Process for the preparation of an extracellular vesicle composition, extracellular vesicle composition and use thereof
Patent Information
- Application Number
- AE202602763
- Authority / Receiving Office
- AE · AE
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2024-02-19
- Filing Date
- 2025-02-18
Smart Images
Figure ABST_ABST
Abstract
Description
process for the preparation of aN Extracellular vesicle composition, extracellular vesicle composition and use thereOF FIELD OF THE INVENTIONThe present invention relates to a process for the preparation of a composition comprising extracellular vesicles and to such a composition comprising extracellular vesicles. The invention further relates to the use of such a composition comprising extracellular vesicles or exosomes. BACKGROUND OF THE INVENTIONExtracellular vesicles, in short EVs, are extracellular nanoparticles produced in the endosomal compartment of eukaryotic cells and released from the cells of origin to the environment of the cells of origin. They are enveloped by a lipid bilayer. EVs cannot reproduce themselves. They are found in biological fluids of multicellular organisms, such as in saliva, blood urine etc. EVs carry generic material and proteins from their cell of origin. EVs are sometimes categorized by their size. They are much smaller than cells and show a dimension of approx. 30 to 2000 nm. Exosomes are considered a subclass of EVs and generally show a dimension below 150 nm. Other subclasses are ectosomes or microvesicles (100 to 1000 nm) and apoptotic bodies (500 to 2000 nm). In more recent times, it has been found that EVs which were considered cell debris actually serve as mediators for cell-to-cell communication. With their unique nucleic acids, proteins, and lipids cargo compositions that reflect the characteristics of the cells of origin or producer cells, EVs and more specifically exosomes can be used as cell-free therapeutics. Among exosomes derived from various cellular origins, mesenchymal stem cell-derived exosomes have gained great attention due to their immunomodulatory and regenerative functions. Indeed, many studies have shown anti-inflammatory, anti-ageing and wound-healing effects of MSC-exosomes in various in-vitro and in-vivo models (see Cells 2020, 9,1157; Dae Hyun Ha et al; Mesenchymal Stem / Stroma Cell-Derived Exosomes for Immunomodulatory Therapeutics and Skin Regeneration).A review article by Shengyang Fu et al.; Exosome engineering: Current progress in cargo loading and targeted delivery; NanoImpact 20 (2020) 10026, states that for cargo loading in exosomes, the cargos may be incubated with exosomes or exosomes-secreting cells. Similarly, in a review article, Yaoyao Lu et al; CRISPR-Cas9 delivery strategies with engineered extracellular vesicles; Molecular Therapy: Nucleic Acids Vol.34 December 2023, confirm that EVs function as messengers by exchanging cargo between cells, allowing the transport of various signaling chemicals, such as bioactive lipids, proteins and nucleic acids. The authors classify the methods to load cargo into EVs into endogenous and exogenous post-isolation loading methods. According to the authors, the endogenous methods involve loading the cargo before isolation of the EVs. This is accomplished by adding new genetic material or selected molecules to interact with or modify parent cells, such as drugs, RNAs, DNAs, proteins or select molecules that can be incorporated into their EVs. Exogenous post-isolation loading methods encompass various techniques to load therapeutic cargo into EVs after their isolation. A major advantage of EVs in pharmaceutical applications is that they can be sterilized and produced as an off-the-shelf product, while MSCs themselves cannot, and that they show less safety issues as compared to cell-based therapy.However, the isolation of EVs or exosomes has shown to be a complex process. While ultracentrifugation techniques have been applied, they appear rather aggressive to the EVs. Research work is still ongoing which focusses on suitable methods to separate EVs from other contaminants present in the culture medium, including certain proteins such as lipoproteins for instance. Other techniques have been suggested, such as size-exclusion chromatography, immunoaffinity chromatography and asymmetric-flow field-flow fractionation or combination of above-mentioned techniques.WO2023 / 056272 discloses EV isolation methods comprising tangential flow filtration, possibly combined with upstream centrifugation. EP-4144836-A1 discloses a method for producing stem cell-derived EVs comprising culturing of stem cells, and a step of isolation of EVs from the culture medium through multiple centrifugation steps. WO2017 / 218964 also suggests to isolate EVs from the cell culture medium.It has been suggested to increase the yield or efficacy of EV production by subjecting the cells of origin or the cells of production to a stress which promotes the EV secretion. Such a stress may be generated by subjecting the cells of origin to starvation conditions during a given period of time.WO2022 / 204955 discloses a method for preparing exosomes derived from induced pluripotent progenitor cells (iPSCs) or from iPSC-derived cells, comprising (i) culturing the iPSCs or iPSC-derived cells in a culture medium, (ii) obtaining a cell culture containing a first supernatant and adherent cells when the cells reach at least 80% confluence, collecting the first supernatant and treating the adherent cells with trypsin or trypsin substitute in a medium, (iii) collecting the treated cells and adding same to the first supernatant to obtain a mixture, (iv) obtaining a second supernatant and cell pellets by giving the mixture a heat chock and then centrifuging same, (v) filtering the second supernatant at least once to obtain exosomes. WO2019 / 002608 and WO2020 / 136362 seek to generate a stress by subjecting the medium containing the cells of origin to a turbulent flow which exerts hydrodynamic stress, such as shear stress, on the relevant cells. WO2022 / 008652 discloses a method for the manufacturing of a composition of EVs derived from mesenchymal stromal cells, comprising culturing and expanding mesenchymal stromal cells in a serum-free and xeno-free medium comprising purified human serum albumin and human transferrin, collecting cell supernatant comprising EVs, filtering said supernatant to obtain EVs and concentrating said EVs, e.g. by filtration, size exclusion chromatography or ultracentrifugation. Fangfang Ni et al; in “Efficient preparation of high-purity and intact mesenchymal stem cell-derived extracellular vesicles”, Anal Bioanal Chem. 2024 Feb 14. doi: 10.1007 / s00216-024-05193-0. Epub ahead of print; PMID: 38355844; make use of an EV isolation method using nanoporous membrane based resonators. The disclosed method still is complex.US2019 / 328792 discloses a method for obtaining EVs derived from stem cells wherein the method comprises removing fibrinogen from the heparin-free culture medium, and removing vesicles from the culture medium, culturing the stem cells in said culture medium, and isolating the stem cell-derived EVs from the medium.US11801268B2 discloses isolation of EVs derived from cardiosphere-derived cells. Latter are cultured at 100% confluence, washed with PBS and incubated in PBS-free medium during 15 days. Thereafter, the supernatant is collected, centrifuged and filtered. The EVs are used for the treatment of diseases affecting the eye.Cheng et al. : « Development of a rinsing separation method for exosome isolation and comparison to conventional methods”, 1 January 2019, XP093194405, compare three methods for EV isolation from mice bone marrow derived mesenchymal cells.Warnecke et al.: “First-in-human intracochlear application of human stromal cell-derived extracellular vesicles”, Journal of Extracellular Vesicles, vol. 10, n° 8, 1 June 2021, XP093194360, discloses the isolation of MSC-EVs derived from umbilical cord tissue from conditioned medium by tangential flow filtration and diafiltration. The EVs are isolated by ultracentrifugation and the pellets are resuspended in Ringer Lactate. The EVs are used for treatment of a patient with a cochlear implant.AREVALO-TURRUBIARTE MAGDALENA et al.: « Extracellular vesicles from equine mesenchymal stem cells decrease inflammation markers in chondrocytes in vitro”, Equine Veterinary Journal., Vol. 54, N° 6, 24 November 2021, pages 1133-1143, discloses the cultivation of stem cells wherein the culture medium is centrifuged and filtered, followed by ultracentrifufation in order to obtain EVs.SOUKUP ROBERT et al.: “Characterisation of Extracellular Vesicles from Equine Mesenchymal Stem Cells”, International Journal Of Molecular Sciences, Vol 23, n° 10, 23 May 2022, page 5858, discloses the isolation of EVs from the stem cell culture medium by ultracentrifugation and / or size exclusion chromatography. It is emphasized that the EV preparations are free from contaminating proteins (see bottom page 8 / 18).It has also been found that MSC-derived EVs can transfer miRNAs to target cells, modulating various physiological processes including cell proliferation, differentiation, and immune responses. For instance, studies have demonstrated that MSC-EV miRNAs contribute to osteoblastic differentiation, angiogenesis, and the modulation of inflammatory responses. The therapeutic potential of MSC-derived miRNAs extends to various pathological conditions. In the context of musculoskeletal disorders, EV-miRNAs have been found to exhibit teno- and chondro-protective properties, as well as the ability to induce M2 macrophage polarization and promote regulatory T cells. Furthermore, MSC-EV miRNAs have shown promise in suppressing fibrosis by preventing fibroblast differentiation into myofibroblasts. There, however, still is a need for efficient production methods that generate EVs of consistent quality.There further still is a need for a simple method allowing for collection of secretome with significantly reduced or no contaminating aspects.There also is a need for production methods that produce EVs in high quantities and quality which satisfies the requirements in pharmaceutical applications. It is well-known that such applications require compliance with demanding GLP and GMP regulations and other regulations relating to pharmaceutical products. SUMMARY OF THE INVENTIONThe present invention now provides a process for the preparation of a extracellular vesicle comprising composition, the process comprising the steps of:(i) growing stem cells in a culture medium, thus generating a cell population; (ii) rinsing the cells obtained in an isotonic electrolyte solution;(iii) separating the cells from the medium used in step (ii) using a gentle mechanical stimulation and retaining the medium, the separated cells being discarded; (iv) possibly separating remaining undesirable components, cells and cell debris from the medium of interest retained at step (iii) using a centrifugation at 1000 – 3000 g during a time period of 1 to 30 minutes. In an efficient and / or optimized process, the stem cells are advantageously grown to near confluency, preferably to near 80% confluency, as is commonly managed in known cell culture techniques.Depending on the cell growing technique applied, it may be advantageous to dissociate the stem cells obtained in step (i) from their support and possibly also to separate them from the medium used in the previous steps.It has been found advantageous to use modified stem cells, including but not limited to cells modified by interaction with selected molecules, such as drugs, RNAs, DNAs, proteins, or genetically modified cells, which all generate extracellular vesicles of interest which advantageously reflect the modification to the relevant cell of origin used in step (i). The culture medium for the stem cells may advantageously be a xenofree base medium or a medium comprising FBS and / or platelet lysate, depleted of contaminants. As an example, it may comprise DMEM-F12, more preferably supplemented with 10% platelet enriched plasma and Heparin. Heparin may be added to the medium in a concentration ranging from 1 to 2,5 IU / ml, preferably from 1.2 to 2.1 IU / ml. Too high a heparin concentration is likely to initiate cell differentiation.Step (i) as well as the optional dissociation of stem cells from their growing support are generally known to the person skilled in the art. For dissociation of the cells including detachment from their support, said cells may be placed in a dissociation medium or a dissociation medium may be added to the culture medium. Dissociation media are known in the art. The choice of suitable dissociation medium may depend on the cell culture used. In a 2D-culture for instance, such dissociation medium may comprise a trypsin, a trypsin derivative or a trypsin equivalent agent, a serine-based derivative showing trypsin-like enzymatic activity or EDTA. In the case of a 3D-culture, the dissociation medium may comprise a nattokinase, possibly in combination with the above dissociation agents. The dissociation action of nattokinase, trypsin, derivatives or equivalents thereof may be neutralized after the cells are detached, by admixture of PBS and / or of an isotonic electrolyte solution to the cell containing medium. Said admixture may comprise addition of PBS and / or of an isotonic electrolyte solution at a suitable period of time to the cell containing medium and / or the presence thereof ab initio in the dissociation medium. The cells or cell population obtained, either directly in step (i) or after an optional step of dissociation and separation from growing medium, is retained for further treatment in step (ii), that is a rinsing of the relevant cells or cell population in isotonic electrolyte solution. As a result, the following steps intended to collect and concentrate the EVs are significantly simplified and facilitated. While prior art processes for the production of EVs generally collect the EVs in a complex medium requiring ultrafiltration or ultracentrifugation in order to concentrate the EVs, such processing steps are not required when carrying out the process of the invention.The optional step of dissociation and separation from the growing medium may comprise subjecting the culture medium to a gentle centrifugation. In accordance with the present invention, such gentle centrifugation may consist in a centrifugation at 100 – 400 g, preferably at 150 – 350 g, more preferably at 200 – 300 g during a time period of 1 – 30 min, preferably 5 – 20 min, or 5 – 15 min, more preferably around 10 min. The culture medium, possibly replaced by or modified by a separation medium, and from which the stem cells or cell population have been separated, is not retained.Step (iii) advantageously comprises subjecting the cell-containing isotonic electrolyte solution to a gentle mechanical stimulation designed to favor secretion of EVs. Such gentle mechanical stimulation may advantageously consist in a gentle centrifugation. Under the conditions of the present invention, “gentle centrifugation” is understood to mean a centrifugation at 100 – 400 g, preferably at 150 – 350 g, more preferably at 200 – 300 g during a time period of 1 – 30 min, preferably 5 – 20 min, or 5 – 15 min, more preferably around 10 min.While gentle centrifugation has shown to be a preferred method for extraction of the desirable EVs per the invention, other gentle methods may be used, such as a combination of a mechanical stimulation by ultrasound or agitation with filtration.The isotonic electrolyte solution used in step (ii) may advantageously comprise an alkalising agent, such as lactate for instance. Preferably, the isotonic solution comprises a lactate concentration from 10 to 40 mmol / l and an osmolarity of from 200 to 300 mosm / l. Ringer lactate is an example of such an isotonic solution. It is also known as Hartman solution or sodium lactate solution and consists in a mixture of sodium chloride, sodium lactate, potassium chloride and calcium chloride in water. Interestingly, the stem cells discarded in step (iii) may still be further used in therapeutical applications or for other uses, including culturing and production of EVs.According to a preferred embodiment, last step (iv) effected on the medium essentially without cells, consists in a centrifugation at 1000 to 3000 g, more preferably at 1500 to 2500 g, more particularly at 1800 to 2200 g, during a time period of 1 to 30 minutes, more preferably of 5 to 20 minutes or of 5 to 15 minutes, more particularly of approx. 10 minutes. It has been found that a centrifugation step as defined is particularly suited to separate undesirable components while maintaining in the mixture, i.e. in the supernatant, the desired EVs and certain desirable proteins, such as immunomodulatory proteins and other cell originating proteins and peptides.It has to be emphasized that the invention process is a simple process which is easy to carry out and to control.Moreover, the invention process appears to be a gentle process which is the less aggressive versus the EVs sought to be obtained, and therefore generates high quality EVs. Clearly process steps which are aggressive towards EVs, like ultracentrifugation, are avoided.The invention process allows for the preparation of EVs showing particle sizes comprised between 100 and 250 nm. In the case of equine cells serving as donor cells or originating cells in step (i) of the invention process, EVs showing particle sizes comprised between 100 and 200 nm may be obtained. In the case of human stem cells serving as donor cells or originating cells in step (i) of the invention process, EVs showing particle sizes comprised between 150 and 250 nm may be obtained. Depending on the number of cells cultured up front and possibly on the number of culture cycles, the EVs may be obtained in concentrations ranging from 1 x 107 to 10 x 109 particles / ml, particularly from 1 x 108 to 1 x 109, more particularly from 0.10 x 109 to 1 x 109 particles / ml, or from 0.25 x 109 to 0.50 x 109 particles / ml. The product obtained, thus the EV composition, expresses characteristic markers, such as markers selected from CD9, CD63, CD81, CD29, CD49e, CD105 and CD44, depending on the mammalian source. It is interesting to note that the concentration of EVs may be controlled as desired by suitable addition of isotonic solution. Higher concentrations may be of interest in certain specific applications, such as the treatment of particularly acute pathologies. It has been found that the EV containing Ringer lactate solution can advantageously be stored at -20 °C to -80 °C for extended periods of time and that the EVs collected remain stable. In comparison to prior art EV solutions which require long term storage at temperatures as low as a temperature comprised between -80 °C and -200 °C, the product obtained by the invention process thus shows a significant advantage.The cells discarded in step (iii) may advantageously be reused in cell culture, or in a further cycle of the process above described. It has been found that the gentle process of the invention is the least aggressive v. the cultured cells which hence remain a acceptable source for cell culture.The invention process generates a composition comprising EVs, more specifically exosomes, production cell-originating immunomodulatory proteins and other cell originating proteins and peptides in suspension in isotonic solution. The invention composition hence comprises EVs in a simple ready-to-use buffer solution. Because of the gentle processing conditions applied in the invention process, useful proteins originating from the production cell, such as immunomodulatory proteins and / or peptides and other useful and / or desirable proteins and / or peptides, remain in the product obtained and may be of interest in pharmaceutical treatments. In contrast, prior art techniques for isolation of EVs generate a composition which no longer comprises proteins and / or peptides from the culture medium.It has been found that the invention composition shows no or significantly less contaminating aspects than prior art EV compositions and further shows interesting pharmaceutical effects, such as for instance immunomodulatory properties. As will be shown herein below, the invention compositions also comprise certain desirable miRNAs of interest, such as EV-derivable miRNAs, which are believed to sustain the pharmaceutical effect of said compositions.The invention therefore provides a composition comprising EVs for use in treating one or more of the disorders, such as Systemic Inflammation Response Syndrome (SIRS); Acute Respiratory Distress Syndrome (ARDS), acute or degenerative conditions within the systems composing the mammalian body, such as the circulatory system, the digestive system, the immune system, the integumentary system, the musculosceletal system, the nervous system, the reproductive system, the respiratory system and the urinary system. More specifically the invention composition may be used to treat one or more disorders of several organs such as liver, lung, heart, uterus, brain, eye, skin, bone, tendon and cartilage, in mammalian subjects. Said differently, the invention provides a method of treating at least one or more of the said disorders, in mammalian subjects. The relevant method of treatment may comprise the administering to the relevant mammalian subject, of a therapeutically effective amount of the composition of the invention or of a pharmaceutical composition comprising the invention composition and another pharmaceutical active and / or a pharmaceutically acceptable carrier and / or extender. In particular, unexpected pharmaceutical effects have been evidenced on immunomodulation, more specifically on myeloperoxidase (MPO).Other pharmaceutical effects of the invention composition have been shown in regenerative and healing processes. They promote tissue repair across various models, including liver fibrosis, autoimmune keratitis or uveoretinitis and myocardial infarction. They have also demonstrated to promote angiogenesis and accelerate wound healing. They further sustain anti-infective effects in relevant treatments.Suitable galenic forms may be designed for topical or systemic application. Pharmaceutical compositions containing the invention composition possibly together with other pharmaceutical actives or pharmaceutically acceptable carrier and / or extender may obviously be adapted for appropriate administration to the patient in need, in accordance to known techniques. Examples are subcutaneous or intravenous administration formulae.An interesting galenic form is a gel formulation comprising a invention composition as described above a composition obtained as per the method described above together with platelet lysate.Further details of the invention will become apparent herein below. BRIEF DESCRIPTION OF THE FIGURESThe present invention is illustrated by the following figures which are to be considered for illustrative purposes only and in no way limit the invention to the embodiments disclosed therein:Figure 1: shows a schematic representation of the process steps; Figure 2: is a schematic representation of the SIEFED experiment referred to in Example 3; andFigure 3: compares a keratitis affected eye of two equine subjects (left and right pictures) before and after treatment with an invention composition of EVs in Ringer lactate (top and lower pictures);Figure 4: shows the effect of the supernatant obtained after a 2000 g centrifugation in a SIEFED test; andFigure 5: shows the effect of exosome-free supernatant and resuspended exosomes as obtained after ultracentrifugation, in a SIEFED test. DETAILED DESCRIPTION OF THE INVENTIONAs used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. By way of example, “a cell” refers to one or more than one cell.The terms "comprising", “comprises” and “comprised of” as used herein are synonymous with "including", “includes” or "containing", “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps.The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.The term "about" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of + / -10% or less, preferably + / -5% or less, more preferably + / -1% or less, and still more preferably + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" refers is itself also specifically, and preferably, disclosed.All references cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings of all references herein specifically referred to are incorporated by reference. Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.For general methods relating to the invention, reference is made to well-known textbooks, including, e.g., “Molecular Cloning: A Laboratory Manual, 2nd Ed.” (Sambrook et al., 1989), Animal Cell Culture (R. I. Freshney, ed., 1987), the series Methods in Enzymology (Academic Press), Gene Transfer Vectors for Mammalian Cells (J. M. Miller & M. P. Calos, eds., 1987); “Current Protocols in Molecular Biology and Short Protocols in Molecular Biology, 3rd Ed.” (F. M. Ausubel et al., eds., 1987 & 1995); Recombinant DNA Methodology II (R. Wu ed., Academic Press 1995), incorporated by reference herein.For further elaboration of general techniques useful in the practice of this invention, the practitioner can refer to standard textbooks and reviews in cell biology, tissue culture, and embryology. Included are “Teratocarcinomas and embryonic stem cells: A practical approach” (E. J. Robertson, ed., IRL Press Ltd. 1987); “Guide to Techniques in Mouse Development” (P. M. Wasserman et al. eds., Academic Press 1993); “Embryonic Stem Cell Differentiation in Vitro” (M. V. Wiles, Meth. Enzymol. 225:900, 1993); “Properties and uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy” (P. D. Rathjen et al., al.,1993). Differentiation of stem cells is reviewed, e.g., in Robertson. 1997. Meth Cell Biol 75: 173; and Pedersen. 1998. Reprod Fertil Dev 10: 31, and Ūsas et al., 2011, incorporated by reference herein. General techniques in cell culture and media collection are outlined in Large Scale Mammalian Cell Culture (Hu et al. 1997. Curr Opin Biotechnol 8: 148); Serum-free Media (K. Kitano. 1991. Biotechnology 17: 73); Large Scale Mammalian Cell Culture (Curr Opin Biotechnol 2: 375, 1991), incorporated by reference herein.The term “stem cell” refers generally to an unspecialised or relatively less specialised and proliferation-competent cell, which is capable of self-renewal, i.e., can proliferate without differentiation, and which or the progeny of which can give rise to at least one relatively more specialised cell type. The term encompasses stem cells capable of substantially unlimited self-renewal, i.e., wherein the progeny of a stem cell or at least part thereof substantially retains the unspecialised or relatively less specialised phenotype, the differentiation potential, and the proliferation capacity of the mother stem cell, as well as stem cells which display limited self-renewal, i.e., wherein the capacity of the progeny or part thereof for further proliferation and / or differentiation is demonstrably reduced compared to the mother cell. By means of example and not limitation, a stem cell may give rise to descendants that can differentiate along one or more lineages to produce increasingly relatively more specialised cells, wherein such descendants and / or increasingly relatively more specialised cells may themselves be stem cells as defined herein, or even to produce terminally differentiated cells, i.e., fully specialised cells, which may be post-mitotic. The term “mesenchymal stem cell” or “MSC” as used herein refers to a mammalian adult, mesoderm-derived stem cell that is capable of generating cells of mesenchymal lineages, typically cells of two, preferably of three or more mesenchymal lineages, e.g., osteocytic (bone), chondrocytic (cartilage), myocytic (muscle), tendonocytic (tendon), fibroblastic (connective tissue), adipocytic (fat) and stromogenic (marrow stroma) lineage. Commonly, but without limitation, a cell may be considered MSC if it is capable of forming cells of each of the adipocytic, chondrocytic and osteocytic lineages, using standard, art-accepted differentiation conditions and cellular phenotype evaluation methods, e.g., as described in Pittenger et al. 1999 (Science 284: 143-7) or Barberi et al.,2005 (PLoS Med 2: e161), and Ūsas et al., 2011. The term MSC also encompasses the progeny of MSC, e.g., progeny obtained by in vitro or ex vivo propagation of MSC obtained from a biological sample of a subject.The term “isolating” with reference to a particular component denotes separating that component from at least one other component of a composition from which the former component is thereby “isolated”. The term “isolated” used in relation to any cell, group of cells or a cell population also implies that such cell, group of cells or cell population does not form part of an animal body. The ISCT determined precisely the qualities cells must possess to be defined as mesenchymal stem cells (MSCs) as follows: the cells must be plastic-adherent, positive for the markers CD73, CD90 and CD105, negative for the markers CD14 (or CD11b), CD34, CD45, CD79a (or CD19) and MHC-II, and must exhibit the ability to differentiate into cells of mesodermal origin such as osteoblasts, chondroblasts and adipocytes (Dominici et al., 2006). The use of other MSC markers such as CD29 or CD44 was also reported (Pittenger et al., 1999). The ISCT criteria were extended to the invention herein. The mammalian MSC cells hence are defined in that they express or co-express (i.e., are positive for) at least the mesenchymal marker CD105, and preferably also one or more of the following markers: CD44 and CD90. The mammalian MSC cells are also defined in that they express or co-express (i.e., are positive for) one or more of the following microRNAs: miR-128, miR-133B, miR-218 or miR-802. The mammalian MSC cells are also defined in that they do not express miR-656.Throughout this specification "co-express" intends to cover the meaning "comprising co-expression of" such that the cells can express other markers or microRNAs in addition to the particular recited markers or microRNAs characterising the cells.The terms microRNA, miRNA, miR or eca-miR are used herein interchangeably, and refer to 19-25 nucleotides mature non-coding RNAs or precursors thereof, or fragments thereof, derived from endogenous genes of living organisms such as animals. Mature microRNAs are processed from longer hairpin-like precursors termed pre-microRNAs (pre-miRs) having a length of approximately 75 nucleotides. Where a cell is said to be positive for a particular marker or microRNA, this means that a skilled person will conclude the presence or evidence of a distinct signal, e.g., antibody-detectable or detection by reverse transcription polymerase chain reaction, for that marker or microRNA when carrying out the appropriate measurement, compared to suitable controls. Where the method allows for quantitative assessment of the marker or microRNA, positive cells generate a signal that is significantly different from and higher or stronger than the control, e.g., but without limitation, at least 1.5-fold higher than such signal generated by control cells, e.g., at least 2-fold, at least 4-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold higher or even higher.The expression of cell-specific markers can be detected using any suitable immunological technique known in the art, such as immuno-cytochemistry or affinity adsorption, Western blot analysis, FACS, ELISA, etc., or by any suitable biochemical assay of enzyme activity, or by any suitable technique of measuring the quantity of the marker mRNA, e.g., Northern blot, semi-quantitative or quantitative RT-PCR, etc. The expression of microRNAs may be determined, for example, with an assay for global gene expression (e.g. using a microarray assay for microRNAs expression profiling analysis, a ready-to-use microRNA qPCR plate or RNA sequencing) or by specific detection assays, for example, but not limited to, quantitative PCR, quantitative reverse-transcription (real-time) PCR (qRT-PCR), locked nucleic acid (LNA) real-time PCR, or northern blotting. In particular, the measurement of the expression of a microRNA may be carried out with an oligonucleotide probe specific for the detection of said microRNA. Said oligonucleotide probe may bind directly and specifically to the microRNA, or may specifically reverse transcribe said microRNA. Alternatively, said oligonucleotide probe may bind a cDNA obtained from said microRNA. Said oligonucleotide probe may also amplify a cDNA obtained form said microRNA.Nucleic and amino acid sequence data for marker proteins listed in this disclosure are generally known and can be obtained from public databases such as, among others, from the NIH “Protein Reviews on the Web” database (http: / / mpr.nci.nih.gov / prow / ), the NIH “Entrez Gene” database (http: / / www.ncbi.nlm.nih.gov / sites / entrez?db=gene) or the Uniprot / Swissprot database (http: / / www.expasy.org / ). Suitable detection reagents and methods for said markers can be designed either on the basis of such sequence information or, more commonly, are available commercially (e.g., labelled monoclonal antibody reagents).The skilled person is well aware that microRNAs may be referred to by different names, or synonyms.The MSC cells may further display certain morphological features, such as any one or more of adherence to tissue culture plastic; growth in monolayers; and mononuclear ovoid, stellate or spindle shape with round to oval nuclei having prominent nucleoli.The term "cell population" generally refers to a grouping of cells. A cell population may consist of or may comprise at least a fraction of cells of a common type, or having characteristics in common. Such characteristics may include, without limitation, morphological characteristics, potential for differentiation (e.g., pluripotent, multipotent, unipotent, etc.; e.g., if multipotent or unipotent, ability to differentiate towards specific cell types), or the presence and / or level of one, two, three or more cell-associated markers, e.g., surface antigens. Such characteristics may thus define a cell population or a fraction thereof. Preferably, such a cell population is mesenchymal stem cell population, more preferably a substantially homogenous population of mesenchymal stem cells.The expression “mammal” or “mammalian” refers to all mammals, including, but not limited to, domestic and farm animals, zoo animals, sport animals, pet animals, companion animals and experimental animals, such as, for example, mice, rats, hamsters, rabbits, dogs, cats, guinea pigs, cattle, cows, sheep, horses, pigs and primates, e.g., monkeys and apes, but also humans. Preferred mammals are horses, dogs, or cats.The term “subject” encompasses all mammals as defined above. Cell culture comprises techniques that allow cells to grow outside their natural environment. Depending on cell type, cell proliferation will occur in suspension or by adhesion to a support thanks to adhesion factors added to the growing medium. Cell growth will take place thanks to a medium in which the cells are contained and which provides all nutritive elements required for the cell’s wellbeing. The medium also comprises antibiotics, antifungal, and other components. It is generally known that cell culture requires appropriate temperature, gas mixture, pH, glucose concentration, growth factors and nutrients. Growth factors are generally derived from animal blood, such as fetal bovine serum (FBS), fetal bovine calf serum, equine serum or porcine serum. Serum does not contain white blood cells nor red blood cells nor clotting factor (fibrinogens). It still includes the proteins not involved in blood clotting and all the electrolytes, antibodies, antigens, hormones etc. Serum is also believed to protect the cells against physical chocs as it is a viscous liquid. Serum is further known to control the osmotic balance between the cells and the external medium and to control chemical parameters like protease inhibition. Other cell culture methods are known; reference is made in this respect to WO2021 / 165451 for instance.The term “xeno-free” cell culture medium is understood to mean that the relevant cell culture medium does not comprise any ingredient or component derived from a mammal species other than the one from which the cells are cultured. Nattokinase is an enzyme extracted from natto food which is fermented in the presence of Bacillus subtilis var. natto which in turn produces the enzyme. The exact chemical structure may slightly vary depending on bacterial line and / or fermentation conditions. Trypsin is a serine-based enzyme generated in mammalian pancreas and which hydrolyses peptide bonds, more specifically of adhesion proteins which bond cell walls to a synthetic support. It is commonly used in cell culture for dissociation of grown cells. The precise chemical structure may vary depending on the source. Moreover, serine-based derivatives showing trypsin-like enzymatic activity have been prepared that are more suited for selected applications. Synthetic variants showing trypsin-like enzymatic activity are also available on the market. Unless specified otherwise, the term “trypsin” covers natural variants, derivatives and synthetic variants.The terms “equivalent concentration of x cells / ml” used in connection with the supernatant is understood to mean the concentration of EVs and other components in the supernatant resulting from x cells / ml used as starting cell suspension for secretion of EVs.In equine medicine, tendinitis and osteoarthritis are very frequent pathologies and unfortunately have a poor prognosis. In fact, musculoskeletal injuries are the most common source of injuries for competing horses. Although it is well known that (almost) adult tissues have some tissue-specific progenitor cells, these are often not sufficient for an efficient repair. Thus, effective regenerative medicine requires an exogenous input which should both be able to repair the lesion as well as to coordinate the healing process. Turning now to Figure 1, same is intended to exemplify the invention process with equine muscle derived mesenchymal stem cells. Stem cells may be cultured in a culture medium as is generally known at the present state of art. The relevant stem cells may originate from mammalian tissue, such as umbilical cord, umbilical cord blood, placenta, Wharton’s jelly, peripheral blood, adipose tissue, bone marrow, dental pulp, skeletal muscle tissue or other tissues. In the present example, equine muscle derived stem cells have been obtained as per WO2015 / 091210.Different known culture media may be used as appropriate. In the present example, the stem cells have been grown in DMEM-Ham’s F12 (Dulbecco’s modified Eagle Medium) base medium supplemented with 10% by volume equine platelet rich plasma and 1.4 IU / ml Heparin. Said culture medium is xenofree. Preferably, the stem cells are grown to near 80% confluence. After the culturing step, the stem cells obtained may be dissociated or detached from their support and may also be dissociated from one another. To that effect, the cultured stem cells are washed with a dissociation composition. Trypsin is well known to that effect. In the example, a particularly gentle synthetic trypsin alternative, more specifically TrypLe (sourced from Gibco) has been used to cleave relevant bonding of the stem cells to one another and to their support. The reactions are stopped by addition of PBS buffer. The obtained mixture may then be subjected to a gentle or mild centrifugation. As an example, a centrifugation at approx. 300 g can be applied during approx.10 minutes. The supernatant is to be discarded and the residue comprising the stem cells is resuspended in isotonic electrolyte solution, such as Ringer lactate. In an alternative embodiment of the invention process, and depending on the growing technique used, the cells grown in step (i) may be directly rinsed with isotonic electrolyte solution, such as Ringer lactate.The obtained mixture of cells in electrolyte solution is subjected to mild centrifugation, such as a centrifugation at approx. 300 g during approx. 10 min, which stimulates the secretion of EVs from the cells into the medium. The residue or deposit or pellet, that is the cells, is discarded and the supernatant comprising the EVs is retained as product of interest. If so required, it may be subjected to yet another centrifugation at approx. 2000 g for approx. 10 min in order to eliminate undesirable components and cell debris. The discarded cells may be reused in cell culture, or even returned to the cell culture step (i), or subjected to cryopreservation.It is within the experience and skills of the skilled person to vary the above centrifugation conditions as appropriate. Clearly though, the final composition comprises EVs in a simple buffer solution and can be used directly as such in or as a pharmaceutical composition. The invention process is simple and comprises steps that are well known per se and easy to operate. The processing conditions are mild and as a result, high quality stable EVs are obtained. The invention process does not comprise any process step that is rather aggressive towards EVs. More specifically, the invention process does not include any ultracentrifugation step.The invention process allows for the preparation of EVs showing particle sizes comprised between 50 and 250 nm. These EVs may be obtained in concentrations ranging from 1 x 107 to 10 x 109 particles / ml, particularly from 1 x 108 to 1 x 109, more particularly from 0.10 x 109 to 1 x 109 particles / ml, or from 0.25 x 109 to 0.50 x 109 particles / ml. Higher concentrations may be of interest in specific applications, such as the treatment of particularly acute pathologies. It has been found that the EV containing Ringer lactate solution can advantageously be stored at -20 °C to -80 °C for extended periods of time and that the EVs collected remain stable over extended periods of time. In comparison to prior art EV solutions which require long term storage at temperatures as low as -80 °C to -200 °C, the product obtained by the invention process thus shows a significant advantage.The invention process generates a composition comprising EVs, including exosomes, production cell-originating immunomodulatory proteins and other cell-originating proteins and / or peptides or miRNAs in suspension in isotonic solution; here Ringer lactate. The product obtained is of interest in pharmaceutical treatments. As stated above, the composition resulting from the invention process shows unexpected pharmaceutical effects and may be used for treating one or more of the disorders, such as Systemic Inflammation Response Syndrome (SIRS); Acute Respiratory Distress Syndrome (ARDS), acute or degenerative conditions within the systems composing the mammalian body, such as the circulatory system, the digestive system, the immune system, the integumentary system, the musculoskeletal system, the nervous system, the reproductive system, the respiratory system and the urinary system. More specifically the invention composition may be used to treat one or more disorders of several organs as liver, lung, heart, uterus, brain, eye, skin, bone, tendon and cartilage, in mammalian subjects. The method of treatment may comprise the administering to the relevant mammalian subject, of a therapeutically effective amount of the composition of the invention or of a pharmaceutical composition comprising the invention composition and another pharmaceutical active and / or a pharmaceutically acceptable carrier and / or extender. The invention composition may be in a form designed for topical or systemic application (subcutaneously or intravenously). Pharmaceutical compositions containing the invention composition together with other pharmaceutical actives or pharmaceutically acceptable carrier and / or extender may obviously be adapted for appropriate administration to the patient in need, in accordance with known techniques. ExamplesExample 1: Material and methods – Cell culture and medium compositionMicrobiopsy procedures were performed on standing, awake horses. Microbiopsy specimens were obtained from triceps brachii muscles (long head, at the intersection of a vertical line extending from the tricipital crest and a line between the scapulo- and radio-humeral joints) of each horse (n=3).Microbiopsy specimens were collected with a 14-gauge microbiopsy needle and a microbiopsy pistol. Briefly, the sampling site was shaved (one cm square) and aseptically prepared. Each sample (approximately 10 to 20 mg of tissue) was collected at a depth of 5 cm in the long head of the triceps brachii muscle, through a skin incision of + / - 2 mm made with the tip of a scalpel blade nr 11 or a trocar equipped with a mandrel. Closure of the skin incision was not necessary and the whole microbiopsy procedure was completed within 15 minutes. Each microbiopsy specimen was carefully dissected (trying to keep as much as possible only muscular tissue, hence eliminating fibrous tissue parts). Microbiopsy specimens were washed several times in phosphate salt buffer solution (10 mM PBS solution at pH=7.4 containing 137mM NaCl and 2.7mM KCl), in order to eliminate red blood cells and other potential impurities. The microbiopsy specimens then were cut in small pieces (size of the tip of the scalpel blade). Culture preparation was performed by use of sterile equipment, under a streamline flow hood. Each piece was placed individually into the 16 central wells of a 24-mutliwell dish, each well comprising 400µl of culture medium. The culture medium consisted in a mixture of 500mL DMEM-F12 (Lonza) supplemented with 10% of equine platelet rich plasma (PRP) and Heparin at a final concentration of 1,44IU / mL into the whole medium. PRP is obtained thanks to a GMP plasmapheresis COM.tec from Fresenius Kabi. Pooches of 100 to 800 mL are generated and stored in 50 ml Falcon.The multi-well dish was then incubated at 37°C under controlled atmosphere (5% CO2) for several days. A migration of cells occurred. Example 2: Preparation of EV based compositionAt the end of each cell culture expansion and before cryopreservation step, mesenchymal stem cells are detached from the flask with TrypLe and after 5 minutes, PBS (phosphate buffer saline) is used to stop the TrypLe activity. A first centrifugation at 300g for 10 min is performed to collect cells in the pellet. Then, supernatant is discarded and cells are resuspended with Ringer Lactate to obtain a final concentration of 100,000 cells / mL. A second centrifugation at 300g for 10min is performed in order to obtain the secretome of these cells. After that, cells (in the pellet) are cryopreserved and the supernatant is collected. A final centrifugation of the supernatant consists in eliminating debris at 2000g for 10 min. At the end of the process, a cell-free supernatant containing extracellular vesicles (EVs) is obtained in a Ringer lactate solution. Example 3: Evaluation of EV composition on myeloperoxidase (MPO) activityIn order to test myeloperoxidase (MPO) activity, an enzyme released by neutrophils during a period of stress, a SIEFED experiment (Specific immunoextraction followed by enzymatic detection) was performed. Figure 2 is a schematic representation of the experiment. It is based on a plaque coated with antibodies against equine MPO. A defined equivalent concentration of 100,000 cells / mL of the EV containing lactate solution was added into the wells and a known concentration of MPO was added. A 2h-incubation at 37°C was performed before revealing the fluorescence. The invention EV composition with an equivalent concentration in the supernatant of 100000 cells / mL can inhibit at least 75% of MPO activity (n=8). Example 4: Evaluation of EV composition on Lymphocyte proliferationIsolation of mononuclear cells from peripheral blood8 ml of EDTA-stabilized whole blood from a healthy equine subject was obtained aseptically and further diluted 1:1 with Dulbecco’s Mg2+ / Ca2+ free Phosphate Buffered Saline (PBS). 8 ml of PBS diluted blood was layered onto 7 ml of Ficoll-Paque PLUS (GE Healthcare) and centrifuged during 40 minutes without brakes (18 °C, 400 g). Buffy coat containing mononuclear cells was carefully transferred to a new tube and washed twice with PBS for 15 minutes (500 g, 18° C). Afterwards cells were washed one more time following low speed centrifugation to get rid of the platelets (100 g, 10 minutes, 18°C). Cell pellet (PBMC, peripheral blood mononuclear cells) was reconstituted with RPMI 1640, supplemented with 10% FBS, 5 mM of L-Glutamine, 0.1 mM of 2-Mercaptoethanol (and optionally Amphotericin B and Penicillin / streptomycin mixture). Lymphocyte stimulationTo stimulate the lymphocytes, PBMC were plated in a culture flask (T-175) overnight and then stimulated with phytohemagglutinin PHA-L (Roche) at a concentration of 5 µl / ml. They were finally cultured in 24-well plate at a density of 0,5 x 106 cell / ml for 72 h. Lymphocyte proliferation In order to study the effect of EV containing Ringer lactate solution on lymphocytes we seeded stimulated PBMC at a density of 500000 cells / well of a 6-well plate and co-cultured them during 72 hours with the following different equivalent concentrations in cells of the EV containing lactate solution: 100 000, 50 000, 40 000, 30 000 and 20 000 cells / ml. The diluent was Ringer Lactate Hartmann solution (B. Braun vetcare).The results were obtained by visual assessment of the proliferation of the stimulated PBMC with a manual count of the cells in each experimental condition. The difference in cell counts between the stimulated PBMC in contact or not with the invention and the unstimulated ones was then translated in percentage of inhibition of the lymphocyte proliferation. ResultsTable 1 below shows the results obtained. Table 1. The evolution of the quantity of stimulated PBMC after 3 days of incubation with different equivalent in cells of the EV containing lactate solution and the percentage of inhibition of their proliferation.Equivalent in cells of the EV containing lactate solution( / mL)Quantity of stimulated PBMC after 72h incubation with the EV containing lactate solution(cells / mL)Calculated percentage of inhibition of proliferation of PBMC0272,000 / 20,000122,00055.1%40,000100,00063.2%50,000100,00063.2%100,00017,50093.5% At an equivalent concentration of 20,000 MSCs / mL, a 55.1% inhibition of PBMC proliferation was observed (Table 1). This level of inhibition aligns closely with the decrease observed in the cell population of the negative control, where non-activated PBMC— which inherently lack the ability to proliferate without external activation—display a similar decline.At an equivalent concentration of 100,000 MSCs / mL, a 93.5% inhibition of PBMC proliferation was shown (Table 1). Example 5: Characterization of generated EVsIn order to evidence that the EV containing lactate solution composition actually contains a large amount of EVs (and among them exosomes), a flow cytometry analysis was performed. The invention composition comprising EVs in Ringer lactate was prior-incubated with magnetic balls containing antibodies against CD9, CD63 and CD81 to isolate EVs. The specific EVs, fixed onto these magnetic balls, were then incubated with a mix of different secondary antibodies against 39 different CD proteins (detection cocktail). The emitted fluorescence is a measure of the quantity of EVs expressing specific CD proteins amongst the 39 of the detection cocktail. The EVs contained in the composition expressed CD9, CD63, CD81, CD29, CD49e, CD105, and CD44 in different proportion depending of their origin, human or equine. To determine the amount and quality of EVs (and among them exosomes), an analysis with a Nanosight device was performed. This tool measures nanoscale particles by laser scattering in the range of 10 nm -1000 nm. Particle–by–particle analysis assures high resolution size and concentration data with visual confirmation. The equine EVs of interest contained in the composition have a mean (arithmetic) size of 207,03 ± 4,27nm with a high peak at 200nm and a mean concentration of 4,42x108 ± 2,02x107 particles / mL. Regarding human EVs, the mean size is 234,27 ± 6,97nm with a mean concentration of 2,57x108± 3,40x106 particles / mL. Example 6: Therapeutical application of the invention composition against IMMK IMMK (Immune Mediated Keratitis) is a nonulcerative keratitis of idiopathic origin commonly diagnosed in veterinary ophthalmology. IMMK is characterized by varying degrees of conjunctival hyperaemia, cellular infiltration, corneal vascularization, corneal oedema, calcific degeneration, and fibrosis. IMMK usually necessitates long-term medical treatment. Existing therapeutic approaches focus on reducing the inflammatory reaction and typically encompass a combination of local corticosteroid, non-steroidal anti-inflammatory drug, or cyclosporine administration, alongside systemic administration of steroidal or non-steroidal anti-inflammatory drugs. Therapy can be challenging, as the disease often becomes increasingly refractory to medical treatment and prone to recurrence over time. In addition, prolonged use of steroidal anti-inflammatory treatment can lead to corneal degeneration. Clinical studies8 Horses diagnosed with IMMK and admitted to the Veterinary Teaching Hospital of the University of Liège (Belgium) were included in this study. The inclusion criteria required a non- or partial response to the currently recommended treatment which consisted of topical glucocorticoids and topical cyclosporine. The treatment had to be administered for at least three months before considering the case as refractory. The invention composition consisted in a Ringer lactate solution comprising EVs secreted from equine MSCs (obtained according to WO2015 / 091210) and prepared in accordance with the invention process at a concentration of 3-5 x 109 EVs / ml. The severity of the disease was assessed by a scoring system (see Table 2). The invention treatment consisted of the administration of the invention composition at a rate of 2 drops 3 times a day for 30 days. 5 cases are reported in Table 3. None of the horses experienced ocular discomfort. Conjunctival hyperaemia improved in all cases. All cases exhibited a reduction in cellular infiltration after the first week. All eyes responded positively to the therapy, with a significant reduction of the total lesion score. For illustrative purposes, figure 3 shows two cases (left and right pictures) before and after (top and lower pictures) the invention treatment. The positive effects observed by the administration of the invention treatment is explained by the potent immunomodulatory effects of extracellular vesicles. Despite the small sample size, these findings provide valuable insights into the potential benefits of the invention composition for managing IMMK. Table 2: Severity scoring systemComfort(blepharospasm,lacrimation)0Normal. No blepharospasm, no lacrimation 1Eyelashes downwards, no lacrimation 2Partially closed eye, no lacrimation 3Totally closed eye, lacrimation Conjunctival hyperemia0Bulbar conjunctiva is normal. Small, pale pink vessels may be observed, primarily at, or adjacent to, the limbus 1Pink-to-red bulbar conjunctival vessels with minimal branching are visible extending 1-3 mm posteriorly from the limbus toward the conjunctival fornix 2Prominent red bulbar conjunctival vessels with multiple branches are visible extending from the limbus to the conjunctival fornix 3Red-to-dark red, engorged bulbar conjunctival vessels with extensive branching and / or tortuosity are visible extending from the limbus to the conjunctival fornix. The conjunctiva between large vessels may have a flushed pink-to-red appearance. Corneal opacity severity(cellular infiltration / fibrosis)0Normal cornea. Appears with the slit lamp as having a bright gray line on the epithelial surface and a bright gray line on the endothelial surface with a marble-like gray appearance of the stroma 1Minimal loss of corneal transparency. With diffuse illumination, the underlying anterior segment structures are clearly visible, although corneal opacity is apparent to an experienced observer 2Mild loss of corneal transparency. With diffuse illumination, the underlying anterior segment structures are visible, although there is a reduction in the ability to appreciate their detail. 3Moderate loss of corneal transparency. With diffuse illumination, there is a greater inability to see the details of the underlying anterior segment structures than with a score of 2, but the observer is still able to score aqueous flare, iris vessel congestion, observe for pupillary response, and note particular changes. 4Severe loss of corneal transparency. With diffuse illumination, the underlying anterior segment structures cannot be seen so that the evaluation of aqueous flare, iris vessel congestion, pupillary response, and lenticular changes is not possible. Corneal vascularization0Normal 1Ghost or inactive blood vessels 2Localized, active blood vessels 3Diffuse, active blood vessels Calcific degeneration0Normal cornea with no area of corneal calcification 1Rare, focal mineralization 2Diffuse mineralization 3Dense plaque of mineralization Table 3: Test ResultsHistory of the cases:EX1: first attack in November 2022. Received cyclosporine-dexamethasone during 2 months. Good response. Treatment stopped in May 2023. Recurrence in September 2023.EX2: Affected during 2 years. Received terramycin.EX3: Diagnosis April 2019. Received Metacam cyclosporine BID dexamethasone BID. Resolved July 2019. Put on cyclosporine BID. More problems until approx. March 2023: Recurrence despite cyclosporine. Received terracortril BID 1 week early September with no effect.EX4: Lesion for 3 months with no response to antibiotic treatmentEX5: Doiagnosed in 2009. Frequent recurrences in winter, controlled in summer. In case of crisis: dexamethasone and finadyne, then cyclosporine. Keratitis remained under control until January 2023 when a new attack was detected and treatment with dexamethasone, diclofenac, Trafloxal and tacrolimus was initiated. Recurrence on gradual cessation of treatment. ExSex Age Eye dateBefore treatmtAfter treatmtEvolution 1mare13rightOct 7, 202363Good after 1 month – slight inflammation persists2mare9leftOct 17, 202373Good after 2 weeks – moderate inflammation persists3gelding16rightSept 30, 202361Very good (inflammatory component almost resolved)4mare7rightOct 29, 202374Weak after 1 month (slight reduction in swelling) increased application to 4 x daily5stallion27leftJune15, 2023104Excellent – scar tissue only. No recurrence since discontinuation of treatment Median score6.53 Example 7: Pharmaceutical gel compositionEquine platelet lysate has been sourced from a GMP plasmapheresis (COM.tec from Fresenius). The initial platelet concentration was of 8 x 1011 platelets in a 800 ml batch. The batch has been divided into 5 ml platelet lysate, thus 1 x 109 platelet / ml, in 50 ml falcons. An invention composition was prepared as per examples 1 and 2 above. At the end of the process, a cell-free supernatant containing extracellular vesicles (EVs) and production cell-originating immunomodulatory proteins is obtained in a Ringer lactate solution. The Ringer lactate had an equivalent concentration of 100000 cells / ml with a protein concentration of 0,1 to 20 µg / ml, preferably from 1 to 10 µg / ml.The platelet lysate and the EV containing composition were combined. Gel formation has been observed after approx. 90 seconds. The gel obtained thus comprised platelets which may serve as growth factors and EVs combined with immunomodulatory proteins. The gel has been applied on external as well as internal wounds and has shown to accelerate the wound healing process. Example 8: Effect on myeloperoxidase (MPO) activity of EV composition in comparison to the composition obtained at the end of Example 2The same protocol as used in Examples 1 and 2 was followed. The supernatant obtained after centrifugation at 2000 g at the end of Example 2 was further centrifuged during 30 minutes at 10000 g. The obtained pellet containing cell debris was discarded and the supernatant is recovered for an ultracentrifugation during 60 minutes at 100000 g. The exosome-free supernatant (exo-free) is recovered and its volume is determined. The exosome-containing pellet is resuspended in Ringer lactate at a volume equivalent to the one determined for the supernatant. The effect on myeloperoxidase (MPO) activity of the exosome-free supernatant and of the exosomes resuspended in Ringer lactate are determined in a SIEFED test, in comparison to a 100% control (maximum activity: MPO-containing Ringer lactate) and to a negative control (just Ringer lactate with no MPO added) – see the results shown in Figure 5. For comparison purposes, Figure 4 shows the effect of the supernatant obtained according to the invention method, after a 2000 g centrifugation, in a SIEFED test. As can be seen from a comparison of both figures, the inhibitory effect of the invention EV composition is far stronger than that of exosomes resuspended in Ringer lactate after ultracentrifugation. Example 9: Determination of miRNA profile in compositions of the invention MiRNAs play a crucial role in post-transcriptional regulation of gene expression. These molecules are abundantly present in MSC-derived EVs, particularly in small extracellular vesicles (s-EVs) with diameters ranging from 40 to 120 nm. The miRNA cargo of MSC-derived EVs is representative of the parental cell status and is specific to the cell type and cellular state. Research has shown that MSC-derived EVs can transfer miRNAs to target cells, modulating various physiological processes including cell proliferation, differentiation, and immune responses. The protocols of Examples 1 and 2 were followed to prepare invention EV compositions based on non-activated (non-modified) mesenchymal stem cells originating from 5 horses. Total RNA was then extracted using the miRNeasy Serum / Plasma Kit (Qiagen, ref. 217184) on a QIAcube automated workstation (QIAGEN) to ensure reproducibility.RNA concentration and purity were assessed using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific), with a minimum required input of 100 ng per sample. RNA integrity was evaluated using the Agilent 2200 TapeStation with RNA ScreenTapes, ensuring a RINe score > 6 for all samples.Thereafter, miRNA libraries were prepared using the miRNA-seq Library Prep Kit from Illumina, optimized for low-input total RNA. Sequencing was performed on an Illumina platform with a single-end 1×75 bp read configuration.miRNA sequences were identified based on annotated reference files (gtf) from the Ensembl database for human and horse genomes. Expression profiles were analyzed using miRBase Targets (https: / / www.mirbase.org / ) to predict potential miRNA targets.For product characterization, differentially expressed miRNAs were identified based on their relative abundance across samples. The most overexpressed miRNAs were selected as candidates of interest for further analysis and validation.Following the above methods, more than 200 miRNAs were identified. The relevant detection levels were determined and the mean as well as the standard deviation (SD) were calculated. A summary of the data is shown below:miRNA Mean (Expression) Standard Deviationlet-7-b 2,427,328 761,298 mir-151-a 607,409 195,844 let-7-G 559,755 259,872 let-7-e 425,816 203,517 let-7-d 409,077 147,422 mir-21 403,747 298,301 mir-143 338,880 134,189 mir-320 245,862 90,183 mir-26-b 180,188 107,065 let-7-c 164,462 87,189 mir-93 163,203 98,314 mir-29-a 126,514 106,682 mir-34a 123,307 112,586 mir-31 122,995 57,801 mir-30-d 115,926 56,529 The miRNAs 1-7 may be classified as “very high expression” with a mean > 250,000;The miRNAs 8-15 may be classified as “high expression” with a mean > 100,000; andThe let-7 microRNA family may serve as interesting biomarkers; their expression is at high level and shows low variability across samples. Their expression level is higher than 100,000, preferably higher than 150,000; with expression levels higher than 400,000 for the four first ones (let-7-d,e,g,b).
Claims
1. A process for the preparation of a extracellular vesicle comprising composition, the process comprising the steps of:(i) growing stem cells in a culture medium, thus generating a cell population; (ii) rinsing the cells obtained in an isotonic electrolyte solution;(iii) separating the cells from the medium used in step (ii) by way of a gentle mechanical stimulation, and retaining the medium, the separated cells being discarded; (iv) possibly separating remaining undesirable components, cells and cell debris from the medium of interest retained at step (iii) by way of a centrifugation at 1000 – 3000 g during a time period of 1 to 30 minutes. 2. The process of claim 1 wherein the stem cells obtained in step (i) are dissociated from their support and possibly separated from their medium used in step (i). 3. The process of claim 1 or 2 wherein the stem cells originate from mammalian tissue, such as umbilical cord, umbilical cord blood, placenta, Wharton’s jelly, peripheral blood, adipose tissue, bone marrow, dental pulp, skeletal muscle tissue or other tissues. 4. The process of claim 1 or 2 wherein the stem cells are modified stem cells which are modified by interaction with selected molecules or genetically modified stem cells. 5. The process of any of claims 1 to 4 wherein the culture medium for the stem cells consists in a xenofree base medium or a medium comprising FBS and / or platelet lysate, depleted of contaminants. 6. The process of claim 5 wherein the culture medium for the stem cells comprises DMEM-F12, more preferably supplemented with 10% platelet enriched plasma and Heparin. 7. The process of any of claims 1 to 6 wherein the stem cells are grown to near confluence, preferably to near 80 % confluency. 8. The process of any of claims 2 – 7 wherein the dissociation of the stem cells from their growing support is carried out by addition of a dissociation composition selected from dissociation compositions comprising nattokinase, EDTA, Trypsin, a Trypsin derivative, a Trypsin equivalent agent, a serine-based derivative showing trypsin-like enzymatic activity or a synthetic derivative showing trypsin-like activity. 9. The process of claim 8 wherein the dissociation of the cells from their support is carried out in the presence of synthetic Trypsin variants, such as TrypLe. 10. The process of any of claims 2 – 9 wherein the dissociation action of the dissociation composition is neutralized by admixture into the cell containing medium of PBS and / or of an isotonic electrolyte solution. 11. The process of claim 2 wherein the stem cells dissociated from their support are separated from the growing medium and this separation step consists in a centrifugation at 100 - 400 g, preferably at 150 – 350 g, more preferably at 200 – 300 g, during a time period of 1 – 30 min, preferably 5 – 20 min or 5 – 15 min. 12. The process of any of claims 1 – 11 wherein step (iii) consisting in subjecting the cell containing isotonic electrolyte solution to a gentle mechanical stimulation, consists in a gentle centrifugation. 13. The process of any of claims 1 – 12 wherein step (iv) consists in a centrifugation at 1500 to 2500 g, preferably at 1800 to 2200 g, during a time period of 5 to 20 minutes or of 5 to 15 minutes, preferably of approx. 10 minutes. 14. A composition comprising EVs originating from production stem cells and showing particle sizes comprised between 100 and 250 nm, and immunomodulatory production cell-originating proteins in isotonic solution. 15. The composition of claim 14 wherein the EVs are present in a concentration of from 1 x 107 to 10 x 109 particles / ml, particularly from 1 x 108 to 1 x 109, more particularly from 0,10 x 109 to 1 x 109 particles / ml. 16. The composition of any of claims 14 or 15 characterised by the expression of one or more of the miRNAs selected from the group (i) let-7-b, mir-151-a, let-7-G, let-7-e, let-7-d, mir-21, mir-143, mir-320, mir-26-b, let-7-c, mir-93, mir-29-a, mir-34-a, mir-31, mir-30-d, each preferably at a level higher than 100,000; or(ii) let-7-b, mir-151-a, let-7-G, let-7-e, let-7-d, mir-21, mir-143, each preferably at a level higher than 250,000, more preferably higher than 300,000; or(iii) let-7-b, let-7-G, let-7-e, let-7-d, let-7-c, each preferably at a level higher than 100,000, more preferably higher than 150,000; or(iv) let-7-b, let-7-G, let-7-e, let-7-d, each preferably at a level higher than 400,000. 17. A pharmaceutical composition comprising EVs obtained from stem cells, showing particle sizes comprised between 100 and 250 nm, and immunomodulatory production cell-originating proteins, in isotonic solution, possibly further comprising a pharmaceutically acceptable carrier or extender or an additional pharmaceutically active ingredient, for use in treating one or more of the disorders within the systems composing the mammalian body, such as the circulatory system, the digestive system, the immune system, the integumentary system, the musculoskeletal system, the nervous system, the reproductive system, the respiratory system and the urinary system, more specifically one or more disorders of several organs, such as liver, lung, heart, uterus, brain, eye, skin, bone, tendon and cartilage, in mammalian subjects. 18. The composition of any of claims 16 – 17, which is a gel composition and comprises a composition according to any of claims 14 – 15 or a composition obtained as per the process of any of claims 1 - 13 and platelet lysate. 19. Method of treatment of one or more of the disorders within the systems composing the mammalian body, such as the circulatory system, the digestive system, the immune system, the integumentary system, the musculoskeletal system, the nervous system, the reproductive system, the respiratory system and the urinary system, more specifically one or more disorders of several organs, such as liver, lung, heart, uterus, brain, eye, skin, bone, tendon and cartilage, in mammalian subjects, the relevant method of treatment comprising the administering to the relevant mammalian subject, of a therapeutically effective amount of a composition according to any of claims 14 - 18.