the claims are to be construed broadly based on the terms used in the claims and are not limited to the examples described herein or during the prosecution of the application as non-exclusive.

[0064] The described embodiments and examples of the present disclosure are intended to be illustrative rather than limiting, and are not intended to represent all embodiments or examples of the present disclosure. Accordingly, the scope is not limited by the specific embodiments and examples described herein. The basic novel features applicable to various specific embodiments of the present disclosure have been shown, described, and pointed out. However, various omissions, substitutions, and changes in the details of the disclosed methods may become apparent and may be made by those skilled in the art without departing from the spirit of the present disclosure. For example, it is explicitly intended that all combinations of method steps that perform substantially the same function in substantially the same way to achieve the same result are within the scope of the present disclosure. Further, it should be recognized that any method steps shown and / or described in connection with any disclosed form or embodiment of the present disclosure may be incorporated into any other form or embodiment disclosed, described, or suggested as a general matter of design choice. Further, any of the steps disclosed herein can be repeated. Further, various changes and modifications can be made, both literally and legally in equivalents recognized as such, without departing from the spirit or scope of the present disclosure, as described in the following claims.

Explanation of Signs

[0065] 100 Method 110 Step 120 Step 130 Step 140 Step 150 Step 160 Step 170 Step 180 Step 190 Step

Claims

**Claim 1** A method for growing isolated mitochondria, comprising: selecting a source containing mitochondria and stem cells; extracting the stem cells and mitochondria from the source, and separating the stem cells and mitochondria into a first pool and a second pool, respectively; incubating the extracted mitochondria and transferring them to the extracted stem cells to generate packed stem cells; expanding the packed stem cells in a bioreactor; adjusting the conditions of the bioreactor environment to be favorable for the growth of the mitochondria of the packed stem cells; converting the packed stem cells into megakaryocytes; isolating mitochondria from the megakaryocytes; and applying a coating to the mitochondria after isolation from the megakaryocytes. **Claim 2** The method according to claim 1, wherein the source is placental tissue containing mitochondria and stem cells. **Claim 3** The method according to claim 1, wherein the source is bone marrow containing mitochondria and stem cells. **Claim 4** The method according to claim 1, wherein the source is adipose tissue containing mitochondria and stem cells. **Claim 5** The method according to claim 1, wherein the coating contains asialoorosomucoid (AsOR). **Claim 6** The method according to claim 5, wherein the coating further contains poly-L-lysine. **Claim 7** The method according to claim 6, wherein the coating further contains listeriolysin O (LLO). **Claim 8** The method according to claim 1, further comprising the step of storing the coated mitochondria. **Claim 9** The method according to claim 8, wherein the storing step includes suspending the mitochondria in a cryoprotectant. **Claim 10** The method according to claim 9, wherein the cryoprotectant contains trehalose. **Claim 11** The method according to claim 9, wherein the cryoprotectant contains phosphate buffered saline (PBS). **Claim 12** The method according to any one of claims 1 to 11, wherein the coating is applied at a ratio of 1 μg of coating per 128 μg of bioreactor-grown mitochondria. **Claim 13** The method according to claims 1 to 11, wherein the coating is applied at a ratio that is at least twice the ratio of 1 μg of coating per 128 μg of bioreactor-grown mitochondria. **Claim 14** The method according to any one of claims 1 to 13, further comprising the step of administering a therapeutic amount of the coated mitochondria to a subject.

15. The method according to claim 1, wherein the source is peripheral blood containing platelet-derived extracellular vesicles (PEVs), and the PEVs contain mitochondria.

16. The step of obtaining blood from one or more donors; The step of adding an anticoagulant and a buffer to the blood to form a mixture; The step of separating the mixture into a supernatant and platelet-rich plasma (PRP); The step of collecting the PRP; The step of releasing extracellular vesicles from platelets in the PRP by stimulating the collected PRP; and The method according to claim 15, further comprising the step of collecting the extracellular vesicles as PEVs.

17. The method according to claim 16, wherein the collected PRP is stimulated by immune complexes in the presence of Ca 2+ .

18. The method according to claim 17, wherein the immune complex contains heat-aggregated IgG.

19. The concentration of heat-aggregated IgG is from 0.1 mg / mL to 2.5 mg / mL, and the concentration of Ca 2+ is from 1 mM to 25 mM, the method according to claim 18.

20. The method according to any one of claims 16 to 19, wherein the anticoagulant is anticoagulant citrate dextrose (ACD).

21. The method according to any one of claims 16 to 20, wherein the buffer is a Tyrode buffer with a pH from 6 to 7.

22. A method for growing isolated mitochondria, comprising: The step of selecting a source containing mitochondria and stem cells; The step of extracting the stem cells and mitochondria from the source, and separating the stem cells and mitochondria into a first pool and a second pool, respectively; The step of incubating the extracted mitochondria and transferring them to the extracted stem cells to produce packed stem cells; The step of expanding the packed stem cells in a bioreactor; The step of adjusting the conditions of the bioreactor environment to be favorable for the growth of mitochondria in the packed stem cells; The step of converting the packed stem cells into megakaryocytes; and The method comprising the step of isolating mitochondria from the megakaryocytes.

23. The method according to claim 22, further comprising the step of applying a coating to the mitochondria after isolation from the megakaryocytes.

24. The method according to claim 22 or 23, wherein the source is placental tissue containing mitochondria and stem cells.

25. The method according to claim 22 or 23, wherein the source is bone marrow containing mitochondria and stem cells.

26. The method according to claim 22 or 23, wherein the source is adipose tissue containing mitochondria and stem cells.

27. The method according to any one of claims 23 to 26, wherein the coating contains asialoorosomucoid (AsOR).

28. The method according to claim 27, wherein the coating further comprises poly-L-lysine.

29. The method according to claim 27 or 28, wherein the coating further comprises listeriolysin O (LLO).

30. The method according to any one of claims 23 to 29, further comprising the step of storing the coated mitochondria.

31. The method according to claim 30, wherein the step of storing comprises suspending the mitochondria in a cryoprotectant.

32. The method according to claim 31, wherein the cryoprotectant comprises trehalose.

33. The method according to claim 31, wherein the cryoprotectant comprises PBS.

34. The method according to any one of claims 23 to 33, wherein the coating is applied at a ratio of 1 μg of coating per 128 μg of bioreactor-grown mitochondria.

35. The method according to claims 23 to 33, wherein the coating is applied at a ratio that is at least twice the ratio of 1 μg of coating per 128 μg of bioreactor-grown mitochondria.

36. The method according to any one of claims 23 to 35, further comprising the step of administering a therapeutic amount of the coated mitochondria to a subject.

37. wherein the source is peripheral blood comprising platelet-derived extracellular vesicles (PEVs), and the PEVs contain mitochondria, according to claim 22 or 23.

38. the step of obtaining blood from one or more donors; the step of adding an anticoagulant and a buffer to the blood to form a mixture; the step of separating the mixture into a supernatant and platelet-rich plasma (PRP); the step of collecting the PRP; the step of releasing extracellular vesicles from the platelets in the PRP by stimulating the collected PRP; and the method according to claim 37, further comprising the step of collecting the extracellular vesicles as PEVs.

39. The method according to claim 38, wherein the collected PRP is stimulated by immune complexes in the presence of Ca 2+ .

40. The method according to claim 39, wherein the immune complex comprises heat-aggregated IgG.

41. The concentration of heat-aggregated IgG is from 0.1 mg / mL to 2.5 mg / mL, and Ca 2+ The method according to claim 40, wherein the concentration of is from 1 mM to 25 mM.

42. The method according to any one of claims 38 to 41, wherein the anticoagulant is anticoagulant citrate dextrose (ACD).

43. The method according to any one of claims 38 to 42, wherein the buffer is a Tyrode's buffer having a pH from 6 to 7.

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