Nutritional composition made from isolation and characterization of extracellular vesicle subpopulations from xenogenic tissues and organs

Abstract

The present invention provides a method for preparing a nutritional composition of xenogenic EVs from tissues / organs, comprising steps of collecting tissue / organ sample from fetal or adult mammal (101), keeping the collected sample in buffered saline in cold (102), mincing it into small pieces (103) and then mixing them with collagenase and deoxyribonuclease (104) followed by incubation and agitation (105), filtrating the incubated sample pieces for obtaining a filtrated liquid suspension containing extracellular fluid having EVs (106), differentially centrifuging the liquid suspension to remove remaining cells and tissue debris and obtain a supernatant (107), filtering the supernatant for collecting a crude fraction of large EVs (108) and centrifuging the supernatant for enriching it into a fraction of smaller EVs (109), characterized in that the separated EVs are fetal EV cargo or sinusoidal EVs directly isolated from the collected samples, wherein these EVs have regenerative potency, and the cells remain intact during EV isolation.

Description

[0001] NUTRITIONAL COMPOSITION MADE FROM ISOLATION AND CHARACTERIZATION OF EXTRACELLULAR VESICLE SUBPOPULATIONS FROM XENOGENIC TISSUES AND ORGANS FIELD OF INVENTION The present invention generally relates to a nutritional composition of xenogenic tissue- or organ-derived extracellular vesicles and the method for preparation of such. BACKGROUND OF THE INVENTION Extracellular vesicles have been gaining prominence in medicine due to their potential therapeutic applications. These nano-sized vesicles, secreted by a diversity of cell types, can encase and transport various bioactive molecules, ranging from lipids, proteins to mRNA and non-coding RNAs. Notably, the composition of EVs mirrors the physiological state of their originating cells, enabling them to mediate a wide array of biological functions. Specifically, recent research has underscored the potential of EVs in treating and managing various medical conditions such as osteoarthritis, skin and corneal defects, retinal ischemia, stroke, ligament, and tendon injuries. These EVs, enriched with regenerative potentials, may foster the healing and regeneration of damaged tissues. Extracellular vesicles are small lipid bilayer-enclosed structures released by cells into their surrounding environment. This diverse group of vesicles includes exosomes, microvesicles, and apoptotic bodies, each differing in size, biogenesis, and contents. These vesicles are produced by both normal and diseased cells, and they are found in various body fluids such as blood, urine, saliva, breast milk, and cerebrospinal fluid. EVs are now recognized as important mediators of cell-to-cell communication. They carry a cargo of proteins, lipids, and nucleic acids that can be delivered to other cells, influencing the recipient cells' behavior. Given their roles in cell signaling and ability to reflect the physiological state of their cell / tissue / organ of origin, EVs are increasingly considered valuable sources of supporting biological functions of the primary physiological function of their origin. The potential uses of EVs are broad. One of them is therapeutic applications. EVs can potentially deliver drugs, RNA interference molecules, or other therapeutic agents directly to specific tissue or cell types. Besides that, EVs are used in regenerative medicine and tissue repair. The regenerative properties inherent in some EVs suggest they might aid in tissue repair and regeneration, making them candidates for novel regenerative therapies. With advancements in isolation and characterization techniques, the biomedical field has enthusiastically embraced exosome and EV research, particularly in areas like disease diagnostics, therapeutics, and drug delivery systems. In disease diagnostics, EVs are recognized as promising biomarkers due to their presence in various body fluids and ability to reflect their parent cells' status. Therapeutically, EVs are explored for their natural delivery system capabilities, carrying beneficial molecules to specific target cells, thereby paving the way for targeted therapy. More recently, there has been growing interest in the use of exosomes and EVs in the field of nutrition and dietary supplements. Their potential to transport beneficial compounds, like antioxidants, amino acids, and other bioactive elements, across biological barriers in the body has been proposed. This could enhance the delivery and bioavailability of various beneficial dietary compounds, potentially enhancing health benefits. Furthermore, considering the role of EVs in immune modulation and inflammation regulation, they could have potential applications in managing conditions like obesity, diabetes, and other lifestyle diseases. EVs are secreted by cells and are present in nearly all bodily fluids, including those consumed as part of a regular diet, such as milk. These vesicles contain diverse biological molecules—proteins, lipids, and nucleic acids—integral to cellular communication and function. They can be used as carriers of bioactive compounds that could exert physiological benefits when ingested. Research into milk-derived EVs, for instance, has attracted interest because milk is a natural source of these vesicles and is already a substantial part of human nutrition. Milk EVs are thought to resist digestion and could be absorbed in the gastrointestinal tract, potentially influencing various biological processes such as immune responses or aiding in neonatal development. Studies suggest that these naturally occurring vesicles in milk might play a role in nurturing the infant's immune system and intestinal tract development. EVs as nutritional supplements may offer several advantages. One of them is bioavailability. EVs have a natural ability to cross biological barriers, which could potentially improve the delivery and absorption of nutrients or bioactive compounds. Besides that, the targeting capabilities of EVs could be harnessed to deliver specific compounds to certain tissues or cells in the body. EVs also contain a host of biologically active molecules contributing to health benefits such as anti-inflammatory effects, modulation of immune responses, and more. However, while the potential of EVs is vast, it is essential to note that standardizing EV isolation techniques, ensuring product safety, and conclusively demonstrating the health benefits are critical goals. Effective and scalable methods to isolate EVs from food sources or produce them in vitro while retaining their bioactive properties are essential. The isolation method must ensure purity and consistency, minimizing contaminants that could impact safety or efficacy. At present, the isolation of EVs is carried out from body fluids or from the supernatant of diverse cell culture sources. Currently, other methods are used to isolate EVs, where each has its own set of advantages and disadvantages. These methods include: i) differential ultracentrifugation. This is the most commonly used method for EV isolation. It involves multiple rounds of centrifugation and ultracentrifugation to pellet the EVs. However, the process can cause vesicle aggregation and contamination with other particles, requiring specialized equipment; ii) size-based methods. These include ultrafiltration, size-exclusion chromatography, and nanoparticle tracking analysis. They are generally easier to perform and less time-consuming than ultracentrifugation. However, they may not adequately separate EVs from other similarly sized particles, leading to sample contamination; iii) precipitation methods. They use water-excluding polymers to precipitate the EVs out of solution. These methods are easy to implement without special equipment but may lead to co- precipitation of contaminants; iv) immunoaffinity capture. This method uses antibodies to capture EVs based on the presence of specific surface proteins. It allows for a more particular isolation of EV subtypes, but the availability and effectiveness of specific antibodies could be a limitation. v) microfluidics-based methods. These newer techniques allow for isolation of EVs based on their physical and biological properties, but they require specialized equipment, and the methods are still being standardized. These existing methods face limitations such as low yield, lack of purity, possible structural alteration of the vesicles due to the isolation process, the need for specialized equipment, and the time-consuming nature of some approaches. Moreover, a lack of method standardization across studies leads to difficulty in comparing results from different research. The present invention, which aims to isolate and characterize EVs from tissues and organs, contributes significantly to this innovative field. Before this invention, the isolation of EVs from bodily fluids or cell culture supernatants had progressed considerably with several methods being developed. However, the isolation of EVs directly from solid tissues and organs posed more significant challenges due to the complexity of the tissue matrix and the potential contamination from cell culture media. This invention provides a comprehensive method to isolate and characterize extracellular vesicles from solid tissues, which could significantly help advance clinical practice. A United States prior art document, US2020289580A1 discloses a method of treating different forms of injuries via administering a therapeutically effective amount of EVs to the subject. This prior art uses exosomes (a type of EV) obtained from cardio- sphere-derived cells (CDC-EVs), where cardio-sphere-derived cells are adult stem cells with regenerative potency. In this invention, the exosomes are resuspended in sterile PBS with or without human serum albumin, indicating the usage of PBS in the operating procedure. Besides that, during the preparation of CDCs, the heart biopsies were minced into small fragments and briefly digested with collagenase. The specific methodology listed in this prior art to purify the collected exosomes includes ultracentrifugation and filtration based on size exclusion. However, this invention did not use a tissue chopper or an agitator in the process. The method used in this invention treats ocular inflammation and chemical injuries of the eye. A Japan prior art document, JP2022009194A also uses EVs in the treatment process, specifically EVs derived from non-transformed human neural progenitor (NP) cells. The cells where the EVs in this invention are derived from are either embryonic or adult stem cells with regenerative potency. In this invention, the exosomes are resuspended in PBS, indicating the usage of PBS in the operating procedure. Besides that, this invention uses enzymes in the EV separation process. The specific methodology listed in this invention to purify the collected exosomes includes centrifugation and filtration. The method disclosed in this invention serves as a novel method of treating spinal cord injury, stroke, traumatic brain injury, and neurodegenerative diseases. This shows that the method in this invention is used to treat injuries or issues closely related to injuries. However, this invention does not disclose a tissue chopper, nor an agitator in the operating procedure. Neither of the prior arts above involves a selection process directly from tissues and organs of xenogenic sources. Furthermore, no optional gentle freezing step for the highest preservation of cell viability of the tissue / organ for intermediate storage is used. For this optional purpose, the tissue / organ is frozen gently in containers in pre- cooled liquid alcohol at -85°C, corresponding to a freezing rate of 1°C per second. In procedures of the prior arts listed above, cell culture steps alter the biological function and cause media contamination, which is excluded in the present invention. Furthermore, no tissue chopper for gentle tissue mincing is applied in the preparation steps of the prior arts. The mild mechanical disruption of the tissue brought by the tissue chopper provides a larger surface area for enzymatic digestion of the extracellular matrix. In addition, no agitator is involved in their process for a better enzymatic degradation of the extracellular matrix between the cells to dissolve the extracellular particles between the cells in the tissue. This will slow down the operating procedure and result in insufficient yield, making the usage of tissue / organs without cell culturing steps and using tissue chopper and agitators a pioneer innovation in the preparation process of nutritional composition involving xenogenic organ-derived EVs. SUMMARY OF THE PRESENT INVENTION The present invention features a method for preparing a nutritional composition of xenogenic extracellular vesicles (EVs) from tissues or organs, comprising steps of: collecting a sample of tissues or organs from a fetal or adult mammal; keeping the collected sample in a buffered saline in cold; mincing the sample into small pieces with a tissue chopper for increasing the surface area of the sample; mixing the minced sample pieces with collagenase and deoxyribonuclease, wherein the mixture is incubated and agitated; filtrating the incubated sample pieces for obtaining a filtrated liquid suspension containing extracellular fluid having the EVs; differentially centrifuging the liquid suspension for removing remaining cells and tissue debris and for obtaining a supernatant; filtering the supernatant for collecting a crude fraction of large EVs; and centrifuging the supernatant for enriching the supernatant into a fraction of smaller EVs, characterized in that the separated EVs are fetal EV cargo or sinusoidal EVs that are directly isolated from the collected samples, wherein these EVs have regenerative potency, and the cells remain intact during the isolation of the EVs. Preferably, the tissues are collected from an organ, which is a brain, heart, lung, liver, kidney or placenta. Optionally, a gentle freezing step for the highest preservation of cell viability of the tissue / organ for intermediate storage is used. For this optional purpose, the tissue / organ is frozen gently in containers in pre-cooled liquid alcohol at -85°C, corresponding to a freezing rate of 1°C per second. Before further processing of the tissue / organ, a gentle thawing at 4°C for 5-10 hours depending on tissue / organ size is performed. Preferably, the dimension of the minced sample pieces is approximately 2 × 2 × 2 mm. Preferably, the collagenase is collagenase D. Preferably, the deoxyribonuclease is DNase I. Preferably, the incubation process is performed for 20-30 minutes at approximately 37°C. Preferably, the agitation process in a shaking water bath at 37°C is performed at a speed of 20 rpm for allowing better isolation of EVs from the sample pieces. Preferably, the liquid suspension is differentially centrifuged at 300 g for 10-15 minutes, and then at 2000 g for 20 minutes. Preferably, the supernatant is centrifuged at 20000 g for 15-20 minutes for removing fractions of large EVs. Preferably, the step of filtering the supernatant is done with a 0.22 μm pore size polyethersulfone filter. Preferably, the supernatant is centrifuged at 100,000 g for 90 minutes for removing fractions of small EVs. The present invention consists of features and a combination of parts from now on fully described and illustrated in the accompanying drawings; it being understood that various changes in the details may be made without departing from the scope of the invention or sacrificing any of the advantages of the present invention.

[0002] BRIEF DESCRIPTION OF THE DRAWINGS To further clarify various aspects of some embodiments of the present invention, a more particular description of the invention will be rendered by referencing specific embodiments, which are illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. The invention will be described and explained with additional specificity and detail through the accompanying drawings in which: Figure 1 illustrates a flowchart for the method for preparing the nutritional composition of xenogenic extracellular vesicles (EVs) from tissues or organs. Reference numerals 101 – step of collecting a sample of tissues or organs from a fetal or adult mammal 102 – step of keeping the collected sample in buffered saline in cold 103 – step of mincing the sample into small pieces with a tissue chopper 104 – step of mixing the minced sample pieces with collagenase and deoxyribonuclease 105 – step of incubating and agitating the mixture 106 – step of filtering the incubated sample pieces to obtain a filtrated liquid suspension containing the extracellular fluid having EVs 107 – step of differentially centrifuging the liquid suspension for removing remaining cells and tissue debris and for obtaining a supernatant 108 – step of filtering the supernatant for collecting a crude fraction of large EVs 109 – step of centrifuging the supernatant for enriching the supernatant into a fraction of smaller EVs DETAILED DESCRIPTION OF THE INVENTION The present invention generally relates to a nutritional composition of xenogenic extracellular vesicles (EVs). In particular, the present invention describes a method for preparing a nutritional composition of xenogenic organ-derived extracellular vesicles, where the EVs are fetal EV cargo or sinusoidal EVs directly isolated from the collected samples. From now on, the method for preparing a nutritional composition of xenogenic extracellular vesicles according to the present invention will be described in detail with reference to Figure 1 according to the preferred embodiments. It is to be understood that limiting the description to the preferred embodiments of the invention is merely to facilitate discussion of the present invention, and it is envisioned without departing from the scope of the appended claims. In the preferred embodiment of the present invention, the method for preparing a nutritional composition of xenogenic extracellular vesicles (EVs) from tissues or organs comprises steps of collecting a sample of tissues or organs from a fetal or adult mammal (101). Examples of mammals are mice, lambs, sheep, pigs, and rabbits. Preferably, the tissues are collected from an organ (101), which is a brain, heart, lung, liver, kidney or placenta. Alternatively, the organ is a skeletal organ, such as bones, ligaments, joints, tendons, and cartilage; digestive organs such as mouth, pharynx, esophagus, stomach, intestines, pancreas, gallbladder, mesentery, colon, rectum, anus; respiratory organ, such as nose, trachea, larynx, bronchi, bronchioles; urinary organ, such as kidneys, ureter, bladder, urethra; male reproductive organs, such as testicles, epididymis, vas deferens, prostate, penis, scrotum; female reproductive organs, such as Fallopian tube, ovary, uterus, vagina, vulva; endocrine organs such as glands; circulatory organs such as heart, arteries, veins, capillaries; lymphatic organ, such as lymphatic vessel, lymph nodes, bone marrow, thymus, spleen; nervous system organs, such as spinal cord, nerves; sensory organs such as eye, ear, skin, nose, and tongue. After the sample is collected, it is kept in a buffered saline in the cold (102), preferably on ice. The type of buffered saline used is preferably phosphate-buffered saline (PBS). This is because PBS is a buffer solution that can imitate the osmolarity, ion concentration, and pH of body fluids; it is isotonic concerning body solutions, thus diminishing the risk of causing tissue damage. Alternatively, normal saline can also be used. Optionally, a gentle freezing step for the highest preservation of cell viability of the tissue / organ for intermediate storage is used. For this optional purpose, the tissue / organ is frozen gently in containers in pre-cooled liquid alcohol at -85°C, corresponding to a freezing rate of 1°C per second. Before further processing of the tissue / organ, a gentle thawing at 4°C for 5-10 hours depending on tissue / organ size is performed. Next, the collected sample is minced into small pieces with a tissue chopper (103) and then mixed with enzymes, such as collagenase and deoxyribonuclease (104), wherein the mixture is incubated and agitated (105). The sample is minced into small pieces with a tissue chopper (103) to increase the sample's surface area for the enzymatic reaction of the collagenase and deoxyribonuclease. Preferably, the dimension of the minced sample pieces is approximately 2 × 2 × 2 mm to ensure a better performance of the enzymes. The collagenase and deoxyribonuclease used are preferably collagenase D and DNase I, respectively. Alternatively, collagenase A, B or C can be used for the collagenase, and DNase II can be used for the deoxyribonuclease. The incubation process is performed for 20-30 minutes at approximately 37°C. As 37°C is the typical body temperature for mammals, this temperature is optimal to facilitate enzyme functioning and other biological processes. Besides that, by maintaining a constant incubation temperature, thermal stress on the sample pieces, which can destroy the tissue structure, can be avoided. An agitator is used to shake the incubated mixture to enhance the activity of the collagenase and deoxyribonuclease, accelerating the process. The agitation process in a shaking water bath at 37°C is performed at a speed of 20 rpm to allow better isolation of EVs from the sample pieces in which they are entrapped. Although 20 rpm speed is relatively slow, it can achieve the desired result while providing several benefits such as i) diminished operating cost following the reduction of horsepower requirement; ii) lower capital expenditures due to smaller components needed for the low-speed agitator such as smaller gear drives, motors, and shafts; and iii) decreased maintenance expense due to less wear and tear of agitator parts and lower torque. After the incubation and agitation (105), the incubated sample pieces are filtrated to obtain a filtrated liquid suspension containing the extracellular fluid having EVs (106). Preferably, a 0.2-1.0 µm pore filter is used for the filtration. This pore size range is within the sub-micropore classification, in which particles with a pore size between 0.1 and 1.0 µm are classified. This type of filtration is known as microfiltration. Microfiltration is used in the present invention to remove the larger tissue debris from the solution. After the microfiltration step, the liquid suspension is differentially centrifuged to remove the remaining cells and tissue debris and to obtain a supernatant (107). This differential centrifugation occurs in two stages, where both occur at different operating conditions. The first stage is performed at 300 g for 10-15 minutes, while the second stage is performed at 2000 g for 20 minutes. The first stage eliminates remaining cells and tissue debris, while the second stage pellets the EVs. This is because the separation speed of a particle is directly proportional to the particle’s density. The larger the particle’s density, the faster the separation of the particle from the mixture. Therefore, when a centrifuge spins, the denser particles will travel to the bottom as they have larger mass, hence inertia has a stronger effect on them. Thus, a smaller relative centrifuge force (g) is sufficient to separate the denser particles from the mixture. After separating the denser particles, the close centrifugal force is increased to separate the lighter particles. After the differential centrifugation is performed on the liquid suspension, a supernatant is obtained. The supernatant is centrifuged at 20000 g for 15-20 minutes to remove fractions of large EVs. Preferably, the supernatant is further filtered with a 0.22 μm pore size polyethersulfone (PES) filter, which removes the most significant elements and collects the crude fraction of large EVs (108). This supernatant is also subjected to a centrifugation process to enrich it into a fraction of smaller EVs (109). The centrifugation of the supernatant occurs at 100,000 g for 90 minutes. Optionally, the EV fractions can be run through an iodixanol density gradient or cushion for further purification. The vesicles will position themselves at their respective densities within the gradient, allowing for the isolation of purer EV subpopulations. After isolation, EVs can be characterized based on size and morphology using techniques like nanoparticle tracking analysis and cryo-electron microscopy, and their content can be analyzed by proteomics, lipidomics, or transcriptomics. The presence of EV-specific markers can also be detected to confirm and identify the isolated EVs. Different EV subpopulations can be distinguished and characterized using a combination of electron microscopy, proteomics, and RNA profiling. Each of these techniques has its own advantages. Electron microscopy, especially cryo-electron microscopy, provides high-resolution images of EVs, allowing for the identification of their overall morphology and size. Different types of EVs may have distinct morphological features which can help in their characterization. On the other hand, extracellular vesicles typically carry a distinctive set of proteins depending on their cell of origin and their biological function. This unique protein profile can be analyzed using various proteomic techniques, such as mass spectrometry. Identifying these proteins can help characterize the EVs and even allow for the differentiation between subtypes of EVs. Besides that, extracellular vesicles carry diverse RNA species, including mRNA, miRNA, lncRNA, and others. Analyzing the RNA content of EVs using high-throughput sequencing technologies (like RNA-seq) can provide insights into the parent cell's activity. It could potentially yield important information about the EVs' functional roles. The RNA profile can also contribute to EV subtype differentiation, offering a tool for EV characterization. Together, morphology obtained by electron microscopy, protein profile via proteomics, and RNA content analysis form a comprehensive characterization of EV subpopulations. The liquid fractions of EVs can be stored at -20°C. Alternatively, the EVs can be lyophilized and later resuspended in a saline solution. The resulting final product of the isolated EVs is a nutritional composition which can be used in veterinary and human applications. It can be applied nasally, sublingually, conjunctivally, cutaneously / dermally, and also subcutaneous or parenteral administration, such as into the joint and tissue for use in the treatment of osteoarthritis, skin defects, corneal defects, vision, and ligament injuries to support regeneration and healing. These EVs could also be incorporated into nutritional supplements to promote human health and recovery. These supplements could support overall health resilience, tissue repair, and immune function to support health or individuals recovering from an illness. A preferred embodiment of the present invention applies fetal EV cargo or sinusoidal EVs, which are directly isolated from their source (fresh) instead of intracellular EVs or cultured cells or tissues. These EVs retain their regenerative properties, and the cells from which they are derived remain fully intact after isolation. This method allows for the efficient extraction of EVs, preserving their core features, meaning that they can more thoroughly embody their biological roles once administered as therapeutic agents. The presence of specific marker proteins can be used to verify the identity of these isolated EV subpopulations. Xenogenic fetal and adult organ-derived tissue EVs have several advantages over EVs from single cell culture, such as i) immune privilege. Fetal EVs are immune- privileged, meaning they have reduced potential to induce an adverse systemic immune response when administered to the host. This can lead to their prolonged survival and function in the host without the risk of acute immune rejection, a common problem associated with conventional EVs; ii) bioactivity. Fetal and adult EVs carry bioactive compounds and beneficial nutrients that may promote nourishment and repair of the organism, enhancing their therapeutic potential; iii) physiological relevance. Being directly isolated from organ tissues, fetal and adult EVs retain a composition similar to in vivo conditions, making them more physiologically relevant compared to EVs derived from cultured cells, which can be contaminated by the culture medium (FBS, insulin, undefined medium components); and iv) versatility. Fetal and adult organ-derived tissue EVs encompass a diversity of cell types within the organ. Hence, they can reflect a broader range of biological functions native to the organ, unlike EVs from single-cell cultures that are limited to the capabilities of a specific cell type. The advantages of the approach in the present invention are significant. These advantages include: i) regenerative potency. The isolation of specific EVs (fetal EV or sinusoidal) directly from tissues, which have notable regenerative properties, gives raised therapeutic potential; ii) immune privilege. Fetal EVs are immune-privileged, reducing the likelihood of adverse immune reactions in host bodies; iii) extraction efficiency. This method ensures a high yield of intact and functional EVs; iv) physiological relevance. As the EVs are not cultured, the isolation maintains their physiological characteristics and function, enhancing their therapeutic relevance. Xenogenic extracellular vesicles have the unique property of transport encapsulating and transporting various nutrients, such as proteins, lipids, and nucleic acids, which are essential for the body's functioning. As a nutritional supplement, the xenogenic EVs could deliver these nutrients directly into cells, bypassing some of the traditional barriers that limit the effectiveness of many nutrient supplements. The fetal EV cargo or sinusoidal EVs, as mentioned in this protocol, are immune-privileged, meaning they will not trigger a significant immune response in the host body, making them an acceptable delivery vehicle. Moreover, based on their origin, these EVs could have specific beneficial properties. For instance, the fetal EV cargoes could transport bioactive compounds and nutrients beneficial to nourishing and repairing tissues. Sinusoidal EVs originating from liver sinusoidal cells could have immune-enhancing effects and could be used to strengthen immune health. Therefore, using this method to prepare a nutritional composition of xenogenic extracellular vesicles could be a step toward designing more effective, targeted dietary supplements. There are several notable novel and inventive points of the present invention. For instance, this present invention involves intact fetal tissues / organs from mammals, while the prior arts involved cell culture medium from which the EVs are isolated. Besides that, the present invention involves gentle mincing of the tissue / organ with a tissue chopper and using agitator in their process, which are unique in comparison with the prior arts. Other than that, the present invention involves enzymatic digestion of the extracellular matrix of the intact cells of the tissue / organ, which is an innovative step. Furthermore, the final product of the isolated EVs is a nutritional composition, which is uncommon in biotechnology. The present invention may be embodied in other specific forms without departing from its essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. Therefore, the scope of the invention is indicated by the appended claims rather than by the preceding description. All changes that come within the meaning and range of equivalency of the claims are to be embraced within their scope. Throughout this specification, unless the context requires otherwise, the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element or group of elements, but not the exclusion of any other element or group of elements. Thus, in the context of this specification, the term “comprising” is used in an inclusive sense and therefore should be understood as meaning “including principally, but not necessarily solely.”

Claims

CLAIMS 1. A method for preparing a nutritional composition of xenogenic extracellular vesicles (EVs) from tissues or organs, comprising steps of: collecting a sample of tissues or organs from a fetal or adult mammal (101); keeping the collected sample in a buffered saline in cold (102); mincing the sample into small pieces with a tissue chopper (103) for increasing the surface area of the sample; mixing the minced sample pieces with collagenase and deoxyribonuclease (104), wherein the mixture is incubated and agitated (105); filtrating the incubated sample pieces for obtaining a filtrated liquid suspension containing extracellular fluid having the EVs (106); differentially centrifuging the liquid suspension for removing remaining cells and tissue debris and for obtaining a supernatant (107); filtering the supernatant for collecting a crude fraction of large EVs (108); and centrifuging the supernatant for enriching the supernatant into a fraction of smaller EVs (109), characterized in that the separated EVs are fetal EV cargo or sinusoidal EVs that are directly isolated from the collected samples, wherein these EVs have regenerative potency, and the cells remain intact during the isolation of the EVs.

2. The method for preparing a nutritional composition of xenogenic extracellular vesicles from tissues or organs as claimed in claim 1, wherein the tissues are collected from an organ (101), which is a brain, heart, lung, liver, kidney or placenta.

3. The method for preparing a nutritional composition of xenogenic extracellular vesicles from tissues or organs as claimed in claim 1, wherein the dimension of the minced sample pieces is approximately 2 × 2 × 2 mm.

4. The method for preparing a nutritional composition of xenogenic extracellular vesicles from tissues or organs as claimed in claim 1, wherein the collagenase is collagenase D.

5. The method for preparing a nutritional composition of xenogenic extracellular vesicles from tissues or organs as claimed in claim 1, wherein the deoxyribonuclease is DNase I.

6. The method for preparing a nutritional composition of xenogenic extracellular vesicles from tissues or organs as claimed in claim 1, wherein the incubation process is performed for 20-30 minutes at approximately 37°C.

7. The method for preparing a nutritional composition of xenogenic extracellular vesicles from tissues or organs as claimed in claim 1, wherein the agitation process in a shaking water bath at 37°C is performed at a speed of 20 rpm for allowing better isolation of EVs from the sample pieces.

8. The method for preparing a nutritional composition of xenogenic extracellular vesicles from tissues or organs as claimed in claim 1, wherein the liquid suspension is differentially centrifuged at 300 g for 10-15 minutes, and then at 2000 g for 20 minutes.

9. The method for preparing a nutritional composition of xenogenic extracellular vesicles from tissues or organs as claimed in claim 1, wherein the supernatant is centrifuged at 20000 g for 15-20 minutes for removing fractions of large EVs.

10. The method for preparing a nutritional composition of xenogenic extracellular vesicles from tissues or organs as claimed in claim 1, wherein the step of filtering the supernatant is done with a 0.22 μm pore size polyethersulfone filter.

11. The method for preparing a nutritional composition of xenogenic extracellular vesicles from tissues or organs as claimed in claim 1, wherein the supernatant is centrifuged at 100,000 g for 90 min for removing fractions of small EVs.

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