Method for producing stem cell-derived extracellular vesicles with increased yield

Abstract

The present invention relates to a novel method for producing extracellular vesicles which increases the production of extracellular vesicles by applying uniform shear stress to stem cells. By using a bioreactor for producing extracellular vesicles of the present invention and the method for producing extracellular vesicles with improved yield of the present invention, a greater number of extracellular vesicles per stem cell can be effectively obtained, and thus the present invention can be widely used as a more economical production method in various treatment and research fields using extracellular vesicles.

Description

Method for producing stem cell-derived extracellular vesicles with increased yield

[0001] The present invention relates to a novel method for producing extracellular vesicles, which increases the production of extracellular vesicles by applying uniform shear stress to stem cells.

[0002] Extracellular vesicles can be classified into various names such as exosomes, microvesicles, ectosomes, microparticles, membrane vesicles, nanovesicles, and outer membrane vesicles based on their origin, secretion mechanism, and size. For example, mammalian extracellular vesicles are classified into exosomes, which are created when multivesicular endosomes (MVEs) mature, the endosomal membrane inwardly moves to form intraluminal vesicles, and then the multivesicular endosomes fuse with the cell surface to secrete the intraluminal vesicles out of the cell, and microvesicles, which are secreted out of the cell when the plasma membrane protrudes outward and separates. They are approximately 50 to 1000 nm in size, have a phospholipid bilayer structure, which is the structure of the cell membrane, and are characterized by containing cytoplasmic components such as mRNA, DNA, and proteins.

[0003] These extracellular vesicles are fundamental cellular tools for metabolism, metabolite transport, enzyme storage, and chemical reactions. They also mediate intercellular signaling by transferring mRNA, miRNA, and proteins between cells. Because of their high delivery efficiency and the ability to protect the target substance from the external environment, extracellular vesicles are widely used in biological experiments. Stem cell-derived extracellular vesicles, in particular, are effective in disease treatment, cell proliferation, and wound regeneration, and are also widely used in research on disease diagnosis and drug delivery.

[0004] However, the production of extracellular vesicles, which contain a low percentage of cell secretions, is extremely low, limiting the development of therapeutics. To address this issue, active research is being conducted on methods such as growing multiple cells in the same space to increase the overall concentration of secreted cells, or treating cells with substances that promote extracellular vesicle secretion. However, these methods do not improve the yield of extracellular vesicles themselves, and are limited by cost. Therefore, there is a need for new methods to increase the production of extracellular vesicles.

[0005] In other words, there is a need to develop a new method that increases the production yield by increasing the number of extracellular vesicles produced per single cell without negatively affecting the properties of the extracellular vesicles.

[0006] Accordingly, the inventors of the present invention completed the present invention by confirming that when stem cells are cultured by applying uniform shear stress to the stem cells, the number of extracellular vesicles secreted from the stem cells effectively increases while conducting research to increase the production yield of extracellular vesicles obtained from stem cells.

[0007]

[0008] Accordingly, the purpose of the present invention is to provide a method for producing extracellular vesicles with improved yield, including a step of applying uniform shear stress to stem cells and performing perfusion culture; and a bioreactor for producing extracellular vesicles that can be used in the method.

[0009] Another object of the present invention is to provide an extracellular vesicle with enhanced efficacy manufactured using the above method or the above microchannel.

[0010] In order to solve the above problems, the present invention provides a method for producing extracellular vesicles with improved yield, comprising: 1) a step of attaching and culturing stem cells in a U-shaped three-dimensional microchannel; 2) a step of perfusion culturing while applying uniform shear stress to the stem cells; and 3) a step of isolating extracellular vesicles secreted from stem cells cultured under the shear stress conditions.

[0011] In addition, the present invention provides a bioreactor for producing extracellular vesicles, comprising: a housing having a predetermined volume; a plurality of microchannels formed inside the housing and having a U-shape; a supply means connected to one end of the microchannels and supplying a fluid; a discharge means connected to the other end of the microchannels and discharging a fluid; and a peristaltic pump connected to the supply means and the discharge means and controlling the flow of the fluid so as to form a uniform perfusion, and a controller controlling the peristaltic pump; wherein the microchannels having a U-shape have a curved area of ​​7 to 10% of the entire channel area, and the curvature diameter and the distance between lines are the same.

[0012] The present invention also provides an extracellular vesicle manufactured by the above method.

[0013] By using the bioreactor for producing extracellular vesicles and the method for producing extracellular vesicles with improved yield of the present invention, a greater number of extracellular vesicles can be effectively obtained per stem cell, and thus, the method can be widely utilized as a more economical production method in various therapeutic and research fields using extracellular vesicles.

[0014] Figure 1 relates to a method for producing a microchannel of the present invention. Figure 1 A shows a method for forming a mold of a U-shaped channel, and Figure 1 B shows a method for forming a 3D printing mold of a U-shaped microchannel.

[0015] Figure 2 shows the results of calculating the Reynolds number and shear stress range for selecting cell culture conditions.

[0016] Figure 3 is a diagram showing the results of confirming the change in shear stress according to the curve design of various microchannels.

[0017] Figure 4 is a schematic diagram of a microchannel cell culture method of the present invention for producing extracellular vesicles by applying uniform shear stress.

[0018] Figure 5 is a diagram showing the results of confirming the alignment of cultured cells according to shear stress.

[0019] Figure 6 is a diagram showing the results of confirming cell survival according to shear stress.

[0020] Figure 7 shows the size distribution of extracellular vesicles secreted from MSCs according to shear stress conditions and the results of analysis of extracellular vesicle production efficiency (mean ± SD (n = 4; **:p< .01), one-way ANOVA followed by Tukey's post hoc test).

[0021]

[0022] The present invention relates to a method for producing extracellular vesicles with improved yield and a bioreactor for producing extracellular vesicles.

[0023] Using the extracellular vesicle production method and bioreactor for extracellular vesicle production of the present invention, extracellular vesicles with uniform characteristics can be obtained at a high yield from stem cells. In the present invention, as a method for increasing the yield of extracellular vesicles, shear stress conditions that allow stem cells attached within microchannels to be applied with uniform shear stress without causing them to fall off and without affecting the survival of the stem cells, and a microchannel shape for this purpose were developed.

[0024] Below, it is explained in more detail.

[0025] The present invention relates to a method for producing extracellular vesicles with improved yield, comprising: 1) a step of culturing stem cells by attaching them to a U-shaped three-dimensional microchannel; 2) a step of culturing them by perfusion while applying a uniform shear stress to the stem cells; and 3) a step of isolating extracellular vesicles secreted from stem cells cultured under the shear stress conditions.

[0026] The above step 1) is a step of attaching and culturing stem cells in a U-shaped 3D microchannel.

[0027] In the present invention, the U-shaped three-dimensional microchannel is present in multiple numbers inside a housing having a predetermined volume, and a supply means for supplying fluid is provided at one end of the microchannel, and a discharge means for discharging fluid is provided at the other end.

[0028] The above U-shaped three-dimensional microchannel may be characterized by having the same curvature diameter and the same distance between lines. More specifically, in FIG. 3 of the present invention, it is possible to confirm the change in shear stress according to the change in the distance between the curvature diameter and the lines, and the structure in the form of a sinusoid rather than a U-shape. In the present invention, by using a U-shaped three-dimensional microchannel in which the curvature diameter and the same distance between the lines, the effect of minimizing the deviation in shear stress and maximizing the cross-sectional area was achieved.

[0029] In particular, the U-shaped three-dimensional microchannel of the present invention preferably has a curved area of ​​less than 10% of the entire channel, and can occupy 1 to 10%, 2 to 9%, and can prevent turbulence from occurring in the straight-curved portion and apply uniform shear stress to stem cells attached within the channel.

[0030] The U-shaped three-dimensional microchannel of the present invention can be manufactured with a diameter to which cells can be evenly attached, and can be a microchannel having a diameter of preferably 100 to 800 μm, more preferably 400 to 600 μm.

[0031] The U-shaped three-dimensional microchannel of the present invention can be manufactured by a 3D printing method, but is not limited thereto. In addition, it can be manufactured using any hydrogel that is not toxic to cells and to which cells can adhere, but is preferably manufactured using at least one hydrogel selected from the group consisting of collagen, decellularized extracellular matrix (dECM), gelatin, matrigel, methacrylated gelatin (GelMA), hyaluronic acid, alginate, fibrin, chitosan, and agarose, and more preferably, it can be manufactured using methacrylated gelatin (GelMA).

[0032] The attachment culture step of step 1) above is performed for 12 to 48 hours, preferably 12 to 24 hours, and is performed without perfusion. Specifically, stem cells of passages 4 to 10 are injected into the channel for culture, and the culture can be performed while rotating 180° every hour to achieve cell attachment.

[0033]

[0034] The present invention includes 2) a step of perfusion culturing while applying uniform shear stress to the stem cells.

[0035] More specifically, the present invention enables uniform extracellular vesicles to be obtained by applying uniform shear stress to stem cells attached within a microchannel.

[0036] In order to achieve the above perfusion culture, in the present invention, the cell culture solution can be supplied into the microchannel at a rate of 50 to 150 μl / min, preferably 75 to 120 μl / min, thereby applying a shear stress to the stem cells of 0.01 to 15 dyn / cm. 2 It can be adjusted to a range. At this time, it is preferable that the U-shaped three-dimensional microchannel has a diameter of 100 to 800 μm.

[0037] The above shear stress is 15 dyn / cm 2 If it exceeds 0.01 dyn / cm, the stem cells attached to the microchannel may fall off, inhibiting the production of extracellular vesicles. 2 If the shear stress is less than 0.04, the stem cells are not given the appropriate shear stress stimulus, and thus the effect of increasing the production rate of extracellular vesicles cannot be achieved. In the present invention, the shear stress is set to 0.04 to 12 dyn / cm 2 It was confirmed that when uniformly applied, stem cells are stimulated to a level that stimulates microvascular blood flow in the body, so that stem cells do not die, and extracellular vesicles of similar size on average can be obtained. In particular, in the present invention, the concentration is from 0.04 to 12 dyn / cm 2 It was confirmed that when shear stress conditions were applied, an increase in extracellular vesicle production of at least 1.5 to at most 51 times could be achieved compared to the shear stress-untreated group.

[0038] The perfusion culture in step 2) above can be cultured for 36 to 84 hours, preferably 36 to 80 hours, and more preferably 52 to 72 hours.

[0039] The stem cell of the present invention may include, without limitation, a stem cell capable of producing an extracellular vesicle, and may be at least one type of stem cell selected from the group consisting of mesenchymal stem cells, pluripotent stem cells, induced pluripotent stem cells, and embryonic stem cells, or a stem cell derived from one type selected from the group consisting of umbilical cord blood, umbilical cord, bone marrow, fat, placenta, Wharton jelly, amniotic fluid, amniotic membrane, and tonsils.

[0040] The present invention includes a step of 3) separating extracellular vesicles secreted from stem cells cultured under the above shear stress conditions; in the step, a process of separating and harvesting extracellular vesicles secreted by stem cells is performed.

[0041] The above separation harvesting process can use any method known in the art without limitation.

[0042]

[0043] In addition, the present invention relates to a bioreactor for producing extracellular vesicles, comprising: a housing having a predetermined volume; a plurality of microchannels formed inside the housing and having a U-shape; a supply means connected to one end of the microchannels and supplying a fluid; a discharge means connected to the other end of the microchannels and discharging a fluid; and a peristaltic pump connected to the supply means and the discharge means and controlling the flow of the fluid so as to form a uniform perfusion, and a controller controlling the same; wherein the microchannels having a U-shape preferably have a curved area of ​​less than 10% of the entire channel, and may occupy 1 to 10%, 2 to 9%, and the curvature diameter and the distance between lines are the same.

[0044] The microchannel for producing extracellular vesicles of the present invention can be referred to in the form of FIGS. 3 and 4.

[0045] The microchannel of the present invention may be manufactured by 3D printing. To manufacture the microchannel of the present invention, a U-shaped channel mold is first formed, and then a hydrogel that is non-toxic to cells and to which cells can be attached, preferably one or more hydrogels selected from the group consisting of collagen, decellularized extracellular matrix (dECM), gelatin, Matrigel, methacrylated gelatin (GelMA), hyaluronic acid, alginate, fibrin, chitosan, and agarose, more preferably methacrylated gelatin (GelMA), is poured into the manufactured mold and hardened.

[0046]

[0047] The shape of the U-shaped three-dimensional microchannel is determined so that uniform perfusion can be applied to the stem cells to be attached within the channel. The microchannel has a diameter of 0.1 to 0.8 mm, and the width and height of the device can be appropriately selected so that the desired uniform perfusion can be applied. For example, a microchannel having a diameter of 0.1 to 0.8 mm, a width of 15 to 25 mm, and a height of 10 to 20 mm can be used. The microchannel of the present invention may have a curved area of ​​1 to 10% of the entire channel. In a preferred embodiment, the present invention has a total length of 3.87 cm. 2A microchannel with a surface area of ​​0.6 mm in diameter, 20 mm in width, and 16 mm in height was used, and it was designed so that the curved area was only 8% of the total. At this time, the curvature diameter was set equal to the distance between lines.

[0048] The bioreactor of the present invention includes a plurality of microchannels having a U shape, and the plurality of microchannels means a form in which straight microchannels and U-shaped curved microchannels are repeatedly and continuously connected, and a U-shaped curved pipe member having a curvature at the end of the straight pipe part is connected in a form protruding outward. The straight portions of the U-shaped microchannels form mutually parallel lines, and a curved pipe part having the same curvature radius is connected at the end of the straight portion, and a straight portion is connected again at the end of the curved portion, thereby forming a U shape once again. The curvature of the curved portions of all microchannels is the same.

[0049] The controller of the present invention controls the peristaltic pump to form a constant shear stress in the microchannel, and controls the flow rate so that turbulence does not occur in the straight and curved sections of the U-shaped microchannel. The controller of the present invention induces a uniform shear stress of blood flow level in the stem cells attached in the microchannel at a rate of 0.01 to 15 dyn / cm. 2 The flow of fluid can be controlled so that shear stress is generated.

[0050] By using a U-shaped three-dimensional microchannel of this type, turbulence in the straight-curve section can be minimized and shear stress can be generated uniformly.

[0051] A plurality of microchannels having a U-shape are formed in parallel within the housing of the present invention, and both ends of the microchannels are connected to a supply means for supplying fluid and a discharge means for discharging fluid, respectively. The supply means is connected to a peristaltic pump at the other end through a pipe through which the fluid flows. A medium reservoir may be located adjacent to the peristaltic pump. The medium reservoir may be adjacent to the discharge means through a pipe through which the fluid flows.

[0052] The present invention also provides an extracellular vesicle manufactured by the above method.

[0053] The extracellular vesicles of the present invention may be characterized by secreting or expressing more useful components than stem cell-derived extracellular vesicles obtained without shear stress stimulation. For example, the extracellular vesicles of the present invention may be extracellular vesicles with an increased expression pattern of useful miRNAs or increased secretion of useful cytokines.

[0054]

[0055] Hereinafter, the present invention will be described in detail through examples. The following examples are intended solely to illustrate the present invention more specifically. It will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0056]

[0057] Example 1. Fabrication of channels for extracellular vesicle production

[0058] 1.1 Fabrication of U-shaped microchannels

[0059] To fabricate a U-shaped channel, a PDMS (Polydimethylsiloxane) mold was manufactured. Specifically, the mold was manufactured by mixing PDMS base and curing agent (SYLGARD 184 silicone elastomer, Dow Corning, Midland, MI, USA) in a 10:1 ratio.

[0060] A schematic diagram of the manufacturing of a U-shaped channel is shown in Fig. 1.

[0061] As shown in Figure 1A, the manufactured PDMS was attached to a frame made of TPU (thermoplastic polyurethane elastomer) to support the mold and placed in a vacuum chamber to remove air bubbles. Thereafter, the PDMS was cured at 80°C for at least 6 hours to solidify and sterilized with ultraviolet light through ethanol.

[0062] Igacure 2959 (Sigma, St. Louis, MO, USA) was prepared as a 0.05% PBS / Igacure (PI) solution using phosphate-buffered saline (PBS, Cytiva, Marlborough, USA) as a photoinitiator. GelMA was dissolved in PI solution to a concentration of 10% at 40°C and filtered through a 0.22 μm PES membrane filter (Sartorius, Göttingen, Germany).

[0063] Afterwards, as shown in Fig. 1B, 1.54 ml of 10% Gelma hydrogel was dispensed into the PDMS mold and 86.5 mW / cm 2It was cured by exposing to ultraviolet rays for 30 seconds. At this time, the inlet and outlet of the channel were connected, and stainless steel was connected to fix the gelma to prevent it from slipping. The U-shaped channel was printed using a 0.6 mm nozzle using a 3D printer, and 35 wt% pluronic F-127 (Sigma) was used as a sacrificial ink at 30°C. After printing, 1.54 ml of 10% gelma was additionally dispensed to perform cross-linking. After that, 4°C PBS was flowed through a syringe pump to remove pluronic F-127. After that, uncured gelma was washed and removed using 4°C PBS.

[0064] 1.2 Setting channel conditions using microchannels

[0065] To establish conditions for producing extracellular vesicles by applying uniform shear stress to stem cells, microchannel conditions were established. Stem cells that form extracellular vesicles are highly sensitive and their characteristics can be easily influenced by various variables. Therefore, it is important to apply uniform shear stress to stem cells, and this uniform shear stress can lead to the formation of uniform extracellular vesicles. When the microchannel is ideally formed, it has a circular tubular shape, and the shear stress (τ) can be calculated using the following formula.

[0066]

[0067] (Equation 1)τ = 4μQ / (L / 2)³π

[0068] μ: dynamic viscosity of the fluid

[0069] Q: Flow rate

[0070] L: Diameter of the cylinder, i.e. diameter of the microchannel

[0071]

[0072] Considering that the fluid dynamic properties of the cell media used in this experiment are similar to those of water, assuming water at 20℃ as the fluid, μ has a value of 0.001 [kg / (m·s)]. Since microchannel technology has a diameter in the micron unit, it mainly has laminar flow. Unlike turbulent flow, laminar flow has a regular flow, and since each section is not irregular in time or space, consistent results can be expected in all sections. When producing extracellular vesicles by attaching cells, the experiment must be conducted within the range of laminar flow to maintain consistent quantitative determination and therapeutic substance content of the extracellular vesicles, and thus, it is essential to limit the laminar flow range. The constant value that is an important variable in describing laminar and turbulent flow of a fluid is called the Reynolds number, and the Reynolds number in a circular tube can be calculated using the following formula.

[0073] (Formula 2)

[0074] Re =uL / v

[0075] u: velocity of the fluid

[0076] ν: kinematic viscosity

[0077]

[0078] The Reynolds number in a circular tube calculated through Equation 2 can be classified as laminar flow when it is lower than 2300 and turbulent flow when it is higher than 4000. Therefore, in order to obtain consistent results for extracellular vesicles, it was determined that the characteristics of the microchannel and experimental conditions needed to be specified so that the Reynolds number was lower than 2300. The diameter values ​​ranging from 0.1 to 5 mm, which is the range commonly used for microchannels, were used as the standard, and the maximum flow rate of a syringe pump generally used in laboratories, 11 ml / min, was substituted to calculate the Reynolds number and the shear stress at that time, which are shown in Table 1.

[0079]

[0080] Diameter [mm] 0.1 0.2 0.5 1 2 5 1 0 1 0 Reynolds number 2 3 2 5 1 1 6 2 4 6 5 2 3 2 1 1 6 4 6 2 3 2 0 Maximum shear stress [dyn / cm 2 ]187102339149.718.72.3390.150.0191.871E-5

[0081]

[0082] As shown in Table 1, it can be confirmed that the Reynolds number is distributed within laminar flow under all diameter conditions, even though the shear stress and Reynolds number calculated using the maximum flow rate each have the maximum values.

[0083] On the other hand, laminar flow can provide uniform conditions to all cells, but it is necessary to limit the shear stress range to control stem cell differentiation and avoid a harsh culture environment for cells. The maximum shear stress that does not detach cells and does not significantly modulate their behavior is 20 dyn / cm. 2 It has laminar flow and shear stress is limited to 20 dyn / cm. 2 The conditions that do not exceed this were calculated. The results of calculating the Reynolds number and shear stress range for selecting cell culture conditions are shown in Figure 2.

[0084] When calculating the Reynolds number through the above results, it was confirmed that laminar flow was present within the flow rate range of the syringe pump.

[0085] Additionally, the maximum shear stress is 20 dyn / cm. 2 When limited to , the fluid velocity limit according to the diameter of the microchannel was confirmed. It was confirmed that the fluid velocity limit decreases as the diameter increases, and even for a small diameter of 0.2 mm, a velocity of approximately 50 mm / s can be covered, and it was confirmed again that the microchannel design of the present invention is available in a considerably wide range of dimensions and is controllable.

[0086] In summary of the above results, the Reynolds number is less than 2300 and 20 dyn / cm 2The diameter of the microchannel was selected to be 0.2 to 1 mm, which is an appropriate range for providing a uniform shear stress.

[0087]

[0088] 1.3 U-shaped microchannel selection

[0089] To expand the average area of ​​a microchannel system, a curved U-shaped design was applied. When using a U-shaped channel, a problem of turbulence occurring in the straight-curved section may occur, but in the present invention, the maximum shear stress is 20 dyn / cm, which was selected in Example 1.2. 2 When shear stress is applied, it is expected that microchannels with very small dimensions can maintain laminar flow. To this end, a structure was designed to minimize the area of ​​the curved portion to eliminate possible variables, and the change in shear stress according to the curved design of the microchannel is shown in Figure 3.

[0090] As shown in Fig. 3, it was found that the shear stress changed depending on the shape of the microchannel, but when a U-shaped shape was used as in Fig. 3, 2, and the distance between the curvature diameter and the line was made the same, it was confirmed that the cross-sectional area could be maximized while the deviation in shear stress was small.

[0091] In summary of the above results, a total of 3.87 cm 2 A 3D printed U-shaped channel with a surface area of ​​0.6 mm was designed, and a microchannel with a diameter of 0.6 mm, a width of 20 mm, and a height of 16 mm with a curved area of ​​only 8% of the total was used in subsequent experiments to ensure uniform shear stress distribution.

[0092]

[0093] Example 2. Stem cell culture using a U-shaped channel

[0094] 2.1 Shear stress conditions and stem cell culture

[0095] One day before the cell culture experiment, the microchannels were conditioned with cell culture medium (Dulbecco's Modified Eagle's Medium (DMEM; Life Technologies, Carlsbad, CA), 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), and 1% antibiotic-antimycotic (anti-anti; Invitrogen). 5 x 10 passage 6 mesenchymal stem cells (MSC; Lonza, Basel, Switzerland, Lot. 0000690228) were used. 6 cells / ml (3.2 x 10 5 DMEM, 10% exo-free FBS, 1% anti-anti, and 4% dextran were added to cells and injected into the channel at 100 μl / min. After that, the cell was rotated 180° every hour for 6 hours to complete cell attachment, and then cultured under each shear stress condition. A schematic diagram of microchannel cell culture is shown in Fig. 4.

[0096] MSC is from 0.04 to 12 dyn / cm 2 The cells were cultured for 72 hours under a range of shear stress conditions, and the microchannel cell patterns at each shear stress are shown in Figure 5.

[0097] As shown in Figure 5, it was confirmed that the cultured cells aligned in the direction of the shear stress as the shear stress became stronger.

[0098]

[0099] 2.2 Analysis of cell viability according to shear stress

[0100] To determine cell viability according to shear stress, shear stress was uniformly applied for each condition, and the cells were cultured for 4 days, after which fluorescent staining was performed with calcein AM and ethidium homodimer-1. The results are shown in Figure 6.

[0101] As shown in Fig. 6, 0.04 dyn / cm 2 12 dyn / cm 2 It was confirmed that cells survived uniformly without cell death under all conditions.

[0102] That is, by uniformly applying the shear stress under the above conditions, the stem cells can be maintained in a well-attached state to the channel and the stem cells can be stimulated to a level that stimulates microvascular blood flow in the body without causing cell death.

[0103]

[0104] 2.3 Analysis of extracellular vesicle characteristics according to shear stress

[0105] Since it was confirmed that cells were cultured without death under all shear stress conditions tested in Example 2.2, extracellular vesicles secreted from stem cells cultured using the method of Example 2.1 were isolated. The size distribution and production rate of the isolated extracellular vesicles were confirmed. The size and concentration of the extracellular vesicles produced under each condition were measured using qNano analysis. qNano is a device that can measure the concentration and size of nanoparticles using tunable resistive pulse sensing technology. The nanopore used in the experiment was NP200, and at least 500 particles were measured for all samples. Calibration was performed with CPC100 (100 mm, 1.4E+13 particles / ml, IZON Ltd), and the measurement results are shown in Figure 7. As a control group, a 2D MSC experimental group cultured using a conventional culture method using a 2D culture flask was used.

[0106] As shown in Figure 7A, 2D MSC, 0.04 dyn / cm 2 , 2 dyn / cm 2 , 7 dyn / cm 2 and 12 dyn / cm 2The average sizes of extracellular vesicles measured in the groups were confirmed to be 125, 126.7, 127.6, 130.1, and 141.5 nm, respectively. As the magnitude of shear stress increased, the size of the extracellular vesicles tended to increase, but on average, extracellular vesicles of similar sizes were released.

[0107] As shown in Figure 7B and Table 2, the efficiency of extracellular vesicle production from MSCs under shear stress showed that the secretion amount increased as the magnitude of shear stress increased.

[0108] dyn / cm 2 2D MSC0.042712Particles / cell3.4 × 10 5 6.3 × 10 5 9.4 × 10 5 9.6 × 10 5 1.73×10 7 2D MSC vs. multiplier -1.92.82.850.8

[0109] Through the above results, it was confirmed that when a uniform shear stress is applied to stem cells using a U-shaped microchannel, extracellular vesicles with uniform characteristics can be obtained at a high yield.

Claims

1. 1) Step of attaching and culturing stem cells in a U-shaped 3D microchannel; 2) A step of perfusion culturing while applying uniform shear stress to the stem cells; and 3) A method for producing extracellular vesicles with improved yield, comprising: a step of separating extracellular vesicles secreted from stem cells cultured under the above shear stress conditions.

2. A method for producing extracellular vesicles with improved yield in the first paragraph, wherein the U-shaped three-dimensional microchannel has the same curvature diameter and distance between lines.

3. A method for producing extracellular vesicles with improved yield in the first paragraph, wherein the U-shaped three-dimensional microchannel has a curved area of ​​1 to 10% of the entire channel.

4. A method for producing extracellular vesicles with improved yield, wherein the U-shaped three-dimensional microchannel in the first paragraph has a diameter of 100 to 800 μm.

5. A method for producing extracellular vesicles with improved yield, wherein the U-shaped three-dimensional microchannel is made of at least one hydrogel selected from the group consisting of collagen, decellularized extracellular matrix (dECM), gelatin, matrigel, methacrylated gelatin (GelMA), hyaluronic acid, alginate, fibrin, chitosan, and agarose.

6. A method for producing extracellular vesicles with improved yield, wherein, in paragraph 4, the perfusion culture is performed by supplying a cell culture solution to a U-shaped three-dimensional microchannel at a rate of 50 to 150 μl / min.

7. In the first paragraph, the shear stress is 0.01 to 15 dyn / cm 2 A method for producing extracellular vesicles with improved yield, characterized by applying .

8. A method for producing extracellular vesicles with improved yield, wherein the stem cells in paragraph 1 are at least one selected from the group consisting of mesenchymal stem cells, pluripotent stem cells, induced pluripotent stem cells, and embryonic stem cells.

9. A method for producing extracellular vesicles with improved yield, wherein the stem cells in paragraph 1 are stem cells derived from one species selected from the group consisting of umbilical cord blood, umbilical cord, bone marrow, fat, placenta, Wharton jelly, amniotic fluid, amniotic membrane, and tonsils.

10. A method for producing extracellular vesicles with improved yield, wherein the attachment culture in step 1) is performed for 12 to 48 hours.

11. A method for producing extracellular vesicles with improved yield, wherein the perfusion culture in step 2) is performed for 36 to 84 hours in the first paragraph.

12. A housing having a predetermined volume; A plurality of microchannels formed inside the housing and having a U shape; A supply means connected to one end of the above microchannel for supplying fluid; A discharge means connected to one end of the other side of the micro channel to discharge fluid; and A peristaltic pump connected to the above supply means and discharge means and controlling the flow of fluid so that uniform perfusion is formed, and a controller controlling the same; A bioreactor for producing extracellular vesicles, wherein the microchannel having the above U-shape has a curved area of ​​1 to 10% of the total channel area, and the curvature diameter and the distance between lines are the same.

13. A bioreactor for producing extracellular vesicles, wherein the microchannel has a diameter of 0.1 to 0.8 mm in the 12th paragraph.

14. A bioreactor for producing extracellular vesicles, wherein the microchannel is manufactured by 3D printing in the 12th paragraph.

15. A bioreactor for producing extracellular vesicles, wherein in paragraph 12, the controller controls the flow rate so that turbulence does not occur in the straight and curved sections of the U-shaped microfluidic channel.

16. Extracellular vesicles manufactured by any one of the methods of claims 1 to 11.

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