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

Abstract

The present invention relates to a method of manufacturing a composition of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs), said method comprising the steps of: culturing and expanding MSCs in a serum-free and xeno-free medium comprising purified human serum albumin and human transferrin; collecting cell supernatant comprising EVs in said cell supernatant; filtering said cell supernatant to obtain EVs; and concentrating said EVs, preferably by ultrafiltration. In a second and further aspect, the invention relates to a composition comprising EVs and its clinical use.

Description

Technical Field

[0001] This invention relates to extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) and compositions containing said vesicles, as well as methods for obtaining the latter. Background Technology

[0002] EVs are lipid bilayer-separated particles naturally released by cells, but unlike cells, they are not replicable. EV diameters range from near the physically smallest possible monolayer liposome (approximately 20 to 30 nanometers) to over 10 micrometers, but the vast majority are smaller than 200 nm. They transport large quantities of proteins, nucleic acids, lipids, metabolites, and even organelles derived from parent cells. Most cells studied to date are believed to release EVs, including some bacteria, fungi, and plant cells surrounded by cell walls. Various EV subtypes have been proposed and defined differently based on size, biogenetic pathway, transporter, cellular origin, and function, leading to different historical nomenclature, including terms such as exosome, microvesicle, and extranuclear granule.

[0003] EVs can be used for therapeutic purposes, such as delivering nucleic acids or other transporters to diseased tissues and cells. Parallel to this growing interest are the formation of companies and funding programs focused on developing EVs as biomarkers or disease therapies, the establishment of the International Society for Extracellular Vesicles (ISEV), and the founding of the Journal of Extracellular Vesicles.

[0004] With the growing interest in using EVs for therapeutic purposes, the demand for clinical-grade EVs has also increased. For clinical application, EVs need to be manufactured in a reproducible and controlled manner. Good Manufacturing Practices (GMP) for manufacturing EV-containing compositions are crucial to ensure that each batch is identical and of consistent quality. GMP is particularly challenging when cell-derived therapies are involved.

[0005] Methods for producing exosomes derived from mesenchymal stromal cells (MSCs) are known, for example, Reka AgnesHaraszti et al., 2018. Furthermore, Diem Huong Huang et al., 2020, described MSC-derived exosomes, i.e., exosomes produced under specific conditions, in terms of their regenerative potential for skin tissue. Various culture media can be used for MSC production, such as the serum-free and heterologous-free media disclosed in Lucas G. Chase et al., 2012. Antoine Monsel et al., 2016, further described the use of MSC secretory proteomes and extracellular vesicles (EVs) in the treatment of lung diseases. The data presented regarding their use in humans are mostly speculative and highlight several problems with existing techniques, such as low reproducibility and low yield. Furthermore, this literature warns of potential problems with large-scale MSC culture for EV production using bioreactors. Finally, Youngja Park et al., 2011, mentioned the use of albumin in the treatment of lung diseases.

[0006] However, several challenges remain before EVs can be widely used clinically. Developing a platform for the reliable and reproducible production, storage, and handling of clinical-grade EVs remains a challenge. Before introducing MSC-derived EV therapies into clinical practice, MSC-derived EV drugs and subsequent clinical trials require addressing a range of scientific, regulatory, technical, and mechanistic issues.

[0007] Therefore, there is a need for a solution that allows for the stable, reproducible, low-cost, and large-scale production of clinical-grade MSC-derived EVs under GMP regulations. Furthermore, EV products with good stability and commercially attractive shelf lives are also required. Summary of the Invention

[0008] This invention provides a method for the stable and reproducible production of EVs from MSCs under GMP conditions conforming to the International Council for Harmonisation of Technical Requirements for Quality (ICH) 2020. The resulting EV composition is a clinical-grade cGMP product that is easy to use in clinical trials and patients and exhibits good stability.

[0009] Therefore, the present invention provides a manufacturing method and a composition described herein. Preferred embodiments are also described herein.

[0010] The product can have a variety of clinical applications and therapeutic effects. The uses of the product are described herein. Due to the long-term stability of the composition, this product can be used in a variety of clinical applications. Attached Figure Description

[0011] Figure 1An embodiment of an MSC amplification unit and an EV processing unit that can be used to produce the EVs of the present invention is shown.

[0012] definition

[0013] Unless otherwise defined, all terms (including technical and scientific terms) used in disclosing this invention have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance includes definitions of terms to better understand the teachings of this invention.

[0014] As used herein, the following terms have the following meanings: Unless the context clearly specifies otherwise, the terms “a,” “an,” and “the” used herein refer to the singular and plural pronouns, respectively. For example, “a compartment” means one or more compartments.

[0015] As used herein, “approximately” refers to a measured value such as a parameter, quantity, duration, etc., and is intended to include 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, such variations apply to the disclosed invention to date. However, it should be understood that the value referred to by the modifier “approximately” is itself specifically disclosed.

[0016] As used herein, “comprising” is synonymous with “including” or “containing” and is an open-ended term that specifies the presence of subsequent items such as components, and does not exclude or exclude the presence of additional, unlisted ingredients, features, elements, components, or steps known in the art or disclosed herein.

[0017] The range of values ​​listed by endpoints includes all numbers and fractions contained within that range, as well as the listed endpoints.

[0018] Unless otherwise defined, the expressions “% by weight,” “percentage by weight,” “%wt” or “wt%” used herein and throughout the specification mean the relative weight of the various components based on the total weight of the formulation.

[0019] While the terms “one or more” or “at least one”, such as one or more or at least one of a group of components, are self-explanatory, by further example, the term specifically refers to any one of the said components, or any two or more of the said components, such as ≥3, ≥4, ≥5, ≥6 or ≥7, or at most all of the said components.

[0020] Unless otherwise defined, all terms (including technical and scientific terms) used in disclosing this invention have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance includes definitions of terms to better understand the teachings of this invention. The terms or definitions provided herein are merely illustrative.

[0021] Throughout this specification, "one embodiment" means that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Therefore, the phrase "in one embodiment" throughout different places in this specification does not necessarily refer to the same embodiment, but may refer to the same embodiment. Furthermore, in one or more embodiments, particular features, structures, or characteristics may be combined with each other in any suitable manner that is obvious to those skilled in the art. Moreover, while some embodiments described herein include some features included in other embodiments but not others, combinations of features from different embodiments remain within the scope of the invention and form different embodiments, as understood by those skilled in the art. For example, any claimed embodiments may be used in any combination herein.

[0022] For the purposes of this invention, the term "extracellular vesicle" or "EV" should be understood as micron- or nanon-sized particles secreted in vivo and in vitro by different types of cells, including proteins bound to said EV. EVs contain proteins, growth factors, miRNAs, and other molecules encapsulated in lipid globules. EVs can be classified according to size and intracellular origin. Exosomes are a subtype of EV, typically smaller than 0.1 micrometers. Exosomes originate from multivesicular bodies, a type of secondary endosomal compartment that is secreted through fusion of the multivesicular body with the plasma membrane. Another subtype of EV is exfoliated vesicles (also known as microvesicles), a heterogeneous group of membrane vesicles released directly from the cell membrane by disrupting the cortical cytoskeleton, reaching up to 1 micrometer in size. All types of vesicles secreted by cells are generally referred to as EVs.

[0023] The term “bound to EV” in relation to a substance means that the substance a) is attached to or bound to the surface of the EV (by any means, such as covalent or non-covalent bonding), preferably in the form of non-covalent bonds; b) is attached to or bound to the surface of the EV; or c) is internalized into the EV.

[0024] The substance that binds to EV can be any type of substance, including, but not limited to, the following molecules: amino acids, proteins, peptides, nucleic acids such as DNA and RNA (e.g., non-coding RNA, miRNA, mRNA), sugars, carbohydrates, fats, vitamins, growth factors, pro-angiogenic 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.

[0025] The term "cell culture medium" or "culture medium" refers to an aqueous solution containing nutrients and other defined components that can be used for cell maintenance or growth.

[0026] The term "serum-free" cell culture medium refers to a cell culture medium that does not contain animal or human serum, plasma, or hemolymph. The serum-free medium may contain components processed from or derived from blood, serum, or plasma, such as albumin, transferrin, low-density lipoprotein, and hormones. It may also contain other biological components, preferably known and fully reproducible components and concentrations, but not serum, plasma, or hemolymph (e.g., growth factors, hormones, and carrier proteins).

[0027] The term "heterogeneous" cell culture medium should be understood as a cell culture medium that does not contain any components directly derived from non-human animals or recombinant components made from non-human animal DNA sequences.

[0028] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to the subject and does not eliminate the biological activity and properties of the administered composition. For example, pharmaceutically acceptable carriers can serve as stabilizers and / or excipients. Examples of carriers are, but are not limited to, propylene glycol, saline, emulsions, and mixtures of organic solvents with water.

[0029] The term "sufficient amount" refers to an amount sufficient to produce the desired measurable effect, such as an amount sufficient to alter protein expression properties.

[0030] The term "therapeutic effective dose" refers to the amount that can effectively improve the symptoms of a disease. A therapeutic effective dose can also be a "preventive effective dose," since prevention can be considered treatment.

[0031] The term "treatment" refers to therapeutic and preventative measures aimed at preventing or alleviating a target pathological condition or disease. Those who require treatment include those who already have the disease, those who are susceptible to the disease, and those who want to prevent the disease.

[0032] The term "composition" refers to a composition at any stage of the manufacturing process, including the final pharmaceutically acceptable product and any intermediates in the process.

[0033] The term "treatment" refers to reversing, preventing, improving, or inhibiting the progression of a disease, symptom, or condition, or one or more symptoms of a disease, symptom, or condition. As used herein, "treatment" can also refer to reducing the likelihood or incidence of a disease, symptom, or condition in a mammal compared to an untreated control group or to the same mammal before treatment. For example, as used herein, "treatment" can refer to preventing a disease, symptom, or condition and may include delaying or preventing the onset of a disease, symptom, or condition, or delaying or preventing the symptoms of a disease, symptom, or condition. As used herein, "treatment" can also refer to reducing the severity of a disease, symptom, or condition or alleviating the symptoms of such a disease, symptom, or condition before the onset of the disease, symptom, or condition. Such prevention or reduction of the severity of a disease, symptom, or condition before the onset of the disease involves administering the compositions of the present technology described herein to a subject who does not have a disease, symptom, or condition at the time of administration. As used herein, "treatment" can also refer to preventing the recurrence of a disease, symptom, or condition or one or more symptoms of such a disease, symptom, or condition. As used in this article, the terms “therapeutic,” “treatment,” and “in therapy” refer to the therapeutic acts defined above.

[0034] As used in this article, the term "fibrosis" refers to the formation of an excessive amount of fibrous connective tissue in an organ or tissue during a repair or reaction process.

[0035] For the purposes of this invention, the term "mesenchymal stromal cells" or "MSCs" should be understood as matrix adherent cells capable of differentiating into various cell types. MSCs are derived from bone marrow, umbilical cord cells, adipose tissue, amniotic fluid, mammary glands, and blood. Detailed Implementation

[0036] This invention relates to a method for producing EVs from MSCs, the composition obtained by said method, and its therapeutic uses.

[0037] In a first aspect, the present invention relates to a method for manufacturing a composition of EVs derived from MSC. More specifically, the method includes the following steps: -MSCs were cultured and expanded in a serum-free and heterologous medium containing purified human serum albumin and human transferrin.

[0038] - Collect cell supernatant containing EV; - Filter the cell supernatant to obtain EVs; and - Concentrate the EV, preferably by ultrafiltration.

[0039] MSCs are cultured and expanded in a container, preferably a bioreactor, more preferably a stirred tank bioreactor, for use in the production of EVs. For this purpose, MSCs can be grown and expanded in a serum-free and heterologous medium supplemented with human serum albumin and transferrin. By culturing and expanding the MSCs using a serum-free and heterologous growth medium, unwanted contaminants in the EVs can be avoided. Such contaminants can interfere with the further clinical use of the resulting EV product. Serum and platelet lysates typically contain albumin, and cell growth is often affected by the albumin level in the culture medium. However, as with any other component in serum and platelet lysates, albumin levels are variable. For obtaining clinical-grade EVs, the use of serum and / or platelet lysates should be avoided.

[0040] However, albumin is a key component of the final product because it contributes to the stability and functionality of the EV. In the context of this invention, "stability" of products such as EVs refers to the maintenance by which a particular formulation or product, under specific conditions (e.g., a container or closed system), remains within predetermined ranges of physical, chemical, microbiological, toxicological, and functional specifications or characteristics over a given period of time. Non-limiting examples of such parameters include particle number, particle size, and leakage and activity of internal components.

[0041] Because exogenous albumin contributes to the stability of EVs, it should be present in the culture medium. In one embodiment, the culture medium therefore contains human albumin, which is recombinant or purified albumin, such as albumin purified from human plasma. In a further embodiment, the concentration of the albumin present in the culture medium is from 1 g / L to 5 g / L. The latter concentration has proven particularly useful for obtaining a final product with excellent stability.

[0042] The cell growth medium further comprises transferrin, preferably recombinant or purified transferrin, such as transferrin purified from plasma. Transferrin constitutes a broad microheterogeneous group of single-chain glycoprotein isotypes with a molecular weight of approximately 78 kDa to 80 kDa. Transferrin is a suitable physiological pathway for providing iron to cultured cells because it promotes the storage and transport of extracellular iron. Transferrin has been reported to aid in the cellular uptake of EVs in vivo, for example, see WO2013084001. Furthermore, similar to albumin, transferrin helps stabilize the final EV product. In one embodiment, the transferrin is present in the growth medium at a concentration between 50 mg / L and 100 mg / L, preferably between 55 mg / L and 100 mg / L, preferably between 50 mg / L and 70 mg / L, and most preferably between 55 mg / L and 70 mg / L. The latter concentration is considered optimal for the quality of the EV product.

[0043] This invention enables both albumin and transferrin to be present in the final product, wherein most of the albumin and transferrin will bind to the EV.

[0044] In a further embodiment, the culture medium is a basic mixture of inorganic metal salts, nutrients such as amino acids and vitamins, with a pH in the range of 7.0 to 7.4, for example, as known from the composition of Dulbecco's Modified Eagle Medium (DMEM) or DMEM / F-12. The medium may also contain glutamine and glutamate, or precursors thereof such as L-alanyl-L-glutamine, glucose at a level not exceeding 4500 mg / L, and one or more fatty acids.

[0045] In a further embodiment, the composition comprises human growth factors, such as, but not limited to, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), human platelet-derived growth factor (PDGF, such as 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 found that this growth factor synergistically with other growth factors to promote MSC growth and proliferation. In one embodiment, the culture medium further comprises PDGF and TGF-β1.

[0046] PDGF is a regulator of cell growth and division, binding to platelet-derived growth factor receptor (PDGFR). In chemical terms, PDGF is a dimeric glycoprotein composed of two A (-AA) chains, two B (-BB) chains, or two (-AB) chains. It has been shown that PDGF-AB can bind to the α-receptor and β-receptor subunits of PDGF, thereby forming PDGF α-β receptor dimers and PDGF α-α receptor dimers. In the context of this disclosure, PDGF includes PDGF-BB, PDGF-AB, and PDGF-AA.

[0047] Transforming growth factor-β1 (TGF-β1) is a pleiotropic cytokine that stimulates the proliferation of mesenchymal stem cells (MSCs), and also affects the immunomodulatory properties of MSCs and modifies their secretory proteome, giving them pro-inflammatory or anti-inflammatory properties.

[0048] In one embodiment, the PDGF is PDGF-BB. In one embodiment, the PDGF-BB level is between about 1 ng / ml and 150 ng / ml. In another embodiment, the PDGF-BB level is between about 7.5 ng / ml and 120 ng / ml. In another embodiment, the PDGF-BB level is between about 15 ng / ml and 60 ng / ml. In another embodiment, the PDGF-BB level is at least about 10 ng / ml. In another embodiment, the PDGF-BB level is at least about 15 ng / ml. In another embodiment, the PDGF-BB level is at least about 20 ng / ml. In another embodiment, the PDGF-BB level is at least about 21 ng / ml. In another embodiment, the PDGF-BB level is at least about 22 ng / ml. In another embodiment, the PDGF-BB level is at least about 23 ng / ml. In another embodiment, the PDGF-BB level is at least about 24 ng / ml. In another embodiment, the PDGF-BB level is at least about 25 ng / ml. In another embodiment, the PDGF-BB level is at least about 26 ng / ml. In another embodiment, the PDGF-BB level is at least about 27 ng / ml. In another embodiment, the PDGF-BB level is at least about 28 ng / ml. In another embodiment, the PDGF-BB level is at least about 29 ng / ml. In another embodiment, the PDGF-BB level is at least about 30 ng / ml. In another embodiment, the PDGF-BB level is at least about 31 ng / ml. In another embodiment, the PDGF-BB level is at least about 32 ng / ml. In another embodiment, the PDGF-BB level is at least about 33 ng / ml. In another embodiment, the PDGF-BB level is at least about 34 ng / ml. In another embodiment, the PDGF-BB level is at least about 35 ng / ml. In another embodiment, the PDGF-BB level is at least about 36 ng / ml. In another embodiment, the PDGF-BB level is at least about 37 ng / ml. In another embodiment, the PDGF-BB level is at least about 38 ng / ml. In another embodiment, the PDGF-BB level is at least approximately 39 ng / ml. In yet another embodiment, the PDGF-BB level is at least approximately 40 ng / ml.

[0049] In another embodiment, the PDGF is PDGF-AB. In one embodiment, the PDGF-AB level is between about 1 ng / ml and 150 ng / ml. In another embodiment, the PDGF-AB level is between about 7.5 ng / ml and 120 ng / ml. In another embodiment, the PDGF-AB level is between about 15 ng / ml and 60 ng / ml. In another embodiment, the PDGF-AB level is at least about 10 ng / ml. In another embodiment, the PDGF-AB level is at least about 15 ng / ml. In another embodiment, the PDGF-AB level is at least about 20 ng / ml. In another embodiment, the PDGF-AB level is at least about 21 ng / ml. In another embodiment, the PDGF-AB level is at least about 22 ng / ml. In another embodiment, the PDGF-AB level is at least about 23 ng / ml. In another embodiment, the PDGF-AB level is at least about 24 ng / ml. In another embodiment, the PDGF-AB level is at least about 25 ng / ml. In another embodiment, the PDGF-AB level is at least about 26 ng / ml. In another embodiment, the PDGF-AB level is at least about 27 ng / ml. In another embodiment, the PDGF-AB level is at least about 28 ng / ml. In another embodiment, the PDGF-AB level is at least about 29 ng / ml. In another embodiment, the PDGF-AB level is at least about 30 ng / ml. In another embodiment, the PDGF-AB level is at least about 31 ng / ml. In another embodiment, the PDGF-AB level is at least about 32 ng / ml. In another embodiment, the PDGF-AB level is at least about 33 ng / ml. In another embodiment, the PDGF-AB level is at least about 34 ng / ml. In another embodiment, the PDGF-AB level is at least about 35 ng / ml. In another embodiment, the PDGF-AB level is at least about 36 ng / ml. In another embodiment, the PDGF-AB level is at least about 37 ng / ml. In another embodiment, the PDGF-AB level is at least about 38 ng / ml. In another embodiment, the PDGF-AB level is at least approximately 39 ng / ml. In yet another embodiment, the PDGF-AB level is at least approximately 40 ng / ml.

[0050] In another embodiment, the PDGF is PDGF-AA. In one embodiment, the PDGF-AA level is between about 1 ng / ml and 150 ng / ml. In another embodiment, the PDGF-AA level is between about 7.5 ng / ml and 120 ng / ml. In another embodiment, the PDGF-AA level is between about 15 ng / ml and 60 ng / ml. In another embodiment, the PDGF-AA level is at least about 10 ng / ml. In another embodiment, the PDGF-AA level is at least about 15 ng / ml. In another embodiment, the PDGF-AA level is at least about 20 ng / ml. In another embodiment, the PDGF-AA level is at least about 21 ng / ml. In another embodiment, the PDGF-AA level is at least about 22 ng / ml. In another embodiment, the PDGF-AA level is at least about 23 ng / ml. In another embodiment, the PDGF-AA level is at least about 24 ng / ml. In another embodiment, the PDGF-AA level is at least about 25 ng / ml. In another embodiment, the PDGF-AA level is at least about 26 ng / ml. In another embodiment, the PDGF-AA level is at least about 27 ng / ml. In another embodiment, the PDGF-AA level is at least about 28 ng / ml. In another embodiment, the PDGF-AA level is at least about 29 ng / ml. In another embodiment, the PDGF-AA level is at least about 30 ng / ml. In another embodiment, the PDGF-AA level is at least about 31 ng / ml. In another embodiment, the PDGF-AA level is at least about 32 ng / ml. In another embodiment, the PDGF-AA level is at least about 33 ng / ml. In another embodiment, the PDGF-AA level is at least about 34 ng / ml. In another embodiment, the PDGF-AA level is at least about 35 ng / ml. In another embodiment, the PDGF-AA level is at least about 36 ng / ml. In another embodiment, the PDGF-AA level is at least about 37 ng / ml. In another embodiment, the PDGF-AA level is at least about 38 ng / ml. In another embodiment, the PDGF-AA level is at least approximately 39 ng / ml. In yet another embodiment, the PDGF-AA level is at least approximately 40 ng / ml.

[0051] Heterogeneous and serum-free culture media can be prepared by combining individual components or can be selected from (arbitrarily adapted) commercial products based on the guidance herein. For example, commercially available serum-free and heterogeneous culture media can be used, optionally after modification according to the optional features and / or preferences disclosed herein.

[0052] In one embodiment, the culture medium may be equipped with a buffering system. pH adjustment is crucial for optimal culture conditions and is typically achieved using one of two buffering systems (natural or chemical).

[0053] In a natural buffer system, gaseous CO2 reacts with CO3 in the culture medium. -- / HCO3 - The content of [unspecified components] needs to be kept balanced. Culture media with natural buffering systems require a 5-10% CO2 atmosphere, typically maintained using a CO2 incubator. Natural buffering systems are inexpensive and non-toxic. Chemical buffers using zwitterionic HEPES exhibit excellent buffering capacity in the pH range of 7.2 to 7.4 and do not require a controlled gas environment. HEPES are relatively expensive and toxic to certain cell types at higher concentrations. HEPES has been shown to significantly enhance the sensitivity of culture media to phototoxic effects caused by exposure to fluorescent light.

[0054] In one embodiment, the culture medium may include phenol red as a pH indicator, which can continuously monitor the pH. During cell growth, the culture medium changes color due to pH variations caused by metabolites released by the cells. At low pH values, phenol red turns the culture medium yellow, while at higher pH values, it turns the culture medium purple. At pH 7.4, the culture medium is bright red, which is the optimal pH for cell culture.

[0055] In another embodiment, the culture medium may contain inorganic salts. These inorganic salts help maintain osmotic pressure balance and aid in regulating membrane potential by providing sodium, potassium, and calcium ions.

[0056] The culture medium may contain amino acids, preferably essential amino acids. L-Glutamine is an essential amino acid and is particularly important. L-Glutamine provides nitrogen to NAD, NADPH, and nucleotides and serves as a secondary energy source for metabolism. L-Glutamine is an unstable amino acid that can transform into forms that cells cannot use over time, and therefore should be added to the culture medium just before use. Caution should be exercised when adding L-Glutamine above the level required by the original culture medium formulation, as degradation of L-Glutamine can lead to ammonia accumulation, which can have detrimental effects on some cell lines. L-Glutamine concentrations in mammalian cell cultures can range from 0.68 mM in Medium 199 to 4 mM in Dulbecco's modified Eagle medium. Invertebrate cell cultures can contain up to 12.3 mM of L-Glutamine. Supplements such as glutamax are more stable and can replace glutamine for long-term cultures without changing or replenishing the medium, providing a more stable glutamine concentration during this period.

[0057] Non-essential amino acids can also be added to the culture medium to replace those amino acids that are depleted during growth. Supplementing the culture medium with non-essential amino acids can stimulate cell growth and prolong cell viability.

[0058] Carbohydrates in the form of sugars are the primary source of energy. Most culture media contain glucose and galactose; however, some media contain maltose and fructose.

[0059] The culture medium may further contain fatty acids, lipids, vitamins, and trace elements.

[0060] Trace elements are often supplemented into serum-free culture media to replace those that are normally present in serum. Trace elements such as copper, zinc, selenium, and tricarboxylic acid intermediates are trace chemical elements required for normal cell growth.

[0061] In one embodiment, antibiotics can be used to control the growth of bacterial and / or fungal contaminants, such as a mixture of penicillin and streptomycin, and / or other compounds, such as, but not limited to, amphotericin B, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, levofloxacin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, penicillin, polymyxin, puromycin, rifampin, spectinomycin, streptomycin, tetracycline, tylosin, and giomycin.

[0062] In one embodiment, the MSCs can be grown and expanded on microcarriers or microbeads present in the bioreactor. Such microcarriers or microbeads are known in the art and are commercially available. In one embodiment, the microcarriers or microbeads 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 microbeads are negatively charged.

[0063] Once the MSCs reach the desired concentration and / or confluence, the cell supernatant containing the EVs is collected for further processing. In the context of this invention, the cell supernatant is a cell culture medium in which MSCs can be grown and expanded. In one embodiment, when the MSCs reach at least 40 x 10⁻⁶ ppm... 6 The supernatant was collected at the lowest possible cell / L concentration. Cell concentration and viability can be determined by cell counting, for example, using a hematology analyzer such as a Bürker counting chamber and trypan blue staining. Under the culture conditions described above, the MSCs should produce at least 0.25 x 10⁻⁶ cells per ml of culture medium during a 18-24 hour culture period. 9 EVs.

[0064] The term "particle" as used above can refer to any particle with a particle size preferably between 0.05 micrometers and 0.22 micrometers, wherein the particle is an EV. Other examples of particles can be protein or peptide aggregates. The particles are derived from MSCs or cell culture media used for culturing and expanding MSCs. Therefore, the particles can be any particles commonly present in the cell culture media or MSCs, or any particles that are a portion of the cell culture media or MSCs.

[0065] Preferably, when using the method described herein, at least 90% of the particles with a diameter between 0.05 micrometers and 0.22 micrometers are EVs.

[0066] In subsequent steps, the cell supernatant is filtered to remove contaminants present in the cell culture medium. This contributes to the purity and stability of the final product. In a preferred embodiment, the filtration includes at least two filtration steps. In one embodiment, at least one of the filtration steps is dead-end filtration. In a further embodiment, both filtration steps are performed by dead-end filtration. Preferably, at least one filtration step serves as product sterilization to comply with GMP specifications as defined above. In some cases, continuous dead-end filtration has been found necessary for adequate removal of impurities from the supernatant. In some cases, it has been found that single filtration often results in clogging of the filter used and reduces the purity and quantity of the final product.

[0067] In the first filtration step, the cell supernatant is filtered by dead-end filtration. In a further preferred embodiment, the filtration is performed by placing the supernatant on a filter with a sieve size of 1 to 5 micrometers, more preferably a filter with a sieve size of 1 to 3 micrometers. In one embodiment, the filtration is performed by dead-end filtration, preferably in a closed system having a peristaltic pump that provides a constant flow rate through the filter, preferably 100 ml / min.

[0068] In one embodiment, the filtrate from the first filtration is passed through a second filter, the pore size of which is smaller than that of the filter used in the first filtration step. Preferably, this second filtration step is a sterilization step to ensure the final product complies with the GMP specifications defined above. In a more preferred embodiment, the pore size of the filter is less than 1 micrometer, more preferably between 0.05 micrometers and 1 micrometer, even more preferably between 0.1 micrometers and 0.5 micrometers, and most preferably between 0.1 micrometers and 0.22 micrometers. In one embodiment, the second filtration step is performed by dead-end filtration, preferably in a closed system interconnected with the first filtration step, with a constant flow rate through the filter provided by a peristaltic pump, preferably 100 ml / min.

[0069] The filtrate from the filtration step will contain the EV of the present invention. In the final step, the EV will be washed and concentrated. Washing and concentration can be performed using conventional methods in the art, such as membrane filtration, microfiltration, or ultrafiltration. Concentration is the process of removing liquid from a solution while retaining solute molecules.

[0070] Membrane filtration is a commonly used separation technique in life science laboratories. Based on membrane porosity, membrane filtration can be classified as microfiltration or ultrafiltration. Microfiltration membranes typically have pore sizes between 0.1 μm and 10 μm and are commonly used for clarification, sterilization, and removal of particulates, or for cell collection. Ultrafiltration membranes have much smaller pore sizes, ranging from 0.001 μm to 0.1 μm, and are used to concentrate and desalinate dissolved molecules (proteins, peptides, nucleic acids, carbohydrates, and other biomolecules), exchange buffers, and crude fractions. Ultrafiltration membranes are typically classified according to their molecular weight cutoff (MWCO), rather than their pore size. There are two main types of membrane filtration, which can be done using microfiltration or ultrafiltration membranes: 1) Direct-flow filtration (DFF), also known as "dead-end" filtration, applies the feed flow perpendicular to the membrane surface and attempts to allow 100% of the fluid to pass through the membrane, and 2) Tangential-flow filtration (TFF), also known as cross-flow filtration, in which the feed flow passes parallel to the membrane surface, with a portion passing through the membrane (permeate) and the remainder (residue) being recycled back into the feed container.

[0071] Preferably, the washing and concentration are performed using cross-flow filtration (TFF). TFF, or cross-flow filtration, is a method in which the feed stream flows parallel to the membrane surface. Applied pressure forces a portion of the feed stream through the membrane (filtrate or permeate), while the remainder (permeate) is recycled back into the feed container.

[0072] In one embodiment, the filtrate from one or more dead-end filtration steps is used in a TFF concentration step. In a further preferred embodiment, the TFF has a molecular weight cutoff of 100 kDa and removes most (but not all) of particles and components with molecular weights below 100 kDa into the TFF permeate. Therefore, the final composition in the permeate removes free (i.e., non-EV-bound) components, elements, or substances with molecular weights below 100 kDa. These components, elements, or substances can be any particles, such as proteins or peptides typically present in or part of the culture medium. The permeate is recirculated in the TFF apparatus until the desired permeate concentration is achieved. During recirculation, the permeate can be washed with a washing medium or washing buffer, preferably a salt buffer, to remove unwanted components. The concentrated residue can then be collected in a collection container, such as, but not limited to, a collection bag or cryovial, and stored at below 10°C, preferably at 4°C, or frozen at -20°C to -196°C, preferably at -40°C to -196°C, more preferably at -80°C to -196°C.

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

[0074] In one embodiment, MSCs are freshly isolated from one of the aforementioned tissues and further expanded 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 this invention. Briefly, tissues such as umbilical cords can be enzymatically digested, for example by enzymes such as collagenase and / or trypsin, washed, and centrifuged. The resulting precipitate is then inoculated into a suitable cell culture medium in a culture flask and incubated 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 can enhance attachment and cell growth. In another embodiment, the culture flask may contain microcarriers or microbeads, for example 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 percentage of confluence, preferably more than 80%, after which the cells are collected.

[0075] During passage, the cultured cells detach from and dissociate from the culture medium and from each other. Cell detachment and dissociation can be performed in a manner commonly known in the art, such as by enzymatic treatment with proteases (e.g., trypsin, collagenase, such as type I, II, III or IV, dispersin, streptomycin, 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 of these treatments.

[0076] A suitable cell detachment and dispersion method should ensure the desired degree of cell detachment and dispersion while retaining the majority of viable cells in the culture medium. Preferably, detachment and dissociation of the cultured cells will yield a substantial proportion of cells as individual viable cells (e.g., at least 50%, 70%, 80%, or 90% or more). Residual cells may be present in cell clusters, each containing a relatively small number of cells (e.g., between 1 and 100 cells on average).

[0077] Next, the detached and dissociated cells (usually as a cell suspension in isotonic buffer or culture medium) can be replated onto a substrate (to which the cells can adhere) and then cultured in the aforementioned culture medium to allow for further proliferation. These cells can then be cultured in groups of 10 to 10... 5 cells / cm 2The cells are cultured by re-laying at a density between approximately 1 / 16 and 1 / 2, preferably between approximately 1 / 8 and 1 / 2, and more preferably between approximately 1 / 4 and 1 / 2. The division ratio represents the fraction of passaged cells seeded into an empty (usually new) culture vessel with the same surface area as the vessel from which the passaged cells were obtained. The culture vessel, and the surface allowing cell adhesion to the culture vessel, and the type of cell culture medium, may be the same as or different from those initially used and described above.

[0078] In a preferred step, the cells are then seeded and expanded in a larger container. Once the desired cell count is reached, the cells can be directly transferred to a bioreactor for further expansion and EV harvesting, or a suitable number of cells can be cryopreserved for later use. Cryopreservation of the MSCs is typically performed in the presence of one or more cryoprotectants. These agents prevent cell damage during freeze-thaw cycles and are generally known to those skilled in the art. Examples of cryoprotectants include, for instance, DMSO mixed with cell culture medium. The cells can then be cryopreserved in suitable containers or cryopreservation bags.

[0079] In a further step, the MSCs (which may be freshly isolated or cryopreserved) can be further amplified in a bioreactor, preferably a closed stirred tank bioreactor. The cells can be amplified in the bioreactor under the suitable amplification conditions described above (serum-free and heterologous culture medium-free). In one embodiment, the container or cryopreservation bag as described above is designed to be aseptically connected to the bioreactor, thus eliminating the need for a (laminar flow) laminar flow hood. This eliminates the need for a (highly sterile) cleanroom during the EV production process.

[0080] The above-described method can be performed in a purification system for EVs, comprising an MSC cell expansion unit and a processing unit for EVs. The two units, along with their respective compartments and / or components, are fluidly connected to each other, allowing cell culture medium and materials to flow from one unit to the other. The fluid connection will be achieved using conventional methods in the art, such as tubes, pipes, valves, and pumps. In one embodiment, the MSC cell expansion unit will include a bioreactor (preferably a stirred bioreactor) and a cooling chamber. The cooling chamber will be equipped with means for cooling the ambient temperature and materials within the cooling chamber to below 10 degrees Celsius (e.g., 4 degrees Celsius). In a preferred embodiment, the cooling chamber is a refrigerator. The cooling chamber may be equipped with tanks and containers for storing fresh cell culture medium to be transferred to the bioreactor and cell supernatant from the bioreactor, respectively. For this purpose, the outlet of the cell culture medium container or tank will be fluidly connected to the inlet of the bioreactor. A pump, preferably a peristaltic pump, will be present to ensure proper media flow. The outlet of the bioreactor will be fluidly connected to the inlet of the cell supernatant container in the cooling chamber. Similarly, a (peristaltic) pump may be present to ensure product flow. With the current setup, the supernatant from the bioreactor can flow to a cell supernatant collection container or tank, while fresh culture medium can be supplied to the bioreactor. The bioreactor is preferably a stirred bioreactor. Preferably, the fresh culture medium is at least at room temperature or more preferably preheated by any heating device known in the art, such as an incubator, preferably preheated to about 37°C. Stirring can be achieved by conventional methods in the art, such as by a magnetic stirrer or a stirrer installed in the bioreactor. The bioreactor can be supplied with CO2 or can be located in a CO2 incubator. In the latter embodiment, the bioreactor will be equipped with a device that allows gas exchange. Sensors can be present in the cell expansion unit to monitor various processes. In one embodiment, sensors can be present to measure the temperature, humidity, and CO2 levels in the bioreactor and / or CO2 incubator. Sensors can also be present to monitor the conditions of the cooling chamber, such as its temperature.

[0081] Once the required number of MSCs are reached, a cell supernatant containing EVs is obtained from the bioreactor and stored at a temperature below 10°C, for example, 4°C. Once an appropriate volume of cell supernatant is obtained, it is transferred to the EV processing unit. In one embodiment, this appropriate volume is transferred to the EV processing unit in batches. In an alternative embodiment, the volume is transferred continuously to the EV processing unit. The EV processing unit is preferably equipped with one or more filtration devices, membrane filtration devices such as TFF concentrators, and storage compartments for the final product (in the order described herein). These devices and compartments are fluidly connected, and liquid can flow from one device to another, preferably in a closed-loop system. In a further preferred embodiment, the EV processing unit includes a first filtration device with a sieve pore size between 1 micrometer and 5 micrometers, more preferably between 1 micrometer and 3 micrometers. The filtrate from the first filtration is then transferred to a second filtration device with a sieve pore size smaller than that used in the first filtration step. In a preferred embodiment, the sieve pore size of the filter device is less than 1 micrometer, more preferably between 0.05 micrometers and 1 micrometer, even more preferably between 0.1 micrometers and 0.5 micrometers, and most preferably between 0.1 micrometers and 0.22 micrometers. In one embodiment, the filter device allows dead-end filtration, preferably carried out in a closed system interconnected with the first filtration step, wherein a peristaltic pump provides a constant flow rate through the filter, preferably 100 ml / min.

[0082] After filtration, the filtrate is transferred to a TFF (Thin Film Filter) device. For this purpose, a final filtration device is refluidically connected to the TFF. These devices are typically classified according to their molecular weight cutoff (MWCO). The molecular weight cutoff (MWCO), or nominal molecular weight cutoff (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 molecular weight cutoff of 100 kDa. The TFF will be used to wash, concentrate, and further purify the sample. From the TFF, the concentrated and purified sample will be transferred to a filling and final unit to obtain the final product. In one embodiment, the final product will be collected in bags that can be connected to a filling device for aliquoting the sample into final containers. Alternatively, it can be manually aliquoted into the final containers.

[0083] For example, when using the production method described above, the minimum concentration from the bioreactor is at least 40 x 10⁻⁶. 6 From 100-fold concentrated supernatant of MSCs per liter, at least 1 x 10⁻⁶ cells / liter can be obtained per milliliter of the final product composition. 11 Individual particles. The confluence of MSCs is up to 80%. Preferably, approximately 8 x 10⁸ particles are inoculated per 0.5 L bioreactor. 6MSCs were cultured and fully expanded. Subsequently, the MSCs began releasing EVs from the supernatant, which were collected every 24 hours, for example, in culture bags. In this example, the contents of four culture bags were combined, filtered (using a 0.22-micron sieve size in this example), and concentrated 100x to obtain at least 1 x 10⁻⁶ saturates per milliliter. 11 Each particle.

[0084] The “particle” used in the above examples can be any particle with a diameter between 0.05 micrometers and 0.22 micrometers, wherein the particle is an EV. Other examples of particles can be protein or peptide aggregates. The particles are derived from MSCs or cell culture media used for culturing and expanding MSCs. Therefore, the particles can be any particles normally present in the cell culture media or MSCs, or any particles that are a portion of the cell culture media or MSCs.

[0085] Preferably, when using the method described herein, at least 90% of the particles with a diameter between 0.05 micrometers and 0.22 micrometers are EVs.

[0086] In another aspect, the present invention also relates to a composition comprising EVs derived from MSCs. In a preferred embodiment, the composition has a size of less than 1 µm and a human albumin concentration between 10 g / L and 30 g / L.

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

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

[0089] Optical techniques are typically used to determine the size and number of EVs. In one embodiment of the invention, the particle size of the EVs is measured using a nanoparticle tracking analyzer (NTA), a preferred method for quantifying and determining the size of nanoparticles suspended in a liquid buffer. In another, further preferred embodiment, the particle size is measured using a tunable resistive pulse sensor (TRPS), which is used as a reference for the NTA. In yet another, or further preferred embodiment, the particle size is measured using a high-resolution flow cytometer.

[0090] The composition of the present invention obtained by the above method will contain human albumin at a concentration between 10 g / L and 30 g / L, more preferably between 10 g / L and 20 g / L, even more preferably between 15 g / L and 20 g / L, and most preferably between 12 g / L and 16 g / L. The albumin concentration can be measured by colorimetry or by ELISA with an anti-albumin antibody. In one embodiment, the colorimetric method is the bromocresol green assay.

[0091] The albumin is derived from the cell culture medium of MSCs. While albumin could theoretically be considered a contaminant in the production process since it is not produced by MSCs, it has been surprisingly found that it is actually necessary to ensure the stability and functionality of the final product, thereby ensuring the stability and functionality of the compositions of this invention. The required concentration of albumin acts to some extent as a drug stabilizer and activity enhancer. Forced removal of albumin has been found to reduce product activity (Hyungtaek Jeon et al., 2020).

[0092] Preferably, the albumin is a clinical-grade product so that it can be used in animals and / or humans.

[0093] In one embodiment, at least 90% of the albumin present in the composition binds to the EV in the composition.

[0094] Human albumin has a molecular weight of approximately 66 kDa. Therefore, albumin that is not bound to EVs will be removed by TFF treatment, leaving albumin in the composition that is bound to 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 albumin in the composition is bound to EVs.

[0095] In one embodiment, the composition further comprises transferrin. The transferrin level in the composition is preferably between 25 mg / L and 95 mg / L, more preferably between 55 mg / L and 75 mg / L. In one embodiment, the transferrin level in the composition is preferably between 60 mg / L and 600 mg / L, more preferably between 100 mg / L and 500 mg / L, and more preferably between 150 mg / L and 450 mg / L. Transferrin is described to play a role in particle transport in cell membranes, and is said to play a role in the stability of EVs and the uptake of EVs by cells in vivo. Therefore, the presence of transferrin is known to have a positive impact on the further use of the EV composition.

[0096] In one embodiment, at least 90% of the transferrin present in the composition binds to the EV in the composition.

[0097] Human transferrin has a molecular weight of approximately 80 kDa. Therefore, TFF treatment removes a portion of the transferrin that does not bind to the EV, leaving the remaining transferrin in the composition bound to the 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 transferrin in the composition binds to the EV.

[0098] The concentrations of albumin, transferrin, and other proteins can be measured using methods conventional in the art, such as ELISA.

[0099] In one embodiment, the composition comprises less than 5% of a free component with a molecular weight of less than 100 kDa.

[0100] The term "free component" refers to an ingredient or component in the final product that is not bound to the EV and is therefore free in the final product.

[0101] By ensuring that the composition contains less than 5% of free components with a molecular weight below 100 kDa (e.g., obtainable by filtration through a TFF filter with a molecular weight cutoff of 100 kDa), it is ensured that the composition is adequately identified and complies with GMP. Most components of MSC cell growth media have a molecular weight below 100 kDa. When such small components remain in the final product, they can be considered contaminants and are therefore preferably kept to a minimum. In a further preferred embodiment, the composition contains less than 4%, less than 3%, less than 2%, and less than 1% of free components.

[0102] In a further embodiment, at least 60% of the EV will contain annexin V.

[0103] Annexins are calcium-dependent phospholipid-binding proteins that enhance the anti-inflammatory properties of EVs upon binding. Some annexins, such as annexin V, are associated with pro-inflammatory activity. Studies have shown that the binding of annexin V to EVs increases the anti-inflammatory activity of EVs. Annexin V has a molecular weight of approximately 37 kDa.

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

[0105] In one embodiment of the invention, the composition may further comprise one or more second therapeutic agents. As used herein, a therapeutic agent refers to any agent that can be used to prevent, treat, and / or control the disease discussed herein. Such agents may be intravesical (i.e., contained within the EV) or bound to the EV. Suitable therapeutic agents are known to those skilled in the art and may include non-coding RNA, miRNA, mRNA, growth factors, pro-angiogenic molecules, cardioprotective enzymes, antibodies, anti-inflammatory molecules, anti-fibrotic molecules, antioxidant molecules, pro-neurogenic molecules, antiviral molecules, etc. In some embodiments, isolated EVs are used together with a 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 to treat or control certain lung diseases, including but not limited to pulmonary hypertension, include oxygen, anticoagulants such as warfarin (Coumadin); diuretics such as furosemide (Lasix®) or spironolactone (Aldactone®); calcium channel blockers; potassium, such as K-dur®; cardiotonics such as digoxin; vasodilators such as nifedipine (Procardia®) or diltiazem (Cardizem®); endothelin receptor antagonists such as bosentan (Tracleer®) and letairis®; prostacyclin analogs such as eprostadil (Flolan®), treprostol sodium (Remodulin®, Tyvaso®), and iloprost (Ventavis®); and PDE-5 inhibitors such as sildenafil (Revatio®) and tadalafil (Adcirca®).

[0107] Preferably, the composition is formulated as a liquid. It can be stored via vials, IV bags, ampoules, cartridges, inhalers such as liquid inhalers, nebulizers, or powder inhalers, and pre-filled syringes. In addition to being the active ingredient or EV of the pharmaceutical product, the liquid formulation may also contain a variety of compounds to ensure the stability of the active pharmaceutical ingredient during subsequent storage. These include solubilizers, stabilizers, buffers, tension modifiers, fillers, viscosity enhancers / viscosity reducers, 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. Prior to administration, the lyophilized composition is reconstituted into a liquid. This can be done by combining, mixing, and then injecting a liquid diluent with the lyophilized powder. Reconstitution typically requires a reconstitution and delivery system to ensure the drug is properly mixed and administered.

[0109] In a preferred embodiment, the composition is aqueous. In one embodiment of the invention, the EV can be formulated into a composition further comprising a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier or diluent is selected to maintain the activity and properties of the EV of the invention. A pharmaceutically acceptable carrier is a pharmaceutically acceptable material, composition, or carrier involved in the delivery or transport of a preventive or therapeutic active agent, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable," meaning it is compatible with other components of the formulation and does not cause harm to the subject. Some examples of materials that can be used as pharmaceutically acceptable carriers include sugars, such as lactose, glucose, and sucrose; glycols, such as propylene glycol; polyols, such as glycerol, 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; ethanol; phosphate buffer; and other non-toxic, compatible substances used in pharmaceutical formulations.

[0110] The composition can be stored at 4°C, more preferably by cryopreservation, wherein the composition is frozen between -20°C and -196°C, more preferably between -40°C and -196°C, and even more preferably between -80°C and -196°C. The freezing procedure is preferably a rapid freezing procedure or a vitrification procedure to ensure that the product retains its activity during freezing and after thawing. Another freezing procedure can be controlled-rate freezing, preferably compensating for the exothermic reaction at the crystallization point to obtain better product stability. For the latter procedure, a controlled-rate freezer can be used.

[0111] In one embodiment, the composition is suitable for administration by injection, intravenous administration, inhalation, endotracheal infusion, systemic infusion, or intranasal infusion. The composition may also be formulated for external use, alone or in combination with hydrogels, polymers, or polymer medical devices to slowly release EVs.

[0112] In some embodiments, the composition is suitable for intravenous administration. In some embodiments, the composition is suitable for administration to the lungs or trachea of ​​a subject. In some embodiments, the composition is formulated for administration by inhalation. In some embodiments, the composition is formulated for aerosol administration. In some embodiments, the separated EV is administered using a nebulizer. In some embodiments, the separated EV is administered using an endotracheal cannula.

[0113] In some embodiments, the isolated EV is applied or formulated together with a surfactant, preferably a pulmonary surfactant. The surfactant is preferably selected in a manner that does not affect the stability of the composition. In some embodiments, the pulmonary surfactant is an isolated, naturally occurring surfactant. In some embodiments, the pulmonary surfactant is derived from bovine or porcine lungs. In some embodiments, the pulmonary surfactant is a synthetic surfactant. Pulmonary surfactants are lipoprotein mixtures used to maintain the patency of the lung airways (e.g., by preventing alveolar walls from sticking together). Pulmonary surfactants may consist of: phospholipids, such as dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine (PC), phosphatidylglycerol (PG); cholesterol; and proteins, such as SP-A, B, C, and D. Pulmonary surfactants may be derived from naturally occurring sources, such as bovine or porcine lung tissue. Examples include Alveofact™ (from bovine lung lavage fluid), Curosurf™ (from chopped porcine lung), Infasurf™ (from calf lung lavage fluid), and Survanta™ (from chopped bovine lung containing other components including DPPC, palmitic acid, and tripalmitoylglycerol). Lung surfactants can also be synthetic. Examples include Exosurf™ (composed of DPPC with cetyl alcohol and tyloxapol), Pumactant™ or Artificial Lung Expansion Compound (ALEC) (composed of DPPC and PG), KL-4 (composed of DPPC, palmitoyl oleoyl phosphatidylglycerol, palmitic acid, and a synthetic peptide mimicking SP-B), and Venticute™ (composed of DPPC, PG, palmitic acid, and recombinant SP-C). Lung surfactants are available from commercial suppliers.

[0114] The present invention also includes packaged and labeled pharmaceutical products. The product or kit comprises a suitable unit dosage form in a suitable vessel or container, such as a glass vial or plastic ampoule or other sealed container. The unit dosage form should be suitable for pulmonary delivery, such as an aerosol. Preferably, the product or kit also includes instructions on how to use the product, including how to administer it. The instructions may also contain informational material advising physicians, technicians, or subjects on how to properly prevent or treat the disease or condition. In other words, the product includes instructions indicating or recommending a dosing regimen, including but not limited to actual dosage, monitoring procedures, and other monitoring information.

[0115] As with any pharmaceutical product, the packaging materials and containers are designed to maintain the product's stability during storage and transportation, and may contain desiccants to ensure stability.

[0116] This kit may contain EVs in a sterile aqueous suspension, which can be used directly or diluted with physiological saline for intravenous or nebulizer administration, or diluted or used in combination with a surfactant for intratracheal administration. Therefore, the kit may also contain diluents or thinners, such as saline or surfactants. The kit may also include lung delivery devices, such as nebulizers, or disposable components, such as mouthpieces, nosepieces, or masks.

[0117] In a final aspect, the present invention also relates to the use of the above-described compositions. More specifically, the compositions are suitable for therapeutic and preventative uses. The present invention is contemplated for the prevention and treatment of specific diseases. Disease prevention means reducing the likelihood of the disease manifesting itself and / or delaying the onset of the disease. Disease treatment means reducing or eliminating the symptoms of the disease. Therefore, the present invention also contemplates providing a method for treating and / or preventing a disease in a subject.

[0118] The subjects are preferably human subjects, but certain aspects of the invention can be performed on any subject from whom they may potentially benefit, including human subjects, agricultural livestock (e.g., cattle, pigs, etc.), precious animals (e.g., horses), 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 one embodiment, the composition of any of the above embodiments is used for the prevention or treatment of lung diseases. In one embodiment, the lung disease may be inflammatory lung disease, pulmonary vascular disease, or 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 a preferred embodiment, the viral infection is influenza, SARS-CoV-1, MERS, or SARS-CoV-2. In another embodiment, the acute lung injury is related to sepsis or acute respiratory distress syndrome (ARDS).

[0120] These diseases also include pulmonary vascular diseases that may not have an inflammatory component. Other lung diseases that can be treated according to the invention include acute lung injury, which may be related to sepsis or ventilation. An example of the latter is acute respiratory distress syndrome.

[0121] Pulmonary hypertension is a lung disease characterized by pulmonary artery blood pressure that is much higher than normal. Symptoms include shortness of breath, chest pain (especially during physical activity), weakness, fatigue, fainting, mild headache (especially during exercise), dizziness, abnormal heart sounds and murmurs, jugular venous distension, fluid retention in the abdomen, legs and ankles, and cyanosis of the nail beds.

[0122] Bronchopulmonary dysplasia (BPD) is a condition that afflicts newborns or premature newborns who require oxygen or are on a ventilator, especially very premature newborns (e.g., those born before 32 weeks of gestation). It is also known as neonatal chronic lung disease. Causes of BPD include mechanical injury (e.g., from ventilation), oxygen toxicity (e.g., from oxygen therapy), and infection. Over time, the disease may progress from non-inflammatory to inflammatory. Symptoms include cyanosis, chronic cough, tachypnea, and shortness of breath. Subjects with BPD are more susceptible to infections such as respiratory syncytial virus (RSV). Subjects with BPD may develop pulmonary hypertension.

[0123] Acute respiratory distress syndrome (ARDS), also known as respiratory distress syndrome (RDS) or adult respiratory distress syndrome, is a condition caused by lung injury or acute illness. Lung injury can be caused by ventilation, trauma, burns, and / or aspiration. Acute illness can be infectious pneumonia or sepsis. It is considered a severe acute lung injury and is often fatal. 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 inhaled oxygen (FiO2) of less than 200 mmHg in the presence of bilateral infiltrates on chest X-ray. A PaO2 / FiO2 ratio of less than 300 mmHg with bilateral infiltrates indicates acute lung injury, which is often a precursor to ARDS. Symptoms of ARDS include shortness of breath, tachypnea, and confusion due to hypoxemia.

[0124] Idiopathic pulmonary fibrosis is characterized by unexplained scarring or thickening of the lungs. It most commonly occurs in people aged 50 to 70. Its main symptoms include shortness of breath, frequent cough (usually dry), chest pain, and decreased activity levels.

[0125] In some cases, prevention and / or treatment may include the use of EVs alone or in combination with one or more second agents or active ingredients. Mechanical interventions may also be administered to the subject, such as ventilation with or without external oxygen supply.

[0126] Subjects may be those suffering from lung diseases (or conditions) suitable for treatment with the EV of this invention, or they may be those at risk of developing such diseases (or conditions). Subjects include newborns, particularly those born at low gestational age. As used herein, a human newborn refers to a person from birth to approximately 4 weeks of age. As used herein, a human infant refers to a person from approximately 4 weeks of age to approximately 3 years of age. As used herein, low gestational age refers to birth (or delivery) earlier than the normal gestational age for a given species. A full human gestation is approximately 40 weeks, and its range can be from 37 weeks to more than 40 weeks. Low gestational age (similar to preterm birth) in humans is defined as birth before 37 weeks of gestational age, including birth at shorter gestational ages (e.g., before 36 weeks, 35 weeks, 34 weeks, 33 weeks, 32 weeks, 31 weeks, 30 weeks, 29 weeks, 28 weeks, 27 weeks, 26 weeks, or 25 weeks of gestation). Such preterm infants are typically treated as newborns; however, this invention contemplates treating them beyond the newborn stage, reaching childhood and / or adulthood. Some subjects may have a genetic predisposition to certain forms of lung disease (such as pulmonary hypertension), and these subjects may also be treated according to the present invention.

[0127] For newborns, especially those of low gestational age, this invention considers administering EVs at 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 critical cases, MSC exosomes are administered within 1 hour of birth.

[0128] This disclosure further considers the administration of EVs even in the absence of symptoms of lung disease (e.g., but not limited to BPD).

[0129] In one embodiment, the EV-containing composition of the present invention can be used to treat COVID-19, more specifically, COVID-19-induced pneumonia or acute pneumonia. COVID-19 is a novel infectious disease caused by severe acute respiratory syndrome that attacks the human respiratory system and lung epithelial tissue. It has been reported that a subset of patients are at higher risk of developing more severe symptoms of COVID-19. Major complications include pneumonia, acute respiratory distress syndrome (ARDS), multiple organ failure, septic shock, and death.

[0130] It has been shown that COVID-19 (also known as SARS-CoV-2, or caused by SARS-CoV-2) involves different mechanisms of lung infection, which can develop into acute respiratory distress syndrome (ARDS) potentially triggered by cytokine storm, multiple organ failure, septic shock, and thrombosis. In contrast, bacterial pneumonia is a common lung infection in which air sacs throughout or in parts of the lung become inflamed and filled with fluid, pus, and cellular debris, primarily caused by viruses, fungi, or bacteria, and is usually treated with antibiotics. In other cases, cardiovascular complications, elevated liver enzymes reflecting liver damage, and neurological manifestations may occur. In children, if the infection progresses, it develops into pediatric multisystem inflammatory syndrome, with symptoms similar to Kawasaki disease, which can be fatal. Based on current data, children account for a small percentage of reported cases, with approximately 1% in children under 10 years of age and 4% in children between 10 and 19 years of age.

[0131] The composition containing EV provides multi-target therapeutic effects, with its primary mode of action being the inhibition of the inflammatory process.

[0132] EV targets multiple lung injury mechanisms, including high inflammatory response and cytokine storm, fibrosis, oxidative stress induced by (mechanical) ventilation, and epithelial cell apoptosis due to viral activity and inflammatory response.

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

[0134] In another embodiment, the composition of any of the above embodiments is used for the prevention or treatment of inflammatory bowel disease (IBD), such as Crohn's disease or ulcerative colitis. IBD is a group of diseases that cause inflammation of the gastrointestinal tract (GI), the two most common being ulcerative colitis (UC) and Crohn's disease (CD). UC is a disease that causes chronic inflammation and ulceration in the inner lining of the deepest part of the colon and rectum. CD can develop anywhere in the digestive tract and can penetrate deep into the affected tissues. A symptom of CD is the development of anal fistulas. Both diseases are similar in that they both cause abdominal pain, severe diarrhea, fatigue, and weight loss.

[0135] In cases of pulmonary disease, the preferred administration method is intratracheal infusion or inhalation. In cases of neurological disease, systemic infusion, intranasal infusion, or inhalation are preferred. In cases of Crohn's disease fistula, ulcer, or cartilage repair, local injection is preferred. In cases of treating wound healing, burns, and / or ulcers, EV is preferably administered externally to the body, preferably in conjunction with a hydrogel or polymer medical device for slow EV release.

[0136] The EV of the present invention is administered in an effective amount. An effective amount refers to the amount of the formulation that, when used alone, produces the desired result. This absolute amount depends on many factors, including the material chosen, whether it is administered in a single or multiple dose, and individual patient parameters including age, physical condition, body size, weight, and stage of disease. These factors are well known to those skilled in the art and can be resolved through routine experiments. The dosage may also vary depending on the subject administering the composition.

[0137] In one embodiment of the invention, the EV is 10 9 EVs / kg up to 10 12 The dosage administered to the patient is preferably 10 EVs / kg. 10 EVs / kg up to 10 12 The dosage for the patient is EVs / kg. In a further embodiment, the dosage range for children (aged 0 months to 12 years) is 10. 9 EVs / kg up to 10 11 EVs / kg. The range of doses for adolescents and adults is 10 in adults. 9 EVs / kg up to 10 12 EVs / kg.

[0138] In one embodiment of the invention, the EV is approximately 10 per patient. 10 To about 10 12 EV dosage is administered to treat CD anal fistula.

[0139] This disclosure also considers repeated administration of the EV, including two, three, four, five or more administrations. In some cases, the EV can be administered continuously. Depending on the severity of the disease being treated, repeated or continuous administration can occur over several hours (e.g., 1 to 2 hours, 1 to 3 hours, 1 to 6 hours, 1 to 12 hours, 1 to 18 hours, or 1 to 24 hours), several days (e.g., 1 to 2 days, 1 to 3 days, 1 to 4 days, 1 to 5 days, 1 to 6 days, or 1 to 7 days), or several weeks (e.g., 1 to 2 weeks, 1 to 3 weeks, or 1 to 4 weeks). If repeated but not continuous administration is used, the time between two administrations can be several hours (e.g., 4 hours, 6 hours, or 1 hour), 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 time between two administrations can be the same or different. For example, if the symptoms of the disease seem to be worsening, EVs can be administered more frequently, and once the symptoms stabilize or improve, the frequency of EV administration can be reduced.

[0140] In some cases, low-dose EVs can be repeatedly administered intravenously. Therefore, this disclosure contemplates both repeated administration of low-dose EVs and single administration of high-dose EVs. The low-dose range can be 10... 10 Up to 10 11 EV per kilogram or per local infusion, but not limited to this, while the range of high doses can be 10. 11 Up to 10 12 EV per kilogram or per local infusion. It should be understood that single or repeated administration of low or high doses of EV should be considered depending on the severity of the disease, the subject's health status, and the route of administration.

[0141] EVs can be administered via any route of delivery to the lungs or gastrointestinal tract. Systemic administration routes such as intravenous bolus and continuous infusion are suitable. More direct routes such as intranasal administration, intratracheal administration (e.g., via intubation), and inhalation (e.g., aerosols via the nose or mouth) are also considered in this invention and may be more suitable in certain cases where rapid action is required. As used herein, an aerosol is a liquid suspension dispersed in a gas as small particles, and includes fine mists or sprays containing such particles. As used herein, aerosolization is the process of generating an aerosol by converting a liquid suspension into small particles or droplets. This can be done using aerosol delivery systems such as pressurized packs or nebulizers. Nebulizers include air jet nebulizers (i.e., pneumatic), ultrasonic nebulizers, and vibrating mesh nebulizers, using suitable propellants such as, but not limited to, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gases. In addition to nebulizers, other lung delivery devices include, but are not limited to, metered-dose inhalers (MDIs) and dry powder inhalers (DPIs). Capsules and vials made from gelatin, for example, for use in inhalers or blowpipes, can be formulated to contain lyophilized exosomes and a suitable powder matrix, such as lactose or starch.

[0142] When whole-body delivery of EVs is required, the EVs can be formulated for parenteral administration via injection, such as by bolus or continuous infusion. Injectable formulations can be in single-dose form, such as ampoules or multi-dose containers, with or without preservatives.

[0143] The composition may be in the form of an aqueous suspension, solution, or emulsion on an oily or aqueous carrier, and may contain formulation agents such as suspending agents, stabilizers, and / or dispersants. Suitable lipophilic agents or carriers include fatty oils, such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides. Aqueous injectable suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase solubility. Additionally, exosomes may be in lyophilized or other powder or solid form for use with a suitable carrier (e.g., sterile, pyrogen-free water) prior to use.

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

[0145] The invention will now be described in more detail with reference to non-limiting embodiments.

[0146] Description of embodiments and / or accompanying drawings

[0147] Example 1 - Separation of EVs from MSC

[0148] Umbilical cord (UC)-derived MSCs obtained from umbilical cord tissue or Wharton's jelly are amplified in an amplification unit comprising a stirred bioreactor (2) fluidly connected to a cell culture medium supply container (1). (See reference...) Figure 1 The following numbers represent: 1. Cell culture medium container 2. Stirred bioreactor 3. Cell supernatant container 4. Refrigerator 5a, 5b, 5c. Peristaltic pump 6. First Filtering Unit 7. Second Filtering Unit 8. TFF box 9. Final Product Cells proliferated in the presence of three-dimensional microcarriers in the bioreactor (2) and in a heterologous and serum-free medium containing 3 g / L of purified human albumin. The medium also contained 60 mg / L of recombinant purified transferrin.

[0149] MSCs were amplified and grown in bioreactor (2) until they reached a density of at least 40 x 10⁻⁶ in bioreactor (2). 6 The minimum cell / L concentration and the maximum MSC confluence were 80%. Cell concentration was measured by cell counting and fluorescent labeling of MSCs attached to microbeads. At this stage, the cells were ready to begin secreting EVs. For this purpose, fresh serum-free and heterologous medium was diluted 1:10 from basal medium (DMEM / high glucose / phenol red-free / glutamaxx, ThermoScientific) and added to the cell culture medium. After 24 hours, the system was ready to begin collecting EVs. For this purpose, conditioned cell culture medium or cell supernatant from bioreactor (2) was pumped into cell supernatant container (3), which was placed in a refrigerator (4) set at 4°C. This supernatant contained EVs produced by MSCs. The supernatant collected in container (3) was then pumped by peristaltic pump (5c) to a first filtration unit (6) with a 2-micron filter. The supernatant was filtered via dead-end filtration in a closed system with a peristaltic pump (5c) providing a constant flow rate of 100 ml / min through the filter (6). The flow-through from the first filtration step was then filtered in a second filtration unit (7), fluidly connected to the first filtration unit (6) and having a 0.2-micron filter. Filtration was performed via dead-end filtration in a closed system with a peristaltic pump (5c) providing a constant flow rate of 100 ml / min through the filter (7). The flow-through containing EV was collected in a 1-liter bag and then stored at 4°C.

[0150] In the final step, the flow-through solution is introduced into a TFF cartridge with a molecular weight cutoff of 100 kDa, which is fluidly connected to dead-end filter units (6, 7). The EV-containing residue in the TFF cartridge (8) is washed with saline buffer and concentrated to a final volume of 10 ml using a peristaltic pump (5c) at a speed of 300 rpm. The final product (9) is collected from the TFF (8) and frozen below -80°C or stored in cryovials, bags, or other suitable containers at 4°C.

[0151] The final product is analyzed to ensure its quality. Quality control includes measuring the concentration of albumin bound to EVs, and particle analysis using techniques such as colorimetric analysis and ELISA, nanoparticle tracking analyzer (NTA), tunable resistive pulse sensor (TRPS), electron microscopy, and / or RAMAN microscopy to ensure the purity and stability of the product. The product sample is tested for the presence of endotoxins and / or mycoplasma. Other quality control measures include qualitative chromatography, mass spectrometry, ELISA, sequencing, qRT-PCR, and activity assays, such as in vitro T-cell, B-cell, and macrophage polarization.

[0152] Under the above culture conditions, during the MSC culture period of 18 to 24 hours, the MSCs will produce at least 0.25 x 10⁻⁶ ppm of culture medium per ml. 9 Each particle.

[0153] This experiment was performed using MSCs derived from UC, but it can also be replicated using MSCs derived from other tissues or sources, including but not limited to mammary gland, bone marrow, Wharton's jelly, (umbilical cord) blood, peripheral blood, amnion, adipose tissue, dental pulp, fallopian tubes, liver tissue, and lung tissue.

[0154] Example 2: Measuring the albumin concentration of EV products

[0155] The albumin concentration of the EV product obtained by the method described in Example 1 was analyzed. Albumin concentration was measured using two different methods: -Colorimetric measurement - ELISA In short, bromocresol green is used to assess albumin concentration in the sample via a colorimetric reaction. Concentration is measured using an ELISA assay with an anti-human albumin antibody.

[0156] The albumin concentration in the final product, measured by colorimetric reaction, was 16 g / L. The concentration measured by ELISA was approximately 17 g / L.

[0157] Albumin has a molecular weight of approximately 66 kDa, and any proteins not expected to bind to EVs are removed by a 100 kDa TFF (transfer-free protein). Therefore, the remaining albumin is assumed to bind to EVs.

[0158] Example 3: Measurement of transferrin concentration in EV products

[0159] The transferrin concentration of the EV product obtained by the method described in Example 1 was analyzed. The transferrin concentration was quantified using an ELISA (Abcam) according to the manufacturer's protocol.

[0160] The transferrin concentration in the final product, measured by ELISA, was 70 mg / L.

[0161] Example 4: Purification of clinical-grade culture medium for EV production

[0162] Experiments were conducted to evaluate the concentration of contaminant particles in different culture media used for EV production. The following culture media were tested: 1. A heterologous and serum-free culture medium containing 0.3 g / L purified human albumin and 7 mg / L recombinant purified transferrin (diluted approximately 1:10 with DMEM medium compared to the medium used in Example 1). 2. Basic DMEM medium without supplements (Thermo Scientific / Lonza) 3. Supplement culture media containing undefined products (Thermo Scientific / Lonza) 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.

[0163] EVs were produced using one of the above-described culture media, following the method described in Example 1. Quality control results showed that culture medium 2 had the fewest contaminant particles among the different media produced; however, this medium does not support long-term cell culture. Culture medium 3 had the highest number of contaminant particles. Culture medium 1 had a contaminant particle count between the other media, but it supported long-term cell culture.

[0164] Example 5: Cell culture in clinical-grade culture medium for EV production

[0165] This experiment demonstrates the cell number, cell growth curve, and cell viability in the following culture media: 1. A heterologous and serum-free culture medium containing 3 g / L purified human albumin and 60 mg / L recombinant purified transferrin (similar to the culture medium used in Example 1). 2. Basic DMEM medium without supplements (Thermo Scientific / Lonza) 3. Supplement culture media containing undefined products (Thermo Scientific / Lonza)

[0166] method

[0167] In the first experiment, MSCs were expanded in the culture system described in the first embodiment, in the presence of culture medium 1, 2 or 3, until the cells reached 70% to 80% confluence.

[0168] Subsequently, in the second experiment, the cells that had been expanded in media 1, 2 and 3 in the first experiment were renewed using the same media.

[0169] In the third experiment, the cells expanded in culture medium 1 in the first experiment were replaced with a mixture of culture medium 1 and culture medium 2 in a 2:8 ratio. In the same experiment, the cells expanded in culture medium 3 in the first experiment were replaced with a mixture of culture medium 3 and culture medium 2 in a 2:8 ratio.

[0170] In the fourth experiment, the cells that were expanded in media 1, 2 and 3 in the first experiment will be replaced with media 2.

[0171] In experiments 2, 3, and 4, cell culture lasted for at least 5 days, with the culture medium being refreshed every 24 hours. The culture medium was refreshed by removing 100% of the old medium and adding the same volume of fresh medium.

[0172] result

[0173] The first experiment demonstrated that MSCs attached to the microcarriers in media 1 and 3, but not in media 2. MSCs were able to grow / expand in media 1 and 3, but could not maintain their activity in media 2. Only cells cultured in media 1 and 3 were used in subsequent experiments.

[0174] In Experiment 2, cells cultured in undiluted medium 1 and undiluted medium 3 experienced rapid overgrowth within 24 to 48 hours, followed by cell death. In Experiment 3, cell cultures using diluted medium 1 and diluted medium 3 maintained very slow growth, with cells surviving for at least 5 days. In Experiment 4, cell cultures where medium 2 replaced mediums 1 and 3 survived for only 2 days before cell death.

[0175] Example 6: EV concentration and efficiency in clinical-grade culture media

[0176] This experiment demonstrates the EV concentrations in the following culture media: 1. A heterologous and serum-free culture medium containing 0.3 g / L purified human albumin and 7 mg / L recombinant purified transferrin (diluted approximately 1:10 with DMEM medium compared to the medium used in Example 1). 2. Basic DMEM medium without supplements (Thermo Scientific / Lonza) 3. Supplement culture media containing undefined products (Thermo Scientific / Lonza) Amplify MSCs as described above, and amplify MSCs in one of the culture media as described above. Culture for at least 5 days, with all culture media refreshed every 24 hours.

[0177] The EV production protocols of Examples 1 and 5 were used. Culture medium 1 yielded the highest concentration of cell-secreting granules (which were EVs), calculated as total granules minus the granules present in fresh culture medium. In particular, in this example, at least 95% and up to 99% of the granules were secreted by MSCs (EVs).

[0178] Example 7: EV production efficiency of a 3D bioreactor system

[0179] This experiment demonstrates the EV production efficiency of the following system: i) 3D culture systems, such as Figure 1 As shown, and described in Example 1 ii) 2D culture system MSCs were expanded using medium (i) in 3D culture systems (0.5 L and 1 L stirred bioreactors) and 2D multilayer culture flasks, followed by EV production using diluted medium (i). Cell number, medium volume, footprint, and EV yield were calculated and extrapolated to larger bioreactor volumes. Compared to the 2D culture system, the 3D bioreactor approach offered a smaller footprint, lower medium consumption, and higher EV yield per batch.

[0180] Example 8: EV separation efficiency using a TFF system

[0181] This experiment demonstrates the EV purification efficiency using the following method: i) For example, in the TFF system used in the final concentration step, the molecular weight cutoff is 100 kDa. ii) Size exclusion chromatography iii) Ultracentrifugation When compared to SEC, TFF concentration can provide significantly higher EV recovery rates. TFF yields comparable EV yields, but the EV in the latter is compromised, resulting in lower quality. Furthermore, UC is not suitable for GMP / large-scale production.

[0182] Example 9: Activity of EVs intended for the treatment of BPD in in vitro experiments

[0183] EVs were produced according to Example 1 and used to treat the main symptoms of BPD: a) Inflammation EV internal testing Fluorescently labeled EVs were co-cultured with human peripheral blood mononuclear cells (PBMCs). Flow cytometry was performed to quantify the immune cell population with internalized labeled EVs. The following labels were investigated: anti-CD4, anti-CD8, anti-CD11c, anti-CD14, anti-CD19, anti-CD56, and anti-CD15. Neutrophils were analyzed at 0-1-6-12-24 hours and 6 hours later. EVs produced by fibroblasts were used as a negative control.

[0184] Lymphocyte migration test EVs and PBMCs were co-cultured in a trans-well system. Cell migration was studied targeting the SDF-1 chemokine. Cell phenotypic analysis and quantification were performed using flow cytometry.

[0185] B-cell test Human peripheral blood mononuclear cells stimulated with EVs and CpG were co-cultured. Cell proliferation and differentiation into plasma cells were studied, and phenotypic analysis and quantification were performed using flow cytometry. Cytokines and antibodies were quantified by ELISA.

[0186] T-cell test EVs were co-cultured with human peripheral blood mononuclear cells and the T cell activator CD3 / CD28 beads. Flow cytometry was used to quantify CD4+ T cell proliferation and apoptosis. The Treg / Teff ratio was calculated. Flow cytometry was used to quantify Treg proliferation. The following cytokines were quantified using ELISA: IL10, TGF-β, galactagogue-1, HGF, PGE2, GM-CSF, IL2, TNF-α, and IFN-γ.

[0187] CD cell testFlow cytometry was used to investigate the activation of CD3 cells co-cultured with EVs. The upregulation of co-stimulatory molecules CD80 and CD86, as well as the maturation marker CD83, was quantified using flow cytometry. The following cytokines were quantified using ELISA: IL6, IL8, IL12, CCL3, CCL4, IL10, and TGF-β. Phagocytic function was assessed using flow cytometry (incubated with FITC-glucan). Migration was assessed using trans-well assays and flow cytometry.

[0188] Macrophage test EVs were co-cultured with M1-stimulated (LPS) and M2-stimulated (IL4 / IL13) macrophages. The M1 / M2 ratio was calculated using flow cytometry. The following cytokines were quantified using ELISA: IL6, TNFα, IFNγ, IL1β, IL12, IL10, VEGF, MCP1, TGF-β, and FGF, and confirmed by qPCR gene expression analysis. Trans-well assays (towards fMLP) were performed and quantified by flow cytometry.

[0189] NK cell test EVs were co-cultured with NK cells derived from PBMCs. The following cytokines were quantified using ELISA: TNFα and IFNγ. Maturation markers CD27, CD11b, CD107a, IFN-γ, and cell proliferation were quantified using flow cytometry.

[0190] b) Fibrosis

[0191] Experiments were conducted on normal human lung fibroblasts and human epithelial cells using commercial fibrosis assays. Human lung fibroblasts were co-cultured with EVs delivered at various doses and concentrations at different time points. Subsequently, alpha-SMA and type I collagen / fibronectin were analyzed.

[0192] In another experiment, the effect of EVs on epithelial-mesenchymal transition (EMT) in primary human bronchial epithelial cells was determined. EMT was studied using a commercial assay according to the manufacturer's protocol. In one experiment, experimental primary cells derived from healthy tissue or patients with idiopathic pulmonary fibrosis were stimulated with TGF-β to induce EMT, with unstimulated cells serving as a control. Subsequently, human epithelial cells were co-cultured with EVs delivered at various doses and concentrations at different time points. EMT was detected and quantified using FACS and the expression of E-cadherin and fibronectin.

[0193] In another experiment, the effect of EVs on fibroblast-to-fibroblast (FMT) conversion in primary human bronchial fibroblasts was determined. FMT was studied using a commercial assay according to the manufacturer's protocol. In one experiment, experimental primary cells derived from healthy tissue or patients with idiopathic pulmonary fibrosis were stimulated with TGF-β to induce FMT, with unstimulated cells serving as a control. Subsequently, human fibroblasts were co-cultured with EVs delivered at various doses and concentrations at different time points. FMT was detected and quantified using α-smooth muscle actin markers.

[0194] c) Oxidative stress and apoptosis

[0195] The tests and cell lines used to study the characterization of fibrosis were used to quantify apoptosis and oxidative stress (ELISA, flow cytometry).

[0196] Oxidative stress was induced using H2O2 or other reagents. Cells were exposed to oxidative stress at different time points, using varying doses of oxidative stress inducers. Subsequently, cells were co-cultured with EVs delivered at multiple doses and concentrations at different time points. The direct effects of oxidative stress were measured by detecting and quantifying reactive oxygen species (ROS), while indirect effects were measured by measuring nucleic acid damage, lipid peroxidation, and protein oxidation. Direct and indirect biomarkers of oxidative stress were detected and quantified using ELISA and FACS.

[0197] The assay will detect and quantify apoptosis in response to oxidative stress using commercial assay protocols according to the manufacturer's specifications. In one experiment, LIVE / DEAD™ (ThermoFisher) will be used to detect cell viability using FACS.

[0198] In other experiments, annexin V immunofluorescence staining will detect early apoptosis and quantify it using FACS.

[0199] In other experiments, early apoptosis will be detected by quantifying activated caspase-3 and caspase-7 using FACS.

[0200] Example 10: Activity of EV in in vitro studies, intended for the treatment of Crohn's disease

[0201] This experiment demonstrated the activity of the EV produced according to Example 1 in treating the main symptoms of Crohn's disease. In addition to the experiments described in Examples 9(a) to (c), angiogenesis assays were also performed using the EV.

[0202] In one experiment, EVs were co-cultured with HUVEC cells on Matrigel, and tube formation was analyzed. Experiments were performed using human HUVEC cells and a commercially available angiogenesis tube formation assay. Cells were cultured in a 3D environment, i.e., a hydrogel, to mimic the properties of the natural extracellular matrix required for angiogenesis. EVs were delivered at various doses and concentrations at different time points. Analysis was performed using immunolabeling and fluorescent labeling of HUVEC cells, as well as measurements of tube length and branching in response to EVs.

[0203] Example 11: Biodistribution of EVs in an in vivo BPD model

[0204] This experiment assessed the biodistribution of EVs generated in Example 1 in an in vivo BPD model.

[0205] In vivo nonclinical studies were conducted using well-established and widely used animal models of pulmonary dysplasia (BPD) – namely, newborn rats exposed to hyperoxia (O'Reilly et al., 2014; Thébaud B, 2018). This model simulates the condition of artificially ventilated preterm infants because the lungs of newborn rats are immature at birth (late tubular / early cystic stage), reaching the alveolar stage only around day 5 postnatal. Rats achieve full alveolarization around day 30 postnatal, and it is known that early postnatal exposure of full-term rats to hyperoxia disrupts alveolar development, increases alveolar macrophages, and negatively impacts pulmonary angiogenesis. Therefore, structurally, the lungs of full-term neonatal rodents are roughly equivalent to those of preterm human newborns born between 24 and 28 weeks of age (Porzionato A et al., 2019; Porzionato A et al., 2021).

[0206] In a hyperoxia-induced BPD neonatal rat model, the biodistribution of intratracheal (IT) EV products was determined as follows.

[0207] The EVs of the product were stained with a lipophilic fluorescent marker (DiR iodide [1,1-octadecyl-3,3,3,3-tetramethylindole tricarbonine iodide]) to assess the biodistribution of the product after in vivo administration.

[0208] A total of 40 wild-type Sprague-Dawley rat larvae were used. Twenty larvae were exposed to normoxic conditions, while the remaining 20 larvae were exposed to hyperoxic conditions as discussed above. On day 7 postnatally, 20 larvae (10 hyperoxic and 10 normoxic) received an IT administration of the EV product at a dose of 1 x 10⁻⁶. 9 Each particle is equivalent to 1 x 10 11 / kg BW. Twenty control juvenile mice (10 hyperoxia and 10 normoxia) received PBS control injections. IT injection was chosen to directly and locally apply the EV product to the site of lung injury, with the ultimate goal of translating this process into clinical practice.

[0209] The systemic distribution was assessed by fluorescence analysis, thereby evaluating the dye concentration in various organs of 10 young mice in each group at different time points (3 hours and 24 hours) after injection.

[0210] The test results showed that in juvenile mice exposed to normoxic conditions, the EV product was evenly distributed across different organs 3 hours after injection, with the main signal observed in the inguinal lymph nodes. Under normoxic conditions, signals could be detected in the lymph nodes (groin and axilla) 24 hours after injection. In these juvenile mice, the tagged EV product was rapidly cleared from the lungs and distributed throughout the body. The signal was particularly concentrated in the lymph nodes (groin and axilla), which was the only area where it remained within 24 hours.

[0211] In young mice exposed to hyperoxia, at 3 hours post-injection, most signals in the lungs and other organs were completely absent. Almost all signals were detected in the axillary and inguinal lymph nodes. This suggests that under hyperoxia conditions, the uptake of EV product from the application site is faster. In hyperoxia-treated young mice, at 24 hours post-injection, most signals were detected in the axillary lymph nodes (no signal was detected in the inguinal lymph nodes).

[0212] It should be noted that these results are novel because lymph nodes have never been assessed as a potential target tissue in EV biodistribution studies. These results clearly demonstrate that lymph nodes are a major site of EV product accumulation and therefore represent a fundamental effector site driving the immune response to EV product administration.

[0213] Example 12: EV activity in an in vivo BPD model

[0214] This experiment demonstrates the activity of the EV generated according to Example 1 in an in vivo BPD model. The model described by Porzionato et al., 2018, was used.

[0215] In short, with ethics committee approval, 30 wild-type Sprague-Dawley rats were used in the study. The hyperoxia exposure method was established by different research teams and published in several studies (Grisafi et al., 2012, 2013; Marconi et al., 2014; Porzionato et al., 2012, 2018). Experiments were conducted on pups held in boxes under continuous oxygen monitoring. Experimental animals were exposed to 60% oxygen for 2 weeks and treated intratracheally with the EV product of this invention. Control animals were exposed to 60% oxygen for 2 weeks and treated with intratracheal administration of a physiological solution (placebo). Normo-oxygen control animals were exposed to 21% oxygen for 2 weeks and treated intratracheally with either the EV product of this invention or a physiological solution. EV infusions were administered on postpartum days 3, 7, and 10.

[0216] The efficacy of the EV product with single or multiple IT injections was tested. At three different time points, the product was administered at a dose of 6.4 x 10⁻⁶. 9 One particle per application, equivalent to 3.19 x 10 11 Up to 5.71 x 10 11 Particles / kg BW.

[0217] Then, the lungs of the animals were fixed according to the protocol described by Porzionato et al., 2018, and lung volume was measured according to Scherle's method (Scherle, 1970). Histological and immunohistochemical analyses were performed on the lung sections. Tissue contractility factors were calculated according to the protocol described by Porzionato et al., 2018. Stereoscopic analysis was performed according to the description of Porzionato et al., 2018, quantifying the following parameters: i) the volume fraction of alveolar gas space and alveolar septa, ii) the total volume of alveolar gas space and alveolar septa, iii) the surface area density of gas space, iv) the total area of ​​alveolar gas space, v) the total lung volume, vi) the number of alveoli and the mean alveolar volume, etc.

[0218] In particular, the efficacy of EV products administered via IT was evaluated using morphological, cellular fluorescence, and qRT-PCR analyses to determine the recovery from hyperoxia-induced lung injury and the inflammatory response following treatment. Morphological analyses included lung volume estimation, histological assessment, immunohistochemical analysis, immunofluorescence analysis of myofibroblasts, immunofluorescence analysis of alveolar type 2 epithelial cells, quantification of samples stained with immunofluorescence, Alcian blue staining and quantification, protein carbonylation detection, stereographic analysis of alveolar formation, morphological determination of the arterial muscular layer, and morphological analysis of microvessel density and macrophage populations.

[0219] All histopathological and morphological assessments were performed in a single-blind manner, referencing the experimental group. All animals completed the treatment cycle, which included three injections of either IT control (carrier only) or EV product solution on days 3, 7, and 10 postnatally.

[0220] Postnatal exposure to hyperoxia leads to deterioration of all listed histomorphological parameters due to alveolar destruction. Among these parameters, the reduction in alveolar surface area was statistically significant in the hyperoxia group. Diaphragmatic thickness also increased significantly, likely due to inflammatory processes (Table 1).

[0221] Table 1: Morphological parameters of alveoli

[0222] Multiple comparisons (Bonferroni test) showed that, for alveolar area, the hyperoxia group had statistically significant differences compared to both the normoxia group and the hyperoxia + EV product group, thus demonstrating the therapeutic effect of the EV product. For septal thickness, there was no statistically significant difference between the hyperoxia group and the hyperoxia + EV product group.

[0223] Quantification of lung surfactant-associated protein C (SFTPC)-positive cells using immunofluorescence, a specific marker of lung type II epithelial cells (ATII), showed a significant decrease in ATII following hypoxia-induced injury. Treatment with the EV product increased the number of SFTPCs in lung tissue. Alcian blue staining, a marker of glycosaminoglycan production, yielded similar results. Furthermore, the effect of the EV product on protein carbonylation, a well-established marker of ROS (reactive oxygen species)-induced protein oxidative damage resulting from hyperoxia and the ensuing inflammatory process, was tested. The results indicated increased damage under hyperoxia conditions, which was reversed by EV product administration. No increase in mortality was observed in the EV product treatment group, demonstrating the safety of intratracheal administration.

[0224] These results demonstrate the efficacy of EV products in treating bronchopulmonary dysplasia (BPD) in a mature 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 BPD development.

[0225] Example 13: EV activity in an in vivo model of Crohn's disease

[0226] This experiment aimed to demonstrate the activity of the EV produced according to Example 1 in an in vivo model of Crohn's disease. Briefly, this experiment was performed on female 8-week-old B57BL / 6J mice. Colitis was induced by adding 3% sodium lauryl sulfate (SDS) to drinking water, administered randomly for 5 days. This is a well-established and widely used animal model, cited hundreds of times in PubMed (review reference: Kawada et al., 2007). All animals were treated ethically. The animals were subdivided into three experimental groups.

[0227] Group 1 (n=4, normal control): Daily intraperitoneal (ip) injection of 0.2 ml PBS (carrier only) from day 1 to day 5; Group 2 (n=5, induced colitis): Same as Group 1, but with 3% SDS added to drinking water.

[0228] Group 3 (n=4, induction of colitis and treatment with EV): Same as Group 2, but with MSC-EV suspended in 0.2 ml PBS added via the IP route.

[0229] On day 6, the animals were euthanized using CO2. The colon was removed, a portion of the tissue was fixed in formalin for subsequent histological analysis, and another portion was immediately frozen in liquid nitrogen and then stored at -80°C for subsequent RNA extraction.

[0230] Dosage and route of administration of EV

[0231] The EV is separated and applied according to the above procedure.

[0232] Administration route: From day 0 to day 5, administer EV suspended in 0.2 ml PBS daily via intraperitoneal (ip) route.

[0233] Intestinal injury assessment

[0234] animal weight

[0235] Disease activity index (fecal assessment; see Tanaka F, 2008)

[0236] - Histopathological markers of inflammation (Flogosis)

[0237] - Titration of inflammatory mediator expression in colon tissue extracts: TNFα, IL6, IL-1β and Cox2 were detected by RT-PCR.

[0238] Statistical analysis of results

[0239] Data are expressed as mean ± SD. Differences between groups were analyzed using a t-test, and p < 0.05 was considered statistically significant.

[0240] result

[0241] In summary, the results indicate that the EV is suitable for reducing the inflammatory response in experimental colitis, leading to a reduction in the disease activity index and improvement of common symptoms. EV improved clinical manifestations and inflammatory responses in animal models of intestinal inflammatory disease.

[0242] Example 14: Administration regimen and evaluation of safety and efficacy of EV products for the treatment of human BPD

[0243] The safety and efficacy of the EV product for treating BPD, obtained by the method described in Example 1, were tested in a human clinical trial. Briefly, the study was conducted in preterm newborns at high risk of BPD, with a gestational age of 23 to 28 weeks and a birth weight ≤1500g, who were mechanically ventilated via endotracheal intubation and had an inspired oxygen fraction (FiO2) >25%. The EV product was administered intratracheally. Intratracheal administration of the EV product or the saline control solution did not pose an additional risk, as only newborns receiving invasive ventilation via endotracheal intubation were eligible.

[0244] The study was divided into two phases.

[0245] Phase I: Safety and Tolerance of EV Products

[0246] Phase I included 18 participants, divided into 6 groups of 3 participants each. Each participant received one of three dose levels of the EV product (low dose (LD): 1 x 10⁻⁶). 10 EVs / kg body weight (BW), medium dose (MD): 3 x 10 10 EVs / kg BW, or high dose (HD): 9 x 10 10 EVs / kg BW), and one of two regimens (1 or 3 IT applications, with a 24-hour interval between applications).

[0247] The primary objective was to determine the dose-limiting toxicity (DLT) or maximum tolerated dose (MTD) of a single or multiple IT administration of EV products at three different dose levels.

[0248] The following aspects will be evaluated at this stage: - Acute short-term toxicity of EV products (single or multiple doses at different dose levels) administered via IT at 36 weeks of gestation (PMA) or at discharge.

[0249] - DLT at 6 hours and 24 hours after application of EV product.

[0250] - AEs (adverse events) and SAEs (serious adverse events) reported at different time points (including mortality) up to 36 PMA or discharge (related and unrelated).

[0251] - Mid-term toxicity of EV products is assessed through clinical examinations and blood tests (e.g., liver and kidney function tests, hematopoietic markers, blood pressure, body temperature), lung ultrasound, and echocardiography at several time points until discharge.

[0252] - Number of subjects who were on oxygen and ventilation support 28 days after birth (BPD as defined by Jobe AH et al., 2001).

[0253] - The incidence and severity of BPD following administration of the EV product at week 36 of PMA, according to the case definition of the modified NICHD severity grading (Grade I to IIIA) (based on Higgins RD et al., 2018). These were compared with historically matched cases.

[0254] - Overall health status at the end of the study (EOS) (1-year age-adjusted) after administration of the EV product.

[0255] Mortality rates at -36-week PMA and EOS (1-year age-adjusted).

[0256] Phase IIa: Efficacy of EV Products in Treating BPD

[0257] Phase IIa included 70 participants, divided into two groups of 35 participants each. One group was treated with the EV product at the dose level and regimen selected based on the results of Phase I, while the other group was treated with a saline control solution (placebo group).

[0258] The primary objective was to evaluate the efficacy of the EV product in treating BPD in a randomized, double-blind, placebo-controlled study.

[0259] The following aspects will be evaluated at this stage: - The efficacy of the EV product in reducing the incidence and severity of bronchopulmonary dysplasia (BPD) in patients with PMA at 36 weeks compared to the placebo group (saline). The occurrence and severity of BPD were assessed according to the modified NICHD severity classification (Grade I to Grade IIIA).

[0260] - The number of subjects who received oxygen and ventilation support 28 days after birth.

[0261] - Overall health status at EOS (1-year age-adjusted) after administration of the EV product and in the placebo group.

[0262] - Mortality rates at 36-week PMA and EOS (1-year age-adjusted) in both groups.

[0263] - AEs and SAEs reported at different time points in both groups, up to 36 weeks PMA or discharge (related and unrelated).

[0264] - SAEs reported during passive monitoring in both groups, up to EOS (related and unrelated).

[0265] - Duration of MV / respiratory support in both groups, assessment of ROP, NEC, IVH, sepsis, until 36 weeks of PMA or discharge.

[0266] - Test for immune markers (IL-6, IL-8, TNFα, TGFb1, IL1b, IL1ra) in tracheal aspirate at several time points until the infant is intubated.

[0267] - Assess the neurodevelopmental status of infants in both groups at 1 year of age-adjusted age or EOS.

[0268] This invention is by no means limited to the embodiments described and / or shown in the accompanying drawings. Rather, the methods of this invention can be implemented in many different ways without departing from the scope of the invention.

Claims

1. A method for manufacturing a pharmaceutical composition of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs), the method comprising the following steps: -Culturing and amplifying MSCs in a culture medium that is serum-free and heterologous, the serum-free and heterologous culture 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 cell supernatant from the cultured MSCs, the cell supernatant containing EVs; - Filtering the cell supernatant to obtain EVs, the filtration step being a two-step filtration method, the two-step filtration method comprising filtering the cell supernatant through a first filter with a sieve pore size of 1 micrometer to 5 micrometers, and filtering the filtrate from the first filtration step through a second filter with a sieve pore size of less than 1 micrometer; and subsequently - The filtrate from the filtration step is concentrated by tangential flow filtration with a molecular weight cutoff of 100 kDa using TFF.

2. The method as described in claim 1, wherein, The human albumin and human transferrin are purified from human plasma or are recombinant albumin and recombinant transferrin.

3. The method as described in claim 1 or 2, wherein, During the 18-24 hour period of MSC culture, at least 0.25 x 10⁻⁶ ppm of culture medium was produced per ml of medium. 9 The particles, of which at least 90% have a particle size of 0.05 micrometers to 0.22 micrometers, are EVs.

4. A pharmaceutical composition obtained by the method of any one of claims 1 to 3, and comprising extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs), said composition having at least 1 x 10⁻⁶ EVs per milliliter. 11 1 particle, of which The particles have a particle size of 0.05 micrometers to 0.22 micrometers, wherein at least 90% of the particles are EVs, wherein the human albumin concentration of the composition is 10 g / L to 30 g / L, and wherein at least 90% of the human albumin present in the composition is bound to the EVs.

5. The pharmaceutical composition of claim 4, wherein, The composition contains human transferrin at a concentration of 60 mg / L to 600 mg / L.

6. The pharmaceutical composition of claim 4, wherein, The composition comprises human transferrin and human albumin in a ratio of 2 mg to 60 mg of transferrin per gram of albumin.

7. The pharmaceutical composition according to any one of claims 4 to 6, wherein, The EV size ranges from 50 nm to 300 nm.

8. The pharmaceutical composition according to any one of claims 4 to 6, wherein, The composition is further formulated and / or processed.

9. Use of the pharmaceutical composition of any one of claims 4 to 6 in the preparation of a medicament for treating bronchopulmonary dysplasia (BPD) or colitis.

10. The application as described in claim 9, wherein, The composition is administered to a patient who is an adult, an infant, or a newborn.

11. The application as described in claim 9, wherein, The composition is in 10 9 EV / kg up to 10 12 EV / kg is the dose administered to the patient or for each administration.

Images