Cell-free allogeneic exosome preparation and method of using the same

Abstract

A method of treating or ameliorating symptoms of nerve cell damage in a subject, comprising administering to the subject a therapeutically effective amount of a cell-free allogeneic preparation (UCF or UCF-E) having exosomes, wherein the cell-free allogeneic preparation is derived from pooled human umbilical cord blood, is provided.

Description

Technical Field

[0001] The present technology relates to a cell-free allogeneic exosome preparation for the treatment of nerve cell damage, particularly ischemic brain injury such as stroke or cerebrovascular accident (CVA). The present technology also relates to the use of a cell-free allogeneic exosome (extracellular vesicle: EV) preparation for the manufacture of a medicament for the treatment of nerve cell damage such as stroke and CVA.

[0002] Cross-reference to related applications This application claims the priority of Australian Provisional Patent Application No. 2022902095, filed on July 27, 2022, and the entire disclosure of the provisional patent application is incorporated herein by reference.

Background Art

[0003] Worldwide, stroke is the second leading cause of death and ranks third in terms of disability-adjusted life years lost (DALY), combining deaths and disabilities. The estimated global cost of stroke exceeds $721 billion, which is 0.66% of the world's GDP. From 1990 to 2019, the absolute number of stroke cases increased significantly (the incidence of stroke increased by 70%, the death rate due to stroke increased by 43%, the prevalence of stroke increased by 102.0%, and DALY increased by 143.0%).

[0004] A stroke is a condition in which blood flow to the brain is impaired due to the occlusion or rupture of the arteries leading to the brain, and a part of the brain (nerve) cells suddenly die due to oxygen deficiency. The most common types of stroke are ischemic and hemorrhagic strokes. In ischemic stroke, insufficient oxygen flow to the brain can lead to apoptosis and necrosis of brain tissue, resulting in infarction. Similar to ischemic heart disease, cerebral ischemia can be caused by various factors such as thrombus, thrombosis, embolism, atherosclerotic plaque, or other obstructions in the vascular system. Hypercholesterolemia, hypertension, diabetes, and obesity are risk factors for ischemic stroke. Hemorrhagic (intracerebral hemorrhage) stroke occurs when weakened blood vessels (arteries) rupture and bleed into the surrounding brain. Subsequently, blood accumulates and compresses the surrounding brain tissue, resulting in the loss of normal function or movement of a part of the body controlled by the damaged area. There are mainly two types of hemorrhagic stroke. Intracerebral hemorrhage occurs when bleeding occurs into the brain from blood vessels weakened by aneurysms, and arteriovenous malformation (AVM) and subarachnoid hemorrhage occur when bleeding occurs between the brain and the membranes covering the brain.

[0005] The onset of a stroke, regardless of its cause, results in nerve cell death, particularly at the site of occlusion or hemorrhage. Furthermore, biochemical reactions in the vascular system that occur after the onset of a stroke can lead to edema, hemorrhagic infarction, and further damage to nerve tissue. Neurological damage and nerve cell death caused by a stroke can debilitate the subject physically and mentally. In particular, a stroke can cause problems with emotional control, consciousness, sensory perception, speech, hearing, vision, cognition, movement, mobility, and may also cause paralysis.

[0006] The current treatment for ischemic stroke, tissue plasminogen activator (tPA or PLAT), is effective but only when administered within 3 hours of the onset of ischemia. Therefore, treatment with tPA remains inadequate for patients who cannot be diagnosed and treated within that time. Additionally, tPA is not appropriate for patients suffering from hemorrhagic stroke who require, alternatively, interventional radiology or neurosurgical procedures (to stop bleeding and reduce intracranial pressure), and further, drug treatment to suppress swelling, prevent seizures, and relieve pain. Considering the limitations of tPA and the high mortality rate of stroke patients, there is an obvious and unmet need to reduce mortality, restore neurological function, and widen the current treatment window.

[0007] The present invention is based on the inventors' finding that acellular allogeneic exosome preparations such as human umbilical cord plasma (UCF) containing exosomes or exosomes derived only from human umbilical cord plasma (UCF-E) are useful for the treatment of stroke.

Summary of the Invention

[0008] In a first aspect, a method of treating or ameliorating symptoms of nerve cell damage in a subject, the method comprising administering to the subject, in a therapeutically effective amount, an acellular allogeneic preparation (UCF or UCF-E) comprising exosomes, consisting of acellular human umbilical cord plasma (UCF) or exosomes derived only from human umbilical cord plasma (UCF-E), wherein the acellular allogeneic preparation (UCF or UCF-E) comprising exosomes is derived from pooled human umbilical cord blood, the nerve cell damage is ischemic brain injury, hemorrhagic brain injury, or traumatic brain injury, and UCF or UCF-E is first administered 3 to 90 days after the nerve cell damage, is provided.

[0009] The ischemic brain injury or hemorrhagic brain injury may be a stroke.

[0010] In some embodiments, no immunosuppressant is administered.

[0011] In some embodiments, UCF or UCF-E is administered intrathecally.

[0012] Cell-free human umbilical cord plasma preparation (UCF) and a preparation consisting only of umbilical cord plasma exosomes (UCF-E) are derived from plasma characterized in that the levels of one or more of GM-CSF, HGF, MIP-1b (CCL4) and PDGF-bb are at least 20% higher than the levels in normal adult serum.

[0013] Cell-free human umbilical cord plasma preparation (UCF) and a preparation consisting only of human umbilical cord plasma exosomes (UCF-E) are derived from plasma characterized in that the level of IP-10 (CxCL10) is at least 60% of the level in normal adult serum.

[0014] Cell-free allogeneic umbilical cord preparations containing exosomes (UCF and UCF-E) may contain exosomes having an average, median or mode particle diameter of about 30 - 150 nm or about 75 - 95 nm.

[0015] In one embodiment, a cell-free allogeneic umbilical cord plasma (UCF) containing exosomes or a preparation of umbilical cord exosomes only (UCF-E) is first administered 3 - 30 days after neuronal injury, preferably 3 - 10 days after neuronal injury, more preferably 7 or 8 days after neuronal injury.

[0016] In some embodiments, a cell-free allogeneic umbilical cord plasma (UCF) containing exosomes or a preparation of umbilical cord exosomes only (UCF-E) is first administered 7 - 10 days after neuronal injury.

[0017] In some embodiments, the acellular allogeneic UCF or UCF-E preparation is administered to a subject periodically. For example, the acellular allogeneic UCF or UCF-E preparation may be administered weekly, every two weeks, every three weeks, monthly, or combinations thereof. Alternatively, the dosing schedule may be daily, every five days, every ten days, every fifteen days, every twenty days, every twenty-five days, about every thirty days, or combinations thereof.

[0018] The method may include administering the acellular allogeneic UCF or UCF-E preparation a total of two, three, four, five, six, seven, eight, nine, or ten times.

[0019] The symptoms may be selected from dystonia, aphasia, infarct size, inflammation, white matter loss, gray matter loss, apoptotic cells in the peri-infarct region, and CD16 / CD32 positive cells in the peri-infarct region.

[0020] Administration of the acellular allogeneic UCF or UCF-E preparation may reduce any one or more of dystonia, aphasia, infarct size, systemic inflammation, infarct or inflammation in the peri-infarct region, and gray matter and / or white matter loss in the subject.

[0021] Administration of the acellular allogeneic UCF or UCF-E preparation may reduce either or both the number of apoptotic cells in the peri-infarct region of the subject and the number of CD16 / CD32 positive cells in the peri-infarct region of the subject.

[0022] The method may further include administering an additional therapeutic agent such as an anti-inflammatory agent, steroid, dipyridamole, astaxanthin, dabigatran, losartan, nimodipine, policosanol, rivaroxaban, or ticlopidine, together with the acellular allogeneic UCF or UCF-E preparation.

[0023] In a second aspect, a method of improving the mRS score or NIHSS score of a subject having ischemic brain injury, the method comprising administering to the subject a therapeutically effective amount of a cell-free allogeneic UCF or UCF-E preparation, the cell-free allogeneic UCF and UCF-E preparations being derived from pooled human umbilical cord blood, and UCF or UCF-E being first administered 3 to 90 days after ischemic brain injury, a method is provided.

[0024] In one embodiment, an immunosuppressant is not administered.

[0025] The cell-free allogeneic human umbilical cord blood plasma used to isolate UCF or UCF-E may be characterized in that the level of IP-10 (CxCL10) is at least 60% of the level in the serum of normal adults.

[0026] The cell-free allogeneic exosome preparation may comprise exosomes having an average, median or mode particle diameter of about 30 - 150 nm.

[0027] In one embodiment, the cell-free allogeneic plasma (UCF) containing exosomes or a preparation of exosomes only (UCF-E) is first administered 3 to 30 days after neuronal injury, preferably 3 to 10 days after neuronal injury, more preferably 7 or 8 days after neuronal injury.

[0028] In some embodiments, the cell-free allogeneic UCF or UCF-E is first administered 7 to 10 days after ischemic brain injury.

[0029] In some embodiments, preparations of cell-free allogeneic plasma (UCF) containing exosomes or exosomes only (UCF-E) are administered periodically to a subject. For example, the cell-free allogeneic exosome preparation may be administered weekly, every two weeks, every three weeks, monthly, or combinations thereof. Alternatively, the dosing schedule may be daily, every five days, every ten days, every fifteen days, every twenty days, every twenty-five days, approximately every thirty days, or combinations thereof.

[0030] The method may include administering a preparation of cell-free allogeneic plasma (UCF) containing exosomes or exosomes only (UCF-E) a total of two, three, four, five, six, seven, eight, nine, or ten times.

[0031] In some embodiments, UCF or UCF-E is derived from the blood of a single umbilical cord or pooled blood from multiple umbilical cords. The pooled blood may be umbilical cord blood of the same blood type or umbilical cord blood of different blood types.

[0032] In a third aspect, use in the manufacture of a medicament for treating or ameliorating symptoms of nerve cell damage of a preparation of cell-free allogeneic plasma (UCF) containing exosomes derived from pooled human umbilical cord blood or exosomes only (UCF-E), wherein the nerve cell damage is ischemic brain injury, hemorrhagic brain injury, or traumatic brain injury, and the medicament is first administered 3 to 90 days after the nerve cell damage, is provided.

[0033] In a fourth aspect, use in the manufacture of a medicament for improving the mRS score or NIHSS score of a subject with ischemic brain injury of a cell-free allogeneic plasma preparation (UCF or UCF-E) containing exosomes derived from pooled human umbilical cord blood, wherein the medicament is first administered 3 to 90 days after the ischemic brain injury, is provided.

[0034] [Definitions] Throughout this specification, unless the context clearly requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0035] Throughout this specification, the term "consist of" means consisting only of.

[0036] The term "consisting essentially of" also means including the stated elements, integers or steps, as well as other elements, integers or steps that do not materially alter or contribute to the practice of the invention.

[0037] Any consideration of documents, acts, materials, devices, articles, etc. described in this specification is for the sole purpose of providing context for the present technology. It should not be construed as an admission that any of these, in whole or in part, form part of the prior art base in the field of the present technology that existed prior to the priority date of each claim of this specification or were common general knowledge.

[0038] In this specification, an integer, step or technical feature described in the specification as a singular integer, step or element clearly encompasses both singular and plural integers, steps or elements, unless the context requires a different interpretation or it is clearly stated otherwise.

[0039] In the context of this specification, the terms "a" and "an" are used to refer to one or more (i.e., at least one) of the grammatical objects of the article. By way of example, the description "an element" means one element or more than one element.

[0040] In the context of this specification, the term "about" means including the range of variation above or below a numerical value or value, in accordance with what would be understood by a person of ordinary skill in the art based on common general knowledge in the art, including within the range of normal error or limitations due to the device, rather than being received as an absolute numerical value or value. In other words, the use of the term "about" is understood to refer to a range or approximation that a person of ordinary skill in the art would consider equivalent to the recited value in a context that provides the same function or result.

[0041] The terms "treating", "treatment", and "therapy" are used herein to refer to curative therapy, preventive therapy, palliative therapy, and prophylactic therapy. That is, in the context of the present disclosure, the term "treating" encompasses curing, ameliorating, or alleviating the severity of ischemic brain injury or one or more symptoms associated therewith.

[0042] The term "therapeutically effective amount" or "pharmacologically effective amount" or "effective amount" refers to an amount of an agent sufficient to produce a desired therapeutic or pharmacological effect in a subject being treated. The terms are synonyms and are intended to specify the amount of each agent that achieves the goal of improving the severity of a disease and / or the frequency of its onset by treatment with each agent alone, while avoiding or minimizing adverse side effects, including adverse side effects that are normally associated with other therapies.

[0043] "Subject" includes any human and is used interchangeably with the term "patient".

[0044] The term "nerve cell damage" refers to injury to components of the central or peripheral nervous system. Nerve cell damage may result from physical (including mechanical, electrical, thermal), ischemic, hemorrhagic, chemical, biological, or biochemical injury. Examples of nerve cell damage include ischemic and hemorrhagic stroke, spinal cord, brain, cranial nerve, and peripheral nerve injury.

[0045] In the context of this specification, the terms "administering", "administer", and "administration", and variations of these terms including these, include contacting, applying, delivering, or providing the compounds or compositions of the present invention to a subject by any suitable means.

[0046] One of ordinary skill in the art will understand that the techniques described herein are capable of variations and modifications other than those specifically described. It should be understood that this technology includes all such variations and modifications. To avoid doubt, this technology also includes, individually or collectively, all of the steps, features, and compounds described or shown herein, and all combinations of any two or more of these steps, features, and compounds.

[0047] To understand this technology more clearly, preferred embodiments will be described with reference to the explanations set forth below.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0049] The methods disclosed herein include the administration of cell-free human umbilical cord plasma (UCF) or exosomes derived from cell-free human umbilical cord plasma (UCF-E). The inventors have surprisingly found that the administration of UCF or UCF-E can treat or ameliorate one or more symptoms of neuronal injury such as ischemic brain injury (e.g., stroke).

[0050] Those skilled in the art will appreciate that numerous variations and / or modifications can be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. Accordingly, the embodiments of the invention are to be considered in all respects as illustrative and not restrictive.

[0051] [Preparation of Exosomes] Cell-free plasma for use in the methods described herein can be prepared from pooled human umbilical cord blood. In particular, the plasma is obtained from umbilical cord blood collected at the time of delivery from normal healthy pregnant women. The plasma is not obtained from umbilical cord blood of pregnant women who are at high risk or considered to be at high risk or who have had difficulties during delivery. Specifically, the umbilical cord blood is collected aseptically, pooled, and then centrifuged to separate into plasma, red blood cells, and buffy coat. The plasma and buffy coat are pooled, frozen (e.g., at -80°C), thawed, and centrifuged (e.g., at 3000 g for 20 minutes) to remove cells, membranes, and cell / cell debris. The supernatant is filtered through a 0.1 - 0.22 micron filter (with or without pre-filtration using a 5 micron filter). The resulting hUC-P (umbilical cord factor plasma) preparation containing exosomes may be used immediately or lyophilized.

[0052] In some embodiments, cell-free allogeneic plasma preparations (especially UCF) are characterized by the presence of certain proteins including, for example, at least one of IL-1α, IL-1-β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-12 p70, IL-13, IL-15, IL-17A, eosinophil chemotactic protein (CCL11), G-CSF, GM-CSF, basic fibroblast growth factor (FGF-2), hepatocyte growth factor (HGF), interferon-γ (IFN-γ), interferon-inducible protein 10 (CXCL10), monocyte chemoattractant protein-1 (CCL2), macrophage inflammatory protein-1β (CCL4), platelet-derived growth factor-bb (PDGF-bb), regulated on activation, normal T cell expressed and secreted (RANTES) (CCL5), tumor necrosis factor-α (TNF-α) and vascular endothelial growth factor (VEGF), detected, for example, by ELISA.

[0053] In some embodiments, cell-free allogeneic plasma preparations (UCF plasma) are characterized in that the level of one or more of GM-CSF, HGF, MIP-1b (CCL4) and PDGF-bb is at least 20% higher than the level found in the serum of normal adults. For example, the level may be 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% higher than the level found in the serum of normal adults. Alternatively, exosome preparations (UCF-E) are characterized in that the level of IP-10 (CXCL10) is at least 60% of the level found in the serum of normal adults. For example, the level may be 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% of the level found in the serum of normal adults.

[0054] Exosomes (or extracellular vesicles (EVs)) are released into the bloodstream by cells under normal and pathological conditions and carry RNA, lipids, and proteins from their host cells, which can indicate the molecular composition of the parent cell.

[0055] Cell-free allogeneic exosomes (UCF and UCF-E) are derived from a variety of cells and share major structural and functional proteins such as Rab GTPases, SNAREs, annexins, Alix, Tsg101, and tetraspanins (CD9 and CD63), as well as glycosylphosphatidylinositol anchor molecules, flotillin, cholesterol, sphingomyelin, and hexosylceramide. Lipoprotein markers APOB (chylomicrons, VLDL, IDL, and LDL) and APOE (chylomicrons, VLDL, and HDL) are used to distinguish exosomes from lipoproteins.

[0056] Exosomes can be isolated by any means known in the art, including but not limited to ultracentrifugation, ultrafiltration, size exclusion chromatography, polymer-based precipitation, and affinity capture on antibody-conjugated magnetic beads. For example, conventional methods of EV isolation include differential centrifugation and buoyant density centrifugation, ultrafiltration, size exclusion chromatography, precipitation, and immunoaffinity separation. Size exclusion chromatography uses a biological fluid as the mobile phase against a porous stationary phase and elutes molecules at a rate inversely correlated with their size. That is, larger particles elute first, and then smaller vesicles enter the pores and flow, resulting in a longer path and a longer time to elute. Differential ultracentrifugation is known as a method for separating EV subpopulations by gradually increasing the acceleration. Polyethylene glycol (PEG)-based precipitation uses a solution that promotes the massive aggregation of vesicles encapsulated in the polymer. Immunoaffinity capture isolates specific vesicle populations using antibodies that target exosome surface proteins. In microfluidics technology, exosomes are efficiently captured using a chip with specific binding via antibodies. Ultrafiltration relies on a filter with a specific pore size that produces a filtrate rich in vesicles of the desired size.

[0057] Differential ultracentrifugation performs multiple washing spin steps, which are designed to first remove cells, cell debris, apoptotic bodies, and microvesicles. This is done by gradually separating the pellet and supernatant while increasing the speed, separating at 300 - 400×g for 10 minutes, then at 2000×g, and finally at 10000×g, to separate the supernatant containing a relatively high concentration of exosomes, although microvesicles, lipoprotein fractions, and other protein aggregates are still mixed in. After this step, the sample is spun at 100000 - 200000×g for 70 minutes to 2 hours to sediment the final exosomes. The pellet obtained here can be resuspended in phosphate - buffered saline (PBS) and ultracentrifuged again. In this case, the purity increases, but the yield of isolated exosomes decreases. The size of exosomes seen at this level is 20 - 250 nm, contains RNA and miRNA species, and includes some of the common protein markers associated with exosomes, such as flotillin - 1, alix, TSG101, CD81, CD63, CD9, etc. However, among the exosome population, there are those that do not express these proteins, such as CD81 exosomes, and / or these markers may be seen not only in exosomes but also in other types of EVs.

[0058] The size - exclusion filtration method is by using a membrane with a specific pore size to separate particles of a pre - determined size. First, large particles are removed using filters with pore sizes of 0.8 μm and 0.45 μm to obtain a filtrate relatively rich in exosomes. The obtained exosomes are defined by the maximum and minimum size ranges that pass through the first and last porous filtration membranes. This protocol can be used as a standalone technique but can also be used to complement ultracentrifugation for separating large microvesicles and exosomes.

[0059] In an alternative method, nanofiltration by continuous filtration for isolating exosomes, known as cross-flow filtration or tangential flow filtration, is used. This technique first performs dead-end filtration of cells and debris, together with large vesicles, for example, with a diameter of 1000 nm. Next, filtration based on tangential flow is performed, and impurities (mainly proteins) with a diameter smaller than the membrane cut-off size are removed to the waste chamber. The filtrate containing exosomes is then repeatedly passed through an exclusion filter, thereby concentrating the injected solution. Finally, exosomes can be further fractionated using a track-etched membrane with a specific and consistent pore size, for example, having a diameter of 50 - 250 nm. The recovery of exosomes depends on the type of filter because different membrane types and pore sizes exist. For example, a cellulose membrane with a pore size of 10 kDa is often most efficiently recovered.

[0060] Precipitation based on polyethylene glycol (PEG) promotes the formation of exosome aggregates using an aqueous PEG solution, and the aggregates can then be precipitated by, for example, low-speed centrifugation at 1500×g. The size range of the isolated exosomes is consistent with other methods such as differential ultracentrifugation.

[0061] Exosomes (UCF-E) can be used immediately or lyophilized.

[0062] In some embodiments, cell-free exosome preparations (particularly UCF-E) are characterized by the presence of specific proteins including at least one of CD63 and TSG101 and the absence of lipoprotein markers APOB (chylomicrons, VLDL, IDL, and LDL) and APOE (chylomicrons, VLDL, and HDL).

[0063] In some embodiments, the exosomes have a particle size of about 10 to 200 nm. For example, the particle size may be about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm or about 200 nm. Preferably, the exosomes have a particle diameter of about 30 to 150 nm. For example, the particle diameter may be about 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm or about 150 nm. The particle diameter may be the average particle diameter or the median particle diameter. Alternatively, the particle diameter may be the mode (i.e., the particle diameter most frequently observed in the particle population).

[0064] In some embodiments, lyophilization does not substantially change the particle size distribution of the exosomes.

[0065] The exosomes and plasma preparations containing exosomes are acellular. For example, no cells are observed under light microscopy (or there are no viable cells or cell / cell fragments or membranes).

[0066] [Method] The methods described herein include administering a therapeutically effective amount of human umbilical cord plasma and exosomes (hUC-P or UCF) or exosomes only (hUC-E or UCF-E) to a subject to treat or ameliorate at least one symptom of nerve cell damage.

[0067] The terms "therapeutically effective" or "therapeutic effect" refer to an amount of UCF or UCF-E that alleviates to some extent one or more of the symptoms of nerve cell damage being treated. In other embodiments, the term "therapeutically effective dose" refers to an amount of UCF or UCF-E that, when administered to a subject, is effective to at least partially treat nerve cell damage from which the individual is suffering, or to at least partially improve or ameliorate the symptoms of such damage. As is understood in the art, a therapeutically effective amount of UCF or UCF-E depends on at least one of the mode of administration, the carrier or vehicle employed, the particular damage, other pharmaceuticals and compounds the subject is taking, and the particular characteristics (age, weight, condition, gender, etc.) of the subject to whom the compound is administered.

[0068] UCF or UCF-E can be administered parenterally or nasally. Parenteral administration includes intravenous, intraperitoneal, intracranial, intrathecal, intramuscular, subcutaneous, or intraperitoneal administration, and UCF, UCF-E, or both are formulated as an injection solution.

[0069] UCF, UCF-E, or both are suitable for injection after a final filtration step (e.g., after filtration through a 0.1 - 0.2 micron filter). Alternatively, lyophilized UCF or UCF-E can be reconstituted with any suitable vehicle for injection. Suitable vehicles include water for injection, physiological saline, buffered physiological saline (e.g., phosphate buffered saline or 85% phosphate buffered saline), Ringer's solution, or lactated Ringer's solution. In some embodiments, water or 85% PBS is a preferred vehicle for reconstitution of lyophilized UCF or UCF-E.

[0070] Preferably, the administration is intrathecal or intravenous administration.

[0071] In some embodiments, the amount of cell-free plasma and / or exosome preparation (UCF or UCF-E) or both administered to treat nerve cell damage, as well as the dosing regimen, depend on the age, weight, sex, and medical condition of the subject, the severity of the injury, the route and frequency of administration, the particular compounds employed, and the pharmacokinetic properties of the subject (e.g., adsorption, distribution, metabolism, excretion, etc.). Thus, these can vary widely. Such treatment may be administered at the frequency and for the period that the treating physician deems necessary. One of ordinary skill in the art will understand that the dosing regimen or therapeutically effective amount of UCF, UCF-E, or both administered needs to be optimized for each individual. In some embodiments, UCF or UCF-E may be appropriately administered at a dose of about 0.01 mg to 100 mg per kg of body weight, usually about 0.1 mg to about 50 mg per body weight, depending on the route and frequency of administration.

[0072] The mode of administration is preferably in the form of a single bolus dose or injection of UCF, UCF-E, or both over a period of time, regular dosing, or a combination thereof. The concentration of any dose depends on the frequency of administration.

[0073] In some embodiments, UCF or UCF-E is administered regularly, for example, weekly, every two weeks, every three weeks, monthly, or a combination thereof. Alternatively, UCF or UCF-E can be administered daily, every five days, every ten days, every fifteen days, every twenty days, every twenty-five days, about every thirty days, or a combination thereof.

[0074] In one embodiment, treatment of nerve cell damage includes administering UCF, UCF-E, or both multiple times, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times.

[0075] In some embodiments, treatment begins with the first administration of UCF, UCF-E, or both, at least one day after neuronal injury. For example, treatment can begin about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, about 30 days after, about 60 days after, or at least about 90 days after neuronal injury. In some embodiments, treatment is initiated with the first administration of UCF, UCF-E, or both, about 3, 4, 5, 6, 7, 8, 9, or 10 days after injury, for example, 7 or 8 days after injury.

[0076] UCF and / or UCF-E may be administered together with additional therapeutic agents such as anti-inflammatory agents, steroids, dipyridamole, astaxanthin, dabigatran, losartan, nimodipine, policosanol, rivaroxaban, or ticlopidine.

[0077] In some embodiments, UCF and / or UCF-E may be administered together with one or more therapeutic agents that temporarily disrupt the blood-brain barrier because blood-brain barrier disruption (BBBD) is effective and safe for the delivery of therapeutic agents. Suitable therapeutic agents for BBBD include one or more of mannitol, intracarotid arterial hyperosmolar mannitol (ICAHM), RMP-7 (a synthetic analog of bradykinin), and legadenosine.

[0078] In one embodiment, UCF, UCF-E, or both are not administered with immunosuppressive agents.

[0079] The UCF and UCF-E described herein are useful for treating a range of neuronal injuries including ischemic brain injury, hemorrhagic brain injury, or traumatic brain injury.

[0080] In particular, the UCF and UCF-E described herein are useful for treating ischemic or hemorrhagic cerebral infarction.

[0081] Treatment can result in improvement of one or more symptoms of nerve cell damage. For example, treatment can - dystonia (improvement by a decrease in the frequency or severity of involuntary muscle contractions as compared to the same patient before treatment or a control patient), - aphasia (improvement by an improvement in the ability to understand or formulate language as compared to the same patient before treatment or a control patient), - infarct size (improvement by a reduction in infarct size), - inflammation (improvement by a reduction in inflammation, either systemic or at the infarct site), - reduction of white matter (improvement by suppressing the amount of white matter reduction as a result of the injury), - reduction of gray and white matter (improvement by suppressing the amount of gray and white matter reduction as a result of the injury), - apoptotic cells in the perifocal area (improvement by a decrease in the number of apoptotic cells in the perifocal area), - CD16 / CD32 positive cells in the perifocal area (improvement by a decrease in the number of CD16 / CD32 positive cells in the perifocal area), of one or more.

[0082] Typically, dystonia is considered a movement disorder characterized by abnormal and often repetitive muscle contractions, motor signs that cause movement, posture, or both, mainly persistent or intermittent. However, there is increasing evidence indicating that non - motor elements are important in dystonia, such as abnormalities in sensory and perceptual functions, areas of the psychoneurological, cognitive, sleep, etc. Dystonia can be evaluated by any method known in the art. Such evaluation may include the use of the Unified Dystonia Rating Scale (UDRS), which includes a detailed assessment of the severity of dystonia in an individual's body areas. For example, each body area is evaluated for the severity and duration of dystonia. The proximal and distal limbs are evaluated separately. The evaluations of each body area are summed for an overall assessment of the severity of dystonia. Thus, the UDRS is improved by the methods described herein.

[0083] Aphasia is the inability, or impairment of the ability, to understand and / or produce language, especially as a result of damage to nerve cells (such as a stroke). Aphasia may be informally evaluated (through conversation and observation) by a treating physician, or may be evaluated by combining one or more known tests. These tests include PALPA (Psycholinguistic Assessments of Language Processing), Western Aphasia Battery, Boston Naming Test, Boston Diagnostic Aphasia examination, Aachen Aphasia Test, Minnesota Test for Differential Diagnosis of Aphasia, Acute Aphasia Screening Protocol, Mississippi Aphasia Screening Test, Mount Wilga High Level Language Screening Test, Aphasia Language Performance scales, NIH Stroke Scale, Bedside Evaluation Screening Test, Boston Diagnostic Aphasia Examination, Porch Index of Communicative Ability, Burden of Stroke Scale, Pyramids and Palm Trees, Caulfield Language for Cognition, Quick Assessment forAphasia), Cognitive Linguistic Quick Test, Reitan - Indiana Aphasia Screening Examination, Communication Activities for Daily living, ScreeLing, Communicative Effectiveness Index, Sheffield Screening Test for Language Disorders, Comprehensive Aphasia Test, Sklar Aphasia Scale, Frenchay Aphasia Screening Test, Test for Reception of Grammar, Functional Assessment of Communication Skills for Adults, Aphasia Screening Test, Functional Communication Profile, Ullevaal Aphasia Screening Test, Wechsler Individual Achievement Test, Information Language Processing Screen: ILPS, Western Aphasia Battery, Inpatient Functional Communication interview, Whurr Aphasic Screening Test, Measure ofincluding (Cognitive-Linguistic Abilities). The methods described herein can obtain improved performance in any one or more or any combination of these tests.

[0084] Typically, infarct size (volume) is known in the art and is evaluated using MRI or CT scan methods available to the treating physician. For example, infarct core volume can be evaluated by the Alberta Stroke Program Early Computed Tomography Score (ASPECTS), and this scoring system is applied to images obtained by non-contrast computed tomography (NCCT), post-contrast CT (PCCT), and diffusion-weighted imaging (DWI). DWI stroke volume was measured semi-quantitatively by manually delineating the contour of hyperintense lesions. The infarct core volume was calculated using a threshold of apparent diffusion coefficient of 600×10 -6 mm 2 / s. Intraclass correlation coefficients (ICC) were estimated to evaluate the inter-reader reliability of ASPECTS scoring and DWI stroke volume.

[0085] Inflammatory markers such as C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor (TNF)-α, and fibrinogen increase after neuronal injury such as stroke. These biomarkers are known to be associated with increased mortality, recurrent vascular risk, and unfavorable functional outcomes. These biomarkers can be measured using known methods. The methods described herein can reduce these biomarkers compared to untreated subjects.

[0086] It is well known that damage to nerve cells such as in ischemic stroke causes damage to the gray and white matter. White matter damage is a risk factor for unfavorable neurological outcomes. Most of the damage caused by stroke is present in the subcortical region, and white matter occupies nearly half of the mean infarct volume. White matter is extremely vulnerable to ischemia and often sustains more severe damage than gray matter. Clinical symptoms associated with white matter damage include cognitive dysfunction, affective disorders, sensory-motor disorders such as urinary incontinence and pain, all of which are closely related to the disruption and remodeling of the connectivity of white matter. Damage to white matter (and gray matter) can be non-invasively detected by MRI, and its morphology, metabolism, and function can be evaluated three-dimensionally.

[0087] After acute stroke, multiple immune cells successively invade the brain parenchyma. Peripheral immune cells, including myeloid dendritic cells, monocytes / macrophages, and neutrophils, appear within 1 day after stroke. Subsequently, a slight increase in T lymphocytes and B lymphocytes is detected. When the brain environment is affected by pathophysiological changes such as stroke, infiltrating cells differentiate into macrophages with special functions such as the production of inflammatory mediators and phagocytosis. Infiltrating immune cells mainly differentiate into two phenotypes: the classical inflammatory phenotype (M1) and the alternative anti-inflammatory phenotype (M2). The phenotypic markers of M1 include CD16, CD32, CD86, MHC II, and iNOS, and CD206, Arg1, and Ym1 are identified as phenotypic markers of M2. As shown in this specification, the method of the present invention can reduce the number of CD16 / CD32-positive cells in the perifocal area.

[0088] The Modified Rankin Scale (mRS) is a single-item assessment scale for the comprehensive course of subjects after stroke. This scale is used to classify the level of functional independence based on pre-stroke activities rather than the degree of achievement of specific tasks observed.

[0089] The conventional implementation method of mRS is the guided interview process. The evaluation is performed by asking the patient about daily living activities including outdoor activities. Information regarding neurological disorders in the examination, including aphasia and intellectual disability, is obtained. The mRS score is determined by comprehensively considering all aspects of the patient's physical, mental, and speech.

[0090] One mRS score is determined based on the following criteria (Dromerick, A. W., Edwards, D. F., Diringer, M. N. (2003), Sensitivity to changes in disability after stroke: A comparison of four scales useful in clinical trials, Journal of Rehabilitation Research and Development, 40(1), 1-8.).

[0091]

Table 1

[0092] The method disclosed in this specification can bring about an improvement in the mRS score.

[0093] The National Institutes of Health Stroke Scale (NIHSS) is a tool used to objectively quantify the disability caused by stroke. NIHSS consists of 11 items, and each item scores a specific ability on a scale of 0 to 4. For each item, a score of 0 usually indicates that the function of the specific ability is normal, and the higher the score, the more impaired. The scores of each item are summed to calculate the patient's total NIHSS score. The highest score is 42 points, and the lowest score is 0 points. The method described in this specification can bring about an improvement in the NIHSS score.

[0094]

Table 2

[0095] The items evaluated by NIHSS are as follows.

[0096] 1a. Level of consciousness - A score of 3 is given only if the patient does not move (other than reflexive movements) in response to a noxious stimulus.

[0097] 1b. LOC question - Ask the patient about their age in months and years. The answer must be correct. Patients with aphasia or in a comatose state who cannot understand the question are given 2 points. Patients who cannot speak due to endotracheal intubation, tracheal trauma, severe dysarthria, speech disorder, or other problems not secondary to aphasia are given 1 point.

[0098] 2. Best Gaze - Only horizontal eye movements are examined. Spontaneous or reflexive (oculocephalic) eye movements are scored, but caloric testing is not performed. If the patient has conjugate deviation of the eyes and it can be overcome by spontaneous or reflexive activity, the score is 1. If the patient has isolated peripheral nerve palsy (CN III, IV, VI), the score is 1. Gaze can be examined in all aphasic patients. For patients with eye trauma, bandages, previous blindness, or other visual impairments or visual field defects, examination is performed by reflex movement and the examiner makes the scoring judgment. After establishing eye contact, moving the patient from side to side reveals the presence of partial gaze palsy.

[0099] 3. Visual - Visual field (upper and lower quadrants) examination is performed face-to-face using finger counting and appropriate visual threats. It is necessary to prompt the patient, but if the patient can properly see the side of the moving finger, it can be scored as normal. If one side is blind or enucleated, score the visual field of the remaining eye. Score 1 only if obvious asymmetry, including quadrantanopia, is observed. Score 3 if the patient is blind for any reason. Perform double simultaneous stimulation at this point. If there is extinction, the patient is given 1 point, and the result is also used for the answer to question 11.

[0100] 4. Facial nerve paralysis - Instruct the patient verbally and with gestures to show their teeth, raise their eyebrows, and close their eyes. For patients with poor response or poor understanding, score the symmetry of the pain expression when reacting to a noxious stimulus. If the face cannot be seen due to facial trauma / dressing, tracheal tube or tape, or other physical barriers, remove them as much as possible.

[0101] 5 and 6. Arm and leg movement - For each limb, if known, examine them in order starting from the non-paralyzed arm. Position the limbs appropriately: extend the arm (palm down) at 90 degrees (seated position) or 45 degrees (supine position), and extend the leg at 30 degrees (always tested in the supine position). If the arm drops within 10 seconds or the leg drops within 5 seconds, it is determined as drift. For aphasic patients, prompt them using a sense of urgency in the voice and gestures, but do not use noxious stimuli. Only score "9" if there is a cut or joint fixation of the shoulder or hip joint, and the examiner must clearly write an explanation for scoring "9".

[0102] 7. Ataxia of Limbs - This item aims to find evidence of unilateral cerebellar lesions. Examine with the eyes open. Note that if there is visual impairment, conduct the examination in the healthy visual field. Perform the finger-nose-finger test and the heel-shin test bilaterally, and score ataxia only if it is present in proportion to the decrease in muscle strength. Ataxia is not seen in patients who cannot understand or are paralyzed. The use of non-examinable is not recommended, but in cases of amputation, joint fixation, and some fractures, it may be scored as "9", and the examiner must clearly write an explanation for non-scorability. If the patient is blind, examine by touching the nose from the outstretched arm position.

[0103] 8. Sensation - During the examination, check if the patient shows a pinprick-like sensation or a pain expression, or if a patient with apathy or aphasia makes an avoidance behavior from a harmful stimulus. Only sensory loss caused by stroke is scored as abnormal, and the examiner needs to examine as many body parts (arms (not hands), legs, trunk, face) as necessary to accurately check for unilateral sensory loss. The two points indicating "severe or complete" should be given only if severe or complete sensory loss can be clearly proven. Therefore, patients in a coma or with aphasia will probably be scored 1 or 0. Patients with brainstem stroke with bilateral sensory loss are scored 2. Patients with non-responsive quadriplegia are scored 2. Patients in a lethargic state (item 1a = 3) are arbitrarily scored 2 in this item.

[0104] 9. Best language - Much information regarding comprehension can be obtained in the section before the examination. The patient is instructed to explain what is happening in the picture, name the items on the naming sheet, and read from the list of sentences. The patient is made to name all the items on the naming sheet and read all the phrases on the two reading sheets. Comprehension is judged not only from the responses in this examination but also from the responses to all instructions in the previous general neurological examination. If there are difficulties in the examination due to visual impairment, the patient is instructed to identify, repeat, and speak what is placed in the hand. The intubated patient is instructed to write characters. The comatose patient (Question 1a = 3) optionally gets 3 points in this item. The examiner must select the score for the comatose or uncooperative patient, but the 3 points should only be used when the patient is silent and does not follow a one-step command.

[0105] 10. Dysarthria - When the patient is considered normal, it is necessary to obtain a sufficient sample of speech by having the patient read and repeat the words on the list. In the case of severe aphasia, the clarity of the articulation of spontaneous speech can be evaluated. The item can only be scored as "9" if the patient is intubated or unable to speak due to other physical impairments, but the examiner must clearly write an explanation for the non-scorability. The patient should not be explained why they are undergoing the examination.

[0106] 11. Extinction and attentional disorders - Sufficient information to identify extinction phenomena may have been obtained in the previous examinations. If the patient has severe visual impairment, visual double simultaneous stimulation is impossible, and cutaneous stimulation is normal, the score is considered normal. Even if the patient has aphasia, if they seem to be paying attention to both the left and right, the score is considered normal. Visual spatial neglect and anosognosia can also be regarded as evidence of abnormality. Since it is only scored when there is an abnormality, this item cannot be non-scorable.

Example

[0107] Example 1: Anonymized and pooled umbilical cord serum obtained from a cord blood bank Individual umbilical cord blood (UCB) is collected according to standard American Association of Blood Banks (AABB) protocols. (See Badowski M.S., Harris D.T. (2012) Collection, Processing, and Banking of Umbilical Cord Blood Stem Cells for Transplantation and Regenerative Medicine, Singh S. (eds) Somatic Stem Cells. Methods in Molecular Biology (Methods and Protocols), vol 879).

[0108] An aliquot of umbilical cord blood is collected from each individual and tested for disease and blood type.

[0109] After blood type determination and evaluation for disease-free status, individual samples are pooled and cryopreserved at -80°C.

[0110] Example 2: Preparation of plasma Plasma is prepared from pooled umbilical cord blood by centrifugation.

[0111] Human UCB containing adenine-containing citrate phosphate dextrose as an anticoagulant was obtained from a cord blood bank. In this study, only fresh UCB provided within 24 hours after delivery was used.

[0112] UCB was centrifuged at 1500×g for 10 minutes at room temperature and separated into three layers: plasma (UCB-PL supernatant), a mixture of white blood cells and platelets (intermediate layer), and red blood cells (lower layer).

[0113] The UCB-PL layer was carefully collected, aliquoted, and stored at -80°C until used as UCF.

[0114] Cell-free plasma (UCF) was prepared by centrifuging UCB-PL at 3000 g for 20 minutes.

[0115] The cell counts of each layer and cell-free plasma were counted using a Guava® Muse® Cell Analyzer (manufactured by Luminex). The results are shown in Table 3 and Figure 1, which shows optical microscope images of the intermediate layer, upper layer, cell-free plasma, and water, respectively.

[0116]

Table 3

[0117] Cytokine profiling of cell-free plasma was performed, and the results are shown in Figure 2 with normal human (adult) serum as a control.

[0118] Example 2: Preparation of exosomes Cell-free frozen cord blood plasma samples (UCF) were thawed and pooled. Exosomes (UCF-E) were prepared by combining centrifugation, precipitation, and microfiltration steps. In some cases, cryoprotectants were added before freeze-drying (lyophilizing) the exosomes.

[0119] Lyophilized exosomes were stable for more than 36 months at 4°C. Samples stored at 4°C or room temperature did not change from the initial evaluation. From this finding, exosomes were stable under various conditions.

[0120] Subsequently, exosomes were quantified for the total number of particles by nanoparticle tracking analysis (NTA) using a Nano Sight LM10. Lyophilization did not affect the stability of purified exosomes (Figure 2). The exosome standard, which is a lyophilized exosome standard, has high purity, is easy to reconstitute, and is easy to store and transport at 4°C.

[0121]

Table 4

[0122] Samples of the starting material (acellular plasma), the concentrated product (exosomes), and the final product (freeze-dried exosomes) were characterized by nanoparticle tracking analysis (NTA) using a Nanosight NS300 (Malvern Instruments Ltd). As shown in Table 5, the EV concentration and size distribution were estimated by NTA analysis.

[0123]

Table 5

[0124] These data are shown graphically in Figure 3.

[0125] Example 3: Lyophilization The acellular plasma or exosome preparation was first frozen at -80 °C and immediately placed in a Labconco Freezone 12L console freeze dryer with the condenser maintained at -35 °C and left standing for 24 hours under 100 mTorr (a vacuum strong enough to rapidly freeze water), after which the vial was resealed. Since no heat was applied, no organic solvent or salt was added, and the sample remained frozen under strong vacuum even during drying by the -45 °C condenser, it was lyophilized.

[0126] The following protocol was used to lyophilize the acellular plasma or exosome preparation.

[0127]

Table 6

[0128] The freezing temperature was -40 °C, the additional freezing time was 20 minutes. The set temperature of the condenser was -40 °C and the set temperature of the vacuum was 150 mTorr.

[0129]

Table 7

[0130] The secondary temperature was 30°C.

[0131] Example 4: Reconstitution Protocol 285 mg of lyophilized acellular plasma (UCF) containing exosomes was reconstituted with 5 mL of water for injection. The powder completely dissolved and had the appearance of serum (straw-colored (yellow)). The reconstituted acellular plasma containing exosomes was filtered through a 0.2 μm filter. The sterile-filtered acellular plasma (UCF) containing exosomes can be used immediately or stored at 2 - 8°C for 2 weeks.

[0132] 86 mg of lyophilized exosomes (UCF-E) was reconstituted with modified 0.9% saline (85% saline) - 0.9% normal saline, a solution prepared by diluting 0.85-fold with water for injection (8.5 mL of 0.9% saline: 1.5 mL of water for injection). Before reconstitution, the 85% saline was warmed to 37°C. 5 mL of warmed (37°C) saline was added and the contents were completely dissolved. In some cases, appropriate vortexing may be required and incubation at 37°C may be necessary. The powder completely dissolved and became an off-white colloidal liquid. The reconstituted exosome preparation was a uniform, hazy solution and was filtered through a 0.2 μm filter. The sterile-filtered exosomes (UCF-E) can be used immediately or stored at 2 - 8°C for 2 weeks.

[0133] Example 5: Animal Study of Acellular Plasma and Exosomes in the Treatment of Middle Cerebral Artery Occlusion in Rats Adult male Sprague-Dawley rats (250 - 300 g, n = 84) were purchased from the Experimental Animal Research Center of the Chinese Academy of Medical Sciences (Beijing, China). The housing conditions were a 12-hour light-dark cycle, controlled temperature and humidity, and free access to food and water. The rats were randomly assigned to two cohorts and underwent treatment after transient focal cerebral ischemia and reperfusion.

[0134] Cerebral ischemia was induced by a transient intraluminal vascular occlusion method using the intraluminal filament method. Briefly, under halothane (Lunan Pharmaceutical, Jinan City, China) anesthesia, the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were exposed. The ECA was transected with micro scissors, and a monofilament nylon thread with a diameter of 0.25 mm (Sunbio Biotech, Beijing, China) was inserted into the root of the ECA. The nylon thread was inserted into the ICA until resistance occurred (1.8 - 2.1 cm from the carotid bifurcation). After the occlusion period (120 minutes), the nylon thread was removed. The bifurcation of the external carotid artery was ligated with silk suture to stop bleeding. The skin was sutured to close the neck incision. Rats in both cohorts that underwent middle cerebral artery occlusion (MCAO) were administered recommended doses of buprenorphine and cefazolin for pain relief and infection prevention, respectively. Immunosuppressants were not administered.

[0135] Subsequently, a modified neurological severity score (mNSSs) test was performed after surgery. Rats that underwent transient MCAO with an mNSSs of approximately 10 - 12 were randomly divided into 4 groups (groups A - D, and groups A, B, and C were further subdivided into intracranial subgroups and intraperitoneal subgroups) as shown in Table 8.

[0136] At the early stage of the subacute phase, corresponding to 3 days after pMCAO in rats (7 days after infarction in humans), reconstituted acellular plasma containing exosomes (UCF) or exosomes only (UCF - E above) was administered to rats that underwent permeant MCAO (pMCAO). The treatment regimens are shown in Table 8.

[0137] On the 3rd day after post-ischemia (PID), the MCAO animals were randomly divided into eight experimental groups. In the intraperitoneal administration group, cell-free plasma (UCF) containing exosomes (0.5 mL), exosomes only (UCF-E) (0.5 mL), or physiological saline (0.5 mL) was administered intraperitoneally over 5 minutes. In the intracerebral administration group, three holes (A: 30 μL, B: 30 μL, C: 40 μL - see Table 9) were drilled on the right side of the rat skull, and cell-free plasma (UCF) (0.1 mL), exosomes only (UCF-E) (0.1 mL), or physiological saline (0.1 mL) was stereotactically injected. All targets were selected around the infarct margin.

[0138]

Table 8

[0139]

Table 9

[0140] 5.1 Functional evaluation Modified neurological severity scores (mNSSs) test The mNSS test is a standard and globally recognized method for evaluating the severity and recovery of stroke sequelae. This test is a comprehensive test of motor, sensory, reflex, and balance function tests. The severity of the injury is determined by the cumulative score of all tests. An mNSS score of 13 - 18 indicates severe injury, 7 - 12 indicates moderate injury, and 1 - 6 indicates mild injury. The mNSS test was performed on rats in both cohorts at regular intervals (1d, 7d, 14d, 21d) before ischemia and up to 21 days of reperfusion.

[0141] Cylinder test The cylinder test was used as an indicator of forelimb asymmetry by observing the movement of rats at 3-minute intervals within a transparent plexiglass cylinder with a diameter sufficient for movement and a width of 18 cm and a height of 30 cm that could promote rising and wall exploration. There was a mirror behind the cylinder, allowing the movement of the forelimbs to be observed and recorded while the rat had its back to the examiner. After episodes of rising and wall exploration, the limb that first contacted the ground, or both limbs if they contacted simultaneously, was scored for landing. For both the unimpaired and impaired limbs, the usage rate was calculated relative to the total number of movements. The overall score for limb bias was calculated by subtracting the usage rate of the impaired limb from the usage rate of the unimpaired limb. Wall exploration and landing movements were analyzed separately. Rats were examined 1 to 4 weeks after local ischemia following transplantation.

[0142] Limb-placing test The limb-placing test, which evaluates the sensorimotor integration of the forelimbs and hindlimbs by confirming responses to tactile and proprioceptive stimuli, was performed according to the method described by De Ryck et al. (Brain Research. 1992;573(1):44 - 60) and modified by Puurunen K et al. (Neuropharmacology. 2001;40(4):597 - 606, and Exp Neurol. 2001;167(2):348 - 55). The test was performed on both sides of the body 24 hours after local ischemia. The test consisted of seven limb-placing tasks and was scored by an examiner who was blinded to the treatment group. To detect hindlimb and forelimb impairments, scores of 0: no placement, 1: incomplete or delayed placement, 2: complete, immediate placement were used. For each rat, the average placement score (total of forelimbs and hindlimbs) for each week of the test was calculated.

[0143] TTC staining Rats treated with plasma (UCF) and exosomes (UCF-E) were deeply anesthetized with pentobarbital and decapitated. The brains were removed, placed in an adult rat brain matrix (Kent Scientific Corporation, USA), frozen at approximately -70 °C for about 8 - 10 minutes, and sliced into 2-mm-thick coronal sections. The brain coronal sections were incubated in a 2% 2,3,5-triphenyltetrazolium chloride (TTC) solution in the dark for 30 - 45 minutes, and then images were taken using an Olympus SZX12 research stereomicroscope. The ischemic and non-ischemic regions of the ipsilateral and contralateral hemispheres of each section were traced, and the areas were measured using Image J analysis software (NIH). The total volume of each region of the rat brain was calculated. The percent infarct size of each rat was calculated using the formula: infarct size (%) = { (volume of the contralateral hemisphere) - (volume of the non-ischemic ipsilateral hemisphere)} × 100 / volume of the contralateral hemisphere. This formula takes into account the possibility that brain edema may interfere with the infarct volume. The swelling of the ipsilateral hemisphere of each rat was calculated using the formula: swelling (%) = { (volume of the ipsilateral hemisphere) - (volume of the contralateral hemisphere)} × 100 / volume of the contralateral hemisphere.

[0144] MRI scan Seven days after surgery or treatment, the rats were anesthetized with 1.5% - 2% isoflurane and subjected to MRI. During the MRI procedure, the rats were kept at 37 °C, and an MRI-compatible respiratory sensor was used for animal control. The MRI experiments consisted of three-dimensional (3D) T2-weighted images (T2WI), a series of diffusion-weighted images (DWI) for calculating apparent diffusion coefficient (ADC) maps, and a series of perfusion-weighted images for calculating cerebral blood flow maps. The perfusion experiments were performed using arterial spin labeling without injection of a contrast agent. All MRI experiments were conducted using a BIOSPEC BMT 47 / 40 (Bruker, Ettlingen, Germany) spectrometer operating at 4.7 T, equipped with an 11.2 cm active shield gradient system capable of a gradient strength of 200 mT / m and a rise time of 80 ms. A 7 cm birdcage-type radiofrequency coil was used for transmission and reception. Image analysis of T2WI, DWI, and perfusion-weighted images was performed using ParaVision 3.0.1 (Bruker, Ettlingen, Germany). ADC maps were calculated from the DWI series using the image sequence analysis tool of the ParaVision package. Cerebral blood flow maps were calculated from arterial spin labeling images. This calculation was performed using Matlab 7.3 (MathWorks, Inc., Natick, MA), and the data were expressed as a percentage of the affected brain volume.

[0145] Neuroprotection To evaluate gray and white matter damage, coronal paraffin sections (6 μm) were incubated with MAP2 (Sigma Aldrich) or mouse anti-myelin basic protein (MBP, Sternberger Monoclonals, Lutherville, MD), and binding was visualized using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Brain damage was analyzed at a position corresponding to -1.58 mm from bregma in adult mice by outlining the contours of both hemispheres on whole cross-sectional images using ImageJ software (Rasband WS, ImageJ, NIH, Bethesda, MD; http: / / rsb.info.nih.gov / ij / , 1997 - 2009). The ipsilateral area reduction of MAP2 and MBP was calculated as described.

[0146] Cell death Cell death was detected in the peri-infarct area by TUNEL staining (TdT-FragEL DNA fragmentation detection kit, cancer gene research product). The cell count was performed on one slice collected 1.6 mm posterior to bregma for each rat (n = 10 per group). Using a 40× objective lens and image analysis software (Image-Pro Plus 4.1, Media Cybernetics), the number of positive cells was counted in at least 10 different microscopic fields based on nuclear morphology and darkness.

[0147] Neuroinflammation Activation of microglia was evaluated by measuring its morphological changes. On the 1st and 7th days after treatment, 25 microglia cells were randomly selected in a 300-μm-wide area around the infarct for each of 6 coronal sections (150 microglia cells per rat) at 240-μm intervals (n = 4 per group). The cell nucleus was placed at the center of a grid with 10 concentric circles at 5-μm intervals, and the number of intersections between cytoplasmic processes and grid lines was counted using a 100× oil immersion lens.

[0148] Glia scar GFAP expression at the boundary of the infarct area was quantified by measuring the integrated optical density and area ratio using ImageJ software (National Institute of Mental Health, Bethesda, Maryland).

[0149] Statistical analysis All data were expressed as mean ± SEM. The functional outcome measured in the cylinder rearing test was analyzed using two-way ANOVA with Fisher's least significant difference post hoc test. It was considered statistically significant when P < 0.05. Histological measurements were analyzed using one-way ANOVA with Bonferroni's post hoc test. It was considered statistically significant when P < 0.05.

[0150] 5.2 Results Neurological course The mNSS after MCAO is shown in Figure 4. In all six groups, neurological function gradually recovered over time (see Figure 4). mNSS was significantly higher in animals treated with saline than in those treated with UCF (cell-free plasma containing exosomes) and UCF-E (exosomes only) at 14 days (Control-ip vs UCF-ip, UCF-ic, UCF-E-ip and UCF-E-ip, P < 0.05, or Control-ip vs UCF-ip, UCF-ic, UCF-E-ip and UCF-E-ic, P < 0.05) and 21 days (Control-ip vs UCF-ip, UCF-ic and UCF-E-ip, P < 0.05, or Control-ip vs UCF-ip, UCF-ic, UCF-E-ip P < 0.05, Control-ic vs UCF-E-ic, Control-ip vs UCF-E-ic, P < 0.01) after MCAO (P < 0.05). Higher mNSS indicates higher severity of neurological damage. The score of the UCF-E-ic group (3.5 ± 1.5) was lower at 21 days after treatment compared to the other three treatment groups (UCF-ic group: 4.5 ± 0.7, UCF-ip group: 4.2 ± 0.9, UCF-E-ip group: 4.6 ± 0.3). However, no significant difference was observed between rats administered UCF and UCF-E intracranially and intraperitoneally.

[0151] Using the cylinder score and limb position score, rats that received intracranial and intraperitoneal injections of UCF (cell-free plasma containing exosomes) and UCF-E (exosomes only) on days 14 and 21 showed better functional outcomes than the saline control (Figure 5A - B). Notably, rats administered UCF-E-ic on days 14 and 21 after stroke also had significantly better performance in the behavioral tests than rats administered UCF-ic, UCF-ip, and UCF-E-ip (Figure 5A - B). However, no improvement in the test scores was observed immediately after the start of the behavioral tests.

[0152] Evaluation of infarct volume On the 14th and 21st days after treatment, the infarct volumes of the UCF-ic group and the UCF-E-ip group were significantly lower compared to the saline group and the saline-ip group (UCF-ic vs. control-ic, UCF-ip vs. control-ic, UCF-ic vs. control-ip, UCF-ip vs. control-ip, ** P < 0.05; UCF-E-ic vs. control-ic, UCF-E-ip vs. control-ic, ** P < 0.01, Figure 6). However, no significant difference was observed between the rats administered intracranially with UCF and UCF-E and those administered 7 days later. Rats injected intraperitoneally with UCF or UCF-E had larger lesion sizes on the 14th and 21st days after injection compared to the intracranial UCF or UCF-E injection groups.

[0153] The lesion sizes 1 day and 14 days after the experimental procedure were evaluated by MRI (Figure 7). The MRI scan data were the same as the data of TTC staining.

[0154] Rats injected intraperitoneally with UCF or UCF-E had a higher infarction rate on the 14th and 21st days after injection compared to the group injected intracranially with UCF or UCF-E.

[0155] Neuroprotective effect The effects of treatment with UCF and UCF-E on the lesion size were determined by analyzing the reduction of the ipsilateral gray and white matter (mouse anti-microtubule-associated protein (MAP2)) and white matter (mouse anti-myelin basic protein (MBP)) on the 21st day after cerebral ischemia.

[0156] In the saline-treated mice, the reduction of the ipsilateral MAP2 area was 39.13 ± 3.5% and 37.86 ± 7.1%, and the values did not change between the two groups (ip vs. ic). Treatment with UCF-E (ic and ip) or UCF (ip) significantly reduced the lesion volume compared to saline treatment (as shown in Figure 8, *** P < 0.001, *** P < 0.01, * P < 0.05). There was no significant difference in the reduction of the ipsilateral MAP2 area among the four treatment groups.

[0157] In saline-treated rats, the reduction in the ipsilateral MBP area was 35.26±4.2% and 36.91±3.3% (Figure 8), and there was no difference between the two groups (ip vs ic). Treatment with UCF-E (ic and ip groups) or UCF (ic and ip groups) significantly reduced the lesion volume compared to saline treatment (as shown in Figure 8, *** P<0.001, ** P<0.01, * P<0.05). There was no significant difference in the reduction of the ipsilateral MAP2 area among the UCF-ip group, UCF-ic group, and UCF-E-ip group. However, the reduction in the ipsilateral MAP2 area in UCF-E-ip-treated rats was significantly lower than that in UCF-ip, UCF-ic, and UCF-E-ip-treated rats 21 days after treatment.

[0158] TUNEL staining TUNEL staining was used to identify the number of apoptotic cells in the para-infarct area 14 days after treatment (Figure 9). The average number of TUNEL-positive cells per square millimeter was 467±43.2 (control-ip), 8.25±2.693 (sham), 20.45±10.308 (ischemia), 20.15±9.67 (vehicle), and 10.0±3.974 (treatment). Among the four treatment groups, the number of apoptotic cells was less than that in the two control groups. There was a significant difference in the number of TUNEL-positive cells between the control group and the treatment group (Figure 9). The number of TUNEL-positive cells in the UCF-E-ic treatment group was significantly less than that in the UCF-ip, UCF-ic, or UCF-E-ip treatment group 14 days after treatment.

[0159] Glia scar There was no significant difference in the number of GFAP-positive cells among the groups on the 21st day after treatment (Figure 10).

[0160] Macrophage phenotype To investigate whether the administration of UCF-E or UCF (ip or ic) affects the phenotype of macrophages, the expression profiles of macrophages were evaluated by immunofluorescence staining with CD16 / 32 and arginase-1 (Arg-1). On the 14th day after injury, more arginase-1 positive cells were observed in the four treatment groups compared with the two control groups (all P<0.01). The number of Arg-1 positive cells in the UCF-ip treatment group was significantly higher than that in the UCF-ic, UCF-E-ic, and UCF-E-ip treatment groups on the 14th day after treatment (all P<0.01). Furthermore, in animals receiving UCF-ic, UCF-ip, UCF-E-ic, or UCF-E-ip injection, the number of CD16 / 32 positive cells was significantly decreased compared with that in the animals of the two saline control groups. No significant difference was observed among the four treatment groups. See Figure 11.

[0161] 5.3 Conclusions Both UCF (cell-free plasma containing exosomes) and UCF-E (exosomes only) are safe for intraperitoneal or intracranial injection, and no injection-related complications or deaths were observed.

[0162] Intraperitoneal or intracranial injection of UCF and UCF-E can promote functional recovery after stroke in rats. Although there is no statistical significance between the two (this may be related to the relatively small number of experimental rats and the short observation period), intracranial injection of UCF-E shows a higher score in absolute value.

[0163] The repair mechanisms of UCF and UCF-E may be related to neuroprotection, which is observed from the reduction of the damaged area, the staining of neuron-related markers, and the decrease in apoptosis.

[0164] Since UCF and UCF-E have little effect on gliosis, they have little effect on reducing glial scar formation in the chronic phase.

[0165] UCF and UCF-E have a strong regulatory function on the local immune inflammatory response of the central nervous system.

[0166] 6. Human Trial To observe the safety and initial effects of UCF and UCF-E in humans, a small-scale trial was conducted on 6 volunteers. This trial passed the ethical review of the 7th Medical Center of the PLA General Hospital (number 2021-1368-6) and was strictly carried out in accordance with the ethical review opinions and experimental protocols of the 7th Medical Center of the PLA General Hospital.

[0167] The inclusion criteria for the study were 1. The duration of stroke with motor nerve dysfunction was 3 months or more and 24 months or less. 2. Men and women aged 30 to 65 years. 3. Women had a negative result in the serological pregnancy test and were practicing appropriate contraception or were infertile (more than 2 years had passed since menopause or they had undergone hysterectomy, oophorectomy, or infertility surgery). 4. There was a history of obvious and complete ischemic stroke confirmed by head MRI, with or without cerebral cortical ischemic injury, in the subcortical region supplied by the middle cerebral artery (MCA) or lenticulostriate artery. 5. Modified ranking score: 2, 3, or 4. 6. FMMS score was 55 points or less. 7. When evaluated twice at least 3 weeks before surgery, the NIHSS score fluctuated between positive and negative 4. 8. All follow-ups could be completed and the volunteer was willing to do so. 9. The volunteer was willing and able to receive late physical therapy and rehabilitation therapy. That is.

[0168] The exclusion criteria were 1. Having any psychological or psychiatric disorder that may interfere with the study 2. Being a patient with a history of two or more symptomatic strokes, excluding transient ischemic attacks (TIAs). 3. Being a patient with a history of other severe neurological diseases or nerve injuries 4. Having an infarct area of 8 cm or more in any measurement 5. Having a history of myocardial infarction within three months 6. Being a patient with a history of epilepsy or currently taking anti-epileptic drugs 7. Being a patient who participated in the clinical trial of other pharmaceuticals or medical devices within 30 days 8. Being a patient who has received other cell transfusions in addition to blood transfusions 9. Having a concurrent disease or condition, for example: a. Having a coagulation disorder with an INR of 1.4 or more in preoperative examination b. At the first screening, PRA (Panel Reactive Antibodies) > 20% c. Having active infection before surgery d. Having hypotension that requires hypertension treatment e. Having skin damage in the surgical area f. Having a history of malignant tumor or having a malignant tumor g. Having primary or secondary immunodeficiency h. Having continuous MRI artifacts, unable to obtain clear MRI images before and after surgery or unable to perform MRI examination i. Serum creatinine concentration exceeding 115 μmol / L, liver function (aspartate aminotransferase / AST, total bilirubin, alkaline phosphatase) exceeding twice the upper limit of normal value, hematocrit (%) / hemoglobin (g / L) less than 30 / 10, total white blood cell count less than 4 × 10 9 / L, platelet count less than 100 × 10 9Less than / L, uncontrolled hypertension, systolic blood pressure over 180 mmHg or diastolic blood pressure over 100 mmHg, uncontrolled diabetes (HbA1c over 8%), if gastrointestinal bleeding is detected in the fecal occult blood test, positive for tuberculosis (TB skin test: PPD / tuberculin test), hepatitis B or C, positive for human HIV 10. Being in any of the following states: a. Recent drug or alcohol abuse b. Unstable health condition c. Unstable mental disorders including psychosis and untreated major depression 11. Being in any situation that the investigator or the attending physician determines may affect the participation in the clinical trial or may cause harm to the subject 12. Being in any situation that the surgeon determines may cause postoperative complications 13. Being a patient allergic to tacrolimus or methylprednisolone 14. Being a patient who is unable to participate in scheduled physical and / or specialized treatment or has no intention of participating, and is unable to complete scheduled follow-up or has no intention of doing so 15. Failing to provide the informed consent required in the screening program 16. Taking antiplatelet drugs within 2 weeks before surgery That is.

[0169] The evaluation criteria are shown below.

[0170] Safety evaluation Safety was evaluated by adverse events, clinical tests, vital signs and physical examinations.

[0171] As endpoints of follow-up related to intravenous injection, 30-day survival rate, incision site infection, epilepsy, meningitis, cerebral hematoma, new ischemic stroke, exacerbation and secondary surgery, etc. were reported.

[0172] Other safety endpoints include the incidence of complications related to the following: a. Those related to immunosuppressive therapy, including but not limited to nephrotoxicity (creatinine ≥ 177 μmol / L [2 mg / dL] or baseline creatinine ≥ 2 μmol / L [2 mg / dL]) or renal failure (requiring renal replacement therapy), b. Hyperkalemia or hypomagnesemia due to renal insufficiency, c. Opportunistic infections (bacterial, fungal, or viral, including cytomegalovirus), d. Neurotoxicity of the central nervous system caused by tacrolimus (including posterior leukoencephalopathy syndrome, epilepsy, focal neurological dysfunction, tremors, headache), e. Newly developed glucose intolerance, hyperglycemia, or diabetes, f. Gastrointestinal disorders (including anorexia, nausea, vomiting, diarrhea, and abdominal discomfort), g. Malignant neoplasms (squamous cell carcinoma or malignant lymphoproliferative disorders) and malignant hypertension, h. New complications related to stroke or neurological function, such as increased motor dysfunction (results of motor function scores), increased sensory dysfunction (neurological examination), and poor cognitive test results (using a neuropsychological kit), i. Other complications such as urinary tract infections, deep vein thrombosis (DVT), pulmonary embolism, gastric ulcers, pressure ulcers, respiratory infections (pneumonia or tracheobronchitis), death within 30 days after surgery, and prolonged hospital stay.

[0173] Assessment of efficacy The primary efficacy indicators were as follows: a. Adverse events, b. Serious adverse events (SAE), c. Routine brain MRI (routine images and brain perfusion images), d. Serology and hematology, e. Clinical evaluations of brain injury such as the National Institutes of Health Stroke Scale (NIHSS), Modified Rankin Score (MRS), Fugl-Meyer Stroke Scale, and Cognitive Kits. f. Detection of fluorodeoxyglucose uptake by positron emission tomography (FDG-PET).

[0174] The secondary efficacy indicators were as follows: a. The graft survival rate at the graft site, determined by MRI (with or without the use of a gadolinium-containing contrast agent) and autopsy. b. The efficacy of immunosuppressive therapy, evaluated by the detection of donor-specific HLA antibodies.

[0175] Patient information Three patients who met the inclusion criteria were selected and informed consent was obtained. The demographic and clinical information of the patients is shown in Table 10, and the results after UCF-E administration are shown in Table 11.

[0176] 200 μL of UCF-E was administered intrathecally to one patient, and 400 μL of UCF-E was administered intrathecally to the other two patients.

[0177]

Table 10

[0178]

Table 11

[0179] 7. Ongoing human trials The above patients are part of a large-scale study planned as follows and are currently ongoing.

[0180]

Table 12

[0181]

Table 13

[0182] One patient who received three intrathecal injections (total dose 1.5 ml) of exosomes (UCF-E) four months after hemorrhagic stroke recovered the ability to walk short distances without assistance. The formal evaluation of the patient after exosome injection (UCF-E) has been delayed due to Covid restrictions. Video data taken by the family has been collected.

[0183]

Table 14

[0184]

Table 15

[0185] Six patients were treated with intrathecal exosomes (UCF-E) (Table 16), and three of the six are the patients described in Table 10. These patients showed good improvement after exosome injection (UCF-E), and it is expected that greater improvement will be achieved in patients with higher doses.

[0186]

Table 16

[0187]

Table 17

[0188] After admission, the hematoma was punctured and drained under robotic surgery, and rt-PA was injected to dissolve the hematoma. Three days later, most of the hematoma was drained without bleeding or other complications. On the 7th day after surgery, UCF-E was injected into the intrathecal space. The injection volume was 400 μL or 800 μL, and the injection was repeated on the 14th and 21st days after surgery.

[0189]

Table 18

[0190]

Table 19

[0191] UCF-E was injected into the intrathecal space in all 6 patients without complications. No major hemodynamic or pulmonary complications were observed. At the end of the follow-up, no signs of rebleeding, hemorrhagic infarction, or worsening of intracerebral hemorrhage were found. No seizures were observed. Mild fever (38.50 °C) was observed in 5 patients in the test group within 24 hours after injection. The fever in all 5 patients subsided within 2 days without any pharmacological intervention.

[0192] In addition, regarding the effect of the exosome preparation (UCF-E), tests using a rat model of intracerebral hemorrhage have been conducted according to the following table.

[0193]

Table 20

[0194] The degree of brain atrophy was evaluated on the 36th day after injury in an additional test. The degree of brain atrophy in the UCF-E group was significantly lower than that in the saline control group (p = 0.0893, Figure 12), indicating a neuroprotective effect in the UCF-E treatment group.

[0195] 8. Human case study

[0196]

Table 21

[0197] Treatment method

[0198] After admission, under robot guidance, hematoma puncture and drainage were performed, and rt-PA was injected to dissolve the hematoma. In the reexamination 3 days later, the hematoma was essentially completely drained, and there were no complications such as rebleeding. UCF-E was administered by intrathecal injection into the intervertebral space between the 3rd and 4th lumbar vertebrae (L3-4) on the 7th day after surgery, and the injection volume for this patient was 1600 μL. After releasing the same amount of cerebrospinal fluid, exosomes were directly injected into the intrathecal space at a rate of 400 μL per minute, and the injection was completed in a total of 4 minutes. Thereafter, normal rehabilitation exercises were performed.

[0199]

Table 22

Claims

1. A method for treating or ameliorating symptoms of nerve cell damage in a subject, comprising administering to the subject a therapeutically effective amount of a cell-free allogeneic preparation (cell-free human umbilical cord plasma (UCF) containing exosomes or exosomes only derived from human umbilical cord plasma (UCF-E)), wherein the cell-free allogeneic exosome preparation (UCF or UCF-E) is derived from human umbilical cord blood, the nerve cell damage is ischemic brain injury, hemorrhagic brain injury or traumatic brain injury, and the UCF or the UCF-E is first administered 3 to 90 days after the nerve cell damage.

2. The method according to claim 1, wherein the UCF or the UCF-E is first administered on the 7th to 10th day after the nerve cell damage.

3. The method according to claim 1 or 2, wherein the UCF or the UCF-E is administered intrathecally.

4. The method according to any one of claims 1 to 3, wherein the UCF or the UCF-E is administered to the subject periodically.

5. The method according to claim 4, wherein the administration cycle is weekly, every two weeks, every three weeks, every month or a combination thereof.

6. The method according to claim 5, wherein the administration cycle is daily, every 5 days, every 10 days, every 15 days, every 20 days, every 25 days, about every 30 days or a combination thereof.

7. The method according to any one of claims 1 to 6, wherein the ischemic brain injury or the hemorrhagic brain injury is a stroke.

8. The method according to any one of claims 1 to 7, wherein no immunosuppressant is administered.

9. The method according to any one of claims 1 to 8, wherein the cell-free human umbilical cord plasma (UCF) containing exosomes has at least one level of GM-CSF, HGF, MIP-1b (CCL4) and PDGF-bb that is at least 20% higher than that found in the serum of normal adults.

10. The method according to any one of claims 1 to 8, wherein the cell-free allogeneic preparation of exosome-containing (UCF) and exosomes only (UCF-E) has a level of IP-10 (CxCL10) that is at least 60% of the level found in the serum of normal adults.

11. The method according to any one of claims 1 to 10, wherein the exosomes in the allogeneic preparation (UCF or UCF-E) containing the exosomes contain at least one of CD63 and TSG101 and lack the lipoprotein markers APOB and APOE.

12. The method according to any one of claims 1 to 11, wherein the cell-free allogeneic preparation (UCF and UCF-E) containing the exosomes contains exosomes having an average, median or modal particle diameter of about 30 to 150 nm.

13. The method according to any one of claims 1 to 12, comprising administering the cell-free allogeneic preparation (UCF and UCF-E) containing the exosomes a total of 2, 3, 4, 5, 6, 7, 8, 9 or 10 times.

14. The method according to any one of claims 1 to 13, wherein the symptoms are selected from dystonia, aphasia, infarct size, inflammation, white matter reduction, gray matter reduction, apoptotic cells in the perifocal area of the infarct, and CD16 / CD32 positive cells in the perifocal area of the infarct.

15. The method according to claim 14, wherein administration of the allogeneic preparation (UCF or UCF-E) containing the exosomes reduces dystonia in the subject.

16. The method according to claim 14, wherein administration of the allogeneic preparation (UCF and UCF-E) having the exosomes reduces aphasia in the subject.

17. The method according to claim 14, wherein administration of the allogeneic preparation (UCF and UCF-E) having the exosomes reduces the infarct volume in the subject.

18. The method according to claim 14, wherein administration of the allogeneic preparation (UCF and UCF-E) containing the exosomes reduces systemic inflammation or reduces inflammation in the infarct or perifocal area of the subject.

19. The method according to claim 14, wherein administration of the allogeneic preparation (UCF and UCF-E) containing the exosomes reduces the reduction of gray matter and / or white matter in the subject.

20. The method according to claim 14, wherein administration of the allogeneic preparation (UCF and UCF-E) containing the exosomes reduces the number of apoptotic cells in the perifocal area of the infarct in the subject.

21. The method according to claim 14, wherein administration of an allogeneic preparation (UCF and UCF-E) containing exosomes reduces the number of CD16 / CD32 positive cells in the infarct surrounding area of the subject.

22. The method according to any one of claims 1 to 21, further comprising administering at least one additional therapeutic agent and an allogeneic preparation (UCF and UCF-E) containing exosomes.

23. The method according to claim 22, wherein the additional therapeutic agent is an anti-inflammatory agent, steroid, dipyridamole, astaxanthin, dabigatran, losartan, nimodipine, policosanol, rivaroxaban or ticlopidine.

24. The method according to claim 22 or 23, wherein the additional therapeutic agent is mannitol, intracarotid arterial hyperosmolar mannitol (ICAHM), RMP-7, or legadenoson.

25. A method of improving the mRS score or NIHSS score of a subject with ischemic brain injury, comprising administering to the subject a cell-free allogeneic preparation (UCF or UCF-E) containing a therapeutically effective amount of exosomes, wherein the cell-free allogeneic preparation (UCF or UCF-E) is derived from pooled human umbilical cord blood, and wherein the UCF or UCF-E is first administered 3 to 90 days after the ischemic brain injury.

26. The method according to claim 25, wherein UCF or UCF-E is first administered 7 to 10 days after the ischemic brain injury.

27. The method according to claim 25 or 26, wherein no immunosuppressant is administered.

28. The method according to any one of claims 25 to 27, wherein the allogeneic preparation (UCF or UCF-E) containing exosomes is administered to the subject periodically.

29. The method according to claim 28, wherein the administration cycle is weekly, every two weeks, every three weeks, every month, or a combination thereof.

30. The method according to claim 29, wherein the administration cycle is daily, every five days, every ten days, every fifteen days, every twenty days, every twenty-five days, about every thirty days, or a combination thereof.

31. The method according to any one of claims 25 to 30, comprising administering a cell-free allogeneic preparation (UCF or UCF-E) containing exosomes a total of 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

32. The method according to any one of claims 1 to 31, wherein the UCF or UCF-E is derived from the blood of a single umbilical cord or blood pooled from multiple umbilical cords.

33. The method according to claim 32, wherein the pooled blood is derived from umbilical cord blood of the same blood type or umbilical cord blood of different blood types.

34. Use of a cell-free allogeneic preparation (UCF or UCF-E) having exosomes derived from pooled human umbilical cord blood in the manufacture of a medicament for treating or ameliorating symptoms of nerve cell damage, wherein the nerve cell damage is ischemic brain injury, hemorrhagic brain injury, or traumatic brain injury, and the medicament is first administered 3 to 90 days after the nerve cell damage.

35. Use of a cell-free allogeneic preparation (UCF or UCF-E) having exosomes derived from pooled human umbilical cord blood in the manufacture of a medicament for improving the mRS score or NIHSS score of a subject with ischemic brain injury, wherein the medicament is first administered 3 to 90 days after ischemic brain injury.

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