High-throughput method for producing extracellular vesicles in a baffled rotating vessel

Abstract

This method provides a more efficient way to produce extracellular vesicles from minimal cellular material, including not only individual cells in suspension but also cells in a three-dimensional structure. [Solution] The present invention provides a method for producing extracellular vesicles from producing cells, comprising: a) placing producing cells in a liquid culture medium in a container having a baffle structure inside; b) rotating the container, thereby generating extracellular vesicles from the producing cells; and c) collecting the generated extracellular vesicles, wherein step b) of rotating the container includes a sub-step of repeatedly changing the rotational motion of the container.

Description

Technical Field

[0001] The present invention relates to a method for producing extracellular vesicles from producer cells and a container for carrying out the method.

Background Art

[0002] Currently, extracellular vesicles (EVs) are considered to be an important factor in maintaining homeostasis and contributing to diseases. Extracellular vesicles hold great promise as future cancer treatments and regenerative therapies. Extracellular vesicles are endogenously released by cells in a constitutive or inducible manner. These vesicles transport substances such as lipids, proteins, mRNA, and miRNA and constitute the most advanced intercellular communication pathways throughout the human body. Extracellular vesicles have advantages in sterilization, storage, and shelf life compared to their parental cells and are ideal for clinical applications. As a result, in recent years, EV-based clinical trials have been increasing significantly.

[0003] Most of today's EV production methods rely on time-consuming and low-yield protocols such as spontaneous EV release in complete culture media, and the amount of EVs produced per cell in a few days is less than 500 (extracellular vesicles). Alternative methods include serum starvation and oxygen deprivation, but the increase in EV production is only about twice as much, with an EV production amount within the range of 1,000 per cell after performing it continuously for 2 or 3 days. EV release can also be induced by cell activation, such as vesicle formation induced by TNF-α, but here too, the increase in its yield is only twice that of the spontaneous release yield. According to recent trends based on low-stress hollow fiber bioreactors, the EV yield has increased to 2,000 to 10,000 EVs per cell, but the production time is as long as 10 days.

[0004] For extracellular vesicles to be applied to clinical use, the above-mentioned production problems must first be resolved, and a robust and high-yield EV production method using minimal cell material needs to be developed. Furthermore, it is desirable that the method be able to be used flexibly regardless of the morphology of the producing cells, such as being able to easily handle cells in suspension (such as primary cultured cells derived from biopsy) and producing extracellular vesicles from three-dimensional structures (i.e., physiologically appropriate organoid-like settings). To the best of the inventor's knowledge, no method has yet existed that satisfies these technical bottlenecks.

[0005] Several bioreactors are known that perform cell culture in a three-dimensional configuration.

[0006] For example, JP2007222037 (Patent Document 1) discloses a cell culture centrifuge tube comprising a separation tube, a stopper that seals the upper opening of the separation tube, an opening in the stopper, a nonwoven fabric that allows culture gas to pass through and covers the opening, and at least one baffle formed integrally with the separation tube on the inner wall surface of the separation tube.

[0007] In addition to these, other systems for three-dimensional cell culture, such as the ClinoStar system and CERO 3D, are available on the market.

[0008] However, none of these proposals involve the creation of EVs using bioreactors or from organoids / spheroids.

[0009] Furthermore, several methods have been developed to produce extracellular vesicles with higher yields using spinner flask-type bioreactors, which are used in three-dimensional cell culture.

[0010] For example, FR3091296 (Patent Document 2) discloses a fluid system for filling the lumen (extracellular vesicles) of extracellular vesicles from producing cells with a therapeutic agent or contrast agent, comprising at least one container, a liquid culture medium contained in the container, producing cells, and a liquid culture medium agitator and agitator speed control means adapted to grow the producing cells.

[0011] FR3091295 (Patent Document 3) discloses a fluid system for producing extracellular vesicles from suspended producing cells, comprising at least one container, a liquid culture medium contained in the container, suspended producing cells, a liquid culture medium agitator, and agitator speed control means adapted for growing the suspended producing cells.

[0012] FR3068361 (Patent Document 4) discloses a fluid system for producing extracellular vesicles from producing cells, comprising at least one container, a liquid culture medium contained in the container, and producing cells, as well as microcarriers suspended in the liquid culture medium and a liquid culture medium agitator, wherein the majority of the producing cells adhere to the surface of the microcarriers, and the dimensions of the container and the agitator are adapted to control the turbulence of the liquid culture medium within the container.

[0013] FR3112147 (Patent Document 5) discloses a calibration method for a fluid system that produces extracellular vesicles from producing cells.

[0014] However, the systems described in the above literature are merely repurposed commercial bioreactors and are not specifically designed for EV production. Moreover, the morphology of the produced cells is limited to those on beads in suspension or in two-dimensional cell cultures, making it impossible to produce EVs from spheroids or organoids.

[0015] To elaborate, the above literature raises several concerns. First, while it is feasible to achieve an optimal (10x) EV yield using a large production reactor (typically a 1L reactor) at a rotational speed of around 250 rpm, it requires a state-of-the-art, expensive magnetic stirrer. Reducing the volume of the production reactor (for example, to 0.3L) requires an even higher rotational speed (over 350 rpm), making it even less feasible. Furthermore, increasing the production volume in this way also increases the number of cells required (for example, 100 × 10⁶). 6 It is not realistic for cells to achieve this under the physiological conditions of personalized medicine.

[0016] Generally, miniaturizing a typical mixed tank with agitators or impellers requires reducing the size of the impeller blades. Maintaining the same level of stress on cells necessitates increasing the rotational speed, which is extremely difficult. For example, if the blade diameter is 1 cm, the rotational speed needs to be approximately 5,000 rpm, which is difficult to achieve with a mixed tank and magnetic stirrer, for instance.

[0017] Secondly, when cells are exposed to turbulent regions, cell death can occur. Because turbulent regions are highly heterogeneous, the level of shear stress experienced will differ for each individual cell growing on the beads. Furthermore, this heterogeneity makes it difficult to experimentally evaluate turbulent regions. [Prior art documents] [Patent Documents]

[0018] [Patent Document 1] Japanese Patent Publication No. 2007-222037 [Patent Document 2] French Patent Application Publication No. 3091296 [Patent Document 3] French Patent Application Publication No. 3091295 [Patent Document 4] French Patent Application Publication No. 3068361 [Patent Document 5] French Patent Application Publication No. 3112147

Summary of the Invention

Problems to be Solved by the Invention

[0019] Therefore, there is a desire for a more efficient method of producing extracellular vesicles from a minimum (small amount) of cell material, including not only individual cells in suspension (such as biopsy-derived stem cells) but also three-dimensional structured cells (such as organoids / spheroids).

Means for Solving the Problems

[0020] The present invention relates to the following aspects:

[0021] (Aspect 1) A method for producing extracellular vesicles from producer cells, comprising: a) Disposing producer cells in a liquid medium in a container in which a baffle structure is fixed inside; b) Rotating the container, thereby generating extracellular vesicles from the producer cells; c) Recovering the generated extracellular vesicles; The method comprising a sub-step of repeatedly changing the rotational movement of the container in step b) of rotating the container.

[0022] (Aspect 2) The method according to Aspect 1, wherein the producer cells are selected from human cells and animal cells.

[0023] (Aspect 3) The method according to Aspect 1 or 2, wherein the container rotates about a rotation axis oriented substantially vertically.

[0024] (Aspect 4) The method according to any one of Aspects 1 to 3, wherein the extracellular vesicles are generated from producer cells in the form of individual cells suspended in the liquid medium.

[0025] (Aspect 5) A method according to any one of embodiments 1 to 4, wherein the extracellular vesicle is produced from a spheroid and / or organoid-forming cell;

[0026] (Aspect 6) In the method described in Embodiment 5, further, A preliminary step of growing spheroids and / or organoids outside the container, A method comprising a step a) of placing the producing cells in the container, wherein the step of supplying the grown spheroids and / or organoids to the container;

[0027] (Aspect 7) In the method according to embodiment 5, step a) of placing the produced cells in the container includes a sub-step of supplying individual cells to the container, and the method further includes An intermediate step for generating spheroids and / or organoids from the aforementioned individual cells, A method that includes;

[0028] (Pattern 8) In the method of embodiment 7, further, During the intermediate step of generating spheroids or organoids from the individual cells, the container is preferably rotated at a maximum rotational speed less than the maximum rotational speed of step b). A method that includes;

[0029] (Aspect 9) A method according to any one of embodiments 1 to 8, wherein step b) of rotating the container includes a sub-step of periodically changing the rotational motion of the container;

[0030] (Aspect 10) The method according to embodiment 9, wherein the frequency of changing the rotational motion of the container is 0.01 to 5 Hz, preferably 0.05 to 0.5 Hz;

[0031] (Aspect 11) A method according to any one of embodiments 1 to 10, wherein step b) of rotating the container is performed at a maximum rotational speed of 50 to 6,000 rpm, preferably 600 to 1,600 rpm;

[0032] (Aspect 12) A method according to any one of embodiments 1 to 11, wherein step b) includes a sub-step of repeatedly reversing the rotation direction of the container;

[0033] (Aspect 13) A method according to any one of embodiments 1 to 12, wherein step b) includes a sub-step of repeatedly changing the rotation speed of the container;

[0034] (Aspect 14) A method according to any one of embodiments 1 to 13, wherein step b) includes a sub-step of intermittently rotating the container;

[0035] (Aspect 15) In the method described in any one of embodiments 1 to 14, further, A step of introducing a therapeutic agent or contrast agent into the liquid culture medium, A method that includes;

[0036] (Aspect 16) A method according to any one of embodiments 1 to 15, wherein step c) is performed by removing the liquid medium containing the producing cells from the container and separating the extracellular vesicles from the removed liquid medium, preferably by centrifugation;

[0037] (Aspect 17) A method according to any one of embodiments 1 to 15, wherein step c) is performed by removing the liquid medium from the container without substantially removing the producing cells, and separating the extracellular vesicles from the removed liquid medium, preferably by performing decantation and / or centrifugation in the container before removing the liquid medium;

[0038] (Aspect 18) In the method described in any one of embodiments 1 to 17, further, A process that repeats the cycle of at least steps b) and c) using the same producing cells, A method that includes;

[0039] (Aspect 19) In the method described in aspect 18, in each cycle, - The produced cells are removed from the container, separated from the liquid medium, and placed back into the container with a new liquid medium, or - A method in which the liquid culture medium is removed from the container while the produced cells remain largely inside the container, and a new liquid culture medium is added to the container;

[0040] (Aspect 20) In the method described in Embodiment 19, further, Between two consecutive cycles, there is a resting period during which the producing cells are held in the container without the container being rotated. A method that includes;

[0041] (Aspect 21) A method according to any one of embodiments 1 to 20, wherein the baffle structure includes one or more baffles fixed to the inner surface of the container;

[0042] (Aspect 22) A method according to any one of embodiments 1 to 21, wherein the container has a cylindrical inner wall and a central axis;

[0043] (Aspect 23) A method according to embodiment 22, wherein the baffle structure includes at least one pair of baffles, each having two baffles facing in opposite directions with respect to the central axis;

[0044] (Aspect 24) A method according to embodiment 22 or 23, wherein the baffle structure includes a plurality of baffles extending from the cylindrical inner wall toward the central axis;

[0045] (Aspect 25) A method according to embodiment 24, wherein the baffle does not extend to the central axis;

[0046] (Aspect 26) A method according to embodiment 24, wherein the baffles extend to the central axis, thereby dividing the inside of the container into a plurality of compartments that are in fluid communication with each other;

[0047] (Aspect 27) A method according to embodiment 22 or 23, wherein the baffle structure includes a plurality of baffles extending from the central axis toward the cylindrical inner wall;

[0048] (Aspect 28) A method according to any one of embodiments 21 to 27, wherein some or all of the baffles are solid plates or mesh or perforated plates;

[0049] (Aspect 29) A method according to embodiment 28, wherein some or all of the plates are oriented substantially parallel to the central axis of the container;

[0050] (Aspect 30) A method according to embodiment 28 or 29, wherein some or all of the plates are oriented at an angle of 10° to 80°, preferably 30° to 60°, with respect to the central axis of the container;

[0051] (Aspect 31) A method according to any one of embodiments 1 to 30, wherein the baffle structure includes a plurality of plates fixed to a support column, preferably the support column is oriented parallel to the central axis of the container, and preferably the plates of different orientations are alternately arranged along the support column;

[0052] (Aspect 32) A container for producing extracellular vesicles from producing cells, A baffle structure fixed inside the container, A coupling on the outer surface of the container, configured to be connected to a rotating device, A container equipped with;

[0053] (Aspect 33) A container according to embodiment 32, wherein the baffle structure includes one or more baffles fixed to the inner surface of the container;

[0054] (Aspect 34) A container according to embodiment 32 or 33, wherein the container has a cylindrical inner wall and a central axis;

[0055] (Aspect 35) A container according to embodiment 34, wherein the baffle structure includes at least one pair of baffles, each having two baffles facing in opposite directions with respect to the central axis;

[0056] (Aspect 36) A container according to embodiment 34 or 35, wherein the baffle structure includes a plurality of baffles extending from the cylindrical inner wall toward the central axis;

[0057] (Aspect 37) A container according to embodiment 36, wherein the baffle does not extend to the central axis;

[0058] (Aspect 38) A container according to embodiment 36, wherein the baffles extend to the central axis, thereby dividing the interior of the container into a plurality of compartments that are in fluid communication with each other;

[0059] (Aspect 39) A container according to embodiment 34 or 35, wherein the baffle structure includes a plurality of baffles extending from the central axis toward the cylindrical inner wall;

[0060] (Pattern 40) A container according to any one of embodiments 33 to 39, wherein some or all of the baffles are solid plates, or mesh-like or perforated plates;

[0061] (Aspect 41) A container according to embodiment 40, wherein some or all of the plates are oriented substantially parallel to the central axis of the container;

[0062] (Aspect 42) A container according to embodiment 40 or 41, wherein some or all of the plates are oriented at an angle of 10° to 80°, preferably 30° to 60°, with respect to the central axis of the container;

[0063] (Aspect 43) A container according to any one of embodiments 32 to 42, wherein the baffle structure includes a plurality of plates (4') fixed to a support column, preferably the support column is oriented parallel to the central axis of the container, and preferably the plates (4') of different orientations are alternately arranged along the support column;

[0064] (Aspect 44) A container according to any one of embodiments 32 to 43, wherein the coupling includes one or more grooves or ridges on the outer surface of the container;

[0065] (Aspect 45) In the container according to any one of embodiments 32 to 44, further, Closing cap, A container equipped with;

[0066] (Aspect 46) A container according to any one of embodiments 32 to 45, wherein the container is configured to perform the method described in any one of embodiments 1 to 31;

[0067] (Aspect 47) A method according to any one of embodiments 1 to 31, wherein the container is the container described in any one of embodiments 32 to 46;

[0068] (Aspect 48) A system for producing extracellular vesicles from producing cells, A container according to any one of embodiments 32 to 46, Rotating device and A system comprising the above, wherein the container is configured such that its rotation is fixed to the rotating device by key-connecting the coupling of the container to the corresponding coupling in the rotating device;

[0069] (Aspect 49) A system according to embodiment 48, wherein the rotating device includes a cup configured to receive the container; and

[0070] (Appearance 50) In the system according to embodiment 48 or 49, the rotating device includes a drive mechanism, and the system further comprises Control unit for controlling the drive mechanism, A system comprising, preferably, the control unit being configured to perform step b) of the method according to any one of claims 1 to embodiment 31.

[0071] The present invention can overcome the shortcomings of the prior art. Specifically, the present invention provides a highly efficient method for producing extracellular vesicles from producing cells, and a container for carrying out the method.

[0072] This is achieved by the method comprising the steps of placing production cells in a liquid culture medium into a container having a baffle structure inside, and rotating the container, thereby generating extracellular vesicles from the production cells, wherein the step of rotating the container includes a sub-step of repeatedly changing the rotational motion of the container.

[0073] The process of rotating the container eliminates the need for using a stirring blade or agitator. In this case, the manufacturing volume is determined by the diameter of the container, and the requirement for miniaturization, which was difficult to achieve with a typical stirring tank equipped with a stirring blade or agitator as described above, is met.

[0074] The inventors of the present invention noticed that by repeatedly changing the rotational motion of the container with the baffle structure, the fluid flow inside the rotating container was disturbed, and the amount of extracellular life (EV) produced was significantly higher compared to the amount produced from the same cells when a stirred tank (spinner flask) type bioreactor was operated at its maximum speed.

[0075] Specifically, the present invention offers the following advantages:

[0076] - Higher EV yields can be obtained compared to using a spinner flask-type bioreactor repurposed for cell culture; - This enables low-volume regimes (e.g., effective volumes in the range of 10-120 mL) that are perfectly in harmony with the cell count per unit volume associated with personalized medicine applications; - Controlling flow conditions, i.e., turbulence, becomes easier, enabling a more robust method in terms of EV yield per cell; - Extracellular vesicles can be produced from cells with a three-dimensional structure, such as organoids / spheroids; furthermore, -EV production can be linked to three-dimensional cell culture or spheroid / organoid maturation. [Brief explanation of the drawing]

[0077] [Figure 1] This figure shows an example of the container of the present invention. [Figure 2a] This is a top view of an example of a container equipped with a baffle structure including a solid baffle plate according to the present invention. [Figure 2b] This is an oblique top view of the container shown in Figure 2a, cut along a plane parallel to the central axis of the container. [Figure 3a] This is a top view of another example of a container with a baffled structure including a solid baffle plate according to the present invention. [Figure 3b] Figure 3a is an oblique top view of the container, cut along a plane parallel to the central axis of the container. [Figure 4a] This is a top view of another example of a container with a baffle structure including a mesh-like baffle plate according to the present invention. [Figure 4b] This is a side view of the container shown in Figure 4a, cut along a plane parallel to the central axis of the container. [Figure 4c] Figure 4a is an oblique top view of the container, cut along a plane parallel to the central axis of the container. [Figure 5a] This is a top view of another example of a container with a baffle structure including a plate fixed to a support column according to the present invention. [Figure 5b] This is a side view of the container shown in Figure 5a, cut along a plane parallel to the central axis of the container. [Figure 5c] Figure 5a is an oblique top view of the container, cut along a plane parallel to the central axis of the container. [Figure 6a] This is a top view of another example of a container with a baffle structure including a plate fixed to a support column according to the present invention. [Figure 6b] Figure 6a is a side view of the container, cut along a plane parallel to the central axis of the container. [Figure 6c] Figure 6a is an oblique top view of the container, cut along a plane parallel to the central axis of the container. [Figure 7a]The graph shows the number of extracellular vesicles (EVs) per bead as a function of rotational speed (rpm), using the container of the present invention (blank circle) and a general spinner flask (gray filled circle), as tested in Example 1 described below (vertical axis: number of extracellular vesicles per bead, horizontal bar (black): average value, horizontal axis: rotational speed of the container (blank circle), T0: control (time=0)). [Figure 7b] The metabolic activity of cells as a function of rotational speed (rpm) was tested using the container of the present invention (blank circle) and a general spinner flask (gray filled circle), as tested in Example 1 described below (vertical axis: metabolic activity (%), horizontal bar (black): average value, horizontal axis: rotational speed of the container (blank circle)). [Figure 7c] The following are Western blot results comparing the protein expression of transmembrane extracellular vesicle (EV) markers CD63 and CD9, cytoplasmic matrix EV markers Alix and Ferritin, and the non-EV marker 14-3-3, which is abundant in the soluble fraction, in extracellular vesicles (EVs) prepared using the container of the present invention and a general spinner flask, as tested in Example 1 described below. (The presence of EV markers in the EV fraction and the absence of non-EV markers is evidence of sample purity. Lanes No. 1 to No. 3 (lanes 2 to 7 from the left) correspond to three types of samples using a general spinner flask, and No. 4 (lanes 9 and 11 from the left) corresponds to a sample using the container of the present invention. "CCM" refers to concentrated cell culture medium, "EVs" refers to the extracted EV fraction after size exclusion chromatography, and "MM" refers to molecular weight markers.) [Figure 7d] This is a transmission electron microscope image of extracellular vesicles prepared using the container of the present invention, as tested in Example 1 described below. [Figure 8a]The graph shows the number of extracellular vesicles (EVs) per spheroid composed of mouse mesenchymal stem cells (mMSCs) as a percentage of rotation time (hours), as tested in Example 2 below using the container of the present invention (vertical axis: number of extracellular vesicles per spheroid, horizontal bars: average number of extracellular vesicles produced when the method of the present invention is performed (black) and for a control sample without rotation (white), horizontal axis: rotation time, T0: control for rotation time (hours = 0)). [Figure 8b] This shows the number of extracellular vesicles (EVs) per spheroid composed of cancer cells as a percentage of rotation time (hours), as tested in Example 2 described below using the container of the present invention (vertical axis: number of extracellular vesicles per spheroid, horizontal bar: average number of extracellular vesicles produced, horizontal axis: rotation time, T0: control for rotation time (hours = 0)). [Figure 9a] The graph shows the number of extracellular vesicles (EVs) produced per spheroid after 1 hour of rotation, as tested in Example 4a below, using the container of the present invention, against the rotation speed (rpm) (vertical axis: number of extracellular vesicles per spheroid, horizontal bar (black): average number, horizontal axis: rotation speed (rpm)). [Figure 9b] The graph shows the number of extracellular vesicles (EVs) produced per spheroid after 3 hours of rotation, as tested in Example 4a below, using the container of the present invention, against the rotation speed (rpm) (vertical axis: number of extracellular vesicles per spheroid, horizontal bar (black): average number, horizontal axis: rotation speed (rpm)). [Figure 9c]The protein expression of the transmembrane extracellular vesicle (EV) marker CD63, the cytoplasmic matrix EV marker Synt-1, and the non-EV marker 14-3-3, which is abundant in the soluble fraction, was tested in Example 4a below using the container of the present invention at various rotation speeds (rpm). (The presence of the EV marker and the non-EV marker in the EV fraction is evidence of the purity of the sample. No. 1 (the first and second lanes from the left) corresponds to a rotation speed of 800 rpm, and No. 2 (the third to fifth lanes from the left) corresponds to a rotation speed of 1,600 rpm. "CCM" refers to concentrated cell medium, "Int" refers to the intermediate fraction after size exclusion chromatography, "EVs" refers to the extracted EV fraction after size exclusion chromatography, and "MM" refers to the molecular weight marker.) [Figure 9d] This shows the number of EVs produced per cell in the container of the present invention, as tested in Example 4b described below (vertical axis: number of EVs produced per cell at various rotation speeds (rpm), horizontal axis: rotation time (hours)). [Figure 9e] The graph shows the percentage of cell death at various rotation speeds, with the horizontal axis representing rotation time (hours), using the same container as in Example 4b described later in Figure 9d. [Figure 10a] This is the average number of extracellular vesicles (EVs) per cell from a single (individual) cell, as tested in Example 5a below, using a container equipped with a baffle structure including the solid baffle plate of the present invention (vertical axis: number of extracellular vesicles per cell, horizontal axis: rotation speed (rpm), the two vertical bars on the left: EVs produced from mouse mesenchymal stem cells (mMSCs), the two vertical bars on the right: EVs produced from human mesenchymal stem cells (hMSCs)). [Figure 10b] This shows the number of EVs produced per cell in the container of the present invention, as tested in Example 5b described below (vertical axis: number of EVs produced per cell at various rotation speeds (rpm), horizontal axis: rotation time (hours)). [Figure 10c] The graph shows the percentage of cell death at various rotation speeds, with the horizontal axis representing rotation time (hours) and the vertical axis representing rotation time (hours), using the same container as in Example 5b described later in Figure 10b. [Figure 10d] This shows the protein expression of EV markers CD63 and CD81, cytoplasmic EV marker Synt-1, and non-EV marker 14-3-3 in EV prepared in Example 5b described below ("EVs" refers to the extracted EV fraction after size exclusion chromatography, "EV Int" refers to the intermediate fraction after size exclusion chromatography, and "MM" refers to the molecular weight marker, with bands below Synt-1 corresponding to the high-exposure Synt-1 band). [Figure 11a] The graph shows the number of extracellular vesicles (EVs) produced per spheroid after 1 hour of rotation as a percentage of the rotation speed (rpm), using a container equipped with a baffle structure including the mesh-like baffle plate of the present invention, as tested in Example 6 described below (vertical axis: number of extracellular vesicles per spheroid, horizontal bar (black): average number of extracellular vesicles produced, horizontal axis: rotation speed (rpm)). [Figure 11b] The graph shows the number of extracellular vesicles (EVs) produced per spheroid after 3 hours of rotation, relative to the rotation speed (rpm), using a container equipped with a baffle structure including the mesh-like baffle plate of the present invention, as tested in Example 6 described below (vertical axis: number of extracellular vesicles per spheroid, horizontal bar (black): average number of extracellular vesicles produced, horizontal axis: rotation speed (rpm)). [Figure 12a] The figures represent the number of extracellular vesicles (EVs) produced per spheroid relative to the number of rotational cycles performed, using a container equipped with a baffle structure including the mesh-like baffle plate of the present invention, as tested in Example 7 described below (vertical axis: number of extracellular vesicles produced per spheroid, horizontal axis: number of rotational cycles performed, black filled circle: 800 rpm rotation, blank circle: 1,200 rpm rotation, square: 1,600 rpm rotation). [Figure 12b] This shows the metabolic activity of cells as a function of the number of rotational cycles performed, using a container equipped with a baffle structure including the mesh-like baffle plate of the present invention, as tested in Example 7 described below (vertical axis: metabolic activity of cells, horizontal axis: number of rotational cycles performed, black filled circle: 800 rpm rotation, blank circle: 1,200 rpm rotation, square: 1,600 rpm rotation). [Figure 12c] This represents cell death as a percentage of rotational cycles performed, using a container equipped with a baffle structure including the mesh-like baffle plate of the present invention, as tested in Example 7 described below (vertical axis: cell death (%), horizontal axis: number of rotational cycles performed, black filled circle: 800 rpm rotation, blank circle: 1,200 rpm rotation, square: 1,600 rpm rotation). [Figure 13a] This is the number of extracellular organisms (EVs) produced per cell in a container equipped with a baffle structure including the solid baffle plate of the present invention, as tested in Example 3b described below, using hMSC spheroids formed in the container as the producing cells (vertical axis: number of EVs produced per cell at various rotation speeds (rpm), horizontal axis: rotation time (hours)). [Figure 13b] The vertical axis represents the percentage of cell death at various rotation speeds, as tested in Example 3b described later, using the same container as in Figure 13a, with respect to rotation time (hours). [Figure 13c] This shows the protein expression of EV markers CD63 and CD81, cytoplasmic matrix EV marker Synt-1, and non-EV marker 14-3-3 in EVs prepared in Example 3b described below (lanes "EV1", "EV2", and "EV3" correspond to three independent production lines of EV extracts after size exclusion chromatography; lanes "EV2 Int" and "EV3 Int" correspond to the intermediate fractions of EV2 and EV3 after size exclusion chromatography; lanes "2D" and "3D" correspond to controls (EVs produced by two-dimensional starvation and three-dimensional starvation, respectively); "MM" refers to the molecular weight marker, and bands below Synt-1 correspond to the high-exposure Synt-1 bands). [Figure 14a]This graph shows the expression of EV-specific markers tested in Example 8 (horizontal axis: detected marker, vertical axis: expression level (median fluorescence) relative to immunoglobulin (Ig) expression level, 2D and 3D correspond to controls (EVs produced by two-dimensional starvation and three-dimensional starvation, respectively), A corresponds to EVs produced from individual hMSC cells using the container of the present invention, C1-C3 correspond to EVs produced in the container of the present invention using hMSC spheroids formed in the container as producing cells, and the markers CD9 / CD63 / CD81 correspond to EV-specific proteins). [Figure 14b] This shows the expression of mesenchymal markers tested in Example 8 (horizontal axis: detected marker, vertical axis: expression level (median fluorescence) relative to immunoglobulin (Ig) expression level, 2D and 3D correspond to controls (EVs produced by two-dimensional starvation and three-dimensional starvation, respectively), A corresponds to EVs produced from individual hMSC cells using the container of the present invention, C1-C3 correspond to EVs produced in the container of the present invention using hMSC spheroids formed in the container as producing cells, and markers CD29 / CD44 / CD49e / CD105 / CD146 correspond to mesenchymal markers). [Figure 15a] These are microscopic images showing the germination of endothelial cell spheroids under various conditions tested in Example 9 (endothelial cell spheroids cultured alone (negative control; 0% FBS), 10% FBS and VEGF (positive control; 10% FBS and VEGF), EVs produced by two-dimensional or three-dimensional starvation (controls: comparative examples "2D" and "3D", respectively), EVs produced from individual hMSC cells (A), EVs produced from hMSC spheroids formed outside the container (B), and EVs produced from hMSC spheroids formed inside the container (C)) (1X, 2X, or 4X indicates dose multipliers). [Figure 15b]The graph shows the sprouting of endothelial cell spheroids tested in Example 9 under various conditions (endothelial cell spheroids cultured alone (negative control; 0% FBS), 10% FBS and VEGF (positive control; 10% FBS and VEGF), EVs produced by two-dimensional or three-dimensional starvation (controls: comparative examples "2D" and "3D", respectively), EVs produced from individual hMSC cells (A), EVs produced from hMSC spheroids formed outside the container (B), and EVs produced from hMSC spheroids formed inside the container (C)) (horizontal axis: under various conditions, vertical axis: number of endothelial cell spheroids that sprouted) (1X, 2X, or 4X indicate dose multiples). [Figure 16a] This graph shows the area coverage over time of human dermal fibroblasts (HSF) in a wound healing assay tested in Example 10 (horizontal axis: different time points (hours), vertical axis: area coverage (%) (corresponding to the ratio of the area covered by cells in the scratch to the total area of ​​the scratch), FBS 0%, FBS 1%, FBS 2%, FBS 4%, and FBS 10% correspond to controls cultured with HSF alone, HSF cultured with 1% FBS, HSF cultured with 2% FBS, HSF cultured with 4% FBS, and HSF cultured with 10% FBS, respectively). [Figure 16b] This graph shows the time course of human dermal fibroblast (HSF) scratch wound closure in the wound healing assay tested in Example 10 (horizontal axis: different time points (hours), vertical axis: closure (%) (corresponding to the ratio of the area covered by cells within the scratch wound to the total area of ​​the scratch wound), 2D and 3D represent other controls (HSF cultured in EVs produced by two-dimensional starvation and three-dimensional starvation, respectively). [Figure 16c]This graph shows the time course of wound closure of human dermal fibroblasts (HSF) in the wound healing assay tested in Example 10 (horizontal axis: different time points (hours), vertical axis: degree of closure (%) (corresponding to the ratio of the area covered by cells in the scratch to the total area of ​​the scratch), A corresponds to HSF cultured with EVs produced from individual hMSC cells, B corresponds to HSF cultured with EVs produced from hMSC spheroids formed outside the container, and C corresponds to HSF cultured with EVs produced from hMSC spheroids formed inside the container). [Figure 17] The anti-inflammatory effect of EVs prepared according to the present invention, as tested in Example 11 (horizontal axis: type of treatment, vertical axis: NO concentration (arbitrary unit) in relation to measured absorbance value) (CTL refers to a control (positive control (+), negative control (-), control containing a known anti-inflammatory inhibitor (Inh), A corresponds to EVs produced from individual hMSC cells, B corresponds to EVs produced from hMSC spheroids formed outside the container, and C corresponds to EVs produced from hMSC spheroids formed inside the container, with 0.25X, 0.5X, 1X, or 2X indicating dose multipliers). [Figure 18a] This is an oblique top view of a container, which is an example of a container other than the present invention, as tested in Example 12 described later. [Figure 18b] Figure 18a is a side view of the container shown, cut along a plane parallel to the central axis of the container. [Figure 18c] This shows the number of EVs produced in the containers shown in Figures 18a and 18b, as tested in Example 12 described later (horizontal axis: rotation time (hours), vertical axis (left): number of EVs produced per spheroid, vertical axis (right): number of EVs produced per producing cell). [Figure 19] This is an ultra-high-speed camera image showing the projection velocity of cells produced in the container (lateral cross-section) of the present invention during use (the grayscale on the right shows the projection particle velocity (mm / sec), and the velocity values ​​shown on the trajectory are in the range of less than 20 mm / sec to more than 125 mm / sec depending on the location on the trajectory). [Modes for carrying out the invention]

[0078] The present invention will be described in detail below, but not limited to, the present.

[0079] As used herein, the term "extracellular vesicle" refers to a vesicle that is endogenously released by a producing cell, either constitutively or in an induceable manner. The particle size of extracellular vesicles is generally between 30 nm and 500 nm. Examples of extracellular vesicles include, but are not limited to, exosomes, microvesicles, and apoptotic bodies.

[0080] As used herein, the term "cell" refers to the smallest structural and functional basic unit of an organism that is capable of division and reproduction.

[0081] As used herein, the term “producing cell” refers to a cell capable of secreting extracellular vesicles.

[0082] As used herein, the term “organoid” refers to a cell aggregate that replicates the characteristics of cell self-organization, structure, and signaling interactions that occur in native organs.

[0083] As used herein, the term "spheroid" refers to a cellular structure composed of multiple single cells that grows from one or more cells.

[0084] As used herein, the term “microcarrier” refers to a particulate matrix capable of growing productive cells attached to its surface or within it. The matrix may consist of particles with a maximum particle size of 50 μm to 500 μm, preferably 100 μm to 300 μm, and preferably approximately spherical. The microcarriers are generally beads whose specific gravity is selected to be approximately the same as that of the liquid culture medium for the productive cells, thereby allowing the beads to remain suspended in the liquid culture medium with only gentle mixing.

[0085] As used herein, the term "container" refers to any type of container, such as a cylinder or tank, that holds a liquid culture medium.

[0086] As used herein, the term “vertical” refers to a direction that is perpendicular to the horizontal plane and parallel to the direction of gravity.

[0087] (A container for producing extracellular vesicles from producing cells) The present invention provides a container for producing extracellular vesicles (EVs) from production cells. The container has an internal space for holding a liquid culture medium. The container has a baffle structure inside the container, i.e., inside the internal space. Preferably, the container has a coupling on its outer surface configured to be connected to a rotating device, as will be detailed later.

[0088] A "baffle structure" refers to one or more components fixed inside a container that cause the flow of the liquid inside the container to deviate. Generally, baffle structures promote turbulence within the container.

[0089] Figure 1 shows an example of the container of the present invention.

[0090] The container 1 has a baffle structure (not shown in Figure 1) inside the container, which will be described in detail later, and a coupling 3 on the outer surface of the container that is configured to be connected to a rotating device.

[0091] The container may be made of a material suitable for the production of extracellular vesicles, such as a biocompatible resin, biocompatible polymer, or metal.

[0092] The container can be manufactured by 3D printing, injection molding, blow molding, or compression molding, preferably by 3D printing.

[0093] In some embodiments, the container is not subjected to (or has not undergone) surface treatment.

[0094] Preferably, as shown in the figure, the internal space of the container is defined by a base and a peripheral wall extending from the base. Preferably, the base is substantially flat. Preferably, the baffle structure does not extend over the entire peripheral wall. Preferably, the peripheral wall is smooth, i.e., has no depressions.

[0095] Preferably, the shape of the container is substantially cylindrical (or, at least, the shape of the internal space of the container is substantially cylindrical). In this case, as shown in Figure 1, the peripheral wall becomes a cylindrical inner wall. The central axis of the container can be defined as the axis of the cylinder. Preferably, the axis is perpendicular to the base and the cylinder is a straight cylinder. The cylinder is preferably cylindrical, and more preferably a straight cylinder. In an alternative embodiment, the peripheral wall may be non-cylindrical in shape. For example, if the base is substantially polygonal (such as a square or cuboid), the peripheral wall may be cylindrical in shape, formed by connecting a plurality of flat sections (such as four flat sections) along their respective edges.

[0096] The capacity volume of the container can be adjusted as appropriate, for example, depending on the effective volume, the target number of extracellular vesicles to be produced, etc.

[0097] In some embodiments, the capacity of the container may be 10 mL to 10 L. For example, the capacity may be 10 mL to 5 L, 10 mL to 1 L, 10 mL to 500 mL, or 10 mL to 250 mL.

[0098] The diameter of the container can be adjusted as appropriate depending on the different effective volume. For example, the inner diameter of the container may be 2 to 25 cm. For example, the diameter of the container may be 2 to 20 cm, 3 to 15 cm, or 3 to 10 cm.

[0099] The height of the container (the maximum dimension in the direction parallel to the central axis) can be 1 to 30 cm, preferably 2 to 20 cm, and more preferably 3 to 10 cm.

[0100] The container may be provided with a closure, such as a cap. In this case, the container may have a neck at its top (opposite the base at the bottom) designed to receive the cap for sealing the container.

[0101] The dimensions and shape of the neck may vary depending on the type of container and the cap. Generally, the neck has a smaller diameter than the rest of the container (also referred to as the "body") and includes a shoulder (a transitional section between the body and the neck) which may be a curved or inclined portion of the container from the body to the neck where the diameter changes. The cap may be secured to the container by screwing, bayonet connection, press-fitting, magnetic connection, or the like.

[0102] The coupling may have any geometric shape, as long as it is a suitable geometric shape that allows the rotation of the container to be fixed to the rotating device.

[0103] In some embodiments, the coupling may include one or more grooves or ridges on the outer surface of the container.

[0104] For example, as shown in Figure 1, the coupling 3 may include ridges (relieving or protruding lines) extending along the outer surface of the container (on the back surface of the peripheral wall of the internal space) in a direction parallel to the central axis of the container. Grooves extending along the outer surface of the container in a direction parallel to the central axis of the container are also possible.

[0105] Alternatively, or in addition to the above, the coupling 3 may be provided on the outer surface of the bottom of the container (on the underside of the base in the internal space).

[0106] The baffle structure may extend from the peripheral wall of the container toward the central axis of the container.

[0107] In some embodiments, the baffle structure may include one or more baffles. A “baffle” means a wall that may be substantially flat or substantially curved, but is preferably substantially flat. The baffles are fixed to the inner surface of the container in the internal space of the container. The baffles are preferably fixed to the base and / or the peripheral wall, and preferably integrally formed with the base and / or the peripheral wall. Each baffle functions as an obstruction to the flow of liquid inside the container and is configured to deflect such flow of liquid. When it is stated in this text that A is “fixed” to B, it may mean that it is indirectly fixed (i.e., A is fixed to B through some element), more preferably “directly fixed” (i.e., A is fixed to B without any element interposed between A and B).

[0108] For example, the baffle structure may include one, two, three, four, five, six, seven, eight, nine, or ten baffles fixed to the inner surface of the container.

[0109] If the baffle structure includes two or more baffles, the baffles may be arranged at equal intervals within the container, or they may be arranged at uneven intervals within the container.

[0110] In some embodiments, the baffles may be fixed symmetrically to each other on the inner surface of the container with respect to the central axis of the container.

[0111] The baffles may have the same dimensions (e.g., height, thickness, length, etc.) or they may have different dimensions.

[0112] For example, the height of each baffle may be 1 to 25 cm, preferably 1 to 15 cm, and more preferably 1.5 to 6 cm. As used herein, the term “height” of a baffle refers to the maximum dimension of the baffle in the direction parallel to the central axis.

[0113] The length of each baffle may be 1 to 12.5 cm, preferably 1 to 5 cm, and more preferably 1 to 2.5 cm. As used herein, the term “length” of a baffle refers to the maximum dimension of the baffle perpendicular to the central axis (preferably parallel to the base of the container).

[0114] The thickness of each baffle may be 0.1 to 5 cm, preferably 0.1 to 2.5 cm, and more preferably 0.1 to 0.5 cm. As used herein, the term “thickness” of a baffle refers to a dimension that is smaller than the maximum dimension in each orthogonal direction and perpendicular to the length direction (preferably parallel to the base of the container).

[0115] In some embodiments, the baffle structure includes at least one pair of baffles. Each pair of baffles may have two baffles facing each other in opposite directions with respect to the central axis of the container.

[0116] Preferably, the two baffles of each pair are identical in terms of dimensions.

[0117] For example, the baffle structure may include one pair, two pairs, three pairs, four pairs, or five pairs of baffles facing each other in opposite directions.

[0118] If the baffle structure includes at least two pairs (for example, n pairs) of baffles, the pairs may be arranged such that the baffles (for example, 2n baffles) are equidistant from each other. Alternatively, the pairs may be arranged such that the baffles (for example, 2n baffles) are not equidistant from each other.

[0119] In some embodiments, pairs of baffles may be arranged symmetrically with respect to the central axis of the container.

[0120] The dimensions of the two baffles in each pair may be the same throughout all pairs, or they may differ from pair to pair.

[0121] In some embodiments, the baffle structure may include a plurality of baffles extending from the peripheral wall toward the central axis.

[0122] The baffles may be directly connected to each other. For example, the baffles may extend to the central axis and be connected at the central axis. In this case, the baffle structure may divide the internal space of the container into a plurality of compartments that are fluidly connected to each other.

[0123] In the modified configuration, the baffles are not directly connected to each other (rather, they are only indirectly connected via the base or the peripheral wall). Specifically, the baffles may not extend to the central axis, thereby creating an unobstructed central space within the container. In this case, the baffles may extend at different distances toward the central axis, or they may extend at the same distance toward the central axis.

[0124] In other embodiments, the baffle structure may include a plurality of baffles extending from the central axis of the container toward the peripheral wall of the container.

[0125] In some embodiments, the baffles do not extend to the peripheral wall of the container, thereby creating an unobstructed peripheral space between the baffles and the peripheral wall.

[0126] Some or all of the aforementioned baffles may be plates or walls, i.e., substantially flat elements. These elements may have a single dimension, i.e., a thickness, that is much thinner (e.g., more than 10 or 100 times thinner) than the maximum dimension in each orthogonal direction.

[0127] The plate or wall is preferably substantially flat, but may be curved as a modified example.

[0128] If the plate or wall is substantially flat, the thickness shall be the dimension of the plate perpendicular to the main plane of the plate or wall. The thickness may vary or remain constant throughout the structure. If it varies, the thickness value shall specify the average thickness.

[0129] The plate or wall may be a solid plate or wall, or it may be a mesh or perforated plate or wall. As used herein, the term “mesh plate” refers to a plate formed from intertwined wires or posts (also referred to as a “grid”). As used herein, the term “perforated plate” refers to a plate having openings or holes.

[0130] In the case of a mesh-like or perforated plate or wall, the mesh size (dimensions of the openings / holes), the uniformity of the openings / holes, and the arrangement of the openings / holes can be adjusted as appropriate.

[0131] For example, the dimensions of the openings / holes in a mesh-like or perforated plate or wall (e.g., the diameter in the case of a circular opening / hole, the diagonal in the case of a polygonal opening / hole, etc.) can be 0.1 to 5 cm, preferably 0.1 to 1 cm, and more preferably 0.1 to 0.5 cm.

[0132] The outer shape of the plate or wall (perpendicular to the direction of the thickness) may be approximately quadrangular, rectangular, triangular, trapezoidal, rhombic, pentagonal, hexagonal, octagonal, or more broadly, polygonal, and may be at least partially curved. Specifically, the plate or wall may be approximately perpendicular to the base, and may be oriented approximately parallel to the central axis of the container.

[0133] As a variation, the plate or wall may be oriented substantially perpendicular to the central axis of the container.

[0134] Furthermore, the plate or wall may be oriented at an angle of 10° to 80°, preferably 30° to 60°, with respect to the central axis of the container and / or the base.

[0135] For example, the plate or wall may be oriented at an angle of approximately 10°, 20°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 70°, or 80° with respect to the central axis of the container and / or the base.

[0136] All angle values ​​in this text are absolute values.

[0137] All baffles may or may not be oriented in the same direction.

[0138] In some embodiments, the baffle structure may include a plurality of plates fixed to a support column, preferably the support column oriented parallel to the central axis of the container. Preferably, plates oriented in different directions are arranged alternately along the support column.

[0139] In the following, several possible designs for the container of the present invention will be described in detail with reference to the drawings, but please understand that the design of the container may be modified or further modified to optimize the flow conditions inside the container.

[0140] Referring to Figures 2a and 2b, the container 1 may have a substantially cylindrical shape and may have a baffle structure 2 and a coupling 3 inside the container.

[0141] The container may further have a neck 1′ at the top of the container and a body 1′′. The body 1′′ has a base 7 and a peripheral wall 8, as described above in relation to Figure 1.

[0142] The baffle structure 2 may have three pairs of baffles 4, each having two baffles facing in opposite directions with respect to the central axis of the container. Naturally, other numbers of baffle pairs are also possible.

[0143] In this example, the baffle structure 2 includes a total of six baffles 4 fixed to the inner surface of the container (peripheral wall 8 and / or base 7), and the three pairs are arranged such that the six baffles are spaced equally apart along the circumference of the cylinder.

[0144] All baffles may be solid plates as shown in the diagram, but baffles may also be mesh-like or perforated plates, or a combination of solid plates, mesh-like plates and perforated plates.

[0145] The plate may, but is not limited to, a rectangular shape.

[0146] The baffles may extend from the peripheral wall 8 toward the central axis, but not to the central axis itself (in other words, the baffles do not reach the central axis of the container, creating a central space).

[0147] The two baffles 4 in each pair may be identical within the pair, but their dimensions may differ from pair to pair. For example, the baffles may be plates of the same thickness and length, but their heights may differ from pair to pair. As used herein, the term "height" (of structures such as plates and baffles) refers to the maximum dimension of the structure such as plates and baffles in the direction parallel to the central axis.

[0148] As used herein, the term "length" (of structures such as plates and baffles) refers to the maximum dimension of the structure such as plates and baffles that is perpendicular to the central axis (preferably parallel to the base 7).

[0149] As a variation, the two baffles 4 in each pair may have the same height, length, and thickness (identical to each other), or they may have the same thickness but different height and length, or the same height and thickness but different length, or the same height and length but different thickness, or the same length but different height and thickness, or they may have different height, length, and thickness.

[0150] Some or all of the plates may have a plane that is oriented substantially parallel to the central axis of the container.

[0151] For example, as shown in Figures 2a and 2b, the planes of all plates may be oriented substantially parallel to the central axis of the container.

[0152] As a basic principle, in all embodiments described herein, each edge or corner may be provided with a rounded chamfer or fillet to prevent sharp edges that could induce localized high-shear areas that could damage any biological material present in the container.

[0153] In the examples shown in Figures 2a and 2b, the edges of the baffle 4 that face the central axis (preferably extending parallel to the central axis) are chamfered with a radius (R-chamfer).

[0154] The containers shown in Figures 3a and 3b are the same as the example containers shown in Figures 2a and 2b, except that fillets are also present in the parts connecting the baffle 4 to the peripheral wall 8 and base 7, allowing for a smooth transition from each baffle to the peripheral wall 8 and base 7.

[0155] The arrangement, orientation, and dimensions of the aforementioned baffles may be as defined above.

[0156] Container 1 shown in Figures 4a to 4c is the same as the containers shown in the examples in Figure 2 and Figure 3, except for the baffle structure.

[0157] The baffle structure 2 may include six baffles 4 and a central support 5. The central support may be aligned with the central axis of the container, or it may be fixed to the base 7 of the container.

[0158] The baffles can be made of mesh plates having the same dimensions, as shown in Figures 4a to 4c. However, it should be understood that they may be perforated plates and / or may have different dimensions from each other. Preferably, the plates are substantially flat.

[0159] The baffle 4 may be fixed to the central support 5 and extend from the central support 5 toward the cylindrical inner wall. If the baffle is flat, it may be approximately perpendicular to the base 7.

[0160] The height of the central support column 5 may be greater than the height of the main body 1′′ of the container (the height in the direction parallel to the walls of the container). In other words, the central support column 5 may extend from the base 7 to the neck 1′. The top edge of each baffle (i.e., the edge opposite to the base 7) may be oriented at an angle other than 90° with respect to the central axis (central support column 5), for example, approximately 45°. As a result, the planar outer shape of the plate may be trapezoidal (see Figure 4b).

[0161] However, please note that the height of the central support column 5 may be shortened, and the plane of the baffle may be made into a different shape, such as a rectangular prism.

[0162] The plates may be spaced equally apart with respect to the central axis.

[0163] In Figures 4b and 4c, the baffles do not extend to the peripheral wall 8, creating an unobstructed space between each baffle and the peripheral wall 8. In a modified example, the baffles may extend from the central support column 5 to the peripheral wall 8, thereby dividing the interior of the container into multiple compartments. These compartments may be in fluid communication with each other, for example, through openings in a mesh or perforated plate.

[0164] In some embodiments, the container does not have a central support. In this case, the baffle 4 may extend from the central axis of the container toward the cylindrical inner wall (or to the cylindrical inner wall) (and may be fixed to each other along the central axis), or it may extend from the cylindrical inner wall of the container toward the central axis (or to the central axis).

[0165] In some other embodiments, the baffles 4 may be fixed to the base 7 of the container, creating an unobstructed outer peripheral space and / or an unobstructed central space within the container between the baffles and the peripheral wall.

[0166] The baffle 4 may be oriented radially with respect to the central axis of the container.

[0167] Referring to the embodiments shown in Figures 5a to 5c, container 1 in this example is the same as the container in the examples shown in Figures 2 to 4, except for the baffle structure.

[0168] The baffle structure 2 shown in Figure 5a includes multiple plates 4' fixed to multiple support columns 6.

[0169] Preferably, the support columns 6 are cylindrical in shape and extend along their respective axes. The cross-section of the support columns 6 (perpendicular to the axis) can be circular, polygonal, or the like. In the illustrated embodiment, the cross-section is star-shaped. Such a non-circular shape can further improve the flow characteristics inside the container.

[0170] Preferably, the support columns 6 are parallel to each other, and preferably, the support columns 6 are oriented parallel to the central axis of the container.

[0171] The support columns 6 to which plate 4' is fixed can be fixed to the base 7 of the container (see Figures 5b and 5c). The support columns 6 may be arranged in a sequence, with each sequence supporting multiple separate plates. The structure consisting of the sequence of support columns and the multiple plates supported by the sequence can be referred to as the laminate 2'.

[0172] The number of support columns 6 can be adjusted according to the number and dimensions of plates 4'. For example, the support columns 6 in each array may be arranged in three rows, with each row having five support columns, as shown in Figure 5c. As another example, the support columns in each array may be arranged in one, two, three, four, or five rows, with two, three, four, five, six, seven, eight, nine, or ten support columns per row. The rows may be oriented radially with respect to the central axis of the container, for example.

[0173] Preferably, the plates are flat. The plane of each plate may be oriented at an angle of 10° to 80°, preferably 30° to 60°, with respect to the central axis of the container, and / or at an angle of 10° to 80°, preferably 30° to 60°, with respect to the base 7 of the container.

[0174] For example, as shown in Figure 5c, the plane of each plate facing the central axis of the container may be oriented at an angle of approximately 45° with respect to the central axis of the container.

[0175] Furthermore, the plates 4' may be oriented in different directions, or plates 4' oriented in different directions may be alternately arranged along the support column 6.

[0176] Although this is merely an example, the plates arranged in a continuous line along each column of the arrangement may be oriented symmetrically with respect to a plane (preferably parallel to the base 7 of the container) between the continuous plates. As shown in Figures 5b and 5c, these plates 4' oriented in different directions may be arranged alternately along the columns to form a zigzag pattern.

[0177] The baffle structure 2 may include one or more baffle layers 2'.

[0178] The laminated structures 2' may be spaced at equal distances from each other. As a variation, the laminated structures 2' may be spaced at unequal distances from each other. Specifically, the laminated structures 2' may be arranged at equal intervals around the central axis.

[0179] In some embodiments, the laminates 2' may be arranged symmetrically (in pairs) with respect to the central axis of the container.

[0180] The laminated structures 2' may have the same dimensions or may have different dimensions.

[0181] The baffle structure 2 may have at least one pair of laminates 2', for example, three pairs of laminates 2', each having two laminates facing in opposite directions with respect to the central axis of the container.

[0182] These pairs of laminates 2' may be arranged such that all laminates are equidistant from each other.

[0183] For example, the baffle structure 2 may include three pairs of laminates 2', that is, a total of six laminates 2', and the three pairs may be arranged such that the six laminates are equidistant from each other.

[0184] Each pair of laminated bodies 2' may have the same dimensions within that pair, but may have different dimensions from pair to pair.

[0185] As a variation, the dimensions of each pair of laminates 2′ may differ.

[0186] A region free of obstacles may exist near the central axis. In some cases, plate 4' may be fixed to the peripheral wall 8. In other cases, a region free of obstacles may exist in the annular region between the laminate 2' and the peripheral wall 8.

[0187] Referring to Figures 6a to 6c, container 1 in this example is the same as the container shown in Figures 5a to 5c, but with the following modifications.

[0188] As shown in Figure 6a, in addition to the container shown in Figures 5a to 5c, the container is further equipped with a central support column 5.

[0189] The central support column 5 is preferably aligned with the central axis of the container and can be fixed to the base 7 of the container.

[0190] Plate 4' can be fixed to a single row of support columns 6 and a central support column 5.

[0191] Each laminate 2' may extend from the central support towards or to the peripheral wall 8. In some cases, the plate 4' may be fixed to the peripheral wall 8. In other cases, there may be an unobstructed space in the annular region between the laminate 2' and the peripheral wall 8.

[0192] (A system for producing extracellular vesicles (EVs) from producing cells) The present invention further provides a system for producing extracellular vesicles (EVs) from producing cells.

[0193] The system includes the container and rotating device defined above.

[0194] The container is configured such that its rotation is fixed to the rotating device by key-connecting the coupling of the container to the corresponding coupling of the rotating device. If the coupling of the container has a groove, the coupling of the rotating device may have a corresponding edge. If the coupling of the container has an edge, the coupling of the rotating device may have a corresponding groove.

[0195] In some embodiments, the rotating device may include a rotating body such as a cup configured to receive the container. The coupling in the rotating device may be located on the inner surface of the cup that is in contact with the outer surface of the container.

[0196] Alternatively, or in combination therewith, the rotating device may include a fixing mechanism within the rotating body that maintains the container in a fixed position, such as fastening means using screws or press-fit engagements.

[0197] In some embodiments, the outer shape of the container is non-circular and the cup has a corresponding shape, thereby ensuring that the rotation of the container is securely fixed to the cup without requiring coupling on the outer surface of the container or on the inner surface of the cup. For example, the container may have one or more flat outer surfaces in addition to the base (for example, the container may have an overall rectangular parallelepiped shape), or it may have an ellipsoidal surface. In this case, the cup will have a complementary shape such that one or more outer surfaces of the container and one or more inner surfaces of the cup are in secure contact.

[0198] In some embodiments, the rotating device may include a drive mechanism and a control unit that controls the drive mechanism.

[0199] The rotating body (for example, a cup) can be operated by the drive mechanism. The rotating device may have a fixed portion which may include the drive mechanism and the control unit.

[0200] Preferably, the control unit may be configured to perform the rotation of the container (described later) required by the method of the present invention.

[0201] The control unit may comprise one or more processing means connected to a storage medium, and a computer program including instructions stored in the storage medium for performing various processes which will be described in detail later. The control unit may receive input from sensors of the rotating device or sensors associated with the rotating device and / or input from a user. The control unit may issue commands to the drive mechanism as a result of processing the input data. In some embodiments, part or all of the control unit may be located in a separate computing device instead of the rotating device.

[0202] (Method for producing extracellular vesicles (EVs) from producing cells) The present invention further provides a method for producing extracellular vesicles from producing cells.

[0203] This method, a) A step of placing the cells produced in a liquid culture medium into a container having a baffle structure inside, b) A step of rotating the container, thereby generating extracellular vesicles from the producing cells, c) A step of collecting the extracellular vesicles that have been generated, The invention comprises a sub-step (b) in which the container is rotated, and the sub-step (b) includes repeatedly changing the rotational motion of the container.

[0204] The method of the present invention is performed ex vivo.

[0205] The container may be one of those defined above. The container may be rotated by the rotating device described above.

[0206] By rotating the container, the liquid culture medium inside the container flows. Preferably, no liquid culture medium is added or removed during rotation. Preferably, the container is not fluidically connected to a supply line and / or a recovery line within its internal space.

[0207] Preferably, the producing cells move freely due to the flow of the liquid culture medium. In other words, it is preferable that the producing cells are contained in the majority of the liquid culture medium and are not fixed to the walls of the container, as shown in Figure 19.

[0208] Preferably, the rotation of the container is such that the flow of the liquid inside the container becomes turbulent. Preferably, a turbulent region characterized by a Reynolds number greater than 2,000 or greater than 7,000, preferably greater than 10,000, most preferably greater than 12,000, greater than 15,000, or greater than 20,000 can be achieved while minimizing shear stress on cells in the liquid. At the point when a stationary region occurs, i.e., when the average velocity of the liquid inside the container is equal to the velocity of the container, the global Reynolds number can be estimated overall as Re = R × V / v (wherein R is the inner radius of the container, V is the velocity of the container's perimeter wall, and v is the kinematic viscosity of the liquid). At the point in time when a transition region occurs, i.e., when the rotational speed changes or the direction of rotation reverses, the Reynolds number can be locally estimated as Re = W × V' / v (wherein W is the characteristic dimension of the baffle body (such as the radial length of the baffle plate), V' is the relative velocity between the liquid and the baffle body, and v is the kinematic viscosity of the liquid). Preferably, for at least a portion of the duration of step b), the global Reynolds number and / or local Reynolds number defined above are within any of the ranges listed above.

[0209] As shown in Figure 19, the produced cells can move toward the container at speeds that can reach maximum values ​​of 25 mm / second or more, 50 mm / second or more, 75 mm / second or more, 100 mm / second or more, or 125 mm / second or more.

[0210] Preferably, there are no stirring blades or other rotating or stirring bodies (such as magnetic stirrs) inside the container, meaning that the flow of the liquid is caused solely by the rotation of the container itself.

[0211] In step b), preferably, the container is closed (for example, by using the closure described above). In this step, the container may be substantially filled with the liquid culture medium (i.e., it may have no (or almost no) gas headspace). In a modified example, gas headspace may be present. Preferably, if present, the volume of gas headspace in the container may be less than 20%, less than 10%, less than 5%, less than 2%, or less than 1% of the volume of liquid in the container.

[0212] In some embodiments, the cells produced may be selected from human cells, animal cells, and combinations thereof.

[0213] In some embodiments, the cells produced may be human cells, preferably healthy human cells. In some embodiments, the cells produced may not be human embryo-derived cells, and in particular may not be human embryonic stem cells.

[0214] As a variation, the cells produced may be pathological cells, such as tissue-derived cells and / or cancer cell lines such as A673 cells or HeLa cells.

[0215] In some embodiments, the cells produced may be animal cells, preferably mouse cells, such as mouse MSC (mouse mesenchymal stem cell) cells.

[0216] In some embodiments, the cells produced may be stem cells, particularly induced pluripotent stem cells or compound pluripotent cells. For example, the stem cells may be selected from compound pluripotent mesenchymal cells, genetically modified cells, human umbilical vein endothelial cells (HUVECs), or primary cultured cells.

[0217] In other embodiments, the producing cells may be cells from a cell line, preferably a human monocyte line or a human cell line derived from B lymphocyte-derived hematopoietic cells, more preferably THP-1 cells or Raji cells.

[0218] In some embodiments, the producing cells may be cells of an isogenic lineage, that is, the producing cells may be producing cells derived from a certain subject, and the extracellular vesicles produced by these producing cells may be administered to or used ex vivo by the same subject or another subject (for the prevention or treatment of a disease).

[0219] In some embodiments, the extracellular vesicles are administered to a subject (to prevent or treat a disease), but the producing cells do not necessarily have to originate from the subject. In this case, the producing cells may be cells of an allogeneic lineage, i.e., cells of the same species as the subject. As a variation, the producing cells may be cells of a heterogeneous lineage, i.e., cells of a different species than the subject.

[0220] The subject is preferably a human, but may also be an animal.

[0221] The aforementioned cells produced may adhere to the culture medium or they may not adhere to the culture medium (also called suspension cells).

[0222] In the case of adherent, prolific cells, the culture medium may consist of microcarriers suspended in a liquid culture medium.

[0223] In some embodiments, the producing cells may be adherent producing cells that have been detached from the culture medium and placed in a suspension state by an appropriate treatment selected from, for example, enzymatic treatment, chemical treatment, mechanical treatment, or a combination thereof.

[0224] Preferably, the producing cells are in the form of individual cells suspended in a liquid culture medium. "Individual cells suspended in a liquid culture medium" means that the cells are separate from each other.

[0225] In some other embodiments, the producing cells may be in the form of cell aggregates. The term "cell aggregates" refers to an aggregate of a plurality of producing cells that adhere to each other.

[0226] In that case, the producing cells are preferably in the form of spheroids and / or organoids.

[0227] In some embodiments, when considering a single cell as the producer, the concentration of the producing cells in the liquid medium in the container when the extracellular vesicles are produced is 1,000 to 1,000,000 cells per milliliter, preferably 10,000 to 500,000 cells per milliliter, more preferably 50,000 to 200,000 cells per milliliter, and even more preferably 100,000 to 150,000 cells per milliliter.

[0228] In some embodiments, when the producing cells are adhered to microcarriers (beads), the number of cells per bead is 1 to 50, preferably 10 to 20. The concentration of beads is 100 to 50,000 beads per milliliter, preferably 1,000 to 20,000 beads per milliliter, more preferably 4,000 to 8,000 beads per milliliter.

[0229] In some embodiments, when the producing cells are cell aggregates such as spheroids or organoids, the average particle size of the aggregate is 50 μm to 5 mm, preferably 100 μm to 500 μm, and the number of cells per aggregate is 100 to 1,000,000, preferably 1,000 to 100,000. The concentration of the aggregates is 10 to 10,000 aggregates per milliliter, preferably 50 to 2,000 aggregates per milliliter, more preferably 100 to 500 aggregates per milliliter.

[0230] It is well known that the structure and composition of extracellular vesicles, particularly in terms of the membrane markers and components present within them, vary depending on the producing cell and the method used to create it.

[0231] In some embodiments, the average particle size of extracellular vesicles produced according to the present invention is 40-500 nm, preferably 65-200 nm, and more preferably 80-110 nm.

[0232] The average particle size of extracellular vesicles can be measured by interferometry alone, or by a combination of interferometry and fluorescence, such as with ExoView® R100 (NanoView Bioscience). Alternatively, the average particle size can be measured by individual particle tracking (or nanoparticle tracking analysis (NTA)) using, for example, NanoSight NS300 (Malvern Panalytical).

[0233] The liquid culture medium used in this invention for the production of extracellular vesicles can be a general liquid culture medium such as FBS (fetal bovine serum), (serum-free) DMEM (Dulbecc's modified Eagle medium), or serum-free medium.

[0234] The container rotates around its axis of rotation.

[0235] More preferably, if the container has the central axis defined above, the axis of rotation corresponds to the central axis of the container.

[0236] In some preferred embodiments, the axis of rotation is oriented substantially vertically (parallel to the direction of gravity).

[0237] In some embodiments, the flow trajectory of the liquid inside the container is directly visualized during rotation. In such a configuration, a high-speed camera can track the movement of beads (e.g., fluorescent beads) (or cells or particles attached to such beads or labeled with fluorescent markers) on the plane of the rotating container as the container rotates. This allows for thorough analysis of the shear stress experienced by the cells during rotation, resulting in superior control of the flow conditions.

[0238] In some embodiments, step b) of rotating the container is performed at a maximum rotational speed of 50 to 6,000 rpm, preferably 600 to 1,600 rpm. In some embodiments, the maximum rotational speed may be 50 to 100 rpm, 100 to 200 rpm, 200 to 300 rpm, 300 to 600 rpm, 600 to 1,000 rpm, 1,000 to 1,600 rpm, 1,600 to 3,000 rpm, or 3,000 to 6,000 rpm.

[0239] Step b) includes a sub-step that repeatedly changes the rotational motion of the container. This means that the container does not rotate at a constant speed throughout step b). The rotational speed of the container changes multiple times during step b).

[0240] In some embodiments, step b) may include a sub-step that periodically changes the rotational motion of the container. This means that a certain pattern of rotational motion is repeated multiple times at a constant frequency.

[0241] The frequency at which the rotational motion of the container is changed may be 0.01 to 5 Hz, preferably 0.05 to 0.5 Hz.

[0242] For example, step b) may include a sub-step that repeatedly (e.g., periodically) reverses the direction of rotation (clockwise to counterclockwise or vice versa).

[0243] Alternatively, or in addition to, step b) may include a sub-step of repeatedly (for example, periodically) changing the rotation speed of the container.

[0244] Alternatively, or in addition to the above, step b) may include a sub-step of intermittently rotating the container. This means that there are rest sequences in step b) in which the container does not rotate. The rotation sequences alternate with the rest sequences. Subsequent rotation sequences may be characterized by the same direction of rotation or by different directions of rotation.

[0245] The duration of step b) may be, for example, 1 to 5 hours, or approximately 1, 2, 3, 4, or 5 hours.

[0246] In some embodiments, the extracellular vesicles may be generated from spheroid and / or organoid-forming cells.

[0247] In some embodiments, the method may further include a preliminary step of growing spheroids and / or organoids outside the container, and step a) of placing the produced cells inside the container includes a secondary step of supplying the grown spheroids and / or organoids into the container.

[0248] In this field, preliminary steps for growing spheroids and / or organoids outside the container are well known, such as the hanging drop method, microwell-based methods, scaffold-based methods, and agitation-based methods.

[0249] In other embodiments, step a) of placing the production cells in the container may include a sub-step of supplying individual cells to the container, and the method may further include an intermediate step (before step b) of generating spheroids or organoids from the individual cells.

[0250] During the intermediate process of generating spheroids or organoids from the individual cells, the intermediate process may include a sub-process of rotating the container, preferably at a maximum rotation speed less than the maximum rotation speed of step b).

[0251] The rotation speed of the intermediate process can be set to 20 - 200 rpm, preferably 50 - 100 rpm. During this intermediate process, the rotation operation can be maintained constantly. As a variant, the rotation operation may be repeatedly changed as described above in relation to step b).

[0252] In some embodiments, step c) of collecting the generated extracellular vesicles may be carried out by taking out the liquid medium containing the producer cells from the container and separating the extracellular vesicles from the taken-out liquid medium.

[0253] The extracellular vesicles can be separated from the taken-out liquid medium by common methods such as centrifugation, filtration, size exclusion chromatography, immunoaffinity-based separation, decantation, any combination thereof, etc.

[0254] As another embodiment, step c) may be carried out by taking out the liquid medium from the container without substantially removing the producer cells and separating the extracellular vesicles from the taken-out liquid medium by any of the above-described separation methods, preferably centrifugation.

[0255] In this case, preferably, decantation and / or centrifugation are carried out in the container itself, and then the liquid medium is taken out.

[0256] The recovered extracellular vesicles (e.g., the supernatant containing extracellular vesicles in the case of separation by decantation and / or centrifugation) can be counted by an individual particle tracking method (or nanoparticle tracking analysis (NTA)) using, for example, NanoSight NS300 (Malvern Panalytical).

[0257] The recovered extracellular vesicles (e.g., the supernatant containing extracellular vesicles in the case of separation by centrifugation) may be observed and / or counted by cryo-TEM.

[0258] In some embodiments, the method may further include a step of repeating the cycle of at least step b) rotating the container and step c) collecting the extracellular vesicles produced, using the same producing cells.

[0259] For example, in each cycle, the producing cells may be removed from the container, separated from the liquid medium (for example, by centrifugation), and placed back into the container with a fresh liquid medium.

[0260] As a variation, in each cycle, the liquid culture medium may be removed from the container while leaving the produced cells largely inside the container (for example, by centrifugation), and new liquid culture medium may be added to the container.

[0261] The method may further include a rest period between two consecutive cycles.

[0262] During the resting period, the producing cells can be retained in the container without the container being rotated.

[0263] During the aforementioned resting period, the produced cells may be held without rotation in a general cell culture apparatus such as a cell culture flask.

[0264] The produced cells may be kept rotating within the container (this may allow the cells to grow during the rest period). If the container is rotated during the rest period, the rotation may be constant. The rotation speed (if constant) or the maximum rotation speed (if not constant) shall be less than the rotation speed during the cycle of step b). The rotation speed for all resting steps may be 20 to 200 rpm, preferably 50 to 150 rpm.

[0265] For example, the method may have 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles. The duration of each cycle (step b) and step c), and optionally step a), may be between 1 and 5 hours, for example, approximately 1, 2, 3, 4, or 5 hours. The duration of the rest period between subsequent cycles may be between 30 minutes and 24 hours, for example, approximately 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 10 hours, 15 hours, 20 hours, 24 hours, etc.

[0266] In some embodiments, the method may further include a step of introducing a therapeutic agent or contrast agent into the liquid culture medium.

[0267] The step of introducing a therapeutic agent or contrast agent into the liquid culture medium may be performed before and / or between and / or after steps a) to c).

[0268] The aforementioned therapeutic agents may be any agents capable of preventing, inhibiting, or halting the symptoms and / or progression of infectious diseases, autoimmune diseases, fibrous diseases, inflammatory diseases, neurodegenerative diseases, cardiovascular diseases, cancer, or any other diseases disclosed below.

[0269] The contrast agent may be any substance used to improve the visibility of specific organs, tissues, cells, or physiological processes during medical imaging examinations.

[0270] (Uses of the extracellular vesicles produced) The present invention may further relate to extracellular vesicles produced by the method of the present invention as described above or by using the container of the present invention.

[0271] More specifically, the present invention may further relate to the use of such extracellular vesicles for imaging and / or therapeutic and / or diagnostic purposes, such as in personalized medicine and / or immunotherapy and / or regenerative medicine and / or cell therapy and / or treatment of tumors, infectious diseases, inflammatory diseases, immune diseases, metabolic diseases, cancerous diseases, genetic diseases, degenerative diseases, or sequelae of surgery or trauma.

[0272] For this purpose, the extracellular vesicles produced according to the present invention may be used as vectors or carriers for delivering at least one therapeutic agent and / or contrast agent, for example, by being administered to a subject requiring such delivery.

[0273] In a preferred embodiment, the extracellular vesicles according to the present invention may be obtained from physiologically appropriate organoids / spheroids and may be used, for example, in personalized medicine.

[0274] In some embodiments, the extracellular vesicles according to the present invention may be obtained from THP-1 producing cells or lymphocytes and may be used, for example, in immunotherapy and / or cancer treatment.

[0275] In another embodiment, the extracellular vesicles may be derived from mesenchymal stem cells (MSCs) and may be used in regenerative medicine for processes such as angiogenesis and wound healing.

[0276] In another embodiment, the extracellular vesicles may be used to treat inflammatory diseases, or to treat tumors, infectious diseases, immune diseases, metabolic diseases, cancerous diseases, genetic diseases, degenerative diseases, or sequelae of surgery or trauma, due to their anti-inflammatory properties. [Examples]

[0277] (Preparation of cells grown on microcarrier beads) Cytodex 1 dextran microcarrier beads (200 μm) (GE Healthcare) were suspended in phosphate-buffered saline (PBS) to a bead concentration of 10 g / L and sterilized by autoclaving. Next, the PBS bead suspension was re-diluted with Dulbecco's modified Eagle medium (DMEM) prepared with 1% penicillin / streptomycin to a bead concentration of 2.5 g / L. Then, to ensure oxygen supply to the medium, this bead suspension was incubated at 37°C for 2 hours.

[0278] After counting the cells, the cells were added to a bead / culture medium mixture at a concentration of approximately 10 cells / bead. This mixture was subjected to 23 cycles of gentle mixing (30-60 rpm) for 3 minutes each, followed by a 45-minute standing period to ensure cell adhesion. One hour after the start of these cycles, fetal bovine serum (FBS) was added to the culture medium until the FBS concentration reached 10%. The microcarrier beads were then gently mixed (30-60 rpm) for 2-3 days before being made available for use.

[0279] (Microwell spheroid formation) Spheroids were formed using a custom-made 3D-printed stamp, which consisted of an array of micropillars (250-750 per stamp) with a diameter and height of 200 μm each, within a microwell. First, a 2% agarose PBS solution (4 mL) was added to each well of a 6-well plate. Then, the 3D-printed micropillar stamp was immediately used to print the mold directly onto the agarose, and it was allowed to solidify for 15 minutes.

[0280] Next, when the stamp was removed, an array with the dimensions of the micropillars appeared. The 6-well plate was sterilized by exposing it to UV light for 30 minutes, and then cells (1,000 cells per well) were seeded by a single centrifugation cycle at 1,200 rpm for 3 minutes. The seeded cells in the wells were placed in an incubator (37°C) to allow for cell growth, spheroid formation, and maturation.

[0281] Depending on the experimental requirements, spheroids were collected one or three days after maturation.

[0282] (Spheroid formation on non-adhesive flasks) The T75 cell culture flasks were pre-coated with a 2% agarose PBS solution to prevent cell adhesion. The pre-treated flasks were left at 4°C for 30 minutes to ensure that the agarose was completely gelled.

[0283] Confluent cells were detached from a T75 flask, seeded in a 1:1 ratio onto the T75 flask covered with agarose, and matured in an incubator at 37°C.

[0284] Cells were interacted and aggregated in a forced suspension state to induce spheroid formation in the flask, and the formed spheroids were collected one or three days after maturation under the above conditions.

[0285] (Preparation of extracellular vesicles in the container of the present invention) All containers (capacity: 20-80mL) were custom-made using 3D printing.

[0286] The container was connected to an EC45 motor (397172 (Maxon)) and an ESCON50 / 5 servo controller (409510 (Maxon)). This device was operated using ESCON Studio software provided by the vendor (operating speed range = 100 to 2,000 rpm). The controller was driven by a DC power supply and connected to a waveform generator to control the rotation speed.

[0287] In the experiments conducted, the rotation speed was set to 400 rpm, 600 rpm, 800 rpm, 1,200 rpm, or 1,600 rpm, and the rotation direction reversal frequency was set to 0.05 Hz and 0.1 Hz.

[0288] Single-cell, microcarrier bead, or spheroid samples were thoroughly washed with serum-free DMEM without phenol red after counting, resuspended in an appropriate effective volume, and placed inside the corresponding containers.

[0289] Each container was sealed with a vent cap, and the rotating system was placed inside an incubator (37°C) for the entire duration of the experiment.

[0290] The supernatant containing the produced extracellular vesicles was collected and examined for viability and morphology.

[0291] (Preparation of extracellular vesicles in a spinner flask) Cells grown on microcarrier beads were thoroughly washed with serum-free DMEM without phenol red, and then placed in a spinner flask (500 mL) equipped with a magnetic stirrer and placed inside an incubator (37°C).

[0292] The rotation speed of the magnetic stirrer in the flask used to produce extracellular vesicles was set to 200 rpm (resulting in a final volume of 350 mL).

[0293] (Quantification, qualitative analysis, and purification of extracellular vesicles) The samples recovered using the above preparation method were centrifuged at 2,000 g and 4°C for 10 minutes and resuspended in fresh, serum-free DMEM without phenol red. Nanoparticle tracking analysis of the processed sample suspension was performed using NanoSight NS300 (Malvern Panalytical) or Videodrop to obtain the number and particle size distribution of the extracellular vesicles produced.

[0294] Next, the samples were filtered (Centricon 70 100kDa (Merck Millipore)), purified using a qEV column (qEVoriginal / 70nm Gen 2 Column (Izon Science)), and then subjected to cryo-TEM observation or Western blot analysis.

[0295] (cell metabolic activity analysis) Cell viability was evaluated by measuring the metabolic activity of recovered cell and spheroid samples after rotating the container or spinner flask using the alamarBlue® metabolic assay (DAL1100 (Invitrogen)).

[0296] Following the manufacturer's instructions, the collected samples were stained in a 96-well plate with a 1:10 dilution of the reagent using complete DMEM, incubated at 37°C for 2 hours, and then analyzed using an EnSight® multimode plate reader (fluorescence excitation wavelength = 570 nm, emission wavelength = 585 nm).

[0297] (Cytotoxicity analysis) We quantified cell damage caused by rotation during extracellular vesicle formation using the ToxiLight® bioassay kit (LT017-117 (Lonza)).

[0298] The sample recovered from the container of the present invention or the sample recovered from the spinner flask was subjected to centrifugation and processing, and 20 μL of the supernatant was cultured at room temperature for 5 minutes using 100 μL of AK (adenylate kinase) detection reagent as instructed by the distributor.

[0299] Separately, a positive control for cell death was constructed using a ToxiLight 100% lysis reagent kit, following the instructions provided by the vendor. The bioluminescence signal was then quantified using an EnSight multimode plate reader.

[0300] (Western blot analysis) Exosome protein markers were detected using anti-CD63, anti-CD81, and anti-CD9 antibodies via standard Western blotting procedures, and cytoplasmic matrix markers were detected using anti-Alix and anti-ferritin antibodies. Anti-14-3-3 antibodies were used against soluble xenobiotic proteins. Anti-CD29, anti-CD44, anti-CD49e, anti-CD105, and anti-CD146 markers were used against mesenchymal proteins.

[0301] (Example 1: EV production from stem cells grown on beads in a container equipped with a baffle structure including a solid baffle plate) A container having the configuration shown in Figures 3a and 3b (a container equipped with a baffle structure including a solid baffle plate) was fabricated as described above in the section "Preparation of extracellular vesicles in the container of the present invention."

[0302] The experimental conditions were as follows: Producing cells: Mouse mesenchymal stem cells (mMSCs) grown on microcarrier beads. Bead concentration: 6000 per 1 mL Container capacity: 60mL Rotation time (process b): 3 hours Rotation parameters: Rotation speed = 0~1,200 rpm, Rotation frequency = 0.1 Hz

[0303] Under the conditions described above, extracellular viability (EV) production was performed at various rotation speeds, and the EV production in the container of the present invention and the EV production in a general spinner flask with magnetic stirring at the highest setting were compared using the same cells grown on beads of the same concentration.

[0304] The spinner flask experiment was carried out as described above in the section "Preparation of extracellular vesicles in a spinner flask."

[0305] Furthermore, after each rotation condition, the metabolic activity of the cells was quantified as described above in the "Cellular Metabolic Activity Analysis" section.

[0306] Quantification was performed using NTA.

[0307] Figures 7a and 7b show the number of extracellular vesicles (EVs) produced per bead and metabolic activity, respectively, on the vertical axis against rotation speed (rpm), using a container with a baffle structure including a solid baffle plate (blank circle) and a typical spinner flask stirred at maximum stirring speed (gray filled circle on the right). The horizontal axis represents rotation speed (rpm), and the vertical axis represents the number of extracellular vesicles (EVs) produced per bead and metabolic activity, respectively. The horizontal bars (black) represent the average value calculated over multiple experiments.

[0308] When the container of the present invention was used at 600 rpm, the effective volume was significantly reduced, and the number of extracellular vesicles produced was about the same as the number produced using a typical spinner flask.

[0309] When the container of the present invention was used at 800 rpm and 1200 rpm, the effective volume was significantly reduced, while the yield of extracellular vesicles increased compared to a typical spinner flask.

[0310] Furthermore, it was found that using the container of the present invention resulted in better metabolic activity of cells after each rotation experiment compared to when using a spinner flask.

[0311] As shown in Figure 7c, Western blotting of protein expression confirmed the presence of EV surface markers CD63 and CD9, as well as Alix and ferritin (cytoplasmic matrix proteins), in the EV extract prepared using the container of the present invention.

[0312] However, these proteins were not observed (or were barely observed) in the EV extract prepared using a spinner flask.

[0313] In all cases, the non-EV marker 14-3-3 (marker for impurities and foreign substances) was not observed.

[0314] In other words, Figure 7c demonstrates that the integrity of the extracellular vesicles prepared according to the present invention was maintained, and this integrity was better maintained than when a typical spinner flask was used. The maintenance of extracellular vesicle integrity was also observed in the transmission electron microscope image shown in Figure 7d, demonstrating that the morphological structure was intact.

[0315] (Example 2: EV production from mMSC spheroids and tumor organoids in a container equipped with a baffle structure including a solid baffle plate) The same container as in Example 1 was used.

[0316] The experimental conditions were as follows: Producing cells: Spheroids consisting of mMSCs and spheroids (tumor organoids) consisting of A-673 cancer cells. Spheroid particle size: 80-150 μm Spheroid concentration: 100-200 per 1 mL (Average) cell count per spheroid: 500 (mMSC) and 2,000 (A673) Rotation time (process b): 1 hour, 2 hours, or 3 hours Rotation parameters: Rotation speed = 800 rpm, Rotation frequency = 0.1 Hz

[0317] As described above in the section "Spheroid Formation in Microwells," extracellular vesicles were prepared from mMSC spheroids and A673 spheroids prepared in agarose microwells over 3 days of maturation, using the container of the present invention under the conditions described above.

[0318] The generated extracellular vesicles (EVs) were quantified using NTA.

[0319] Figures 8a and 8b show the number of extracellular vesicles (EVs) per spheroid composed of mouse mesenchymal stem cells (mMSCs) and the number of extracellular vesicles (EVs) per spheroid composed of A673 cancer cells, respectively, against rotation time (hours) on the horizontal axis, using the container of the present invention. The horizontal bars represent the average value over multiple experiments. The white horizontal bar corresponds to a control sample without rotation. T0 corresponds to the control with time = 0.

[0320] Overall, using A673 spheroids as the producing cells resulted in an increased EV yield compared to using mMSC spheroids. This is because A673 cancer cells inevitably produce more extracellular vesicles.

[0321] In the container of the present invention, the yield of EV increased as the rotation time was increased.

[0322] (Example 3a: EV fabrication from mMSC spheroids formed in a container equipped with a baffle structure including a solid baffle plate) The same container as in Example 1 was used.

[0323] The experimental conditions were as follows: Producing cells: Mouse stem cells (mMSCs) Spheroid particle size: 80-150 μm Spheroid concentration: 100-200 per 1 mL (Average) number of cells per spheroid: 500-1000 Rotation time: 2 hours or 4 hours Rotation parameters: For EV production, rotation speed = 800 rpm, rotation frequency = 0.1 Hz; for spheroid maturation, rotation speed = 80 rpm

[0324] As the cells to be produced, a suspension of mMSC cells was directly supplied into a container and subjected to rotation at 80 rpm for 3 days, thereby directly forming spheroids within the container.

[0325] Subsequently, the rotation speed was increased to 800 rpm, and the production of extracellular vesicles was induced by rotational stimulation for 2 or 4 hours.

[0326] Imaging analysis of spheroids after 2 or 4 hours of rotational stimulation (using the LIVE / DEAD® Cell Imaging Kit (R37601 (Invitrogen®))) demonstrated that, like the cells grown on beads, the spheroids themselves did not undergo large-scale cell death.

[0327] The manufactured EVs were quantified using NTA.

[0328] The results are shown in Table 1:

[0329] [Table 1]

[0330] The amount of extracellular vesicles produced per spheroid was remarkably high compared to the usual amount per bead (in mMSCs), which is at most 500,000 or far less.

[0331] In short, the present invention provides an all-in-one solution for producing extracellular vesicles from therapeutic cells with a physiological three-dimensional structure.

[0332] (Example 3b: EV fabrication from hMSC spheroids formed in a container equipped with a baffle structure including a solid baffle plate) The EV was prepared in the same manner as in Example 3a, except that some conditions were changed as follows: Producing cells: Human stem cells (hMSCs) Rotation time: 0-3 hours Rotation parameters: For EV fabrication, rotation speed = 0-1,600 rpm, rotation frequency = 0.2 Hz

[0333] The generated EVs were quantified using Videodrop. Cell death was also examined as described in the "Cytotoxicity Analysis" section.

[0334] The results are shown in Figures 13a, 13b, and 13c.

[0335] Figure 13a shows the amount of EVs produced per producing cell at various rotation speeds over various time periods. Note that Figure 13a shows the number of EVs produced per producing cell (not per spheroid) detected by Videodrop (not NTA), compared to the numbers listed in Table 1 of Example 3a. The number of EVs per producing cell in this example = 13,000 is (considering that Videodrop detects approximately four times fewer objects than NTA, and that each spheroid contained approximately 500 cells) 2 × 10⁶ in Example 3a. 7 It is equivalent to an EV.

[0336] As shown in Figure 13a, the number of EVs per cell increased as the rotation speed increased and the rotation time increased.

[0337] Figure 13b shows that the rotation conditions can be easily adjusted to ensure that the impact on cell death is significantly reduced.

[0338] As shown in Figure 13c, Western blotting of protein expression confirmed the absence of the non-EV marker 14-3-3 in the EV extracts (EV1-EV3), thus demonstrating the purity of the samples. The presence of EV markers CD63 and CD81, as well as the cytoplasmic EV marker Synt-1, in the EV extracts (EV1-EV3) confirmed the integrity of the EVs produced according to the present invention using hMSC spheroids formed in the container as the producing cells, as is evident from the controls (2D and 3D).

[0339] In summary, Example 3a demonstrates that the present invention provides an all-in-one solution for producing extracellular organisms (EVs) from physiologically structured, three-dimensional therapeutic cells.

[0340] (Example 4a: EV fabrication from spheroids formed outside a container, within a container equipped with a baffle structure including a solid baffle plate) The same container as in Example 1 was used.

[0341] The experimental conditions were as follows: Producing cells: spheroids of hMSCs (human hTERTs) Spheroid particle size: 80-250 μm Spheroid concentration: 50-250 per 1 mL Rotation time: 1 hour or 3 hours Rotation parameters: Rotation speed = 0~1,600 rpm, Rotation frequency = 0.05 Hz

[0342] Spheroids were prepared on a two-dimensional agarose flask with a 1-day maturation period, as described in the section on "Spheroid Formation on Non-Adhesive Flasks."

[0343] Quantification was performed using NTA.

[0344] Figures 9a and 9b show the number of extracellular vesicles (EVs) per spheroid after 1 hour of rotation and the number of extracellular vesicles (EVs) per spheroid after 3 hours of rotation, respectively, using the container of the present invention, with the horizontal axis representing the rotation speed (rpm). The horizontal bar (black) represents the average value over multiple experiments.

[0345] As shown in Figures 9a and 9b, the amount of extracellular vesicles (EVs) produced increased with rotation speed, and after 3 hours of rotation at 1,600 rpm, the number of extracellular vesicles per spheroid increased to 20,000,000.

[0346] Figure 9c shows that the EV surface marker CD63 is present in both the 800 rpm and 1,600 rpm EV extracts according to the present invention. The amount of CD63 detected was higher in the 800 rpm extract than in the 1,600 rpm extract. The presence of the cytoplasmic matrix EV marker Synt-1 was also observed. It was also confirmed that the non-EV marker 14-3-3 (marker for impurities and foreign substances) was not present in the EV extract.

[0347] In other words, the results in Figure 9c demonstrate that the integrity of the extracellular vesicles produced according to the present invention was maintained even at high rotational speeds such as 1,600 rpm.

[0348] Importantly, after a 3-hour rotation process, when cell metabolic activity and cell death were examined as described in the sections on "Cellular Metabolic Activity Analysis" and "Cytotoxicity Analysis," it was found that increasing the rotation speed had little effect on cell metabolic activity. While the cell death rate increased at 1,600 rpm, the impact on cell death was similarly small. See Table 2.

[0349] [Table 2]

[0350] (Example 4b: EV fabrication from spheroids formed outside a container, within a container equipped with a baffle structure including a solid baffle plate) The EV was fabricated in the same manner as in Example 4a, except that the rotation parameters were changed as follows: Rotation time: 0-3 hours Rotation parameters: Rotation speed = 0~1,600 rpm, Rotation frequency = 0.2 Hz

[0351] The extracellular vesicles produced were quantified using Videodrop.

[0352] The results are shown in Figures 9d and 9e. The number of EVs per produced cell in this example = 20,000 is (considering that Videodrop detects approximately four times fewer objects than NTA, and that each spheroid contained 200-300 cells) the same as in Example 4a, 2 × 10 7 It is equivalent to an EV.

[0353] From these results, it can be seen that as the rotation speed increases and the rotation time increases, the number of EVs per producing cell increases (Figure 9d), and that the rotation conditions can be easily adjusted to reliably reduce the impact on cell death (Figure 9e).

[0354] (Example 5a: EV production from single (individual) cells in a container equipped with a baffle structure including a solid baffle plate) The same container as in Example 1 was used.

[0355] The experimental conditions were as follows: Producing cells: Mouse mesenchymal stem cells (mMSCs) and human mesenchymal stem cells (hMSCs) Rotation time: 3 hours Rotation parameters: Rotation speed = 800 rpm, Rotation frequency = 0.1 Hz

[0356] Mouse and human mesenchymal stem cells were detached, resuspended to a concentration of 150-350,000 cells / mL, and subjected to rotation under the above conditions.

[0357] Quantification was performed using NTA.

[0358] Figure 10a shows the average number of extracellular vesicles (EVs) per cell from a single (individual) cell using the container of the present invention. The vertical axis represents the number of extracellular vesicles per cell, and the horizontal axis represents the rotation speed (rpm). The two vertical bars on the left correspond to extracellular vesicles produced from mouse mesenchymal stem cells (mMSCs), and the two vertical bars on the right correspond to extracellular vesicles produced from human mesenchymal stem cells (hMSCs).

[0359] The results show that the present invention increases EV yield even with the simplest single-cell level configuration.

[0360] (Example 5b: EV production from single (individual) cells in a container equipped with a baffle structure including a solid baffle plate) Except for changing the rotation parameters as described below, EVs were prepared in the same manner as in Example 5a using individual hMSC cells as the producing cells: Rotation time: 0-3 hours Rotation parameters: Rotation speed = 0~2,000 rpm, Rotation frequency = 0.2 Hz

[0361] The extracellular vesicles produced were quantified using Videodrop.

[0362] The results are shown in Figures 10b and 10c.

[0363] Figure 10b shows that the number of EVs per producing cell did not change much when the rotation speed exceeded 800 rpm, but increased as the rotation speed and rotation time increased.

[0364] Figure 10c shows that the rotation conditions can be easily adjusted to ensure that the impact on cell death is significantly reduced.

[0365] The EVs prepared as described above at 600 rpm and 0.5 Hz for 2 hours were subjected to Western blotting. The results are shown in Figure 10c.

[0366] Figure 10c confirms the absence of the non-EV marker 14-3-3 in the EV extract (EV), thus demonstrating the purity of the sample. The presence of EV markers CD63 and CD81, as well as the cytoplasmic matrix EV marker Synt-1 (see high exposure) in the EV extract (EV) confirms the integrity of the EVs produced according to the present invention using individual hMSCs as producing cells.

[0367] (Example 6: EV fabrication from spheroids in a container equipped with a baffle structure including a mesh-like baffle plate) A container with the structure shown in Figures 4a to 4c (a container equipped with a baffle structure including a mesh-like baffle plate) was fabricated as described above in the section "Preparation of extracellular vesicles in the container of the present invention."

[0368] The experimental conditions were as follows: Producing cells: spheroids of hMSCs Spheroid particle size: 80-250 μm Spheroid concentration: 100-200 per 1 mL Container capacity: 60mL Rotation time: 1 hour or 3 hours Rotation parameters: Rotation speed = 0~1,600 rpm, Rotation frequency = 0.05 Hz

[0369] Spheroids were prepared on a two-dimensional agarose flask with a 1-day maturation period, as described in the section on "Spheroid Formation on Non-Adhesive Flasks."

[0370] Figures 11a and 11b show the number of extracellular vesicles (EVs) per spheroid after 1 hour of rotation and the number of extracellular vesicles (EVs) per spheroid after 3 hours of rotation, respectively, with the horizontal axis representing the rotation speed (rpm). The horizontal bar (black) represents the average number of extracellular vesicles produced.

[0371] The amount of EVs produced increased proportionally with the rotational speed, demonstrating efficiency similar to that of a container with a baffle structure including a solid baffle plate.

[0372] After 3 hours of rotation, we examined cell metabolic activity and cell death as described in the sections on "Cell Metabolic Activity Analysis" and "Cytotoxicity Analysis." We found that increasing the rotation time and speed slightly increased cell death, as shown in Example 4 (data not shown), but increasing the rotation speed had little effect on cell metabolic activity and cell viability.

[0373] (Example 7: Multi-step EV fabrication from spheroids in a container equipped with a baffle structure including a mesh-like baffle plate) The same container as in Example 6 was used.

[0374] The experimental conditions were as follows: Producing cells: spheroids of hMSCs Spheroid particle size: 80-250 μm Spheroid concentration: 140 ± 60 spheroids per 1 mL (Average) number of cells per spheroid: 500-2,000 Container capacity: 60mL Rotation / Rest Time: 4 cycles of 1 hour rotation, with a 30-minute interval between each cycle (120 rpm) Rotation parameters: Rotation speed = 800 rpm, 1,200 rpm, or 1,600 rpm; Rotation frequency = 0.05 Hz

[0375] Spheroids were prepared on a two-dimensional agarose flask with a 1-day maturation period, as described in the section on "Spheroid Formation on Non-Adhesive Flasks."

[0376] NTA was used to quantify EVs.

[0377] Extracellular vesicles were prepared under the above conditions. After 1 hour of rotation, the collected samples were centrifuged over 3 minutes at 1,200 g and 4°C. The spheroid precipitate was immediately resuspended in a container with fresh, serum-free DMEM (without phenol red), and placed in a rotating incubator at 37°C for 30 minutes before being subjected to the next rotation cycle. This was repeated four times.

[0378] The number of extracellular vesicles produced at each cycle stage is shown in Figure 12a.

[0379] Figure 12a shows the number of extracellular vesicles (EVs) produced per spheroid on the vertical axis, with the horizontal axis representing the number of rotational cycles performed.

[0380] A black filled circle represents a rotation of 800 rpm, an empty circle represents a rotation of 1,200 rpm, and a square represents a rotation of 1,600 rpm. The same notation is used in Figures 12b and 12c.

[0381] Figure 12a shows that the amount of extracellular vesicles produced increased with more cycles. The increase in the number of extracellular vesicles became more pronounced as the rotation speed increased.

[0382] Furthermore, cellular metabolic activity and cell death were measured after each cycle. These are shown in Figures 12b and 12c.

[0383] Figures 12b and 12c show the number of rotational cycles performed on the horizontal axis and the metabolic activity and cell death of cells on the vertical axis, respectively.

[0384] Figure 12b shows that while metabolic activity at 1,600 rpm was slightly lower than at 800 rpm and 1,200 rpm, the impact on metabolic activity was small at 800 rpm, 1,200 rpm, and 1,600 rpm.

[0385] Furthermore, it was found that metabolic activity remained constant even when the number of cycles was increased, and that repeated production of extracellular viable cells (EVs) from the same producing cells did not have a significant impact on the metabolic activity of the producing cells.

[0386] Figure 13c shows that while cell death at 1,600 rpm was slightly higher than at 800 rpm and 1,200 rpm, the impact on cell viability was small at 800 rpm, 1,200 rpm, and 1,600 rpm.

[0387] The results show that cell death remained relatively constant even when the number of cycles was increased, and that repeated production of extracellular viability (EVs) from the same producing cells did not have a significant impact on the cell viability of the producing cells.

[0388] (Example 8: Quality of an EV manufactured according to the present invention) EVs were prepared from individual hMSCs or from hMSC spheroids formed within a container.

[0389] EV preparation from hMSC spheroids formed in a container was carried out in the same manner as in Example 4a, except that the rotation speed, rotation frequency, and rotation time were fixed at 600 rpm, 0.2 Hz, and 2 hours, respectively.

[0390] EV fabrication from individual hMSCs was carried out in the same manner as in Example 5a, except that the rotation speed, rotation frequency, and rotation time were fixed at 600 rpm, 0.2 Hz, and 2 hours, respectively.

[0391] For control, EV generation was also performed using common methods involving two-dimensional or three-dimensional starvation.

[0392] The quality of the EVs produced in this manner was determined using the MACSPlex Exosome kit (Miltenyi Biotec, Auburn, California) according to the manufacturer's protocol.

[0393] The EVs, mixed with beads coated with a specific antibody, were examined using a MACSQuant cytometer (Miltenyi Biotec) equipped with MACSQuantify software.

[0394] The results are shown in Figures 14a and 14b. The results show that EVs produced according to the present invention (EV(A) produced from individual hMSCs, or produced from hMSC spheroids placed in a container (corresponding to three independent production lines C1, C2, and C3)) show similar levels of protein expression compared to controls (2D and 3D, corresponding to EVs produced by two-dimensional starvation and three-dimensional starvation, respectively).

[0395] More specifically, as exosome-specific markers, markers CD63 and CD81 were present on the produced exosomes (A and C1-C3) prepared according to the present invention, as observed under classical starvation production conditions (2D and 3D). Marker CD9 was absent because it is not expressed by hMSCs.

[0396] The mesenchymal markers CD29, CD44, CD49e, CD105, and CD146 were also well expressed in EVs prepared according to the present invention.

[0397] (Example 9: Angiogenic capacity of EVs produced according to the present invention) The angiogenic capacity of EVs produced according to the present invention was investigated using human umbilical vein endothelial cells (HUVECs).

[0398] It is known that germination can be induced when HUVEC spheroids are treated with serum proteins such as vascular endothelial growth factor (VEGF) or FBS.

[0399] HUVEC was cultured as a three-dimensional spheroid in agarose swells, as explained in the section on "Spheroid Formation in Microwells," and then introduced into a collagen matrix.

[0400] EVs were prepared in the same manner as in Example 3b (from hMSC spheroids formed inside the container), in the same manner as in Example 4 (from hMSC spheroids formed outside the container), and in the same manner as in Example 5 (from individual hMSC cells), except that the rotation parameters were as follows: Rotation time: 2 hours Rotation speed: 600 rpm Rotation frequency: 0.2Hz

[0401] As shown in Figures 15a and 15b, germination was observed after treating endothelial cell spheroids with EV under various conditions, including 0% FBS (negative control), 10% FBS and VEGF (1x or 2x dose) (positive control), EV produced under two-dimensional or three-dimensional starvation (1x or 2x dose) (controls designated as Comparative Examples "2D" and "3D," respectively), and EV (A) produced from individual hMSC cells prepared as described above according to the present invention, EV (B) produced from hMSC spheroids formed outside the container, and EV (C) produced from hMSC spheroids formed inside the container (1x, 2x, or 4x dose).

[0402] Figure 15a shows that in the presence of EVs (various doses of A, B, and C) prepared according to the present invention, germination was observed to a similar degree or even more pronounced degree compared to treatment with 10% FBS or 2x / 4x VEGF, or conventional starvation-produced EVs (1x or 2x 2D).

[0403] Figure 15b shows the number of germinated cells measured using common image analysis software. The results show that EVs produced according to the present invention are more efficient at angiogenesis than FBS and VEGF under all conditions (A, B, and C with 1x, 2x, and 4x doses). In some cases, particularly EVs (B) produced from hMSC spheroids formed outside the container and EVs (C) produced from hMSC spheroids formed inside the container, germinated more than EVs produced by conventional two-dimensional or three-dimensional starvation methods (2D and 3D).

[0404] In summary, the above results demonstrate the angiogenic capacity of EVs produced according to the present invention.

[0405] (Example 10: Wound healing effect of EV prepared according to the present invention) The wound-healing activity of EVs produced according to the present invention was investigated using immortalized human fibroblasts (fHDF / TERT166) (HSF).

[0406] As EVs, EVs (A) were prepared from individual hMSC cells, EVs (B) from hMSC spheroids formed outside the container, and EVs (C) from hMSC spheroids formed inside the container, in the same manner as in Example 9.

[0407] HSF cells were seeded in a 96-well plate. A physical void (scratch) was created within the monolayer, and the culture medium was replaced with a new medium containing EV prepared as described above. The closure of the void by cell migration was monitored and quantified over time using general image software.

[0408] Overall, the extravasation embryos (EVs) prepared according to the present invention (A, B, and C; see Figure 16c) improved the wound healing process. More specifically, the EVs prepared according to the present invention (A, B, and C) showed significantly improved action compared to controls (HSF treated with 1% FBS, 2% FBS, and 4% FBS; see Figure 16a), and slightly improved action compared to other controls (HSF treated with EVs (2D and 3D) prepared by conventional two-dimensional or three-dimensional starvation methods; see Figure 16b). Furthermore, they showed comparable action to treatment with 10% FBS (Figure 16a).

[0409] (Example 11: Anti-inflammatory effect of EV produced according to the present invention) The anti-inflammatory effect of EVs prepared according to the present invention was investigated using a general nitric oxide (NO) measurement assay.

[0410] It is known that treating mouse macrophage cells (RAW264.7) with lipopolysaccharide (LPS) induces the formation of nitric oxide (NO), which is an inflammatory response.

[0411] As EVs, EVs were prepared in the same manner as in Example 9: EV(A) from individual hMSC cells, EV(B) from hMSC spheroids formed outside the container, and EV(C) from hMSC spheroids formed inside the container.

[0412] Mouse macrophage cells were treated with lipopolysaccharide (LPS) and then with EV cells prepared as described above.

[0413] The results are shown in Figure 17.

[0414] EVs (A, B, and C) prepared according to the present invention significantly reduced NO formation in a dose-dependent manner compared to the positive control (CTL+). More specifically, EVs prepared according to the present invention showed anti-inflammatory effects comparable to those of treatment with known anti-inflammatory inhibitors at twice the dose (2X).

[0415] From the above results, it can be seen that the EV produced according to the present invention exhibits anti-inflammatory effects.

[0416] (Example 12: EV production in a container other than the present invention) A container with the configuration shown in Figures 18a and 18b was manufactured using 3D printing.

[0417] The surface structure of the container consisted of 638 wells spaced approximately 1.4 mm apart (density: 1 mm²). 2 (Approximately 0.65 wells per well). The dimensions of each well were as follows: Depth: 1.25mm Diameter: Diameter of the opening = 0.53 mm, diameter of the base = 0.27 mm (therefore, the surface of the opening protrudes slightly) Angle: 42.5° relative to the axis of the cylinder

[0418] In other words, the container did not have a baffle structure according to the present invention that would cause the flow of liquid inside the container to deviate, but rather had microwells on its inner surface that could accept cells.

[0419] The experimental conditions were as follows: Producing cells: hMSC Spheroid particle size: 80-150 μm Spheroid concentration: 100-200 per 1 mL Rotation time: 0 hours, 3 hours, 6 hours, and 24 hours for EV production; 48 hours for maturation. Rotation parameters: For EV production, rotation speed = 200 rpm, rotation frequency = 0.2 Hz; for spheroid maturation, rotation speed = 60 rpm

[0420] 1,000,000 cells were seeded in the same container and subjected to rotation at a speed of 200 rpm for 1 hour. Subsequently, the rotation speed was reduced to 60 rpm and subjected to a maturation period of 48 hours to form spheroids in the wells of the cylinder.

[0421] After a maturation period, we began producing EVs at 200 rpm and 24 hours.

[0422] The results are shown in Figure 18c.

[0423] The number of EVs produced per spheroid is 1 to 2 × 10⁻¹⁶ per spheroid. 5 The range of EVs and the number of EVs produced per cell ranged from 50 to 150 EVs per cell.

[0424] This represents a 100-fold decrease compared to the yield observed in EV production according to the present invention (see, for example, Examples 3a, 3b, etc.).

[0425] This dramatic decrease is likely due to the fact that the stress experienced by the spheroids at the bottom of the well (where they are sown and remain) was minimal and not sufficient to induce the high yield of extravalent vegetables (EVs) that can be obtained with the container of the present invention.

[0426] From the above results, it can be seen that EV production using a container equipped with the baffle structure of the present invention significantly improves yield compared to containers without such a baffle structure.

Claims

1. A method for producing extracellular vesicles from producing cells, a) A step of placing the cells produced in a liquid culture medium into a container in which a baffle structure is fixed inside, b) A step of rotating the container, thereby generating extracellular vesicles from the producing cells, c) A step of collecting the extracellular vesicles that have been generated, A method comprising a step b) in which the step of rotating the container includes a sub-step of repeatedly changing the rotational motion of the container.

2. A method according to claim 1 or 2, wherein the container rotates about a rotation axis that is oriented substantially vertically.

3. A method according to claim 1 or 2, wherein the extracellular vesicles are produced from a producing cell in the form of a spheroid and / or organoid.

4. In the method according to claim 3, - The method further comprises a preliminary step of growing spheroids and / or organoids outside the container, and step a) of placing the produced cells inside the container includes a sub-step of supplying the grown spheroids and / or organoids to the container, or - A method comprising a step a) of placing the production cells in the container, the step of supplying individual cells to the container, and the method further comprising an intermediate step of generating spheroids and / or organoids from the individual cells.

5. A method according to any one of claims 1 to 4, wherein step b) of rotating the container includes a sub-step of periodically changing the rotational motion of the container, preferably, step b) includes a sub-step of repeatedly reversing the direction of rotation of the container and / or a sub-step of repeatedly changing the rotational speed of the container and / or a sub-step of intermittently rotating the container.

6. The method according to claim 5, wherein the frequency of changing the rotational motion of the container is 0.01 to 5 Hz, preferably 0.05 to 0.5 Hz.

7. A method according to any one of claims 1 to 6, wherein step b) of rotating the container is performed at a maximum rotational speed of 50 to 6,000 rpm, preferably 600 to 1,600 rpm.

8. The method according to any one of claims 1 to 7, further, A process that repeats the cycle of at least steps b) and c) using the same producing cells, A method that includes [a certain feature].

9. A container (1) for producing extracellular vesicles from producing cells, A baffle structure (2) fixed inside the container, A coupling (3) on the outer surface of the container, configured to be connected to a rotating device, A container equipped with [the necessary features / features].

10. A container according to claim 9, wherein the baffle structure (2) includes one or more baffles (4) fixed to the inner surface of the container.

11. A container according to claim 9 or 10, wherein the container (1) has a cylindrical inner wall (8) and a central axis.

12. A container according to claim 11, wherein the baffle structure (2) includes a plurality of baffles (4) extending from the cylindrical inner wall (8) toward the central axis, and / or a plurality of baffles (4) extending from the central axis toward the cylindrical inner wall (8).

13. A container according to any one of claims 10 to 12, wherein some or all of the baffles (4) are solid plates or mesh or perforated plates.

14. A container according to any one of claims 10 to 12, wherein the baffle structure (2) includes a plurality of plates (4') fixed to a support column (6), preferably the support column (6) is oriented parallel to the central axis of the container, and preferably the plates (4') of different orientations are alternately provided along the support column (6).

15. A system for producing extracellular vesicles from producing cells, A container according to any one of claims 10 to 14, Rotating device and A system comprising the above, wherein the container is configured such that its rotation is fixed to the rotating device by key-connecting the coupling of the container to the corresponding coupling in the rotating device.

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