Platelet-Derived Extracellular Vesicles for Treating Cardiogenic Shock and Sepsis

Abstract

The subject can be treated for cardiogenic shock and / or sepsis, or symptoms thereof, using platelet-derived extracellular vesicles (PEVs) or mitrettes containing PEVs. The PEVs contain mitochondria. The PEVs can be collected by a process comprising 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, stimulating the collected PRP, thereby expelling extracellular vesicles from the platelets within the PRP, and collecting the extracellular vesicles as PEVs. The PEVs can also be isolated from supplier cells grown in a bioreactor and suspended in a buffer for storing the PEVs.

Description

Technical Field

[0001] Incorporation by reference of applications claiming priority In any PCT request filed in connection with this application, all applications in which a foreign or domestic priority claim is identified are hereby incorporated by reference into this specification.

[0002] The invention disclosed and claimed herein relates to methodologies for treating indications of cardiogenic shock and / or sepsis, and more particularly, generally relates to such methodologies involving the delivery of mitlets (including platelet-derived extracellular vesicles (PEVs) containing mitochondria).

Background Art

[0003] Mitochondria are membrane-bound intracellular organelles that have their own DNA (mtDNA) and their own machinery for synthesizing RNA and proteins. Mitochondria are found in almost all eukaryotic cells, and their number and location vary depending on the cell type.

[0004] In eukaryotic cells, mitochondria perform a number of essential roles, such as pyruvate oxidation, the Krebs cycle, and the metabolism of amino acids, fatty acids, and steroids. The main function of mitochondria is to generate energy as adenosine triphosphate (ATP) by the electron transport chain and the oxidative phosphorylation system (the "respiratory chain"). Additional processes in which mitochondria are involved include thermogenesis, calcium ion storage, calcium signaling, programmed cell death (apoptosis), and cell proliferation. Mitochondria have been shown to play a role in cellular control and signaling events (e.g., especially the control of Ca 2+ flux, oxidative stress, and energy-related signaling).

[0005] One manifestation of cardiovascular disease is cardiogenic shock, which occurs in approximately 5 - 8% of ST-segment elevation myocardial infarction (STEMI) cases and 2 - 3% of non-STEMI cases. The tendency of the incidence rate of cardiogenic shock due to acute myocardial infarction is shown in Figure 1. This corresponds to approximately 50,000 cases of cardiogenic shock per year in the United States, and the mortality rate exceeds 40%. Therefore, cardiogenic shock is one of the major causes of death in patients with acute myocardial infarction.

[0006] The pathophysiology of cardiogenic shock includes acute myocardial ischemia, which causes myocardial dysfunction. From myocardial dysfunction, there is a potential for a devastating vicious cycle of decreased cardiac output and decreased blood pressure to manifest, which causes further coronary ischemia and impairment of myocardial tissue contractility. Such a process, as shown in Figure 2, when added to infarction-induced tissue damage as well as genetic and environmental risk factors, may trigger a cytokine storm (i.e., systemic inflammatory response syndrome (SIRS)). A cytokine storm is characterized by enhanced activation of inflammatory signaling pathways, which causes massive release of pro-inflammatory cytokines and may lead to sepsis. As shown in Figure 2, major tissue damage due to acute myocardial infarction associated with cardiogenic shock may trigger a cytokine storm, but this is a self-perpetuating cycle that leads to systemic perfusion decline progressing to multiple organ failure, progressive cardiac dysfunction, and often death.

[0007] Furthermore, 5 - 7% of patients with cardiogenic shock also develop sepsis. In patients with cardiogenic shock complicated by sepsis, the risk of poor outcomes increases. Systemic perfusion decline induced by the cytokine storm and increased production of nitric oxide (NO) may cause vascular endothelial damage and circulatory organ dysfunction. Excessive production of NO leads to persistent vasodilation (Olwal et al., "Parallels in Sepsis and COVID-19 Conditions: Implications for Managing Severe COVID-19." Front Immunol. 2021;12:602848). Also, vascular endothelial damage makes blood vessels more prone to leakage and may provide a route for opportunistic pathogens, such as bacteria, viruses, fungi, or parasites, to invade the circulatory system. The mortality rate of patients with cardiogenic shock complicated by sepsis exceeds 40%.

[0008] In addition, the pneumonia condition caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and called coronavirus disease 2019 (COVID-19) is characterized by many pathophysiological and clinical similarities to sepsis. As shown in Figure 3, in severe cases of COVID-19, it is correlated with high levels of pro-inflammatory cytokines measured in the bloodstream, such as interleukin (IL) 6 (IL-6), IL-10, tumor necrosis factor α (TNFα), colony-stimulating factor (CSF), and interferon-inducible protein 10 (IP10), but not limited to these. Similar to the case of sepsis in patients with cardiogenic shock, sepsis caused by COVID-19 may also cause sepsis with the potential to lead to multiple organ failure and tissue damage. Furthermore, the prolonged COVID-19 state (COVID-19 symptoms observed in individuals suspected or confirmed to be infected with SARS-CoV-2 that persist for at least 2 months after the onset of COVID-19 and up to 3 months) may make patients more susceptible to cardiogenic shock and subsequent sepsis.

[0009] Some have approached the problem of treating cardiogenic shock with emergency revascularization. As shown in Figure 4, since about 80% of the deaths due to cardiogenic shock occur within 30 days, early aggressive treatment is recommended. TRIUMPH trial investigators, "Effect of tilarginine acetate in patients with acute myocardial infarction and cardiogenic shock: the TRIUMPH randomized controlled trial." JAMA. 2007;297(15):1657-1666. Also as shown, survivors at 90 days have a very good prognosis over the next several months. Data from the TRIUMPH trial suggest that if an effective early aggressive treatment method can be developed, it will have a great impact on the long-term outcomes of patients with cardiogenic shock and potentially those with COVID-19. Mitochondrial transfer technology (collecting mitochondria from an external source and transferring them into the body) has been developed in recent years by several major universities. Mitochondrial transfer is generally less invasive than other approaches, but finding a mitochondrial source to treat the above condition and preparing that mitochondria for transfer has long been a problem.

Prior Art Documents

Patent Documents

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Patent Document 1

Patent Document 2

Non-Patent Documents

[0011]

Non-Patent Document 1

[0012] The methods disclosed herein each have several aspects, but one of them is not involved in its desirable characteristics alone. Without limiting the scope of the claims, some prominent characteristics are briefly discussed here. Many other embodiments are also considered, including embodiments having fewer, additional, and / or different components, steps, characteristics, objectives, benefits, and advantages. The components, aspects, and steps can be configured and ordered differently. After considering this discussion, especially after reading the section entitled "Modes for Carrying Out the Invention," it will be understood how the characteristics of the devices and methods disclosed herein provide advantages over other known devices and methods. [Means for Solving the Problems]

[0013] In one aspect, the disclosed technology is a method for treating a condition or its symptoms in a subject having the condition, the method comprising platelet-derived extracellular vesicles (PEVs) containing mitochondria, wherein the PEVs are obtained by a process 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, stimulating the collected PRP to thereby expel extracellular vesicles from the platelets in the PRP, and collecting the extracellular vesicles as PRP, obtaining mitorets, administering an effective amount of the mitorets to the subject to thereby treat the condition or its symptoms. In some embodiments, the PEVs are collected at a location different from the location where the treatment is being performed.

[0014] In some embodiments, the condition includes cardiogenic shock. In some embodiments, the condition includes sepsis. In some embodiments, the condition includes a virus-induced disease. In some embodiments, the virus includes a coronavirus. In some embodiments, the coronavirus includes severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In some embodiments, the disease is coronavirus disease 2019 (COVID-19). In some embodiments, the condition includes cardiogenic shock, sepsis, and a virus-induced disease. In some embodiments, the disease is COVID-19. In some embodiments, the disease is post-COVID-19. In some embodiments, COVID-19 precedes cardiogenic shock, and cardiogenic shock precedes sepsis.

[0015] In some embodiments, the administering step includes injecting an effective amount of mitret into the subject to treat the condition. In some embodiments, the collected PRP is stimulated with immune complexes in the presence of Ca2+. In some embodiments, the immune complexes include heat-aggregated IgG. In some embodiments, the collected PRP is stimulated by freeze-thaw cycles. In some embodiments, the concentration of heat-aggregated IgG is from about 0.1 mg / mL to about 2.5 mg / mL, and the concentration of Ca2+ is from about 1 mM to about 25 mM. In some embodiments, the anticoagulant is citrate dextrose (ACD) of the anticoagulant. In some embodiments, the buffer is Tyrode's buffer at about pH 6 to about pH 7. In some embodiments, the separating step is performed by centrifugation. In some embodiments, the blood is stored for 4 days or more. In some embodiments, the blood is stored for up to 1 year.

[0016] In some embodiments, during and / or after the step of administering mitret into the subject, mitret contacts at least one cell of the subject. In some embodiments, after mitret contacts the cell, mitret translocates intracellularly. In some embodiments, the effective amount corresponds to the amount of mitret that has translocated intracellularly, and that amount ranges from about 3 mitrets / cell to about 100 mitrets / cell. In some embodiments, mitret is frozen during storage. In some embodiments, the frozen mitret is stored in combination with a cryoprotective substance. In some embodiments, the cryoprotective substance is selected from the group consisting of saccharides, oligosaccharides, and polysaccharides.

[0017] In a second aspect, the disclosed technology is a method of treating a condition or its symptoms in a subject having the condition, the method comprising: obtaining a population of extracellular vesicles (PEVs) containing mitochondria from a source; suspending the PEVs in a buffer for storage; and administering an effective amount of the PEVs into the subject to thereby treat the condition or its symptoms. In some embodiments, the source comprises cells, which can be cells selected from the group consisting of stem cells such as placental stem cells, umbilical cord stem cells, adipose tissue-derived stem cells, and induced pluripotent stem cells. The cells can also be selected from the group consisting of hepatocytes, blood cells, stem cells, or any cells obtained from a donor. In some embodiments, the source comprises a tissue selected from the group consisting of the liver, bone marrow, placenta, adipose tissue, or any tissue obtained from a donor.

[0018] In some embodiments, the obtaining step comprises growing the source in a bioreactor and isolating the PEVs from the source grown in the bioreactor. In some embodiments, the obtaining step further comprises coating the PEVs after the isolating step. In some embodiments, the PEVs are collected at a site different from the site where the treatment is to be performed.

[0019] In some embodiments, the condition includes cardiogenic shock. In some embodiments, the condition includes sepsis. In some embodiments, the condition includes a virus-induced disease. In some embodiments, the virus includes a coronavirus. In some embodiments, the coronavirus includes SARS-CoV-2. In some embodiments, the disease is COVID-19. In some embodiments, the condition includes cardiogenic shock, sepsis, and a virus-induced disease. In some embodiments, the disease is COVID-19. In some embodiments, COVID-19 precedes cardiogenic shock, and cardiogenic shock precedes sepsis. In some embodiments, the administering step includes injecting an effective amount of mitolactol into the subject to treat the condition.

[0020] In some embodiments, the buffer contains a cryoprotectant. In some embodiments, the cryoprotectant is selected from the group consisting of saccharides, oligosaccharides, and polysaccharides. In some embodiments, the buffer contains a hydrogel. In some embodiments, the hydrogel has temperature-dependent hydrophilicity and hydrophobicity.

[0021] In some embodiments, a mitolet is provided, the mitolet comprising platelet-derived extracellular vesicles (PEVs) containing mitochondria for use in the treatment of cardiogenic shock and / or sepsis, or symptoms thereof, the PEVs being collected by a process comprising 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, stimulating the collected PRP to thereby expel extracellular vesicles from the platelets within the PRP, and collecting the extracellular vesicles as PEVs. In some embodiments, the PEVs are collected at a site different from the site at which the treatment is to be performed. In some embodiments, the mitolet is used in treating cardiogenic shock. In some embodiments, the mitolet is used in treating sepsis. In some embodiments, cardiogenic shock and / or sepsis is caused by a virus. In some embodiments, the virus includes a coronavirus. In some embodiments, the coronavirus includes severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In some embodiments, the PEVs are used in co-treating cardiogenic shock, sepsis, and diseases caused by a virus. In some embodiments, the disease is COVID-19.

[0022] In some embodiments, the collected PRP is stimulated with immune complexes in the presence of Ca2+. In some embodiments, the immune complexes include heat-aggregated IgG. In some embodiments, the collected PRP is stimulated by freeze-thaw cycles. In some embodiments, the concentration of heat-aggregated IgG is from about 0.1 mg / mL to about 2.5 mg / mL, and the concentration of Ca2+ is from about 1 mM to about 25 mM. In some embodiments, the anticoagulant is citrate dextrose (ACD) of the anticoagulant. In some embodiments, the buffer is Tyrode's buffer at about pH 6 to about pH 7. In some embodiments, the separating step is performed by a centrifuge.

[0023] In some embodiments, the blood has been stored for 4 days or more. In some embodiments, the blood has been stored for up to 1 year. In some embodiments, the effective amount of mitotret is in the range of about 3 mitotrets / cell to about 100 mitotrets / cell. In some embodiments, the mitotret is frozen during storage. In some embodiments, the frozen mitotret is stored in combination with a cryoprotectant. In some embodiments, the cryoprotectant is selected from the group consisting of saccharides, oligosaccharides, and polysaccharides.

[0024] In some embodiments, a PEV is provided, the PEV comprising mitochondria for use in the treatment of cardiogenic shock and / or sepsis, or symptoms thereof, the PEV being isolated from donor cells suspended in a buffer for storing the PEV and grown in a bioreactor, the donor cells obtained being selected from the group consisting of placental stem cells, umbilical cord stem cells, adipose tissue-derived stem cells, hepatocytes, blood cells, bone marrow, and induced pluripotent stem cells. In some embodiments, the PEV is coated. In some embodiments, an effective amount of the PEV is administered into a subject as shown. In some embodiments, the PEV is used in treating cardiogenic shock. In some embodiments, the PEV is used in treating sepsis. In some embodiments, cardiogenic shock and / or sepsis is caused by a virus. In some embodiments, the virus includes a coronavirus. In some embodiments, the coronavirus includes severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In some embodiments, the PEV is used in co-treating cardiogenic shock, sepsis, and diseases caused by a virus.

[0025] In some embodiments, the disease is COVID-19. In some embodiments, the buffer includes a cryoprotectant. In some embodiments, the cryoprotectant is selected from the group consisting of saccharides, oligosaccharides, and polysaccharides. In some embodiments, the buffer includes a hydrogel. In some embodiments, the hydrogel has temperature-dependent hydrophilicity and hydrophobicity.

[0026] The characteristics of the examples of the present disclosure will become apparent by referring to the following detailed description and the drawings. Like reference numerals correspond to similar but perhaps not identical components. For the sake of brevity, reference numerals or characteristics having functions already described may or may not be described in association with other drawings in which they appear.

Brief Description of the Drawings

[0027]

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Mode for Carrying Out the Invention

[0028] In the above "Summary of the Invention" section, "Detailed Description of the Invention" section and the following claims, specific features of the present invention are referred to. It is understood that the disclosure of the present invention within this specification includes all possible combinations of such specific features. For example, if a specific feature is disclosed in the context of a particular aspect or embodiment of the present invention, or a particular claim, that feature can, to the extent possible, be combined with and / or in the context of other specific aspects or embodiments of the present invention and is generally usable in the present invention.

[0029] Mitochondrial dysfunction is a potential factor in multiple diseases including cardiovascular diseases, cancer, Alzheimer's disease, diabetes, vision loss, and frailty. In some aspects of the present invention, mitochondria are transplanted to treat these and other diseases and conditions. Finding a source of mitochondria for transplantation is a challenge. As with any donor organ, mitochondria from young, healthy donors are in short supply. Some diseases or injuries can be cured by autologous mitochondria (e.g., taken from leg muscles within one's own body), however, in many other diseases, due to age or mutations in mitochondrial DNA (mtDNA), the quality of the "patient's" mitochondria is reduced. In such patients, donor mitochondria are a preferred solution. In addition, newly isolated mitochondria rapidly die within minutes of isolation and also have the potential to induce an immune response when introduced naked into the bloodstream, reducing their effectiveness as a therapy. Therefore, it is advantageous to find a convenient and readily available source of mitochondria for immediate donation (encapsulated, at the same time, in a coating, vesicle, or media suspension (or any combination of these) that protects it from the immune system).

[0030] As shown in FIG. 5, some embodiments relate to a method 100 for extracting platelet-derived mitochondria-containing extracellular vesicles (PEVs). In some embodiments, method 100 includes: 1) obtaining blood from a donor in step 102; 2) adding an anticoagulant and a buffer to the blood to form a mixture in step 104; 3) separating the mixture into a supernatant and platelet-rich plasma (PRP) in step 106; 4) collecting the platelet-rich plasma (PRP) in step 108; 5) stimulating the collected platelets in step 110; and collecting the PEVs in step 112. In some embodiments, mitotretes contain PEVs.

[0031] Platelets derived from human blood contain, on average, 4-5 mitochondria, which are released into extracellular vesicles when the platelets are activated. Platelet-derived mitochondria-containing extracellular vesicles are referred to herein as PEVs. This PEV is typically larger (>400 nM) than other platelet extracts or lysates (30-100 nM) and is not as well-known, although other sizes may also apply.

[0032] PEVs can donate their mitochondria to neighboring cells and, as shown in FIGS. 14A-14D (shown in FIGS. 10A-10B, 11, and 12), can increase the respiratory activity of cells that absorb the PEVs, and thus have been shown to be able to regenerate tissue and cure some age-related diseases. PEVs have several advantages for early commercialization: In particular, PEVs can be extracted from donor platelets that would otherwise have to be discarded as “expired”; PEVs provide another excellent and medically effective use for platelets that would otherwise be disposed of; PEVs can be collected in most blood banks (which already have all the required skilled personnel, clean handling practices, and required equipment and are already adjacent to hospitals, making it possible to produce PEV products that may be available for international use very quickly). PEVs are various platelet infusions and are thus more likely to be included and are tested by medical professionals already familiar with blood transfusion therapy. Furthermore, PEVs can be prepared for local infusion into various internal anatomical regions to treat various clinical disorders while using already commercially available delivery devices.

[0033] In some embodiments, non-limiting examples of delivery devices include a hollow barrel that forms at least an internal space, a plunger connected to and fitted within the hollow barrel, and a needle connected to the barrel, the needle including a space that is continuous with the internal space of the hollow barrel when the needle is connected to the barrel. Both the plunger and the needle can be connected directly or indirectly to the hollow barrel. The syringe is constructed to deliver PEV and / or naked mitochondria into the eye or vitreous body. In some embodiments, the syringe is constructed to deliver PEV and / or naked mitochondria subcutaneously. In some embodiments, the syringe is constructed to deliver PEV and / or naked mitochondria intravenously. In some embodiments, the syringe is constructed to deliver PEV and / or naked mitochondria to a subretinal site. In some embodiments, the syringe is constructed to deliver PEV and / or naked mitochondria into the peritoneal cavity (intraperitoneal injection). In some embodiments, the syringe is constructed to deliver PEV and / or naked mitochondria systemically to a patient (enteral or parenteral).

[0034] In some embodiments, a non-limiting example of a delivery device is a port delivery system that provides for sustained release of PEV and / or naked mitochondria (either of which can optionally be used in combination with other therapeutic agents) via intravitreal or subretinal delivery. The port delivery system is described in U.S. Patent No. 9,968,603, the disclosure of which is incorporated herein by reference.

[0035] As another non-limiting example in some embodiments, the ORBIT™ subretinal delivery system, which could be devised for the delivery of PEVs, was developed by Gyroscope Therapeutics. Another delivery system relates to providing a PEV (which is then placed in the affected eye) on a contact lens. Also, the PEV may be embedded within a gel-like material and placed within a “microneedle” as described by Lee et al., Advanced Functional Materials, doi.org / 10.1002 / adfm.202000086 (2020), the disclosure of which is incorporated herein by reference. Many other such delivery systems are known and could be devised for delivering PEVs.

[0036] In some embodiments, the blood is derived from a mammalian subject. According to another embodiment, the mammalian subject is a human subject. According to another embodiment, the mammalian subject is selected from the group consisting of human, horse, dog, cat, mouse, rat, cow, and sheep. Each possibility represents an individual embodiment of the invention. According to another embodiment, the PEV of the present invention is derived from mammalian cells. According to another embodiment, the mammalian cells are human cells. According to another embodiment, the PEV is derived from cells in culture. According to another embodiment, the PEV is derived from tissue.

[0037] According to another embodiment, the PEV is derived from cells or tissues selected from the group consisting of human placenta, human placental cells grown in culture, and human blood cells. According to another embodiment, the PEV of the present invention is derived from cells or tissues selected from the group consisting of placenta, hepatocytes, placental cells grown in culture, and blood cells. According to another embodiment, the naked mitochondria can be isolated from cells or tissues selected from the group consisting of donor liver, bone marrow, placenta, human placental cells, or any other tissue. According to another embodiment, the naked mitochondria can be isolated from cells grown in culture or from tissues grown in culture selected from the group consisting of donor liver, bone marrow, placenta, human placental cells, or any other tissue.

[0038] As used herein, the phrase "naked mitochondria" refers to mitochondria isolated from cells or tissues. In some embodiments, the cells are cells grown in culture. In some embodiments, the tissues are tissues grown in culture. The naked mitochondria can be suspended in a cryogenic buffer, a hydrogel, a pharmaceutically acceptable liquid medium having the ability to support the naked mitochondria, or a buffer solution containing a saccharide. In some embodiments, the hydrogel is biocompatible, biodegradable, and has the ability to support the naked mitochondria. In some embodiments, the hydrogel can be thermosensitive, which includes temperature-dependent hydrophilicity and hydrophobicity. In some embodiments, the hydrogel is biocompatible, biodegradable, has the ability to support the naked mitochondria, and is thermosensitive (thermosensitivity includes hydrogels having temperature-dependent hydrophilicity and hydrophobicity).

[0039] As used herein, the phrase "cells grown in culture" or "tissue grown in culture" refers to a plurality of cells or tissue, respectively, grown in a liquid, semi-solid, or solid medium outside of the organism from which the cells or tissue are derived. According to some embodiments, the cells grown in culture are cells grown in a bioreactor. By way of non-limiting example, cells can be grown in a bioreactor, followed by isolation of PEV from the cells. According to another non-limiting example, cells can be grown in a bioreactor, followed by isolation of mitochondria from the cells. In some embodiments, the mitochondria isolated from cells grown in a bioreactor are naked mitochondria. By way of non-limiting example, tissue can be grown in a bioreactor, followed by isolation of PEV from the cells of the tissue.

[0040] In some embodiments, the blood is of mouse origin. In some embodiments, mouse blood is used to test the feasibility of the PEV extraction method. In some embodiments, the blood is of human donor origin.

[0041] Once the blood is obtained, an anticoagulant and a buffer are added to prevent the blood from thickening and solidifying. In some embodiments, the anticoagulant is ACD (20%). In some embodiments, the buffer is a 40% Tyrode's buffer having a pH of about 6 to about 7, preferably pH 6.5.

[0042] After adding an anticoagulant and a buffer to the blood, the mixture is then separated into a supernatant and platelet-rich plasma (PRP). In some embodiments, the separation is by centrifugation. Plasma is the liquid portion of whole blood. Plasma is mainly composed of water and proteins and provides a medium for red blood cells, white blood cells, and platelets to circulate throughout the body. Platelets are blood cells that bring about blood clotting and other necessary growth and healing functions. After centrifugation of the mixture, the blood cells form a pellet that accumulates at the bottom of the tube. The pellet is called platelet-rich plasma (PRP) and contains concentrated platelets.

[0043] Next, a buffer is added to the collected PRP to resuspend the platelets. The platelets are then activated or stimulated. There are many ways to activate platelets. Any of several substances including carbon radioisotopes, prostaglandins, serotonin, adenosine triphosphate, collagen, l-lactate dehydrogenase, thrombin, magnesium, adenosine, calcium, and heat-aggregated antibodies can be used for this purpose. In some embodiments, the platelets are activated by freeze-thaw cycles. As used herein, the term "freeze-thaw cycle" refers to freezing the mitochondria of the present invention at a temperature below 0°C, maintaining the mitochondria at a temperature below 0°C for a defined period, and thawing the mitochondria at room temperature or body temperature or any temperature above 0°C. The term "room temperature" as used herein refers to a temperature of 18°C to 25°C. The term "body temperature" as used herein refers to a temperature of 35.5°C to 37.5°C, preferably 37°C.

[0044] In another embodiment, mitochondria that have been subjected to freeze-thaw cycles were frozen at a temperature of at least -70°C. In another embodiment, mitochondria that have been subjected to freeze-thaw cycles were frozen at a temperature of at least -20°C. In another embodiment, mitochondria that have been subjected to freeze-thaw cycles were frozen at a temperature of at least -4°C. In another embodiment, mitochondria that have been subjected to freeze-thaw cycles were frozen at a temperature of at least 0°C. According to another embodiment, the freezing of mitochondria is stepwise. According to some embodiments, the freezing of mitochondria is by flash freezing. As used herein, the term "flash freezing" refers to rapidly freezing mitochondria by exposing them to extremely low temperatures.

[0045] In another embodiment, mitochondria that have been subjected to freeze-thaw cycles were frozen for at least 30 minutes before thawing. According to another embodiment, the freeze-thaw cycle includes freezing partially purified functional mitochondria for at least 30, 60, 90, 120, 180, 210 minutes before thawing. Each possibility represents an individual embodiment of the present invention. In another embodiment, mitochondria that have been subjected to freeze-thaw cycles were frozen for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 24, 48, 72, 96, 120 hours before thawing. Each freezing time represents an individual embodiment of the present invention. In another embodiment, mitochondria that have been subjected to freeze-thaw cycles were frozen for at least 4, 5, 6, 7, 30, 60, 120, 365 days before thawing. Each freezing time represents an individual embodiment of the present invention. According to another embodiment, the freeze-thaw cycle includes freezing partially purified functional mitochondria for at least 1, 2, 3 weeks before thawing. Each possibility represents an individual embodiment of the present invention. According to another embodiment, the freeze-thaw cycle includes freezing partially purified functional mitochondria for at least 1, 2, 3, 4, 5, 6 months before thawing. Each possibility represents an individual embodiment of the present invention.

[0046] According to another embodiment, mitochondria that have undergone freeze-thaw cycles were frozen in a freezing buffer. According to another embodiment, mitochondria that have undergone freeze-thaw cycles were frozen in an isolation buffer. As used herein, the term "isolation buffer" refers to a buffer in which the mitochondria of the present invention are partially purified. In a non-limiting example, the isolation buffer contains 200 mM sucrose, 10 mM Tris-MOPS, and 1 mM EGTA. According to some embodiments, BSA (bovine serum albumin) is added to the isolation buffer during the partial purification period. According to some embodiments, 0.2% BSA is added to the isolation buffer during the partial purification period. According to some embodiments, HSA (human serum albumin) is added to the isolation buffer during the partial purification period. According to some embodiments, 0.2% HAS is added to the isolation buffer during the partial purification period. According to other embodiments, HSA or BSA is removed from the mitochondria of the present invention after partial purification. Each possibility represents an individual embodiment of the present invention. Without being bound by any mechanism or theory, freezing the mitochondria in the isolation buffer eliminates the need to replace the isolation buffer with a freezing buffer before freezing or to replace the freezing buffer during thawing, resulting in time and isolation process savings.

[0047] According to another embodiment, the freezing buffer contains a cryoprotectant. According to some embodiments, the cryoprotectant is a saccharide, oligosaccharide, or polysaccharide. Each possibility represents an individual embodiment of the present invention. According to another embodiment, the saccharide concentration in the freezing buffer is a saccharide concentration sufficient to act to maintain mitochondrial function. According to another embodiment, the isolation buffer contains a saccharide. According to another embodiment, the saccharide concentration in the isolation buffer is a saccharide concentration sufficient to act to maintain mitochondrial function. According to another embodiment, the saccharide is sucrose. According to another embodiment, the saccharide is a saccharide other than trehalose. Without being bound by any theory or mechanism, mitochondria frozen in a freezing buffer or isolation buffer containing sucrose exhibit an oxygen consumption rate comparable to or higher than that of control mitochondria that have not been subjected to freeze-thaw cycles or that have been frozen in a freezing buffer or isolation buffer without sucrose after thawing.

[0048] According to some embodiments, when a saccharide is added to the mitochondrial composition of the present invention at a sufficient concentration, such addition acts to maintain mitochondrial function. According to another embodiment, the saccharide concentration sufficient to act to maintain mitochondrial function is a concentration of 100 mM to 400 mM, preferably 100 mM to 250 mM, and most preferably 200 mM to 250 mM. Each possibility represents an individual embodiment of the present invention. According to another embodiment, the saccharide according to the present invention is sucrose. According to some embodiments, the saccharide of the present invention is a saccharide other than trehalose. According to some embodiments, the saccharide of the present invention is a saccharide other than mannitol.

[0049] According to another embodiment, the saccharide concentration in the composition of the present invention is 100 mM to 150 mM. According to another embodiment, the saccharide concentration in the composition of the present invention is 150 mM to 200 mM. According to another embodiment, the saccharide concentration in the composition of the present invention is 100 mM to 200 mM. According to another embodiment, the saccharide concentration in the composition of the present invention is 100 mM to 400 mM. According to another embodiment, the saccharide concentration in the composition of the present invention is 150 mM to 400 mM. According to another embodiment, the saccharide concentration in the composition of the present invention is 200 mM to 400 mM. According to another embodiment, the saccharide concentration in the composition of the present invention is at least 100 mM. According to another embodiment, the saccharide concentration in the composition of the present invention is at least 200 mM. Without being bound by any theory or mechanism of action, saccharide concentrations less than 100 mM may not be sufficient to maintain mitochondrial function.

[0050] In some embodiments, the stimulant is heat-aggregated IgG.

[0051] Stimulated platelets are centrifuged to remove residual platelets or cells. The supernatant containing PEV is then collected.

[0052] According to some embodiments, the PEV is derived from a subject in need thereof. According to another embodiment, the PEV is derived from a subject different from the subject in need thereof. According to another embodiment, the PEV is derived from the same subject as the subject to which the PEV is administered. According to another embodiment, the PEV is derived from a subject different from the subject to which the PEV is administered. According to another embodiment, the PEV of the present invention is derived from a source selected from autologous, allogeneic, and xenogeneic. Each possibility represents an individual embodiment of the present invention. As used herein, autologous mitochondria refers to mitochondria derived from the same subject as the subject being treated. As used herein, allogeneic mitochondria refers to mitochondria derived from a subject different from the subject being treated but of the same species. As used herein, xenogeneic mitochondria refers to mitochondria derived from a subject different from the subject being treated and of a different species. According to another embodiment, the PEV of the present invention is derived from a donor. According to some embodiments, the donor is an allogeneic donor. According to some embodiments, the donor is an autologous donor.

[0053] As used herein, the term "subject in need thereof" refers to a subject suffering from or at risk of suffering from a condition that would benefit from enhanced mitochondrial function. In some embodiments, the condition includes a retinal disease or condition. In some embodiments, the condition includes cardiogenic shock. In some embodiments, the condition includes sepsis. In some embodiments, the condition includes COVID-19. Each possibility represents an individual embodiment of the present invention. According to some embodiments, the "subject in need thereof" is a subject suffering from a condition that could benefit from apoptosis-promoting activity. By way of non-limiting example, a condition that could benefit from apoptosis-promoting activity is cancer. According to another embodiment, the subject in need thereof is a mammal. According to another embodiment, the subject in need thereof is a human. According to another embodiment, the subject in need thereof is selected from the group consisting of human, horse, dog, cat, mouse, rat, cow, and sheep.

[0054] Some embodiments relate to a method of transducing platelet-derived mitochondria-containing extracellular vesicles (PEVs) into cells. The method includes 1) extracting PEVs from blood, and 2) incubating the PEVs with cells for a time sufficient to transduce the PEVs into the cells. The step of incubating the cells can be in vitro or in vivo, with the latter (in vivo) shown in FIG. 9.

[0055] In certain in vivo embodiments, the step of incubating the PEVs with cells includes injecting the PEVs into the subject's blood, cerebrospinal fluid, pleural fluid, pericardial fluid, peritoneal fluid / ascites, synovial fluid, saliva, or any other body fluid. This can be accomplished in a variety of ways, including the use of appropriate catheters, such as intra-arterial or intrathecal catheters. The PEVs can also be introduced into a specific organ or tissue of the subject, such as the subject's eye or retina. For this purpose, the PEVs can be delivered via intravitreal, intravenous, or intra-arterial injection.

[0056] According to another embodiment, as shown in FIG. 6, a method 200 for treating an eye disorder or symptoms of an eye disorder in a patient in need thereof is provided, the method including the step of obtaining platelet-derived extracellular vesicles (PEVs) containing mitochondria. The PEVs are collected by a process including, in step 202, obtaining blood from one or more donors, in step 204, adding an anticoagulant and a buffer to the blood to form a mixture, in step 206, separating the mixture into a supernatant and platelet-rich plasma (PRP), in step 208, collecting the PRP, in step 210, stimulating the collected PRP, thereby expelling extracellular vesicles from the platelets within the PRP, and in step 212, collecting the extracellular vesicles as PEVs. Many of these steps associated with the collection of PEVs are described in other embodiments and thus they are similar. In some embodiments, the method further includes, in step 214, administering an effective amount of the PEVs to the patient's eye, thereby treating the eye disorder. In some embodiments, the PEVs are collected at a site different from the site where the treatment is to be performed. In some embodiments, the PEVs are collected on-site or off-site.

[0057] As used herein, the term "on-site" refers to the location where the administration step is being performed or is scheduled to be performed. The location can be in the same room, office, or ward as the room, office, or ward where the administration step is being performed or is scheduled to be performed. The location can be within the same building as the building where the administration step is being performed or is scheduled to be performed. The location can be within the same building complex including multiple buildings, with at least one of the multiple buildings being the location where the administration step is being performed or is scheduled to be performed. The building complex can have the same partner (business or organization), or at least one of the multiple buildings can have a different partner.

[0058] As used herein, the term "off-site" refers to an external location that is distant from the location where the administration step is being performed or is scheduled to be performed. The external location can be a room or laboratory that is distant from the building and building complex (if the building is part of a building complex) where the administration step is being performed or is scheduled to be performed.

[0059] In some embodiments, the blood has been stored for about 4 days or more. In some embodiments, the blood has been stored for about 1 year. In some embodiments, the PEV is frozen during storage. In some embodiments, the frozen PEV is stored in combination with a cryoprotectant. In some embodiments, the cryoprotectant is selected from the group consisting of saccharides, oligosaccharides, and polysaccharides. In some embodiments, the anticoagulant is citrate dextrose (ACD) of the anticoagulant. In some embodiments, the buffer is Tyrode's buffer at about pH 6 to about pH 7, preferably about pH 6.5. In some embodiments, the separation is performed by centrifugation. In some embodiments, in the case of the stimulating step, the collected PEV is in the presence of Ca 2+ and is stimulated by immune complexes. In some embodiments, the immune complexes contain heat-aggregated IgG. The concentration of heat-aggregated Ig used in the stimulating step is preferably about 0.1 mg / mL to about 2.5 mg / mL, more preferably about 0.5 mg / mL. The concentration of Ca 2+ used in the stimulating step is about 1 mM to about 25 mM, more preferably about 5 mM. In some embodiments, the collected PRP is stimulated by freeze-thaw cycles.

[0060] In some embodiments, after the PEV is administered intravitreally, the PEV contacts at least one cell of the eye. In some embodiments, after the PEV contacts the cell, the PEV translocates intracellularly. As used herein, the terms "contact with" and "contacting with" mean that a composition containing mitochondria is located in close proximity to a cell such that it is sufficient to induce at least the intracellular translocation of mitochondria into the cell.

[0061] In some embodiments, the effective amount for treating an eye disorder corresponds to the amount of PEV that has translocated internally, and that effective amount ranges from about 3 PEVs / cell to about 100 PEVs / cell for at least one cell, as shown with respect to RPEC and bEND in FIGS. 13A - 13B. In some embodiments, the effective amount corresponds to the amount of PEV that has translocated internally, and that effective amount is about 3 PEVs / cell, about 10 PEVs / cell, about 30 PEVs / cell, or about 100 PEVs / cell for at least one cell. The effective amount will vary depending on the route of administration, the possibility of co - administration with another therapeutic agent, the possibility of co - utilization with another therapeutic treatment, or method, the type of delivery device used, and the utilization of any excipients, as will be recognized by those of ordinary skill in the art.

[0062] In some embodiments, the eye disorder to be treated is age - related macular degeneration (AMD). AMD is an eye disorder that is one of the leading causes of vision loss, particularly in developed countries, and has a prevalence of up to approximately 40%. AMD is characterized by mitochondrial dysfunction that affects the retina, and this dysfunction is caused by oxidative stress from reactive oxygen species (ROS). In AMD, ROS are produced at high levels in RPE cells, causing damage to mtDNA. If the mtDNA repair mechanism is insufficient, this damage can accumulate over time to the point of causing mitochondrial cell death and, ultimately, RPE cell death. Since RPE cells support the health of photoreceptors, RPE cell death leads to the death of photoreceptors supported by RPE cells, causing vision loss.

[0063] In some embodiments, the eye disorder to be treated is retinitis pigmentosa (RP). RP is a hereditary disorder of the eye that causes severe visual impairment and is characterized by rod degeneration. In some embodiments, the eye disorder is Leber's hereditary optic neuropathy (LHON), a hereditary mitochondrial genetic disorder that manifests due to three major mtDNA mutations found in 90% of cases. From this mtDNA mutation, LHON mainly affects retinal ganglion cells (RGCs), causing their degeneration and leading to vision loss. In some embodiments, the eye disorder is diabetic retinopathy, which is characterized by dysfunction of endothelial cells of retinal microvessels and supporting cells of the retina, such as Müller cells. In diabetic retinopathy, dysfunction of endothelial cells causes an increase in their permeability, which can lead to vascular leakage. This vascular leakage can cause peripheral edema and thus may result in other related retinal diseases, such as diabetic macular edema. In some embodiments, the eye disorder is glaucoma.

[0064] In some embodiments, the cells that receive the PEV include retinal pigment epithelial cells. In some embodiments, the cells that receive the PEV include retinal ganglion cells. In some embodiments, the cells that receive the PEV are located around the macula of the retina of the eye.

[0065] By complexing a specific receptor or coating that promotes "homing" to a specific cell type with the PEV, specific tissues and organs can be specifically targeted. For example, U.S. Patent No. 10,537,594, the content of which is incorporated herein by reference, exemplifies the use of the asialoglycoprotein (AsG) receptor system for targeting mitochondria to hepatocytes. Similar systems can be used to target other tissues or organs.

[0066] To promote the intracellular translocation of mitochondria both in vitro and in vivo, various techniques can be employed. A high level of mitochondrial translocation is thought to be achievable in certain diseased tissues or the elderly (in this case, the tissue is in a state of severe mitochondrial deficiency). Cells in an energy-deficient state are expected to activate chemical pathways that enable easier translocation. When free mitochondria or mitochondria-filled stem cells are floating, the cells signal their need and / or phagocytose the mitochondria. It is also possible to adjust the timing, frequency, and duration to take up mitochondria at a higher level to ensure a continuous supply of a large amount of mitochondria in the bloodstream for rapid availability. Direct injection of mitochondrial substances into organs via an arterial shunt can also be employed to accurately place them. For example, the hepatic artery can be used to concentrate mitochondria in the liver to preferentially regenerate liver tissue. By doing this appropriately, processes where mitochondria may be diluted or reverse flow, such as clonal expansion, can be omitted.

[0067] Diathermy and exercise by the subject may also promote the uptake of mitochondria by cells. Exercise causes skeletal muscle to create more mitochondria. This is expected to cause cells to receive more transplants. Research suggests that this effect can also be induced by heating muscle with RF radio energy or ultrasound for 2 - 4 hours per session.

[0068] Calorie restriction / fasting by the subject may also promote the uptake of mitochondria. Tests have shown that fasting clearly causes changes, fission / fusion, or mitophagy of mitochondria. Perhaps by repeating fasting with infusions several times, cells are induced to receive some extra mitochondria and share new mtDNA with the mitochondrial network more rapidly.

[0069] Other techniques may also be employed to enhance mitochondrial uptake. For example, partial poisoning, chemotherapy, or hypoxia that induces autophagy, followed by multiple repetitions of infusion, may also be employed. In addition, metformin, melatonin, or other drugs are thought to stimulate mitochondrial uptake by cells. By using drugs to shut down the ability of mitochondria to regenerate themselves through cloning, cells can be forced to rely solely on infused mitochondria.

[0070] Some embodiments relate to a method of increasing cellular respiration. The method includes transducing a cell with an isolated PEV based on the methods described herein, and generating ATP from the PEV.

[0071] The isolated PEV contains functional mitochondria. In some embodiments, the term "functional mitochondria" refers to mitochondria that consume oxygen. In another embodiment, the functional mitochondria have an intact outer membrane. Other embodiments include mitochondrial fragments, mitochondrial DNA, or segments thereof, and mRNA encoding mitochondrial gene products. In some embodiments, the functional mitochondria are intact mitochondria. In another embodiment, the functional mitochondria consume oxygen at an increased rate over a long period of time. In another embodiment, mitochondrial functionality is measured by oxygen consumption. In another embodiment, the oxygen consumption of mitochondria can be measured by any method known in the art. According to some embodiments, the functional mitochondria are mitochondria that exhibit an increased rate of oxygen consumption in the presence of substrates such as ADP and glutamate, malate, or succinate, but not limited thereto. Each possibility represents an individual embodiment of the present invention. In another embodiment, the functional mitochondria are mitochondria that produce ATP. In another embodiment, the functional mitochondria are mitochondria that have the ability to produce their own RNA and proteins and are self-replicating structures. In another embodiment, the functional mitochondria produce mitochondrial ribosomes and mitochondrial tRNA molecules.

[0072] As used herein, the term "intact mitochondria" refers to mitochondria that include an outer membrane and an inner membrane, an intermembrane space, cristae (formed by the inner membrane), and a matrix. In another embodiment, the intact mitochondria contain mitochondrial DNA. In another embodiment, the intact mitochondria contain active respiratory chain complexes I-V embedded in the inner membrane. In another embodiment, the intact mitochondria consume oxygen.

[0073] According to another embodiment, the integrity of the mitochondrial membrane can be determined by any method known in the art. In a non-limiting example, the integrity of the mitochondrial membrane is measured using a tetramethylrhodamine methyl ester (TMRM) or tetramethylrhodamine ethyl ester (TMRE) fluorescent probe. Each possibility represents an individual embodiment of the present invention. Mitochondria observed under a microscope and showing TMRM or TMRE staining have an intact outer mitochondrial membrane.

[0074] As used herein, the term "mitochondrial membrane" refers to a mitochondrial membrane selected from the group consisting of the inner mitochondrial membrane, the outer mitochondrial membrane, or a combination thereof.

[0075] In some embodiments, the functional mitochondria are partially purified mitochondria. As used herein, the term "partially purified mitochondria" refers to mitochondria separated from other cellular components, where the weight of the mitochondria, as exemplified by Hartwig et al., Proteomics, 2009, (9):3209-3214 (the disclosure of which is incorporated herein by reference), accounts for 20-80%, preferably 30-80%, most preferably 40-70% of the combined weight of the mitochondria and other subcellular fractions. Each possibility represents an individual embodiment of the present invention.

[0076] According to another embodiment, the partially purified mitochondria do not contain intact cells. According to another embodiment, the composition of the present invention does not contain intact cells. According to another embodiment, the composition of the present invention does not contain mitochondrial aggregates or agglomerates, or cell debris, or components larger than 5 μm. Each possibility represents an individual embodiment of the present invention. According to another embodiment, the composition of the present invention lacks particulate matter greater than 5 μm. As used herein, the term "particulate matter" refers to intact cells, cell debris, aggregates of mitochondria, aggregates of cell debris, or combinations thereof. Each possibility represents an individual embodiment of the present invention. As used herein, a composition lacking exogenous particulate matter greater than 5 μm has a concentration of particulate matter greater than 5 μm of 1 μM or less, preferably less than 0.5 μM, and most preferably less than 0.1 μM.

[0077] According to some embodiments, intact cells, cell debris, or aggregates are removed from the composition of the present invention. According to some embodiments, as exemplified below herein, the composition of the present invention is filtered through a filter with a pore size of 5 μm or less to remove any intact cells, cell debris, or aggregates. Without being bound by any theory or mechanism, based on the methods of the present invention, the use of a composition containing mitochondrial aggregates can reduce efficacy and even be harmful to the subject. According to another embodiment, the composition of the present invention does not contain liposomes or any other particulate carriers. Each possibility represents an individual embodiment of the present invention.

[0078] According to another embodiment, the weight of the mitochondria in the partially purified mitochondria accounts for at least 20% of the combined weight of the mitochondria and other subcellular fractions. According to another embodiment, the weight of the mitochondria in the partially purified mitochondria accounts for 20% to 40% of the combined weight of the mitochondria and other subcellular fractions. According to another embodiment, the weight of the mitochondria in the partially purified mitochondria accounts for 40% to 80% of the combined weight of the mitochondria and other subcellular fractions. According to another embodiment, the weight of the mitochondria in the partially purified mitochondria accounts for 30% to 70% of the combined weight of the mitochondria and other subcellular fractions. According to another embodiment, the weight of the mitochondria in the partially purified mitochondria accounts for 50% to 70% of the combined weight of the mitochondria and other subcellular fractions. According to another embodiment, the weight of the mitochondria in the partially purified mitochondria accounts for 60% to 70% of the combined weight of the mitochondria and other subcellular fractions. According to another embodiment, the weight of the mitochondria in the partially purified mitochondria accounts for less than 80% of the combined weight of the mitochondria and other subcellular fractions.

[0079] According to another embodiment, there is provided a method of treating a condition or its symptoms in a subject having the condition, the method comprising obtaining a mitorette comprising platelet-derived extracellular vesicles (PEVs) containing mitochondria, and administering an effective amount of the mitorette into the subject, thereby treating the condition or its symptoms. In some embodiments, the condition includes cardiogenic shock. In some embodiments, the condition includes sepsis. In some embodiments, the condition includes a disease caused by a virus. In some embodiments, the virus includes a coronavirus. In some embodiments, the coronavirus includes SARS-CoV-2. In some embodiments, the disease is COVID-19. In some embodiments, the condition includes cardiogenic shock, sepsis, and a disease caused by a virus. In some embodiments, the disease is COVID-19. In some embodiments, the disease is post-COVID-19. In some embodiments, COVID-19 precedes cardiogenic shock, and cardiogenic shock precedes sepsis.

[0080] Similar to other embodiments, the PEVs are collected by 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, stimulating the collected PRP, thereby excluding extracellular vesicles from the platelets in the PRP, and collecting the extracellular vesicles as PEVs. In some embodiments, the PEVs are collected at a site different from the site where the treatment is performed.

[0081] In some embodiments, the administering step includes injecting an effective amount of mitret into the subject to treat the condition. In some embodiments, the injecting step includes a local injection. In some embodiments, the local injection includes an intracardiac injection. In some embodiments, the injecting step includes an injection via an intramyocardial infusion catheter. In some embodiments, the injecting step includes a systemic injection. In some embodiments, the injection includes an enteral injection. In some embodiments, the injecting step includes a parenteral injection. In some embodiments, the injecting step includes an intravenous injection.

[0082] Similar to other embodiments, the collected PRP is stimulated by immune complexes in the presence of Ca2+. In some embodiments, the immune complexes include heat-aggregated IgG. In some embodiments, the collected PRP is stimulated by freeze-thaw cycles. In some embodiments, the concentration of heat-aggregated IgG is from about 0.1 mg / mL to about 2.5 mg / mL, and the concentration of Ca2+ is from about 1 mM to about 25 mM. In some embodiments, the anticoagulant is citrate dextrose (ACD) of the anticoagulant. In some embodiments, the buffer is Tyrode's buffer at about pH 6 to about pH 7. In some embodiments, the separating step is performed by a centrifuge.

[0083] In some embodiments, the blood has been stored for 4 days or more. In some embodiments, the blood has been stored for up to 1 year. In some embodiments, mitret is frozen during storage. In some embodiments, the frozen mitret is stored in combination with a cryoprotective substance. In some embodiments, the cryoprotective substance is selected from the group consisting of saccharides, oligosaccharides, and polysaccharides.

[0084] In some embodiments, during and / or after the step of administering mitoret to a subject, mitoret contacts at least one cell of the subject. In some embodiments, after mitoret contacts the cell, mitoret translocates intracellularly. In some embodiments, the effective amount corresponds to the amount of mitoret that has translocated intracellularly and ranges from about 3 mitorets / cell to about 100 mitorets / cell.

[0085] According to another embodiment, as shown in FIG. 7, in a subject having a condition, a method 300 for treating the condition or its symptoms, comprising: obtaining, in step 302, a PEV containing mitochondria from a source; suspending the PEV in a buffer for storing the PEV in step 308; and administering, in step 312, an effective amount of the PEV into the subject, thereby treating the condition or its symptoms. In some embodiments, the source obtained in step 302 comprises cells selected from the group consisting of placental stem cells, umbilical cord stem cells, adipose tissue-derived stem cells, hepatocytes, blood cells, stem cells, or any cells obtained from a donor. In some embodiments, the source comprises a tissue selected from the group consisting of liver, bone marrow, placenta, adipose tissue, or any tissue obtained from a donor.

[0086] In some embodiments, the obtaining step comprises growing the source in a bioreactor in step 304 and isolating the PEV from the source grown in the bioreactor in step 306. In some embodiments, the obtaining step further comprises coating the PEV in step 310 after the isolating step 306. In some embodiments, the PEV is collected at a site different from the site where the treatment is performed.

[0087] In some embodiments, the condition includes cardiogenic shock. In some embodiments, the condition includes sepsis. In some embodiments, the condition includes a disease caused by a virus. In some embodiments, the virus includes a coronavirus. In some embodiments, the coronavirus includes SARS-CoV-2. In some embodiments, the disease is COVID-19. In some embodiments, the disease is post-COVID-19. In some embodiments, the condition includes cardiogenic shock, sepsis, and a disease caused by a virus. In some embodiments, the disease is COVID-19. In some embodiments, COVID-19 precedes cardiogenic shock, and cardiogenic shock precedes sepsis. In some embodiments, the administering step includes injecting an effective amount of mitoret into the subject to treat the condition.

[0088] In some embodiments, the buffer includes a cryoprotective substance. In some embodiments, the cryoprotective substance is selected from the group consisting of saccharides, oligosaccharides, and polysaccharides. In some embodiments, the buffer includes a hydrogel. In some embodiments, the hydrogel has temperature-dependent hydrophilicity and hydrophobicity.

[0089] In some embodiments, a mitoret is provided, the mitoret comprising platelet-derived extracellular vesicles (PEVs) containing chondria for use in treating cardiogenic shock and / or sepsis, or symptoms thereof, the PEVs being collected by a process comprising 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, stimulating the collected PRP, thereby excluding extracellular vesicles from platelets within the PRP, and collecting the extracellular vesicles as PEVs. In some embodiments, the PEVs are collected at a site different from the site at which the treatment is performed. In some embodiments, the mitoret is used in treating cardiogenic shock. In some embodiments, the mitoret is used in treating sepsis. In some embodiments, cardiogenic shock and / or sepsis is caused by a virus. In some embodiments, the virus includes a coronavirus. In some embodiments, the coronavirus includes severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In some embodiments, the PEVs are used in co-treating cardiogenic shock, sepsis, and a disease caused by a virus. In some embodiments, the disease is COVID-19.

[0090] In some embodiments, the collected PRP is stimulated with immune complexes in the presence of Ca2+. In some embodiments, the immune complexes include heat-aggregated IgG. In some embodiments, the collected PRP is stimulated by freeze-thaw cycles. In some embodiments, the concentration of heat-aggregated IgG is from about 0.1 mg / mL to about 2.5 mg / mL, and the concentration of Ca2+ is from about 1 mM to about 25 mM. In some embodiments, the anticoagulant is anticoagulant citrate dextrose (ACD). In some embodiments, the buffer is Tyrode's buffer at about pH 6 to about pH 7. In some embodiments, the separating step is performed by a centrifuge.

[0091] In some embodiments, the blood has been stored for more than 4 days. In some embodiments, the blood has been stored for up to 1 year. In some embodiments, the effective amount of mitoret is in the range of about 3 mitorets / cell to about 100 mitorets / cell. In some embodiments, the mitoret is frozen during storage. In some embodiments, the frozen mitoret is stored in combination with a cryoprotectant. In some embodiments, the cryoprotectant is selected from the group consisting of saccharides, oligosaccharides, and polysaccharides.

[0092] In some embodiments, the PEV is provided as shown in FIG. 8, the PEV contains mitochondria for use in the treatment of cardiogenic shock and / or sepsis, or symptoms thereof, and in step 408, the PEV is suspended in a buffer for storing the PEV, and in step 406, it is isolated from the source cells grown in a bioreactor in step 404, and the source cells obtained in step 402 are selected from the group consisting of placental stem cells, umbilical cord stem cells, adipose tissue-derived stem cells, hepatocytes, blood cells, bone marrow, and induced pluripotent stem cells. In some embodiments, the PEV is coated in step 410. In some embodiments, as shown in step 412, an effective amount of the PEV is administered into a subject. In some embodiments, the PEV is used in treating cardiogenic shock. In some embodiments, the PEV is used in treating sepsis. In some embodiments, cardiogenic shock and / or sepsis is caused by a virus. In some embodiments, the virus includes a coronavirus. In some embodiments, the coronavirus includes severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In some embodiments, the PEV is used in co-treating cardiogenic shock, sepsis, and diseases caused by a virus.

[0093] In some embodiments, the disease is COVID-19. In some embodiments, the buffer contains a cryoprotectant. In some embodiments, the cryoprotectant is selected from the group consisting of saccharides, oligosaccharides, and polysaccharides. In some embodiments, the buffer contains a hydrogel. In some embodiments, the hydrogel has temperature-dependent hydrophilicity and hydrophobicity.

[0094] The following examples are presented to enable a more complete understanding of the present invention. The specific techniques, conditions, materials, ratios, and reported data described to illustrate the principles of the present invention are exemplary and should not be construed as limiting the scope of the present invention.

Example

[0095] This example illustrates that PEV is collected from the mouse blood described in some embodiments.

[0096] The following steps were performed:

[0097] 1. Blood was collected from a mouse donor. In this particular protocol, the donor was a male DsRed mouse (a transgenic mouse expressing the red fluorescent protein variant DsRed). MST under the control of the chicken β-actin promoter was ligated to the cytomegalovirus (CMV) immediate early enhancer. Here, 3 × 1 mL (1 mL / mouse) of blood was used.

[0098] 2. ACD (20%) was added as an anticoagulant, and 40% Tyrode's buffer (pH 6.5) was also added to the blood. The blood mixture (20% ACD + 40% Tyrode's buffer (TB) (pH 6.5)) was then centrifuged at 500 g for 3 minutes. PRP and the buffy coat were then collected and centrifuged at 300 g for 2 minutes.

[0099] 3. PRP was collected and after adding 20% ACD + 10 mM EDTA, a centrifugation step was performed at 1300 g for 5 minutes.

[0100] 4. Each pellet was suspended in 0.1 mL of TB (pH 6.5), and 0.9 mL of TB (pH 7.4) was added.

[0101] 5. Platelets were pooled, counted using a cellometer, and diluted to 10e8 / mL in TB 7.4.

[0102] 6. A total of 900 million platelets were obtained and 5 mM CaCl 2 was added before stimulation.

[0103] 7. Platelets were stimulated overnight (16 hours) at room temperature with 0.5 mg / mL heat-aggregated IgG. Heat-aggregated IgG was prepared by aggregating human IgG (25 mg / mL, MPBIO) at 62°C for 1 hour.

[0104] 8. Stimulation was stopped by adding 10 mM EDTA.

[0105] 9. The stimulated platelets were centrifuged at 300 g for 5 minutes to remove residual platelets or cells.

[0106] 10. The supernatant was collected and PEV was analyzed using a flow cytometer.

[0107] 11. The remaining platelets were evaluated and the contamination was less than 1%.

[0108] 12. The obtained PEV was diluted three times with PBS and centrifuged at 18°C, 18000 g for 90 minutes.

[0109] 13. The pellet was resuspended in 0.3 mL of PBS and PEV was counted by flow cytometry. The concentration was estimated to be 1.5×10e9 PEV / ml.

[0110] 14. By tagging the PEV with the CD41 tag, the PEV can be counted in a flow cytometer. When tagged in this way, the PEV accounts for approximately 40% of the whole (CD41 + PEV). A dot plot representing the PEV population is illustrated in Figure 9. (DsRed = PEV).

Example

[0111] This example illustrates that the PEV can be internalized by the retinal cells described in some embodiments.

[0112] 1. Immortalized mouse retinal pigment epithelial cells (RPEC) and brain endothelial cells (bEND) were seeded overnight before PEV incubation (approximately 20,000 cells / well (RPEC) and 16,000 cells / well (bEND)).

[0113] 2. The PEV was collected from mouse donors according to the procedure outlined in Example 1.

[0114] 3. In Prigrow III supplemented with 1% penicillin - streptomycin (pH 7.4) and 1%, 5%, or 10% FBS (non - heat - inactivated), the RPEC was pre - incubated for either 3, 18, 24, or 36 hours with or without PEV (approximately 3, 10, 30, or 100 mitochondria + PEV per cell).

[0115] 4. In DMEM supplemented with 1% penicillin - streptomycin (pH 7.4) and 1%, 5%, or 10% FBS (non - heat - inactivated), the bEND was pre - incubated for 24 hours with or without PEV (approximately 3, 10, 30, or 100 mitochondria + PEV per cell).

[0116] 5. The RPEC and bEND were washed and then introduced into XF medium supplemented with 2 mM glutamine, 1 mM pyruvate, and 8 mM D - glucose, and 1% FBS (pH 7.4).

[0117] 6. After washing, RPEC and bEND in XF medium were centrifuged at 37 °C and 300 g for 5 minutes and 60 minutes without including CO 2 .

[0118] Figures 10A - 10B are confocal images showing the stained nuclei 502 [DAPI (4',6 - diamidino - 2 - phenylindole)], and the cell membranes 504 of RPEC 500, and PEV 510 (DsRed). As shown in Figures 10A - 10B (40× and 20× magnifications respectively), most of the PEV 510 is internalized by RPEC 500. As shown in Figure 11, this internalization can be stable for well over 24 hours.

[0119] To verify mitochondrial internalization in RPEC 600, as shown in Figure 12, X - Z and Y - Z scans of the PEV - derived fluorescently labeled mitochondria 610 (presented here in orange) were performed while using a confocal microscope. Here, the X - Z plane is perpendicular to the Y - Z plane. The X - Z and Y - Z scans show how the highest intensity from the point source of the fluorescently labeled mitochondria (see arrowheads) is positioned within RPEC 600. The nucleus 502 of RPEC600 was stained with DAPI. As shown in Figure 12, the cell membrane 604 of RPEC600 was stained with CellMask™. The results of this internalization are shown in Figure 13A. Figure 13B shows that bEND also internalizes mitochondria delivered by PEV. Therefore, these results demonstrate that PEV is capable of delivering durable mitochondria into RPEC and bEND.

[0120] To evaluate the activity of mitochondria delivered by PEV, a Seahorse XF assay (testing for mitochondrial stress) was performed on RPECs pre-incubated with PEV for 24 hours. While using 5% FBS, which is considered a preferable level for the serum used (higher serum levels such as 10% FBS may induce cell division), the oxygen consumption rate (OCR) was measured for RPECs (PEV0, 3, 10, 30, or 100 particles / cell) between different pre-incubation parameters as presented in step 3 of this example.

[0121] Regarding the OCR readings in FIGS. 14A - 14D, FIG. 14A shows similar (if not slightly lower) basal respiration levels between RPECs pre-incubated with PEV and those not (control). Basal respiration level refers to the energy requirement of RPECs under baseline conditions. FIG. 14B shows an increase in the spare respiratory capacity of RPECs when pre-incubated with PEV, suggesting an improvement in the ability of RPECs to respond to energy requirements (i.e., an improvement in cell adaptability or flexibility). FIG. 14C is thought to show that in some RPECs pre-incubated with PEV, ATP production is slightly enhanced. FIG. 14D shows that proton leak is not a problem for RPECs that internalized PEV compared to those that did not.

Example

[0122] The protocol for the pre - clinical trial of sepsis is summarized in Table 1 below. The subjects used in this specification for this example are mice. Sepsis is induced in the abdomen of mice not belonging to the sham group. Treatment groups 1 and 2 receive an infusion of 120 μL of mitret through intravenous injection (IV) at doses of 1X and 5X, respectively. Treatment groups 3 and 4 receive an infusion of 120 μL of isolated mitochondria from liver tissue through IV at doses of 1Y and 5Y, respectively. The doses X and Y in this experiment may be the same or different. The positive and negative controls receive infusions of an antibiotic cocktail and saline, respectively. In the mice within the sham group, sepsis is not induced and no reagent administration is received.

[0123] The frequency of the IV infusion is daily for 3 consecutive days (12 hours after sepsis induction, day 1, and day 2). The endpoints include a cytokine panel that measures cytokine levels over time for each group, the mortality rate over time for each group, and a muscle strength decline chart. The readings of the endpoints include the baseline (day 0, 12 hours after sepsis induction, day 1, day 3, day 7, day 10, and day 14).

[0124]

Table 1

Example

[0125] Based on several criteria, patients 18 years of age or older are included in the study. The included patients are diagnosed with acute ST - elevation myocardial infarction (STEMI) that includes clinical symptoms of elevated troponin and electrocardiogram abnormalities consistent with acute STEMI. Furthermore, the included patients show measurement criteria suggesting cardiogenic shock (the criteria include: (1) SBP < 90 mmHg, low SVC O 2 saturation (< 70%); and (2) elevated lactate and pulmonary congestion, or elevated CVP exceeding 12 mmHg). In addition, the patients within this study have moderate to severe left ventricular systolic dysfunction defined as a left ventricular ejection fraction (LVEF) < 35% when measured by echocardiogram.

[0126] Patients to be excluded from the trial include those diagnosed with severe chronic obstructive pulmonary disease (COPD) with FEV1 < 1 L and FEV1 / FVC < 70% (FEV1: Forced expiratory volume in 1 s; FVC: Forced Vital Capacity). Other exclusion criteria include a history of organ transplantation; active malignancy (excluding local skin cancer); having a progressive cardiogenic shock state with multiple organ failure (persistent lactate level elevation exceeding 2, oliguria or anuria, mechanical ventilation, or an increase in abnormal liver function test values (LFT) exceeding 3 times the normal limit), and pregnant women.

[0127] The trial design (the baseline echocardiogram (ECG) of the included patients is measured and blood is drawn for the first measurement) is shown in Figure 16. These patients are randomized to receive either mitret injection or placebo injection. The administration of the injection can be local or systemic. Blood samples are taken on the 2nd, 5th, 10th, and 30th days after injection and used to measure the intensity of the inflammatory response associated with cardiogenic shock. Follow-up ECGs are performed on the 10th and 30th days. Markers, such as IL-1 (superfamily), IL-6, IL-8, TNF-α, C-reactive protein (CRP), soluble adhesion molecules, complement system, etc., can be used to identify the inflammatory response.

[0128] The primary endpoints include the change from baseline and side effects of the inflammatory cytokine panel during the first 10 days after infusion of mitret. The secondary endpoints include the survival rate at discharge, the survival rate at 90 days, the change in left ventricular function on the 10th day (or at discharge if discharged before the 10th day) and 30th day, and any signs of major adverse cardiac events (MACE).

[0129] In FIG. 17, the intensity of the inflammatory response is measured based on a cytokine panel measured between a randomized group administered mitret and a randomized group administered placebo. In this example, the administration routes of mitret and placebo are the same, both being IV. Although IV administration was used in this example, other administration routes are possible, including, among others, intra-arterial administration, intraspinal administration, intracerebroventricular administration, intraperitoneal administration, and intraosseous administration. As shown, on the 4th day after the diagnosis of cardiogenic shock, the mitret group shows a dramatic decrease in the intensity of the inflammatory response compared to the placebo group. Also, on the 10th day after the diagnosis of cardiogenic shock, the difference in the intensity of the inflammatory response between the two groups further widens, indicating that the intensity of the inflammatory response in the mitret group has decreased to less than 1 / 3 of that in the placebo group.

[0130] Therefore, these results indicate the effectiveness of early and aggressive mitret treatment, that is, the reduction in the incidence probability of cytokine storm, in reducing the intensity of the inflammatory response caused by cardiogenic shock. Therefore, as shown in FIG. 18, by performing early and aggressive clinical treatment based on mitret, the probability of sepsis and sepsis-related death caused by cardiogenic shock can be significantly reduced.

[0131] The described embodiments and examples of the present disclosure are not restrictive but are for illustrative purposes and are not intended to represent any and all embodiments or examples of the present disclosure. Accordingly, the scope is not limited by the specific embodiments or examples described herein. Although the basic novel features of the present disclosure are presented, described, and emphasized as applied to various specific embodiments, various omissions, substitutions, and changes may become apparent in the details of the disclosed methods, and it will be understood that these may be made by those skilled in the art without departing from the spirit of the present disclosure. For example, all combinations of method steps that perform substantially the same function in substantially the same way to achieve the same result are expressly intended to be within the scope of the present disclosure. In addition, it should be recognized that method steps presented or described in connection with any form or embodiment disclosed in the present disclosure may be incorporated into any other disclosed or described or suggested form or embodiment as a general matter of design choice. Further, various modifications and variations can be made without departing from the spirit or scope of the present disclosure as set forth in the following claims, and these can be made legally as literal and legal equivalents.

Explanation of Signs

[0132] 100 Method 102 Step 104 Step 106 Step 108 Step 110 Step 112 Step 114 Step 200 Method 202 Step 204 Step 206 Step 208 Step 210 Step 212 Step 214 Step 300 Method 302 Step 304 Step 306 Step 308 Step Project 310 Project 312 Method 400 Project 402 Project 404 Project 406 Project 408 Project 410 Project 412 500 RPEC Stained nucleus Cell membrane 510 PEV 600 RPEC Cell membrane Fluorescently labeled mitochondria

Claims

1. A method for treating cardiogenic shock and / or sepsis, or symptoms thereof, in a subject having cardiogenic shock and / or sepsis, comprising: obtaining a mitorette comprising platelet-derived extracellular vesicles (PEVs) containing mitochondria, wherein the PEVs are obtained by: 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; stimulating the collected PRP to thereby expel extracellular vesicles from the platelets within the PRP; collecting the extracellular vesicles as the PEVs; and obtaining the mitorette by; administering an effective amount of the mitorette into the subject to thereby treat the cardiogenic shock and / or sepsis, or symptoms thereof. A method comprising the steps.

2. The method according to claim 1, wherein the PEVs are collected at a site different from the site at which the treatment is to be performed.

3. The method according to claim 1 or 2, wherein the subject has cardiogenic shock.

4. The method according to any one of claims 1 to 3, wherein the subject has sepsis.

5. The method according to any one of claims 1 to 4, wherein the cardiogenic shock and / or sepsis is caused by a virus.

6. The method according to claim 5, wherein the virus includes a coronavirus.

7. The method according to claim 6, wherein the coronavirus includes severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

8. The method according to claim 1 or 2, wherein the subject has cardiogenic shock, sepsis, and a disease caused by a virus.

9. The method according to claim 8, wherein the disease is COVID-19.

10. The method according to claim 9, wherein the COVID-19 precedes the cardiogenic shock, and the cardiogenic shock precedes the sepsis.

11. The method according to any one of claims 1 to 10, wherein the administering step includes injecting the effective amount of the mitorette into the subject to treat the condition.

12. The collected PRP is stimulated with immune complexes in the presence of Ca 2+ The method according to any one of claims 1 to 11, wherein the method is stimulated with immune complexes in the presence of Ca

13. The method according to claim 12, wherein the immune complex includes heat-aggregated IgG.

14. The method according to any one of claims 1 to 11, wherein the collected PRP is stimulated by freeze-thaw cycles.

15. The concentration of the heat-aggregated IgG is from about 0.1 mg / mL to about 2.5 mg / mL, and the concentration of the Ca 2+ is from about 1 mM to about 25 mM, the method according to claim 13.

16. The method according to any one of claims 1 to 15, wherein the anticoagulant is citrate dextrose (ACD) as an anticoagulant.

17. The method according to any one of claims 1 to 16, wherein the buffer is Tyrode's buffer at about pH 6 to about pH 7.

18. The method according to any one of claims 1 to 17, wherein the separating step is carried out by centrifugation.

19. The method according to any one of claims 1 to 18, wherein the blood has been stored for 4 days or more.

20. The method according to claim 19, wherein the blood has been stored for up to 1 year.

21. The method according to any one of claims 1 to 20, wherein the mitorette contacts at least one cell of the subject during and / or after the step of administering the mitorette into the subject.

22. The method according to claim 21, wherein the mitorette translocates intracellularly after contacting the cell.

23. The method according to claim 22, wherein the effective amount corresponds to the amount of translocated mitorette in the range of about 3 mitorettes / cell to about 100 mitorettes / cell.

24. The method according to claim 19 or 20, wherein the mitorette is frozen during storage.

25. The method according to claim 24, wherein the frozen mitorette is stored in combination with a cryoprotectant.

26. The method according to claim 25, wherein the cryoprotectant is selected from the group consisting of saccharides, oligosaccharides, and polysaccharides.

27. A method for treating cardiogenic shock and / or sepsis, or symptoms thereof, in a subject having cardiogenic shock and / or sepsis, comprising: administering an effective amount of PEV containing mitochondria into the subject to thereby treat the condition or its symptoms, wherein the PEV is obtained by a method comprising: obtaining donor cells selected from the group consisting of placental stem cells, umbilical cord stem cells, adipose tissue-derived stem cells, hepatocytes, blood cells, bone marrow, and induced pluripotent stem cells, wherein the PEV contains mitochondria; growing the donor cells in a bioreactor; isolating the PEV from the bioreactor; and suspending the isolated PEV in a buffer for storing the PEV obtained by the method. Method.

28. The method according to claim 27, wherein the PEV is coated.

29. The method according to claim 27 or 28, wherein the PEV is collected at a site different from the site where the treatment is performed.

30. The method according to any one of claims 27 to 29, wherein the subject has cardiogenic shock.

31. The method according to any one of claims 27 to 29, wherein the subject has sepsis.

32. The method according to any one of claims 27 to 31, wherein the cardiogenic shock or sepsis is caused by a virus.

33. The method according to claim 32, wherein the virus includes a coronavirus.

34. The method according to claim 33, wherein the coronavirus includes SARS-CoV-2.

35. The method according to any one of claims 27 to 34, wherein the subject has cardiogenic shock, sepsis, and a disease caused by a virus.

36. The method according to claim 35, wherein the disease is COVID-19.

37. The method according to claim 36, wherein the COVID-19 precedes the cardiogenic shock, and the cardiogenic shock precedes the sepsis.

38. The method according to any one of claims 27 to 37, wherein the step of administering includes injecting the effective amount of mitoret into the subject to treat the condition.

39. The method according to any one of claims 27 to 38, wherein the buffer includes a cryoprotective substance.

40. The method according to claim 39, wherein the cryoprotective substance is selected from the group consisting of saccharides, oligosaccharides, and polysaccharides.

41. The method according to any one of claims 27 to 40, wherein the buffer includes a hydrogel.

42. The method according to claim 41, wherein the hydrogel has temperature-dependent hydrophilicity and hydrophobicity.

43. A mitoret containing platelet-derived extracellular vesicles (PEV) containing mitochondria for use in the treatment of cardiogenic shock and / or sepsis, or symptoms thereof, wherein the PEV is a step 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; Stimulating the collected PRP to thereby eliminate extracellular vesicles from platelets within the PRP; Collecting the extracellular vesicles as the PEV; The mitorette collected thereby.

44. The mitorette according to claim 43, wherein the PEV is collected at a site different from the site where the treatment is performed.

45. The mitorette according to claim 43 or 44, for use in the treatment of cardiogenic shock.

46. The mitorette according to claim 43 or 44, for use in the treatment of sepsis.

47. The mitorette according to any one of claims 43 to 46, wherein the cardiogenic shock and / or sepsis is caused by a virus.

48. The mitorette according to claim 47, wherein the virus includes a coronavirus.

49. The mitorette according to claim 48, wherein the coronavirus includes severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

50. The mitorette according to any one of claims 43 to 49, for use in the simultaneous treatment of cardiogenic shock, sepsis, and diseases caused by a virus.

51. The mitorette according to claim 50, wherein the disease is COVID-19.

52. The collected PRP is stimulated with immune complexes in the presence of Ca 2+ The mitorette according to any one of claims 43 to 51, wherein the mitorette is stimulated with immune complexes in the presence of 2+ .

53. The mitorette according to claim 52, wherein the immune complex includes heat-aggregated IgG.

54. The mitorette according to any one of claims 43 to 51, wherein the collected PRP is stimulated by freeze-thaw cycles.

55. The concentration of the heat-aggregated IgG is from about 0.1 mg / mL to about 2.5 mg / mL, and the concentration of the Ca 2+ is from about 1 mM to about 25 mM, the mitotret of claim 53.

56. The mitorette according to any one of claims 43 to 55, wherein the anticoagulant is citrate dextrose (ACD) of an anticoagulant.

57. The mitorette according to any one of claims 43 to 56, wherein the buffer is Tyrode's buffer of about pH 6 to about pH 7.

58. The mitorette according to any one of claims 43 to 57, wherein the separating step is performed by a centrifuge.

59. The mitorette according to any one of claims 43 to 58, wherein the blood has been stored for 4 days or more.

60. The mitorette according to claim 59, wherein the blood has been stored for up to 1 year at most.

61. The mitorette according to any one of claims 43 to 60, wherein the effective amount of the mitorette is in the range of about 3 mitorettes / cell to about 100 mitorettes / cell.

62. The mitorex according to any one of claims 43 to 61, which is frozen during storage.

63. The mitorex according to claim 62, wherein the frozen mitorex is stored in combination with a cryoprotective substance.

64. The mitorex according to claim 63, wherein the cryoprotective substance is selected from the group consisting of saccharides, oligosaccharides, and polysaccharides.

65. A mitochondrial-containing PEV for use in the treatment of cardiogenic shock and / or sepsis, or symptoms thereof, wherein the PEV is suspended in a buffer for storing the PEV and isolated from donor cells grown in a bioreactor, and the donor cells are selected from the group consisting of placental stem cells, umbilical cord stem cells, adipose tissue-derived stem cells, hepatocytes, blood cells, bone marrow, and induced pluripotent stem cells.

66. The PEV according to claim 65, which is coated.

67. The PEV according to claim 65 or 66, for use in the treatment of cardiogenic shock.

68. The PEV according to claim 65 or 66, for use in the treatment of sepsis.

69. The PEV according to claim 68, wherein the cardiogenic shock and / or sepsis is caused by a virus.

70. The PEV according to claim 69, wherein the virus includes a coronavirus.

71. The PEV according to claim 70, wherein the coronavirus includes severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

72. The PEV according to any one of claims 68 to 71, for use in the simultaneous treatment of cardiogenic shock, sepsis, and a disease caused by a virus.

73. The PEV according to claim 72, wherein the disease is COVID-19.

74. The PEV according to any one of claims 65 to 73, wherein the buffer contains a cryoprotective substance.

75. The PEV according to claim 74, wherein the cryoprotective substance is selected from the group consisting of saccharides, oligosaccharides, and polysaccharides.

76. The PEV according to any one of claims 65 to 75, wherein the buffer contains a hydrogel.

77. The PEV according to claim 76, wherein the hydrogel has temperature-dependent hydrophilicity and hydrophobicity.

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