Methods of isolating umbilical cord blood plasma products, tissues, and cellular exosomes, compositions, and methods of use

Abstract

Described herein are new methods for fractionating and isolating platelets, platelet and extracellular vesicle-derived growth factors, exosomes, globulins, fibrinogen, and albumin, and methods of using the isolated platelets, platelet and extracellular vesicle-derived growth factors, exosomes, globulins, fibrinogen, and albumin to regenerate tissue in a subject, to treat fibrinogenemia or coagulation defects in a subject, to treat ischemia and hypoxia, to treat dry eye syndrome in a subject, an orthopedic disease, or a dental disease. Also described herein are growth media for culturing mammalian (e.g., human) cells.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 942,063, filed November 29, 2019, the entire contents of which are incorporated herein by reference. Invention Field

[0003] The present invention relates generally to methods, compositions and methods of using platelets, exosomes, fibrinogen, albumin, globulins and exosomes isolated from umbilical cord blood, peripheral blood and other biological fluids and tissues, including agents and methods for culturing mammalian cells and treating diseases and conditions. Background of the Invention

[0005] Umbilical cord blood (UCB) is the blood of an infant that remains in the placenta after birth. Produced in the placenta, UCB is the richest source of stem cells outside of the placenta itself, more abundant than any adult tissue, including bone marrow. UCB plasma is the extracellular fluid of UCB, placenta, and fetus, containing proteins, lipid factors, and exosomes that carry signals between the placenta, uterus, mother, and infant. Approximately 5% of UCB is collected from over 30 million newborns worldwide each year. Most UCB contains monocytes, including stem cells.

[0006] UCB plasma contains platelets, growth factors (VEGF, G-CSF, EGF, and FGF), pro-inflammatory cytokines (IL-2, IL-6, IFN-γ, TNF-α), and other factors that promote human cell survival and growth (Ehrhart, et al., 2018). EVs are cytoplasmic vesicles released from cells into the extracellular environment. Platelets are small, nucleus-free cells (2-3 μm). Platelets produced by cells called megakaryocytes contain growth factors and cytokines packaged into granules. When activated, platelets release these cytokines and growth factors. Human platelets have long been used in the growth media for culturing human cells, as well as in the treatment of orthopedic and dental problems and dry eye syndrome.

[0007] Cell culture media require growth factors and cytokines to support and stimulate cell growth and differentiation. Traditionally, cells are grown in fetal bovine serum (FBS) obtained from pregnant dairy cows. Human cells grown in FBS take up bovine serum albumin, which triggers an immune response when the cells are transplanted into the human body. Due to widespread milk consumption, many people have antibodies against bovine serum albumin. Cells grown in FBS may express bovine serum albumin, which activates immune rejection upon transplantation. For this reason, the U.S. Food and Drug Administration (FDA) requires the use of "xeno-free" media to culture cells intended for transplantation into humans.

[0008] Platelets are the “first responders” in the blood. Platelets can be produced by bone marrow stem cells. The most common source of human growth culture medium is human platelet lysate. Platelet lysate is also used to promote joint and tooth healing, and to treat diseases such as dry eye syndrome, which affects more than 200 million people worldwide. Many companies sell human platelet lysate for cell culture (Klatte-Schulz et al., Int J Mol Sci, 2018.19(1)). Most platelet lysates are prepared from adult plasma, which is collected by centrifuging adult peripheral blood to obtain platelet-rich plasma (PRP), monocytes (MNC), and red blood cell fractions (RCF). Many groups and companies that manufacture PRP combine MNC and PRP. Most platelets end up in the plasma layer, but are sticky and may get trapped in the MNC layer or RCF. Often, most devices that collect platelets from adult blood combine MNC and PRP, resulting in MNC waste. Frozen platelets showed reduced activation and impaired signal transduction (Waters et al., Transfusion, 2017.57(12):p.2845-2857). Standards for platelet lysates have not yet been established (Astori, G., et al., Stem Cell Res Ther, 2016.7(1):p.93).

[0009] Exosomes are small endosomes, ranging from 50 to 150 nm in diameter. They are produced from multivesicular bodies (MVBs) through inward budding and possess surface proteins that target specific cells. MVBs fuse with the cell's extracellular membrane to release the exosomes into the extracellular space. Cells communicate with each other via exosomes.

[0010] UCB exosomes exert significant beneficial therapeutic and growth effects on cells, as well as anti-immune, regenerative, and other effects on the brain, heart, and other organs and tissues. UCB exosomes are a primary means of communication between the placenta and the developing fetus and may contain transcription factors, mRNA, and μRNA that stimulate cell growth and differentiation. Placental exosomes possess anti-immune properties, enabling cells to secrete and express molecules that shut down immune cells.

[0011] Plasma also contains fibrinogen, coagulation factors, and albumin and globulins (accounting for more than 50% and 40% of plasma proteins, respectively). Plasma fibrinogen and coagulation factors can be used to treat patients with fibrinogenemia and coagulation disorders, and can also be used as surgical glue when combined with thrombin. Albumin is an important excipient for cell suspensions. Albumin is a carrier molecule for many hormones, cytokines, and drugs. Globulins include all types of immunoglobulins (IgA, IgG, IgM, etc.) and other globulins that can act as signal transduction molecules. Fibrinogen is usually separated by cryoprecipitation, while albumin is usually extracted by chemical methods.

[0012] Traditional methods for isolating exosomes are inefficient and labor-intensive, requiring prolonged ultracentrifugation (greater than 100,000 g) over a sucrose gradient for extended periods (greater than 18 hours). The centrifugal forces associated with ultracentrifugation and the osmotic forces of the sucrose gradient can disrupt exosomes and release their contents into the extracellular fluid. Other methods for isolating exosomes include antibody-coated magnetic beads and extraction with hydrophobic components.

[0013] Therefore, more effective and efficient methods are still needed to isolate platelets, exosomes, fibrinogen, albumin, globulin, and EV from tissues, blood, urine, saliva, eye fluid, and other bodily fluids in order to use these components to enhance the ability of culture media to support the growth and differentiation of human and other cells, as well as for the treatment of various diseases. Summary of the Invention

[0014] This article describes novel methods for fractionating and isolating platelets, platelet- and extracellular vesicle-derived growth factors, exosomes, globulins, fibrinogen, and albumin, and methods for using isolated platelets, platelet- and extracellular vesicle-derived growth factors, exosomes, globulins, fibrinogen, and albumin to regenerate tissue in subjects, treat fibrinogenemia or coagulation defects, and treat dry eye syndrome, orthopedic or dental conditions. Some of the methods described herein include isolating and freeze-drying platelets from platelet-rich plasma (PRP) to obtain platelet-depleted plasma (PDP), which is an excellent source of growth factors and exosomes for cell culture. These methods include extracting more than 90% exosomes from PDP and isolating and freeze-drying albumin, globulins, and clotting proteins from the remaining plasma. Methods for activating PDP are also described herein. In the experiments described below, the method for activating PDP as described herein produced a cell culture medium containing 1% PDP, which was comparable to or better than 10% fetal bovine serum (FBS) in stimulating HeLa cell growth. Growth media for culturing mammalian (e.g., human) cells are also described herein.

[0015] This article also describes methods for preparing PDPs from UCB and activating UCB PDPs using concentrated CaCl2 solution to stimulate cell growth in cell culture. Methods for extracting exosomes from UCB PDPs include cold (e.g., 4°C) extraction of exosomes using sonicated polyethylene glycol (PEG). These methods are by far the most efficient methods for extracting exosomes from biological fluids, recovering over 90% of exosomes from small (1 ml) to large (several liters) volumes of fluid. This article describes novel clinical and laboratory uses of UCB PDPs and exosomes, including improving implantation of fertilized eggs for in vitro fertilization, treating neonatal anemia, treating hypoxic-ischemic cerebral encephalopathy, treating dry eye syndrome, and many other applications.

[0016] Therefore, this paper describes a method for isolating platelets and extracellular vesicles (EVs) from a tissue source. The method includes: (i) providing a tissue source of platelets and EVs; (ii) centrifuging the tissue source at a first speed followed by centrifugation at a second speed higher than the first speed to separate a platelet-rich plasma (PRP) layer containing plasma, platelets, and EVs; (iii) filtering the PRP layer with a microporous filter containing pores with a diameter of 0.22 μm to separate platelets and particles with a diameter greater than 220 nm from the PRP layer, producing a plasma filtrate consisting substantially of isolated platelets and particles with a diameter less than about 220 nm attached to the microporous filter; (iv) freeze-drying the microporous filter in a container under conditions that release growth factors from the isolated platelets; and (v) processing the plasma filtrate to separate EVs with a diameter less than about 220 nm. In several embodiments of the method, EVs with a diameter less than 220 μm in step (v) comprise exosomes with a diameter of 50-150 μm. In several embodiments of the method, the container is stored at -20°C or lower after step (iv). The method may further include: (vi) adding culture medium to the container to thaw and dissolve growth factors, thereby producing a growth solution containing growth factors; and (vii) removing the growth solution from the container. In several embodiments of the method, the tissue source may be, for example, UCB, umbilical cord, umbilical cord lining, umbilical cord stromal cells (Wharton's jelly), amniotic fluid, amnion, placenta, and peripheral blood. In embodiments of the method, a microporous filter removes particles with a diameter greater than 220 nm. In several embodiments of the method, the container may comprise a closed system.

[0017] This document also describes a method for culturing mammalian cells. The method involves culturing a population of mammalian cells in a culture medium containing a growth solution prepared according to the methods described herein. In several embodiments, the mammalian cell population is human cells. In several embodiments, the tissue source is UCB.

[0018] This article further describes cell culture media containing growth solutions prepared by the methods described herein.

[0019] This document further describes a method for producing platelet-depleted plasma (PDP). The method includes: (i) acquiring and thawing undepleted platelet-rich plasma (UCB); (ii) centrifuging the UCB to produce a platelet-rich plasma (PRP) fraction; (iii) separating the PRP fraction; and (iv) removing platelets by centrifuging platelet-poor-plasma (PPP) or filtering the PDP and cell debris, as well as extracellular vesicles larger than 220 nm, to produce a PDP fraction containing citrate phosphate-dextrose (CDP), exosomes, and plasma proteins. In some embodiments, the method further includes activating the PDP fraction by adding CaCl2 to the PDP fraction to precipitate CPD. In some embodiments, 6 M CaCl2 is added. In some embodiments, 5 M CaCl2 is added to achieve a concentration of 100 mM to precipitate CPD. In some embodiments, varying amounts of CaCl2 are added to precipitate CPD. This article also describes a method for obtaining the PDP fraction and activating the PDP fraction by adding CaCl2 (e.g., about 5 M or about 6 M CaCl2 to achieve a concentration of 100 mM) to precipitate the CPD.

[0020] This article also describes a method for isolating one or more of exosomes, globulins, fibrinogen, and albumin from a tissue source. The method includes: (i) providing a tissue source of exosomes, globulins, fibrinogen, and albumin; (ii) filtering the tissue source using a microporous filter containing pores with a diameter of 0.22 μm to produce a plasma filtrate containing exosomes, globulins, fibrinogen, albumin, and CPD; (iii) adding the plasma filtrate to a solution containing polyethylene glycol (PEG) in a first centrifuge tube, such that the exosomes are partitioned into PEG vesicles; (iv) cooling the first centrifuge tube to 2°C to 8°C; (v) centrifuging the first centrifuge tube, such that the exosomes and PEG form a first precipitate and a first supernatant containing albumin, globulins, CPD, and fibrinogen; and (vi) transferring the first supernatant to… The exosomes and PEG are transferred to a second centrifuge tube and the first precipitate containing exosomes and PEG is frozen for storage. (vii) The precipitant is mixed with the first supernatant to precipitate CPD. (viii) The mixture of precipitant and first supernatant is filtered to remove CPD, producing a second supernatant free of CPD. (ix) The second supernatant is cooled to 2°C to 8°C to form a cryoprecipitate containing fibrinogen. (x) The cryoprecipitate is centrifuged in a third centrifuge tube to form a second precipitate and a third supernatant. (xii) The third supernatant containing albumin is removed, and the cryoprecipitate containing fibrinogen is freeze-dried. (xii) The third supernatant containing albumin is freeze-dried. In several embodiments of the method, approximately 80% to approximately 95% of the exosomes from tissue source are isolated. In several embodiments of the method, approximately 90% of the exosomes from tissue source are isolated. In several embodiments of the method, exosomes, fibrinogen, and albumin are isolated. In several embodiments of the method, in step (iv), the first centrifuge tube is cooled under conditions in which globulin cross-links fibrinogen and exosomes in the plasma filtrate are partitioned into PEG and CPD is precipitated by a precipitating agent. In several embodiments of the method, in step (v), the first centrifuge tube is centrifuged at a speed of about 1000 to about 5000 revolutions per minute (RPM) for about 5 minutes to about 30 minutes. In several embodiments of the method, the precipitating agent is CaCl2. In several embodiments of the method, the tissue source may be, for example, UCB, umbilical cord, umbilical cord lining, umbilical cord stromal cells (Wharton's jelly), amniotic fluid, amnion, placenta, and peripheral blood.

[0021] This article further describes a method for generating exosome-rich plasma (ERP). The method involves adding plasma or a saline solution (e.g., PBS) to an exosome precipitate obtained by the method for isolating exosomes as described herein.

[0022] This document further describes a method for culturing mammalian cells. The method includes culturing a population of mammalian cells in a culture medium containing exosomes and / or albumin isolated by the method described herein. In several embodiments, the mammalian cell population is human cells. In several embodiments, the tissue source is UCB.

[0023] This document also describes a cell culture medium comprising exosomes and / or fibrinogen and / or albumin isolated by the methods described herein. This document also describes a cell culture medium comprising a growth solution containing exosomes isolated according to the methods described herein or activated PDPs produced by the methods described herein.

[0024] This article also describes a pharmaceutical composition comprising platelets and EVs isolated by the methods described herein, or exosomes, fibrinogen and / or albumin isolated by the methods described herein, PDPs generated by the methods described herein, or ERPs generated by the methods described herein.

[0025] This document further describes a method for regenerating tissues (e.g., cardiovascular tissue, central nervous system tissue, eye tissue, etc.) in a subject (e.g., a human subject). The method includes administering an effective amount of exosomes isolated by the method described herein to the subject. In several embodiments, the subject is a person suffering from at least one disease, such as tissue damage, brain degeneration, central nervous system degeneration, and cardiovascular degeneration.

[0026] This article further describes a method for treating fibrinogenemia or coagulation defects in subjects. The method involves administering an effective amount of fibrinogen isolated by the method described herein to a subject suffering from fibrinogenemia or coagulation defects.

[0027] This article further describes a method for treating a subject with dry eye syndrome, orthopedic disease, or dental disease. The method includes administering to a subject suffering from dry eye syndrome, orthopedic disease, an effective amount of platelets separated by the method described herein, or PDPs or ERPs generated by the method described herein.

[0028] This article also describes a method for culturing mammalian cells. The method comprises culturing a population of mammalian cells in a culture medium containing a growth solution comprising exosomes isolated according to the method described herein or activated PDPs generated by the method described herein.

[0029] This article also describes a method for treating hypoxia, ischemia, or stroke in subjects. The method involves administering exosomes isolated according to the method described herein or PDPs generated by the method described herein to subjects suffering from hypoxia, ischemia, or stroke.

[0030] This document further describes a method for stimulating uterine angiogenesis in a subject (e.g., a woman) with this need. The method includes administering a PDP or an ERP generated by the method described herein into the uterine cavity of the subject. In several embodiments, a composition comprising a PDP or an ERP may be administered to the subject.

[0031] To aid in understanding the detailed description of the compositions and methods of this disclosure, several specific definitions are provided to facilitate the explicit disclosure of various aspects of this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0032] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include the plural, unless the context clearly specifies otherwise. Unless otherwise stated, the terms “comprising,” “including,” “containing,” or “having,” and variations thereof, are intended to cover the items listed thereafter and their equivalents, as well as other subject matter.

[0033] The phrases “in one implementation,” “in various implementations,” “in some implementations,” etc., are used repeatedly. Such phrases do not necessarily refer to the same implementation, but they may refer to the same implementation unless the context otherwise indicates.

[0034] The terms “and / or” or “ / ” mean any one of the items, any combination of items, or all of the items associated with the term.

[0035] The term "about" can refer to a variation of ±5%, ±10%, ±20%, or ±25% of a specific value. For example, in some embodiments, "about 50%" can cover a variation of 45% to 55%. For integer ranges, the term "about" can include one or two integers larger and / or smaller than the integer stated. Unless otherwise stated herein, the term "about" is intended to include values ​​(e.g., weight percentages) close to the range that are equivalent in terms of functionality of a single ingredient, composition, or embodiment.

[0036] As used herein, when referring to a set of items, the term "each" is intended to describe an individual item in the set, but not necessarily every single item in the set. Exceptions may occur if explicitly stated or otherwise specified in the context.

[0037] The terms “platelet-free plasma” and “PDP” refer to plasma from which platelets have been partially or completely removed. The terms “activated PDP” and “aPDP” refer to platelet-free plasma that has been treated with calcium ions to precipitate citrate-phosphate-dextrose (CPD) and other chemicals (which bind to and reduce calcium ion concentration to a level sufficient to prevent blood clotting), thereby restoring calcium ion activity to a level sufficient to support cell growth.

[0038] As used in this article, the terms "red blood cell fraction" and "RCF" refer to the fraction of centrifuged peripheral blood or umbilical cord blood containing red blood cells.

[0039] The terms "platelet-rich plasma" and "PRP" refer to plasma containing platelets. Plasma should contain as many platelets as or more than normal blood or cord blood (UCB).

[0040] As used in this article, the terms "ERP" and "exosome-rich plasma" refer to plasma containing exosomes at a higher concentration than normal blood. ERP differs from PRP in that ERP contains exosomes and few or no platelets, while PRP contains both platelets and exosomes.

[0041] As used herein, the term "purified" refers to a compound or entity (e.g., a cell) that has been separated from other compounds or entities. The compound or entity (e.g., a protein, peptide, cell) may be partially purified, substantially purified, or pure.

[0042] A compound or entity is considered pure when it is removed from substantially all other compounds or entities (i.e., preferably at least about 90%).

[0043] The terms “isolated” and the phrase “biopure” refer to substances that are substantially or substantially free of the components that are usually found with them in their natural state.

[0044] The terms “patient,” “subject,” and “individual” are used interchangeably in this document and refer to a mammalian (e.g., human) subject to treatment, diagnosis, and / or from which a biological sample is obtained. Subjects may be affected by conditions such as anemia, hypoxia-ischemia, stroke, hypo-endometria, or autoimmune conditions (e.g., multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis).

[0045] As used herein, the term "therapeutic agent" is intended to encompass any molecule, cell, chemical entity, composition, drug, or biological agent that can cure, heal, alleviate, relieve, alter, remedy, improve, enhance, or influence a disease, its symptoms, or a susceptibility to it. The term "therapeutic agent" includes polypeptides, peptides, cells, organic or inorganic molecules, natural or synthetic compounds, etc.

[0046] As used herein, the terms “treatment” and “therapeutic approach” mean the application or administration of a therapeutic agent to a patient, or to a separate tissue derived from a patient who has a disease or condition, symptoms of a disease or condition, or susceptibility to a disease or condition, with the aim of curing, healing, alleviating, relieving, altering, remedying, improving, enhancing, eliminating, or otherwise affecting the disease or condition, symptoms of a disease or condition, or susceptibility to a disease or condition. Treatment includes pharmaceutical compositions that can inhibit, reduce, decrease, or eliminate adverse (e.g., physical) symptoms, symptoms, diseases, or complications arising from or related to a disease, such as anemia, hypoxia-ischemia, stroke, dry eye syndrome, fibrinogenemia or coagulation disorders, orthopedic diseases, dental diseases, hypoendometrial hypoplasia, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, etc.

[0047] The phrases “therapeutic effective amount” and “effective dose” mean an amount sufficient to produce a therapeutically (e.g., clinically) desired outcome; such outcome could be stimulation of angiogenesis and blood flow (e.g., stimulation of endometrial growth), improvement of neurological recovery in a subject (e.g., mammals, including humans) from hypoxia and / or ischemia (e.g., hypoxic-ischemic encephalopathy), suppression of the immune system in a subject with an autoimmune condition, treatment of fibrinogenemia or coagulation defects in a subject, treatment of dry eye syndrome in a subject, treatment of orthopedic diseases in a subject, treatment of dental diseases in a subject, etc.

[0048] As used in this article, "spinal cord injury" and "SCI" ​​refer to damage to the spinal cord caused by trauma (including prolonged compression (greater than 20 minutes), contusion (greater than 0.5 m / sec), and local transection or severance) as well as non-traumatic causes (such as inflammation, infection, edema (swelling), toxins, hypoxia-ischemia due to cardiac arrest or aortic aneurysm, spinal stenosis (narrowing), demyelination, and tumor infiltration). Spinal cord compression arises from herniated discs, bone displacement, tumor growth, and adhesions leading to spinal cord indentation. Contusion is a rapid indentation of the spinal cord at a speed approaching or exceeding 0.5 m / sec, causing rapid tissue displacement to stretch and destroy cellular processes, including dendrites and axons. Local transection or severance is due to a penetrating wound invading the spinal cord. Inflammation can arise from autoimmune inflammatory diseases such as lupus, invasion of inflammatory cells (leukocytes) or infectious cells (bacteria, fungi, parasites, viruses), meningeal infections (meningitis), and loss of cerebrospinal fluid (CSF) flow due to adhesions or leakage. Toxins from many sources, including bacteria and fungi, can damage the spinal cord. Infections include tuberculosis, rabies, viruses, and fungi. Demyelination may occur due to the attack of toxins and autoimmune activity on oligodendrocytes. Tumor infiltration can occur in metastatic or intrinsic tumors of the central nervous system. When applied to spinal cord injury, the term "acute" refers to the early stage of injury or other events, while the term "chronic" refers to the sustained stable state following injury.

[0049] As used in this article, "hypoxic-ischemic encephalopathy" in the brain and spinal cord refers to reduced oxygenation and blood flow to the brain. The most common causes of hypoxia and ischemia in the brain or spinal cord are carotid artery occlusion due to aortic dissection or trauma, cardiac arrest, or aortic occlusion. In newborns, umbilical cord constriction during delivery and prolonged passage through the birth canal can lead to hypoxic-ischemic encephalopathy.

[0050] As used in this article, "stroke" refers to brain damage caused by the blockage, bleeding, or embolism of blood vessels in or leading to the brain. For example, cardiac arrest is not considered a "stroke," but rather hypoxic-ischemic stroke caused by the heart ceasing to pump blood. The incidence of stroke varies, ranging from 30 to 120 per 100,000 people per year in the 35-44 age group and from 670 to 970 per 100,000 people in the 65-70 age group. In the United States, approximately 800,000 people experience their first or recurrent stroke each year, and about 3% (approximately 7 million) of adults have already had a stroke (https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC3250269 / ). More than 80 million people worldwide suffer from stroke-related long-term neurological disorders.

[0051] As used in this article, "macular degeneration" refers to the degeneration of retinal neurons called ganglionic cells. The most common type is age-related macular degeneration (AMD), a leading cause of blindness worldwide, affecting more than 200 million people globally. In the United States, approximately 11 million people have macular degeneration. Other causes of macular degeneration include macular ischemia, which includes eye conditions related to diabetes and other causes of ganglionic cell loss. Treatments for macular ischemia include prevention of macular degeneration, regeneration of the optic nerve, or replacement of already degenerated retinal ganglionic neurons. It is important to note that other treatments include replacing the pigmented retinal epithelial cells that nourish the visual cells of the retina. Umbilical cord blood plasma and exosomes can stimulate the growth of retinal ganglia.

[0052] As used herein, the terms “dry eye syndrome” and “DES” refer to conditions in which eye drops fail to provide adequate or suitable lubrication to the eye. DES is associated with many inflammatory conditions, such as Sjogren's syndrome, rheumatoid arthritis, and collagen vascular disease. Most treatments for DES involve artificial tear replacement, surgical interventions to prevent eye discharge, and immunosuppressants such as cyclosporine A to suppress the immune response. Various components of umbilical cord blood (UCB), including umbilical cord blood exosomes and protoplasmic peptide decomposition (PDP), may be beneficial for DES.

[0053] As used in this article, the term "endometrial atrophy" refers to a thinning of the uterine lining. Normally, the endometrium is about 1 cm thick. However, in older women (over 35 years of age), postmenopausal women, women with ovarian dysfunction, women using oral contraceptives long-term, and women using tamoxifen, the endometrial thickness is less than 5 mm. In women with endometrial atrophy, in vitro fertilization (IVF) and implantation of fertilized eggs often fail. Umbilical cord blood exosomes contain vascular factors, including vascular endothelial growth factor (VEGF), which stimulate angiogenesis and endometrial growth to restore endometrial thickness for IVF.

[0054] The above summary is not intended to define every aspect of this disclosure, and other aspects are described in other parts, such as in the detailed description below. The entire document is interconnected as a whole, and it should be understood that all combinations of the features described herein are taken into account, even if such combinations of features do not appear together in the same sentence, paragraph, or section of this document. Other features and advantages of the invention will become apparent from the detailed description below. However, it should be understood that while the detailed description and specific embodiments show specific implementations of this disclosure, they are given by way of example only, as various changes and modifications within the spirit and scope of this disclosure will be apparent to those skilled in the art to which this detailed description pertains.

[0055] Detailed description of the attached diagram

[0056] Figure 1 The effects of adding UCB platelet-rich plasma (PRP) on HeLA cells with and without CaCl2 activation are shown. The left side of the figure shows the effects of 10% fetal bovine serum (FBS) and 10% adult human serum (HS). When PRP is applied to untreated medium, the effect on cell number at a concentration of 5% is similar to that of 10% FBS or 10% HS. However, higher concentrations of 10% or 20% PRP inhibited cell growth. After CaCl2 treatment, the addition of 5%, 10%, and 20% PRP (sample PRP 30) showed similar growth effects to 10% FBS or 10% HS at 7 days. Adding CaCl2 to PRP resulted in platelet precipitation. On the far left, after centrifuging (rotational sedimentation) of the CaCl2-treated PRP to allow the precipitate to settle, cell growth recovered to the effect seen without CaCl2 treatment. The data showed that adding 1% PRP to the culture medium significantly enhanced (tenfold) the growth-stimulating effect, equivalent to that of 10% FBS or 10% HS. However, higher concentrations of PRP, such as 10% and 20%, were observed to be less stimulating to growth than 1% PRP, 10% FBS, or 10% HS, suggesting that PRP contains substances that inhibit HeLA cell growth. This species is likely citrate-phosphate-dextrose (CPD), which binds calcium ions and reduces extracellular calcium ion activity to levels below those required for cell growth. Interestingly, centrifugation of the precipitate (likely CPD) restored the growth effect to levels before the addition of CaCl2. The addition of CaCl2 may activate platelets and release growth factors from them, thus contributing to cell growth.

[0057] Figure 2The graph shows the citrate concentrations in PRP and PPP after adding increased amounts of 5M CaCl2 (ranging from NT (untreated) to 20mM, 100mM, 250mM, and 500mM calcium concentrations). To assess the effect of adding 20–100mM CaCl2 on plasma citrate concentration, citrate levels (mM) were determined by ELISA. The leftmost portion of the graph (29PRP) shows NT with approximately 90mM citrate. Adding 20mM CaCl2 reduced citrate to 70mM, and adding 100mM CaCl2 reduced citrate to less than 10mM. In another sample (31PRP), adding 100mM CaCl2 reduced citrate to 20mM. In the third PPP sample (62PPP), adding 100mM CaCl2 resulted in no citrate. In the fourth PPP sample (64PPP), adding 100mM CaCl2 resulted in citrate less than 5mM. In the fifth PPP sample (65PPP), the addition of 100 mM CaCl2 resulted in citrate levels less than 10 mM. CPD-A1 was a citrate-phosphate-dextrose (CPD) solution placed in the collection bag.

[0058] Figure 3 The study demonstrates the precipitation of CPD from PRP and PPP with or without the addition of CaCl2 (NT), or with the addition of sufficient 5M CaCl2 (equivalents of 20mM, 50mM, and 100mM Ca concentrations). Two PRP (38PRP, 39PRP) and two PPP (61PPP, 67PPP) samples were treated with equivalents of 20, 50, and 100mM CaCl2. Increasing CaCl2 to 100mM resulted in citrate levels less than 5mM in the two PRP samples and less than 10mM in the two PPP samples. CPDA-1 is a positive control containing citrate-phosphate-dextrose.

[0059] Figure 4The growth of HeLa cells in cultures containing 1% to 10% activated platelet-depleted plasma (aPDP) was shown by adding 5M CaCl2 to an equivalent amount of 100mM. S1 refers to PDPs collected in October 2017 and then cryopreserved at -20°C until August 2020. S2 refers to PDPs collected in November 2019 and then cryopreserved at -20°C until August 2020. PDPs were thawed and activated by adding a solution equivalent to 100mM 5M CaCl2. FBS was a positive control, and 0% FBS was a negative control. Cell proliferation is expressed as cell counts starting at 3000 cells in 1%, 2.5%, 5%, and 10% aPDP at days 1, 2, 3, and 7 after initial culture.

[0060] Figure 5 This study demonstrates the effect of CaCl2 activation on HeLa cell growth using platelet-rich plasma (PRP) and platelet-poor plasma (PPP). PPP refers to plasma centrifuged to remove platelets. PRP does not require activation at lower concentrations (1-5%), but 10% PRP inhibits growth. In the presence of platelets, PRP at concentrations of 1%, 2.5%, and 5.0% increases cell count, and it appears that only 10% PRP is inhibited. The addition of 100 mM CaCl2 increases the growth rate. In the absence of platelets, 10% PPP does not stimulate as much cell growth without CaCl2 activation.

[0061] Figure 6 The number of human umbilical cord liner (HCL) mesenchymal cells grown in media containing PRP or PDP is shown. PDP is platelet-depleted plasma prepared by filtration through a microporous filter with 200 nm pores. Neither PRP nor PDP was as effective as 10% FBS in stimulating HCL mesenchymal cell proliferation. The effect of untreated PRP on 5% and 10% PRP concentrations was delayed and smaller compared to 10% FBS. After treatment with 100 mM CaCl2, aPDP improved cell growth only in media containing 5% and 10% aPDP. Untreated PDP was not effective in stimulating cell proliferation, but CaCl2-treated PDP was more effective.

[0062] Figure 7Growth of normal human dermal fibroblasts (NHDF) was observed. Untreated PPP moderately supported growth, but CaCl2-treated PDP improved growth at 5% and 10% PPP concentrations, with cell growth exceeding that of 10% FBS and a commercial growth factor mixture (GF). “Umbilical cord plasma” was purchased from Stem Cell Tech and had limited effect on cell growth. Neither FBS nor GF alone was as effective as CaCl2-treated PPP. However, FBS+GF stimulated growth to 44,000 cells at 7 days.

[0063] Figure 8 These are images of dot blot analysis of common exosome markers used in UCB exosomes. Positive responses include ICAM, ANXAS, TSG101, CD63, CD81, and FLOT1.

[0064] Figure 9 The effects of PDP and exosomes on HeLa cell culture are shown. On the right are the effects of PDP: 1%, 2.5%, 5%, and 10%, obtained from S1 and S2 umbilical cord blood plasma samples obtained since October 2017 and November 2019 and cryopreserved at -20°C, which were then used to promote HeLa cell growth. Even at 1%, aPDP significantly stimulated HeLa cell growth. On the left are various concentrations of exosomes (from 1.2 x 10⁻⁶). 8 Particle size up to 1.2x10 10 The application of PEG stimulated HeLa cell growth. PEG alone did not stimulate much growth. Invention Details

[0066] This disclosure describes a method for separating platelet-rich plasma (PRP) from UCB and peripheral blood.

[0067] This disclosure describes a method for isolating, activating, and cryopreserving UCB and peripheral blood.

[0068] This disclosure describes methods for generating platelet-depleted plasma (PDP) from UCB and for “activating” UCB PDP to stimulate cell growth in culture.

[0069] This disclosure also describes novel methods for the highly efficient isolation of exosomes from UCB, other biological fluids (including peripheral blood, cerebrospinal fluid (CSF), and urine), and conditioned media from cell cultures.

[0070] This disclosure also describes efficient methods for isolating globulins, albumins, and fibrinogen from UCB and peripheral blood.

[0071] This disclosure also describes cell culture media and methods for culturing mammalian (e.g., human) cells using freeze-dried platelets, PDPs, and exosomes.

[0072] This disclosure also describes pharmaceutical compositions comprising platelets, exosomes, albumin, and fibrinogen isolated by the methods described herein.

[0073] This disclosure also describes methods for regenerating tissue in subjects, for treating fibrinogenemia or coagulation defects in subjects, and for treating dry eye syndrome, orthopedic or dental diseases in subjects.

[0074] The following sections define the plasma components associated with the methods, kits, and compositions described herein.

[0075] UCB plasma components

[0076] Plasma is the clear extracellular fluid of UCB and peripheral blood. Plasma differs from serum, which is the supernatant of clotted blood. To prepare serum, blood is clotted, which results in the breakdown of blood cells. When the clot is centrifuged, the remaining supernatant is a pale yellow liquid containing both extracellular and intracellular fluid. In contrast, plasma is the supernatant of anticoagulated blood, prevented from clotting by chemicals such as citrate-phosphate-dextrose (CPD) or heparin, and contains only extracellular contents (including the anticoagulant) but not the intracellular contents of the clotted cells.

[0077] Depending on the hematocrit of the blood, the volume of UCB plasma ranges from 40-60% of the blood volume. When separated by centrifugation, the plasma contains platelets. Platelets are relatively light and tend to separate from the plasma after centrifugation, but due to their stickiness, up to 50% of platelets may become trapped in the RCF or MNC layer. If staged centrifugation is performed, where the blood is spun and agitated at a relatively low speed (100x g) to transfer platelets from one layer to the next, a second centrifugation at a higher speed can result in up to 80% of platelets being localized in the plasma layer.

[0078] platelets

[0079] Plasma with a high concentration of platelets is called platelet-rich plasma (PRP). Platelets are 2-3 μm in diameter and are filled with particles of growth factors and cytokines, which are released when platelets are activated. There are approximately 200,000 platelets per milliliter of adult blood. UCB platelet counts vary but should approximate the platelet count of adult blood. Platelet production is regulated by thrombopoietin. Each megakaryocyte produces approximately 1,000-3,000 platelets in its lifetime. An average of 100 billion (10^10) platelets are produced daily. 11Platelets are the "first responders" to injury. Tissue damage causes platelets to adhere to blood vessels, activating and releasing factors that attract stem cells and inflammatory / immune cells. Platelet lysates are used as a source of growth factors for cell culture.

[0080] exosomes

[0081] Exosomes are released by the placenta and UCB cells, particularly monocytes, macrophages, and platelets. They range in diameter from 30 nm to 150 nm. Larger EVs typically contain waste products from the cell. Exosomes form within multivesicular bodies (MVBs) within the cell. Exosomes carry "cargo," including μRNAs and transcription factors that signal to the uterus and fetus. Cells communicate with each other via exosomes. Exosomes differ from other EVs in that they possess an endosomal sorting complexes for transport (ESCRT) or soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor (SNARE) system for docking with cells. When exosomes dock with these receptors, they load their contents into the cell. The exosome membrane also contains cholesterol, sphingomyelin, ceramides, and phosphatidylserine, which distinguishes them from liposomes. Exosomes can contain both beneficial and harmful factors, including signals that lead to apoptosis.

[0082] plasma proteins

[0083] Four classes of proteins dominate umbilical cord plasma. The first is albumin, which accounts for more than half of plasma proteins (55.2%, 2.8-4.5 g / dL) with a molecular weight of 69 kDa. The second is fibrinogen, a precursor protein of fibrin that forms clots. Plasma also contains prothrombin, a precursor protein of thrombin that catalyzes clot formation. The third class is globulins, namely α-1-globulins (5.3%, including α1-antitrypsin, TBG, transcortin, etc.), α-2-globulins (8.6%, including hepatoglobulins, seruloplasmin, α2-macroglobulins, etc.), β-globulins (13.4%, including β1-transferrin, β-lipoprotein, etc.), and γ-globulins (11.0%, including antibodies IgA, IgG, and IgM). The total serum globulin concentration is approximately 0.42 g / dL. The fourth class is fibrinogen (6.5%), a blood clotting protein. Fibrinogen has a molecular weight of 340 kDa and six polypeptide chains. It can be converted into fibrin by thrombin and other coagulated fibrin.

[0084] Methods for separating and cryopreserving platelets from blood

[0085] In several implementations of platelet separation, the first stage of centrifugation should be performed at a low speed, producing approximately 100g, for 10-20 minutes. Adjust the speed to achieve the minimum centrifugation time required to separate RCF from MNC. The red color of RCF clearly distinguishes RCF and MNC through the "buffy coat layer" of MNC. Remove RCF from the bottom of the tube. The remaining MNC and PRP are sometimes referred to as white blood (WB). Agitation loosens the platelets adhering to the MNC to allow them to enter the PRP.

[0086] In several implementations of platelet separation, the second-stage centrifugation is performed at a higher speed, producing 500-1000 x g, for 20-30 minutes to separate the MNC and PRP. The MNC and PRP can be distinguished by light scattering. The PRP is removed from the top of the centrifuge to concentrate or separate the platelets. To concentrate the platelets, the PRP is placed in a separate centrifuge tube and centrifuged at high speed to 1000-3000 g's, causing the platelets to settle to the bottom of the tube. Removing the supernatant leaves a more concentrated platelet suspension at the bottom of the tube.

[0087] Filtration can be used to separate platelets from PRP. If the sample contains cell debris, EVs, and exosomes in the PRP, filtration can be performed in stages. Pre-filtration using a 1 μm filter helps clean the mixture, followed by a microporous filter with 0.22 μm pores. Alternatively, the PRP contains a mixture that can pass directly through the microporous filter with 0.22 μm pores. The microporous filter will capture all platelets, except for bacteria and most viruses. Platelets have a diameter of 2-3 μm and can pass through this filter.

[0088] In the methods described herein, the term "processing" refers to adding plasma filtrate to a solution containing PEG to isolate exosomes. Such a method may further include the steps of adding culture medium to a container to thaw and dissolve growth factors, thereby producing a growth solution containing growth factors, and removing the growth solution from the container. This growth solution can then be used in cell culture media and methods described in more detail below.

[0089] A method for isolating one or more of exosomes, globulins, fibrinogen, and albumin from a tissue source includes: providing a tissue source of exosomes, globulins, fibrinogen, and albumin; filtering the tissue source using a microporous filter having pores with a diameter of 0.22 μm to produce a plasma filtrate containing exosomes, globulins, fibrinogen, albumin, and CPD; adding the plasma filtrate to a solution containing PEG (e.g., sonicated PEG) in a first centrifuge tube, such that the exosomes are partitioned into PEG vesicles; cooling the first centrifuge tube to 2°C to 8°C (e.g., about 4°C); centrifuging the first centrifuge tube, such that the exosomes and PEG form a first precipitate and a first supernatant, and a second... A first supernatant contains albumin, globulin, CPD, and fibrinogen; the first supernatant is transferred to a second centrifuge tube, and a first precipitate containing exosomes and PEG is frozen for storage; a precipitant is mixed with the first supernatant to precipitate CPD; the mixture of precipitant and first supernatant is filtered to remove CPD, resulting in a second supernatant without CPD; the second supernatant is cooled to 2°C to 8°C (e.g., about 4°C) to form a cryoprecipitate containing fibrinogen; the cryoprecipitate is centrifuged in a third centrifuge tube to form a second precipitate and a third supernatant; the third supernatant containing albumin is removed, and the cryoprecipitate containing fibrinogen is freeze-dried; and the third supernatant containing albumin is freeze-dried. In this method, after separating exosomes by PEG, the remaining PPP is used to separate albumin, fibrinogen, globulin, and coagulation factors.

[0090] In methods for separating one or more of exosomes, globulins, fibrinogen, and albumin from a tissue source, any suitable tissue source (e.g., UCB) containing one or more of exosomes, globulins, fibrinogen, and albumin can be used. In typical methods, the tissue source is filtered (filtered) using a microporous filter having pores with a diameter of 0.22 μm. In several embodiments, any suitable filter that can separate particles larger than about 220 nm in diameter from the tissue source can be used. In several embodiments, the solution containing PEG can be a 10% PEG solution. In some embodiments, sonicated PEG is used. Sonicated PEG can be produced or commercially available using known methods. When the first centrifuge tube is centrifuged to form a first precipitate of exosomes and PEG and to form a first supernatant, the centrifugation speed is typically about 3000 g (typically about 1000 to 5000 rpm) and the centrifugation is performed for about 20 minutes (typically in the range of 5 to 30 minutes). In several embodiments, after this centrifugation, the first supernatant contains albumin, globulins, CPD, and fibrinogen. In this method, the precipitant is typically CaCl2, but any suitable precipitant can be used. After separating the exosomes, fibrinogen, and albumin, they are stored separately under appropriate conditions.

[0091] The disclosed method can be used to separate platelets, EVs, exosomes, globulins, fibrinogen, and albumin from UCB. Umbilical cord blood is an attractive source of platelets, EVs, exosomes, globulins, fibrinogen, albumin, as well as PRP, PDP, aPDP, etc.

[0092] The disclosed method can also be applied to isolate platelets, EVs, exosomes, globulins, fibrinogen, and albumin from tissues other than UCB. Non-limiting examples of such sources include peripheral blood, umbilical cord, umbilical cord stromal cells (Wharton's jelly), amniotic fluid which may include amniotic cells, placenta, umbilical cord lining, urine, and menstrual blood.

[0093] Plasma products are produced from platelet-rich plasma (PRP), which is generated by separating red blood cells (RBCs) or red blood cell fractions (RCFs) from monocytes (MNCs) and PRP (also known as white blood). The standard method for separating red blood cells (RBCs) from MNCs and PRP (called white blood) from UCBs and adult blood is to centrifuge the blood slowly to separate the RCFs from the WBs, and then centrifuge at a higher speed to separate the MNCs from the PRPs.

[0094] The following examples illustrate methods for separating PRP using two types of centrifuges:

[0095] 1. UCB can be centrifuged in a horizontal rotor with multiple tubes in a swinging stand. After 30 minutes, red blood cells (RBCs) will settle to the bottom of the tube to form erythrocyte fraction (RCF). Initial centrifugation is performed at a low speed, typically 900 rpm (11 cm rotor radius), producing approximately 100 x g, to separate the RCF from the MNCs and PRP or WB. Remove the RCF from the bottom of the tube and agitate the remaining MNCs and PRP so that any platelets adhering to the MNC layer can flow into the plasma. Centrifugation at 2000-2500 rpm (producing 492-769 x g) for 20-30 minutes will separate the MNC and PRP layers, which can then be removed individually.

[0096] 2. UCBs can be centrifuged in a rotary centrifuge. Red blood cells (RBCs) will form an outer layer of RCF against the inner surface of the cylinder, MNCs will form a middle layer, and PRP will form the innermost central zone of the cylinder. An upward piston in the cylinder pushes the rotating mixture upwards, while the central tube collects the cells. The rotary cylinder can be reversed during rotation to agitate the cells, causing attached platelets to detach and migrate into the plasma. PRP emerges first. A light scattering sensor can be used to determine when nucleated cells emerge and to replace the collection container to collect MNCs. Finally, RCF can be easily detected based on the red hemoglobin color of the RBCs.

[0097] In practice, a continuous two-stage centrifugation method with shaking between stages produces MNCs with less than 5% hematocrit and PRPs with more than 80% platelets. RFCs and MNCs are then cryopreserved separately. PRPs can be efficiently and rapidly separated into several components with important therapeutic effects, particularly freeze-dried platelets (FDP), platelet-depleted plasma (PDP), exosomes, exosome-rich plasma (ERP), as well as plasma globulins, albumins, and clotting proteins. The centrifugation method can be modified to separate tissue components and other biological fluids into processable components.

[0098] Filtering PRP using a microporous filter with 0.22 μm pores will extract 99% of platelets from PRP, along with most bacteria and viruses. The resulting sterile filtrate is called platelet-depleted plasma (PDP). Depending on the source, tissue solutions or PRP may require centrifugation or pre-filtration to avoid clogging the microporous filter. While filtration has long been used to separate platelets (cells and other biological components), the novelty of the described method lies in freeze-drying (lyophilization) the platelets to break down their particles into soluble proteins.

[0099] Filters containing freeze-dried platelet powder can be stored in capped, sterile microporous filters, sealed in plastic, and stored for years at -20°C. When stimulation of cell growth in culture is needed, culture medium can be injected through the filter, which dissolves the freeze-dried particulate chemicals and sterilizes the filtrate. The number of platelets on each filter can be estimated using platelet concentration and the volume of PRP filtered.

[0100] The filtrate produced by microfiltration of PRP is called platelet-depleted plasma (PDP). In other embodiments, centrifugation can be used to separate platelets. At high-speed centrifugation (greater than 2500 x g) of PRP, platelets will form a precipitate. Since centrifugation may not precipitate all platelets, the supernatant of centrifuged PRP is platelet-depleted plasma (PPP), not PDP. Figure 5 As shown. Freeze-dry the precipitate or store it in a sealed vial.

[0101] Passing phosphate-buffered saline or cell culture medium through a microporous filter containing lyophilized platelets will extract soluble proteins, including growth factors and cytokines. Using a 0.22 μm microporous filter offers several advantages. First, sterile microporous filters are widely available and inexpensive. Second, the 0.22 μm microporous filter removes most cell debris and captures EVs larger than 0.22 μm. Third, lyophilized microporous filters containing platelets can be stored at -20°C or lower without degradation. Finally, to use lyophilized platelets, the user simply injects room temperature culture medium through the filter to dissolve the frozen proteins, and the filtrate is sterile.

[0102] Most commercial microporous filters have a sterile housing designed to fit syringes with a Luer lock. The filter can be freeze-dried directly within its housing, capped, vacuum-sealed with a plastic wrapping membrane, and stored at -20°C. Injecting culture medium through the filter dissolves the freeze-dried growth factors and cytokines directly into the culture medium container. In other embodiments, the freeze-dried filter can be placed in a sterile vial. Culture solution is injected into the vial to dissolve the filter material, producing a stock solution of growth factors and cytokines, which are then injected into the culture medium. Another method is to remove the filter from its housing and place it in a vial, then vacuum-seal it for more efficient storage.

[0103] Freeze-dried platelets offer several advantages over traditional methods that involve freezing and thawing platelets 3-4 times to lyse them. Platelets are activated upon contact with collagen and von Willebrand factor, thrombin, or negatively charged surfaces such as glass. Platelets possess protease-activating receptors (PARs), which can be activated via multiple pathways (Yun SH, et al. (2016). Biomed. Res. Intl. 9060143). Traditional freeze-thaw methods require hours and are energy-intensive. The final product is a frozen or wet plasma of partially lysed platelets, which may not be as durable for cryopreservation as freeze-dried material. Freeze-dried material can be rapidly and easily quantified by weight.

[0104] Activate PDPs for use as a growth medium in cell culture.

[0105] UCB contains a high concentration of the anticoagulant citrate-phosphate-dextrose (CPD). Both citrate and phosphate bind to calcium ions, reducing calcium ion activity in the culture medium and inhibiting cell growth, which requires calcium ions. When UCB is added to the culture medium at a concentration higher than 5% (ml / dL), the amount of CPD will reduce the extracellular calcium concentration, which is sufficient to inhibit cell growth. For this reason, calcium ions must first be precipitated by PDP or PRP before being added to the culture medium to stimulate cell growth.

[0106] Some researchers add calcium chloride (CaCl2) to PDPs or PRPs to precipitate CPD. This is sometimes referred to as “activation” of the PDP or PRP. Adding CaCl2 not only precipitates CPD but also other calcium-binding proteins and platelets. An unusually high amount of CaCl2 must be added to the PDP or PRP to precipitate enough CPD to “activate” the PDP or PRP to stimulate cell growth. To determine the amount of 5M CaCl2 solution added to the PDP or PRP, the citrate concentration in the PDP or PRP is measured (using an ELISA with an antibody that binds to citrate), while equivalent amounts of 20mM, 50mM, and 100mM CaCl2 concentrations are added. Figure 3 As shown, the amount of 5M CaCl2 added up to 100 mM is necessary to reduce the citrate concentration to near zero.

[0107] Adding CaCl2 to PRP causes many other calcium-binding proteins and molecules, as well as platelets, to precipitate. For this reason, platelets should be filtered before adding CaCl2, and the precipitate should be removed by centrifugation or filtration before use in cell culture media. Activated PDP is very effective in stimulating cell growth, even at concentrations of 1% or lower in the culture medium. Figure 1-5The effects of different aPDP concentrations on HeLa cell culture were shown. Activated PDP also promoted the growth of human umbilical cord liner cells (…). Figure 6 ) and normal human dermal fibroblasts ( Figure 7 ) growth.

[0108] Methods for isolating and cryopreserving exosomes from platelet-depleted plasma

[0109] Platelet-depleted plasma (PDP) obtained through filtration or platelet-poor plasma (PPP) obtained through centrifugation contains exosomes and plasma proteins, providing the basic material for exosome extraction. Traditional methods for isolating exosomes from plasma and tissues are inefficient. These methods fall into two categories:

[0110] 1. Ultracentrifugation. Exosomes are membrane-enclosed extracellular vesicles containing mRNA, μRNA, and nuclear factors. Exosomes have proteins on their surface that bind to receptors on target cells and load their contents into the cells. Due to their low density, similar to plasma, exosomes require ultracentrifugation at 100,000 x g for 24–48 hours in a hyperosmolar solution (sucrose gradient) to separate them from plasma. The osmotic pressure and centrifugal force of the solution damage exosomes, reducing the number of recoverable, intact exosomes.

[0111] 2. Exosomes are partitioned into hydrophobic environments. Surface proteins bind water and increase the solubility of exosomes in aqueous environments. If water is removed from the exosome surface, the exosomes will preferentially partition into more hydrophobic environments, such as polyethylene glycol (PEG). PEG is partially soluble in water, ethanol, acetonitrile, benzene, and dichloromethane, but insoluble in more hydrophobic environments, such as ether or hexane.

[0112] Commercial kits for exosomes use PEG, but collect less than 20% of exosomes. In 2016, Deregibus et al. (Deregibus, et al., 2016) used protamine to bind negatively charged proteins and capture more exosomes (Genschmer, et al., 2019). Fujifilm 1 Using magnetic beads to capture exosomes via phosphatidylserine receptors (Miyanishi, et al., 2007; Nakai, et al., 2016).

[0113] Most commercial kits prepare a 10% PEG solution (10 ml PEG to 90 ml phosphate-buffered saline) and mix it using a vortex apparatus. The solution appears cloudy due to the PEG bubbles. In these kits, PDP containing exosomes is then added to the 10% PEG solution. Typically, 10-20% of the exosomes will partition into the PEG after one or two hours at room temperature. The mixture is centrifuged at 2500 x g, and due to the high molecular weight of PEG, the high density of exosomes in the PEG will form a precipitate at the bottom of the tube. The supernatant is removed, and PBS or PDP is added to the precipitate to obtain exosome-rich plasma (ERP).

[0114] The novel method described in this paper differs in several aspects. For example, sonication is used to break down PEG into microvesicles with a much larger surface-to-volume ratio, which significantly increases the interaction between exosomes and vesicles. For instance, mixing 50 ml of PEG (10,000 molecular weight) into 50 ml of PBS yields a 50% PEG solution. This solution is turbid under light because PEG is in the form of bubbles with a diameter greater than 340 nm. Sonication of the mixture rapidly clarifies the solution because the sonicated microvesicles are smaller than 340 nm in diameter and therefore invisible.

[0115] Another example involves cooling the solution to 4°C and storing it in a refrigerator overnight. As the temperature approaches 0°C, water tends to leave the exosome surface to participate in ice formation, thereby reducing the solubility of exosomes in the aqueous phase and increasing exosome migration into PEG microbubbles. After 8 hours at 4°C, more than 90% of the exosomes had partitioned into the PEG.

[0116] Another example involves centrifuging the PEG mixture at 2500x g for 30 minutes, removing the supernatant, and then freezing the exosomes in the PEG to -170°C. PEG solutions are used in automotive antifreeze to prevent ice formation that could damage exosomes. When the cryoprecipitate of exosomes (exosome precipitate) thaws at room temperature, PBS or PDP is added to produce exosome-rich plasma with an appropriate exosome concentration.

[0117] Methods for isolating globulins and albumin / fibrinogen from exosome-depleted plasma

[0118] Arranged by prevalence, exosome-depleted plasma (EDP) mainly contains albumin, globulin, and fibrinogen.

[0119] i. Albumin is the most abundant protein in umbilical cord blood and peripheral blood, accounting for more than 50% of plasma proteins. Growth factors, hormones, and cytokines are bound to albumin.

[0120] ii. Globulins include immunoglobulins, such as IgA, IgG, and IgM, which are frequently used to treat autoimmune diseases. Many globulins are signaling molecules. Some globulins are cryoglobulins, which bind to and cross-link with other globulins.

[0121] iii. Fibrinogen, a clotting protein, is widely available from peripheral blood and should not differ significantly from adult fibrinogen. However, it can be easily removed by inducing clot formation and centrifuging the clot. This can be accomplished by adding thrombin and calcium.

[0122] Because the second step in exosome isolation involves cooling the PDP to 4°C overnight and centrifuging the mixture to form a PEG precipitate, most of the globulins will be cooled to the point of gel formation. The globulin gel can be poured into vials, followed by the transfer of the albumin-fibrinogen solution into the vials, and both the gel and albumin-fibrinogen can be freeze-dried. The PEG precipitate containing exosomes (exosome precipitate) can then be placed in vials and frozen.

[0123] If the user wants to obtain albumin without fibrinogen, this can be done by adding thrombin solution and inducing centrifugation, centrifuging the clot, and using the supernatant containing mainly albumin. PDP can, of course, be used directly as a growth medium and added directly to the medium, like FBS. Exosomes, albumin, and freeze-dried platelets can also be added to the medium individually or together.

[0124] In other embodiments, all of the above-mentioned components of plasma globulin and albumin-fibrinogen can be used for research and clinical treatment.

[0125] Methods of culturing mammalian cells using UCB plasma fractions

[0126] An embodiment of the method for culturing mammalian cells includes culturing a population of mammalian cells in a cell culture medium containing a growth solution prepared according to the method described above. The growth solution may contain PRP, PDP, lyophilized platelets, exosomes, and / or albumin and / or growth factors. In the method, the mammalian cells are typically human cells, and the tissue source is typically UCB. The methods and growth solutions described herein can be used to culture any type of mammalian cell, including, as non-limiting examples, HeLa cells, mesenchymal stem cells, normal dermal cells, hepatocytes, and fertilized eggs.

[0127] The term "culture" refers to maintaining cells under conditions in which they can proliferate. For example, cells are cultured in a medium containing a UCB component and additional growth factors or cytokines (i.e., a mixture of growth factors or cytokines). Methods for culturing and growing mammalian cells are well known in the art.

[0128] According to the methods, cell culture media, and growth solutions described herein, CaCl2-treated UCB PDP can be used as a culture medium for culturing human cells for transplantation. UCB PDP treated with an equivalent of 100 mM CaCl2 is an effective and safe growth adjuvant for human cell culture. UCB is more sterile than adult serum, which can be infected with many diseases. This is partly due to the maternal-placental barrier excluding many maternal infections from the fetus during pregnancy. Collected UCB units are tested for many common diseases, and maternal blood is also tested for antibodies against common diseases. UCB PDP is likely the safest source of human plasma for use in the growth of transplanted cells.

[0129] In some implementations, some primary human cells may require the addition of additives to the growth medium described herein. In such implementations, the UCB PDP growth solution described herein serves as an excellent base for the addition of additional factors. This approach provides an economical and efficient allogeneic-free growth medium for growing human cells for transplantation. For cells that do not grow well in activated PDP, the addition of platelets may be helpful. For certain cells, such as UCB and bone marrow cells, certain growth factors, cytokines, and other factors (e.g., lithium—see U.S. Patent 8,852,938, which is incorporated herein by reference) may be useful.

[0130] UCB has several key advantages over FBS obtained from cattle, such as as a source of growth factors for cell culture. First, FBS has become increasingly expensive due to "mad cow disease," or bovine spongiform encephalopathy (BSE), affecting cattle in the Americas, Europe, and Africa. Currently, the only certified sources of BSE-free FBS are Australia and New Zealand. If BSE were to occur in either of these countries, the global supply of FBS could be severely reduced. However, UCB PDP is available because over 30 million babies are born each year. The average size of a unit of cord blood is 80 ml, and the plasma volume is approximately 40 ml, or about 1.2 billion liters of UCB plasma, which is more than sufficient to meet the needs of cell culture. In India, the predominantly Hindu population, who consider cows sacred, abhors the use of FBS for cell culture, as this would require harvesting serum from 7 million calves of pregnant cows. With 90 million babies born daily in India, this is more than enough to meet the cell culture industry's demand for all growth factors. India currently collects only about 5% of its cord blood. If 20% of umbilical cord blood or 3.6 million ml of plasma were collected in India daily, it would be more than sufficient to meet the current global demand for FBS in cell culture. Since the method described in this paper will replace 10% FBS with 1-5% UCB PDP, it is estimated that UCB PDP could replace FBS at a cost of $20 / ml; the current cost of FBS is approximately $5 / ml.

[0131] Compositions and treatment methods

[0132] Platelets, exosomes, globulins, fibrinogen, and albumin isolated using the methods described above have beneficial effects in many conditions and can be applied in many ways. For example, exosomes promote the repair of tissue damage, including neurodegeneration and autoimmune diseases. UCB globulin can be used as an alternative to intravenous immunoglobulin therapy. Fibrinogen and coagulation factors are used to treat patients with coagulation defects and can be used as surgical glue. Albumin is a useful excipient for transporting cells.

[0133] This disclosure also provides pharmaceutical compositions comprising freeze-dried platelets, exosomes, fibrinogen, and albumin isolated by the methods described above. The pharmaceutical compositions may contain a suitable medium for delivering platelets, exosomes, fibrinogen, and / or albumin to a subject in need. In some embodiments, the compositions may also contain a cryopreservative and / or a pharmaceutically acceptable carrier.

[0134] Therefore, this disclosure provides a method for regenerating tissue in a subject. The method includes administering to the subject an effective amount of exosomes isolated by the method described herein. In this method, the subject is typically a person suffering from at least one of the following conditions: tissue damage, degeneration, central nervous system injury and degeneration, and cardiovascular degeneration.

[0135] This disclosure also provides a method for treating fibrinogenemia or coagulation defects in a subject. The method includes administering to a subject suffering from fibrinogenemia or coagulation defects an effective amount of fibrinogen isolated by the method described herein.

[0136] This disclosure further provides a method for treating a subject with dry eye syndrome, orthopedic disease, or dental disease. The method includes administering to a subject suffering from dry eye syndrome, orthopedic disease, an effective amount of platelets separated by the method described herein.

[0137] Dosage and frequency of administration will depend on clinical signs confirming remission, with at least one, or more preferably, a reduction or absence of acute-phase clinical signs known to those skilled in the art. More generally, dosage and frequency will depend in part on the pathological signs of the disease condition or symptom to which the composition is intended for treatment, as well as the reduction of clinical and subclinical symptoms. Those skilled in the art will understand that dosage and administration regimens can be adjusted based on the age, sex, physical condition, benefits of the conjugate, side effects in the treated patient or mammalian subject, and the physician's judgment.

[0138] As used herein, the term "subject" refers to a vertebrate, and in some exemplary respects, to a mammal. Such mammals include, but are not limited to, rodent mammals such as mice and rats, and lagomorph mammals such as rabbits, carnivorous mammals including felines (cats) and canines (dogs), even-toed ungulates including bovines (cows) and suidae (pigs), or odd-toed ungulates including equines (horses), primates such as ceboids or simoids (monkeys), and ape mammals (humans and apes). In several exemplary respects, the mammal is a mouse. In many more exemplary respects, the mammal is a human.

[0139] As used herein, the term "application" refers to the delivery of exosomes, fibrinogen, or platelets via various routes, including but not limited to oral, intranasal, intraocular, intravenous, intraosseous, intraperitoneal, intraspinal, intramuscular, intra-articular, intraventricular, intracranial, intralesional, intratracheal, intrathecal, subcutaneous, intradermal, transdermal, or transmucosal administration.

[0140] As used herein, the term "effective amount" or "therapeutic effective amount" refers to an amount that results in a measurable improvement in at least one symptom or parameter of a particular disease. Therapeutic effective amounts of the aforementioned plasma exosomes, platelets, extracellular vesicles, fibrinogen, and coagulation factors can be determined by methods known in the art. Effective amounts for treating a disease can be determined by empirical methods known to those skilled in the art. The exact amount administered to a patient will vary depending on the state and severity of the disease and the patient's physical condition. Measurable improvement in any symptom or parameter can be determined by those skilled in the art or reported by the patient to a physician. It should be understood that any clinically or statistically significant reduction or improvement in any symptom or parameter of the aforementioned disease is within the scope of this invention. Clinically significant reduction or improvement means perceptible to the patient and / or physician.

[0141] Pharmaceutical compositions can be prepared by mixing a therapeutically effective amount of exosomes, fibrinogen, or platelets, along with optional other active agents / compounds, with a pharmaceutically acceptable carrier. A carrier is a diluent, excipient, or medium that is administered with the therapeutic agent. Carriers can take various forms, depending on the route of administration. Carriers can be sterile liquids, such as water and oil. Water or aqueous solutions, saline solutions, and aqueous solutions of dextrorotatory glucose and glycerol are preferred as carriers, especially for injectable solutions. Suitable pharmaceutical carriers are described in EW Martin's "Remington's Pharmaceutical Sciences," 18th edition. For example, compositions can be prepared by mixing with conventional pharmaceutical excipients and by conventional preparation methods. Excipients can be mixed with disintegrants, solvents, granulating agents, humectants, and binders.

[0142] The phrase "pharmaceutically acceptable" refers to molecular entities and other components of such compositions that are physiologically tolerable when administered to humans and generally do not produce undesirable reactions. Preferably, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other recognized pharmacopoeia for use in mammals, more specifically in humans. Pharmaceutically acceptable salts, esters, amides, and prodrugs are those salts (e.g., carboxylates, amino acid addition salts), esters, amides, and prodrugs that, within reasonable medical judgment, are suitable for use in contact with patient tissues without excessive toxicity, irritation, allergic reactions, etc., in proportion to a reasonable benefit / risk ratio, and effective for their intended use.

[0143] The use of any and all instances or exemplary language (e.g., “for example”) provided herein is intended only to better illustrate the invention and does not constitute a limitation on the scope of the invention, unless otherwise stated. No language in the specification should be construed as indicating that any unclaimed element is essential to the implementation of the invention.

[0144] All methods described herein may be performed in any suitable order unless otherwise stated herein or clearly contradicted by the context. With respect to any method provided, the steps of the method may occur simultaneously or sequentially. When the steps of the method occur sequentially, they may occur in any order unless otherwise stated.

[0145] Where a method comprises a combination of steps, all kinds and every combination or subcombination of steps are covered within the scope of this disclosure unless otherwise stated herein.

[0146] Each publication, patent application, patent, and other reference cited herein is incorporated in its entirety by reference to the extent that it is not inconsistent with this disclosure. Publications disclosed herein are provided only because they were published prior to the filing date of this invention. Nothing herein should be construed as an admission that this invention is not entitled to precedence over such publications. Furthermore, the publication dates provided may differ from the actual publication dates, which may require separate verification.

[0147] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or variations thereof are conceived by those skilled in the art and are included within the spirit and scope of this application and the appended claims.

[0148] UCB platelet-depleted plasma and exosomes

[0149] UCB plasma and exosomes differ from adult blood plasma and exosomes in several important ways. UCBs are produced by the placenta and are the primary means of communication between the placenta, uterus, and fetus, transmitting information through hormones, cytokines, chemokines, and exosomes in the plasma. Therefore, UCB plasma contains factors not found in adult blood. UCBs also contain more and more diverse stem cells and progenitor cells than adult blood, including a higher proportion of monocytes. Monocytes are progenitor cells of macrophages and dendritic cells. These cells secrete factors and exosomes different from those in adult blood. UCB platelets and plasma can be used as an alternative to FBS as a growth medium for the culture of human mesenchymal stem cells (MSCs). UCB plasma is known to contain very high concentrations of transforming growth factor β-1 (TGF-β1), vascular endothelial growth factor (VEGF), platelet-derived growth factors (PDGF-AAA, PDGF-BBB), and endothelial growth factor (EGF), as well as chemokines (RANTES, GRO, MCP-1, Eotaxin, Fractakline) and immune factors (IL-12p70, IFNy, IL-4, IL-17A, IL-10, and IL-13). Many of these factors are used in specific culture media for culturing human MSCs. The main problem with using UCB plasma as a growth medium is the presence of CPD in cord blood. CPD is an anticoagulant that binds to and reduces calcium ion activity sufficiently to cause blood clotting to cease. Reduced calcium ion activity also leads to cell growth arrest. This is why UCB plasma only promotes growth when sufficient CaCl2 is used to restore calcium to the levels required for cell growth. In the experiments described below, the addition of 20 mM CaCl2 was insufficient to bind and precipitate CPD in the culture, while the equivalent addition of 100 mM CaCl2 restored calcium ion activity to a level sufficient for cell growth. These results indicate that 1% “activated” PDP stimulates HeLa cell growth to the same extent as 10% FBS.

[0150] The experiments described below show that once calcium ion activity is restored, growth factors, chemokines, and immune factors known to be present in plasma will stimulate cell growth. The experiments also show that exosomes in PDPs promote growth, as 1.2 x 10⁻⁶ per milliliter... 11Adding PDP particles at a concentration of 10% to the culture medium also stimulated cell growth, although to a lesser extent than 10% PDP. Treatment of PDP with an equivalent amount of 100 mM CaCl2 eliminated the cell growth inhibitory effect of PDP and demonstrated that PDP strongly stimulated HeLa cell growth. HeLa cells require growth factors to grow because they will not grow on culture medium alone. Most laboratories currently use FBS as the source of growth medium for culturing cells. Numerous studies have shown that cells grown in FBS express bovine proteins and are immune rejected. The U.S. Food and Drug Administration (FDA) recommends that cells intended for human use should not be grown or stored in media containing animal proteins, especially FBS, because more than half of people have antibodies against FBS due to milk consumption. The only source of human growth factors is lysed platelets from adult blood. The novel approach described in this article incorporates UCB PDP as a better and safer source.

[0151] One example of a condition where UCB exosomes can play a major beneficial role in treatment is hypoxic-ischemic encephalopathy (HIE), a condition affecting approximately 0.4% of infants due to the inability to deliver oxygen to the infant because umbilical cord blood is wrapped around the neck, travels slowly and for a long time through the birth canal, or is severely anemic. Exosomes can cross the blood-brain barrier (BBB) ​​and can be administered intravenously (Perets et al. Nano Lett. 2019; 19(6):3422-31; Osorio-Querejeta et al., Front Mol Neurosci. 2018; 11:434). Several clinical trials (Tsuji et al. Sci Rep. 2020; 10(1):4603) and animal studies have shown that infusion of UCB in newborns with HIE can improve neurological recovery from HIE. Another example of a condition where UCB exosomes can provide a therapeutic benefit is stroke. A third example of a condition where UCB exosomes can be beneficial in treatment is hypo-endometria, meaning a thin endometrial lining, which is a major reason why fertilized eggs cannot implant in women with a thin endometrial lining of less than 0.5 cm. Because UCB exosomes carry many signals that stimulate angiogenesis and blood flow, placing UCB exosomes into the uterine cavity can stimulate endometrial growth. This can be done during oocyte retrieval from the ovary. Concentrated exosome-rich plasma (ERP) can be injected into the uterine cavity one week before implantation. The endometrial lining can be easily monitored by ultrasound. A fourth example of a condition that can be treated with UCB exosomes is autoimmune diseases. UCB exosomes contain many factors that suppress the immune system; therefore, injection of UCB exosomes isolated according to the methods described herein can suppress autoimmune conditions such as multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, and others. Intravenous injection of UCB exosomes can alleviate acute autoimmune attacks. Example

[0152] Example 1

[0153] i. Separation of platelets, EVs, exosomes, albumin, fibrinogen, globulins and coagulation factors

[0154] Examples of novel methods for isolating platelets, EVs, and exosomes from UCB PRP include:

[0155] (a) A method for separating platelets and extracellular vesicles and forming freeze-dried platelets. PRP is filtered through a 0.22 μ filter to capture platelets and extracellular vesicles, which are then freeze-dried as a source of growth factors.

[0156] (b) A method for isolating exosomes and precipitating the anticoagulant CPD. To isolate exosomes, platelet-depleted plasma (PDP) is added to a solution of 10% PEG and 100 mM CaCl2. The exosomes partition into the PEG, while the CaCl2 precipitates the anticoagulant CPD. The solution is cooled to 4°C and centrifuged at 3000 g for 20 minutes.

[0157] (c) A method for separating albumin, fibrinogen, globulins, and coagulation factors. Cooling PPP to 4°C activates temperature-sensitive globulins in plasma to bind and cross-link fibrinogen and fibrin. The cryoprecipitate of fibrinogen, globulins, and other coagulation factors is gel-like. The supernatant containing albumin and other plasma proteins is poured into a separate container for freeze-drying. The cryoprecipitate is aspirated into another container for freeze-drying. The exosome / PEG precipitate is frozen at -20°C, with PEG protecting the exosomes from freeze-drying damage.

[0158] Example 2

[0159] ii. UCB freeze-dried platelets

[0160] In a novel method for separating UCB from PRP, UCB is first centrifuged in stages using a device such as Syngen (Thermogenesis) to separate it into PRP, MNC, and RCF, maximizing platelet concentration in the plasma. The PRP layer is extracted using a 60 ml syringe and filtered through a 0.22 μMillipore filter to remove particles larger than 220 nm in diameter. The Millipore filter containing EVs and platelets is then placed in a sterile container and freeze-dried to release the growth factors. The container can be stored at -20°C.

[0161] In this method, filtration and freeze-drying of platelets are novel. In contrast, most laboratories and industries centrifuge PRP to collect platelets, remove the supernatant, and then repeatedly freeze-thaw the precipitate to release growth factors. Filtration collects platelets containing growth factors and EVs. In the method described herein, freeze-drying releases and concentrates growth factors better than repeated freeze-thaw cycles. This process requires less handling of the sample and can be performed in a completely closed system. To thaw and dissolve the freeze-dried growth factors, culture medium is injected into the container to dissolve the growth factors on the filter, and the resulting growth solution is then removed from the container and placed in a cell culture dish.

[0162] Example 3

[0163] UCB plasma exosomes, fibrinogen, and albumin

[0164] In a novel method for separating plasma exosomes, fibrinogen, and albumin from UCB PRP, PDP was added to centrifuge tubes containing 10% polyethylene glycol (PEG, 10,000 MW) and 100 mM CaCl2 solution and cooled to 4°C. Exosomes partitioned into the PEG, while CaCl2 precipitated the CPD, and cooling activated cold-sensitive globulins in the plasma, which bound fibrinogen to form a cryoprecipitate. After centrifugation at 3000 g for 20 min, exosomes formed a PEG precipitate at the bottom of the tube, a cold-induced globulin gel, and the supernatant contained albumin and other plasma proteins. After discarding the supernatant, the gel-like cryoprecipitate was aspirated and freeze-dried to obtain fibrinogen and coagulation factors. The exosome precipitate could be frozen at -20°C, with PEG used as a cryopreservative.

[0165] This novel method uses a combination of PEG and CaCl2 to separate exosomes and precipitate CPD in a short (20 min) and low-speed centrifugation step. The new method offers the following advantages:

[0166] i. The process precipitates CPD and separates exosomes in a single step. Due to low-speed centrifugation, a large volume can be processed at once, i.e., 60 ml from a single unit of plasma. This is far more efficient than repeatedly spinning small volumes of plasma at 120,000 g for 18 hours.

[0167] ii. PEG will prevent exosome crystallization and cryopreserve the exosomes.

[0168] iii. The process separates fibrinogen and albumin for freeze-drying.

[0169] Example 4

[0170] Exosomes were isolated from UCB and PDP.

[0171] Centrifugation of UCB separates the three main components of UCB: PRP, monocytes (MNCs), and RCF. PRP is separated from the UCB and filtered through a 0.22 μm microporous filter to remove platelets, producing PDP. The filter removes platelets larger than 220 nm in diameter and extracellular vesicles. Exosomes are retained. In one embodiment of the method for producing PDP, UCB is thawed, centrifuged to produce a PRP fraction, the PRP fraction is separated, and platelets are removed by centrifugation of the PPP or by filtering the PDP and cell debris, as well as extracellular vesicles larger than 220 nm, producing a PDP fraction containing CDP, exosomes, and plasma proteins.

[0172] Depending on the platelet concentration in the PRP, the platelet precipitate can be obtained by centrifugation using a 0.22 μm filter. This precipitate is then filtered through a 1.0 μm filter to remove platelets, followed by filtration through a 0.22 μm filter to remove extracellular vesicles larger than 220 nm in diameter. The centrifuged or filtered platelets are then freeze-dried to ensure all growth factors are frozen and soluble in the culture medium.

[0173] Umbilical cord blood was anticoagulated using a CPD that reduces extracellular calcium activity in the cell culture medium. CPD prevents clotting by binding calcium ions and reducing extracellular calcium ion activity below the level required for clotting. This reduction in calcium ion activity is sufficient to inhibit cell growth in the culture. To reduce CPD to a level that does not inhibit cell growth, 20 mM CaCl2 was added to the PRP. CaCl2 binds to the CPD. Results are as follows... Figure 1 As shown.

[0174] Adding 20 mM CaCl2 to PRP has complex effects on HeLa cell growth. Figure 1 In the left-hand panel, 10% FBS and 10% human serum (HS) resulted in rapid cell growth on days 1, 2, 4, and 7 after plating. When UCB PRP was added, the effect of a 5% PRP concentration was delayed, but reached the same level at day 7. However, 10–20% PRP concentrations inhibited cell growth. This can be interpreted as meaning that 5% PRP stimulated the growth of HeLa cells in culture to the same extent as 10% FBS or 10% HS, but higher concentrations of 10–20% PRP inhibited growth because the added CPD was sufficient to reduce extracellular calcium, thus preventing cell growth.

[0175] Adding 20 mM CaCl2 to PRP restored cell growth at 10 mM and 20 mM PRP levels. However, when the CaCl2-treated PRP was centrifuged (swirl-down), growth inhibition at 10% and 20% PRP concentrations was reversed. This is likely because CPD binds calcium ions and reduces extracellular calcium activity, thereby inhibiting cell growth. Adding 20 mM CaCl2 prevented this inhibition, but it was clearly insufficient, as centrifugation of the CaCl2-treated PRP and removal of the precipitate restored the growth inhibition at 10-20% PRP levels. Figure 1 (Rightmost part)

[0176] Effects of untreated and CaCl2-treated PDP and PRP on cell growth in cultures:

[0177] Complex effects of adding 20 mM CaCl2 to PRP and the discovery that vortex precipitate restored 10-20% of the growth inhibition of CaCl2-treated PRP. Figure 1 (Rightmost part) This indicates that 20 mM did not eliminate all CPD. To determine whether CPD has been eliminated, extracellular calcium cannot be measured using a calcium-specific ion-selective electrode because CPD interferes with calcium ion activity assays. Instead, enzyme-linked immunosorbent assay (ELISA) is used to determine citrate concentration. Increasing amounts of CaCl2 are added to raise the CaCl2 concentration to 20, 100, 250, and 500 mM, which will show which amount of CaCl2 addition reduces citrate levels to near zero. Figure 2 These results are shown.

[0178] CaCl2 activation of PRP and PDP:

[0179] Figure 2 The experimental results shown indicate that adding CaCl2 to raise the plasma Ca concentration to an equivalent amount of 100 mM will reduce citrate levels in PRP to low levels and in PPP to near-zero levels. PPP refers to platelets that have been centrifuged to precipitate platelets and the supernatant removed, which may contain some platelet residue. PDP refers to filtered plasma with all platelets removed. Although some citrate may still be present in the plasma after adding an equivalent amount of 100 mM citrate, the amount of citrate is insufficient to reduce the Ca ion concentration to a level sufficient to inhibit cell growth. To minimize the volume added to the solution, the highest concentration of CaCl2 (5 M) was used to add calcium to the plasma.

[0180] Please note that the addition of CaCl2 to precipitate CPD in PRP or PPP is only necessary when PDP is used to provide growth medium for cell culture. If PDP is injected intravenously into the human body, the amount of calcium in the body fluids is more than sufficient to neutralize CPD. Note that even when a comparable amount of CaCl2 (500 mM) is added, it does not eliminate all citrate, indicating that the assay does not accurately measure zero citrate. To confirm, repeat the experiment to measure citrate after adding sufficient CaCl2 to reach 20, 50, and 100 mM, as shown below. Figure 3 As shown.

[0181] Data indicate that adding a substantial amount of CaCl2, 100 mM, was sufficient to reduce citrate levels in PRP samples (38 and 39 PRP) to low levels. However, in PPP samples (61 and 67 PPP), adding sufficient CaCl2 to reach a concentration of 100 mM reduced citrate to less than 10 mM. In untreated (NT) plasma, citrate concentrations in PRP appeared to be close to 60 mM, but those in PPP were closer to 40 mM, suggesting the possible presence of some background citrate activity in PRP.

[0182] The molecular weight of CaCl2 is 110.98 g, 100 mM CaCl2 is 11.098 g / L, and 5 M CaCl2 is 554.9 g / L. For example, if there is 100 ml of plasma, then 1.1098 g of CaCl2 or about 2.0 ml of 5 M CaCl2 will be added to 98 ml of plasma. Most cord blood bags contain CPD, but some contain CPD plus 0.25 mM adenine (CPDA), which can prolong the life of red blood cells.

[0183] A standard 300ml cord blood collection unit contains 27ml of CPD solution (Gibson et al. Bibl Haematol. 1968; 29:758-63), which contains 0.92g sodium citrate (dihydrate) USP, 0.89g dextran glucose (monohydrate) USP, 114.5mg citric acid (monohydrate) USP, and 87.9mg sodium dihydrogen phosphate (dihydrate) USP. The molecular weight of sodium citrate dihydrate is 294.1g / mol, the molecular weight of sodium dihydrogen phosphate (dihydrate) is 156.01g / mol, and the molecular weight of dextran glucose monohydrate is 198.17g / mol.

[0184] Effects of CaCl2-treated PDPs on HeLa cell growth:

[0185] HeLa cells are derived from human cervical cancer cell lines. These cells require FBS to grow. Figure 4 The rightmost figure shows 3,000 cells on day 1, followed by few or no cells on days 2, 3, and 7. With 10% FBS, cells grew to more than 4,000 on day 1, 5,000 on day 2, 15,000 on day 3, and 18,000 on day 7.

[0186] A 1% concentration of S1 aPDP, collected in October 2017 and stored at -20°C for 3 years until August 2017, initially stimulated cell proliferation slowly but caught up by day 7. At 2.5% and 5% concentrations, 2.5% and 5% aPDP were superior to 10% FBS at day 7. At 10% concentration, aPDP was less effective than 10% FBS at day 7.

[0187] At concentrations of 1-5%, S2 aPDP collected in November 2017 and stored for approximately 10 months also stimulated HeLa cell proliferation, exhibiting a delayed response, and by day 7, the results were similar to those of 10% FBS. The data revealed two surprising findings: 1% aPDP effectively stimulated HeLa cell growth, and it was as effective as 10% FBS, regardless of whether the cells were cryopreserved at -20°C for 3 years or stored for 3 years in 2020.

[0188] Table 1: Addition of calcium, pH, and total protein (mg / ml) to four plasma samples. PRP is platelet-rich plasma, and PPP is platelet-anemic plasma, prepared by centrifugation. Note that the addition of CaCl2 lowered the pH of cord blood. In all samples, the addition of CaCl2 increased the weight of total protein (in mg / ml).

[0189]

[0190] Table 1 shows the plasma pH and total protein weight in PRP and PPP samples before and after CaCl2 treatment. Before treatment, the pH was normal (7.4–7.6). After CaCl2 treatment, the pH dropped to 6.4, which may have led to reduced culture growth. See also Figure 5 Experiments showed that in the presence of platelets, PRP concentrations of 1%, 2.5%, and 5.0% increased cell counts, while only 10% PRP appeared to be inhibited. The addition of 100 mM CaCl2 increased the growth rate. In the absence of platelets, 10% PRP appeared to perform poorly without CaCl2 activation.

[0191] Effects of CaCl2 treatment on PRP and PPP cells in human umbilical cord lining:

[0192] HCL cells are mesenchymal cells (CD105+) containing up to 10% mesenchymal stem cells. Low concentrations (1% and 2.5%) of untreated PDP will support HCL growth. Higher concentrations (5% and 10%) of untreated PDP have no effect (see [link to product description]). Figure 6 Activation with 100 mM CaCl2 did indeed improve cell growth, especially at higher PRP concentrations of 5% and 10%.

[0193] These results indicate that PDP at a concentration of 10% and activated with 100 ml CaCl2 has the best effect on the growth of mesenchymal stem cells. The growth effect is not as good as that with 10% FBS. Approximately 6% of the umbilical cord lining cells are Muse cells, and their cell growth rate is not high. On the other hand, PPP activated with 10% CaCl2 only produced half the number of cells at 7 days compared to 10% FBS.

[0194] CaCl2 activation improved cell growth, especially at higher doses of PPP. At concentrations of 1% and 2.5%, untreated PRP was superior to untreated PPP, but not at 5% or 10%. The optimal human growth medium for mesenchymal cells was 10% PPP activated with CaCl2.

[0195] Effects of CaCl2 treatment on PRP and PPP on normal human dermal fibroblasts:

[0196] Fibroblasts clearly require both 10% FBS and growth factors to achieve optimal growth. 10% FBS itself does stimulate growth with a clear dose-response curve, but not as much as 10% PPP activated by CaCl2. Commercially available (untreated) umbilical cord plasma did not significantly improve growth; although the pattern of reduced cell growth at higher umbilical cord plasma concentrations suggests a CPD effect, CaCl2 was not used to activate umbilical cord plasma.

[0197] Human dermal fibroblasts do not grow well in 10% FBS alone or in growth factor (GF) alone. However, the combination of FBS and GF yields the best results at 7 days, producing rapid and maximum cell growth. Figure 7 (Right side). Unlike HeLa cells, human dermal fibroblasts require higher concentrations of PPP and activation by CaCl2 to stimulate cell growth.

[0198] Compared to CaCl2-activated PPP, FBS+GF stimulated cell growth earlier and faster. By day 3, the cell count was more than twice that of cultures using activated PPP. However, by day 7, the cell count of CaCl2-activated PPP was catching up. Growth factors immediately stimulate growth, while exosomes require time to stimulate cells to produce growth factors.

[0199] Activated PPP and PDF may primarily stimulate growth via exosomes. Exosomes require time to stimulate cultured cells to produce their own growth factors, therefore growth curves may lag by several days. This early growth variability is minimized if freeze-dried platelets are used to supplement cell growth at the beginning.

[0200] Studies verifying the effect of CaCl2-activated PPP on cell growth:

[0201] CaCl2-activated PDPs were tested on a variety of cell types, including HeLa cells, Hep cells (human hepatocytes), NHDF cells (normal human dermal fibroblasts), and MIHA cells (immortalized human hepatocyte line). HeLa cells grown for 4 days in 1% and 5% PPP had similar cell counts compared to cells cultured in 10% FBS and 10% human serum (HS), meaning that 1% and 5% PPP were as good as 10% FBS and 10% HS. Similarly, HeLa cells grown for 7 days in 1%, 2.5%, and 5% PRP had similar or higher cell counts than those in 10% FBS. Hep cells grew as well in 1% PPP as in 1% FBS. NHDF cells grew 10 times more cells in 10% PPP than in 10% umbilical cord plasma (commercial). NHDF and MIHA cells grew as well in 1% FBS as in 1% PPP.

[0202] Example 5

[0203] New and effective exosome isolation method

[0204] This article describes a novel and efficient method for isolating exosomes from UCB PDP by adding PEG (molecular weight 10,000). Exosomes in UCB plasma are soluble because water molecules bind to proteins on the lipid membrane of the exosome surface. Exosomes have lower solubility in water if less water binds to them, and this is more favorable in hydrophobic PEG. PEG is partially soluble in water. When PEG is added to water or phosphate-bubble buffered saline and the mixture is stirred, PEG forms bubbles. If the bubbles are larger than (diameter greater than) the wavelength of visible light, the PEG bubbles can be seen with the naked eye and the solution becomes turbid. However, if they are smaller than the wavelength of visible light, the solution is transparent and clear to the naked eye. Sonicating the PEG solution produces microbubbles with a diameter less than 380 nm. The visible spectrum is from 380 nm to 700 nm. The sonicated solution becomes transparent to the naked eye.

[0205] Lower temperatures reduce the solubility of exosomes in aqueous solutions and increase their preference for PEG. At lower temperatures near the freezing point (e.g., 4°C), water molecules detach from the exosome surface and begin to form ice. When this occurs, the exosomes become less soluble in water and preferentially partition into PEG vesicles. Sonicating the PEG before adding exosome-containing plasma breaks down the PEG vesicles into smaller vesicles with a diameter of less than 380 nanometers. This clarifies the solution, eliminating turbidity. At 4°C, approximately 50% of the plasma exosomes partition into the PEG microvesicles within about 1 hour. The optimal final concentration of the sonicated PEG solution used for exosome extraction is approximately 10%. If the plasma and PEG are subsequently kept at 4°C overnight, 90-95% of the exosomes partition into the PEG.

[0206] The above method can collect up to 10 [units of something] per 10 ml of UCB plasma. 12 These are exosome-sized particles (30-150 nm). This is the number of 10 particles that can be collected from 10 ml of peripheral blood serum. 10 More than 100 times that of exosomes, collected from 10 ml of cell culture medium. 10 More than 100 times that of exosomes. This has several effects. First, 10% PEG is effective for collecting 90-95% of exosomes from 10 ml of umbilical cord plasma. 12 One exosome is more than enough. Secondly, if the PEG suspension is not sonicated or maintained at 4°C, less than 20% of the exosomes will partition into the PEG. Sonication significantly increases the surface area of ​​the PEG microbubbles, allowing for greater interaction between the exosomes and the PEG microbubbles. At 4°C, the solubility of exosomes decreases, making them more likely to migrate into the PEG.

[0207] Centrifugation (2500 x g) of plasma and sonicated PEG solution results in a PEG precipitate at the bottom of the tube containing more than 90% exosomes. If a higher molecular weight PEG, i.e., 10,000, is used, the PEG is heavier than water and will settle to the bottom of the tube after centrifugation at 3000 x g for 30 minutes. By using 10,000 molecular weight PEG and incubating the plasma and PEG solution overnight (e.g., 8 hours), extraction of 95% or more exosomes from UCB plasma was achieved. Although commercially available PEG kits for exosome isolation are available, these kits typically isolate only 20% of exosomes from blood and plasma, as well as conditioned media. The novel method described herein isolates more than 90% of exosomes not only from UCB plasma but also from culture media and adult peripheral plasma.

[0208] In summary, some embodiments of the method for isolating exosomes involve incubating sonicated 10,000 molecular weight PEG (10% concentration) with PDP overnight (8 hours) at 4°C to extract more than 90% of exosomes from plasma into the PEG. Centrifugation at 3000 x g for 30 minutes produces a PEG precipitate containing more than 90% exosomes. Since PEG is a cryopreservative, the PEG precipitate can be directly frozen at -80°C or -180°C.

[0209] Typical protocols for extracting or isolating exosomes from PDPs

[0210] Equipment and reagents:

[0211] Ultrasonic instrument (Fisherbrand) TM 705 Sonic Dismembrator or equivalent

[0212] Refrigerated centrifuges with swinging bucket rotor

[0213] PEG (10kD, Sigma catalog number 81280-1Kg)

[0214] 0.22μm filtration system (Corning catalog number 430767 or equivalent)

[0215] 0.1μm filtration system (Nalgene catalog number 565-0010 or equivalent)

[0216] 50ml conical tube (BD Falcon 352098 or equivalent)

[0217] Filtering brine through a 0.1μm filter unit

[0218] Starting materials

[0219] Frozen UCB PDP or fresh UCB PDP

[0220] process

[0221] A-PEG stock solution:

[0222] • Prepare 50% (w / v) PEG in deionized water

[0223] • Sonicate the PEG solution on ice for 1 hour to obtain a homogeneous aqueous solution (settings depend on volume and instrument recommendations).

[0224] Centrifuge at 5,000 x g for 20 minutes at 4°C.

[0225] • Filter the supernatant using a 0.22 μm filter unit and store at room temperature.

[0226] B-exosome isolation:

[0227] 1. Thaw HUCBPDP on ice or overnight at 4°C.

[0228] 2. Filter the PDP using a microporous (0.22μm) filter.

[0229] 3. Mix the filtered PDP with an appropriate amount of PEG stock solution to a final concentration in the mixture (which has a final PEG concentration of 10%).

[0230] 4. Incubate overnight at 4°C (no stirring required)

[0231] 5. Centrifuge at 3,000 x g for 30 minutes at 4°C.

[0232] 6. Aspirate and preserve the supernatant (exosome-de-etoposome plasma (EDP)).

[0233] 7. Resuspend the exosome precipitate in saline solution with a volume of 1 / 10 of the PDP volume, and dissolve the precipitate using a stirring rod.

[0234] 8. Pack into smaller portions and freeze at -80°C.

[0235] Biological fluids contain billions of exosomes per milliliter. Depending on the method used, the number of particles in serum collected via the P / PEG method ranges from 2.5 x 10⁻⁶. 9 (2.5 billion) to 2.0x10 10 The particle count varies from 20 billion to several hundred million. The particle count ranges from 6 x 10⁻⁶ in saliva. 10 (60 billion) to 2.5 x 10⁻⁶ in conditioned medium derived from human hepatocyte culture. 9 (2.5 billion).

[0236] Table 2: Particle counts, recoveries, and particle size distributions collected using sonicated PEG (molecular weight 10,000) at a 20% PEG concentration in phosphate-buffered saline. PDP = platelet-free plasma. PEG(2h) refers to incubation at 4°C for 2 hours prior to centrifugation, and PEG(8h) refers to incubation overnight at 4°C for 8 hours. D / 10 is the lowest 10% of particle size, D / 50 is the 50th percentile of particle size, and D / 90 is the 90th percentile of particle diameter. Recovery is the ratio of the total PEG(8h) counts to the total PDP counts.

[0237]

[0238] PDPs were filtered using a microporous filter to remove large extracellular vesicles and debris. The particle size distribution in the PDPs was low at the 10th percentile (D / 10) at 90.6 nm and high at the 90th percentile (D / 90) at 171.0 nm. Once the particles were partitioned into PEG, after incubation at 4°C for 2 hours, the D / 10 decreased to 69.5 nm and the D / 90 decreased to 124.8 nm. After incubation at 4°C for 8 hours, the D10 was 67.9 nm and the D90 was 135.8 nm. Counting showed that an order of magnitude more exosomes were collected than previously reported. The number of particles per milliliter of PDP was 8.2 x 10⁻⁶. 10 Or 82 billion / ml. Total count is 820 billion / 10ml. Incubated with 20% PEG, then counted from the PEG precipitate sample, concentration increased to 8.0 x 10⁻⁶. 11 / ml or 800 billion / ml PEG precipitate. The precipitate contained 48.7% exosomes from PDP. After 8 hours of incubation, the particle number almost doubled, indicating a 91% recovery rate of exosomes.

[0239] Microbubbles can play a role in limiting the size of exosomes that can be dispensed into PEG. Extracellular vesicles with diameters exceeding 150 nm were excluded. Filtering with a 0.22 μ filter eliminated all fragments larger than 0.22 μ in the PDP, and the D / 90 decreased from 171.0 to 135.8 nm without further filtration. Microbubbles exclude larger vesicles that are too large to fit. PEG microbubbles are smaller than the wavelength of visible light (350 nm) because sonication removes turbidity caused by PEG bubbles. The total count of exosomes collected from 10 ml of PDP was 750 billion, which far exceeds the count in the study by Deregibus, et al. 2016 (Int J Mol Med. 2016; 38(5):1359-66) (more than 65 billion in saliva). Our informal study of commercial exosome kits showed that the best kits collected 20% of exosomes from PDP, while many kits may have collected less than 10%.

[0240] Example 6

[0241] Identification of biomarkers in UCB exosomes and the effects of exosomes on cell growth

[0242] Exosomes express many biomarkers. Some of the more common exosome biomarkers were selected and subjected to dot blot analysis to determine UCB PDP exosome biomarkers (see [link to study]). Figure 8The most significant biomarker is ICAM. The second most significant biomarker is ANXAS. The fourth is CD81 or TAPA-1 (a target of anti-proliferation antibodies) and tetraspanin, the fifth is CD63, and the sixth is FLOT1.

[0243] UCB exosomes have a significant impact on cell growth, such as Figure 9 As shown. UCB exosomes support the growth of HeLa cells. The exosomes were thoroughly washed and contained no plasma proteins. Note that the PDP already contained exosomes and represents the combined growth-stimulating effect of plasma proteins and exosomes. The negative control was PEG only, and these cultures showed no growth. The positive control was 1-10% PDP and 10% FBS. These data indicate that the exosomes collected by sonicated PEG and cold extraction are viable and support cell growth.

[0244] References

[0245] Deregibus, MC, F. Figliolini, S. D'Antico, PMManzini, C. Pasquino, M. DeLena, C. Tetta, MFBrizzi and G. Camussi, 2016. Charge-based precipitation of extracellular vesicles. Int J Mol Med.38, 1359-1366.38.

[0246] Ehrhart, J., PR Sanberg and S. Garbuzova-Davis, 2018. Plasma derived from human umbilical cord blood: Potential cell-additive or cell-substitutetherapeutic for neurodegenerative diseases. J Cell Mol Med. 22, 6157-6166.22.

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[0250] abbreviation

[0251] aPDP is an activated PDP (by adding CaCl2 to the PDP).

[0252] CaCl2 (calcium chloride)

[0253] CPD (Citrate-phosphate-dextrose)

[0254] CPDA1 is a citrate-phosphate-dextrose solution used for UCB anticoagulation.

[0255] CSF (Cerebrospinal Fluid)

[0256] DES dry eye syndrome

[0257] EGF (Epidermal Growth Factor)

[0258] ERP-rich exosome plasma

[0259] EV extracellular vesicles

[0260] FBS Fetal Bovine Serum

[0261] FDA (Food and Drug Administration, United States)

[0262] FGF (fibroblast growth factor)

[0263] G-CSF Granulocyte Colony-Stimulating Factor

[0264] HeLa Henrietta Lack (from this person's cervical cancer cell lineage)

[0265] HS human serum (adult)

[0266] IFN-γ interferon-γ

[0267] IL-2, interleukin-2, is a cytokine that controls leukocyte activity.

[0268] IL-6, interleukin-6 (IL-6), is a pro-inflammatory cytokine.

[0269] IgA (Immunoglobulin A)

[0270] IgG Immunoglobulin G

[0271] IgM Immunoglobulin M

[0272] IVF (In Vitro Fertilization)

[0273] mM (millimolecular concentration)

[0274] mRNA messenger RNA

[0275] μRNA microRNA

[0276] MNC mononuclear cells

[0277] MVB (Multivesicular Body)

[0278] NT No processing (group)

[0279] PDP (platelet-depleted plasma)

[0280] PEG (Polyethylene Glycol)

[0281] PPP (platelet-rich plasma for anemia) (supernatant of centrifuged PRP)

[0282] PRP (platelet-rich plasma)

[0283] RCF (Red Blood Cell Part)

[0284] S1 PDP sample 1 collected in October 2017

[0285] S2 PDP sample 2 collected in November 2019

[0286] SCI spinal cord injury

[0287] TNF-α (tumor necrosis factor-α)

[0288] UCB cord blood

[0289] VEGF (Vascular Endothelial Growth Factor)

[0290] Other implementation plans

[0291] All features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can be replaced by other features for the same, equivalent, or similar purposes. Therefore, unless explicitly stated otherwise, each disclosed feature is merely an example of some general equivalent or similar features. From the above description, those skilled in the art can readily determine the essential features of the invention and can make various changes and modifications to adapt it to various uses and situations without departing from the spirit and scope of the invention. Therefore, other embodiments are also within the scope of the appended claims.

Claims

1. A method for separating one or more of exosomes, globulins, fibrinogen, and albumin from umbilical cord blood (UCB), the method comprising: i. Provides UCB containing exosomes, globulins, fibrinogen, and albumin; ii. Filter the UCB using a microporous filter containing pores with a diameter of 0.22 μm to produce a plasma filtrate containing exosomes, globulins, fibrinogen, albumin, and citrate-phosphate-dextrose (CPD); iii. The plasma filtrate is added to a solution containing ultrasonically treated polyethylene glycol (PEG) vesicles with a diameter less than 340 nm, and the solution is transferred to a first centrifuge tube, such that the exosomes are dispensed into the PEG vesicles; iv. Cool the first centrifuge tube to 2°C to 8°C for 8 hours or longer; v. Centrifuge the first centrifuge tube at a speed of 1000 to 5000 revolutions per minute for 5 to 30 minutes, so that the exosomes and PEG form a first precipitate and a first supernatant, the first supernatant containing albumin, globulin, CPD and fibrinogen; and vi. Recover the first supernatant and freeze the first precipitate containing exosomes and PEG for storage; The centrifugation mentioned above does not include ultracentrifugation.

2. The method of claim 1, wherein 90% of the exosomes of the UCB are isolated.

3. The method of claim 1, wherein in step (iv), the first centrifuge tube is cooled under conditions in which the globulin crosslinks fibrinogen and exosomes in the plasma filtrate are partitioned into PEG and the precipitating reagent precipitates CPD.

4. A method for producing exosome-rich plasma (ERP), the method comprising the steps of the method of any one of claims 1-3 to obtain an exosome precipitate, and further comprising the step of adding plasma or PBS solution to the exosome precipitate.

5. A pharmaceutical composition comprising exosomes isolated by the method of any one of claims 1-3, or, in addition to exosomes isolated by the method of any one of claims 1-3, further comprising globulins, fibrinogen, and / or albumin isolated by the method of any one of claims 1-3, or comprising ERPs generated by the method of claim 4.

6. Use of exosomes isolated by the method of any one of claims 1-3 in the preparation of a medicament for regenerating tissues in a subject.

7. The use as claimed in claim 6, wherein the subject is a person suffering from at least one of the following diseases: tissue damage, brain degeneration, central nervous system degeneration, and cardiovascular degeneration.

8. Use of the ERP generated by the method of claim 4 in the preparation of a medicament for treating a subject with dry eye syndrome, orthopedic disease, or dental disease.

9. Use of exosomes isolated by the method of any one of claims 1-3 in the preparation of a medicament for treating hypoxia, ischemia or stroke in a subject.

10. Use of the ERP generated by the method of claim 4 in the preparation of a medicament for stimulating uterine angiogenesis in subjects who require it.

11. The method of any one of claims 1-3, further comprising the step of: vii. Mix CaCl2 with the first supernatant to precipitate CPD; viii. Filter the mixture of CaCl2 and the first supernatant to remove the CPD, producing a second supernatant free of CPD; ix. Cool the second supernatant to 2°C to 8°C to form a cryoprecipitate containing fibrinogen; x. Centrifuge the cold precipitate in a further centrifuge tube to form a second precipitate and a third supernatant. xi. Remove the third supernatant containing albumin and freeze-dry the cryoprecipitate containing fibrinogen; and xii. Freeze-dry the third supernatant containing albumin. The centrifugation mentioned above does not include ultracentrifugation.

12. The method of claim 11, wherein exosomes, fibrinogen, and albumin are isolated.

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