Extracellular vesicles (EVs) derived from mesenchymal stromal cells and methods for obtaining such EVs.

Abstract

The present invention relates to a process for producing a composition of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs), the process comprising: culturing and expanding MSCs in a serum-free, xeno-free medium containing purified human serum albumin and human transferrin; harvesting a cell supernatant containing EVs; filtering the cell supernatant to obtain EVs; and concentrating the EVs, preferably by ultrafiltration. In a second and further aspect, the invention is directed to a composition containing EVs and its clinical uses.

Description

[Technical Field]

[0001] The present invention relates to extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs), compositions comprising said extracellular vesicles, and methods for obtaining said extracellular vesicles. [Background technology]

[0002] Exosomes (EVs) are particles surrounded by a lipid bilayer that are naturally released from cells and, unlike cells, do not replicate. EV diameters vary from single-membrane liposomes (approximately 20-30 nanometers), close to the smallest physically possible size, to over 10 microns, but most are less than 200 nanometers. They transport cargo from parent cells, including proteins, nucleic acids, lipids, metabolites, and even organelles. Most cells studied to date, including some bacterial, fungal, and plant cells surrounded by cell walls, are thought to release EVs. Various subtypes of EVs are defined according to their size, biosynthetic pathway, transporter, originating cell, and function, and different names such as exosomes, microvesicles, and ectosomes have been proposed.

[0003] Extracellular vesicles (EVs) can be used for therapeutic purposes, such as delivering nucleic acids and other transporters to diseased tissues and cells. Alongside this growing interest, companies and funding programs have been established focusing on the development of EVs as disease biomarkers or therapies, and the International Society for Extracellular Vesicles (ISEV) has been founded, along with the launch of the Journal of Extracellular Vesicles, a scientific journal specializing in this field.

[0004] As interest in the therapeutic use of extracellular viable cells (EVs) grows, so does the need for clinical-grade EVs. For clinical benefit, EVs must be produced with good reproducibility and control. Good Manufacturing Practices (GMP) are crucial in the production of EV-containing compositions to ensure consistent quality across each batch. GMP is particularly challenging when dealing with cell-derived therapeutics.

[0005] The process for producing exosomes derived from mesenchymal stromal cells (MSCs) is described, for example, in Non-Patent Document 1. Non-Patent Document 2 also describes MSC-derived exosomes produced under specific conditions, focusing on their potential for skin tissue regeneration. Various culture media can be used for MSC culture, among which a serum-free xeno-free culture medium is disclosed in Non-Patent Document 3. Furthermore, Patent Document 4 describes the use of MSC secretomes and extracellular vesicles (EVs) in the treatment of lung diseases. The data presented in this document regarding human use is largely speculative, highlighting several problems in the art, such as low reproducibility and low yield. The document also warns of potential problems when using bioreactors for large-scale MSC culture to produce EVs. Finally, Reference Document 5 mentions the use of albumin in the treatment of lung diseases. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Reka Agnes Haraszti et al., 2018 [Non-Patent Document 2] Diem Huong Huang at al., 2020 [Non-Patent Document 3] Lucas G. Chase et al., 2012 [Non-Patent Document 4] Antoine Monsel et al., 2016 [Non-Patent Document 5] Youngja Park et al., 2011 [Summary of the Invention] [Problems to be Solved by the Invention]

[0007] However, several problems need to be solved before the clinical use of EVs becomes widespread. Developing a platform for quantitatively producing, storing, and handling clinical-grade EVs with high reliability and good reproducibility remains an issue. For pharmaceuticals using MSC-derived EVs and subsequent clinical trials, several scientific, regulatory, technical, and institutional problems need to be solved before introducing MSC-derived EV-based therapies into clinical practice.

[0008] Therefore, there is a need for a protocol that can produce clinical-grade MSC-derived EVs under GMP conditions in a stable, highly reproducible, and cost-effective large-scale manner. Furthermore, there is a need for EV-based products with good stability and a commercially advantageous shelf life. [Means for Solving the Problems]

[0009] The present invention provides a method for enabling the production of EVs from MSCs under GMP conditions in a stable and highly reproducible manner, in accordance with the Pharmaceutical Regulatory Harmonization International Conference (ICH) 2020 GMP Quality Guidelines. The resulting EV composition is a clinical-grade cGMP product, easy to use in clinical trials and for patients, and has good stability.

[0010] For this purpose, the present invention provides the manufacturing process according to claim 1 and the composition according to claim 9. Preferred embodiments are described in claims 2-8 and claims 10-13.

[0011] The said product can be expected to have various clinical uses and therapeutic effects. The use of the said product is described in claims 14-23. [Effects of the Invention]

[0012] Due to the long-term stability of the composition, this product can be used in various clinical applications.

Brief Description of the Drawings

[0013] [Figure 1] An embodiment of the MSC growth unit and EV processing unit that can be used in the production of EVs of the present invention is shown.

Modes for Carrying Out the Invention

[0014] Definitions Unless otherwise defined, all terms used in the disclosure of the present invention, including technical and scientific terms, have the meanings commonly understood by those of ordinary skill in the technical field to which the present invention pertains. As a further guide, definitions of terms are provided to better understand the teachings of the present invention.

[0015] As used herein, the following terms have the following meanings.

[0016] As used herein, "A", "an", and "the" refer to the singular and plural forms, unless the context clearly indicates otherwise. For example, "a compartment" refers to one or more compartments.

[0017] As used herein, "about" when referring to measurable values such as parameters, amounts, time, etc., encompasses variations of ±20% or less, preferably ±10% or less, more preferably ±5% or less, even more preferably ±1% or less, and still more preferably ±0.1% or less of the defined value, as appropriate for the invention disclosed herein. However, it should be understood that the value itself to which the modifier "about" refers is also specifically disclosed.

[0018] As used herein, “has,” “equipped with,” and “includes,” and “composed of,” are synonymous with “has,” “equipped with,” “includes,” or “composed of,” and are, for example, comprehensive or open-ended terms that identify the components listed above and do not exclude or exclude any components, features, elements, members, or processes that are known to those skilled in the art or not disclosed in this description.

[0019] When specifying a numerical range based on an endpoint, it includes not only the specified endpoint but also all numbers and fractions within that range.

[0020] Unless otherwise defined, the terms "weight %", "weight percent", "%wt", or "wt%" throughout this specification refer to the relative weight of each component based on the total weight of the formulation.

[0021] The terms “one or more” or “at least one,” such as one or more or at least one member, are self-explanatory and, as further exemplification, include, in particular, any one of the members, or any two or more of the members, for example, any three or more, four or more, five or more, six or seven or more of the members, and up to all of the members.

[0022] Unless otherwise defined, all terms used in the disclosure of this invention, including technical and scientific terms, have the meaning generally understood by a person of ordinary skill in the art to which this invention pertains. For further guidance, and to better understand the teachings of this invention, terms used herein are defined. Terms or definitions used herein are provided solely to aid in understanding this invention.

[0023] Throughout this specification, any reference to “one embodiment” or “a certain embodiment” means that a particular feature, structure, or characteristic described in relation to that embodiment is included in at least one embodiment of the present invention. Therefore, expressions such as “in one embodiment” or “in a certain embodiment” in various parts of this specification may refer to the same embodiment, though not all of them. Furthermore, in one or more embodiments, certain features, structures, or characteristics may be combined in any suitable manner, as will be apparent to those skilled in the art from this disclosure. Furthermore, as will be understood by those skilled in the art, some embodiments described herein include some features included in other embodiments but do not include others, but combinations of features from different embodiments mean that they are within the scope of the present invention, and do not include other features but form different embodiments. For example, in the claims described below, any of the claimed embodiments may be used in any combination.

[0024] For the purposes of this invention, the term “extracellular vesicle” or “EV” is understood as a micrometer or nanometer-sized particle secreted in vivo and in vitro by different types of cells, containing proteins associated with the EV. EVs include molecules such as proteins, growth factors, and miRNAs encapsulated within lipid spheres. EVs are classified by their size and intracellular origin. Exosomes are a subgroup of EVs, typically smaller than 0.1 microns. Exosomes originate from the polysplenium, a late endosomal compartment, and are secreted by the fusion of the polysplenium with the plasma membrane. Another type of EV is the desolate vesicle (also called microvesicle), a heterogeneous collection of membrane vesicles smaller than 1 micron, released directly from the cell membrane by disruption of the cortical cytoskeleton. All types of vesicles secreted from cells are collectively defined as EVs.

[0025] In relation to a substance, the term "associated with EV(plural)" means that the substance is a) attached to or bonded to the surface layer of the EV (by any type of bond, such as covalent or non-covalent bonds), preferably by non-covalent bonds; and / or b) attached to or bonded within the surface layer of the EV; and / or c) internalized within the EV.

[0026] The substance associated with EV can be any type of substance, including, but is not limited to, molecules such as amino acids, proteins, peptides, nucleic acids such as RNA and DNA (e.g., non-coding RNA, miRNA, mRNA), sugars, carbohydrates, lipids, vitamins, growth factors, angiogenesis-promoting molecules, cardioprotective enzymes, antibodies, anti-inflammatory molecules, anti-fibrotic molecules, antioxidant molecules, pro-neurogenic molecules, and antiviral molecules; and ions such as metal ions or calcium ions.

[0027] The terms "cell medium," "cell culture medium," or "culture medium" refer to an aqueous solution of nutrients and other components of a specified composition that can be used for the maintenance or growth of cells.

[0028] The term "serum-free" cell culture medium means a cell culture medium that does not contain animal or human serum, plasma, or hemolymph. The serum-free medium may contain components that have been processed from or are derived from blood, serum, or plasma, such as albumin, transferrin, low-density lipids, and hormones. It may also contain other biological components (e.g., growth factors, hormones, and carrier proteins) that are not serum, plasma, or hemolymph, preferably known, fully regenerative, and in concentrations.

[0029] The term "xeno-free" cell culture medium is understood to refer to a cell culture medium that does not contain components directly derived from non-human animals, nor recombinant components manufactured from DNA sequences of non-human animals.

[0030] As used herein, the term “pharmaceutically acceptable carrier” means a carrier or diluent that does not cause significant irritation to the subject and does not impair the biological activity and properties of the administered composition. For example, a pharmaceutically acceptable carrier may act as a stabilizer and / or adjuvant. Examples of carriers include, but are not limited to, propylene glycol, physiological saline, emulsions and mixtures of organic solvents and water.

[0031] The term "sufficient amount" means an amount sufficient to produce the desired measurable effect, for example, an amount sufficient to alter the protein expression profile.

[0032] The term "therapeutic dose" refers to the amount of medication that is effective in improving the symptoms of a disease. Since prevention can be considered a form of treatment, the therapeutic dose may also be called the "preventive dose."

[0033] The term "treatment" encompasses both therapeutic and preventive measures, aiming to prevent or delay (reduce) the pathological condition or disorder in question. Those requiring treatment include individuals who already have a disorder, those who are prone to developing a disorder, or those for whom prevention of disorder is desired.

[0034] The term "composition" refers to the composition at any stage of the manufacturing process, including the pharmaceutically acceptable final product and any intermediates in that process.

[0035] The term “treatment” means reversing, preventing, mitigating, or suppressing the progression of a disease, disorder, or condition, or one or more symptoms of a disease, disorder, or condition. As used herein, the term “treatment” may also mean reducing the probability or number of occurrences of a disease, disorder, or condition in a mammal compared to an untreated control population or to the same mammal before treatment. For example, as used herein, the term “treatment” may mean preventing a disease, disorder, or condition, and may include delaying or preventing the onset of a disease, disorder, or condition, or delaying or preventing the symptoms of a disease, disorder, or condition. As used herein, the term “treatment” may also mean reducing the severity of a disease, disorder, or condition, or reducing the symptoms of such a disease, disorder, or condition before it occurs. Such prevention or reduction of severity before the onset of a disease, disorder, or condition is associated with the administration of the compositions of the Technology described herein to subjects who are not suffering from the disease, disorder, or condition at the time of administration. As used herein, “treatment” may further mean preventing the recurrence of a disease, disorder, or condition, or the recurrence of one or more symptoms of such a disease, disorder, or condition. As used herein, the terms “treatment,” “procedure,” and “therapeutic” refer to the act of treating, as defined above.

[0036] As used herein, the term “fibrosis” refers to the formation of excessive fibrous connective tissue in organs or tissues during a repair or reaction process.

[0037] For the purposes of this invention, the term "mesenchymal stromal cells" or "MSCs" shall be understood as stromal-adhering cells capable of differentiating into various cell types. MSCs are derived from bone marrow, umbilical cord cells, adipose tissue, amniotic fluid, mammary gland, blood, etc.

[0038] The present invention relates to a process for producing EVs from MSCs, a composition obtained by the said method, and its therapeutic use.

[0039] In a first embodiment, the present invention relates to a manufacturing process for EV compositions derived from MSCs. More specifically, the process is: - A step of culturing and growing MSCs in serum-free xeno-free medium containing purified human serum albumin and human transferrin, -A step of collecting cell supernatant containing EVs, - A step of filtering the cell supernatant to obtain EV, -The process includes a step of concentrating the EV, preferably by ultrafiltration.

[0040] MSCs are cultured and grown in a container used for the production of EVs, preferably a bioreactor, more preferably a stirred-tank bioreactor. For this purpose, MSCs are cultured and grown in serum-free xeno-free culture medium supplemented with human serum albumin and transferrin. Using serum-free xeno-free medium for culturing and growing MSCs prevents the inclusion of unwanted contaminants in the EVs. Such contaminants may hinder the further clinical use of the resulting EV product. Serum and platelet lysates generally contain albumin, and cell growth is often influenced by the level of albumin in the medium. However, the levels of albumin and other components in serum and platelet lysates vary. To obtain clinical-grade EVs, the use of serum and platelet lysates should be avoided.

[0041] However, albumin is an important component of the final product to support the stability and functionality of the EV. In this context, the term “stability” of a product such as an EV refers to the ability of a particular formulation or product to remain within a predetermined range of values ​​for a predetermined period of time within a specific environment, such as a container or closed system, for parameters such as physical, chemical, microbiological, toxic, and functional specifications or characteristics. Examples of such parameters, but not limited to, include particle count, particle size, leakage of internal components, and activity.

[0042] To enhance the stability of EVs, it is desirable that exogenous albumin be present in the cell culture medium. Therefore, in some embodiments, the medium contains human albumin, either recombinant or purified, such as that purified from human plasma. In further embodiments, the albumin is present in the medium at a concentration of 1 g / L to 5 g / L. The latter concentration has been shown to be particularly useful in obtaining a good and stable final product.

[0043] The cell growth medium further comprises transferrin, preferably recombinant or purified transferrin, such as purified transferrin from plasma. Transferrin forms a broad, micro-heterogeneous population of isotypes of single-chain glycoproteins with an approximate molecular weight of 78-80 kDa. Transferrin is physiologically appropriate as a way to supply iron to cells in culture because it promotes the storage and transport of iron extracellularly. Transferrin has been reported to aid in the uptake of EVs into cells in vivo (see, for example, International Publication 2013 / 084001). In addition to albumin, and similar to albumin, transferrin can also aid in the stabilization of the final EV product. In some embodiments, the transferrin is present in the growth medium at a concentration of 50 mg / ~100 mg / L, preferably 55 mg / L~100 mg / L, preferably 50 mg / L~70 mg / L, preferably 55 mg / L~70 mg / L. The latter concentration proved to be optimal for the quality of EV products.

[0044] This protocol makes it possible to have both albumin and transferrin present in the final product, with the majority of albumin and transferrin associated with the EV.

[0045] In further embodiments, the medium is a basic mixture of nutrients such as inorganic metal salts and amino acids, and vitamins, with a pH in the range of pH 7.0 to 7.4, as known from the composition of Dulbecco's modified Eagle medium (DMEM) or DMEM / F-12. The medium may also contain glutamine or glutamic acid, or its precursors such as L-alanyl-L-glutamine, as well as glucose and one or more fatty acids at levels of 4500 mg / L or less.

[0046] In further embodiments, the culture medium further comprises, but is not limited to, human growth factors such as epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), human platelet-derived growth factor (PDGF, e.g., human PDGF-BB, human PDGF-AB, and PDGF-AA) and / or human TGF-β1, preferably recombinant PDGF-BB and / or TGF-β1. The inventors have also discovered that growth factors act synergistically with other growth factors to promote the growth and proliferation of MSCs. In some embodiments, the culture medium further comprises PDGF and TGF-β1.

[0047] PDGF is a regulator of cell proliferation and division and binds to platelet-derived growth factor receptor (PDGFR). Chemically, PDGF is a dimeric glycoprotein composed of two A chains (-AA), two B chains (-BB), or a combination thereof (-AB). PDGF-AB is known to bind to PDGFα and PDGFβ receptor subunits to form PDGFα-β receptor dimers and PDGFα-α receptor dimers. In the context of this disclosure, PDGF encompasses PDGF-BB, PDGF-AB, and PDGF-AA.

[0048] Transforming growth factor β1 (TGF-β1) is a multifaceted cytokine that stimulates the proliferation of mesenchymal stem cells (MSCs), further influences the immunomodulatory properties of MSCs, and alters their secretome in a direction that is either pro-inflammatory or anti-inflammatory.

[0049] In some cases, PDGF is PDGF-BB. In some cases, the level of PDGF-BB is approximately 1 ng / mL to 150 ng / mL. In other cases, the level of PDGF-BB is approximately 7.5 ng / mL to 120 ng / mL. In other cases, the level of PDGF-BB is approximately 15 ng / mL to 60 ng / mL. In other cases, the level of PDGF-BB is at least approximately 10 ng / mL. In other cases, the level of PDGF-BB is at least approximately 15 ng / mL. In other cases, the level of PDGF-BB is at least approximately 20 ng / mL. In other cases, the level of PDGF-BB is at least approximately 21 ng / mL. In other cases, the level of PDGF-BB is at least approximately 22 ng / mL. In other cases, the level of PDGF-BB is at least approximately 23 ng / mL. In other cases, the level of PDGF-BB is at least approximately 24 ng / mL. In other cases, the level of PDGF-BB is at least approximately 25 ng / mL. In another example, the PDGF-BB level is at least approximately 26 ng / mL. In yet another example, the PDGF-BB level is at least approximately 27 ng / mL. In yet another example, the PDGF-BB level is at least approximately 28 ng / mL. In yet another example, the PDGF-BB level is at least approximately 29 ng / mL. In yet another example, the PDGF-BB level is at least approximately 30 ng / mL. In yet another example, the PDGF-BB level is at least approximately 31 ng / mL. In yet another example, the PDGF-BB level is at least approximately 32 ng / mL. In yet another example, the PDGF-BB level is at least approximately 33 ng / mL. In yet another example, the PDGF-BB level is at least approximately 34 ng / mL. In yet another example, the PDGF-BB level is at least approximately 35 ng / mL. In yet another example, the PDGF-BB level is at least approximately 36 ng / mL. In yet another example, the PDGF-BB level is at least approximately 37 ng / mL. In another example, the PDGF-BB level is at least approximately 38 ng / mL. In yet another example, the PDGF-BB level is at least approximately 39 ng / mL. In yet another example, the PDGF-BB level is at least approximately 40 ng / mL.

[0050] In another example, PDGF is PDGF-AB. In one example, the level of PDGF-AB is approximately 1 ng / mL to 150 ng / mL. In another example, the level of PDGF-AB is approximately 7.5 ng / mL to 120 ng / mL. In yet another example, the level of PDGF-AB is approximately 15 ng / mL to 60 ng / mL. In yet another example, the level of PDGF-BB is at least approximately 10 ng / mL. In yet another example, the level of PDGF-BB is at least approximately 15 ng / mL. In yet another example, the level of PDGF-BB is at least approximately 20 ng / mL. In yet another example, the level of PDGF-BB is at least approximately 21 ng / mL. In yet another example, the level of PDGF-BB is at least approximately 22 ng / mL. In yet another example, the level of PDGF-BB is at least approximately 23 ng / mL. In yet another example, the level of PDGF-BB is at least approximately 24 ng / mL. In yet another example, the level of PDGF-BB is at least approximately 25 ng / mL. In another example, the PDGF-BB level is at least approximately 26 ng / mL. In yet another example, the PDGF-BB level is at least approximately 27 ng / mL. In yet another example, the PDGF-BB level is at least approximately 28 ng / mL. In yet another example, the PDGF-BB level is at least approximately 29 ng / mL. In yet another example, the PDGF-BB level is at least approximately 30 ng / mL. In yet another example, the PDGF-BB level is at least approximately 31 ng / mL. In yet another example, the PDGF-BB level is at least approximately 32 ng / mL. In yet another example, the PDGF-BB level is at least approximately 33 ng / mL. In yet another example, the PDGF-BB level is at least approximately 34 ng / mL. In yet another example, the PDGF-BB level is at least approximately 35 ng / mL. In yet another example, the PDGF-BB level is at least approximately 36 ng / mL. In yet another example, the PDGF-BB level is at least approximately 37 ng / mL. In another example, the PDGF-BB level is at least approximately 38 ng / mL. In yet another example, the PDGF-BB level is at least approximately 39 ng / mL. In yet another example, the PDGF-BB level is at least approximately 40 ng / mL.

[0051] In another example, PDGF is PDGF-AA. In one example, the level of PDGF-AA is approximately 1 ng / mL to 150 ng / mL. In another example, the level of PDGF-AA is approximately 7.5 ng / mL to 120 ng / mL. In yet another example, the level of PDGF-AA is approximately 15 ng / mL to 60 ng / mL. In yet another example, the level of PDGF-AA is at least approximately 10 ng / mL. In yet another example, the level of PDGF-AA is at least approximately 15 ng / mL. In yet another example, the level of PDGF-AA is at least approximately 20 ng / mL. In yet another example, the level of PDGF-AA is at least approximately 21 ng / mL. In yet another example, the level of PDGF-AA is at least approximately 22 ng / mL. In yet another example, the level of PDGF-AA is at least approximately 23 ng / mL. In yet another example, the level of PDGF-AA is at least approximately 24 ng / mL. In yet another example, the level of PDGF-AA is at least approximately 25 ng / mL. In another example, the PDGF-AA level is at least approximately 26 ng / mL. In yet another example, the PDGF-AA level is at least approximately 27 ng / mL. In yet another example, the PDGF-AA level is at least approximately 28 ng / mL. In yet another example, the PDGF-AA level is at least approximately 29 ng / mL. In yet another example, the PDGF-AA level is at least approximately 30 ng / mL. In yet another example, the PDGF-AA level is at least approximately 31 ng / mL. In yet another example, the PDGF-AA level is at least approximately 32 ng / mL. In yet another example, the PDGF-AA level is at least approximately 33 ng / mL. In yet another example, the PDGF-AA level is at least approximately 34 ng / mL. In yet another example, the PDGF-AA level is at least approximately 35 ng / mL. In yet another example, the PDGF-AA level is at least approximately 36 ng / mL. In yet another example, the PDGF-AA level is at least approximately 37 ng / mL. In another example, the PDGF-AA level is at least approximately 38 ng / mL. In yet another example, the PDGF-AA level is at least approximately 39 ng / mL. In yet another example, the PDGF-AA level is at least approximately 40 ng / mL.

[0052] Xeno-free serum-free culture media may be prepared by combining the components, or they may be selected (optionally adapted) from commercially available products based on the guidelines described herein. For example, commercially available xeno-free serum-free media that may be used may be optionally adapted to any of the characteristics and / or conditions (preferences) disclosed herein before use.

[0053] In some embodiments, the culture medium may include a buffer system. pH adjustment is important for optimal culture conditions and is generally performed by either a natural buffer system or a chemical buffer system.

[0054] In natural buffer systems, gaseous CO2 is contained in the culture medium. -- / HCO3 - Equilibrium is reached. Culture media containing the natural buffer system need to be maintained in an air atmosphere containing 5-10% CO2, usually using a CO2 incubator. The natural buffer system is low-cost and non-toxic. Chemical buffering using the zwitterionic HEPES has excellent buffering capacity in the pH range of 7.2-7.4 and does not require control of the gaseous atmosphere. HEPES is relatively expensive and toxic at high concentrations depending on the cell type. In addition, HEPES has been shown to significantly increase the sensitivity of the culture medium to phototoxic effects caused by exposure to fluorescence.

[0055] In one embodiment, the culture medium may contain phenol red as a pH indicator, which allows for continuous monitoring of the pH. During cell proliferation, metabolites released from the cells change the pH and thus the color of the culture medium. With phenol red, the culture medium turns yellow at low pH and purple at high pH. At pH 7.4, which is the optimal pH value for cell culture, the culture medium turns bright red.

[0056] In another embodiment, the culture medium may contain inorganic salts. These inorganic salts help maintain osmotic balance and regulate membrane potential by supplying ions such as sodium, potassium, and calcium.

[0057] The culture medium may contain amino acids, preferably essential amino acids. L-glutamine, an essential amino acid, is particularly important. L-glutamine supplies nitrogen to NAD, NADPH, and nucleotides, and functions as a secondary energy source for metabolism. Since L-glutamine is an unstable amino acid and changes over time into a form unusable by cells, it must be added to the culture medium immediately before use. Ammonia accumulates due to the breakdown of L-glutamine, and ammonia can have harmful effects on some cell lines; therefore, caution is necessary when adding more L-glutamine than the original culture medium composition. The L-glutamine concentration in mammalian cell culture media can vary significantly, from 0.68 mM in 199 medium to 4 mM in Dulbecco's modified Eagles medium. Invertebrate cell culture media may contain as much as 12.3 mM L-glutamine. Additives such as glutamax are more stable and can be used as a substitute for glutamine for long-term culture without requiring medium replacement or addition, and to further stabilize the glutamine concentration during this period.

[0058] Additionally, non-essential amino acids, which decrease during growth, may be added to the culture medium. Supplementing the culture medium with non-essential amino acids promotes cell growth and extends the survival period.

[0059] Carbohydrates in the form of sugars are the primary energy source. Most culture media contain glucose and galactose, but some also contain maltose and fructose.

[0060] The culture medium may further contain fatty acids, lipids, vitamins, and trace elements. Serum-free media are often supplemented with trace elements normally found in serum. Trace elements such as copper, zinc, and selenium, as well as tricarboxylic acid intermediates, are chemical elements required in trace amounts for normal cell growth.

[0061] In some embodiments, antibiotics may be used to control the growth of bacterial and / or fungal contaminants, for example, typically a mixture of penicillin and streptomycin, or / or other compounds such as, but not limited to, amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, levofloxacin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, penicillin, polymyxin, puromycin, rifampicin, spectromycin, streptomycin, tetracycline, tyrosine, and zeosin.

[0062] In one embodiment, the MSCs may grow and proliferate on microcarriers or microbeads placed within the bioreactor. Such microcarriers or beads are known in the art and commercially available. In one embodiment, the microcarriers or beads may be coated with extracellular matrix proteins such as fibronectin, laminin, hyaluronic acid, their mimics, or combinations thereof. In a preferred embodiment, the microcarriers or beads are negatively charged.

[0063] After the MSCs have reached the desired concentration and / or confluence, the cell supernatant containing the EVs is collected and further processed. In this invention, the cell supernatant is the cell medium in which the MSCs were grown and proliferated. In one embodiment, the supernatant contains at least 40 × 10¹⁶ MSCs. 6 The cells are harvested when the minimum concentration of cells / L is reached. The cell concentration and viability can be determined by cell counting methods, such as a Bruker counting chamber and a hematological counter such as trypan blue staining. Under the above culture conditions, the MSCs have a viability of at least 0.25 × 10⁶ per mL of cell medium during the 18–24 hour period in which the MSCs are cultured. 9 It produces individual particles / culture medium.

[0064] The "particles" used above may be any particles, preferably with a particle size of 0.05 to 0.22 microns, but extraepithelial particles (EVs) are an example. Examples of particles include protein or peptide aggregates. The origin of the particles is MSCs or cell culture media for culturing and growing MSCs. Therefore, the particles may be any particles that are normally present in or part of the cell culture media or MSCs.

[0065] Preferably, when using the method described herein, at least 90% of the particles having a particle size of 0.05 to 0.22 microns are EV.

[0066] In a subsequent step, the cell supernatant is filtered to remove contaminants present in the cell culture medium. The latter contributes to the purity and stability of the final product. In a preferred embodiment, the filtration includes at least two stages of filtration. In one embodiment, at least one stage of the filtration is dead-end filtration. In a further embodiment, both stages of the filtration are performed by dead-end filtration. Preferably, at least one stage of the filtration is also used as a sterilization method for the product to comply with the GMP standards defined above. It has been found that in some cases, it is necessary to perform dead-end filtration continuously to sufficiently remove impurities from the supernatant. It has also been found that in some cases, a single filtration often results in clogging of the filter used, leading to a decrease in the purity and quantity of the final product.

[0067] In the first filtration step, the cell supernatant is filtered through a dead-end filter. In a further preferred embodiment, the filtration is carried out by passing the supernatant through a filter with a mesh size of 1 to 5 microns, more preferably 1 to 3 microns. In one embodiment, the first filtration step is This is performed by dead-end filtration in a closed system equipped with a peristaltic pump that supplies a constant flow rate, preferably 100 mL / min, through a filter.

[0068] In one embodiment, the filtrate from the first filtration is passed through a second filter, which in this step has a smaller pore size than the filter used in the first filtration step. Preferably, this second filtration step is a sterilization step in order to make the final product conform to the above GMP standards. In a more preferred embodiment, the mesh size of the filter is 1 micron or less, more preferably 0.05 to 1 micron, even more preferably 0.1 to 0.5 microns, and most preferably 0.1 to 0.22 microns. In one embodiment, the second filtration step is preferably performed by dead-end filtration in a closed system equipped with a peristaltic pump that is interconnected with the first filtration step and supplies a constant flow rate, preferably 100 mL / min, through the filter.

[0069] The filtrate from the filtration step contains EV according to the present invention. In the final step, the EV is washed and concentrated. Washing and concentration may be carried out by conventional means known in the art, such as membrane filtration of either microfiltration or ultrafiltration. Concentration is the process of removing liquid from a solution to obtain solute molecules.

[0070] Membrane filtration is a widely used separation technique in life science research. Membrane filtration is classified into microfiltration and ultrafiltration processes based on the porosity of the membrane. Microfiltration membranes typically have pore sizes of around 0.1 μm to 10 μm and are generally used for clarification, sterilization, and removal of fine particles for cell collection. Ultrafiltration membranes have extremely small pore sizes of 0.001 to 0.1 μm and are used for concentration and desalting of dissolved molecules (proteins, peptides, nucleic acids, carbohydrates, and other biomolecules), buffer exchange, and total fractionation. Ultrafiltration membranes are generally classified by molecular weight cutoff (MWCO) rather than pore size. The main modes of membrane filtration, which can use either microfiltration membranes or ultrafiltration membranes, include two methods: 1) Direct flow filtration (DFF), also known as "dead-end" filtration, in which the supply flow is directed perpendicular to the membrane surface, allowing 100% of the fluid to pass through the membrane; and 2) Tangential flow filtration (TFF), also known as cross-flow filtration, in which the supply flow is directed parallel to the membrane surface, allowing some of the fluid to pass through the membrane (permeate) and the remainder (retainate) to be recirculated to the supply tank.

[0071] Depending on the circumstances, the washing and concentration are carried out by TFF. TFF (cross-flow filtration) is a process in which the feed flow is passed parallel to the membrane surface. When pressure is applied, a portion of the flow passes through the membrane (filtrate or permeate), and the remaining portion (retainate) is returned to the feed tank and recycled.

[0072] In one embodiment, the filtrate from one or more dead-end filtration steps is used in the TFF concentration step. In a further preferred embodiment, the TFF has a cutoff range of 100 kDa, removing particles and components with a molecular weight of less than 100 kDa, transferring most, if not all, of them to the TFF permeate. As a result, the final composition remaining in the retainate has a reduced amount of free components, components, or substances with a molecular weight of less than 100 kDa (i.e., not associated with EVs). These components, components, or substances may be any particles, such as proteins or peptides, that are normally present in or are part of the cell medium. The retainate is recirculated within the TFF apparatus until the desired concentration is reached. During recirculation, the retainate can be washed with a washing medium or washing buffer, preferably a saline washing buffer, to remove unwanted components. The concentrated ritentate can then be collected in a recovery container such as a recovery bag or cryotube, but is not limited to this, and kept at a low temperature of less than 10°C, preferably 4°C, or frozen at -20°C to -196°C, preferably -40°C to -196°C, and more preferably -80°C to -196°C.

[0073] The EV of the present invention is derived from MSCs. The MSCs are of human origin and may be derived from bone marrow, umbilical cord, Wharton's jelly, umbilical cord blood, amniotic membrane, bone marrow, adipose tissue, dental pulp, peripheral blood, fallopian tubes, mammary glands, liver and lung tissue, etc. In a preferred embodiment, the MSCs are derived from umbilical cord (UC-MSC).

[0074] In one embodiment, MSCs are isolated fresh from one of the tissues as described above and further grown in a bioreactor. In another embodiment, cryopreserved MSCs are used. Methods for obtaining MSCs from various sources are generally known in the art and readily applicable to the present invention. Briefly, tissue such as umbilical cord may be enzymatically digested with an enzyme such as collagenase and / or trypsin, washed, and centrifuged. The resulting pellet is plated in a suitable cell medium in a culture flask and cultured under suitable conditions. The culture flask may be coated with extracellular matrix proteins such as fibronectin, laminin, hyaluronic acid, their mimics, or combinations thereof. The latter may promote adhesion and cell growth. In another embodiment, the culture flask may contain coated microcarriers or microbeads coated with extracellular matrix proteins such as fibronectin, laminin, hyaluronic acid, their mimics, or combinations thereof. The culture medium is refreshed until the cells reach a predetermined minimum confluence, preferably 80% or more, and then the cells are harvested.

[0075] During subculturing, the cultured cells are detached to separate them from the culture medium and from each other. Cell detachment and separation can be carried out as is generally known in the art, for example, by enzymatic treatment with proteolytic enzymes (e.g., selected from trypsin, type I, type II, type III or IV collagenases, dispase, pronase, papain, etc.), treatment with divalent ion chelating agents (e.g., EDTA or EGTA), or mechanical treatment (e.g., repeated pipetting using a small-bore pipette or pipette tip), or any combination thereof.

[0076] A suitable method for detaching and dispersing cells must detach and disperse the cells to the desired extent while preserving the majority of viable cells in the culture medium. Preferably, detachment and dissociation of cultured cells yields a significant proportion of cells (e.g., at least 50%, 70%, 80%, 90%, or more) as single viable cells. The remaining cells may exist in cell clusters, each containing a relatively small number of cells (e.g., 1 to 100 cells on average).

[0077] Next, the cells that have been detached and dissociated in this manner (usually as a cell suspension in isotonic buffer or culture medium) may be replated onto a substrate to which cells can adhere, and then cultured in the culture medium described above to further sustain growth. After that, these cells may be divided into 10-10 5 cells / cm 2 The cells may be replated and cultured at a concentration of approximately 1 / 16 to 1 / 2, preferably approximately 1 / 8 to 1 / 2, and more preferably approximately 1 / 4 to 1 / 2. The division ratio indicates the proportion of the subcultured cells that are seeded into an empty (usually new) culture vessel with the same surface area as the vessel from which the cells were obtained. The type of culture vessel, and the type of surface on which the cells are attached to the culture vessel and cell culture medium, may be the same as the one used initially, or it may be different.

[0078] In a preferred procedure, the cells are then seeded into larger recipients and grown. Once the desired number of cells is reached, they may be placed in a bioreactor for further growth and harvesting of extracellular viable cells (EVs), or an appropriate amount of cells may be cryopreserved for later use. Cryoprotection of the MSCs is typically carried out in the presence of one or more cryoprotective agents. Such agents prevent cell damage during freezing and thawing and are generally known to those skilled in the art. Examples of cryoprotective agents include cell culture media, which are used mixed with DMSO. The cells may be cryopreserved in suitable containers or cryobags.

[0079] In a further step, the MSCs (either fresh isolated MSCs or cryopreserved MSCs) may be further grown in a bioreactor, preferably a closed-loop agitated tank bioreactor. The cells are grown in the bioreactor under appropriate growth conditions (serum-free xeno-free medium) as described above. In one embodiment, the container or cryobag used for cryopreservation as described above is designed to be attached to the bioreactor in a sterile state, eliminating the need for a (laminar) flow cabinet. This eliminates the need for a (highly sterile) cleanroom in the EV manufacturing process.

[0080] The above method can be implemented in an EV purification system comprising an MSC cell proliferation unit and an EV processing unit. The two units and their respective compartments and / or components are fluidically connected to each other, allowing cell culture medium and materials to flow from one unit to the other. The fluid connection is achieved by conventional means in the art, such as tubes, pipes, valves, and pumps. In one embodiment, the MSC cell proliferation unit comprises a bioreactor, preferably a stirred bioreactor, and a cooling compartment. The cooling compartment is provided by means of cooling the ambient temperature and the objects within the cooling compartment to a temperature below 10 degrees, such as 4 degrees. In a preferred embodiment, the cooling compartment is a refrigerator. The cooling compartment may include tanks or containers for storing fresh cell culture medium to be transferred to the bioreactor and cell supernatant coming out of the bioreactor, respectively. For this purpose, the outlet of the cell culture medium container or tank is fluidically connected to the inlet of the bioreactor. A pump, preferably a peristaltic pump, is provided to ensure that the medium flows properly. The outlet of the bioreactor is fluidically connected to the inlet of a cell supernatant container in a cooling compartment. In this case as well, a (peristaltic) pump may be provided to ensure the product flows. In this configuration, the supernatant from the bioreactor flows into a cell supernatant recovery container or tank, and fresh culture medium may be supplied to the bioreactor. The bioreactor may optionally be an agitated bioreactor. Preferably, the fresh culture medium is preheated to at least room temperature, or more preferably to about 37°C, by any heating device known in the art, such as an incubator. Agitation may be performed by conventional means in the art, such as a magnetic stirrer or a stirrer installed in the bioreactor. The bioreactor may be supplied with CO2 or may be placed in a CO2 incubator. In the latter embodiment, the bioreactor is equipped with means to enable gas exchange. The cell proliferation unit may be equipped with sensors for monitoring various processes.In one embodiment, sensors are provided to measure the temperature, humidity, and CO2 levels inside the bioreactor and / or CO2 incubator. Additionally, sensors may be provided to monitor the conditions inside the cooling compartment, for example, by monitoring the temperature.

[0081] Cell supernatant containing EVs is supplied from the bioreactor when the MSCs reach a desired amount and stored at a temperature below 10°C, for example, 4°C. Once an appropriate amount of cell supernatant is obtained, the supernatant is transferred to an EV processing unit. In one embodiment, this appropriate amount is transferred to the EV processing unit in a single batch. This is possible after re-pooling multiple batches before processing. In an alternative embodiment, this amount is transferred continuously to the EV processing unit. The EV processing unit preferably comprises one or more filter devices, membrane filtration devices such as TFF concentrators, and a storage compartment for the final product (in the order described herein). These devices and compartments are fluidically connected to each other, allowing for the transport of fluids from one device to another, and optionally forming a closed-loop system. In a more preferred embodiment, the EV processing unit comprises a first filter device with a filter mesh size of 1 to 5 microns, more preferably 1 to 3 microns. The filtrate from the first filtration is then transferred to a second filter device with a smaller mesh size than that used in the first filtration step. In a more preferred embodiment, the mesh size of the filter device is 1 micron or less, more preferably 0.05 to 1 micron, even more preferably 0.1 to 0.5 microns, and most preferably 0.1 to 0.22 microns. In one embodiment, the filter device is preferably interconnected with a first filtration step and enables dead-end filtration carried out in a closed system with a peristaltic pump that supplies a constant flow rate, preferably 100 mL / min, through the filter.

[0082] After filtration, the filtrate is transferred to a TFF device. For this purpose, a final filter device is again fluid-connected to the TFF. These devices are typically classified by molecular weight cut-off (MWCO). The molecular weight cut-off (MWCO) or nominal molecular weight cut-off (NMWCO) is defined as the minimum molecular weight of the solute that is 90% retained by the membrane. In a preferred embodiment, the TFF has a cut-off range of 100 kDa. The TFF is used for washing, concentrating, and further purifying the sample. From the TFF, the concentrated and purified sample is transferred to a filling and final manufacturing unit to obtain the final product. In one embodiment, the final product is collected in a bag connected to a filling device, and the filling device may aliquot the sample for the final recipient. It is also possible to manually perform the aliquoting for the final recipient.

[0083] In one example, when using the manufacturing process as described above, the minimum concentration in the bioreactor is at least 40×10 6 particles / L, and at least 1×10 11 particles per mL of the final product composition can be obtained from the 100-fold concentrated supernatant of MSC. The confluency of MSC was up to 80%. Preferably, about 8×10 6 MSCs are planted in one 0.5 L bioreactor, cultured, and grown completely. Then, the MSCs begin to release EVs into the supernatant, and the supernatant is collected every 24 hours, for example, into a culture bag. In this example, when the contents of the culture bag are pooled 4 times, filtered (using a mesh size of 0.22 microns in this case), and concentrated 100-fold, at least 1×10 11 particles are obtained.

[0084] The “particles” used in the above example may be any particles with a particle size of 0.05 to 0.22 microns, with EV being a notable example. Other examples of particles include protein or peptide aggregates. The origin of the particles is MSCs or the cell medium used to culture and grow MSCs. Therefore, the particles may be any particles that are normally present in or part of the cell medium or MSCs.

[0085] Preferably, when using the method described above, at least 90% of the particles, which have a particle size of 0.05 to 0.22 microns, are EVs.

[0086] In a further embodiment, the present invention also covers compositions containing EVs derived from MSCs, wherein the EVs are less than 1 μm in size and the human albumin concentration of the composition is 10 to 30 g / L.

[0087] In another or further embodiment, the composition according to the present invention contains EVs of less than 1 μm in size derived from MSCs, and contains transferrin and albumin, in a ratio of about 2 to 60 mg of transferrin per 1 g of albumin, preferably about 5 to 55 mg of transferrin per 1 g of albumin, more preferably 10 to 45 mg of transferrin per 1 g of albumin, and most preferably 10 to 40 mg of transferrin per 1 g of albumin.

[0088] In one embodiment, the EV size of the composition is less than 1 μm. In a preferred embodiment, the EV size of the composition is less than about 750 nm, preferably less than 500 nm, preferably less than 400 nm, preferably less than 300 nm. In another or further preferred embodiment, the EV size of the composition is at least 5 nm, more preferably at least 10 nm, more preferably at least 25 nm, more preferably at least 50 nm. In another or further preferred embodiment, the EV size of the composition is 25 to 500 nm, preferably 25 to 400 nm, more preferably about 50 to about 300 nm.

[0089] Optical methods are typically used for measuring and counting the size of extracellular particles (EVs). In one embodiment of the present invention, the particle size of the EVs is measured by nanoparticle tracking analysis (NTA), a preferred method for the quantification and sizing of nanoparticles suspended in a liquid buffer. In another or further embodiment, the particle size is measured using adjustable resistance pulse sensing (TRPS), which is used as a reference method for NTA. In yet another or further embodiment, the particle size is measured using high-resolution flow cytometry.

[0090] The composition according to the present invention, which can be obtained by the process described above, contains human albumin at a concentration of 10 to 30 g / L, more preferably 10 to 20 g / L, more preferably 15 to 20 g / L, or preferably 12 to 16 g / L. The albumin concentration can be measured by a colorimetric method or by an ELISA method using an anti-albumin antibody. In one embodiment, the colorimetric measurement is a bromocresol green assay.

[0091] The source of the albumin is the cell culture medium of the MSCs. Although albumin is not produced by MSCs and therefore theoretically considered a contaminant in this manufacturing process, it has surprisingly become clear that its presence is actually necessary to ensure the stability and functionality of the final product and, consequently, the composition of this invention. At the required concentration, albumin acts partially as a drug stabilizer and activity enhancer. It has been shown that forcibly removing albumin reduces activity (Hyungtaek Jeon et al, 2020).

[0092] Preferably, the albumin is a clinical-grade product that is acceptable for use in animals and / or humans.

[0093] In one embodiment, at least 90% of the albumin present in the composition is associated with the EV in the composition.

[0094] The molecular weight of human albumin is approximately 66 kDa. Therefore, albumin that is not associated with EV is removed by TFF treatment, and the albumin remaining in the composition is associated with EV. In a further preferred embodiment, at least 93%, more preferably 94%, more preferably 95%, more preferably 96%, more preferably 97%, more preferably 98%, and more preferably 99% of the albumin in the composition is associated with EV.

[0095] In some embodiments, the composition also contains human transferrin. The level of transferrin in the composition is preferably 25 mg / L to 95 mg / L, more preferably 55 mg / L to 75 mg / L. In some embodiments, the level of transferrin in the composition is preferably 60 to 600 mg / L, more preferably 100 to 500 mg / L, more preferably 150 to 450 mg / L. Transferrin has been reported to be involved in the movement of particles across cell membranes and is said to be involved in the stability and uptake of EVs into cells in vivo. For this reason, the presence of transferrin is known to have a positive effect on the further use of the EV composition.

[0096] In one embodiment, at least 90% of the transferrin present in the composition is associated with the EV in the composition.

[0097] The molecular weight of human transferrin is approximately 80 kDa. Therefore, transferrin that is not associated with EVs is removed to some extent by TFF treatment, and the transferrin remaining in the composition is associated with EVs. In a further preferred embodiment, at least 93%, more preferably 94%, more preferably 95%, more preferably 96%, more preferably 97%, more preferably 98%, and more preferably 99% of the transferrin in the composition is associated with EVs.

[0098] The concentrations of both albumin and transferrin, as well as other proteins, can be measured by conventional means in the art, such as ELISA.

[0099] In one embodiment, the composition contains less than 5% of free components having a molecular weight of less than 100 kDa.

[0100] The term "free components" refers to components or constituents in the final product that are not associated with extracellular viable (EVs) and are therefore available in a free form within the final product.

[0101] For example, by using TFF filtration with a cutoff value of 100 kDa, the composition is well-defined and compliant with GMP by reducing the amount of free components with a molecular weight (MW) of less than 5%. Most components in the cell growth medium of MSCs have molecular weights of less than 100 kDa. Since such low molecular weight components are considered contaminants if they remain in the final product, it is desirable to minimize their amount. In a further preferred embodiment, the composition contains less than 4%, less than 3%, less than 2%, or less than 1% of free components.

[0102] In further embodiments, at least 60% of the EV contains annexin V.

[0103] Annexin is a calcium-dependent phospholipid-binding protein that enhances the anti-inflammatory properties of extracellular viable (EV) by associating with EV. Some annexins, such as annexin V, are involved in pro-inflammatory activity. However, it has been found that when annexin V associates with EV, the anti-inflammatory activity of EV increases. The molecular weight of annexin V is approximately 37 kDa.

[0104] According to one embodiment of the present invention, the ratio of EV-associated albumin to EV-associated annexin V is 1:4.500 to 1:350.000.

[0105] In some embodiments of the present invention, the composition may further comprise one or more second therapeutic agents. As used herein, therapeutic agents mean any agent that can be used to prevent, treat and / or manage diseases such as those described herein. Such agents may be vesicle-based (e.g., contained within the body of the EV) or associated with the EV. Suitable therapeutic agents are known to those skilled in the art and include non-coding RNAs, miRNAs, mRNAs, growth factors, angiogenic molecules, cardioprotective enzymes, antibodies, anti-inflammatory molecules, anti-fibrotic molecules, antioxidant molecules, neurogenic molecules, antiviral molecules, and the like. In some embodiments, isolated EVs are used in combination with the second agent. In some embodiments, the second agent is a steroid, an antioxidant, or inhaled nitric oxide. In some embodiments, the steroid is a corticosteroid. In some embodiments, the corticosteroid is methylprednisolone or dexamethasone. In some embodiments, the antioxidant is superoxide dismutase.

[0106] Certain secondary therapeutic agents used for the treatment or management of certain lung diseases, including pulmonary hypertension, but not limited to, include oxygen, anticoagulants such as warfarin (coumadin); diuretics such as furosemide (Lasix®) or spironalactone (Aldactone®); calcium channel blockers; potassium such as K-dur®; cardiac stimulants such as digoxin; and vasodilators such as nifedipine (Procardia®) or diltiazem (Cardigem®). Examples include endothelin receptor antagonists such as bosentan (Tracleer®) and ambrisentan (Retyris®); prostacyclin analogs such as poprostenol (Floran®), treprostinil sodium (Limodulin®, Tyvaso®), and iloprost (Bentavis®); and PDE-5 inhibitors such as sildenafil (Revatio®) and tadalafil (Adcirca®).

[0107] The composition is preferably formulated as a liquid. Storage may be carried out by means such as vials, infusion bags, ampoules, cartridges, liquid inhalers, nebulizers or powder inhalers, and pre-filled syringes. In addition to the active ingredient or EV as a pharmaceutical, the liquid formulation may further contain various compounds to ensure that the active drug remains stable after storage. These include solubilizers, stabilizers, buffers, tension modifiers, volume expanders, viscosity improvers / decreasers, surfactants, chelating agents, and adjuvants.

[0108] In another embodiment, the composition may be lyophilized or freeze-dried. The lyophilized formulation may be stored in vials, cartridges, inhalers such as liquid inhalers, nebulizers or powder inhalers, dual-chamber syringes, and pre-filled mixing systems. Before administration, the lyophilized composition is reconstituted as a liquid. In this case, a liquid diluent may be added to the lyophilized powder, mixed, and then injected. Typically, reconstitution requires a reconstitution and delivery system to ensure that the drug is properly mixed and administered.

[0109] In preferred embodiments, the composition is aqueous. In one embodiment of the present invention, the EV is formulated as a composition further comprising a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier or diluent is selected to keep the EV of the present invention viable and to retain its properties. The pharmaceutically acceptable carrier is a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that is involved in the transport or delivery of a drug having prophylactic or therapeutic activity. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and does not harm the subject. Some examples of materials that can function as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; buffers such as magnesium hydroxide, magnesium stearate, and aluminum hydroxide; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer; and other non-toxic, suitable substances used in pharmaceutical formulations.

[0110] The composition may be preserved by cryopreservation, which involves freezing the composition at 4°C, more preferably at -20°C to -196°C, preferably -40°C to -196°C, and more preferably -80°C to -196°C. The freezing procedure is preferably snap freezing or vitrification freezing, which allows the product to exist during the freezing process and after thawing. Alternatively, to enhance the stability of the product, a rate-controlled freezing procedure may be performed, preferably with correction of the exothermic reaction at the crystallization point. In the latter procedure, a rate-controlled freezer can be used.

[0111] In some embodiments, the composition is adapted for administration by injection, intravenous administration, inhalation, intratracheal infusion, systemic infusion, or intranasal infusion. The composition may also be formulated for topical use, either alone or in combination with a hydrogel, polymer, or polymer medical device, to sustain the release of EV.

[0112] In some embodiments, the composition is adapted for intravenous administration. In some embodiments, the composition is adapted for administration to the target lung or trachea. In some embodiments, the composition is formulated for administration by inhalation. In some embodiments, the composition is formulated for administration as an aerosol. In some embodiments, isolated EV is administered using a nebulizer. In some embodiments, isolated EV is administered using an endotracheal tube.

[0113] In some embodiments, isolated EVs are administered or formulated with a surfactant, preferably a pulmonary surfactant. This surfactant is preferably selected so as not to affect the stability of the composition. In some embodiments, the pulmonary surfactant is an isolated, naturally derived surfactant. In some embodiments, the pulmonary surfactant is derived from bovine or porcine lung. In some embodiments, the pulmonary surfactant is a synthetic surfactant. The pulmonary surfactant is a lipoprotein mixture useful for keeping the airways of the lungs open (for example, by preventing adhesion between alveolar walls). The pulmonary surfactant may consist of phospholipids such as dipalmitoylphosphatidylcholine (DPPC), phosphotidylcholine (PC), and phosphotidylglycerol (PG); cholesterol; and proteins such as SP-A, SP-B, SP-C, and SP-D. In some embodiments, the pulmonary surfactant is derived from natural sources such as tissue from bovine or porcine lung. Examples include Alveofact™ (derived from bovine lung lavage solution), Curosurf™ (derived from porcine lung mince), Infasurf™ (derived from calf lung lavage solution), and Survanta™ (bovine lung mince with added components such as DPPC, palmitic acid, and tripalmitine). Lung surfactants may also be synthetic. Examples include Exosurf™ (DPPC with added hexadecanol and tyroxapole), Pumactant™ or Artificial Lung Expanding Compound (ALEC) (containing DPPC and PG), KL-4 (containing DPPC, palmitoyl-oleylphosphatidyl glycol, palmitic acid, and a synthetic peptide mimicking SP-B), and Venticute™ (containing DPPC, PG, palmitic acid, and recombinant SP-C). Lung surfactants can be obtained from commercial distributors.

[0114] Furthermore, the present invention encompasses packaged and labeled pharmaceuticals. The product or kit includes a suitable unit dosage form in a container such as a glass vial or plastic ampoule, or other sealed container. The unit dosage form is preferably adapted for, for example, aerosol administration to the lungs. Preferably, the product or kit further includes instructions on how to use the pharmaceutical, including a method of administration. The instructions may further include informational material that advises healthcare providers, technicians, or subjects on how to appropriately prevent or treat the targeted disease or disorder. In other words, the product includes instructions indicating or suggesting an administration regimen for use, including, but not limited to, actual dosages, monitoring procedures, and other monitoring information.

[0115] As with other pharmaceuticals, packaging materials and containers are designed to protect the stability of the product during storage and transport, and may contain desiccants to ensure stability.

[0116] The kit may contain EV in a sterile aqueous suspension and be used directly, or diluted with ordinary saline for intravenous injection or nebulizer use, or diluted with or in combination with a surfactant for intratracheal administration. Therefore, the kit may contain diluents or drugs such as saline or surfactants. The kit may also contain lung delivery devices such as nebulizers, or disposable components such as mouthpieces, nosepieces, or masks.

[0117] In a final embodiment, the present invention also relates to the use of the compositions described above. More specifically, the compositions are adapted for therapeutic or preventive use. The present invention is intended to prevent and treat specific diseases. Prevention of disease means reducing the likelihood of the disease becoming apparent and / or delaying the onset of the disease. Treatment of disease means reducing or eliminating the symptoms of the disease. Thus, the present invention is also intended to provide methods for treating and / or preventing the diseases of interest.

[0118] The subject is preferably a human subject, but certain embodiments of the present invention can be implemented in any subject that may benefit, such as a human subject, agricultural livestock (e.g., cattle, pigs, etc.), companion animals (e.g., horses), or companion animals (e.g., dogs, cats, etc.). In a preferred embodiment, the subject is a human, preferably a human patient, who may be an adult, an infant, or a newborn.

[0119] In some embodiments, a composition according to any of the above embodiments is used to prevent or treat a lung disease. In some embodiments, the lung disease may be an inflammatory lung disease, a pulmonary vascular disease, or an acute lung injury. More preferably, the inflammatory lung disease is pulmonary hypertension, also known as pulmonary arterial hypertension (PAH), asthma, bronchopulmonary dysplasia (BPD), allergy, idiopathic pulmonary fibrosis, or pneumonia. The inflammatory lung disease may be caused by an infection, such as a viral infection. In preferred embodiments, the viral infection is influenza, SARS-CoV-1, MERS, or SARS-CoV-2. In other embodiments, the acute lung injury is associated with sepsis or acute respiratory distress syndrome (ARDS).

[0120] These diseases also include pulmonary vascular diseases that do not have an inflammatory component. Yet another lung condition that may be treated according to the present invention includes sepsis or acute lung injury that may be associated with ventilation. An example of the latter is acute respiratory distress syndrome.

[0121] Pulmonary hypertension is a lung disease characterized by pulmonary artery blood pressure being significantly higher than normal. Symptoms include shortness of breath, chest pain especially during exercise, weakness, fatigue, fainting, mild headache especially during exercise, dizziness, abnormal heart sounds and murmurs, jugular vein distension, fluid accumulation in the abdomen, legs, and ankles, and blue discoloration of the nail beds.

[0122] Bronchopulmonary dysplasia (BPD) is a disease that affects newborns receiving oxygen or on a ventilator, premature babies, and especially very premature babies (born before 32 weeks of gestation). It is also called neonatal chronic lung disease (BPD). Causes of BPD include mechanical damage from ventilators, oxygen toxicity from oxygen therapy, and infections. This disease can progress from non-inflammatory to inflammatory over time. Symptoms include blue discoloration of the skin, chronic cough, rapid breathing, and shortness of breath. Patients with BPD are susceptible to infections such as respiratory syncytial virus (RSV) infection. Patients with BPD may also develop pulmonary hypertension.

[0123] Acute respiratory distress syndrome (ARDS), also known as respiratory distress syndrome (RDS) or adult respiratory distress syndrome, is a condition resulting from lung injury or acute illness. Lung injury can result from ventilation, trauma, burns, and / or aspiration. Acute illnesses can include infectious pneumonia or sepsis. This is considered a severe case of acute lung injury and is often life-threatening. It is characterized by lung inflammation, impaired gas exchange, release of inflammatory mediators, hypoxemia, and multiple organ failure. ARDS can also be defined as a ratio of arterial oxygen partial pressure (PaO2) to inspired oxygen volume (FiO2) below 200 mmHg, when bilateral infiltration is observed on chest X-ray. A PaO2 / FiO2 ratio of less than 300 mmHg with bilateral infiltration indicates acute lung injury and is often a precursor to RDS. Symptoms of ARDS include shortness of breath, tachypnea, and mental confusion due to decreased oxygen levels.

[0124] Idiopathic pulmonary fibrosis is characterized by scarring or thickening of the lungs for unknown reasons. It most commonly develops in people in their 50s to 70s. Symptoms include shortness of breath, a regular cough (usually a dry cough), chest pain, and decreased activity levels.

[0125] For prevention and / or treatment, EV may be used alone or in combination with one or more second agents or active ingredients, as may be the case. Mechanical interventions, such as ventilation with or without external oxygen administration, may also be performed on the patient.

[0126] The subjects may be those with a lung disease (or condition) that is suitable for treatment with the EV of the present invention, or subjects at risk of developing such a disease (or condition). Such subjects include neonates, particularly neonates born in the early stages of pregnancy. In this specification, a human neonatal is a human being from birth to about four weeks after birth. In this specification, a human infant is a human being from about four weeks after birth to about three years after birth. In this specification, early gestation is defined as a birth (or delivery) that occurs before the normal gestation period of a particular species of organism. In humans, the full gestation period is about 40 weeks, ranging from 37 weeks to 40 weeks or more. In humans, early gestation is defined as a birth before 37 weeks of gestation, similar to premature birth. Therefore, the present invention is intended to prevent and / or treat subjects born before 37 weeks of gestation, or even earlier (e.g., before 36 weeks, before 35 weeks, before 34 weeks, before 33 weeks, before 32 weeks, before 31 weeks, before 30 weeks, before 29 weeks, before 28 weeks, before 27 weeks, before 26 weeks, or before 25 weeks). Normally, such premature infants are treated as neonates, but the present invention is intended to extend treatment beyond the neonatal stage into childhood and / or adulthood. Some subjects may have a genetic predisposition to certain forms of lung disease, such as pulmonary hypertension, and such subjects may also be treated according to the present invention.

[0127] With regard to neonates, particularly those of low gestational age, the present invention intends to administer EV within 4 weeks, 3 weeks, 2 weeks, 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hours, 6 hours, 3 hours, or 1 hour after birth. In some important cases, MSC exosomes are administered within 1 hour after birth.

[0128] This disclosure, without limitation, further intends to administer EV even in cases where there are no symptoms indicating lung disease such as BPD.

[0129] In one embodiment, the composition containing EV according to the present invention may be used to treat COVID-19, more particularly COVID-19-induced pneumonia or acute pneumonia. COVID-19 is an emerging infectious disease caused by severe acute respiratory syndrome that attacks the human respiratory system and epithelial tissue of the lungs. It has been reported that there is a higher risk of developing more severe symptoms in the COVID-19 disease population. Major complications include pneumonia, acute respiratory distress syndrome (ARDS), multiple organ failure, septic shock, and death.

[0130] COVID-19, also known as SARS-CoV-2, has been shown to be associated with a different mechanism of lung infection that can progress to acute respiratory distress syndrome (ARDS) with cytokine storms, multiple organ failure, septic shock, and thrombosis as prodromal symptoms. In contrast, bacterial pneumonia is a common lung infection in which the entire lung or parts of the alveoli become inflamed and filled with fluid, pus, and cellular debris. It is mostly caused by viruses, fungi, or bacteria and is often treated with antibiotics. Other cases may involve cardiovascular complications, elevated liver enzymes due to liver damage, and neurological symptoms. In children, if the infection progresses, it can lead to pediatric multiple organ failure syndrome with symptoms similar to Kawasaki disease, and can be fatal. Current data show that children make up a small proportion of reported cases, with about 1% under 10 years old and about 4% between 10 and 19 years old.

[0131] The composition containing EV provides a multi-target therapeutic effect with the suppression of inflammatory processes as its main mechanism of action.

[0132] EV targets multiple mechanisms of lung injury, including excessive inflammation and cytokine storms, fibrosis, oxidative stress due to (mechanical) ventilation, and apoptosis of epithelial cells due to viral activity and inflammatory responses.

[0133] In another embodiment, the composition containing EV is used as an adjunctive treatment for COVID-19, more particularly for COVID-19-induced pneumonia or acute pneumonia.

[0134] In another embodiment, the composition according to any of the above embodiments is used to prevent or treat inflammatory bowel diseases (IBD), such as Crohn's disease or ulcerative colitis. IBD is a general term for diseases that cause inflammation of the gastrointestinal tract, with ulcerative colitis (UC) and Crohn's disease (CD) being the most common. UC is a disease that causes prolonged inflammation and ulceration of the innermost mucosa of the colon and rectum. CD can occur anywhere in the gastrointestinal tract and can penetrate deep into the affected tissue. A symptom of CD is the development of perianal fistulas. However, these diseases are similar in that they all cause abdominal pain, severe diarrhea, fatigue, and weight loss.

[0135] For lung diseases, the preferred method of administration of EV is intratracheal infusion or inhalation. For neurological diseases, preferred methods are systemic infusion, intranasal infusion, or inhalation. For Crohn's disease fistulas, ulcers, or cartilage repair, local injection is preferred. For wound healing, burns, and / or ulcer treatment, EV is preferably administered externally to the body in combination with a hydrogel or polymer medical device for sustained release of EV.

[0136] The EV of the present invention is administered in an effective dose. An effective dose means the amount of the drug that alone produces the desired result. The absolute dose depends on various factors, including the material selected for administration, whether it is a single or multiple dose, and individual patient parameters such as age, physical condition, body size, weight, and stage of disease. These factors are known to those skilled in the art and can be addressed without going beyond routine experimentation. Furthermore, the dosage may vary depending on the target to whom the composition is administered.

[0137] In one embodiment of the present invention, the EV is the 10 of the patient 9 EV / kg~10 12 It is administered at a dose of EV / kg, or 10% of the patient. 10 EV / kg~10 12It is administered in doses of EV / kg. In further embodiments, the dose is 10 for children (ages from 0 months to 12 years). 9 EV / kg~10 11 The range is EV / kg. For 12-19 year olds and adults, the above dose is 10 9 EV / kg~10 12 It can be within the range of EV / kg.

[0138] In one embodiment of the present invention, the EV is used for the treatment of perianal fistulas of CD, approximately 10 per patient. 10 ~about 10 12 It is administered at the EV dose.

[0139] Furthermore, this disclosure also intends to describe repeated administration of EV, including two, three, four, five or more doses. In some cases, the EV may be administered consecutively. Repeated or consecutive doses may be administered over periods of several hours (e.g., 1-2, 1-3, 1-6, 1-12, 1-18 or 1-24 hours), several days (e.g., 1-2, 1-3, 1-4, 1-5, 1-6 or 1-7 days), or several weeks (e.g., 1-2 weeks, 1-3 or 1-4 weeks), depending on the severity of the condition being treated. If the administration is repeated but not consecutive, the interval between doses may be several hours (e.g., 4 hours, 6 hours or 12 hours), several days (e.g., 1 day, 2 days, 3 days, 4 days, 5 days or 6 days), or several weeks (e.g., 1 week, 2 weeks, 3 weeks or 4 weeks). The intervals between doses may be the same or different. For example, if the symptoms of the disease appear to be worsening, EV may be administered more frequently, and the frequency of EV administration may be reduced once the symptoms stabilize or improve.

[0140] In some cases, low doses of EV may be administered repeatedly intravenously. Therefore, this disclosure intends for repeated administration of low-dose forms of EV and single administration of high-dose forms of EV. Low-dose forms include, but are not limited to, 10 per kilogram or per local infusion. 10 ~10 11 The EV range may be, and the high-dose form is not limited to, 10 per kilogram or per local injection.11 ~10 12 This range may be appropriate. In particular, it will be understood that a single or repeated administration of low-dose or high-dose extravasation (EV) may be intended, depending on the severity of the disease, the health status of the subject, and the route of administration.

[0141] EV may be administered via any route that allows delivery to the lungs or gastrointestinal tract. Suitable routes of systemic administration are intravenous bolus injection or continuous infusion. More direct routes such as intranasal administration, intratracheal administration (e.g., by intubation), and inhalation (e.g., via aerosol from the mouth or nose) are also intended by the present invention and may be more appropriate in some cases, especially when a rapid action is required. As used herein, an aerosol is a suspension of a liquid dispersed as fine particles in a gas, and also includes fine mist or spray containing such particles. As used herein, aerosolization is the process of producing an aerosol by changing a liquid suspension into fine particles or droplets. This may be done using an aerosol delivery system such as a pressurized pack or a nebulizer. Examples of nebulizers, but not limited to, include airflow (i.e., pneumatic), ultrasonic, and vibrating mesh nebulizers, using a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gases. In addition to nebulizers, other devices for delivery to the lungs include, but are not limited to, metered-dose inhalers (MDIs) and dry powder inhalers (DPIs). For use in inhalers or air inhalers, for example, gelatin capsules and cartridges may be formulated containing lyophilized exosomes and a suitable powder base such as lactose or starch.

[0142] EV may be formulated for parenteral administration by infusion, including bolus or continuous infusion, if systemic delivery is desired. Infusion formulations may be provided in unit-dose forms with or without preservatives, such as ampoules or multi-dose containers.

[0143] The composition may take the form of a water-soluble suspension, solution, or emulsion in an oily or aqueous vehicle, and may contain formulations such as suspending agents, stabilizers, and / or dispersants. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides. The aqueous injection suspension may contain a substance that increases the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may contain a suitable stabilizer or agent to improve solubility. Alternatively, the exosomes may be in a lyophilized or other powder or solid form for reconstitution in a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0144] The EVs and compositions described herein are preferably obtained by the methods described above.

[0145] Next, the present invention will be described in more detail with reference to examples that are not limiting. [Examples]

[0146] Example 1 - Isolation of EVs from MSCs MSCs derived from umbilical cord tissue or Wharton's jelly were grown in a growth apparatus equipped with a cell culture medium supply container (1) and a stirred bioreactor (2) that was fluidly connected to it. Referring to Figure 1, the reference numerals are as follows. 1. Cell culture medium container 2. Agitated Bioreactor 3.Cell supernatant container 4. Refrigerator 5a, 5b, 5c. Peristaltic pump 6. First filter unit 7. Second filter unit 8. TFF Cassette 9. Final product

[0147] Cells were grown in the presence of a three-dimensional microcarrier placed inside the bioreactor (2) and in a xenofree serum-free medium containing purified human albumin at a concentration of 3 g / L. This medium further contained recombinant purified transferrin at a concentration of 60 mg / L.

[0148] The minimum concentration inside the bioreactor (2) is at least 40 × 10 6 MSCs were grown and expanded until the cell / L concentration reached a maximum of 80% confluence. Cell concentration was determined by cell counting and fluorescent labeling of MSCs attached to microbeads. At this stage, the cells were ready to begin secreting extracellular viable cells (EVs). For this purpose, fresh serum-free xeno-free medium was diluted 1:10 with basal medium (DMEM / high glucose content / phenol red-free / glutamax, Thermoscientific) and supplied to the cell culture. After 24 hours, EV harvesting could be initiated. For this purpose, the prepared cell culture or cell supernatant in the bioreactor (2) was pumped into a cell supernatant container (3) placed in a refrigerator (4) set to 4°C. This supernatant contained EVs produced by the MSCs. The supernatant collected in container (3) was then pumped by a peristaltic pump (5c) to a first filter unit (6) with a 2-micron filter. The supernatant was filtered by dead-end filtration in a closed system equipped with a peristaltic pump (5c) and supplied with a constant flow rate of 100 mL / min through a filter (6). Subsequently, the permeate from the first filtration step was filtered through a second filter unit (7) which was fluidly connected to the first filter unit (6) and had a 0.2 micron filter. The permeate was filtered by dead-end filtration in a closed system equipped with a peristaltic pump (5c) that supplied a constant flow rate of 100 mL / min through the filter (7). The permeate containing EV was collected in a 1 L bag and then stored at 4°C.

[0149] In the final step, the permeate was passed through a TFF cassette (8) with a cutoff of 100 kDa, which was fluidly connected to a dead-end filter unit (6, 7). The retainate containing EV that passed through the TFF cassette (8) was washed with physiological saline buffer, and the TFF cassette (8) was circulated using a peristaltic pump (5c) at a rotor speed of 300 rpm to concentrate it until the final volume was 10 mL. The final product (9) was recovered from the TFF (8) and frozen at -80°C or below, or stored at 4°C in a cryotube, bag, or other suitable container.

[0150] The aforementioned final product was analyzed to confirm its quality. Quality control included measuring the albumin concentration associated with EVs by colorimetric analysis and ELISA to ensure the purity and stability of EVs, and particle analysis using techniques such as nanoparticle tracking analyzer (NTA), controlled resistance pulse sensing (TRPS), electron microscopy, and / or RAMAN microscopy. The product samples were tested for the presence of endotoxins and / or mycoplasma. Other quality control measures included qualitative chromatography, mass spectrometry, ELISA, sequencing, qRT-PCR, and in vitro activity tests of T cells, B cells, and macrophages.

[0151] Under the culture conditions described above, the MSCs should have at least 0.25 × 10¹⁶ cells per mL of cell medium during the 18-24 hour period of culturing the MSCs. 9 It produces individual particles.

[0152] The above experiments were conducted with MSCs derived from ulcerative colitis (UC), but were repeated with MSCs from other tissues or origins, including, but not limited to, mammary gland, bone marrow, Wharton's jelly, (umbilical cord) blood, peripheral blood, amniotic membrane, adipose tissue, dental pulp, fallopian tube, liver, and lung tissue.

[0153] Example 2: Measurement of albumin concentration in EV products The albumin concentration of the EV product obtained by the method described in Example 1 was analyzed. The albumin concentration was measured by the following two methods. -Colorimetric measurement -ELISA In short, albumin concentration in samples was evaluated by a colorimetric reaction using bromocresol green. Concentration was measured by ELISA using an anti-human albumin antibody.

[0154] The albumin concentration of the final product, as measured by colorimetric reaction, was 16 g / L.

[0155] The concentration measured by ELISA was approximately 17 g / L. The molecular weight of albumin is approximately 66 kDa, and it is expected that albumin not associated with extracellular viable cells (EVs) will be removed by TFF with a cutoff of 100 kDa. Therefore, the remaining albumin is thought to be associated with EVs.

[0156] Example 3: Measurement of transferrin concentration in EV products The transferrin concentration of the EV product obtained by the method described in Example 1 is analyzed. The transferrin concentration is quantified using ELISA (Abcam) according to the manufacturer's protocol.

[0157] The transferrin concentration in the final product, as measured by ELISA (Abcam), was 70 mg / L.

[0158] Example 4: Purity of clinical-grade culture medium for EV production Experiments were conducted to evaluate the concentration of contaminating particles in various culture media used in EV manufacturing. The culture media tested were as follows: 1. Xenofree serum-free medium containing 0.3 g / L purified human albumin and 7 mg / L recombinant purified transferrin (diluted approximately 1:10 with DMEM medium to the medium used in Example 1). 2. Basic DMEM medium (no additives) (Thermo Scientific / Lonza) 3. Culture medium with unspecified products added (ThermoScientific / Lonza)

[0159] Fresh culture medium samples were analyzed using NTA, TRPS, and FACS instruments. Particle size and quantity were quantified. Purified water was used as a control.

[0160] EVs are produced using one of the above-mentioned media, as described in Example 1. Quality control results show that while medium 2 has the fewest contaminated particles in EVs produced using different media, this medium is not suitable for long-term cell culture. Medium 3 has the most contaminated particles. Medium 1 contains an intermediate number of contaminated particles compared to the other media, but is suitable for long-term cell culture.

[0161] Example 5: Cell culture in clinical-grade medium for EV production This experiment shows the cell number, cell proliferation curve, and cell viability in the following conditions. 1. Xeno-free serum-free medium containing 3 g / L purified human albumin and 60 mg / L recombinant purified transferrin (the same medium used in Example 1). 2. Basic DMEM medium (no additives) (Thermo Scientific / Lonza) 3. Culture medium with unspecified products added (ThermoScientific / Lonza)

[0162] method In the first experiment, MSCs are grown in the presence of medium 1, medium 2, or medium 3 in the culture system described above in the first example until the cells reach 70-80% confluence.

[0163] Subsequently, in the second experiment, the cells that were grown in medium 1, medium 2, and medium 3 in the first experiment are refreshed with the same medium.

[0164] In the third experiment, the cells grown in medium 1 in the first experiment are refreshed with medium 1 mixed with medium 2 in a 2:8 ratio. In the same experiment, the cells grown in medium 3 in the first experiment are refreshed with medium 3 mixed with medium 2 in a 2:8 ratio.

[0165] In the fourth experiment, the cells grown in medium 1, medium 2, and medium 3 in the first experiment are refreshed with medium 2.

[0166] In Experiments 2, 3, and 4, cell culture was continued for at least 5 days, and the culture medium was refreshed every 24 hours. Medium refreshment was performed by removing 100% of the old medium and adding the same amount of new medium.

[0167] result In the first experiment, it was confirmed that MSCs in mediums 1 and 3 attached to microcarriers, but this was not observed in medium 2. MSCs could grow / proliferate in both mediums 1 and 3, but could not maintain viability in medium 2. In subsequent experiments, only cells cultured in mediums 1 and 3 were used.

[0168] In Experiment 2, cell cultures in undiluted medium 1 and medium 3 rapidly overgrow within 24-48 hours, after which the cells die. In Experiment 3, cell cultures using diluted medium 1 and medium 3 continue to grow very slowly, and the cells can survive for at least 5 days. In Experiment 4, cell cultures using medium 2 instead of medium 1 and medium 3 survive for only 2 days, after which the cells die.

[0169] Example 6: Concentration and efficiency of EV in clinical-grade culture medium This experiment shows the EV concentration in the following conditions. 1. Xenofree serum-free medium containing 0.3 g / L purified human albumin and 7 mg / L recombinant purified transferrin (diluted approximately 1:10 with DMEM medium to the medium used in Example 1). 2. Basic DMEM medium (no additives) (Thermo Scientific / Lonza) 3. Culture medium with unspecified products added (ThermoScientific / Lonza)

[0170] Grow the MSCs in one of the above-mentioned culture media as described above. Continue culturing for at least 5 days, refreshing all media every 24 hours.

[0171] The EV production protocols of Examples 1 and 5 are used. Medium 1 has the highest concentration of cell-secreted particles (true EVs), determined by subtracting the particles present in fresh medium from the total particles. In particular, in this example, at least 95% and up to 99% of the particles are secreted by MSCs (EVs).

[0172] Example 7: Production efficiency of EVs in a 3D bioreactor system This experiment demonstrates the efficiency of EV generation in the following contexts. i) Three-dimensional culture system as shown in Figure 1 and described in Example 1 ii) Two-dimensional culture system

[0173] MSCs are produced in a three-dimensional culture system (0.5 L and 1 L agitated bioreactors) and a two-dimensional multilayer flask using growth medium (i) followed by diluted medium (i) for EV generation. Cell number, medium volume, footprint, and EV yield are calculated and extrapolated to larger bioreactors. The 3D bioreactor method has a smaller footprint, consumes less medium, and yields higher EV per batch compared to the 2D culture system.

[0174] Example 8: Separation efficiency of EVs using a TFF system This experiment demonstrates the purification efficiency of EV using the following method. i) A TFF system with a cutoff of 100 kDa as the final concentration step. ii) Size exclusion chromatography iii) Ultracentrifugation

[0175] Using TFF concentration yields significantly higher EV recovery rates compared to SEC. While TFF yield is comparable to UC yield, the latter suffers from EV damage and reduced quality. Furthermore, UC is unsuitable for GMP / mass production.

[0176] Example 9: Activity of EV in an in vitro assay intended for use in BPD treatment. EV is manufactured according to Example 1 and used to treat the following major symptoms of BPD.

[0177] a) Inflammation Internalization assay of extracellular vesicles (EVs). Fluorescently labeled EVs are co-cultured with human peripheral blood mononuclear cells (PBMCs). The population of immune cells encapsulating the labeled EVs is quantified by flow cytometry. Anti-CD4, anti-CD8, anti-CD11c, anti-CD14, anti-CD19, anti-CD56, and anti-CD15 markers are examined. Analysis is performed at 0, 1, 6, 12, and 24 hours, except for neutrophils, which are analyzed at 6 hours. EVs produced by fibroblasts are used as a negative control.

[0178] Lymphocyte migration assay. Extracellular lymphocytes (EVs) are co-cultured with PBMCs in a Transwell system. Cell migration in response to the chemokine SDF-1 is examined. Cell phenotypic analysis and quantification are performed by flow cytometry.

[0179] B-cell assay. Extracellular viable cells (EVs) are co-cultured with CpG-stimulated human peripheral blood mononuclear cells. Cell proliferation and differentiation into plasma cells are examined, and phenotypic analysis and quantification are performed by flow cytometry. Cytokine and antibody quantification is performed by ELISA.

[0180] T cell assay. EVs are co-cultured with human peripheral blood mononuclear cells and T cell activator CD3 / CD28 beads. CD4+ T cell proliferation and CD4+ T cell apoptosis are quantified using flow cytometry. The Treg / Teff ratio is determined. Proliferating Tregs are quantified by flow cytometry. Quantification of cytokines such as IL10, TGF-β, galectin-1, HGF, PGE2, GM-CSF, IL2, TNF-α, and IFN-γ is performed by ELISA.

[0181] CD cell assay. CD cell activation is investigated by flow cytometry after co-culturing with EV cells. The upregulation of the costimulatory molecules CD80 and CD86, as well as the maturation marker CD83, is quantified by flow cytometry. The cytokines IL6, IL8, IL12, CCL3, CCL4, IL10, and TGF-β are quantified by ELISA. A phagocytosis assay is performed using flow cytometry (incubation with FITC-dextran). A migration assay is performed using the Transwell assay and flow cytometry.

[0182] Macrophage assay. EVs were co-cultured with macrophages stimulated with M1 (LPS) and M2 (IL4 / IL13). The M1 / M2 ratio was calculated by flow cytometry. Quantification of cytokines IL6, TNFα, IFNγ, IL1β, IL12, IL10, VEGF, MCP1, TGF-β, and FGF was performed by ELISA and confirmed by qPCR gene expression analysis. Transwell migration assay (fMLP direction) was performed and quantified by flow cytometry.

[0183] NK cell assay. Extracellular viable cells (EVs) are co-cultured with PBMC-derived NK cells. TNFα and IFNγ cytokines are quantified by ELISA. CD27, CD11b, CD107a, IFN-γ maturation markers, and cell proliferation are quantified by flow cytometry.

[0184] b) Fibrosis Experiments will be conducted using normal human lung fibroblasts and human epithelial cells with commercially available fibrosis assays. Human lung fibroblasts will be co-cultured with extravasation cells (EVs) delivered at varying doses and concentrations over different time periods. Subsequently, α-SMA and collagen I / fibronectin will be analyzed.

[0185] Another experiment measures the effect of extracellular vein (EV) on epithelial-mesenchymal transition (EMT) in human primary bronchial epithelial cells. EMT is examined using commercially available assays according to the manufacturer's protocol. In one experiment, experimental primary cells derived from healthy tissue or patients with idiopathic pulmonary fibrosis are stimulated with TGF-β to induce EMT, and unstimulated cells are used as controls. Subsequently, human epithelial cells are co-cultured with EV delivered at varying doses and concentrations over different time periods. EMT is detected and quantified using FACS and the expression of E-cadherin and fibronectin.

[0186] Another experiment measures the effect of extracellular viable cells (EVs) on fibroblast-myofibroblast conversion (FMT) in human primary bronchial fibroblasts. FMT is examined using commercially available assays according to the manufacturer's protocol. In one experiment, experimental primary cells derived from healthy tissue or patients with idiopathic pulmonary fibrosis are stimulated with TGF-β to induce FMT, and unstimulated cells are used as controls. Human fibroblasts are then co-cultured with EVs delivered at varying doses and concentrations over different time periods. FMT is detected and quantified using an α-smooth muscle actin marker.

[0187] c) Oxidative stress and cellular apoptosis We will quantify cellular apoptosis and oxidative stress (ELISA, flow cytometry) using assays and cell lines reported in fibrosis research.

[0188] Oxidative stress is induced using H2O2 or other reagents. Cells are exposed to oxidative stress for varying periods of time and with varying amounts of oxidative stress inducers. Subsequently, the cells are co-cultured with extravasation molecules (EVs) delivered at varying doses and concentrations over varying periods of time. The direct effects of oxidative stress are measured by the detection and quantification of reactive oxygen species, and the indirect effects are measured by nucleic acid damage, lipid peroxidation, and protein oxidation. Detection and quantification of direct and indirect markers of oxidative stress are performed using ELISA and FACS.

[0189] Apoptosis in cells in response to oxidative stress will be detected and quantified using commercially available assays according to the manufacturer's protocol. In one experiment, cell viability will be detected by FACS using LIVE / DEAD® (Thermo Fisher).

[0190] In another experiment, early apoptosis was detected using annexin V immunofluorescence staining and quantified using FACS.

[0191] In another experiment, early apoptosis was detected by quantifying activated caspase-3 and caspase-7 using FACS.

[0192] Example 10: Activity of EV in an in vitro assay intended for use in the treatment of Crohn's disease This experiment demonstrates the activity of EV prepared according to Example 1 in the treatment of major symptoms of Crohn's disease. In addition to the experiments described in Examples 9(a) to (c), an angiogenesis assay using EV will also be performed.

[0193] In one experiment, HUVEC cells are co-cultured with EVs on Matrigel to investigate tube formation. The experiment will be conducted using human HUVEC cells and a commercially available angiogenesis tube formation assay. To mimic the properties of the natural extracellular matrix necessary for angiogenesis, the cells will be cultured in a three-dimensional environment such as a hydrogel. EVs will be delivered at different times and in multiple doses and concentrations. Analysis will be performed using immunolabeling and fluorescent labeling of HUVEC cells, as well as measurements of the length and branching of tubes that react to EVs.

[0194] Example 11: Study of the in vivo distribution of extravasation cells (EVs) in an in vivo model of bipolar disorder (BPD). This experiment evaluates the in vivo distribution of extravasation cells (EVs) produced according to Example 1 in an in vivo model of bipolar disorder (BPD).

[0195] In vivo non-clinical studies will be conducted using an established and widely used animal model for BPD, namely neonatal rats exposed to a high-concentration oxygen environment (O'Reilly M et al, 2014; Thebaud B, 2018). Since neonatal rats have immature lung structures at birth (late tubular / early saccular stage) and reach the alveolar stage around 5 days postnatality, this model can be said to mimic the condition of premature infants on artificial respiration. Although rats reach full alveolar formation around 30 days postnatality, it is known that exposure to a high-concentration oxygen environment shortly after birth hinders alveolar development, increases alveolar macrophages, and adversely affects pulmonary vascular formation. Thus, from a structural standpoint, the lungs of neonatal rodents are almost identical to those of premature human neonates born between 24 and 28 weeks of gestation (Porzionato A et al, 2019; Porzionato A et al, 2021).

[0196] In a neonatal rat model of high-concentration oxygen-induced bipolar disorder (BPD), the in vivo distribution of EV products administered intratracheally (IT) will be evaluated as follows.

[0197] The extracellular viability (EV) of the product is stained with a lipophilic fluorescent marker (DiR iodide [1,1-dioctadecyl-3,3,3-tetramethylindotricarbocyanine iodide]) to evaluate the in vivo distribution of the product after in vivo administration.

[0198] Of a total of 40 wild-type Sprague-Dawley rat pups, 20 were exposed to a normal oxygen environment (normoxia), and the remaining 20 were exposed to a high oxygen environment (hyperoxia) as described above. On the 7th day after birth, the 20 pups (10 in the high oxygen environment and 10 in the normal oxygen environment) were subjected to 1 × 10⁶ 9 The EV product of particles is administered via IT, and this is 1 x 10 11 This corresponds to / kgBW. 20 control rat pups (10 in a high-concentration oxygen environment, 10 in a normal-concentration oxygen environment) are infused with PBS control. IT infusion is selected for direct local administration of the EV product to the site of lung damage, with the ultimate goal of applying this method clinically.

[0199] Fluorescence analysis will be used to evaluate the whole-body distribution, thereby assessing the pigment concentration in individual organs of each group of 10 rat pups at different time points (3 hours and 24 hours) after injection.

[0200] The results of this assay revealed that in rat pups exposed to normal oxygen concentration conditions, the EV product was uniformly distributed to various organs 3 hours after injection, with a large signal observed in the inguinal lymph nodes. Under normal oxygen concentration conditions, a signal could be observed in the lymph nodes (inguinal and axillary regions) 24 hours after injection. In these rat pups, the labeled EV product was rapidly excreted from the lungs and distributed throughout the body. The signal was particularly concentrated in the lymph nodes (inguinal and axillary regions), and after 24 hours, it was only observed in these locations.

[0201] In rat pups exposed to a high-concentration oxygen environment, lung signals completely disappeared 3 hours after injection, and signals from other organs also almost completely disappeared. Almost all signals were observed in the axillary and inguinal lymph nodes. This suggests that uptake of the EV product from the injection site is rapid under high-concentration oxygen conditions. In rat pups treated with a high-concentration oxygen environment, almost all signals were observed in the axillary lymph nodes 24 hours after injection (no signals were observed in the inguinal lymph nodes).

[0202] It should be noted that these results are novel, as lymph nodes have never been evaluated as a target tissue in studies of the in vivo distribution of EVs. These results clearly indicate that lymph nodes are the primary site of accumulation of EV products and are the basic effector sites that trigger immunological responses to EV product administration.

[0203] Example 12: EV activity using an in vivo model of BPD This experiment demonstrates the activity of EVs prepared according to Example 1 in an in vivo model of BPD. The model adopted follows the description in Porzionato et al. 2018.

[0204] In short, with the approval of the ethics committee, 30 wild-type Sprague-Dawley rats will be used for the study. Methods for exposure to high-concentration oxygen environments have been established by various research teams and published in multiple publications (Grisafi et al., 2012, 2013; Marconi et al., 2014; Porzionato et al., 2012, 2018). Experiments will be conducted on pup rats raised in a chamber with continuous oxygen monitoring. Experimental animals will be exposed to 60% oxygen for two weeks and treated with the EV product of the present invention by intratracheal administration. Control animals will be exposed to 60% oxygen for two weeks and treated with intratracheal administration of saline (placebo). Control animals in a normal oxygen environment will be exposed to 21% oxygen for two weeks and treated with the EV product of the present invention or saline by intratracheal administration. EV will be injected on days 3, 7, and 10 after birth.

[0205] The efficacy of single or multiple IT infusions of the EV product will be tested. The dosage of this product is 6.4 × 10⁻⁶. 9 The dosage is 3.19 × 10 at three different time points. 11 ~5.71×10 11 This corresponds to the range of particles / kg body weight.

[0206] Subsequently, the lungs of the animals are fixed according to the protocol described in Porzionato et al., 2018, and the lung volume is measured according to Scherle's method (Scherle, 1970). Histological and immunohistological analyses are performed using lung slices. The tissue contraction coefficient is estimated according to the protocol described in Porzionato et al., 2018. Stereoanalysis is performed as described in Porzionato et al., 2018, and several parameters are quantified, including: i) volume fraction of alveolar space and alveolar diaphragm, ii) total volume of alveolar space and alveolar diaphragm, iii) surface area density of space, iv) total surface of alveolar space, v) total lung volume, vi) number of alveoli and mean alveolar volume, and other parameters.

[0207] In particular, the efficacy of EV product IT administration was evaluated by morphological measurements, cytoflometry, and qRT-PCR analysis to establish the recovery and inflammatory response of hyper-oxygen-induced lung injury after treatment. Morphological analyses included lung volume estimation, histological evaluation, immunohistochemical analysis, immunofluorescence analysis of myofibroblasts, immunofluorescence analysis of type II alveolar epithelial cells, quantification of immunofluorescence-stained samples, Alcian blue staining and quantification, protein carbonylation assay, stereochemistry of alveolar formation, morphological measurements of the arterial muscle layer, microvascular density, and morphological measurements of macrophage populations.

[0208] All histopathological and morphological evaluations will be performed blindly on the experimental group. At 3, 7, and 10 days postnatally, all animals will complete a treatment cycle including three IT injections of either the control (vehicle only) or the EV product solution, respectively.

[0209] Postnatal exposure to a high-concentration oxygen environment leads to alveolar destruction, resulting in deterioration of all listed histological parameters. Of these parameters, a statistically significant decrease in alveolar surface area was observed in the high-concentration oxygen exposure group. Furthermore, septal thickness was significantly increased, which may be due to inflammatory processes (Table 1).

[0210] [Table 1]

[0211] Multiple comparisons (Bonferroni test) showed that, regarding alveolar surface, the high-concentration oxygen environment exposure group had a statistically significant difference compared to both the normal-concentration oxygen environment exposure group and the high-concentration oxygen environment exposure + EV product treatment group, indicating that the EV product demonstrated a therapeutic effect. Regarding septal wall thickness, there was no statistically significant difference between the high-concentration oxygen environment exposure group and the high-concentration oxygen environment exposure + EV product treatment group.

[0212] Immunofluorescence quantification of lung surfactant-related protein C (SFTPC)-positive cells, a specific marker for type II lung epithelial cells (ATII), showed a significant decrease in ATII after hypoxia-induced injury. Administration of the EV product increased the amount of SFTPC in lung tissue. Similar results were obtained with Alcian blue staining, a marker of glycosaminoglycan production. Furthermore, the effect of the EV product on protein carbonylation, an established marker of oxidative damage to proteins caused by reactive oxygen species (ROS) resulting from exposure to high-concentration oxygen environments and the associated inflammatory process, was tested. As a result, the damage increased under high-concentration oxygen conditions was reversed by administration of the EV product. No increase in mortality was observed in the EV product-treated group, confirming the safety of intratracheal administration.

[0213] These results demonstrate the efficacy of using EV products in the treatment of bronchopulmonary dysplasia in an established neonatal rat model. Preliminary data suggest that EV products protect the lung parenchyma from oxidative stress and increase surfactant production by AT2 cells, a key factor in the development of BPD.

[0214] Example 13: Evaluation of EV activity using an in vivo model of Crohn's disease This experiment demonstrates the activity of EV prepared according to Example 1 in an in vivo model of Crohn's disease. Briefly, the experiment was conducted using 8-week-old female B57BL / 6J mice. Colitis was induced by adding 3% sodium dodecyl sulfate (DSS) to drinking water and administering it ad libitum for 5 days. This is an established and widely used animal model, cited hundreds of times in PubMed (see Kawada et al., 2007 for a summary paper). All animals were treated in accordance with appropriate ethical standards. The animals were divided into the following three experimental groups. - Group 1 (n=4, normal control): PBS (vehicle only), 0.2 mL administered intraperitoneally (ip) daily from day 1 to day 5. - Group 2 (n=5, colitis induction): The procedure was the same as Group 1, except that 3% DSS was added to the drinking water. - Group 3 (n=4, treatment with colitis induction and EV): The procedure was the same as for Group 2, except that MSC-EV suspended in 0.2 mL of PBS was added via the IP route.

[0215] On the sixth day, the animals are sacrificed using CO2. The large intestine is removed, a portion of the tissue is fixed with formalin for histological analysis, and the remaining tissue is immediately frozen with liquid nitrogen and stored at -80°C for RNA extraction.

[0216] EV dosage and route of administration Isolate the EV using the procedure described above and administer it. Route of administration: EV is suspended in 0.2 mL of PBS and administered intraperitoneally daily from day 0 to day 5.

[0217] - Assessment of intestinal injury animal weight Disease activity indicators (fecal assessment; see Tanaka F, 2008) - Pathological signs of inflammation (flogosis) - Titration of inflammatory mediator expression in colon tissue extracts. Analysis of TNFalfa, IL6, IL-1β, and Co×2 by RT-PCR.

[0218] Statistical analysis of the results Data are presented as mean ± SD. Between-group differences are analyzed using a t-test, with p<0.05 considered statistically significant.

[0219] result In conclusion, the results indicate that EV is suitable for reducing the inflammatory response, lowering the disease activity index, and improving the overall condition in experimental colitis. EV improves clinical presentation and flogistic responses in animal models of intestinal inflammatory disease.

[0220] Example 14: Protocol for administration and evaluation of safety and efficacy of EV product for use in the treatment of BPD in humans. The safety and efficacy of the EV product obtained by the method described in Example 1 for use in the treatment of BPD will be investigated in a human clinical trial. Briefly, the trial will involve premature infants at high risk of BPD, born between 23 and 28 weeks of gestation and weighing 1500g or less at birth, who are intubated and on mechanical ventilation with an inhaled oxygen fraction (FiO2) greater than 25%. The EV product will be administered intratracheally. Since only premature infants undergoing invasive ventilation via an endotracheal tube will be included in the trial, intratracheal (IT) application of the EV product or saline control solution will not pose any further risks.

[0221] This trial is divided into the following two phases. Safety and tolerability of Phase I EV products In Phase I, 18 subjects were divided into six cohorts of three, and each cohort was administered one of three different dosages (low dose (LD): 1 × 10⁻¹⁴). 10 EV / kg body weight (BW), medium dose (MD): 3×10 10 EV / kgBW, or high dose (HD): 9 × 10 10 The EV product is administered using either EV / kgBW or one of two regimens (single or three IT doses, with a 24-hour interval between doses).

[0222] The primary objective is to identify dose-limiting toxicity (DLT) or maximum tolerated dose (MTD) when three different dose levels of EV products are administered as a single or multiple IT dose.

[0223] In this phase, we evaluate several aspects, including the following: - Acute and short-term toxicity when EV products are administered via IT (single or multiple doses with varying dosages) at 36 weeks postmenstruation (PMA) or at discharge. -DLT within 6 hours and 24 hours after administration of the EV product. - AEs (adverse events) and SAEs (serious adverse events) (including fatal cases) (related and unrelated) reported at PMA36 or at different points in time until discharge. - Assessment of the medium-term toxicity of the EV product at multiple points in time leading up to discharge, using clinical and blood tests (liver and kidney function tests, hematopoietic indicators, blood pressure, body temperature, etc.), lung ultrasound, and echocardiography. - Number of subjects who received oxygen therapy and artificial respiration at 28 days of age (BPD definition according to Jobe AH et al., 2001). - Incidence and severity of BPD after administration of EV product at 36 weeks of PMA, according to the modified NICHD severity classification (grades I-IIIA) case definition (based on Higgins RD et al., 2018). This was compared with similar cases in the past. - Overall health status (adjusted age in 1 year) after administration of the EV product at the end of the study (EOS). - Mortality rates at 36 weeks PMA and EOS (adjusted age in 1 year).

[0224] Phase IIa. Efficacy of EV products in BPD treatment. In Phase IIa, 70 subjects will be divided into two groups of 35. One group will receive treatment with the EV product at a dose level and regimen selected based on the results of Phase I, while the other group will receive saline as a control solution (placebo group).

[0225] The primary objective is to evaluate the efficacy of EV products in the treatment of BPD in a randomized, double-blind, placebo-controlled trial.

[0226] In this phase, we evaluate several aspects, including the following: - Results of the efficacy of the EV product compared to the placebo group (saline solution) in terms of the number and severity of BPD cases at 36 weeks PMA. The status and severity of BPD were assessed according to the definition of the modified NICHD severity classification (grades I-IIIA). - For both groups, the number of subjects who required oxygen and those who received mechanical ventilation assistance at 28 days after birth. - Overall health status of the EV product administration group and the placebo group at EOS (adjusted for 1 year of age). - Mortality rates at PMA 36 weeks and EOS (1-year adjusted age) for both groups. - For both groups, AEs and SAEs (related and unrelated) reported at different time points until PMA 36 weeks or discharge. - For both groups, SAEs (related and unrelated) reported through passive surveillance up to EOS. - For both groups, evaluation of the duration of MV / ventilator support until 36 weeks of PMA or discharge, ROP, NEC, IVH, and sepsis. - Examination of immunomarkers (IL-6, IL-8, TNFa, TGFb1, IL1b, IL1ra) in tracheal aspirate at several points in time before intubation of the child. - Assessment of the neurodevelopmental status of infants in both groups at 1 year adjusted age or EOS.

[0227] The present invention is not limited to the embodiments described in the examples and / or illustrated in the figures. Rather, the methods according to the present invention can be carried out in many different ways without departing from the scope of the invention. [Explanation of Symbols]

[0228] 1. Cell culture medium container 2. Agitated Bioreactor 3 Cell supernatant container 4 Refrigerator 5a, 5b, 5c Peristaltic pump 6. First filter unit 7. Second filter unit 8 TFF cassettes 9 Final product

Claims

1. A process for producing a pharmaceutical composition of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs), - Culturing and growing MSCs in serum-free xeno-free medium containing human albumin at a concentration of 1 g / L to 5 g / L and human transferrin at a concentration of 55 mg / L to 100 mg / L, - Collect the cell supernatant containing EV from the cultured MSCs, - A two-step filtration process is used to filter the cell supernatant and obtain EV, which includes a first filtration step of filtering the cell supernatant through a first filter having a mesh size of 1 to 5 μm, and a second filtration step of passing the filtrate obtained in the first filtration step through a second filter having a mesh size of less than 1 μm, and then A process comprising: concentrating the filtrate from the filtration process by a tangential flow filtration (TFF) with a cutoff value of 100 kDa.

2. The process according to claim 1, wherein the human albumin and human transferrin are purified from human plasma or recombinant albumin and recombinant transferrin.

3. During the 18-24 hour period in which the MSCs are cultured, at least 0.25 × 10 per mL of culture medium ９ The process according to claim 1 or 2, wherein particles are produced, and at least 90% of the particles, having a particle size of 0.05 to 0.22 microns, are extravolts (EVs).

4. A pharmaceutical composition containing extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs), At least 1 × 10⁻¹⁶ per 1 mL １１ It contains particles, the particle size of which is 0.05 to 0.22 microns, and at least 90% of the particles are EV. A pharmaceutical composition wherein the human albumin concentration of the pharmaceutical composition is 10 to 20 g / L, the pharmaceutical composition contains human transferrin at a concentration of 60 mg / L to 600 mg / L, and at least 90% of the human albumin present in the pharmaceutical composition is associated with the EV.

5. The pharmaceutical composition according to claim 4, wherein the pharmaceutical composition comprises human transferrin and human albumin in a ratio of 2 to 60 mg of transferrin per 1 g of albumin.

6. The pharmaceutical composition according to claim 4 or 5, wherein the pharmaceutical composition is further formulated and / or processed.

7. A pharmaceutical composition according to any one of claims 4 to 6, for therapeutic or prophylactic use.

8. A pharmaceutical composition according to any one of claims 4 to 7, for use in the prevention or treatment of lung disease.

9. The pharmaceutical composition for use according to claim 8, wherein the lung disease is an inflammatory lung disease, a pulmonary vascular disease, or acute lung injury.

10. The pharmaceutical composition for use according to claim 9, wherein the inflammatory lung disease is pulmonary hypertension, asthma, bronchopulmonary dysplasia (BPD), allergy, pneumonia, or idiopathic pulmonary fibrosis.

11. The pharmaceutical composition for use according to claim 9, wherein the acute lung injury is associated with sepsis or acute respiratory distress syndrome (ARDS).

12. The pharmaceutical composition for use according to claim 9, wherein the inflammatory lung disease is associated with a viral infection.

13. A pharmaceutical composition for use in the treatment of COVID-19, according to any one of claims 6 to 12.

14. A pharmaceutical composition according to any one of claims 4 to 7, for use in the prevention or treatment of inflammatory bowel diseases such as Crohn's disease.

15. The pharmaceutical composition for use according to any one of claims 7 to 14, wherein the therapeutically effective amount of the pharmaceutical composition is administered to a patient, the patient may be an adult, an infant, or a neonatal.

16. The pharmaceutical composition is 10 for the patient or each dose. ９ EV / kg ~ 10 １２ A pharmaceutical composition for use according to any one of claims 7 to 15, administered in a dose of EV / kg.

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