Process for obtaining extracellular vesicles, extracellular vesicles, grafting unit, method for therapeutic treatment and uses of extracellular vesicles

Abstract

The present invention refers to a process for obtaining extracellular vesicles (EVs) isolated from the culture supernatant of human adipose-derived mesenchymal stromal cells (hASCs), comprising: (a) obtaining hASCs from adipose tissue, preferably abdominal; (b) culturing and expanding hASCs; and (c) isolating EVs from the culture supernatant by filtration, followed by polymer precipitation. The present invention also refers to isolated extracellular vesicles, a grafting unit, a therapeutic treatment method and to the uses of the extracellular vesicles.

Description

PROCESS FOR OBTAINING EXTRACELLULAR VESICLES, EXTRACELLULAR VESICLES, GRAFTING UNIT, METHOD FOR THERAPEUTIC TREATMENT AND USES OF EXTRACELLULAR VESICLESRelated Applications

[0001] The present patent application claims the benefit of internal priority to the previous patent application BR 102025002168-4, filed on February 02, 2025, which content is fully incorporated herein.Field of Invention

[0002] The present invention, which is in the field of biotechnology, particularly human bioengineering, refers to processes for obtaining extracellular vesicles (EVs) isolated from the culture supernatant of human adipose-derived mesenchimal stem / stromal cells (hASCs), useful for therapeutic purposes, such as grafting orthopedic and / or dental. The present invention also refers to isolated extracellular vesicles, to a grafting unit, to a therapeutic treatment method and to uses of the extracellular vesicles.Background of the Invention

[0003] Bone grafing, in both dental and orthopedics, is a procedure which aims to restore bone structure which was lost or damaged by diseases, trauma or the use of prosthesis. In this market, it is relevant to identify the challenges and limitations involving the choice of the suitable kind of material and bone grafting technique for the treatment of each individual case. A few of the factors influencing this decision include: availability, cost, biocompatibility, rejection risk, integration capability and bone regeneration, as well as the local morbidity of the donor.

[0004] With the object of developing scar response improvements from surgical reconstructions involving bone grafting, the state of the art is trying to optimize, by means of various studies, the therapeutic response of the patients.

[0005] The first works with cellular cultures of lab-grown stem cells, aiming for its applicability for bone grafts to repair jaw fractures and / or resorption sequelae, highlighted significant problems related to biological safety, with exacerbated tissue inflammatory responses and oncogenic potential. To minimize these risks, studies have suggested a novel acellular approach, employing only soluble factors and / or microvesicles produced by cells. Results from the literature have indicated benefits in the use of an exosome signaling product from stem cells in local bone repair processes, both in terms of maturation speed as well as quality (density) of the newly formed bone tissue, providing an improvement in the qualitative response and time reduction for bone consolidation, generating relevant socioeconomic benefits to the healing process of patients.

[0006] This is because the exosomes carry specific proteins, chemokines, growth factors, mRNAs and microRNAs, among others, with anti-inflammatory, cell differentiation-inducing, regenerative and immunomodulatory functions, playing a fundamental role for intercellular signaling by way of paracrine mechanisms. They act decisively for the uptake / recruitment processes of local cells responsible for the tissue formation and repair processes, thus inducing their proliferation, differentiation, and consequent protein synthesis.

[0007] Thus, it is reported that the role played by exosomes in bone defect repair is multifaceted, including osteogenesis, angiogenesis and inflammation regulation. Specifically, in the bone defect microenvironment, they promote target cell differentiation, angiogenesis promotion so to provide an ideal bone regeneration niche, and inflammation modulation to maintain a moderate immune response.

[0008] Particularly, in bone regeneration, the exosomes derived from mesenchymal stromal cells (MSCs) play a relevant role. MSCs are a heterogeneous population of cells similar to fibroblasts which can be isolated from many human tissues, including, but not limited to, bone marrow, adipose tissue, skeletal muscle, heart, umbilical cord and placenta. MSCs have called the attention of scientists and clinicians due to their differential potential and active participation in tissue repair and regeneration after migrating to the site of tissue injury. When stimulated by suitable signals, MSCs are capable of differentiating into various types of specialized cells, such as adipocytes, osteoblasts and chondrocytes, for example. However, although being reasonably accepted forin vivoallogeneic transplantation, exosomes derived from them have been reported to similarly lead to benefits, without the potential disadvantages.

[0009] MSCs can promote the increase of factors and protein expression related to osteogenesis, such as RUNX2, COL1A1, OPN and ALP. RNA sequencing of exosomes derived from human MSCs suggests that the exosomes include either positively regulated osteogenic miRNAs (Hsa-miR-146a-5p, Hsa-miR503-5p, Hsa-miR-483-3p and Hsa-miR-129-5p) or negatively regulated anti-osteogenic miRNAs (Hsa-miR-32-5p, Hsa-miR-133a-3p and Hsa-miR-204-5p), which activate PI3K / Akt and MAPK signaling pathways. Exosomal miR-1260a released by MSCs, miR-335, miR-140 and miR-375, miR-26a, miR-199a, miR-21 and miR-23a-3p, let-7a-5p, let-7c-5p, miR-328a-5p, miR-31a-5p and miR-150 was also reported in the literature as an osteogenesis promoter. The exosomes promoting osteogenesis involve many pathways, such as BMP / Smad, Wnt / β-catenin signaling and PTEN / PI3K / Akt.

[0010] The adequate blood supply is an important basis for the success of bone regeneration. The effects of exosome-mediated angiogenesis are indicated by the increase of expression of angiogenesis factors, tube formation and others. Mechanistic studies have revealed that exosomal miR-21 not only regulates SPRY2, but also promotes angiogenesis through the miR-21 / NOTCH1 / DLL4 signaling axis. Exosomal miR-1260a derived from MSC also increases angiogenesis by inhibiting COL4A2, and exosomes increase endothelial cell viability and VEGF and ANG-1 expression as well.

[0011] Inflammatory and immune cells are important constituents of the bone defect microenvironment, and a moderate inflammatory response is imperative for repairing bone defects. Therefore, exosomes derived from MSCs play a role in modulating inflammation, promoting the activation and differentiation of macrophages in an M2 profile via the NFκB pathway, and inhibiting the inflammatory response, reducing protein expression of inflammatory cytokines, such as IL-6 and TNF-α.

[0012] Even with the increasing knowledge about the mechanisms by which exosomes contribute to bone regeneration, their usage in this context is naturally a challenge. In part, this is because there are many variants involved in the processes of obtaining said exosomes, and it is not always possible to guarantee the immunomodulation of interest in a given case. Adding another degree of complexity to this technical field, it should be noted that, while some studies apply exosomes directly to the affected site, others are focused on incorporating them into specific materials for this purpose, both aimed to replace and reconstruct bone tissue, for the functional rehabilitation of the individual. Thus, there are many possible therapies that can effectively contribute to clinical improvement, although more optimized and effective processes are still needed.

[0013] In this context, there is room for improvement regarding reconstructive surgical techniques involving bone grafts, which are often essential to achieve good results, but with limitations in terms of the obtained results. Several types of bone substitute materials are currently available for use in these surgeries, mainly acting as structural means for the establishment of local osteo-regenerative processes.

[0014] However, as the research in the tissue engineering field advances, many progresses and results leading to improved clinical outcomes obtained in conventional bone repair surgeries have emerged, with studies suggesting that the association of techniques combining bone substitutes with tissue engineering products, such as growth factors, is very promising to significantly improve these surgical results as obtained, considering bone healing and reconstruction.

[0015] However, despite the many known and used processes in the state of the art for obtaining extracellular vesicles and / or exosomes from mesenchymal stromal cells, many of these processes are not applicable to human treatment, despite beneficial effects have been reported inin vitroand animal models. Similarly, commercial products considered as effective for exosome isolation are limited to scientific research and also expensive. WO 2018 / 130554, for example, aims to provide an improved treatment for bone defects. To this end, it disclosed a method for obtaining extracellular vesicles derived from mesenchymal stromal cells (MSCs), wherein the extracellular vesicles are essentially free of non-MSC-derived vesicles and heparin. MSCs are derived from bone marrow or umbilical cord (preferably umbilical cord), and the method comprises, in summary, maintaining the cells in heparin-free medium until they reach 70% confluence; washing the cells with PBS and changing the medium to a fresh, heparin-free medium depleted from fibrinogen and extracellular vesicles; harvesting the conditioned medium after 48 hours; filtering the medium to remove cellular debris and large vesicles, followed by centrifugation at 30,000 x g for 20 minutes to sediment larger microvesicles; and submitting supernatants to centrifugation at 120,000 x g for 3 hours to sediment the extracellular vesicles of interest. Therapeutic tests were performed in a murine model.

[0016] CN 110403959 aims to provide mesenchymal stem cell exosome preparations and their application for the prevention and control of osteoporosis. To this end, in a preferred embodiment, it started from bone marrow-derived mesenchymal stem cells, which were expanded in exosome-free medium for 3 days. The medium was collected and centrifuged to remove cellular debris from the culture medium. The supernatant was collected and submitted to a series of ultracentrifugations to obtain the exosomes. In the fourth centrifugation, the precipitate was collected, resuspended in PBS and centrifuged again, the precipitate being the isolated exosomes. Tests were performed in a murine model.

[0017] WO 2022 / 008657 aims to provide extracellular vesicles produced in a reproducible and controlled manner, in accordance with “Good Manufacturing Practice” (GMP) for clinical application. To this end, it discloses a general method comprising culturing and expanding mesenchymal stromal cells (MSCs) in a cellular medium, preferably serum-free and xeno-free; collecting the cell supernatant, said cell supernatant comprising extracellular vesicles (EVs); filtering said cell supernatant to obtain EVs; and concentrating said EVs, preferably by ultrafiltration. The preferred source of MSCs is the umbilical cord. Therapeutic tests were performed in murine and human clinical models. EVs could be useful for treating various diseases, with pulmonary and intestinal inflammatory conditions evaluated.

[0018] WO 2022 / 008652 similarly aims to provide extracellular vesicles produced in a reproducible and controlled manner, in accordance with “Good Manufacturing Practice” (GMP) for clinical application. To this end, it discloses a general method comprising culturing and expanding mesenchymal stromal cells (MSCs) in a cellular medium, preferably serum-free and xeno-free, wherein the medium comprises purified human serum albumin and human transferrin; collecting the cell supernatant, said cell supernatant comprising extracellular vesicles (EVs); filtrating said cell supernatant to obtain EVs; and concentrating said EVs, preferably by means of ultrafiltration. The source of MSCs is preferably the umbilical cord. Therapeutic tests were performed in murine and human clinical models. EVs could be useful for the treatment of various diseases, with pulmonary and intestinal inflammatory conditions evaluated.

[0019] WO 2021 / 067421 aims to provide alternative methods for producing clinically relevant quantities of mesenchymal stem cells (MSCs) that do not involve fetal bovine serum or other animal-derived means, supplements or constituents, wherein the MSCs produced can be used more safely to treat appropriate diseases and disorders in humans and other animals. To that end, they disclose a method for preparing MSCs expressing CD10 at a level equal to or higher than 70%, which comprises, in summary, filtering conditioned means from IFP (synovium and infrapatellar fat)-MSC groups cultured for two days in exosome-depleted means to remove debris and large vesicles. Said means were then collected, differentially centrifuged and ultracentrifuged using a sucrose gradient to increase purity. Tests were performed in a murine model for acute synovitis and IFP fibrosis, which would contribute to the pathogenesis and progression of osteoarthritis (OA).

[0020] It should be noted that, despite the above documents refer to one or more aspects of the present invention, they disclose different processes from the present invention in one or more relevant characteristics, which an expert in the art would not be led to disregard, in order to obtain the present process and auxiliary inventions, nor expect, with any reasonable expectation of success, that they would still lead to the obtained results. Furthermore, they present disadvantages, which are minimized and / or solved by the present invention.

[0021] In this context, there is still need in the state of the art to improve the process of obtaining extracellular vesicles, such as exosomes, for human clinical purpose as proposed by the present invention, preferably for orthopedic and / or dental grafting treatment. Therefore, the process of the present invention was disclosed, being liable for use in human clinical practice, and also optimized in relation to the processes used in the state of the art in similar contexts. Therefore, the particular combination of steps of the process as disclosed, unexpectedly, allows to obtain extracellular vesicles compatible with vesicles reached through conventional protocols and / or commercial kits, leading to results as good or betterin vitro, and inin vivobone grafting therapies, with advantages over the commercial and state-of-the-art protocols.

[0022] In particular, conventional MSC cell cultures make use of fetal bovine serum, which, despite being a commonly used supplement for cell development, contributing to an increase in the final protein yield, is also a risk factor for exposure to animal-derived pathogens. In this context, for clinical application, it is necessary to develop protocols that do not use said constituent. Serum-free / xeno-free means do not always successfully lead to the isolation of extracellular vesicles, such as exosomes. Alternative serum-free means, in general, still contain poorly defined factors, which clinical application may be risky. Currently, there are also commercially available xeno-free means. However, the state-of-the-art protocols for the purposes of the present invention comprise disadvantageous steps, particularly with regard to the methods of obtaining MSCs, and / or obtaining exosomes, not being easy, fast and / or effective, including for clinical application.

[0023] In one example, ultracentrifugation is considered the gold standard in obtaining exosomes, as it achieves great purity, a desirable characteristic for clinical application, even though their recovery rate is considered low. Therefore, we again highlight the need to disclose a process to meet the demands as required for clinical application, while ideally overcoming the known disadvantages in the state of the art concerning methods to cultivate and obtain extracellular vesicles. Furthermore, considering the technical challenges involved in translating results obtainedin vitrotoin vivoconditions, the demonstration ofin vivotherapeutic efficacy is particularly desirable.Brief Description of the Invention

[0024] Therefore, it is an object of the present invention to disclose a process for obtaining extracellular vesicles (EVs) isolated from the culture supernatant of human adipose mesenchymal stromal cells (hASCs), comprising:

[0025] a. obtaining hASCs from adipose tissue, preferably abdominal;

[0026] b. culturing and expanding hASCs; and

[0027] c. isolating EVs from the culture supernatant by filtration, followed by polymer precipitation.

[0028] In a preferred embodiment of the process of the present invention, the process is intended for orthopedic treatment and / or dental therapeutic grafting.

[0029] In another preferred embodiment of the process of the present invention, the process is xeno-free.

[0030] In another preferred embodiment of the process of the present invention, step (a) of obtaining hASCs from adipose tissue, preferably abdominal tissue, comprises:

[0031] - obtaining the lipoaspirate;

[0032] - concentrating the lipoaspirate;

[0033] - digesting the concentrated lipoaspirate in a digestion solution;

[0034] - incubating under agitation;

[0035] - concentrating to obtain a precipitate and resuspending the precipitate in culture medium;

[0036] - plating and incubating until about 80 to about 90% confluence, with medium change every 48 h; and

[0037] - dissociating hASCs.

[0038] In another preferred embodiment of the process of the present invention, step (b) of culturing and expanding the hASCs comprises:

[0039] - seeding hASCs as obtained in step (a) and culturing them up to the 4th to 7th passage, preferably to the 5th to 6th passage, with medium change every 48 h.

[0040] In another preferred embodiment of the process of the present invention, in step (c), filtration is made through a 0.22 µm filter, and polymer precipitation is performed by means of 12% polyethylene glycol (PEG).

[0041] In another preferred embodiment of the process of the present invention, polymer precipitation by means of 12% polyethylene glycol (PEG) comprises:

[0042] - incubating the filtrate with 12% PEG, preferably overnight; and

[0043] - centrifuging it, to obtain the EV precipitate, wherein the centrifugation is preferably performed at about 1500 g to about 4500 g, more preferably 3000 g, for about 5 min to about 30 min, more preferably for about 10 min to about 25 min, even more preferably for about 15 min.

[0044] In another preferred embodiment of the process of the present invention, between filtration and polymer precipitation, the filtrate is centrifuged to remove the precipitate from debris, dead cells and / or apoptotic bodies.

[0045] In another preferred embodiment of the process of the present invention, after centrifugation to obtain the EV precipitate, the precipitate is resuspended, preferably in buffered saline solution, more preferably in 1X phosphate-buffered saline (PBS) solution, and the resuspended precipitate is centrifuged to obtain the washed EV precipitate for use, which may still be preferably diluted, wherein the dilution is preferably 1:10, preferably in buffered saline solution, more preferably in 1X phosphate-buffered saline (PBS) solution.

[0046] A second object of the present invention refers to isolated extracellular vesicles obtained by the process as defined herein.

[0047] A third object of the present invention refers to isolated extracellular vesicles obtained by the process as defined herein, wherein the vesicles are predominantly nanovesicles.

[0048] In a preferred embodiment of the vesicles of the present invention, the vesicles are between about 30 nm and about 1000 nm in measurement, preferably between about 30 nm and about 800 nm, more preferably between about 30 nm and about 600 nm, even more preferably between about 30 nm and about 400 nm, most preferably between 30 nm and about 200 nm.

[0049] In another preferred embodiment of the vesicles of the present invention, the vesicles express one or more markers from the group consisting of CD9, CD45, CD63, CD81, CD82, Alix, calnexin, EpCAM, and Rab5.

[0050] A fourth object of the present invention refers to a grafting unit comprising isolated extracellular vesicles, as defined herein, or obtained by the process as defined herein, and a support.

[0051] In a preferred embodiment of the unit of the present invention, the support comprises bone materials and / or bone substitutes, preferably synthetic bone substitutes, such as a combination of hydroxyapatite and beta-tricalcium phosphate, more preferably 60% hydroxyapatite and 40% beta-tricalcium phosphate (HP / β-TCP).

[0052] A fifth object of the present invention refers to a therapeutic treatment method comprising applying isolated extracellular vesicles, as defined herein, or obtained by the process as defined herein, or applying the grafting unit, as defined herein, to a patient in need.

[0053] In a preferred embodiment of the method of the present invention, the therapeutic treatment is orthopedic and / or dental grafting.

[0054] A sixth object of the present invention refers to the use of isolated extracellular vesicles, as defined herein, or obtained by the process as defined herein, for the manufacture of a product for the therapeutic treatment of a patient in need.

[0055] In a preferred embodiment of the use of the present invention, the product is a grafting unit or an EV concentrate.

[0056] A seventh object of the present invention refers to the use of isolated extracellular vesicles, as defined herein, or obtained by the process as defined herein, or of the grafting unit, as defined herein, for the therapeutic treatment of a patient in need.

[0057] In a preferred embodiment of the use of the present invention, the therapeutic treatment is orthopedic treatment and / or dental grafting.

[0058] An eighth object of the present invention refers to isolated extracellular vesicles, as defined herein, or obtained by the process as defined herein, or a grafting unit, as defined herein, for use in the therapeutic treatment of a patient in need.

[0059] In a preferred embodiment of the vesicles of the present invention, the therapeutic treatment is orthopedic treatment and / or dental grafting.Brief Description of the Drawings

[0060] The present invention can also be understood based on the details as presented by the Figures.

[0061] represents the phase-contrast microscopy photomicrograph of hADSCs, in the fourth passage, obtained in both full culture medium (CM) and xeno-free medium (KSR), at 20x magnification.

[0062] shows the growth curve graph (passage X number of cells) according to the culture medium as tested (CM and KSR);shows the cell doubling time graph in exponential phase (passage X PDT (hour)) in the same medium; andshows the hADSC viability graph (passage X viability (%)), with the same medium. There was no statistical difference between the different means (p < 0.05).

[0063] shows the exosome concentration graph by particle size for CM and KSR groups when submitted to protocols 1 and 2 (n=6). The data are average values ± standard error.

[0064] represents the transmission electron micrograph (150k) showing exosomal structures derived from hADSC-KSR, using isolation protocol 1. The arrow shows vesicles in suspension with cylindrical morphology and electron-dense membrane. Scale: 200 nm.

[0065] illustrates the graph detailing the exosomal yield obtained by protocols 1 and 2 in each medium as used (EXO-KSR and EXO-CM). The ANOVA method was used for statistical analysis. There was statistical difference between the different means (p< 0.05). The data are average values ± standard error.

[0066] illustrates radiographic photograph clippings obtained by means of a Western Blot Assay performed with the exosome fraction obtained after hADSC expansion in CM and KSR culture means and protocols 1 and 2. Total proteins were obtained from the reaction of anti-CD63 granules.

[0067] shows exosome biodistribution from murine adipose mesenchymal stromal cells (EXO-mASCs) stained with PKH26 in different tissues, evaluated 1 hour after grafting, at a dose of 6 × 109exosomal particles. The fluorescent signal indicates the location of the exosomes in the analyzed tissues, with the observed distribution pattern being representative and consistent with that identified for the other doses evaluated.

[0068] shows micro-computed tomography (micro-CT) images of the regenerated bone defects, obtained 15 days after treatment, showing the filling of the defects with different formulations and different numbers of exosomal particles associated with the biomaterial.

[0069] shows the graph representing the average and standard deviation for the percentage volume of bone tissue (VBT%) formed within the femoral defects, evaluated 15 days after treatment, as a function of the dose of exosomal particles and the type of treatment applied.Detailed Description of the Invention

[0070] Throughout the present document, unless indicated otherwise, the limits of a range of values are included within that range. Furthermore, the value ranges include all numbers and fractions encompassed within the respective ranges.

[0071] Throughout the present document, the singular forms “a”, “an” and “the” include both the singular and the corresponding plurals, and vice-versa, unless the context clearly dictates otherwise.

[0072] Throughout the present document, the expression “at least one” is equivalent to the expression “one or more”, and means one or more members, or at least one member of a group of members. These terms include any one of ≥ 1, ≥ 2, ≥ 3, ≥ 4, ≥ 5, ≥ 6, ≥ 7 etc. of said members, and up to all said members.

[0073] Throughout the present document, the terms “about” or “approximately”, when referring to a measurable value such as a parameter, a quantity, a temporary duration, and the like, mean variations of the specified value, such as variations of + / -10% or less, preferably + / -5% or less, more preferably + / -1% or less, and even more preferably + / -0.1% or less, to the extent that such variations are reasonably appropriate for each applicable parameter of the invention. It is understood that the value to which the modifier “about” refers to is also specifically, and preferably, disclosed.

[0074] Throughout the present document, words and expressions such as “preferably”, “particularly”, “for example”, “such as”, “like”, “more particularly”, “more preferably”, and the like, as well as their variations, should be interpreted as entirely optional characteristics, preferred embodiments, or possible non-exhaustive examples, without conferring a limiting scope to the present document.

[0075] Throughout the present document, the word “comprises”, and any variations such as “comprise” or “comprising”, should be interpreted as “open terms”, which may imply the inclusion of additional elements or groups of elements that have not been explicitly disclosed, and are not limiting in nature.

[0076] Throughout the present document, the word “consists”, and any variations such as “consist” or “consisting”, should be interpreted as “closed terms”, which may not imply the inclusion of additional elements or groups of elements that have not been explicitly disclosed, and are limiting in nature.

[0077] Unless explicitly indicated otherwise, all acronyms, expressions and / or technical terms should be interpreted as having the meanings as generally used and widely known in the technical field of the present invention. In some cases, terms with commonly understood meanings are defined herein for the purpose of clarity and / or for quick reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from what is generally understood in the state of the art.

[0078] Unless explicitly stated otherwise, the techniques and procedures disclosed or referred to herein are generally well understood and employed using conventional methodology, based on the available literature and the knowledge of an expert in the art. Such techniques are explained and disclosed in the literature, e. g. by Welsh, J. A., Goberdhan, D. C. I., O’Driscoll, L., Buzas, E. I., Blenkiron, C., Bussolati, B., Cai, H., Di Vizio, D., Driedonks, T. A. P., Erdbrügger, U., Falcon-Perez, J. M., Fu, Q. L., Hill, A. F., Lenassi, M., Lim, S. K., Mahoney, M. G., Mohanty, S., Möller, A., Nieuwland, R., Ochiya, T., Sahoo, S., Torrecilhas, A. C., Zheng, L., Zijlstra, A., Abuelreich, S., Bagabas, R., Bergese, P., Bridges, E. M., Brucale, M., Burger, D., Carney, R. P., Cocucci, E., Crescitelli, R., Hanser, E., Harris, A. L., Haughey, N. J., Hendrix, A., Ivanov, A. R., Jovanovic-Talisman, T., Kruh-Garcia, N. A., Ku’ulei-Lyn Faustino, V., Kyburz, D., Lässer, C., Lennon, K. M., Lötvall, J., Maddox, A. L., Martens-Uzunova, E. S., Mizenko, R. R., Newman, L. A., Ridolfi, A., Rohde, E., Rojalin, T., Rowland, A., Saftics, A., Sandau, U. S., Saugstad, J. A., Shekari, F., Swift, S., Ter-Ovanesyan, D., Tosar, J. P., Useckaite, Z., Valle, F., Varga, Z., van der Pol, E., van Herwijnen, M. J. C., Wauben, M. H. M., Wehman, A. M., Williams, S., Zendrini, A., Zimmerman, A. J.; MISEV Consortium; Théry, C., Witwer, K. W.,Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches,J.Extracell. Vesicles, 2024 Feb; 13(2): e12404. doi: 10.1002 / jev2.12404. Erratum in:J.Extracell. Vesicles, 2024 May; 13(5): e12451. doi: 10.1002 / jev2.12451. PMID: 38326288; PMCID: PMC10850029, incorporated herein by reference.

[0079] Throughout this document, all headings and subheadings are used for convenience only and should not be construed as limitations of the present invention.

[0080] The present invention, therefore, refers to a process for obtaining extracellular vesicles (EVs) isolated from the culture supernatant of human adipose mesenchymal stromal cells (hASCs), comprising: (a) obtaining hASCs from adipose tissue, preferably abdominal; (b) culturing and expanding hASCs; and (c) isolating the EVs from the culture supernatant by filtration followed by polymer precipitation.

[0081] In the context of the present invention, EVs can be defined as vesicles that are produced (and obtained) from anin vitroculture of hASCs. Therefore, EVs are independent of their size, which may vary depending on the case, the literature considered, etc. According to the process of the present invention, EVs are preferably nanovesicles, but are not limited to them. Nanovesicles of particular relevance in the context of the present invention are exosomes, which are extracellular nanovesicles formed from multivesicular cell bodies (MVCs). These MVCs originate from the late endosome of the cell and merge themselves with the plasma cell membrane, releasing their contents into the organism. Therefore, in the context of the present invention, EVs are preferably between about 30 nm and about 1000 nm, more preferably between about 30 nm and about 801 nm, even more preferably between about 30 nm and about 600 nm, more preferably between about 30 nm and about 400 nm, most preferably between about 30 nm and about 200 nm.

[0082] Furthermore, in the context of the present invention, EVs express one or more CD9, CD45, CD63, CD81, CD82, Alix, calnexin, EpCAM and Rab5 markers, commonly used to classify exosomes.

[0083] Human adipose mesenchymal stromal cells (hASCs), on the other hand, may be defined as a category of human mesenchymal stem cells (MSCs), which can be easily obtained from adipose tissue and have many of the same regenerative capabilities as other MSCs. MSCs are multipotent somatic stem cells derived from the mesoderm, having the capacity for self-regeneration and differentiation to produce progenitor cells with a wide phenotypic variety, including connective tissues, bone marrow stroma, adipocytes, dermis, and muscles, among others. Furthermore, MSCs can be defined as plastic-adherent fibroblast cells with the potential for osteogenic, chondrogenic, and adipogenic differentiation. Furthermore, they express the cell surface markers CD73, CD90, and CD105 and do not express hematopoietic and endothelial antigens (CD14 or CD11b, CD19 or CD79α, CD34, CD45, HLA-DR). In the context of the present invention, any adipose tissue can be used to obtain hASCs. However, abdominal adipose tissue is especially preferred.

[0084] The step (a) mentioned above, of obtaining hASCs from adipose tissue, preferably abdominal tissue, in the context of the present invention, does not include any surgical method or procedure in the human body, that is, it refers to obtaining the tissue after an invasive procedure has been performed. In other words, the process referred to in the present invention is necessarily anin vitroprocess. In any case, the process that precedes obtaining hASCs from adipose, particularly abdominal tissue, which is tissue collection, can be done by a variety of techniques known in the literature, being of no particular importance in the context of the present invention. However, the collection by minimally invasive liposuction (conventional surgical lipoplasty) is especially preferred for obtaining the abdominal lipoaspirate.

[0085] It is subsequently necessary to process this tissue, which, likewise, can be carried out under a variety of conditions. These steps are therefore included in the preferred scope of the process of the present invention. In a preferred embodiment of the process of the present invention, obtaining hASCs from adipose, preferably abdominal tissue, comprises:

[0086] - obtaining the lipoaspirate;

[0087] - concentrating the lipoaspirate;

[0088] - digesting the concentrated lipoaspirate in a digestion solution;

[0089] - incubating under agitation;

[0090] - concentrating to obtain a precipitate and resuspending the precipitate in culture medium;

[0091] - plating and incubating until about 80 to about 90% confluence, changing the medium every 48 h; and

[0092] - dissociating the hASCs.

[0093] Optional preferred conditions of the above paragraph have been disclosed in the “Examples” section of the present document. However, the experts in the art in the field of the present invention know that small variations in these conditions do not significantly change the results.

[0094] As an example, the lipoaspirate concentration is usually carried out by centrifugation, under conditions sufficient to sediment the cells, which may obviously vary. The digestion solution is typically collagenase. However, other constituents and / or solutions could be used for this purpose, such as trypsin and / or trypsin-EDTA and / or EDTA and / or collagenase, elastase, dispase, or a non-enzymatic solution or non-xenogeneic recombinant enzyme. The time and amount of the digestion solution may vary depending on the used solution– and its concentration –,tissue volume, etc., and can be readily adjusted by an expert in the art, based on his / her knowledge and the available literature, if applicable. It is preferable that the incubation of the tissue with the digestion solution be carried out by means of continuous agitation, preferably at a controlled temperature, such as 37 °C, for example, in a water bath at 37 °C, which usually optimizes digestion. After the stipulated incubation period for digestion, it is concentrated, such as by centrifugation, to obtain a precipitate, the supernatant is discarded, and the precipitate is resuspended in culture medium.

[0095] The culture medium, in the context of the present invention, is preferably a xeno-free medium, like the process as a whole, since the process is primarily aimed at human therapy, preferably for human therapeutic orthopedic treatment and / or dental grafting. Therefore, the expression xeno-free, in the context of the present invention, refers to the lack of direct or indirect exposure to non-human animal constituents. The advantages of the xeno-free medium include the lack of potential contaminants and improved consistency in both performance and quality of the culture medium. Therefore, the terminology xeno-free obviously also implies that the medium is free of serum or plasma of non-human animal origin. There is a variety of commercial culture means for clinical therapy purposes, including those specifically designed for MSC culture. In the context of the present invention, the medium used, and therefore preferred, is “StemPro® MSC SFM XenoFree” (Gibco). However, any other equivalent serum-free, xeno-free means, such as those for MSC culture, could be equally used. Finally, we should remember that the medium, depending on the intended use of the particular aliquot of the cell culture in question, such as for experimental purposes, may be additionally supplemented with one or more suitable antibiotics.

[0096] After resuspending the cells in culture medium, as disclosed above, incubate until reaching between about 80 and about 90%, changing the medium every 48 h. Variations in confluence are acceptable. As an example, the appropriate confluence until hASC dissociation may be between about 72 and about 99%, preferably between about 80 and about 90% confluence. Afterwards, cells are dissociated, preferably with trypsin. The culture conditions are normoxic, in a humidified greenhouse at 37 ºC, 5% CO2.

[0097] hASCs dissociated as disclosed above, according to step (b) of the process, are cultured and expanded. Thus, step (b) preferably involves seeding the hASCs obtained in step (a) and culturing them up to the 4thto 7thpassage, preferably up to the 5thto 6thpassage, with medium change every 48 h, under the same culture conditions disclosed above.

[0098] Finally, step (c) is then carried out to isolate EVs from the culture supernatant by filtration, followed by polymer precipitation. Preferably, the supernatant from the hASC culture, as disclosed above, is filtered through a 0.22 µm filter. Optionally, after filtration, the supernatant is centrifuged in order to remove cellular debris, dead cells and apoptotic bodies. After that, polymer precipitation of the supernatant containing EVs is carried out using 12% polyethylene glycol (PEG). Thus, preferably the filtrate, or the supernatant as obtained, is exposed to 12% PEG, preferably overnight (typically, the mixture is incubated at 4 ºC for 16 h), followed by centrifugation, which is preferably between about 1500 g and about 4500 g, more preferably 3000 g, between about 5 min and about 30 minutes, more preferably between about 10 minutes and about 25 minutes, even more preferably for about 15 minutes, at 4 ºC.

[0099] The PEG-EV sediment on the bottom of the tube is preferably resuspended in a buffered saline solution, more preferably in 1X phosphate-buffered saline (PBS), and centrifuged to obtain the EV precipitate washed for use, which may also be preferably diluted, wherein the dilution is preferably 1:10, preferably in buffered saline solution, more preferably in 1X phosphate-buffered saline (PBS).

[0100] Therefore, the extracellular vesicles isolated by the process of the present invention, as disclosed herein, i.e., both before dilution (EV concentrate, which comprises the washed precipitate) and after dilution, are also part of the present invention. Said EVs can have measurements ranging between about 30 nm and about 1000 nm, preferably between about 30 nm and about 800 nm, more preferably between about 30 nm and about 600 nm, even more preferably between about 30 nm and about 400 nm, with the highest preference between about 30 nm and about 200 nm. However, they are mostly nanovesicles, particularly exosomes, with preferred sizes between about 30 nm and about 200 nm, preferably expressing one or more typical exosome markers in this context, such as CD9, CD45, CD63, CD81, CD82, Alix, calnexin, EpCAM and Rab5.

[0101] We should notice that, although EVs as defined in the present document, or obtained by the process of the present invention, already have desirable characteristics for various therapeutic applications, such as, but not limited to, therapeutic orthopedic treatments and / or direct dental grafting, it is advantageous to apply them to a support, giving rise to the grafting unit of the present invention.

[0102] In the context of the present invention, the support may be defined as any framework or matrix, whether or not combined with one or more additional biological carriers, such as collagen and / or hyaluronic acid, applicable to the preferred purposes of the present invention, i. e. orthopedic treatment and / or dental grafting, aiming to assist and / or promote bone reconstitution. In this sense, the support may or may not be inert, and may be osteogenic, osteoinductive and / or osteoconductive. Preferably, the support is synthetic, more preferably comprising bone materials and / or bone substitutes, preferably synthetic bone substitutes, such as a combination of hydroxyapatite and beta-tricalcium phosphate, more preferably 60% hydroxyapatite and 40% beta-tricalcium phosphate (HP / β-TCP). Therefore, the vesicles obtained according to the disclosed process are added to said bone materials or bone substitutes by means of an extemporaneous preparation for topical use, in the orthopedic therapeutic procedure and / or dental grafting.

[0103] The addition of EVs, as proposed by the present invention, to bone materials or bone substitutes as commonly used in bone grafting surgeries in orthopedics / dentistry for the reconstruction of calcified tissues improves the local tissue regenerative capacity in the short and medium term, both in terms of the speed of maturation / incorporation of these biomaterials into the basal bone, and also accelerating the deposition of local mineral content, aiming at a better quality / density of the newly formed bone tissue.

[0104] Therefore, the present invention also refers to a method for therapeutic treatment comprising the application of the isolated extracellular vesicles, as defined herein, or obtained by the process as defined herein, to a patient in need. Similarly, the present invention also refers to a method for therapeutic treatment, comprising the application of the grafting unit, as defined herein, to a patient in need. Preferably, in both cases, the therapeutic treatment is an orthopedic treatment and / or dental grafting. Also, preferably, in both cases, the treatment is autologous.

[0105] Similarly, the present invention refers to the use of isolated extracellular vesicles, as defined herein, or obtained by the process as defined herein, for the manufacture of a product for therapeutic treatment of a patient in need. The product, in the context of the present invention, is preferably a grafting unit and / or an EV concentrate. The latter may be in any form, be it lyophilized or not, for resuspension and / or dilution, whether or not forming part of a kit.

[0106] Additionally, the present invention refers to the use of isolated extracellular vesicles, as defined herein, or obtained by the process as defined herein, for therapeutic treatment of a patient in need. Similarly, the present invention also refers to the use of the grafting unit, as defined herein, for therapeutic treatment of a patient in need.

[0107] According to the present invention, all uses are preferably for therapeutic orthopedic treatment and / or dental grafting, but not limited to them.

[0108] Finally, the present invention refers to isolated extracellular vesicles, as defined herein, or obtained by the process as defined herein, for use in the therapeutic treatment of a patient in need. And to the grafting unit, as defined herein, for use in the therapeutic treatment of a patient in need. In both cases, likewise, the therapeutic treatment is preferably an orthopedic treatment and / or dental grafting.

[0109] Please note that the characteristics and preferred embodiments of the process of the present invention, as disclosed above, may be equally provided for in the present method and uses, and vice versa.

[0110] The present invention may be further understood based on the non-limiting examples as disclosed below.Examples

[0111] In vitro Assays

[0112] Example 1: hADSC characterization

[0113] The cells were obtained from the human adipose tissue-derived mesenchymal stromal cell bank Poietics® (hADSC – Lonza Poietics Walkersville, MD, USA), isolated from normal adult (non-diabetic) lipoaspirates collected during elective surgical liposuction procedures. Poietics® hADSCs were cryopreserved in the primary passage and guaranteed in 5 passages to express CD13, CD29, CD44, CD73, CD90, CD105 and CD166 and not to express CD14, CD31 and CD45. All cells showed negative results for mycoplasma, bacteria, yeast and fungi, as well as HIV-1, hepatitis B and hepatitis C, which were not detected. A frozen vial containing ~1 × 106cells was thawed at 37 ºC and seeded in a 25 cm2vial (TPP). The cells were cultured in complete medium (CM) – Dulbecco’s modified Eagle (DMEM) (Sigma-Aldrich, San Luis, MI, USA), low glucose content, containing 10% fetal bovine serum (FBS) (Gibco / Thermo Fisher Scientific, Waltham, MA, USA), 100 units / mL penicillin, 100 μg / mL streptomycin (Gibco / Thermo Fisher Scientific, Waltham, MA, USA), 50 mg / L gentamicin (Sigma-Aldrich) and 2.5 mg / L fungizone (Sigma-Aldrich) (pH 7.4) and cultured at 37 ºC in a humidified incubator with 5% CO2. After 24 h, debris and non-adherent cells were carefully removed. When adherent cells reached 80% confluence (passage 1: P1), hADSCs were dissociated with xeno-free trypsin CTS® TrypLE® Select Enzyme and seeded in vials at a density of 5 × 104cells / 75 cm2(passage 2: P2). Cell density was determined by manually counting the number of cells in each passage under the conditions disclosed above.

[0114] Example 2: Influence of xeno-free culture medium on morphology, cell Population Doubling Time (PDT), and viability of hADSCs

[0115] Qualitative morphological analysis of hADSCs was routinely performed by phase microscopy observation with an inverted microscope (Axiovert 25; Zeiss, Hallbergmoos, Germany) and a photography using a digital camera (AxioCam MRc, Zeiss) and AxioVision 3.1 software (Zeiss).

[0116] The cell Population Doubling Time (PDT) and hADSC viability were evaluated. To assess the efficiency of the xeno-free culture medium, the number of accumulated cells in the culture was compared using the complete conventional medium (CM-SFB) – DMEM + 10% SFB or xeno-free medium (KSR) – Knockout® Serum Replacement, under the same conditions disclosed above (37 ºC; 5% CO2). In the second passage P2, cells were dissociated, washed with PBS, counted and seeded (triplicate) in 75 cm2vials (TPP) at a density of 5 × 104cells. Three of the six vials received 16 mL complete culture medium (CM). The remaining vials underwent sequential adaptation by changing CM to KSR medium (24 h: 75% CM + 25% KSR; 48 h: 50% CM + 50% KSR; 72 h: 25% CM + 75% KSR; 96 h: 100% KSR).

[0117] After the adaptation protocol, cells were seeded in 6-well plates (TPP), 104cells / well, in triplicate, with their respective culture means (CM or KSR) at P3 (37 ºC; 5% CO2). When the cells reached 90% confluence, they were again dissociated with xeno-free trypsin and subcultured one more time until P6. In each subculture, hADSCs were counted in a hematocytometer chamber. PDT and accumulated cell numbers were calculated by the following equation: T x (log (2) / log (q2) – log (q1)), where T is the cell culture time;q1, the initial number of cells; andq2, the final number of cells.

[0118] For the cell viability assay, the number of stained (non-viable) cells was counted over the total number of cells (stained cells + unstained cells), expressed as a percentage. For this, 25 µL of the cell suspension were added to 75 µL of 0.4% Trypan blue solution (Sigma Chemical, St. Louis, MO, USA). From each homogenized sample (triplicate), 10 µL were transferred to the hemocytometer (Neubauer chamber), using a manual volume counter. To determine cell concentration, the cells in the four quadrants were counted, multiplied by the correction factor (10,000), to finally obtain the number of cells per milliliter of sample.

[0119] Example 3: Preparation of the supernatant for isolation of exosome-enriched EVs from hADSC-CM

[0120] In 75 cm2vials, the hADSC cell culture medium, on the fourth passage, at 70-80% confluence, was removed and new medium was added, consisting of DMEM + 10% vesicle-depleted FBS (triplicate). After 48 h, the supernatant was collected and submitted to Protocols 1 and 2 for exosome-enriched EV isolation.

[0121] Example 4: Preparation of the supernatant for exosome-enriched EV isolation from hADSC-KSR

[0122] In 75 cm2vials (triplicate), the hADSC cell culture medium, on the fourth passage, at 70-80% confluence, was simply renewed, giving continuity to the xeno-free medium (KSR) – Knockout® Serum Replacement, under the same conditions as hADSC-CM (37 ºC; 5% CO2). After 48 h, the supernatant was collected and submitted to Protocols 1 and 2 of isolation exosome-enriched EV isolation.

[0123] Example 5: Isolation from hADSC supernatant

[0124] Protocol 1: vesicle isolation and concentration, as disclosed by Example 15.

[0125] Protocol 2: use of the commercial reagent Total Exosome Isolation (from cell culture medium) – Invitrogen®. The reagent is added to the collected cell supernatant (500 µL / mL) and the solution is incubated overnight between 2 ºC and 8 ºC. Precipitated exosomes are recovered by standard centrifugation at 10,000 x g for 60 minutes. The sediment is then diluted (1:10) in 1×PBS.

[0126] Example 6: EV characterization from hADSC supernatant relative to the total protein quantification

[0127] The determination of total proteins in the supernatant was carried out using the Lowry method (Lowry et al, 1951). The principle of the method is based on a reaction between CuSO4and amino groups in an alkaline medium, resulting in the formation of Cu+ions. These formed ions then reduce a mixture of phosphotungic and phosphomolybdic acids (Folin-Ciocalteu reagent) to form tungstate and molybdate with absorption at a wavelength of 750 nm. For this purpose, a standard curve (0 – 20 μL / mL) of Bovine Serum Albumin (BSA) was used to calculate the total protein concentration by linear regression.

[0128] Example 7: EV characterization from hADSC supernatant relative to particle size and polydispersity index (PDI)

[0129] Photon correlation spectroscopy was used to measure particle size and polydispersity index (PDI). The hADSC-derived EV suspension (50 μL) from the different culture medium supernatants and isolation protocols was diluted in 1 mL PBS. All analyses were performed in triplicate by using a Malvern Nano-ZS90® (Malvern Instruments, Marvin City, United Kingdom) at 25 ºC.

[0130] Example 8: EV characterization from hADSC supernatant relative to transmission electron microscopy (TEM) analysis

[0131] Vesicles were identified by transmission electron microscopy (TEM) and photon correlation spectroscopy. Using a direct identification technique to assess the purity and diameter size of the vesicles, after isolation, they were suspended at 10 μL, at 1 mg / mL protein, aliquoted onto a carbon film-coated grid and dried at room temperature. Uranyl acetate (Merck KGaA, Darmstadt, DE, USA) was used as a contrast agent (Rohden et al, 2021). The sample was analyzed by TEM 120Kv (JEM 1200 Ex11-JEOL, Tokyo, Japan).

[0132] Example 9: EV characterization from hADSC supernatant relative to exosomal marker analysis by Western Blot

[0133] Exosomes were lysed in Lane Marker Reducing Sample Buffer (Pierce, Thermo Scientific, Waltham, MA, USA) separated on 12% SDS / PAGE gels (Bio-Rad, Hercules, CA, USA), always applying 10 μg protein per column, and transferred to a nitrocellulose membrane using the Transblot Turbo System (Bio-Rad). The membrane was incubated at 4 ºC overnight with CD63 antibody (TS63, Thermo Scientific). After washing, a corresponding secondary antibody (1:10,000, anti-mouse, ThermoFisher Scientific) was added for 1 hour at room temperature.

[0134] Example 10: Statistical Analysis

[0135] Statistical analysis was performed using GraphPad Prism 8 software. The differences between the groups were assessed using a one-way variance analysis (ANOVA) and Bonferroni post-hoc test (differences between the 2 groups, Student’s t-test). Normal distribution and variance homogeneity were considered.P-values < 0.05 were considered statistically significant.

[0136] In vivo assays

[0137] Example 11: hASC isolation

[0138] The collected adipose tissue (20 mL), obtained by minimally invasive liposuction (conventional surgical lipoplasty), was centrifuged (15000 rpm for 6 to 10 minutes), obtaining approximately 10 mL of concentrated adipose tissue, which was added to 10 mL digestion solution at 1.5 mg / mL collagenase (Collagenase NB 6 GMP Grade 17458, Serva GmbH, Heidelberg, Germany), diluted in StemPro MSC SFM XenoFree medium (Gibco), and kept under continuous agitation for 30 minutes at 37 ºC in a water bath. Then, it was centrifuged at 15000 rpm for 7 minutes. The supernatant was discarded and the precipitate was resuspended in StemPro MSC SFM XenoFree medium with 1% gentamicin and plated, incubated under normoxic conditions in a humidified greenhouse at 37 ºC and 5% CO2. The culture medium was changed every 48 h. When they reached between about 80 and 90% confluence, cells were dissociated with TrypLE™ Express Enzyme trypsin (3 mg / mL in StemPro MSC SFM XenoFree), without phenol red. Stromal cells were cultured under normoxic conditions in a humidified greenhouse at 37 ºC and 5% CO2.

[0139] Example 12: hASC characterization

[0140] Cell characterization by immunophenotyping and differentiation assays (Example 3) is in accordance with the criteria and recommendations by the International Society for Cell and Gene Therapy (ISCT). Therefore, to control the process, hASCs were characterized by surface molecule immunophenotyping, as follows: positive for CD73, CD90 and CD105 and negative (≤ about 2%) for CD14 or CD11b, CD19 or CD79α, CD34, CD45, HLA-DR (BD monoclonal antibodies, New Jersey, USA), using flow cytometry (FACSCalibur® flow cytometer), according to routine techniques.

[0141] Example 13: Differentiation assays in in vitro multilineages

[0142] To induce hASC differentiation, cells between the 4thand 5thpassages were seeded at a concentration of 105cells / cm2and cultured until they reach between 70 and 80% confluence. Then, for chondrogenic differentiation, the cells were cultured for 14 days in the presence of the StemPro® chondrogenic differentiation kit (Gibco®, New York, USA), according to the methodology proposed by the manufacturer; to induce osteogenic differentiation, cells under the same conditions were cultured for 21 days in the presence of the StemPro® osteogenic differentiation kit (Gibco®, New York, USA); and for adipogenic differentiation, hASCs were cultured for 21 days in the presence of the StemPro® adipogenic differentiation kit (Gibco®, New York, USA).

[0143] The extent of differentiation was determined microscopically by the appearance of lipid vacuoles stained with Oil Red O (Sigma-Aldrich®, Missouri, USA) in adipocytes, calcium deposits stained with Alizarin Red (Sigma-Aldrich®, Missouri, USA) produced by osteocytes, or proteoglycans stained with Alcian Blue (Sigma-Aldrich®, Missouri, USA) synthesized by chondrocytes.

[0144] Example 14: Quality control

[0145] Quality control of the conditioned medium for hASCs was also carried out in accordance with the standards for pre-clinical assays, as shown by [Table 1] below, and methods which are known and widely used for this purpose.

[0146] Quality ControlResultMethodSterilitynegativeMicrobial detection system, such as BACT / ALERT® 3DGram stainingnegativeMicroscopyEndotoxinless than 100 EU / mLLimulus assay (Limulus Amebocyte Lysate)MycoplasmanegativeqPCRExogenous virusnegativePCR with random primersParticle analysislabel quantity ± 20%NTA (Nano Tracking Analysis)

[0147] Example 15: Vesicle isolation

[0148] In 182 cm2vials, the medium conditioned by hASCs, between the 5thand 6thpassages, with confluence between 70 and 80%, was collected and submitted to the extracellular vesicle isolation protocol from autologous hAScs. hASc supernatants were filtered by using 0.22 μm filters and centrifuged at 6000 g for 15 minutes at 4 ºC, to remove cellular debris, dead cells and apoptotic bodies. The resulting supernatant was exposed to 12% polyethylene glycol (mixing the supernatant in a solution of 10 mL StemPro MSC SFM XenoFree + 4.3 mL PEG 6000 (Sigma-Aldrich, St. Louis, MO, USA) at 40% in PBS). The mixture was incubated at 4 ºC for 16 h (overnight), followed by centrifugation at 3000 g for 15 minutes at 4 ºC. The PEG-exosome sediment at the bottom of the tube was resuspended in 5 mL PBS and centrifuged again at 3000 g for 20 minutes at 4 ºC. The top liquid layer was discarded and the sediment was diluted (1:10) in 1X PBS.

[0149] Example 16: Exosome characterization

[0150] This was performed according toMinimal information for studies of extracellular vesicles 2018(MISEV2018). All samples were diluted in double-filtered PBS (dfPBS) at least 1:10 to obtain particles within the target readout range between 10 and 100 particles per frame on a NanoSight NS300 (Malvern Panalytical Inc., Malvern, UK). Using a manual injection system, five 30-second videos were prepared for each sample at 21 ºC. In addition to exosome quantification and sizing by using the particle counter NanoSight NS300, exosome protein markers were analyzed by Western Blotting. The exosomal concentrate was lysed, homogenized, and then centrifuged at 13000 g for 5 minutes at room temperature. The amount of total protein was measured from the supernatant and CD09, CD45, CD63, CD81, CD82, Alix, calnexin, EpCAM and Rab5 (biochemical analysis) were evaluated, in addition to morphological analysis.

[0151] After exosome characterization, they were also submitted to the quality tests indicated above ([Table 1]) for clinical use.

[0152] Results

[0153] Example 17: Morphology, cell Population Doubling Time (PDT) and hADSC viability

[0154] Mesenchymal stem cells of adipogenic origin had a typical fibroblastoid morphology, with a central nucleus and a few extensions. They showed high replicative rates and adhered to the plastic bottles of cell culture, both in complete culture medium (CM) and in xeno-free medium (KSR) ().

[0155] Both means had their proliferative and viability potential compared.shows the number of viable cells throughout the cell culture of the CM and KSR groups. It is possible to observe an increasing curve considering the moment when the cells were plated and the end of the experiment at P5. hADSCs showed an average of 2.85 x 104± 1.28 x 103for CM cells and 3.03 x 104± 1.93 x 103for KSR cells (P3-P5). There was no significant difference in the number of viable cells between the two means.

[0156] The medium for hADSC expansion must ensure that these cells can proliferate well enough to reach a sufficient number of cells in a given culture period for clinical applications. The population doubling time (PDT) is an MSC growth kinetics index, wherein the lower the PDT, the faster the cell growth rate. The growth coefficient for CM was 0.486 (doubling time: 19h58min) and for KSR was 0.5521 (17h39min). The growth coefficient value was obtained from linear regression adjusted by the Napierian Logarithm (log2.7) of the number of cells. The growth coefficient was higher in KSR and presented lower doubling time, showing good capacity to support mesenchymal stem cell growth (). There was no significant difference in viability between the cells cultured in the two means.

[0157] Example 18: EV isolation and characterization

[0158] For vesicle extraction, all samples were submitted to both established protocols. Afterwards, EV size and concentration were evaluated. Submitted to isolation protocol 1, the exosomes of the EXO-CM group presented an average size of 85 ± 23.1 nm and EXO-KSR, 109.6 ± 26.6 nm; in protocol 2, EXO-CM presented an average of 69.3 ± 8.8 nm and EXO-KSR 68.3 ± 6.2 nm. No significant differences were observed in the different culture means. The polydispersity index (PDI) was 0.386, indicating a monodisperse sample (PDI <1). The particles obtained by both protocols are within ranges characterizing the presence of exosomes ().

[0159] Exosomes were successfully isolated and positively detected, and their purity was confirmed by using transmission electron microscopy. EXO-KSR analysis disclosed the presence of microvesicles which were morphologically consistent with exosomes in terms of size and shape ().

[0160] The protein yield from exosomes according to each sample was also performed. The highest protein concentration was observed in the EXO-CM (45.3 + 4.6 μg / mL) and EXO-CM (47.1 + 9.2 μg / mL) supernatant samples, respectively performed by using protocols 1 and 2 ().

[0161] In order to confirm exosome isolation, the presence of CD63 protein was analyzed by using Western Blot. Both microvesicles isolated from CM and KSR supernatant expressed the CD63 marker, which is characteristic of exosomes. Labeling was positive, regardless of the adopted protocol ().

[0162] When analyzed together, results show that the process of the present invention, which is duly applicable to clinical protocols, leads to effectively obtaining extracellular vesicles, particularly exosomes. In particular, it was disclosed that, by the process of the present invention (its particular combination of steps), hADSCs cultured in xeno-free medium presented fusiform morphology and a high proliferative rate, similar to that of cells cultured in DMEM medium supplemented with FBS. When observing morphology and size of the extracellular vesicles obtained after the two adopted protocols, no statistical difference was observed between the groups. Exosomes were isolated and detected by transmission electron microscopy, showing capsule structure and shape consistent with literature reports, with positive labeling for CD63. Finally, statistical analysis of the protein detection assay results showed no significant differences in the amount of protein obtained by different isolation methods. However, the results show a difference in the protein amount obtained as a function of the different means. Lower yield obtained in the EXO-KSR group appears to be a purer yield, free of contaminants and with potential for safe clinical use.

[0163] In vivo assays in rats – Biodistribution and therapeutic efficacy evaluation

[0164] Example 19: Isolation, expansion and characterization of murine mesenchymal stromal cells

[0165] Wistar Kyoto lineage rats, 80 days old (n=2), were euthanized by isoflurane inhalation (4–5%) and prepared under aseptic conditions. Adipose tissue was collected by pre-retroumbilical median laparotomy in a conical tube (15 mL), washed in Hank’s solution, fragmented and submitted to enzymatic digestion in 3 mL type I collagenase solution (1.5 mg / mL) (Gibco), prepared in DMEM supplemented with sodium bicarbonate (Sigma) and HEPES (Sigma), in the lack of serum, under incubation at 37 °C for 45 to 60 minutes.

[0166] After mechanical dissociation, homogenization with the aid of a Pasteur pipette and centrifugation in 10 mL K-SFM culture medium (Kit-Serum Free Medium, Gibco®, New York, USA) (400 x g for 10 minutes), the cellular material was resuspended in 4 mL of the same culture medium and seeded in culture plates. Cultures were maintained in a humidified greenhouse at 37 °C and 5% CO2until reaching minimum confluence of 80%, at which point they were submitted to successive passages by trypsinization (between 0.25 and 0.5% trypsin / EDTA in HBSS without Ca2+and Mg2+) and expanded in culture vials, according to cell growth kinetics.

[0167] The characterization of mesenchymal stromal cells included a qualitative morphological analysis by phase microscopy, routinely performed during cell culture, as well asin vitroexpansion capacity evaluation.

[0168] Cell quantification was performed by the Trypan blue exclusion method, allowing the determination of the number of viable cells. Cell expansion was evaluated based on the growth area occupied throughout successive passages, making it possible to obtain a growth curve representing cell proliferative ability.

[0169] The cells were trypsinized, centrifuged and incubated for 30 minutes at 4ºC with murine antibodies specific for Sca-1, CD11b, CD31, CD29, CD44, CD45, CD90, and MHC-II, conjugated to phycoerythrin (PE) or fluorescein isothiocyanate (FITC) (BD Pharmingen, San Diego, CA). Data acquisition was performed on a FACSCalibur flow cytometer (Becton Dickinson), equipped with a 488 nm laser, collecting at least 10,000 events per sample, using CELLQuest software. Data analysis was conducted using WinMDI 2.8 software.

[0170] In the fourth passage, cells were cultured for four weeks in 12-well plates in complete medium supplemented with 10-8M dexamethasone, 2.5 µg / mL bovine pancreatic insulin, 100 mM indomethacin and 5 mM rosiglitazone, to induce adipogenic differentiation. The adipocyte phenotype was evaluated by staining with Oil Red O, disclosing the presence of lipid vacuoles.

[0171] For osteogenic differentiation, cells were maintained for four weeks in 12-well plates in complete medium supplemented with 10-5M dexamethasone, 100 µg ascorbic acid and 10 mM β-glycerophosphate. Osteoblastic activity was evaluated by staining with Alizarin Red S, disclosing the deposition of calcium-rich extracellular matrix.

[0172] Mesenchymal stromal cells derived from adipose tissue showed sustained growthin vitroover 30 days and typical fibroblast morphology at passage 4. Under inducing conditions, cells efficiently differentiated into adipocytes, forming lipid vacuoles stained by Oil Red O, and into osteoblasts, with the deposition of calcium-rich extracellular matrix as shown by Alizarin Red S. Immunophenotyping by flow cytometry confirmed the characteristic phenotypic profile of mASCs (cells positive for CD29, CD90 and CD44, and negative for CD11b, CD31, MHC-II), according to the expression of the analyzed markers in comparison with isotype controls.

[0173] Example 20: Exosome isolation and characterization from supernatant (medium conditioned by mASCs)

[0174] The isolation protocol as used was substantially equivalent to the already disclosed protocol, differing in terms of cell origin (murine cells (mASCs) versus human cells (hASCs)), culture scale, number of passages and used medium, although being, in both cases, serum-free medium, considering the particularities of each cell type.

[0175] The characterization of extracellular vesicles was performed according to theMinimal Information for Studies of Extracellular Vesicles 2018(MISEV2018) guidelines. Samples were diluted in double-filtered PBS (dfPBS) at a minimum ratio of 1:10, so to obtain particle concentration within the ideal reading range between 10 and 100 particles per frame on the NanoSight NS300 system (Malvern Panalytical Inc., Malvern, United Kingdom). Using a manual injection system, five 30-second videos per sample were acquired at a temperature of 21 °C.

[0176] Vesicle quantification and size distribution were determined by nanoparticle tracking analysis (NTA) on the NanoSight NS300. Additionally, molecular characterization of exosomes was performed by Western blotting for the markers CD9, CD63 and CD81, as well as for calnexin as a negative control. For this analysis, the exosomal concentrate was lysed, homogenized and centrifuged at 13,000 × g for 5 minutes at room temperature, with the total protein concentration being determined from the supernatant.

[0177] The results of the NTA analysis showed that the isolated extracellular vesicles presented size distribution which is predominantly compatible with the exosomal range (between 30 and 100 nm), while the protein analysis confirmed the presence of the positive exosomal markers CD9, CD63 and CD81 and the lack of the negative marker calnexin, confirming the successful isolation and the acquisition of exosome-enriched nanoparticles.

[0178] Dose, efficiency and biodistribution of EXO-mASCs associated to hydroxyapatite β-tricalcium phosphate

[0179] Example 21: EXO-mASC labeling

[0180] To determine the administered dose and evaluate EXO-mASC biodistribution, exosomes were labeled with the fluorescent membrane coloring agent PKH26 (Sigma-Aldrich). Initially, the PBS solution containing the exosomes was submitted to ultracentrifugation at 100,000 × g for 2 hours at 4 °C, and the sediment was resuspended in the diluent provided by the fluorescent labeling kit. The previously filtered PKH26 coloring agent (4 mM) was then mixed to the exosome suspension in a 1:1 ratio, remaining in incubation for 5 minutes, followed by the addition of 5% bovine serum albumin (BSA) to stop the reaction.

[0181] To remove the excess unincorporated coloring agent, labeled exosomes were washed three times with PBS, 5 mL PBS were subsequently added and submitted to a new ultracentrifugation at 100,000 × g for 2 hours at 4 °C, discarding the supernatant. Finally, the pellet containing labeled exosomes was resuspended in PBS (0.5 mL) and filtered through a 0.2 μm filter, so to remove any possible coloring agent aggregates.

[0182] Labeled exosomes were administered into the right femur of the animals, which had been previously anesthetized with isoflurane at a concentration between 2.5 and 3.0%, under an oxygen flow between 0.8 and 1.0 L / min.

[0183] Example 22: Surgical procedure (bone defect) and treatments

[0184] In vivoexperiments were conducted in male Wistar Kyoto lineage rats, approximately 90 days old and with an average body weight of 250 ± 25 g. The animals were anesthetized with isoflurane, at a concentration between 4% and 5% for induction and maintained between 2% and 3% during the procedure, in association with intraperitoneally administered ketamine (weight × 0.0009) and xylazine (weight × 0.0006).

[0185] After anesthetic induction, the thigh region was shaved and the skin was properly disinfected. A lengthwise incision of about 3 cm was made, with the dissection of tissues in successive planes until exposing the periosteum, which was incised with a scalpel. Then, a monocortical bone lesion was produced on the femur, about 2.0 mm in diameter and 2.5 mm deep, using a carbide-type drill under abundant irrigation with sterile 0.9% sodium chloride physiological solution.

[0186] Femurs were treated according to the experimental protocol corresponding to each group. At the end of the procedure, the surgical planes were sutured, the animals were properly identified, and postoperative analgesia was given with tramadol hydrochloride (12 mg / kg), subcutaneously administered every 8 hours, for 48 hours.

[0187] The animals were distributed into two experimental groups: the EXO+HA group, in which the right femur received 2.5 mg hydrated hydroxyapatite homogenized in 5 µL PBS containing 2 × 109exosomal particles, and the HA group, in which the left femur received 2.5 mg hydrated hydroxyapatite homogenized in 5 µL PBS, without exosomes. Each group consisted of six animals.

[0188] The animals were distributed into experimental groups, wherein the right femur was treated with exosome-linked hydroxyapatite (EXO+HA), while the left femur received only hydroxyapatite (HA). In the EXO+HA group, 2.5 mg hydrated and homogenized hydroxyapatite in 5 µL PBS were administered, containing, according to the experiment, 2 × 109, 4 × 109or 6 × 109exosomal particles. In the HA control group, the left femur received 2.5 mg hydrated and homogenized hydroxyapatite in 5 µL PBS, with no exosomes; this mass was defined based on the volume of the critical defect (7.8 mm3) and HA concentration (1 g / 3.13 cm3). Each experimental condition consisted of six animals.

[0189] The exosome distribution was analyzed at different time intervals after administration (1 h, 2 h, 6 h, 12 h and 24 h). For tissue collection, animals (n = 3 per group) were anesthetized with ketamine hydrochloride (90 mg / kg; 450 µL / kg) and xylazine hydrochloride (10 mg / kg; 300 µL / kg), both intraperitoneally administered, and submitted to transcardiac perfusion with PBS by using a peristaltic pump, followed by perfusion with 4% paraformaldehyde (PFA), both at a flow rate of 10 mL / min, totaling 100 mL. After perfusion, the organs of interest (liver, pancreas, spleen, kidneys and heart) were dissected, immersed in 4% PFA, and stored at 4 °C for a maximum period of seven days.

[0190] Tissue sections with 20 µm thickness were obtained by vibratome and incubated for 5 minutes, in the dark, with a solution containing 1 µg / mL of Hoechst 33342 coloring agent to label cell nuclei. The sections were subsequently washed with PBS (four times), assembled on coverslips and analyzed by confocal laser scanning microscopy, acquiring 8-bit grayscale images using an Olympus FV1000 microscope.

[0191] The biodistribution of PKH26-stained EXO-mASCs was evaluated one hour after grafting into femoral defects at doses of 2 × 109, 4 × 109and 6 × 109exosomal particles. In all analyzed doses, a fluorescent signal was detected in the main organs evaluated, including the liver, pancreas, spleen, kidneys and heart, suggesting rapid and systemic exosome dispersion from the application site. Increasing the dose has not changed the overall distribution pattern, confirming that EXO-mASCs show early and widespread biodistribution in peripheral tissues, as early as in the first hour after administration.

[0192] After 15 days, the animals (n = 3 per group) were euthanized and the femurs were dissected, preserving the regenerated bone tissue, with the samples fixed in 10% formaldehyde for analysis by micro-computed tomography (micro-CT). Micro-CT allowed for quantification of the percentage volume of newly formed bone tissue (VTO%) within the femoral defects treated with β-tricalcium phosphate hydroxyapatite alone or associated to different exosome doses.

[0193] The control group treated only with biomaterial showed the lowest VTO% average (13.67%), while the association between exosomes and the biomaterial resulted in an increase in the volume of newly formed bone at all investigated doses, with VTO% of 20.95% for 2 × 109particles, 21.11% for 4 × 109particles and 23.43% for 6 × 109particles. These results suggest that the incorporation of EXO-mASCs into the biomaterial enhances bone regeneration and a dose-dependent response trend, with greater bone formation observed at the highest exosome concentration after 15 days.

[0194] Therefore, we conclude that the EV isolation method of the present invention is a viable alternative, improved for clinical application, also duly promoting greater bone regeneration.Bibliography

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Claims

Process for obtaining extracellular vesicles (EVs) isolated from the culture supernatant of human adipose-derived mesenchymal stromal cells (hASCs), comprising: (a) obtaining hASCs from adipose tissue, preferably abdominal; (b) culturing and expanding hASCs; and (c) isolating EVs from the culture supernatant by filtration, followed by polymer precipitation.Process of claim 1, intended for orthopedic treatment and / or dental grafting.Process of any of claims 1 or 2, wherein the process is xeno-free.Process of any of claims 1 to 3, wherein step (a) of obtaining hASCs from adipose tissue, preferably abdominal tissue, comprises:- obtaining the lipoaspirate;- concentrating the lipoaspirate;- digesting the concentrated lipoaspirate in a digestion solution;- incubating under agitation;- concentrating to obtain a precipitate and resuspending the precipitate in culture medium;- plating and incubating until between 80 and about 90% confluence, changing means every 48 h; and- dissociating the hASCs.Process of any of claims 1 to 4, wherein step (b) of culturing and expanding hASCs comprises:- seeding the hASCs obtained in step (a) and culturing them up to the 4th to 7th passage, preferably up to the 5th to 6th passage, changing means every 48 h.Process of any of claims 1 to 5, wherein, in step (c), filtration is performed through a 0.22 µm filter, and polymer precipitation is made by using 12% polyethylene glycol (PEG).Process of claim 6, wherein the polymer precipitation by means of 12% polyethylene glycol (PEG) comprises:- incubating the filtrate with 12% PEG, preferably overnight; and- centrifuging it to obtain the EV precipitate, wherein centrifugation is preferably performed between about 1500 g and about 4500 g, more preferably 3000 g, for between about 5 min and about 30 min, more preferably between about 10 min and about 25 min, even more preferably for about 15 min.Process of any of claims 6 or 7, wherein, between filtration and polymer precipitation, the filtrate is centrifuged to remove debris, dead cells and / or apoptotic bodies from the precipitate.Process of any of claims 6 to 8, wherein, after centrifugation to obtain the EV precipitate, the precipitate is resuspended, preferably in buffered saline solution, more preferably in 1X phosphate-buffered saline (PBS) solution, and the resuspended precipitate is centrifuged to obtain the washed EV precipitate for use, which may also be preferably diluted, wherein the dilution is preferably 1:10, preferably in buffered saline solution, more preferably in 1X phosphate-buffered saline (PBS) solution.Isolated extracellular vesicles, obtained by the process of any of claims 1 to 9.Isolated extracellular vesicles, obtained by the process of any of claims 1 to 9, which are predominantly nanovesicles.Vesicles of claim 10, which are between about 30 nm and about 1000 nm in size, preferably between 30 nm and about 800 nm, more preferably between 30 nm and about 600 nm, even more preferably between about 30 nm and about 400 nm, most preferably between about 30 nm and about 200 nm.Vesicles of any of claims 10 to 12, expressing one or more markers from the group consisting of CD9, CD45, CD63, CD81, CD82, Alix, calnexin, EpCAM, and Rab5.Grafting unit, comprising the isolated extracellular vesicles of any of claims 10 to 13, or obtained by the process of any of claims 1 to 9, and a support.Unit of claim 14, wherein the support comprises bone materials and / or bone substitutes, preferably synthetic bone substitutes, such as a combination of hydroxyapatite and beta-tricalcium phosphate, more preferably 60% hydroxyapatite and 40% beta-tricalcium phosphate (HP / β-TCP).Therapeutic treatment method, comprising the application of the isolated extracellular vesicles of any of claims 10 to 13, or obtained by the process of any of claims 1 to 9, or the application of a grafting unit of any of claims 14 to 15, to a patient in need.Method of claim 16, wherein the therapeutic treatment is orthopedic and / or dental grafting.Use of isolated extracellular vesicles of any of claims 10 to 13, or obtained by the process of any of claims 1 to 9, intended for the manufacture of a product for therapeutic treatment of a patient in need.Use of claim 18, wherein the product is a grafting unit or an EV concentrate.Use of isolated extracellular vesicles of any of claims 10 to 13, or obtained by the process of any of claims 1 to 9, or of the grafting unit of any of claims 14 to 15, for a therapeutic treatment of a patient in need.Use of any of claims 18 to 20, wherein the therapeutic treatment is orthopedic and / or dental grafting.Isolated extracellular vesicles of any of claims 10 to 13, or obtained by the process of any of claims 1 to 9, or grafting unit of any of claims 14 or 15, intended for use in a therapeutic treatment of a patient in need.Vesicles of claim 22, wherein the therapeutic treatment is orthopedic and / or dental grafting.

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