Roller bottle culture method for stem cells for mass production of extracellular vesicles

Abstract

The present invention relates to a method for culturing stem cells and a method for isolating stem cell-derived extracellular vesicles in which a roller bottle for enhancing the yield and quality of the isolation and purification of extracellular vesicles is used. When exosomes or extracellular vesicles were isolated from a culture medium obtained by culturing the stem cells using the roller bottle method, the yield was significantly increased and high-quality extracellular vesicles highly expressing CD9 and CD81 could be obtained. Accordingly, the method for culturing stem cells using a roller bottle according to the present invention can be used to mass-culture stem cells and separate and purify exosomes and extracellular vesicles with high yield.

Description

Roller bottle culture method of stem cells for mass production of extracellular vesicles

[0001] The present invention relates to a method for culturing stem cells for mass production of extracellular vesicles and a method for isolating stem cell-derived extracellular vesicles.

[0002]

[0003] Extracellular vesicles (EVs) are vesicles released by cells outside the cell. EVs are membrane-bound vesicles with diameters ranging from approximately 20 nm to approximately 5 μm, and include exosomes, ectosomes, microvesicles, microparticles, and apoptotic bodies.

[0004] Among extracellular vesicles, exosomes are the smallest vesicular particles, measuring approximately 50-200 nm in diameter. "Stem Cell Research, vol. 1, no. 2, pp. 129-137, 2008" reported that exosomes, a component of stem cell culture medium measuring 50-200 nm in diameter, are effective in reducing scarring after myocardial infarction.

[0005] Exosomes, which facilitate intercellular communication through the exchange of proteins and RNA, also function to expel unnecessary intracellular substances. Because they contain microRNA (miRNA), they are expected to be useful markers for molecular diagnostics, such as the early detection of diseases like cancer. Exosomes are secreted by nearly all cells as mediators of material transport for the exchange of information between cells, and serve as mediators of immune regulation, cancer metastasis, and signaling between neurons and glial cells.

[0006] While the importance and value of exosomes or extracellular vesicles are being recognized, producing large quantities of them, isolating them, and collecting them poses significant challenges. Exosomes or extracellular vesicles can only be produced by cells, and their secretion is limited to trace amounts. Therefore, the development of methods for mass production of exosomes or extracellular vesicles is essential.

[0007] Despite various efforts to date for suspension culture, such as spheroids (aggregates), microcarriers, and microencapsulation, stem cells are highly dependent on the matrix during cell culture and easily lose their characteristics depending on the culture environment, making it difficult to achieve mass production using existing cell culture methods.

[0008]

[0009] Accordingly, the present inventors sought to develop a method for culturing stem cells to mass-produce exosomes or extracellular vesicles and a method for isolating stem cell-derived extracellular vesicles using the same.

[0010]

[0011] The present inventors have made extensive efforts to develop a method for culturing stem cells and isolating extracellular vesicles in order to mass-produce exosomes or extracellular vesicles. As a result, they discovered that the roller bottle culture method releases extracellular vesicles of small size and uniformity, increases the yield after purification, increases the content of CD9 and CD81 proteins, and effectively suppresses NO production even in small amounts, thereby completing the present invention.

[0012] The purpose of the present invention is to provide a method for culturing stem cells for mass production of extracellular vesicles.

[0013] Another object of the present invention is to provide a method for isolating stem cell-derived extracellular vesicles for mass production of extracellular vesicles.

[0014]

[0015] The present invention relates to a stem cell culture method using a roller bottle for producing stem cell-derived extracellular vesicles and a method for isolating stem cell-derived extracellular vesicles. Culturing stem cells using the above culture method produces exosomes or extracellular vesicles of good quality and high purification yield. Accordingly, the stem cell culture method of the present invention can produce stem cell-derived exosomes or extracellular vesicles in large quantities in a good manufacturing practice (GMP) pharmaceutical manufacturing facility.

[0016]

[0017] Figure 1 illustrates a method of culturing stem cells in a roller bottle. Figure 1(A) illustrates a method of culturing stem cells of the present invention in a roller bottle, and Figure 1(B) is an enlarged image of the cultured stem cells.

[0018] Figure 2 shows the results of comparing the proliferation capacity of stem cells after culturing them in a flask or roller bottle.

[0019] Figure 3 is a graph showing the particle size distribution and number of particles obtained by performing nanoparticle tracking analysis (NTA) on stem cell secondary culture (CM) obtained by culturing stem cells in a serum-free medium in a flask or roller bottle, or on extracellular vesicles (EV) separated and purified therefrom.

[0020] Figure 4 shows the results of measuring the number of nanoparticles and protein content of isolated exosomes (extracellular vesicles) cultured in a flask or roller bottle and the expression of stem cell marker CD105 and exosome markers CD9 and CD81.

[0021] Figure 5 shows the results of culturing stem cells in a flask or roller bottle and comparing their anti-inflammatory effects.

[0022] Figure 6 shows the results of performing NTA (nanoparticle tracking analysis) after 5 days of inoculating companion dog (beagle) adipose tissue-derived stem cells in a flask and roller bottle, replacing the culture medium with DMEM / F12 serum-free medium once a day, and collecting the culture medium (medium) a total of 3 times.

[0023]

[0024] As one embodiment for achieving the above object, the present invention provides a method for isolating stem cell-derived extracellular vesicles for mass production of extracellular vesicles, comprising: a first step of inoculating stem cells into a roller bottle and culturing them in a cell culture medium; a second step of replacing the cell culture medium with a serum-free medium and culturing them in the serum-free medium; and a third step of isolating and purifying extracellular vesicles by performing tangential-flow filtration (TFF).

[0025]

[0026] The method for isolating stem cell-derived extracellular vesicles of the present invention is for mass production of extracellular vesicles, and includes the first step of inoculating stem cells into a roller bottle and culturing them in a cell culture medium.

[0027] In the present invention, the term "stem cell" means an undifferentiated cell that has excellent proliferative capacity and the ability to differentiate into various tissues.

[0028] The above stem cells may be embryonic stem cells, adult stem cells, or induced pluripotent stem cells.

[0029] In the present invention, the term "embryonic stem cell" means a cell that is pluripotent or totipotent and can differentiate into cells of all tissues of an individual, which is cultured in vitro by extracting the inner cell mass from a blastocyst embryo just before the fertilized egg is implanted in the uterus of the mother.

[0030] The embryonic stem cells of the present invention may be derived from humans or animals, and specifically, may include embryonic stem cells of all origins, such as humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice, and rabbits.

[0031] In the present invention, the term "adult stem cell" refers to a stem cell that appears during the developmental stage, when each organ of an embryo is formed, or during the adult stage. Adult stem cells are generally limited in their differentiation potential to cells that constitute specific tissues. For example, bone marrow cells are destined to differentiate into blood cells, skin stem cells are destined to differentiate into skin cells, and olfactory neurons are destined to differentiate into olfactory neurons. These adult stem cells remain in most organs even after adulthood, playing a role in replenishing cell loss that occurs either normally or pathologically.

[0032] The above adult stem cells may be derived from a human or an animal, and include adult stem cells derived from all animals, including humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice, and rabbits.

[0033] The above adult stem cells may be of human or animal tissue origin or origin, and the human or animal tissue may be selected from the group consisting of brain, teeth, liver, ovary, peripheral blood, hair follicle, heart, umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta.

[0034] The above adult stem cells include adult stem cells derived from human or animal tissue, mesenchymal stem cells derived from human or animal tissue, or mesenchymal stem cells derived from induced pluripotent stem cells derived from human or animal tissue.

[0035] The adult stem cells of human or animal tissue origin may be selected from the group consisting of mesenchymal stem cells, endothelial stem cells, hair follicle stem cells, muscle stem cells, ovarian stem cells, cardiac stem cells, hematopoietic stem cells, mammary stem cells, intestinal stem cells, vascular endothelial stem cells, neural stem cells, olfactory nerve stem cells, adipose stem cells, and testicular stem cells.

[0036] As used herein, the term "mesenchymal stem cell" refers to a stem cell with multipotency that can differentiate into cells such as fat, cartilage, bone, muscle, skin, and nerve cells. In adults, these mesenchymal stem cells typically reside in bone marrow, but also exist in other tissues such as umbilical cord blood, peripheral blood, fat, and amniotic fluid.

[0037] The above mesenchymal stem cells are mesenchymal stem cells derived from human or animal tissues, and may be mesenchymal stem cells derived from bone marrow, adipose tissue, umbilical cord tissue, umbilical cord blood, skeletal muscle, peripheral blood, synovial membrane, amniotic fluid, or amniotic membrane, and the above mesenchymal stem cells may be differentiated from induced pluripotent stem cells.

[0038] The term "induced pluripotent stem cell (iPSC)" of the present invention refers to cells that are induced to have pluripotent differentiation ability through an artificial dedifferentiation process from already differentiated cells, such as somatic cells, and can be used with the same meaning as "dedifferentiated stem cell." By inducing dedifferentiation in differentiated cells, they return to their initial undifferentiated state and acquire pluripotency. The dedifferentiation can be induced by introducing and expressing specific genes, such as Sox2, c-Myc, Klf4, Oct-4, or by injecting a dedifferentiation-inducing protein produced in cells into which the specific genes have been introduced.

[0039] The term "pluripotency" of the present invention means the ability to differentiate into tissues or organs derived from the three germ layers that constitute a living body: endoderm, mesoderm, and ectoderm.

[0040] The above induced pluripotent stem cells are derived from human or animal tissues, and specifically include induced pluripotent stem cells derived from all mammals, including humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice, and rabbits.

[0041] In the present invention, “mesenchymal stem cells derived from induced pluripotent stem cells (iPSCs)” means mesenchymal stem cells (iMSCs) differentiated from induced pluripotent stem cells.

[0042] The above term "cell culture medium" refers to a medium that can support the growth and survival of cells in vitro, and includes all media commonly used in the relevant field suitable for cell culture. Examples of the medium may include DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), DMEM / F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), etc., and includes media commonly used for stem cell culture in the relevant field. The medium generally includes a carbon source, a nitrogen source, and trace element components, and may further include amino acids, antibiotics, etc.

[0043] In the present invention, the term "roller bottle" refers to a cylindrical culture bottle. The culture step using a roller bottle is a culture method in which cells are attached to the inside of a cylindrical culture bottle, the bottle is laid down, and rotated in a rotating incubator. It is a method for mass culturing wall-adherent cells, and can increase the production of stem cells with a small amount of culture medium by maximizing the surface area to which stem cells can attach.

[0044] Figure 1 illustrates a method of culturing stem cells in a roller bottle. As shown in Figure 1, the stem cells can be cultured by rotating the roller bottle at a rotation speed of 0.25 to 2.0 rpm. If the rotation speed of the roller bottle is less than 0.25 rpm, the rotation speed is too low, which has the disadvantage of lowering the proliferation rate of the stem cells. In addition, if it exceeds 2.0 rpm, it causes stress to the stem cells, which lowers the attachment or proliferation rate. Therefore, the rotation speed of the roller bottle is appropriately 0.25 to 2.0 rpm.

[0045] In the step of inoculating stem cells into the roller bottle and attaching the cells, the culture bottle (roller bottle) is rotated at 0.25 to 0.3 rpm to attach the stem cells for 0.5 to 1.5 days, specifically 1 day. Thereafter, the culture is rotated at 0.5 to 2.0 rpm, specifically 0.8 to 1.2 rpm, to culture for 3.5 to 4.5 days, specifically 4 days, to proliferate the stem cells.

[0046] In the above culture step 1, the surface area of ​​the roller bottle cm 2 Stem cells can be cultured by inoculating 1,000 to 5,000 cells per cm surface area. Specifically, 2 Stem cells can be cultured by inoculating 1,500 to 4,500 cells per cell. The surface area of ​​the roller bottle used for stem cell culture is 700 to 1,000 cm 2 can be used. In addition, multiple roller bottles can be used for simultaneous culture of stem cells during a single culture, enabling mass production of stem cells. Accordingly, mass production of extracellular vesicles or exosomes isolated and purified from stem cells mass-produced and produced also becomes possible.

[0047] The method for isolating stem cell-derived extracellular vesicles of the present invention includes two steps: replacing a cell culture medium with a serum-free medium and then culturing in the serum-free medium.

[0048] The term "serum-free medium" in this step refers to a conventional cell culture medium from which serum has been removed. Serum commonly used in cell culture, including fetal bovine serum, contains large amounts of exosomes or extracellular vesicles. Therefore, by using a serum-free medium, serum-free exosomes or extracellular vesicles are prevented from being mixed in. Therefore, only exosomes or extracellular vesicles produced by stem cells are included in the culture medium.

[0049] In the second stage of the culture of the present invention, stem cells are cultured in a serum-free medium for 0.5 to 5 days, specifically 1 to 4 days, and then the stem cell culture medium can be obtained up to 3 to 4 times in total. Specifically, the stem cell culture medium can be recovered once after culturing the stem cells in a serum-free medium for 1 day. In addition, the culture medium can be removed, a serum-free medium can be added to the remaining stem cells, and the stem cell culture medium can be additionally recovered after culturing for another day. In addition, the culture medium can be removed again, a serum-free medium can be added to the remaining stem cells, and the stem cell culture medium can be additionally recovered after culturing for another 1 to 2 days. In this way, the stem cell culture medium can be recovered up to 3 to 4 times per roller bottle.

[0050] In this way, the stem cell culture process using serum-free media enables stable production of extracellular vesicles, and the culture medium can be recovered up to 3-4 times. Consequently, the production of exosomes or extracellular vesicles per roller bottle increases, enabling the mass production and isolation of extracellular vesicles through stem cell culture using multiple roller bottles.

[0051] The method for separating extracellular vesicles derived from stem cells of the present invention includes three steps of separating and purifying extracellular vesicles by performing tangential-flow filtration (TFF).

[0052] Step 3 of the present invention is a step of culturing stem cells in a serum-free medium, then recovering the culture solution and isolating and purifying exosomes or extracellular vesicles.

[0053] The term "extracellular vesicles (EVs)" used herein refers to vesicles released by cells into the extracellular space. All cells secrete EVs, a phenomenon conserved throughout evolution, extending from bacteria to humans and plants. EVs facilitate the exchange of materials between cells, including proteins, lipids, and genetic material, and serve as mediators for physiological and pathological signaling.

[0054] Extracellular vesicles are broadly classified into exosomes and microvesicles. Exosomes are intraluminal vesicles, approximately 50-200 nm in size, created when the endosomal membrane of multi-vesicular endosomes inwardly matures, and are secreted when multi-vesicular endosomes fuse with the cell surface. Microvesicles are vesicles, 50-1000 nm in size, secreted outside the cell by protrusion of the plasma membrane.

[0055] Cells produce and secrete extracellular vesicles with specific lipid / protein / nucleic acid compositions depending on their physiological state. The specific composition of an extracellular vesicle determines its function and fate.

[0056] The term "exosome" of the present invention refers to a vesicle composed of a lipid bilayer derived from a cell and constitutes an extracellular vesicle. Exosomes are secreted out of the cell, containing cell-specific proteins, RNA, etc., and deliver proteins and RNA, etc. to other cells. The proteins contained in exosomes are protected by phospholipids in the form of cell membranes, so they do not easily lose activity due to proteolytic enzymes, etc., and can perform a more stable function than simple proteins in a soluble form. In addition, since the lipid bilayer structure easily fuses with the cell membrane, substances within exosomes can be efficiently delivered to cells.

[0057] The above extracellular vesicles or exosomes can be obtained from the culture medium obtained after culturing cells. The extracellular vesicles or exosomes contain RNA, proteins, lipids, and metabolites reflecting the cell type from which the extracellular vesicles or exosomes are derived. The extracellular vesicles or exosomes contain various molecular components (e.g., proteins and RNA) of the cell from which the exosomes are derived.

[0058] The above term "culture medium" refers to a culture obtained by culturing isolated stem cells in a liquid medium that can support growth and survival in a test tube for a certain period of time, or a culture supernatant obtained by removing stem cells from the culture.

[0059] In the present invention, extracellular vesicles or exosomes can be separated and purified by performing tangential-flow filtration (TFF).

[0060] First, in order to remove stem cells from the culture medium of the recovered stem cells, a step of obtaining a first supernatant by centrifugation at 1000 rpm to 3000 rpm for 10 to 30 minutes, specifically at 2000 rpm for 20 minutes; a step of filtering the first supernatant with a filter having a size of 0.1 to 0.3 ㎛, specifically, 0.2 ㎛, to obtain a second culture medium; a step of concentrating the second culture medium using ultrafiltration with a size of 500 to 1000 kda, specifically, a tangential flow filter, until the volume of the culture medium becomes 1 / 100 to 1 / 25, specifically, 1 / 50, to obtain a third concentrate; and a step of exchanging the third concentrate with a phosphate buffer solution is repeated twice to isolate and purify extracellular vesicles or exosomes from the culture medium of the stem cells.

[0061] In the present invention, a hollow fiber filter can be used for the tangential flow filtration.

[0062] In one embodiment of the present invention, in order to remove cells from the recovered culture medium, centrifugation was performed at 2000 rpm for 20 minutes to obtain a first supernatant, and in order to remove cell debris from the first supernatant, the second supernatant was filtered through a 0.2 ㎛ filter to obtain a second culture medium, and the second culture medium was passed through a 750 kda hollow fiber filter (TFF filter) to concentrate until the volume of the culture medium became 1 / 50 to obtain a third concentrate. The third concentrate was buffer-exchanged with a phosphate buffer solution to isolate and purify extracellular vesicles or exosomes from the stem cell culture medium.

[0063] In one embodiment of the present invention, after culturing canine adipose tissue-derived stem cells, which are adult stem cells, in a cell culture medium, and then culturing them in a serum-free medium for 1 day, it was confirmed that the quality of exosomes or extracellular vesicles contained in extracellular vesicles was improved in the recovered cell culture medium compared to the existing flask culture method. Specifically, compared to the existing flask culture method, the exosomes or extracellular vesicles isolated and purified from the stem cell culture medium of the present invention had a larger number of extracellular vesicle particles per unit volume, a smaller and more uniform size, an increased total protein content, and an increased content of extracellular vesicle marker proteins CD9 and CD81, and it was confirmed that even a small amount effectively inhibited NO production.

[0064] In the present invention, the proliferation rate of stem cells by the culture method using the roller bottle is reduced by 20 to 40% compared to the culture method using a flask. Fig. 1 shows a state in which stem cells are cultured in a roller bottle. As shown in Fig. 1, when multiple roller bottles are installed to culture stem cells, mass production of stem cells is possible. In addition, as described above, since the stem cell culture solution can be recovered up to 3 to 4 times by replacing the serum-free medium, the recovery amount of the stem cell culture solution cultured in the serum-free medium increases compared to the conventional flask-using method. Accordingly, although the proliferation rate of stem cells is relatively lower than that of the flask-using method, the production amount of extracellular vesicles to be separated and purified increases.

[0065] In addition, the stem cell culture method using a roller bottle in the present invention also increases the yield (yield) of separation and purification of extracellular vesicles. In addition, it is possible to produce small and uniform exosomes or extracellular vesicles, and the separated and purified extracellular vesicles have a homogeneous diameter of 100 to 300 nm, and specifically, the separated and purified extracellular vesicles have a diameter of 100 to 300 nm or more in 95% or more. In addition, the separated extracellular vesicles are 10 per unit volume of 1 L of culture medium.11 10 inland 13 The exosomes or extracellular vesicles of the present invention contain extracellular vesicles, and the total protein content is 6 to 10 mg per 1 L of culture medium. In addition, since the exosomes or extracellular vesicles of the present invention have a high inhibitory effect on the production of NO, which is utilized as an inflammatory factor, they can be utilized for anti-inflammatory purposes, and thus, the separated and purified exosomes or extracellular vesicles can be applied to anti-inflammatory pharmaceutical compositions or cosmetic compositions.

[0066] In this way, the extracellular vesicles separated and purified in the present invention have the characteristics of being uniform in size, having increased protein concentration or particle number per unit volume, having increased CD9 and CD81 protein expression, or having increased anti-inflammatory effects, compared to a culture method using a flask.

[0067]

[0068] In another aspect, the present invention provides a method for culturing stem cells for mass production of extracellular vesicles, comprising a step of inoculating stem cells into a roller bottle and culturing them.

[0069] In the present invention, the step of inoculating stem cells into a roller bottle and culturing them, and the step of mass production of extracellular vesicles are as described in the method for producing stem cell-derived extracellular vesicles described above.

[0070] In addition, the terms of the present invention, “roller bottle,” “stem cell,” “exosome,” and “extracellular vesicle” are also described above.

[0071] In addition, the matters mentioned in the method for isolating stem cell-derived extracellular vesicles for mass production of the above extracellular vesicles are equally applicable to the method for culturing stem cells for mass production of extracellular vesicles, unless they are contradictory.

[0072]

[0073] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings so that those skilled in the art can easily implement the present invention. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

[0074]

[0075] Example 1: Stem cell culture and exosome isolation

[0076] Stem cells derived from adipose tissue of companion dogs (Beagle dogs) were suspended in DMEM / F12 culture medium containing 10% fetal bovine serum (FBS), 100 units / mL penicillin, and 100 μg / mL streptomycin, and then seeded at 2,000 cells / cm in a flask or roller bottle. 2 The cells were inoculated at a density of 100 μm and cultured in a 37°C, 5% CO2 incubator. Five days after inoculation, the medium was replaced with DMEM / F12 serum-free medium, and 1 day later, the culture medium (medium) was collected.

[0077]

[0078] Example 2: Isolation and purification of exosomes

[0079] Tangential flow filtration (TFF) was used to isolate exosomes from the culture medium recovered in Example 1. Specifically, to remove cells from the recovered culture medium, centrifugation was performed at 2,000 rpm for 20 minutes, and the first supernatant was collected. Then, to remove cell debris from the first supernatant, the second culture medium was filtered through a 0.2 μm pore size filter. This was used as a sample for NTA measurement before exosome isolation. In addition, the second culture medium was concentrated using a 750 kDa hollow fiber filter (TFF filter) until the volume of the culture medium became 1 / 50, and then buffer exchange with 5 times the volume of PBS was performed twice in total, and finally exosomes were isolated and purified.

[0080]

[0081] Example 3: Evaluation of stem cell proliferation capacity according to culture method

[0082] In order to analyze the stem cell proliferation ability according to the culture method in Example 1 above, the number of cells before and after culture was measured using the trypane blue method.

[0083] The results of cell number increase rate according to the above stem cell culture method are shown in Fig. 2. Fig. 2 shows the results of evaluating stem cell proliferation ability after culturing stem cells in a flask or roller bottle. As shown in Fig. 2, depending on the inoculation density and culture period, the cell proliferation rate was approximately 11 times in the general flask method, but approximately 7-8 times in the roller bottle method. It can be seen that the cell proliferation rate in the roller bottle culture is actually lower than in the flask culture.

[0084]

[0085] Example 4: Size analysis and isolation yield evaluation of exosomes

[0086] 4-1. Measurement of exosome size

[0087] In order to analyze the characteristics of exosomes before and after separation in Example 2, the size of nanoparticles was measured using the nanoparticle tracking analysis (NTA: purchased from Malvern), and the results of the nanoparticle tracking analysis (NTA) of the exosomes are shown in Fig. 3.

[0088] Figure 3 is a graph showing the particle size distribution and number of particles obtained by performing nanoparticle tracking analysis (NTA) on the secondary culture (CM) obtained by culturing stem cells in serum-free medium in a flask or roller bottle, or on the extracellular vesicles (EVs) isolated and purified therefrom. As shown in Figure 3, the average sizes of nanoparticles before exosome isolation in the roller bottle and flask culture methods were 177.5 ± 3.5 nm and 240.4 ± 4.07 nm, respectively, indicating that the nanoparticles obtained from the roller bottle culture method were much smaller. In addition, the average sizes of nanoparticles after exosome isolation in the roller bottle and flask culture methods were 152.0 ± 3.1 nm and 163.5 ± 1.2 nm, respectively, indicating that the nanoparticles obtained from the roller bottle culture method were much smaller and more uniform.

[0089]

[0090] 4-2. Measurement of exosome separation yield

[0091] The exosome isolation yield was defined as follows.

[0092] Total number of nanoparticles before exosome separation / Total number of nanoparticles after exosome separation x 100

[0093]

[0094] The exosome isolation yields in the roller bottle and flask culture methods were 23.6% and 12.0%, respectively, confirming that the isolation and purification yield of exosomes obtained from the roller bottle culture method was approximately twice as high (Table 1).

[0095] The average size and purification yield of nanoparticles before and after exosome separation are summarized in Table 1.

[0096] ItemRoller BottleFlaskAverage particle size before separation177.5±3.5 nm240.0±4.07 nmAverage particle size after separation152.0±3.1 nm163.5±1.2 nmPurification yield23.6%12.0%

[0097]

[0098] Example 5. Measurement of exosome number and marker expression

[0099] Since CD9 and CD81 are representative exosome markers, the content of CD9 and CD81 is linear with the content of exosomes, and an increase in CD9 and CD81 content indicates a proportional increase in exosome content. Furthermore, exosome quality can be assessed by measuring the expression of exosome markers per nanoparticle.

[0100] Here, exosome surface protein expression was analyzed using the MACSPlex exosome kit (Miltenyi Biotec, Cat#130-108-813). Specifically, exosomes 5x10 8 The particles were reacted with exosome capture beads overnight in a 4°C refrigerator. After washing once with 1 ml of washing solution, the exosomes were reacted with a cocktail of exosome detection antibodies CD9, CD63, and CD81 at room temperature for 1 hour. After washing twice, the samples were analyzed using a flow cytometer (Cytoflex, BECKMAN COULTER) to measure the expression of stem cell marker CD105 and exosome markers CD9 and CD81 in the isolated exosomes.

[0101] Figure 4 shows the results of measuring the number of nanoparticles and protein content of isolated exosomes, and the expression of stem cell marker CD105 and exosome markers CD9 and CD81. As a result, the productivity (i.e., the number of exosome particles per unit L of culture medium) and protein content (concentration) of exosomes isolated from the roller bottle culture method were significantly higher. In addition, the expression amount of CD105, a stem cell marker, was not significantly different between the exosomes isolated from the roller bottle culture method and the exosomes isolated from the flask culture method, but it was confirmed that the expression of exosome markers CD9 and CD81 was significantly increased (see Figure 4).

[0102]

[0103] Example 6: Measurement of inflammatory response using macrophage cell lines

[0104] Mouse macrophage cell lines, RAW 264.7, were purchased from the Korea Cell Line Bank and cultured. Cells were subcultured in DMEM (ThermoFisher Scientific) containing 10% fetal bovine serum (FBS; ThermoFisher Scientific) and 1% antibiotic-antimycotics (FBS; ThermoFisher Scientific) at 37°C and 5% CO2.

[0105] RAW 264.7 cells were suspended in DMEM medium containing 10% FBS and seeded 7x10 into each well of a multiwell plate. 5 The cells were divided into 10 cells / ml. After 1 hour, exosomes were treated, and 3 hours after exosome treatment, LPS was treated and cultured for 24 hours. The NO contained in the culture medium was measured by absorbance (550 nm) using a NO detection kit (Intron Bio).

[0106] As a result, it was confirmed that exosomes isolated from the roller bottle culture method had a greater nitrite inhibition effect than exosomes isolated from the flask culture method, indicating a greater anti-inflammatory effect.

[0107]

[0108] Example 7: Comparison of the number and size distribution of nanoparticles according to the culture period in serum-free medium.

[0109] As in Example 1, stem cells derived from adipose tissue of a companion dog (Beagle) were inoculated into a roller bottle for 5 days, and the culture medium (medium) was collected three times in total by replacing it with DMEM / F12 serum-free medium once a day, and the number and size distribution of nanoparticles of the secondary stem cell culture medium obtained through the same process as in Example 2 were compared.

[0110] Figure 6 is a graph showing the particle size distribution and number of particles obtained by performing NTA (nanoparticle tracking analysis) on the secondary culture of stem cells obtained by collecting the culture medium (medium) three times in total by replacing it with DMEM / F12 serum-free medium once every day 5 days after inoculating companion dog (beagle) adipose tissue-derived stem cells in a flask and roller bottle.

[0111] As a result, as illustrated in Fig. 6, it was confirmed that the roller bottle culture method showed less decrease in extracellular vesicle production and more uniform particle size after replacing the serum-free medium compared to the conventional flask culture method. Therefore, it can be seen that the stem cell culture method using the roller bottle of the present invention is capable of producing exosomes up to three times in a serum-free medium.

[0112]

[0113] From these results, the present invention has confirmed that the isolation yield and quality of exosomes or extracellular vesicles are improved when stem cells are cultured using roller bottles, and thus exosomes or extracellular vesicles can be mass-produced using existing equipment in a GMP facility.

[0114]

[0115] Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims also fall within the scope of the present invention.

Claims

1. Step 1: Inoculate stem cells into a roller bottle and culture them in cell culture medium; Step 2: replacing the above cell culture medium with a serum-free medium and culturing in the serum-free medium; and A method for isolating stem cell-derived extracellular vesicles for mass production of extracellular vesicles, comprising three steps of isolating and purifying extracellular vesicles by performing tangential-flow filtration (TFF).

2. A method for isolating stem cell-derived extracellular vesicles, wherein the stem cell culture is performed by rotating the roller bottle at a rotation speed of 0.25 to 2.0 rpm in the first paragraph.

3. In the first paragraph, the surface area of ​​the roller bottle cm 2 A method for isolating stem cell-derived extracellular vesicles, which comprises inoculating and culturing 1,000 to 5,000 stem cells per cell.

4. In the first paragraph, the surface area of ​​the roller bottle is 700 to 1000 cm 2 person A method for isolating stem cell-derived extracellular vesicles.

5. A method for isolating stem cell-derived extracellular vesicles, wherein, in the first paragraph, stem cells are cultured simultaneously using multiple roller bottles during a single culture.

6. A method for isolating stem cell-derived extracellular vesicles, wherein the stem cell in paragraph 1 is an embryonic stem cell, an adult stem cell, or an induced pluripotent stem cell (iPSC).

7. A method for isolating stem cell-derived extracellular vesicles, wherein the adult stem cell in paragraph 6 is an adult stem cell derived from human or animal tissue, a mesenchymal stem cell derived from human or animal tissue, or a mesenchymal stem cell derived from an induced pluripotent stem cell derived from human or animal tissue.

8. A method for isolating stem cell-derived extracellular vesicles, wherein the human or animal tissue in paragraph 7 is selected from the group consisting of brain, teeth, liver, ovary, peripheral blood, hair follicle, heart, umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta.

9. A method for isolating stem cell-derived extracellular vesicles, wherein the adult stem cells of human or animal tissue origin in paragraph 7 are selected from the group consisting of mesenchymal stem cells, endothelial stem cells, hair follicle stem cells, muscle stem cells, ovarian stem cells, cardiac stem cells, hematopoietic stem cells, mammary stem cells, intestinal stem cells, vascular endothelial stem cells, neural stem cells, olfactory nerve stem cells, adipose stem cells, and testicular stem cells.

10. A method for isolating stem cell-derived extracellular vesicles, wherein culturing stem cells using the roller bottle in the first paragraph increases the yield of separation and purification of extracellular vesicles.

11. A method for isolating stem cell-derived extracellular vesicles, wherein the separated and purified extracellular vesicles in the first paragraph have a diameter of 100 to 300 nm in 95% or more.

12. In the first paragraph, the separated and purified extracellular vesicles are 10 per unit volume of 1 L of culture medium. 11 10 inland 13 A method for isolating stem cell-derived extracellular vesicles, comprising extracellular vesicles of a dog.

13. A method for isolating stem cell-derived extracellular vesicles, wherein the proliferation rate of stem cells by the culture method using the roller bottle in the first paragraph is reduced by 20 to 40% compared to the culture method using a flask.

14. A method for isolating stem cell-derived extracellular vesicles, wherein, in the first paragraph, the separated and purified extracellular vesicles have a uniform size, an increased protein concentration or particle number per unit volume, an increased expression of CD9 and CD81 proteins, or an increased anti-inflammatory effect, compared to a culture method using a flask.

15. A method for culturing stem cells for mass production of extracellular vesicles, comprising a step of inoculating stem cells into a roller bottle and culturing them.

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