Method for isolating and cryopreserving extracellular vesicles

Abstract

The present invention relates to a method for isolating and cryopreserving extracellular vesicles, the method comprising steps of evaluating functional characteristics of extracellular vesicles, and selecting and cryopreserving extracellular vesicles on the basis of the evaluation. According to the present invention, as described above, an improved method for isolating and cryopreserving extracellular vesicles enables stable securing and long-term storage of extracellular vesicles selected on the basis of functional characteristics, thereby enabling the extracellular vesicles to be used for various medical and research purposes.

Description

Method for isolating and freezing extracellular endoplasmic reticulum

[0001] This patent application claims priority to Korean Patent Application No. 10-2025-0049545, filed with the Korean Intellectual Property Office on April 16, 2025, and the disclosures of said patent application are incorporated herein by reference.

[0002] The present invention relates to a method for isolating and freezing extracellular vesicles, and more specifically, to a method for freezing extracellular vesicles selected by evaluating the functional characteristics of extracellular vesicles isolated from biologically derived materials.

[0003] Extracellular vesicles (EVs) are nano-sized vesicles that all cells secrete into the external environment for intercellular information exchange. Extracellular vesicles (EVs) contain various substances exhibiting biological activity, such as proteins, lipids, nucleic acids, and metabolites, and considering these characteristics, they are currently being actively developed as therapeutic agents for various indications.

[0004] In particular, stem cell-derived extracellular vesicles (EVs) are known to contain various physiologically active factors and have the potential to be utilized as a new therapeutic agent capable of resolving issues such as low survival rates and low differentiation rates when conventional stem cells are injected into the body.

[0005] Meanwhile, various technologies have been developed regarding cryopreservation methods for autologous cells, stem cells, and umbilical cord blood, which involve collecting these from subjects, freezing and storing them, and then thawing them as needed for purposes such as disease treatment. However, even with these cryopreservation methods, there are technical issues where cell viability and activity remain low when the cells are thawed at the time required by the user.

[0006] Although extracellular vesicles (EVs) can resolve issues such as low viability and differentiation rates that occur in cells or stem cells upon thawing after cryopreservation, and allow for the selection of more economical cryopreservation methods compared to cells or stem cells, technological development regarding cryopreservation methods for EVs has not yet been achieved.

[0007] Furthermore, there is a need for a method to isolate and freeze-store extracellular vesicles (EVs) that allows for the isolation of EVs from bio-derived materials—whether autologous from a subject or allogenic from a donor—depending on the intended use of EVs, such as growth promotion or anti-aging, and subsequent thawing and provision to the subject or user when required.

[0008] [Prior Art Literature]

[0009] [Patent Literature]

[0010] Published Patent Application 10-2015-0078123 (Cryopreservation and culture method of human amniotic fluid-derived stem cells)

[0011] As a result of diligent efforts to develop a method for isolating and freezing extracellular vesicles (EVs), the inventors have completed a method for isolating and freezing extracellular vesicles, comprising the steps of isolating extracellular vesicles from a biologically derived material, evaluating the functional characteristics of the extracellular vesicles, selecting the extracellular vesicles based on the functional characteristics, and freezing the selected extracellular vesicles.

[0012] The objective of the present invention is to provide a method for isolating and freezing extracellular vesicles, which evaluates the functional characteristics of extracellular vesicles isolated from biologically derived materials, freezes selected extracellular vesicles, and thaws and provides them when necessary.

[0013] The objects of the present invention are not limited to those mentioned above, and other unmentioned objects and advantages of the present invention may be understood from the following description and will become more clearly known from the embodiments of the present invention. Furthermore, it will be readily apparent that the objects and advantages of the present invention can be realized by the means and combinations thereof set forth in the claims.

[0014] The present invention relates to a method for isolating and cryopreserving extracellular vesicles (EVs), enabling the stable acquisition and long-term storage of EVs so that they can be utilized for various medical and research purposes.

[0015] According to one aspect of the present invention, the present invention provides a method for isolating and storing extracellular vesicles from a biologically derived material, comprising the steps of: isolating extracellular vesicles from a biologically derived material; evaluating the functional characteristics of the extracellular vesicles; and selecting the extracellular vesicles based on the functional characteristics and freezing the selected extracellular vesicles.

[0016] In this specification, the term 'biologically derived material' can be blood, plasma, lymph fluid, aqueous humor, cerebrospinal fluid, etc., and means that if extracellular vesicles can be isolated, it is included in biologically derived material.

[0017] In this specification, the term 'extracellular vesicles (EV)' refers to substances derived from cells that are involved in intercellular information exchange and can be isolated from biologically derived substances such as blood, plasma, and lymph fluid, and includes, but is not limited to, exosomes.

[0018] In this specification, the term 'separation of extracellular vesicles' includes cases where a centrifuge is used based on the size, density, etc. of extracellular vesicles, as well as cases where a filter is used, but is not limited thereto; any method capable of separating extracellular vesicles is not limited thereto.

[0019] Meanwhile, extracellular vesicles (EVs) may be isolated from biological material provided by a recipient or a donor. The 'recipient' refers to a case where autologous extracellular vesicles (EVs) are provided, and the person providing the biological material for isolating the EVs is the same as the person who freezes and thaws the EVs isolated from the biological material for use. Meanwhile, the 'donor' refers to a case where allogenic extracellular vesicles (EVs) are provided, and the person providing the biological material for isolating the EVs is different from the person who freezes and thaws the EVs isolated from the biological material for use.

[0020] In this specification, the term 'functional property' refers to a measurable and reproducible biological response or effect resulting from the action of extracellular vesicles (EVs) on a specific cell, tissue, or biological system.

[0021] In this specification, the term "frozen storage" means that extracellular vesicles (EVs) isolated from biological material are stored under conditions that maintain quality suitable for the purpose of frozen storage.

[0022] In one embodiment of the present invention, the biological material to be used for extracellular vesicle separation can be selected from various biological materials such as blood, plasma, lymph fluid, aqueous humor, and cerebrospinal fluid, thereby enabling the securing of optimal extracellular vesicles according to the purpose of cryopreservation. The process of separating extracellular vesicles from the selected biological material includes a process of efficiently separating extracellular vesicles to suit the purpose of storage, thereby enabling the securing of extracellular vesicles of high purity. Meanwhile, if the biological material used for separating extracellular vesicles is blood, it can be stored at 1°C to 30°C prior to separation to maintain an optimal state.

[0023] In one embodiment of the present invention, the functional characteristics of the extracellular vesicle may include cell proliferation enhancement, cell viability improvement, and anti-fibrotic activity. Cell proliferation enhancement can be viewed as an indicator of growth promotion, and 'growth promotion' refers to increasing the growth rate of cells, tissues, or organs of an organism, or promoting growth-related physiological functions (e.g., protein synthesis, cell proliferation, growth of cartilage and bone tissue, etc.). Growth here may include not only natural growth stages in vivo (childhood, puberty, etc.) but also the regeneration process of damaged tissue, increase in muscle mass, weight gain, or improvement in the thickness and density of specific tissues (e.g., hair, skin, etc.). Furthermore, it includes actions through the induction of expression of physiologically active substances such as growth hormone and insulin-like growth factor (IGF-1) or the activation of signal transduction pathways. Cell viability improvement and anti-fibrotic activity can be viewed as indicators of anti-aging effects, and 'anti-aging' includes actions that delay the biological aging process or inhibit or restore the decline in function of cells, tissues, and organs caused by aging. Specifically, it may include reducing oxidative stress, inhibiting inflammation, maintaining telomere length, inhibiting apoptosis, regulating autophagy, regulating the expression of aging-related genes (SIRT1, FOXO, etc.), and maintaining mitochondrial function.In addition, it may also include the effect of improving externally observable indicators of aging (e.g., skin wrinkles, pigmentation, loss of elasticity, etc.).

[0024] In one embodiment of the present invention, among the functional characteristics, the cell proliferation effect can be evaluated by the increase in the proliferation rate of cartilage cells.

[0025] In one embodiment of the present invention, the effect of increasing cell viability among functional characteristics can be evaluated by increasing viability in aging-induced cells.

[0026] In one embodiment of the present invention, among the functional characteristics, the fibrosis inhibitory effect can be evaluated by the reduction of α-SMA protein expression.

[0027] In one embodiment of the present invention, extracellular vesicles can be isolated after pre-treating a bio-derived material to improve the functional characteristics of the extracellular vesicles. The material for pre-treating the bio-derived material may include at least one of Exendin-4, Phorbol 12-myristate 13-acetate (PMA), Interferon-γ, Tetrandrine, Hyaluronic acid, Substance P, Resveratrol, Rapamycin, Bezafibrate, or Lanifibranor.

[0028] In one embodiment of the present invention, a quality inspection of extracellular vesicles may be performed prior to freezing. The quality inspection ensures the quality of the extracellular vesicles to be frozen by performing tests on appearance, the number and size of extracellular vesicles, a negative test for mycoplasma, a negative test for exotic viruses, a sterility test, an endotoxin test, an identification test, a purity test, a potency test, etc.

[0029] In one embodiment of the present invention, for stable cryopreservation of extracellular vesicles, they are stored at -70°C to -90°C. At this time, a cryopreservation agent may be added considering the purpose of storage and the storage period of the extracellular vesicles, or they may be stored without a cryopreservation agent. The cryopreservation period may be set from 1 month to 120 months to allow for customized storage according to the purpose of storage, and if additional storage is required after storage from 1 month to 120 months, the storage period may be reset to 1 month to 120 months for additional storage.

[0030] The features and advantages of the present invention are summarized as follows.

[0031] (a) The present invention provides an improved method for isolating extracellular vesicles and freezing them.

[0032] (b) When using the method for isolating and freezing extracellular vesicles according to the present invention, it is possible to stably secure and store extracellular vesicles selected based on their functional characteristics for a long period, thereby enabling their use for various medical and research purposes.

[0033] Figure 1 is a diagram of the configuration of the method for separating and freezing extracellular vesicles according to the present invention.

[0034] FIG. 2 shows the quality inspection items before cryopreservation of isolated extracellular vesicles according to the present invention.

[0035] Figure 3 shows an example of examinee information input.

[0036] Figure 4 shows the normal levels of blood cells (red blood cells, white blood cells, platelets) in Asian infants, children, and adolescents.

[0037] Figure 5 shows the normal levels of blood cells (red blood cells, white blood cells, platelets) in Western infants, children, and adolescents.

[0038] Figure 6 is a graph showing the effect of increased chondrocyte proliferation following exosome treatment.

[0039] Figure 7 is a graph showing the effect of increasing the survival rate of aging-induced skeletal muscle cells following exosome treatment.

[0040] Figure 8 shows the reduction in α-SMA protein expression, a fibrosis marker, and the fibrosis inhibitory effect according to concentration in the group treated with exosomes derived from young rat plasma.

[0041] Figure 9 shows the effect of reducing the expression of α-SMA protein, a fibrosis marker, in a group treated with exosomes derived from young rat plasma in liver cells according to the pretreatment substance.

[0042] Figure 10 shows the effect of reducing the expression of α-SMA protein, a fibrosis marker, in a group treated with exosomes derived from young rat plasma in kidney cells according to the pretreatment substance.

[0043] Figure 11 shows the effect of reducing the expression of α-SMA protein, a fibrosis marker, in a group treated with exosomes derived from young rat plasma in cardiac cells according to the pretreatment substance.

[0044] The present invention relates to a method for isolating and cryopreserving extracellular vesicles, and more specifically, to a method for cryopreserving extracellular vesicles selected by evaluating the functional characteristics of extracellular vesicles isolated from a biologically derived material, comprising the steps of: isolating extracellular vesicles from a biologically derived material; evaluating the functional characteristics of the extracellular vesicles; and selecting extracellular vesicles based on the functional characteristics and cryopreserving the selected extracellular vesicles.

[0045] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0046] FIG. 1 is a diagram showing the configuration of a method for providing extracellular vesicle separation and cryopreservation services according to the present invention.

[0047] The method for isolating and freezing extracellular vesicles according to the present invention comprises the steps of: isolating extracellular vesicles from a biologically derived material; evaluating the functional characteristics of the extracellular vesicles; and selecting extracellular vesicles based on the functional characteristics and freezing the selected extracellular vesicles.

[0048] The process of isolating extracellular vesicles from biologically derived materials involves efficiently isolating them for storage purposes, thereby enabling the securing of high-purity extracellular vesicles. This includes, but is not limited to, the use of centrifuges based on the size and density of the extracellular vesicles as well as the use of filters; any method capable of isolating extracellular vesicles may be used.

[0049] The purpose of isolating extracellular vesicles and cryopreserving them may be either growth promotion or anti-aging, and the optimal biological material for extracellular vesicle isolation can be selected depending on the storage purpose. Biological materials capable of isolating extracellular vesicles may include blood, plasma, lymph fluid, aqueous humor, cerebrospinal fluid, etc., but are not limited to any biological material capable of isolating extracellular vesicles. Meanwhile, if the biological material is blood, problems with the yield and function of the isolated extracellular vesicles may occur if a freezing process is performed before isolating them, so it may be stored at room temperature or refrigerated. It is preferable to store it at 1°C to 30°C for less than 24 hours, or to isolate it into serum or plasma within 24 hours and store it frozen (-20°C or lower) or ultra-low temperature frozen (-70°C to -90°C).

[0050] The functional characteristics of extracellular vesicles may include at least one of a cell proliferation-promoting effect, a cell viability-increasing effect, or a fibrosis-inhibiting effect. The cell proliferation-promoting effect is related to growth promotion and can be evaluated by an increase in the proliferation rate of chondrocytes. The cell viability-increasing effect and the fibrosis-inhibiting effect are related to anti-aging, the cell viability-increasing effect can be evaluated by an increase in viability in aging-induced cells, and the fibrosis-inhibiting effect can be evaluated by a decrease in α-SMA protein expression.

[0051] To enhance the functional characteristics of extracellular vesicles, a bio-derived material may be pretreated before the separation of extracellular vesicles. The pretreatment material may include at least one of Exendin-4, Phorbol 12-myristate 13-acetate (PMA), Interferon-γ, Tetrandrine, Hyaluronic acid, Substance P, Resveratrol, Rapamycin, Bezafibrate, or Lanifibranor.

[0052] Quality inspections may be performed on extracellular vesicles selected based on functional characteristics before freezing. Quality inspections may include appearance, number of extracellular vesicles, size of extracellular vesicles, negative test for Mycoplasma, negative test for exotic viruses, sterility test, endotoxin test, identification test, purity test, and potency test. Items related to quality inspection and quality inspection standards for each item are shown in Fig. 2. Meanwhile, the method for isolating and freezing extracellular vesicles according to the present invention may include a thawing step and a quality inspection step after the freezing step, and the quality inspection may be performed using the same items as those used in the quality inspection before freezing. This quality inspection step after thawing allows for quality control by comparing the quality before and after freezing by performing quality inspections on the same items as those used in the quality inspection step for the extracellular vesicles isolated before freezing.

[0053] Extracellular endoplasmic reticulum can be frozen at -70°C to -90°C. Cryopreservatives may be optionally included, or frozen without them. For long-term freezing (more than 1 month), freezing at -80°C is the optimal condition, as this condition is most effective for the morphological stability of extracellular endoplasmic reticulum particles, preservation of protein and RNA content, and maintenance of biological function. Meanwhile, short-term freezing (less than 1 month) is possible at -20°C to 4°C. If the temperature conditions fall outside the above range during long-term or short-term freezing, problems such as an increase in the size of extracellular endoplasmic reticulum and a decrease in biological activity may occur. Furthermore, if cryopreservatives are optionally included, the addition of cryoprotectants such as trehalose may be effective.

[0054] The storage period for extracellular vesicles can be from 1 month to 120 months. Meanwhile, if additional storage is required, the storage period can be reset to 1 month to 120 months for further storage. As can be seen from the freezing conditions below, unlike cells, extracellular vesicles can be stored without problems such as quality degradation even during long-term storage when stored under ultra-low temperature freezing (-70℃ to -90℃). Therefore, in addition to the initial storage period of 120 months (10 years), additional storage for 120 months (10 years) or longer is possible.

[0055] The method for isolating and cryopreserving extracellular vesicles according to the present invention may additionally provide information on the subject or donor, who is the provider of the biological material for isolating the extracellular vesicles, depending on the purpose of storage of the extracellular vesicles. The subject or donor information may include gender, age, weight, height, blood test results, and disease information in addition to basic personal details such as name and resident registration number. Depending on the purpose and duration of storage of the extracellular vesicles, various additional information may be provided in addition to the above information.

[0056] When the purpose of extracellular endoplasmic reticulum storage is growth promotion, in addition to basic information, information such as height at birth, weight at birth, head circumference, father's height, mother's height, target height (calculated as (father's height + mother's height) / 2 + 6.5 cm for boys, and (father's height + mother's height) / 2 - 6.5 cm for girls), whether growth hormone was used and the duration of use, whether growth-related diseases were diagnosed, whether there is a family history of precocious puberty, and whether there is a growth disorder. An example of specific subject information provided when the purpose of extracellular endoplasmic reticulum storage is growth promotion is shown in Figure 3.

[0057] Blood test information may include red blood cell counts, white blood cell counts, and platelet counts, and if the purpose of extracellular vesicle storage is growth promotion, it may include information on IGF-1 (insulin-like growth factor 1) concentration and the ratio of IGF-1 to IGFBP3 (IGF Binding Protein). Normal blood cell (red blood cells, white blood cells, platelets) values ​​for Asian infants, children, and adolescents are shown in Fig. 4, and normal blood cell (red blood cells, white blood cells, platelets) values ​​for Western infants, children, and adolescents are shown in Fig. 5.

[0058] IGF-1 (Insulin-like Growth Factor-1) is a protein hormone primarily produced in the liver that acts as a major mediator of growth hormone, regulating cell proliferation, differentiation, and survival. In particular, IGF-1 acts on chondrocytes in the growth plate to directly influence bone growth and body size development. Rather than existing alone, IGF-1 circulates mostly in the bloodstream bound to IGFBP-3 (Insulin-like Growth Factor Binding Protein-3), a form essential for maintaining IGF-1's stability and regulating biological responses. IGFBP-3 is the primary carrier of IGF-1, indirectly regulating the intensity of growth stimulation by controlling IGF-1's direct binding to receptors. Physiologically, blood concentrations of IGF-1 and IGFBP-3 increase rapidly during puberty, which is closely associated with the growth spurt. Numerous domestic and international studies have shown that the concentrations of IGF-1 and IGFBP-3 reach a peak during early puberty and then gradually decline, with variations depending on age and gender. Growth hormone deficiency, nutritional deficiencies, and chronic diseases reduce IGF-1 and IGFBP-3 levels, and these values ​​are clinically utilized for the diagnosis of growth disorders and the monitoring of responses to growth therapy. In particular, the IGF-1 / IGFBP-3 molar ratio serves as an indicator reflecting the activity of free IGF-1 and is useful for assessing the effects of growth promotion. Furthermore, free IGF-1 exhibits stronger biological activity than its bound form and has been reported to be directly involved in the rapid skeletal growth during puberty. Accordingly, the regulation of IGF-1 and IGFBP-3 activity is utilized as a primary target in the development of biomaterials aimed at growth promotion or in therapeutic approaches utilizing extracellular vesicles.

[0059] When the purpose of extracellular vesicle storage is anti-aging, additional information on muscle mass, muscle strength, inflammation, and hormones—biomarkers related to sarcopenia in the subject or donor—may be included. Muscle mass information may include calf circumference, mid-arm muscle circumference (MAMC), and bioelectric impedance analysis (BIA); muscle strength information may include handgrip strength (IHG) and five-time sit-to-stand test (5STS); inflammation information may include C-reactive protein (CRP) levels; and hormone information may include DHEAS (Dehydroepiandrosterone sulfate) levels. Specific criteria and values ​​considered to be associated with biomarkers and sarcopenia are shown in Table 1 below.

[0060] Biomarkers Related to Sarcopenia and Criteria for Assessing Sarcopenia Meaning of Cut-off Values Muscle Mass Calf circumference: If it is 31 cm or less, muscle mass is considered low. If the calf circumference is smaller than 31 cm, there is a possibility of sarcopenia. Mid-arm muscle circumference (MAMC): Men 21.0–24.9 cm, Women 19.8–23.3 cm. If it is below this range, there is a risk of sarcopenia. Bioelectric Impedance Analysis (BIA): Muscle Mass Index (SMI) criteria: Men < 7.0 kg / m², Women < 5.5 kg / m². If the value is lower than this, muscle mass is insufficient. Muscle Strength Handgrip strength (IHG): Men < 27 kg, Women < 16 kg. If grip strength is lower than this value, muscle strength is considered reduced. Five-time Sit-to-Stand Test (5STS): 5 times Taking more than 15 seconds to stand up (slow speed) -> Possible decline in muscle strength or lower body function Inflammation C-reactive protein (CRP) - Generally, CRP indicates inflammation levels, and chronic inflammation is associated with sarcopenia Hormone DHEAS (Dehydroepiandrosterone sulfate) - DHEAS is an aging-related hormone, and a decrease in levels may be associated with sarcopenia

[0061] Disease information may include growth-related diseases and systemic diseases, and the types of growth-related diseases and reference indicators that may be important information when the purpose of extracellular vesicle storage is growth promotion are as shown in Table 2 below.

[0062] Types of Growth-Related Diseases and Reference Indicators Types of Growth-Related Diseases and Reference Indicators Osteochondrodysplasia - A group of various diseases, among which achondroplasia is the most common. It occurs due to chromosomal abnormalities; the average adult height for males and females is approximately 130 cm and 120 cm, respectively. Chromosomal Abnormalities - Down syndrome, Turner syndrome, Noonan syndrome Intrauterine Growth Retardation (Small for Gestational Age) - Fetal weight or height is below the 3rd percentile (lower 3%), or at least less than an average of 2 standard deviations (SD) based on gestational age. Generally, full-term infants have a birth weight of less than 2,500 g. - In 10–20% of cases, catch-up growth does not occur, often resulting in short stature in adulthood. Syndromes Associated with Short Stature - Prader-Willi syndrome: A genetic disorder; hypotonia, feeding difficulties, and low birth weight are observed in infancy.Low serum growth hormone (IGF-1, IGFBP3) levels - Russell-Silver Syndrome: A genetic disorder characterized by intrauterine growth retardation, postnatal growth failure, precocious puberty, and delayed bone age. - Noonan Syndrome: A genetic disorder characterized by delayed or incomplete puberty, winged neck, and pulmonary artery valve abnormalities. - Idiopathic short stature - Height is 2 standard deviations (Height SDS < -2) or in the 3rd percentile, below the bottom 3%. - No apparent cause for short stature (body type, nutritional intake, and weight are within the normal range). - Genetic (familial) short stature - Explained by the criteria below, but the diagnostic criteria themselves are somewhat ambiguous. - Parents are short, in the 10th percentile (bottom 10%), the onset and progression of puberty are nearly normal, and bone age is within 2 standard deviations of actual age. - The child's final adult height is short but within the target height range based on parental height. - Constitutional growth retardation - Height due to a decrease in growth rate during the first 3 years of life. Drops below the 3rd percentile (bottom 3%), and thereafter maintains a normal growth rate, keeping the growth curve parallel to the 3rd percentile (bottom 3%) - Height age and bone age are delayed by about 2-4 years compared to actual age, and puberty is also delayed - Final adult height is mostly within the normal range, but may be slightly shorter than the target height, and may be even shorter in cases of familial short stature. Growth hormone deficiency - Can be caused by pituitary dysfunction or damage such as brain tumors, congenital malformations, or intracranial radiation exposure, - Also includes idiopathic growth hormone deficiency where the cause cannot be found - Growth rate is less than 4 cm per year or less than 2 standard deviations (bottom 3%) - Diagnosed by growth hormone secretion stimulation tests (insulin loading tests), etc. Kidney disease - Growth impairment related to chronic renal insufficiency up to the time of kidney transplant - Growth hormone therapy is administered in conjunction with the management of chronic renal insufficiency.

[0063] In addition, the above disease information may include hepatitis B (HBV), hepatitis C (HCV), acquired immunodeficiency syndrome (HIV 1 / 2), syphilis, human T-lymphotropic virus (HTLV), cytomegalovirus (CMV), chlamydia, and gonorrhea of ​​the donor.

[0064]

[0065] Experimental Example 1. Increase in chondrocyte proliferation following exosome treatment (growth-promoting effect)

[0066] To confirm the effect of increased cartilage cell proliferation following exosome treatment, the proliferation-promoting effect of human blood-derived exosomes was confirmed by treating cartilage cells with different concentrations of exosomes according to age group.

[0067] Human blood-derived exosomes were used from exosomes isolated from the blood of 7-year-olds (Young Exo(age 7)), 10-year-olds (Young Exo(age 10)), 12-year-olds (Young Exo(age 12)), and adults (adult Exo).

[0068] Chondrocytes were seeded at a density of 3 × 10³ per well in a 96-well plate and cultured in maintenance medium for 24 hours. Subsequently, the medium was replaced with serum-free medium, and blood-derived adult exosomes or age-specific Young exosomes (7, 10, and 12 years) were added at concentrations of 50, 150, and 250 μg / mL for 24 hours. After treatment, CCK-8 solution was added at a volume equal to 1 / 10 of the culture medium and incubated for 3 hours, after which the cell proliferation rate was measured.

[0069] As a result, as shown in Figure 6, the groups treated with human blood-derived Young exosomes (7, 10, and 12 years old) showed an overall tendency for increased chondrocyte proliferation compared to the PBS control group and the adult exosome treatment group. In particular, the most pronounced increase in proliferation was observed in the 7-year-old blood-derived exosome treatment group. Additionally, the proliferation-promoting effect was greater in the high-concentration treatment group than in the low-concentration treatment group, confirming that the chondrocyte proliferation-promoting effect of blood-derived Young exosomes (7, 10, and 12 years old) increased in a concentration-dependent manner.

[0070]

[0071] Experimental Example 2. Increase in the survival rate of aging-induced skeletal muscle cells following exosome treatment (anti-aging effect)

[0072] To confirm the effect of exosome treatment on increasing the survival rate of aging-induced skeletal muscle cells, the effect of improving the survival rate of aging-induced skeletal muscle cells was confirmed by treating aging-induced skeletal muscle cells with human blood-derived exosomes of different age groups at various concentrations.

[0073] Human blood-derived exosomes were used from exosomes isolated from the blood of 10-year-olds (Young Exo(age 10)), 11-year-olds (Young Exo(age 11)), and 14-year-olds (Young Exo(age 14)).

[0074] Skeletal muscle cells were seeded at a density of 3 × 10³ per well in a 96-well plate and cultured in maintenance medium for 24 hours. Subsequently, cellular senescence was induced by treatment with 10 μM palbociclib for 24 hours. After senescence induction, the cells were washed once with PBS and replaced with serum-free medium. Then, blood-derived Young exosomes of different age groups (10, 11, and 14 years) were added at concentrations of 0, 12.5, 25, 50, and 100 μg / mL for 24 hours. After treatment, CCK-8 solution was added to 1 / 10 of the culture volume and incubated for 3 hours, after which cell viability was measured.

[0075] As a result, as shown in Figure 7, when aging-induced skeletal muscle cells were treated with blood-derived Young exosomes (10, 11, and 14 years), the overall cell survival rate increased compared to the PBS control group. In addition, the increase in survival rate was more pronounced in the high-concentration treatment group than in the low-concentration treatment group, confirming that blood-derived Young exosomes (10, 11, and 14 years) improve the survival rate of aging-induced skeletal muscle cells and that the effect is concentration-dependent.

[0076]

[0077] Experimental Example 3. Inhibition of fibrosis in renal tubular cells by exosome treatment (anti-aging effect)

[0078] To confirm the inhibitory effect of exosome treatment on fibrosis in renal tubular cells, exosomes derived from young rat plasma were treated to human renal proximal tubular cells at various concentrations to confirm the inhibitory effect on fibrosis in renal tubular cells. Meanwhile, to evaluate renal fibrosis, which has a high risk of developing in relation to aging, a renal proximal tubular cell model treated with TGF-β1 was used.

[0079] Human kidney proximal tubule cells were seeded in a 6-well plate and cultured in maintenance medium until cell confluency reached 90–100%. Subsequently, fibrosis was induced by treating the cells with 100 ng / mL TGF-β1 and exosomes derived from young rat plasma at concentrations of 0.5, 1, 2, and 4 μg / mL for 24 hours, while simultaneously evaluating the effects of the exosomes. After 24 hours of treatment, cells were harvested and subjected to western blot, and the expression levels of α-SMA protein, a fibrosis marker, were measured.

[0080] As a result, as shown in Figure 8, a tendency for the expression of α-SMA protein, a fibrosis marker, to decrease was observed in the group treated with exosomes derived from young rat plasma compared to the group treated with PBS. In addition, as the concentration of exosome treatment increased, α-SMA expression gradually decreased, confirming that exosomes derived from young rat plasma inhibit the fibrosis response of kidney cells in a concentration-dependent manner.

[0081]

[0082] Experimental Example 4. Inhibition of Fibrosis in Liver Cells by Exosome Treatment (Anti-aging Effect)

[0083] To confirm the inhibitory effect of exosome treatment on liver cell fibrosis, human liver cells were treated with exosomes derived from young rat plasma. Additionally, to verify that the functional characteristics of exosomes are enhanced through pretreatment with bio-derived materials for exosome isolation, exosomes isolated after treating young rat plasma cells with the pretreatment substance were treated with human liver cells to compare the inhibitory effect on liver cell fibrosis. Meanwhile, a liver cell model treated with TGF-β1 was used to evaluate liver fibrosis, which carries a high risk of occurrence associated with aging.

[0084] Human hepatocytes were seeded in a 6-well plate and cultured in maintenance medium until cell confluency reached 90-100%. Subsequently, fibrosis was induced by treatment with 10 ng / mL TGF-β1 for 24 hours. Then, exosomes derived from young rat plasma and young rat plasma cells were treated with exosomes pretreated with hyaluronic acid (HA) (HA-Young Exo), rapamycin (Rapa-Young Exo), and resveratrol (Res-Young Exo), respectively, at a concentration of 4 μg / mL for 24 hours. After treatment, cells were harvested and subjected to western blot analysis, and the expression level of α-SMA protein, a fibrosis marker, was measured.

[0085] As a result, as shown in Figure 9, there were differences in the inhibitory effect of exosomes on liver fibrosis depending on the pretreatment conditions of young rat plasma cells. In particular, it was confirmed that the expression of α-SMA protein, a fibrosis marker, decreased to a level similar to that of the control group in the group treated with exosomes pretreated with resveratrol (Res-Young Exo).

[0086]

[0087] Experimental Example 5. Inhibition of Fibrosis in Kidney Cells by Exosome Treatment (Anti-aging Effect)

[0088] To confirm the inhibitory effect of exosome treatment on renal cell fibrosis, exosomes derived from young rat plasma were treated to human renal proximal tubule cells to verify the inhibitory effect. In addition, to confirm that the functional characteristics of exosomes are enhanced through pretreatment with bio-derived materials for exosome isolation, exosomes isolated after treating young rat plasma cells with the pretreatment material were treated to human renal proximal tubule cells to compare the inhibitory effect on fibrosis. Meanwhile, a renal cell model treated with TGF-β1 was used to evaluate renal fibrosis, which has a high risk of occurrence associated with aging.

[0089] Human kidney proximal tubule cells were seeded in 6-well plates and cultured in maintenance medium until cell confluency reached 90–100%. Subsequently, fibrosis was induced and the effects of exosomes were evaluated by treating the cells with 100 ng / mL TGF-β1 along with young rat plasma-derived exosomes (Young Exo), exosomes pretreated with hyaluronic acid (HA) (HA-Young Exo), exosomes pretreated with rapamycin (Rapa-Young Exo), and exosomes pretreated with resveratrol (Res-Young Exo) at a concentration of 4 μg / mL for 24 hours. After treatment, cells were harvested and subjected to western blot analysis, and the expression levels of α-SMA protein, a fibrosis marker, were measured.

[0090] As a result, as shown in Figure 10, all exosomes exhibited antifibrotic effects. In particular, it was confirmed that the expression of α-SMA protein, a fibrosis marker, was significantly reduced in the group treated with exosomes pretreated with resveratrol (Res-Young Exo).

[0091]

[0092] Experimental Example 6. Inhibition of Fibrosis in Cardiac Cells by Exosome Treatment (Anti-aging Effect)

[0093] To confirm the inhibitory effect of exosome treatment on cardiac cell fibrosis, human cardiac fibroblasts were treated with exosomes derived from young rat plasma to verify the inhibitory effect. In addition, to confirm that the functional characteristics of exosomes are enhanced through pretreatment with bio-derived materials for exosome isolation, exosomes isolated after treating young rat plasma cells with the pretreatment material were treated with human cardiac fibroblasts to compare the inhibitory effect on fibrosis. Meanwhile, a cardiac cell model treated with TGF-β1 was used to evaluate cardiac fibrosis, which has a high risk of developing in relation to aging.

[0094] Human cardiac fibroblasts were seeded in a 6-well plate and cultured in maintenance medium until cell confluency reached 90-100%. Subsequently, fibrosis was induced by treating with 10 ng / mL TGF-β1 for 24 hours, and then young rat plasma-derived exosomes (Young Exo), exosomes pretreated with hyaluronic acid (HA) (HA-Young Exo), exosomes pretreated with rapamycin (Rapa-Young Exo), and exosomes pretreated with resveratrol (Res-Young Exo) were treated at a concentration of 4 μg / mL for 24 hours to induce fibrosis while simultaneously evaluating the effects of the exosomes. After treatment, cells were harvested and western blot was performed, and the expression level of α-SMA protein, a fibrosis marker, was measured.

[0095] As a result, as shown in Figure 11, all exosomes exhibited antifibrotic effects. In particular, it was confirmed that the expression of α-SMA protein, a fibrosis marker, was significantly reduced in the group treated with exosomes pretreated with resveratrol (Res-Young Exo).

[0096] The present invention described above is not limited by the aforementioned embodiments and attached drawings, as various substitutions, modifications, and changes are possible within the scope of the technical concept of the present invention without departing from the technical spirit of the present invention to those skilled in the art.

[0097] The present invention relates to a method for isolating and freezing extracellular vesicles, and more specifically, to a method for freezing extracellular vesicles selected by evaluating the functional characteristics of extracellular vesicles isolated from biologically derived materials.

Claims

1. A method for isolating and storing extracellular vesicles from biologically derived material, A step of isolating extracellular vesicles from biologically derived material; A step of evaluating the functional characteristics of the above extracellular vesicles; and A step of selecting extracellular vesicles based on the above functional characteristics and freezing the selected extracellular vesicles; A method for isolating and freezing extracellular endoplasmic reticulum including 2. In Paragraph 1, A method in which the above-mentioned biological material includes blood, plasma, lymph fluid, aqueous humor, or cerebrospinal fluid.

3. In Paragraph 1, The above functional characteristics include at least one of a cell proliferation promoting effect, a cell viability increasing effect, or a fibrosis inhibiting effect.

4. In Paragraph 3, The above cell proliferation-promoting effect is evaluated by the increase in the proliferation rate of chondrocytes.

5. In Paragraph 3, A method in which the above-mentioned effect of increasing cell viability is evaluated by increasing viability in aging-induced cells.

6. In Paragraph 3, The above fibrosis inhibitory effect is evaluated by a method of reducing α-SMA protein expression.

7. In Paragraph 1, A method for performing pretreatment to improve the functional characteristics of the above extracellular vesicles.

8. In Paragraph 7, A method comprising at least one of the above-mentioned pretreatment material, Exendin-4, Phorbol 12-myristate 13-acetate (PMA), Interferon-γ, Tetrandrine, Hyaluronic acid, Substance P, Resveratrol, Rapamycin, Bezafibrate, or Lanifibranor.

9. In Paragraph 1, A method for performing a quality inspection of extracellular vesicles before the above-mentioned frozen storage.

10. In Paragraph 9, The above quality inspection method includes at least one of the following: appearance, number of extracellular vesicles, size of extracellular vesicles, mycoplasma negative test, exogenous virus negative test, sterility test, endotoxin test, identification test, purity test, and potency test.

11. In Paragraph 1, The above freezing method is a freezing method of -70°C to -90°C in the absence of a freeze-preservative.

12. In Paragraph 1, A method comprising the step of thawing the above-mentioned frozen extracellular vesicles and re-evaluating their functional properties.

Images