EP4689049A2

EP4689049A2 - Method for high throughput production of extracellular vesicles in baffled rotating vessel

Info

Publication number: EP4689049A2
Application number: EP24716131.8A
Authority: EP
Inventors: Claire WILHELM; Jose Efrain PEREZ; Elliot THOUVENOT; Giacomo GROPPLERO
Original assignee: Centre National de la Recherche Scientifique CNRS; Institut Curie; Sorbonne Universite
Current assignee: Centre National de la Recherche Scientifique CNRS; Institut Curie; Sorbonne Universite
Priority date: 2023-03-28
Filing date: 2024-03-27
Publication date: 2026-02-11
Also published as: WO2024200570A3; WO2024200570A2; JP2026512415A; KR20260002799A

Abstract

The invention relates to a method of producing extracellular vesicles from producer cells, comprising the steps of: a) placing producer cells in a liquid medium in a vessel comprising a baffle structure inside the vessel; b) rotating the vessel so as to generate extracellular vesicles from the producer cells; and c) collecting the generated extracellular vesicles; wherein the step b) of rotating the vessel comprises repeatedly changing the rotational motion of the vessel.

Description

METHOD FOR HIGH THROUGHPUT PRODUCTION OF EXTRACELLULAR VESICLES IN BAFFLED ROTATING VESSEL

TECHNICAL FIELD

The present invention relates to a method of producing extracellular vesicles from producer cells, and to a vessel for performing the method.

TECHNICAL BACKGROUND

Extracellular vesicles (EVs) are now recognized as key players in maintaining homeostasis and contributing to diseases. They hold tremendous potential for future cancer and regenerative therapies. Extracellular vesicles are endogenously released by cells in a constitutive or inducible manner. These vesicles transport materials including lipids, proteins, mRNAs and miRNAs, and constitute the most advanced far-reaching intercellular communication pathway in our body. Compared to their mother cells, extracellular vesicles bring unique benefits in terms of sterilization, storage and shelf-life, making them ideal for clinical applications. Consequently, there has been a significant increase in EV- based clinical trials in recent years.

Most of the current methods for the production of EVs rely on low-yield, time-consuming protocols, such as spontaneous EV release in a complete culture medium, resulting in a low EV production of less than 500 extracellular vesicles per cell over days. Serum starvation or oxygen deprivation are alternative methods, but the increase in EV production is about 2-fold, producing EVs in the range of 1000 per cell, in 2 to 3 consecutive days. Cell activation can also trigger EV release, such as with TNF-a induced vesiculation, but again the yield is only 2-fold higher than that of spontaneous release. A recent trend based on a low- stress, hollow-fiber bioreactor achieves an increased EV yield of 2000-10000 EVs/cell; however, the production time is rather long, over 10 days.

For extracellular vesicles to be used for clinical applications, these production issues need to be solved first, and robust and high-yield EV production methods need to be developed using minimal cell materials. Production should also be versatile with respect to the configuration of the producer cells, including ease of working with cells in suspension (such as primary cells from biopsies) and the possibility to produce extracellular vesicles from a 3D configuration, i.e., physiologically relevant, organoid-like settings. To the best of the inventors’ knowledge, a method that satisfies these technological bottlenecks does not yet exist.

Several bioreactors for cell culture in the 3D configuration are known.

For example, document JP200722203 discloses a cell-culturing and centrifuging tube comprising a separation tube and a stopper for sealing the upper opening of the separation tube, an opening in the stopper, a culture gas- penetrable nonwoven fabric to cover the opening, and at least one baffle on the inner wall surface of the separation tube, which is integrally formed with the separation tube.

There also exist commercially available systems for 3D cell culture, such as ClinoStar system and CERO 3D.

However, none of them proposes using the bioreactors for EV production, or EV production from organoids/spheroids.

Some methods for producing an increased yield of extracellular vesicles, using a spinner flask bioreactor used for 3D cell culture, have also been developed.

For example, document FR3091296 discloses a fluidic system for loading a therapeutic or imaging agent into the lumen of extracellular vesicles (extracellular vesicles) from producer cells, comprising at least one vessel, a liquid medium contained by the vessel, producer cells, a liquid medium agitator and agitator speed control means adapted for the growth of the producer cells.

Document FR3091295 discloses a fluidic system for producing extracellular vesicles from suspended producer cells, comprising at least one vessel, a liquid medium contained by the vessel, suspended producer cells, a liquid medium agitator, agitator speed control means adapted for growth of the suspended producer cells.

Document FR3068361 discloses a fluidic system for producing extracellular vesicles from producer cells, including at least one container, a liquid medium contained by the container and producer cells, which also includes microcamers suspended in the liquid medium, the majority of producer cells being adherent to the surface of the microcarriers, and a liquid medium agitator, the agitator and the dimensions of the container being adapted to control a turbulent flow of the liquid medium in the container.

Document FR3112147 discloses a method for calibrating a fluidic system for producing extracellular vesicles from producer cells.

However, in these documents, the system is simply a repurposed commercial bioreactor, and is not designed specifically for EV production. In addition, the configuration of the producer cells is limited to 2D culture on beads or cells in suspension, and thus does not enable production from spheroids or organoids.

Specifically, these documents raise several concerns. Firstly, a large production reactor, typically a reactor of 1 L, is employed at a rotation speed of around 250 rpm for reaching an optimal (10-fold) EV yield, which requires, while achievable, advanced and expensive magnetic agitators. Reducing the production reactor volume (for example, to 0.3 L) requires higher rotating speeds (over 350 rpm), which are even less attainable. Moreover, such large production volumes require a large number of cells (e.g., 100 x 10⁶ cells), which is not feasible to obtain under physiological conditions in personalized medicine.

Generally, reducing the size of a conventional stirred tank with an impeller or an agitator is quite difficult because it would require reducing the size of the blades of the impeller or the agitator, which would in turn require increasing the speed of rotation to maintain the same level of stress on the cells. For example, with a blade diameter of 1 cm, the rotation speed would need to be approximately 5000 rpm, which is difficult to achieve with, for example, a stirred tank and a magnetic stirrer.

Secondly, the turbulent regime that the cells are exposed to can cause cell death. Due to a highly heterogeneous turbulent regime, individual cells growing on beads experience different levels of shear stress. This heterogeneity also makes it difficult to assess the turbulent regime experimentally.

Thus, there is a need for a more efficient method for producing extracellular vesicles from minimal cell materials (low volume), which include not only individual cells in suspension (such as stem cells from biopsies) but also cells in a 3D configuration (such as organoids/spheroids).

SUMMARY OF THE INVENTION

The present invention relates to the following items.

Item 1 . A method of producing extracellular vesicles from producer cells, comprising the steps of : a) placing producer cells in a liquid medium in a vessel comprising a baffle structure fixed inside the vessel; b) rotating the vessel so as to generate extracellular vesicles from the producer cells; and c) collecting the generated extracellular vesicles; wherein the step b) of rotating the vessel comprises repeatedly changing the rotational motion of the vessel. Item 2. The method of Item 1 , wherein the producer cells are selected from human cells and animal cells.

Item 3. The method of Item 1 or 2, wherein the vessel rotates around a rotation axis which is substantially vertically oriented.

Item 4. The method of any one of Items 1 to 3, wherein the extracellular vesicles are generated from producer cells in the form of individualized cells suspended in the liquid medium.

Item 5. The method of any one of Items 1 to 4, wherein the extracellular vesicles are generated from producer cells in the form of spheroids and/or organoids.

Item 6. The method of Item 5, comprising a preliminary step of growing spheroids and/or organoids outside of the vessel, and wherein step a) of placing the producer cells in the vessel comprises supplying the grown spheroids and/or organoids to the vessel.

Item 7. The method of Item 5, wherein step a) of placing the producer cells in the vessel comprises supplying individualized cells to the vessel, the method further comprising an intermediate step of generating spheroids and/or organoids from the individualized cells.

Item 8. The method of Item 7, comprising rotating the vessel during the intermediate step of generating spheroids and organoids from the individualized cells, preferably at a maximum speed of rotation which is less than a maximum speed of rotation during step b).

Item 9. The method of any one of Items 1 to 8, wherein the step b) of rotating the vessel comprises periodically changing the rotational motion of the vessel.

Item 10. The method of Item 9, wherein the frequency of changing the rotational motion of the vessel is from 0.01 to 5 Hz, preferably from 0.05 to 0.5 Hz.

Item 11 . The method of any one of Items 1 to 10, wherein the step b) of rotating the vessel is carried out at a maximum rotational speed of 50 to 6000 rpm, preferably 600 to 1600 rpm.

Item 12. The method of any one of Items 1 to 11 , wherein step b) comprises repeatedly reversing the rotational direction of the vessel.

Item 13. The method of any one of Items 1 to 12, wherein step b) comprises repeatedly changing the rotational speed of the vessel.

Item 14. The method of any one of Items 1 to 13, wherein step b) comprises intermittently rotating the vessel. Item 15. The method of any one of Items 1 to 14, further comprising a step of introducing a therapeutic agent or an imaging agent into the liquid medium.

Item 16. The method of any one of Items 1 to 15, wherein step c) is carried out by withdrawing the liquid medium including the producer cells from the vessel, and separating the extracellular vesicles from the withdrawn liquid medium, preferably by centrifugation.

Item 17. The method of any one of Items 1 to 15, wherein step c) is carried by withdrawing the liquid medium from the vessel without substantially withdrawing the producer cells, and separating the extracellular vesicles from the withdrawn liquid medium; wherein, preferably, decantation and/or centrifugation in the vessel is carried out prior to withdrawing the liquid medium.

Item 18. The method of any one of Items 1 to 17, further comprising repeating cycles of at least steps b) and c), using the same producer cells.

Item 19. The method of Item 18, wherein, at each cycle:

- the producer cells are withdrawn from the vessel, separated from the liquid medium, and placed again in the vessel together with a fresh liquid medium; or

- the liquid medium is withdrawn from the vessel, the producer cells substantially remaining within the vessel, and a fresh liquid medium is added to the vessel.

Item 20. The method of Item 19, further comprising a time interval of rest between two subsequent cycles, wherein the producer cells are kept in the vessel without rotation of the vessel.

Item 21. The method of any one of Items 1 to 20, wherein the baffle structure comprises one or more baffles fixed to an internal surface of the vessel.

Item 22. The method of any one of Items 1 to 21 , wherein the vessel comprises a cylindrical inner wall and a central axis.

Item 23. The method of Item 22, wherein the baffle structure comprises one or more pairs of baffles, each pair of baffles comprising two diametrically opposed baffles relative to the central axis.

Item 24. The method of Item 22 or 23, wherein the baffle structure comprises a plurality of baffles extending from the cylindrical inner wall towards the central axis.

Item 25. The method of Item 24, wherein the baffles do not extend to the central axis.

Item 26. The method of Item 24, wherein the baffles extend to the central axis, thereby dividing the inside of the vessel into a plurality of compartments, the compartments being in fluid communication with one another. Item 27. The method of Item 22 or 23, wherein the baffle structure comprises a plurality of baffles extending from the central axis towards the cylindrical inner wall.

Item 28. The method of any one Items 21 to 27, wherein part or all of the baffles are solid plates, or are meshed or perforated plates.

Item 29. The method of Item 28, wherein the part or all of the plates are oriented substantially parallel to the central axis of the vessel.

Item 30. The method of Item 28 or 29, wherein part or all of the plates are oriented at an angle from 10° to 80°, preferably from 30° to 60° relative to the central axis of the vessel.

Item 31 . The method of any one of Items 1 to 30, wherein the baffle structure comprises a plurality of plates fixed on struts, the struts being preferably oriented parallel to the central axis of the vessel, wherein, preferably, differently oriented plates alternate along the struts.

Item 32. A vessel for producing extracellular vesicles from producer cells, wherein the vessel comprises a baffle structure fixed inside the vessel, and a coupling on an external surface of the vessel configured to be coupled to a rotating apparatus.

Item 33. The vessel of Item 32, wherein the baffle structure comprises one or more baffles fixed to an internal surface of the vessel.

Item 34. The vessel of Item 32 or 33, wherein the vessel comprises a cylindrical inner wall and a central axis.

Item 35. The vessel of Item 34, wherein the baffle structure comprises one or more pairs of baffles, each pair of baffles comprising two diametrically opposed baffles relative to the central axis.

Item 36. The vessel of Items 34 or 35, wherein the baffle structure comprises a plurality of baffles extending from the cylindrical inner wall towards the central axis.

Item 37. The vessel of Item 36, wherein the baffles do not extend to the central axis.

Item 38. The vessel of Item 36, wherein the baffles extend to the central axis, thereby dividing the inside of the vessel into a plurality of compartments, the compartments being in fluid communication with one another.

Item 39. The vessel of Item 34 or 35, wherein the baffle structure comprises a plurality of baffles extending from the central axis towards the cylindrical inner wall.

Item 40. The vessel of any one of Items 33 to 39, wherein part or all of the baffles are solid plates, or are meshed or perforated plates. Item 41 . The vessel of Item 40, wherein part or all of the plates are oriented substantially parallel to the central axis of the vessel.

Item 42. The vessel of Item 40 or 41 , wherein part or all of the plates are oriented at angle from 10° to 80°, preferably from 30° to 60° relative to the central axis of the vessel.

Item 43. The vessel of any one of Items 32 to 42, wherein the baffle structure comprises a plurality of plates (4’) fixed on struts, the struts being preferably oriented parallel to the central axis of the vessel, wherein, preferably, differently oriented plates (4’) alternate along the struts .

Item 44. The vessel of any one of Items 32 to 43, wherein the coupling comprises one or more grooves or ridges on an external surface of the vessel.

Item 45. The vessel of any one of Items 32 to 44, comprising a closing cap.

Item 46. The vessel of any one of Items 32 to 45, configured for carrying out the method of any one of Items 1 to Item 31 .

Item 47. The method of any one of Items 1 to 31 , wherein the vessel is according to one of Items 32 to Item 46.

Item 48. A system for producing extracellular vesicles from producer cells, comprising a vessel according to one of Items 32 to 46, and a rotating apparatus, the vessel being configured to be rotationally fixed to the rotating apparatus by keying the coupling of the vessel to a corresponding coupling on the rotating apparatus.

Item 49. The system of Item 48, wherein the rotating apparatus comprises a cup configured for receiving the vessel.

Item 50. The system of Item 48 or 49, wherein the rotating apparatus comprises a drive mechanism, the system further comprising a control unit for controlling the drive mechanism, and wherein the control unit is preferably configured for implementing step b) of the method of any one of claims 1 to Item 31.

The present invention makes it possible to overcome the drawbacks of the prior art. In particular, the present invention provides an efficient method of producing extracellular vesicles from producer cells, and a vessel for performing the method.

This is achieved because the method comprises placing producer cells in a liquid medium in a vessel comprising a baffle structure inside the vessel, and rotating the vessel so as to generate extracellular vesicles from the producer cells, wherein the step of rotating the vessel comprises repeatedly changing the rotational motion of the vessel. The step of rotating the vessel eliminates the need to use an impeller or an agitator. In this case, the vessel diameter determines the production volume, satisfying the need for miniaturization, which has been difficult to achieve with a conventional stirred tank with an impeller or an agitator, as explained above.

The present inventors have discovered that, by repeatedly changing the rotational motion of the vessel with a baffle structure, the flow inside the rotating vessel is disrupted, resulting in significantly higher EV production in comparison with the production from the same cells in a stirred tank (spinner flask) bioreactor operating at maximum regime.

In particular, the present invention provides the following advantages:

- a higher EV yield can be obtained compared with repurposed spinner flask bioreactors used for cell culture;

- a low-volume regime (for example, a working volume in the range of 10 to 120 mL) is possible, which is perfectly in line with the number of cells per unit of volume associated with personalized medicine applications;

- control of the flow conditions and thus the turbulent regime, is easier, allowing a more robust methodology in terms of the EV yield per cell;

- extracellular vesicles can be produced from cells in a 3D configuration, such as organoids/spheroids;

- EV production can be coupled with 3D cell culture or spheroid and organoid maturation.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows one example of a vessel of the invention.

Figure 2a shows a top view of an example of a vessel having a baffle structure comprising solid baffle plates of the invention.

Figure 2b shows a diagonal top view of the vessel shown in Figure 2a, cut along a plane parallel to the central axis of the vessel.

Figure 3a shows a top view of another example of a vessel with a baffle structure comprising solid baffle plates of the invention.

Figure 3b shows a diagonal top view of the vessel shown in Figure 3a, cut along a plane parallel to the central axis of the vessel.

Figure 4a shows a top view of another example of a vessel with a baffle structure comprising meshed baffle plates of the invention.

Figure 4b shows a side view of the vessel shown in Figure 4a, cut along a plane parallel to the central axis of the vessel.

Figure 4c shows a diagonal top view of the vessel shown in Figure 4a, cut along a plane parallel to the central axis of the vessel. Figure 5a shows a top view of another example of a vessel with a baffle structure comprising plates fixed on struts of the invention.

Figure 5b shows a side view of the vessel shown in Figure 5a, cut along a plane parallel to the central axis of the vessel.

Figure 5c shows a diagonal top view of the vessel shown in Figure 5a, cut along a plane parallel to the central axis of the vessel.

Figure 6a shows a top view of another example of a vessel with a baffle structure comprising plates fixed on struts of the invention.

Figure 6b shows a side view of the vessel shown in Figure 6a, cut along a plane parallel to the central axis of the vessel.

Figure 6c shows a diagonal top view of the vessel shown in Figure 6a, cut along a plane parallel to the central axis of the vessel.

Figure 7a shows the number of extracellular vesicles (EVs) per bead as a function of the rotation speed (in rpm), using a vessel of the invention (white circles) and a conventional spinner flask (grey circles), as tested in Example 1 below. The y-axis represents the number of extracellular vesicles per bead. The black bars represent average values. The x-axis represents, for the white cercles, the rotational speed of the vessel. TO corresponds to the control (at time 0).

Figure 7b shows the metabolic activity of cells as a function of the rotation speed (in rpm), using a vessel of the invention (white circles) and a conventional spinner flask (grey circles), as tested in Example 1 below. The y-axis represents the metabolic activity (%). The black bars represent average values. The x-axis represents, for the white cercles, the rotational speed of the vessel.

Figure 7c shows the results of a western blot comparing the protein expression of the transmembrane extracellular vesicle (EV) markers CD63 and CD9, of the cytosolic EV markers Alix and Ferritin and of a non-EV marker enriched in the soluble fraction, 14-3-3, of extracellular vesicles (EVs) produced using a vessel of the invention and a conventional spinner flask, as tested in Example 1 below. The presence of the EV markers and the absence of the non- EV marker in the EV fraction is proof of the samples purity. No. 1 to 3 (lanes 2 to 7 from the left) correspond to three samples using the conventional spinner flask; and No. 4 (lanes 9 and 11 from the left) to the sample using the vessel of the present invention. “CCM” refers to a concentrated cell medium; “EVs” refers to the extracted EV fraction after the size exclusion chromatography; and “MM” refers to a molecular weight marker.

Figure 7d shows a transmission electron microscopy image of extracellular vesicles produced using a vessel of the present invention, as tested in Example 1 below. Figure 8a shows the number of extracellular vesicles (EVs) per spheroid produced from mouse mesenchymal stem cells (mMSC), as a function of the rotation time (in hours), using a vessel of the invention, as tested in Example 2 below. The y-axis represents the number of extracellular vesicles per spheroid. The bars represent average numbers of produced extracellular vesicles, when the method of the invention is implemented (in black) and for control samples with no rotation (in white). The x-axis represents the duration of the rotation. TO corresponds to the control for the rotation duration (at time 0).

Figure 8b shows the number of extracellular vesicles (EVs) per spheroid produced from cancer cells as a function of the rotation time (in hours), using a vessel of the invention, as tested in Example 2 below. The y-axis represents the number of extracellular vesicles per spheroid. The bars represent average numbers of produced extracellular vesicles. The x-axis represents the duration of the rotation. TO corresponds to the control for the rotation duration (at time 0).

Figure 9a shows the number of extracellular vesicles (EVs) produced per spheroid as a function of the rotation speed (in rpm) after a 1-hour rotation, using a vessel of the invention, as tested in Example 4a below. The y-axis represents the number of extracellular vesicles per spheroid. The black bars represent average numbers. The x-axis represents the rotation speed in rpm.

Figure 9b shows the number of extracellular vesicles (EVs) produced per spheroid as a function of the rotation speed (in rpm) after a 3-hour rotation, using a vessel of the invention, as tested in Example 4a below. The y-axis represents the number of extracellular vesicles per spheroid. The black bars represent average numbers. The x-axis represents the rotation speed in rpm.

Figure 9c shows the protein expression of the transmembrane extracellular vesicle (EV) marker CD63, of the cytosolic EV marker Synt-1 and of a non-EV marker enriched in the soluble fraction, 14-3-3, of extracellular vesicles (EVs) produced using a vessel of the invention at different rotation speeds (rpm), as tested in Example 4a below. The presence of the EV markers and the absence of the non-EV marker in the EV fraction is proof of the sample’s purity. No. 1 (lanes 1 and 2 from the left) corresponds to a rotation speed of 800 rpm, and No. 2 (lanes 3 to 5 from the left) to a rotation speed of 1600 rpm. “CCM” refers to a concentrated cell medium; “Int” refers to an intermediate fraction after size exclusion chromatography; “EVs” refers to the extracted EV fraction after the size exclusion chromatography; and “MM” refers to a molecular weight marker.

Figure 9d shows the number of EVs produced per cell in a vessel of the invention, as tested in Example 4b below. The y-axis represents the number of EVs produced per cell at varying rotation speeds (in rpm), and the x-axis represents the rotation duration (in hours).

Figure 9e shows the cell death of cells (in %) at different rotation speeds on the y-axis as a function of the rotation duration (in hours) on the x-axis, as tested in Example 4b below, using the same vessel as in Figure 9d.

Figure 10a shows the average number of extracellular vesicles (EVs) per cell from single (individualized) cells, using a vessel having a baffle structure comprising solid baffle plates of the invention, as tested in Example 5a below. The y-axis represents the number of extracellular vesicles per cell. The x-axis represents the rotation speed in rpm. The two bars on the left correspond to EVs produced from mouse mesenchymal stem cells (mMSC) and the two bars on the right correspond to EVs produced from human mesenchymal stem cells (hMSC).

Figure 10b shows the number of EVs produced per cell in a vessel of the invention, as tested in Example 5b below. The y-axis represents the number of EVs produced per cell at varying rotation speeds (in rpm), and the x-axis represents the rotation duration (in hours).

Figure 10c shows the cell death of cells (in %) at different rotation speeds on the y-axis as a function of the rotation duration (in hours) on the x-axis, as tested in Example 5b below, using the same vessel as in Figure 10b.

Figure 10d shows the protein expression of EV markers CD63 and CD81 , a cytosolic EV markers Synt-1 and a non-EV marker 14-3-3 of EVs produced as in Example 5b below. “EVs” refers to the extracted EV fraction after the size exclusion chromatography; “EV Int” refers to an intermediate fraction after size exclusion chromatography; and “MM” refers to a molecular weight marker. The bands below Synt-1 corresponds to the bands of Synt-1 with a higher exposition.

Figure 11a shows the number of extracellular vesicles (EVs) produced per spheroid as a function of the rotation speed (in rpm) after a 1-hour rotation, using a vessel having a baffle structure comprising meshed baffle plates of the invention as tested in Example 6 below. The y-axis represents the number of extracellular vesicles per spheroid. The black bars represent average numbers of produced extracellular vesicles. The x-axis represents the rotation speed (in rpm).

Figure 11b shows the number of extracellular vesicles (EVs) produced per spheroid as a function of the rotation speed (in rpm) after a 3-hour rotation, using a vessel having a baffle structure comprising meshed baffle plates of the invention as tested in Example 6 below. The y-axis represents the number of extracellular vesicles per spheroid. The black bars represent the average numbers of produced extracellular vesicles. The x-axis represents the rotation speed (in rpm). Figure 12a shows the number of extracellular vesicles (EVs) produced per spheroid as a function of the number of rotation cycles performed, using a vessel having a baffle structure comprising meshed baffle plates of the invention, as tested in Example 7 below. The y-axis represents the number of extracellular vesicles produced per spheroid. The x-axis represents the number of the rotation cycles performed. The black circles represent the rotation at 800 rpm; the white circles represent the rotation at 1200 rpm; and the squares represent the rotation at 1600 rpm.

Figure 12b shows the metabolic activity of cells as a function of the number of rotation cycles performed, using a vessel having a baffle structure comprising meshed baffle plates of the invention, as tested in Example 7 below. The y-axis represents the metabolic activity of cells. The x-axis represents the number of the rotation cycles performed. The black circles represent the rotation at 800 rpm; the white circles represent the rotation at 1200 rpm; and squares represent the rotation at 1600 rpm.

Figure 12c shows the cell death of cells as a function of the number of rotation cycles performed, using a vessel having a baffle structure comprising meshed baffle plates of the invention, as tested in Example 7 below. The y-axis represents the cell death of cells in %. The x-axis represents the number of the rotation cycles performed. The black circles represent the rotation at 800 rpm; the white circles represent the rotation at 1200 rpm; and squares represent the rotation at 1600 rpm.

Figure 13a illustrates the number of EVs produced per cell in a vessel having a baffle structure comprising solid baffle plates of the invention, using as producer cells hMSC spheroids formed in the vessel, as tested in Example 3b below. The y-axis represents the number EVs produced per cell at varying rotation speeds (in rpm), and the x-axis represents the rotation duration (in hours).

Figure 13b shows the cell death of cells (in %) at different rotation speeds on the y-axis as a function of the rotation duration (in hours), as tested in Example 3b below, using the same vessel as in Figure 13a.

Figure 13c shows the protein expression of the EV markers CD63 and CD81 , a cytosolic EV marker Synt-1 and a non-EV marker 14-3-3 of EVs produced as in Example 3b below. Lanes “EV1”, “EV2” and “EV3” correspond to three independent production lines of EVs extracts after the size exclusion chromatography; lanes “EV2 Int” and “EV3 Int” correspond to intermediate fractions of EV2 and EV3 after size exclusion chromatography; lanes “2D” and “3D” correspond to the control (EVs produced by 2D starvation and 3D starvation, respectively); and “MM” refers to a molecular weight marker. The bands below Synt-1 corresponds to the bands of Synt-1 with a higher exposition.

Figure 14a and Figure 14b show the expression of EV-specific markers and mesenchymal markers, as tested in Example 8. The x-axis represents markers detected, and the y-axis represents the expression (the median florescence) relative to the expression level of immunoglobulin (Ig). 2D and 3D correspond to the control (EVs produced by 2D starvation and 3D starvation, respectively); A corresponds to EVs produced from individualized hMSC cells using a vessel of the invention, and C1 to C3 correspond to EVs produced in a vessel of the invention, using, as producer cells, hMSC spheroids formed in the vessel. Markers CD9/CD63/CD81 correspond to EV-specific proteins (Fig. 14a) and markers CD29/CD44/CD49e/CD105/CD146 correspond to mesenchymal markers (Fig. 14b).

Figure 15a and Figure 15b show the sprout formation of endothelial spheroids under various conditions, as tested in Example 9. Figure 15a shows microscopic images, and Figure 15b shows the number of endothelial spheroids that underwent sprout formation on the y-axis across different conditions shown on the x-axis. The endothelial spheroids were cultured alone (negative control, FBS 0%); 10% FBS and VEGF (positive control, FBS 10% and VEGF); EVs produced by 2D or 3D starvation (control as a comparative example, “2D” and “3D”, respectively), and EVs produced from individualized hMSC cell (A), EVs produced from hMSC spheroids formed outside the vessel (B), and EVs produced from hMSC spheroids formed inside the vessel (C). 1X, 2X or 4X indicates the number of doses administered.

Figure 16a, Figure 16b and Figure 16c show the area coverage of a scratch wound in human skin fibroblasts (HSF) over time in a wound healing assay, as tested in Example 10. The x axis represents different time points (in hours), and the y-axis represents the area coverage in % (corresponding to the area covered by cells in the scratch wound divided by the total area of the scratch wound). FBS 0%, FBS 1%, FBS 2%, FBS 4%, and FBS 10% correspond to the controls in which HSFs are cultured alone or with 1 %, 2%, 4%, or 10% FBS; 2D and 3D correspond to another control (HSFs cultured with EVs produced by 2D starvation and 3D starvation, respectively); A corresponds to HSFs cultured with EVs produced from individualized hMSC cell; B corresponds to HSFs cultured with EVs produced from hMSC spheroids formed outside the vessel, and C corresponds to HSFs cultured with EVs produced from hMSC spheroids formed inside the vessel. Figure 17 shows the anti-inflammatory activity of EVs prepared according to the invention, as tested in Example 11. The x-axis represents the type of treatment. CTL refers to the control (positive control (+), negative control (-), a control with a known anti-inflammatory inhibitor (Inh); A corresponds to EVs produced from individualized hMSC cell, B corresponds to EVs produced from hMSC spheroids formed outside the vessel, and C corresponds to EVs produced from hMSC spheroids formed inside the vessel. 0.25X, 0.5X, 1X or 2X indicates the number of doses administered. The y-axis represents the concentration of NO (in arbitrary unit) with respect to the measured absorbance value.

Figure 18a and Figure 18b show one example of a vessel which is not in accordance with the invention, as tested in Example 12 below. Figure 18a shows a diagonal top view of the vessel, and Figure 18b shows a side view of the vessel shown in Figure 18a, cut along a plane parallel to the central axis of the vessel.

Figure 18c shows the number of EVs produced in the vessel of Figure 18a and Figure 18b, as tested in Example 12 below. The x-axis represents the rotation duration (in hours, and the y-axis represents the number EVs produced per spheroid (left) and the number EVs produced per producer cell (right).

Figure 19 shows ultrafast camera images illustrating the projected velocities of producer cells in a vessel (transverse cross-section) of the invention, in use. The grayscale on the right indicates the projected particle velocity (mm/s). Velocity values shown on the trajectories range from less than 20 mm/s to more than 125 mm/s depending on the portions of the trajectories.

DESCRIPTION OF EMBODIMENTS

The invention will now be described in more detail without limitation in the following description.

The term “extracellular vesicle” as used herein refers to a vesicle that is endogenously released by a producer cell in a constitutive or inducible manner. An extracellular vesicle generally has a diameter of from 30 nm to 500 nm. Examples thereof include, but are not limited to, exosomes, microvesicles and apoptotic bodies.

The term “cell” as used herein refers to the smallest fundamental structural and functional unit of living organisms, which can divide and multiply.

The term “producer cell” as used herein refer to a cell that is capable of secreting extracellular vesicles.

The term “organoid” as used herein refers to an agglomeration of cells that recapitulates aspects of cellular self-organization, architecture and signaling interactions present in a native organ. The term “spheroid” as used herein refers to a cellular structure consisting of more than one single cell, which has initially developed from a single or from multiple cells.

The terms “microcarrier” as used herein refers to a particulate matrix that allows the growth of producer cells adherent on its surface or within it. The matrix may be comprised of particles, preferably substantially spherical particles, having a maximum diameter of between 50 pm and 500 pm, and preferably between 100 pm and 300 pm. The microcamers are generally beads whose density is chosen to be substantially close to that of the liquid culture medium of the producer cells, thereby allowing the beads to remain suspended in the liquid culture medium by gentle mixing.

The term “vessel” as used herein refers to any type of container for containing liquid medium, such as a tube or a tank.

The term “vertical” or “vertically” as used herein refers to a direction which is perpendicular to the plane of the horizon and parallel to the direction of gravity.

Vessel for producing extracellular vesicles from producer cells

The present invention provides a vessel for producing extracellular vesicles (EVs) from producer cells. The vessel has an internal space for holding liquid medium. The vessel comprises a baffle structure inside the vessel, i.e. within the internal space. Preferably, the vessel comprises a coupling on an external surface configured to be coupled to a rotating apparatus as will be described in more detail below.

By “baffle structure” is meant one or more elements which are fixed within the vessel and which deviate the flow of liquid within the vessel. The baffle structure generally promotes turbulent flow in the vessel.

Fig. 1 shows one example of a vessel of the invention.

The vessel 1 comprises a baffle structure inside the vessel (not shown in Fig. 1), which will be explained in detail later, and a coupling 3 on an external surface of the vessel configured to be coupled to a rotating apparatus.

The vessel may be made of a suitable material for the production of extracellular vesicles, e.g., a biocompatible resin, a biocompatible polymer, or a metal.

The vessel may be manufactured by 3D printing, injection molding, blow molding, or compression molding, preferably by 3D printing.

In some embodiments, the vessel has not been subjected to (or has not undergone) any surface treatment. Preferably, and as illustrated, the internal space of the vessel is structurally delimited by a base and a peripheral wall extending from the base. The base is preferably substantially planar (flat). Preferably, the baffle structure does not cover the entirety of the peripheral wall. Preferably, the peripheral wall is smooth, i.e. does not include wells.

Preferably, the shape of the vessel is substantially cylindrical (or at least the shape of the internal space of the vessel is substantially cylindrical). In this case, the peripheral wall is a cylindrical inner wall, as shown on Fig. 1. The central axis of the vessel can then be defined as the axis of the cylinder. Preferably, the axis is perpendicular to the base and the cylinder is a right cylinder. Preferably, the cylinder is a circular cylinder, more preferably a right circular cylinder. In alternative embodiments, the peripheral wall may be in the shape of a non-circular cylinder. For example, if the base is substantially in the shape of a polygon (such as a square or a rectangle), the peripheral wall may be in the shape of a cylinder composed of a number of planar sections joined along respective edges thereof (such as four planar sections).

The capacity volume of the vessel may be suitably adjusted depending on, for example, the working volume, and the target number of the extracellular vesicles to be produced.

In some embodiments, the vessel may have a capacity of from 10 mL to 10 L. For example, the capacity of the may be from 10 mL to 5 L, from 10 mL to 1 L, from 10 mL to 500 mL, or from 10 mL to 250 mL.

The diameter of the vessel may be suitable adjusted to accommodate different working volumes. For example, the internal diameter of the vessel may be from 2 to 25 cm, For example, the diameter of the vessel may be from 2 to 20 cm, from 3 to 15 cm, or from 3 to 10 cm.

The height (maximal dimension in the direction parallel to the central axis) of the vessel may be from 1 to 30 cm, preferably from 2 to 20 cm, and more preferably from 3 to 10 cm.

The vessel may be provided with a closure element, such as a cap. In this case, the vessel may comprise a neck at the top part of the vessel (opposite the base at the bottom part), which is designed to receive the cap to seal the vessel.

The size and shape of the neck may vary depending on the type of vessel and the cap. Generally, the neck has a smaller diameter than the rest of the vessel (also referred to as “body” of the vessel), having a shoulder (transition between the body and the neck), which may be a curved or sloping part of the vessel where the diameter changes from the body to the neck. The cap may be fixed to the vessel by a threaded engagement, by a bayonet connection, by friction fitting, by a magnetic connection or the like.

The coupling may have any suitable geometry, provided that the geometry allows the vessel to be rotatably fixed to the rotating apparatus.

In some embodiments, the coupling may comprise one or more grooves or ridges on the external surface of the vessel.

For example, as shown in Fig. 1 , the coupling 3 may comprise a ridge (a raised or protruding line) running along the external surface of the vessel in the direction parallel to the central axis of the vessel (on the surface across from the peripheral wall in the internal space). The same is possible with a groove running along the external surface of the vessel in the direction parallel to the central axis of the vessel.

Alternatively or additionally, the coupling 3 may be provided on a bottom external surface of the vessel (across from the base in the internal space).

The baffle structure may extend from the peripheral wall of the vessel, towards the central axis of the vessel.

In some embodiments, the baffle structure may comprise one or more baffles. By "baffle” is meant a wall, which can be either substantially planar or curved, and preferably is substantially planar. The baffles are fixed in the internal space of the vessel, to an internal surface of the vessel. Preferably, they are fixed to the base and/or to the peripheral wall and are preferably integrally formed with the base and/or the peripheral wall. Each baffle acts as an obstacle to the flow of liquid within the vessel and is configured to deflect such flow of liquid. In the present text, when A is said to be “fixed to” B, it may mean “indirectly fixed to” (/.e. A is fixed to B via an intermediate element); or, more preferably, “directly fixed to” (/.e. A is fixed to B without any intermediate element between A and B).

For example, the baffle structure may comprise one baffle, two baffles, three baffles, four baffles, five baffles, or six baffles, seven baffles, eight baffles, nine baffles or ten baffles which is/are fixed to an internal surface of the vessel.

When the baffle structure comprises two or more baffles, the baffles may be regularly spaced within the vessel or may be non-regularly spaced within the vessel.

In some embodiments, the baffles may be fixed on the internal surface of the vessel symmetrically relative to the central axis of the vessel.

The baffles may have either the same dimensions (e.g., height, thickness, length), or different dimensions from each other.

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

The length of each baffle may be from 1 to 12.5 cm, preferably from 1 to 5 cm, and more preferably from 1 to 2.5 cm. The term “length” for a baffle as used herein refers to the maximum dimension of the baffle perpendicular to the central axis (and preferably parallel to the base of the vessel).

The thickness of each baffle may be from 0.1 to 5 cm, preferably from 0.1 to 2.5 cm, and more preferably from 0.1 to 0.5 cm. The term “thickness” for a baffle as used herein refers to the dimension which is smaller than the maximum dimensions in the orthogonal directions and which is perpendicular to the length direction (and preferably parallel to the base of the vessel).

In some embodiments, the baffle structure comprises one or more pairs of baffles. Each pair of baffles may comprise two diametrically opposed baffles relative to the central axis of the vessel.

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

For example, the baffle structure may comprise one pair, two pairs, three pairs, four pairs, or five pairs of the diametrically opposed baffles.

When the baffle structure comprises two or more pairs of baffles (for example, n pairs), the pairs may be arranged such that the baffles (for example, 2n baffles) are spaced apart equidistantly from each other. Alternatively, the pairs may be arranged such that the baffles (for example, 2n baffles) are spaced apart non-equidistantly from each other.

In some embodiments, the pairs of baffles may be symmetrically arranged relative to the central axis of the vessel.

The two baffles in each pair may have either the same dimensions among all pairs, or different dimensions among pairs.

In some embodiments, the baffle structure may comprise a plurality of baffles extending from the peripheral wall towards the central axis.

The baffles may be directly joined together. For example, the baffles may extend to the central axis and be joined at the central axis. In this case, the baffle structure may divide the internal space of the vessel into a plurality of compartments, which are in fluid communication with one another.

Alternatively, the baffles are not directly joined together (but are only indirectly joined via the base or the peripheral wall). In particular, the baffles may not extend up to the central axis, thus leaving an unobstructed central space in the vessel. In this case, the baffles may extend a different distance towards the central axis, or the baffles may extend the same distance towards the central axis. In other embodiments, the baffle structure may comprise a plurality of baffles extending from the central axis of the vessel towards the peripheral wall of the vessel.

In some embodiments, the baffles do not extend to the peripheral wall of the vessel, thus leaving an unobstructed peripheral space between the baffles and the peripheral wall.

Part or all of the baffles may be plates or walls, i.e. substantially flat elements. These elements may have one dimension, namely the thickness, which is much smaller (such as at least 10 times or 100 times smaller) than the maximum dimensions in the orthogonal directions.

The plates or walls are preferably substantially planar but may alternatively be curved.

When a plate or wall is substantially planar, the thickness is the dimension of the plate perpendicular to the main plane of the plate or wall. The thickness may vary or be constant across the structure. If it varies, then any thickness values are meant to designate the average thickness.

The plates or walls may be solid plates or walls, or may be meshed or perforated plates or walls. The term “meshed plate” as used herein refers to a plate made from interlocking wires or struts (also referred to as a “grid”). The term “perforated plate” as used herein refers to a plate comprising openings or holes.

In the case of meshed or perforated plates or walls, the mesh size (size of the openings/holes), the uniformity of the openings/holes, the arrangement of the openings/holes can be adjusted suitably.

For example, the size of the openings/holes (for example, a diameter in the case of circular openings/holes or a diagonal in the case of polygonal openings/holes) in a meshed or perforated plate or wall may be from 0.1 to 5 cm, preferably from 0.1 to 1 cm, and more preferably from 0.1 to 0.5 cm.

The plates or walls may have an outer shape (perpendicular to the direction of the thickness) which may be substantially square, rectangular, triangular, trapezoidal, diamond, pentagonal, hexagonal, octagonal, more generally polygonal, or which may be at least partly curved. The plates or walls may in particular be substantially perpendicular to the base and may be oriented substantially parallel to the central axis of the vessel.

Alternatively, the plates or walls may be oriented substantially perpendicular to the central axis of the vessel.

The plates or walls may be also be oriented at an angle from 10° to 80°, preferably from 30° to 60° relative to the central axis of the vessel, and/or relative to the base. For example, the plates or walls may be oriented at an angle of approximately 10°, 20°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 70°, or 80° relative to the central axis of the vessel and/or relative to the base.

All angle values in the present text are absolute values.

All baffles may be oriented similarly or not.

In some embodiments, the baffle structure may comprise a plurality of plates fixed on struts, the struts being preferably oriented parallel to the central axis of the vessel. Preferably, differently oriented plates alternate along the struts.

Below, several possible designs of the vessel of the invention will be explained more in detail by reference to the drawings, but it is understood that the design of the vessel can be configured and further modified to achieve optimal conditions flow conditions within the vessel.

Making reference to Fig. 2a and Fig. 2b, the vessel 1 may be substantially cylindrical in shape and may comprise a baffle structure 2 inside the vessel and a coupling 3.

The vessel may further comprise a neck T at the top part of the vessel and a body 1”. The body 1” comprises the base 7 and peripheral wall 8 as described above in connection with Fig. 1

The baffle structure 2 may comprise three pairs of baffles 4, each pair of baffles comprising two diametrically opposed baffles relative to the central axis of the vessel. A different number of pairs of baffles is of course possible.

In this example, the baffle structure 2 comprises six baffles 4 in total fixed to an internal surface of the vessel (the peripheral wall 8 and/or the base 7), and the three pairs are arranged such that the six baffles are regularly spaced apart along the circumference of the cylinder.

All of the baffles may be solid plates as shown, but the baffles may be also meshed or perforated plates, or a combination of solid plates, meshed plates and perforated plates.

The plates may have, but not limited to, a rectangular shape.

The baffles may extend from the peripheral wall 8 towards the central axis, without extending to the central axis (in other words, the baffles do not reach all the way to the central axis of the vessel and leave an unobstructed central space).

The two baffles 4 in each pair may be identical within the pair, but the dimensions may differ from pair to pair. For example, the baffles may be plates having the same thickness and the same length, but may differ in height among the pairs. The term “height” as used herein (for a plate, baffle or other structure) refers to the maximum dimension of the plate, baffle or other structure in the direction parallel to the central axis. The term “length” as used herein (for plate, baffle or other structure) refers to the maximum dimension of the plate, baffle or other structure perpendicular to the central axis (and preferably parallel to the base 7).

Alternatively, the two baffles 4 in each pair may have, among the pairs, the same height, length, and thickness (identical among pairs); or the same thickness but different heights and lengths; or the same height and thickness but a different length; or the same height and length but a different thickness; or the same height but different thickness and length; or the same length but different height and thickness; or different height, length, and thickness.

The planes of part or all of the plates may be oriented substantially parallel to the central axis of the vessel.

For example, as shown in Fig. 2a and 2b, the planes of all of the plates may be oriented substantially parallel to the central axis of the vessel.

As a general remark, in all embodiments described therein, rounding or fillets may be provided at each edge or corner in order to avoid sharp edges which may induce local high shear areas which may damage any biological material present in the vessel.

In the example of Fig. 2a and 2b, the edges of the baffles 4 facing towards the central axis and preferably running parallel to the central axis are rounded.

The vessel shown in Fig. 3a and Fig. 3b is the same as the vessel of the example shown in Fig. 2a and Fig. 2b, except that there is in addition a fillet in the area where the baffles 4 are connected to the peripheral wall 8 and to the base 7, allowing for a smoother transition from each baffle to the peripheral wall 8 and the base 7.

The arrangement, the orientation, and the dimension of the baffles may be as defined above.

The vessel 1 shown in Fig. 4a to Fig. 4c is the same as the vessel of the examples shown in Fig. 2 and Fig. 3, except for the baffle structure.

The baffle structure 2 may comprise six baffles 4 and a central strut 5. The central strut may be aligned with the central axis of the vessel and may be fixed to the base 7 of the vessel.

As shown in Fig. 4a to Fig. 4c, the baffles may be meshed plates having the same dimensions, but it is understood that they may be also perforated plates and/or may have different dimensions from each other. The plates are preferably substantially planar.

The baffles 4 may be fixed to the central strut 5 and extend from the central strut 5 towards the cylindrical inner wall. If planar, the baffles may be substantially perpendicular to the base 7. The central strut 5 may have a height which is longer than the height of the body 1” of the vessel (the height in in the direction parallel to the wall of the vessel). In other terms, the central strut 5 may extend from the base 7 up to the area of the neck T. The top edge of each baffle (i.e. the edge opposite the base 7) may be oriented at an angle different from 90°, such as approximately 45°, relative to the central axis (the central strut 5). As a result, the planes of the plates may have a trapezoid outer shape (see Fig. 4b).

However, it is understood that the central strut 5 may have a shorter height, and the planes of the baffles may also have another outer shape, such as a rectangle.

The plates may be regularly spaced apart around the central axis.

In Fig. 4b and Fig. 4c, the baffles do not extend to the peripheral wall 8, leaving an unobstructed space between each baffle and the peripheral wall 8. Alternatively, the baffles may divide the inside of the vessel into a plurality of compartments by extending from the central strut 5 to the peripheral wall 8. The compartments may be in fluid communication with one another through, for example, the openings of the meshed or perforated plates.

In some embodiments, the vessel does not comprise a central strut. In this case, the baffles 4 may extend from the central axis of the vessel towards (or to) the cylindrical inner wall (and they may be fixed together along the central axis) or may extend from of the cylindrical inner wall of the vessel towards (or to) the central axis.

In some other embodiments, the baffles 4 may be fixed on the base 7 of the vessel, leaving an unobstructed peripheral space between the baffles and the peripheral wall and/or an unobstructed central space in the vessel.

The baffles 4 may be oriented radially relative to the central axis of the vessel.

Making now reference to the embodiment shown in Fig. 5a to 5c, the vessel 1 of this example is the same as the vessel of the examples shown in Fig. 2 to Fig. 4, except for the baffle structure.

As shown in Fig. 5a, the baffle structure 2 comprises a plurality of plates 4’ fixed on multiple struts 6.

The struts 6 are preferably cylindrical in shape and extend along respective strut axes. Their cross-section (perpendicular to the strut axis) may be circular, polygonal or other. In the illustrated embodiments, the cross-section is starshaped. Such a non-circular shape may further improve flow characteristics within the vessel. The struts 6 are preferably parallel and are preferably oriented parallel to the central axis of the vessel.

The struts 6 on which the plates 4’ are fixed may be fixed on the base 7 of the vessel (see Fig. 5b and Fig. 5c). They may be arranged in arrays, each array supporting a different plurality of plates. The structure composed of an array of struts and the plurality of plates supported by this array can be referred to as a stack 2’.

The number of struts 6 may be adjusted depending on the number or the dimension of the plates 4’. For example, the struts 6 in each array may be arranged in three rows, each row having five struts, as shown in Fig. 5c. As other examples, the struts may be arranged in one, two, three, four or five rows, with two, three, four, five, six, seven, eight, nine or ten struts per row, in each array. The rows may for example be oriented radially relative to the central axis of the vessel.

The plates are preferably planar. The planes of each plate may be oriented at an angle from 10° to 80°, preferably from 30° to 60° relative to the central axis of the vessel; and/or may be oriented at an angle from 10° to 80°, preferably from 30° to 60° relative to the base 7 of the vessel.

For example, as shown in Fig. 5c, the plane of each plate facing the central axis of the vessel may be oriented at an approximately angle from 45° relative to the central axis of the vessel.

The plates 4’ may be also differently oriented, and the differently-oriented plates 4’ may alternate along the struts 6.

Merely by way of example, successive plates along each array of struts may have a symmetrical orientation, relative to a plane between these successive plates (which is preferably parallel to the base 7 of the vessel). These differently- oriented plates 4’ may alternate along the struts, as shown in Fig. 5b and Fig. 5c, forming a zig zag pattern.

The baffle structure 2 may comprise one or more baffle stacks 2’.

The stacks 2’ may be spaced apart equidistantly from each other. Alternatively, the stacks 2’ may be spaced apart non-equidistantly from each other. They may in particular be regularly spaced around the central axis.

In some embodiments, the stacks 2’ may be symmetrically arranged relative to the central axis of the vessel (by pairs).

The stacks 2’ may have either the same dimensions, or different dimensions from each other. The baffle structure 2 may comprise one or more pair of stacks 2’, for example, three pairs of stacks 2’, each pair comprising two diametrically opposed stacks relative to the central axis of the vessel.

The pairs of stacks 2’ may be arranged such that all the stacks are spaced apart equidistantly from each other.

For example; the baffle structure 2 may comprise three pairs of stacks 2’, thus in total six stacks 2’, and the three pairs may be arranged such that the six stacks are spaced apart equidistantly from each other.

The stacks 2’ in each pair may have the same dimensions within the pair, but may have different dimensions from one pair to the other.

Alternatively, the stacks 2’ in each pair may have different dimensions.

An unobstructed area around the central axis may be present. In some cases, the plates 4’ may be fixed to the peripheral wall 8. In other cases, an unobstructed space may be present in an annular area between the stacks 2’ and the peripheral wall 8.

Making reference to Fig. 6a to 6c, the vessel 1 of this example is the same as the vessel of the examples shown in Fig. 5a to 5c, with the following modifications.

As shown in Fig. 6a, in addition to the vessel shown Fig. 5a to 5c, the vessel further comprises a central strut 5.

The central strut 5 is preferably aligned with the central axis of the vessel and may be fixed to the base 7 of the vessel.

The plates 4’ may be fixed to the struts 6 of one array and to the central strut 5.

Each stack 2’ may extend from the central strut towards or to the peripheral wall 8. In some cases, the plates 4’ may be fixed to the peripheral wall 8. In other cases, an unobstructed space may be present in an annular area between the stacks 2’ and the peripheral wall 8.

System for producing extracellular vesicles (EVs) from producer cells

The present invention also provides a system for producing extracellular vesicles (EVs) from producer cells.

The system comprises a vessel as defined above, and a rotating apparatus.

The vessel is configured to be rotationally fixed to the rotating apparatus by keying the coupling of the vessel to a corresponding coupling on the rotating apparatus. If the coupling of the vessel comprises grooves, the coupling on the rotating apparatus may comprise corresponding ridges. If the coupling of the vessel comprises ridges, the coupling on the rotating apparatus may comprise corresponding grooves.

In some embodiments, the rotating apparatus may comprise a rotating element such as a cup configured for receiving the vessel. The coupling on the rotating apparatus may be present on an internal surface of the cup which is in contact with an external surface of the vessel.

Alternatively or in combination, the rotating apparatus may comprise a securing mechanism for maintaining the vessel rotatably fixed within the rotating element, comprising for example tightening means using screws, or a friction fit engagement.

In some embodiments, the outer shape of the vessel is non circular and the cup has a corresponding shape, thus ensuring that the vessel is rotationally fixed relative to the cup without any requirement for respective couplings on the external surface of the vessel and on the internal surface of the cup. For example, the vessel may comprise one or more planar external surfaces in addition to the base (e.g. the vessel may have an overall cuboid shape), or may comprise an ellipsoidal surface. In this case, the cup has a complementary shape so as to ensure contact between one or more external surfaces of the vessel and one or more internal surfaces of the cup.

In some embodiments, the rotating apparatus may comprise a drive mechanism and a control unit for controlling the drive mechanism.

The rotating element (e.g. cup) may be actuated by the drive mechanism. The rotating apparatus may comprise a stationary part which may include the drive mechanism and the control unit.

Preferably, the control unit may be configured for implementing the rotation of the vessel as required by the method of the invention (which will be explained below).

The control unit may comprise one or more processors coupled to a storage medium, as well as a computer program comprising instructions stored thereon, for performing the various steps described in more detail below. The control unit may receive input from sensors in or associated with the rotating apparatus and/or input from the user. The control unit may process the input data and, as a result, provide instructions to the drive mechanism. In some embodiments, part or all of the control unit may be provided not in the rotating apparatus itself but in a separate computing device.

Method for producing extracellular vesicles (EVs) from producer cells The present invention also provides a method of producing extracellular vesicles from producer cells.

The method comprises: a) placing producer cells in a liquid medium in a vessel comprising a baffle structure inside the vessel, b) rotating the vessel so as to generate extracellular vesicles from the producer cells; and c) collecting the generated extracellular vesicles, wherein the step b) of rotating the vessel comprises repeatedly changing the rotational motion of the vessel.

The method of the invention is performed ex vivo.

The vessel may be as defined above. The vessel may be rotated owing to the rotating apparatus described above.

By rotating the vessel, a flow of liquid medium within the vessel is achieved. Preferably, no addition of liquid medium and no withdrawal of liquid medium takes place during the rotation. Preferably, the vessel does not comprise a feeding line and/or a collecting line flu idical ly connected to the internal space of the vessel.

Preferably, the producer cells move freely under the effect of the flow of liquid medium. In other terms, preferably, the producer cells are comprised in the bulk of the liquid medium, and are not fixed to a wall of the vessel, as shown in Figure 19.

The rotation of the vessel is preferably such that the flow of liquid with the vessel is turbulent. Preferably, a turbulent regime characterized by a Reynolds number of greater than 2,000, or of greater than 7,000, preferably greater than 10,000 and most preferably greater than 12,000 or greater than 15,000 or greater than 20,000 may be obtained while the shear stress on cells inside the liquid can be kept minimal. At a point in time when there is a stationary regime, i.e. the average velocity of liquid in the vessel is equal to the velocity of the vessel, the global Reynolds number can be overall estimated as Re = R x V / v, wherein R is the internal radius of the vessel, V is the velocity of the vessel peripheral wall and v is the kinematic viscosity of the liquid. At a point in time when there is a transitional regime, i.e. a change in speed of rotation or a reversal of the rotational direction, the Reynolds number can be locally estimated as Re = W x V' / v, wherein W is a characteristic dimension of a baffle element (such as the radial length of a baffle plate), V’ is the relative velocity between the liquid and the baffle element, and v is the kinematic viscosity of the liquid. Preferably, the global Reynolds number and/or a local Reynolds number as defined above is within one of the ranges cited above during at least part of the duration of step b). The producer cells may move relative to the vessel at a velocity which can reach a maximum value of at least 25, at least 50, at least 75, at least 100, or at least 125 mm/s, as shown in Figure 19.

Preferably, there is no impeller in the vessel nor any other rotating or agitation element (such as a magnetic agitator), so that the flow of liquid is solely effected by the rotation of the vessel itself.

During step b), the vessel is preferably closed (such as by using the closure element described above). The vessel may be substantially filled with liquid medium during this step, i.e. may comprise no (or substantially no) gaseous headspace. In alternative variations, a gaseous headspace may be present. Preferably, the volume of gaseous headspace in the vessel, if present, may be less than 20%, or less than 10%, or less than 5%, or less than 2%, or less than 1 %, relative to the volume of liquid in the vessel.

In some embodiments, the producer cells may be selected from human cells, animal cells, and combinations thereof.

In some embodiments, the producer cells may be human cells, preferably healthy human cells. In some embodiments, the producer cells are not human embryonic derived cells and in particular are not human embryonic stem cells.

Alternatively, the producer cells may be also pathological cells, for example cells derived from tissues and/or cancerous lines such as A673 cells or HeLa cells.

In some embodiments, the producer cells may be animal cells, preferably murine cells, for example murine MSC (murine mesenchymal stem cells) cells.

In some embodiments, the producer cells may be stem cells, in particular induced pluripotent stem cells, or multipotent cells. By way of example, the stem cells may be selected from multipotent mesenchymal cells, genetically modified cells, umbilical cord vein endothelial cells (HUVEC) or primary cells.

In other embodiments, the producer cells may be cell line cells, preferably human monocyte line or human line of cells of hematopoietic origin derived from B lymphocytes, more preferably THP-1 cells or Raji cells.

In some embodiments, the producer cells may be isogenic cells, i.e., they are derived from a subject, so that the extracellular vesicles produced by said producer cells, can then be administered to the subject or another subject (in order to prevent or treat a disease) or can otherwise be used ex vivo.

In some embodiments, the extracellular vesicles may be administered to a subject (in order to prevent or treat a disease) but the producer cells are not derived from this subject. In this case, the producer cells may be allogeneic cells, i.e. from the same species as the species of the said subject. Alternatively, the producer cells may be xenogeneic cells, i.e., from a species different from the species of said subject.

The subject is preferably human but can also be an animal.

The producer cells may be either adherent to a culture medium or nonadherent to a culture medium (also referred to as suspension cells).

In the case of adherent producer cells, the culture medium can be composed of microcamers which themselves are suspended in a liquid culture medium.

In some embodiments, the producer cells may be adherent producer cells detached from their culture medium and put in suspension, for example by a suitable treatment selected from an enzymatic treatment, a chemical treatment, a mechanical treatment or a combination thereof.

Preferably, the producer cells are in the form of individualized cells suspended in the liquid medium. By “individualized cells suspended in the liquid medium” is meant that the cells are separate from each other.

In some other embodiments, the producer cells may be in the form of cell aggregates. The term “cell aggregates” refers to an assembly of a plurality of producer cells that adhere to each other.

Preferably, the producer cells are then in the form of spheroids and/or organoids.

In some embodiments, when single cells are considered as producers, the concentration of the producer cells in the liquid medium in the vessel when the extracellular vesicles are generated is from 1000 to 1 million cells per milliliter, preferably from 10000 to 500000 cells per milliliter, more preferably from 50000 to 200000 cells per milliliter, even more preferably from 100000 to 150000 cells per milliliter.

In some embodiments, when producer cells are adherent on microcarriers (beads), the number of cells per bead is from 1 to 50, preferably from 10 to 20. The concentration of beads is from 100 to 50000 beads per milliliter, preferably from 1000 to 20000 beads per milliliter, more preferably from 4000 to 8000 beads per milliliter.

In some embodiments, when producer cells are cell aggregates, encompassing spheroids and organoids, the aggregates have an average diameter of from 50 pm to 5 mm, preferably from 100 pm to 500 pm; the number of cells per aggregate is from 100 to 1 million, preferably from 1000 to 100000. The concentration of aggregates is from 10 to 10000 aggregates per milliliter, preferably from 50 to 2000 aggregates per milliliter, more preferably from 100 to 500 aggregates per milliliter. It is well known that the structure and composition of the extracellular vesicles varies depending on the producer cells and on the production method thereof, in particular in terms of the membrane markers and constituents present on these vesicles.

In some embodiments, the extracellular vesicles produced according to the present invention have an average diameter of from 40 to 500 nm, preferably from 65 to 200 nm, more preferably from 80 to 110 nm.

The average diameter of the extracellular vesicles may be measured by interferometry alone or in combination with fluorescence, using ExoView™ R100 (manufactured by NanoView Bioscience), for example. Alternatively, the average diameter may be measured by an individual particle tracking method (or NTA for Nanoparticle Tracking Analysis), using, for example, NanoSight NS300 (manufactured by Malvern Panalytical).

The liquid medium used in the invention for the production of extracellular vesicles may be a conventional liquid medium, such as FBS (fetal bovine serum), (serum-free) DMEM (Dulbecco's Modified Eagle Medium), or serum-free media.

The vessel rotates around a rotation axis.

Most preferably, when the vessel has a central axis as defined above, the rotation axis corresponds to the central axis of the vessel.

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

In some embodiments, direct visualization of flow trajectories of the liquid within the vessel is carried out during the rotation. In such a configuration, it is possible to track the movement of (e.g. fluorescent) beads (or of cells or particles tied to such beads or labeled with fluorescent markers) in a plane of the rotating vessel, as it rotates, with an ultrafast camera. This can provide thorough analysis of the shear stress experienced by the cells during the rotation, resulting is a better control of the flow conditions.

In some embodiments, step b) of rotating the vessel is carried out at a maximum rotational speed of 50 to 6000 rpm, preferably 600 to 1600 rpm. In some embodiments, the maximum rotational speed may from 50 to 100 rpm; or from 100 to 200 rpm; or from 200 to 300 rpm; or from 300 to 600 rpm; or from 600 to 1000 rpm; or from 1000 to 1600 rpm; or from 1600 to 3000 rpm; or from 3000 to 6000 rpm.

Step b) comprises repeatedly changing the rotational motion of the vessel. This means that the vessel does not rotate at a constant speed during the entirety of step b). The speed of rotation of the vessel changes multiple times during step b). In some embodiments, step b) may comprise periodically changing the rotational motion of the vessel. This means that a certain pattern of rotational motion is repeated multiple times with a certain frequency.

The frequency of changing the rotational motion of the vessel may be from 0.01 to 5 Hz, preferably from 0.05 to 0.5 Hz.

For example, step b) may comprise repeatedly (e.g. periodically) reversing the rotational direction (from clockwise to counterclockwise and conversely).

Alternatively or additionally, step b) may comprise repeatedly (e.g. periodically) changing the rotational speed of the vessel.

Alternatively or additionally, step b) may comprise intermittently rotating the vessel. This means that there are resting sequences within step b) when the vessel does not rotate. Rotating sequences alternate with resting sequences. Successive rotating sequences may be characterized by the same rotational direction or by different rotational directions.

The duration step b) may be for example from 1 hour to 5 hours, e.g., approximately 1 , 2, 3, 4 or 5 hours.

In some embodiments, the extracellular vesicles may be generated from producer cells in the form of spheroids and/or organoids.

In some embodiments, the method may further comprise a preliminary step of growing spheroids and/or organoids outside of the vessel, and the step a) of placing the producer cells in the vessel comprises supplying the grown spheroids and/or organoids to the vessel.

The preliminary step of growing spheroids and/or organoids outside of the vessel is well known in the domain, for example, using hanging drop methods, microwell-based methods, scaffold-based methods, or agitation-based methods.

In other embodiments, the step a) of placing the producer cells in the vessel may comprise supplying individualized cells to the vessel, and the method may further comprise an intermediate step of generating spheroids and organoids from the individualized cells (before step b)).

The intermediate step may comprise rotating the vessel during the intermediate step of generating spheroids and organoids from the individualized cells, preferably at a maximum speed of rotation which is less than a maximum speed of rotation during step b).

The rotation speed during the intermediate step may be from 20 to 200 rpm, preferably from 50 to 100 rpm. During this intermediate step, the rotation motion may remain constant. Alternatively, repeated changes of rotational motion may take place, as describe above in connection with step b). In some embodiments, step c) of collecting the generated extracellular vesicles may be carried out by withdrawing the liquid medium including the producer cells from the vessel, and separating the extracellular vesicles from the withdrawn liquid medium.

The extracellular vesicles may be separated from the withdrawn liquid medium by conventional methods, such as by centrifugation, filtration, sizeexclusion chromatography, immunoaffinity-based separation, decantation, and any combination thereof.

In other embodiments, step c) may be carried by withdrawing the liquid medium from the vessel without substantially withdrawing the producer cells, and separating the extracellular vesicles from the withdrawn liquid medium, by any separation method as described above, preferably centrifugation.

In this case, decantation and/or centrifugation in the vessel itself is preferably carried out prior to withdrawing the liquid medium.

The collected extracellular vesicles (for example, a supernatant including extracellular vesicles in the case of separation by decantation and/or centrifugation) may be counted by an individual particle tracking method (or NTA for Nanoparticle Tracking Analysis), using, for example, NanoSight NS300 (manufactured by Malvern Panalytical).

The collected extracellular vesicles (for example, a supernatant including extracellular vesicles in the case of separation by centrifugation) may be also observed and/or counted by transmission electron cryo-microscopy (cryo-TEM).

In some embodiments, the method may further comprise repeating cycles of at least step b) of rotating the vessel and step c) of collecting the generated extracellular vesicles, using the same producer cells.

For example, at each cycle, the producer cells may be withdrawn from the vessel; separated from the liquid medium (by way of, for example, centrifugation); and placed again in the vessel together with fresh liquid medium.

Alternatively, at each cycle, the liquid medium may be withdrawn from the vessel, the producer cells substantially remaining within the vessel (by way of, for example, centrifugation), and fresh liquid medium may be added to the vessel.

The method may further comprise a time interval of rest between two successive cycles.

The producer cells may be kept in the vessel without rotation of the vessel during the time interval of rest.

The producer cells may be also kept in a conventional cell culture apparatus, such as a cell culture flask, without rotation during the time interval of the rest. The producer cells may be also kept in the vessel in rotation (this may enable cell growth during the time interval of the rest). If the vessel rotates during rest, the rotation may be constant. The rotation speed (if constant) or the maximum rotation speed (if not constant) is less than the rotation speed during the cycles of step b). The rotation speed during any rest step may be from 20 to 200 rpm, preferably from 50 to 150 rpm.

For example, the method may comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 cycles. The duration of each cycle (optionally step a) and steps b) and c)) may be from 1 hour to 5 hours, e.g., approximately 1 , 2, 3, 4 or 5 hours. The duration of the time interval of rest between successive cycles may be from 30 min to 24 hours, for example, approximately 30 min, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 10 hours, 15 hours, 20 hours, 24 hours.

In some embodiments, the method may further comprise a step of introducing a therapeutic agent or an imaging agent into the liquid medium.

The step of introducing a therapeutic agent or an imaging agent into the liquid medium may be performed before, during and/or after the steps a) to c).

The therapeutic agent can be any agent that can prevent, inhibit, or arrest the symptoms and/or progression of an infectious, an autoimmune disease, a fibrotic disease, an inflammatory disease, a neurodegenerative disease, or a heart and vascular disease, cancer or any other disease disclosed below.

The imaging agent may be any substance that is used to enhance the visibility of specific organs, tissues, cells or physiological processes during medical imaging examinations.

Application of the produced extracellular vesicles

The present invention may also relate to extracellular vesicles produced by the method of the invention or using the vessel of the invention, as described above.

In particular, the present invention may also relate to the use of such extracellular vesicles for imaging purposes and/or for therapeutic purposes and/or for diagnostic purposes, such as personalized medicine, immunotherapy, regenerative medicine, cell therapy, and/or the treatment of tumors, infectious diseases, inflammatory diseases, immunological diseases, metabolic diseases, cancerous diseases, genetic diseases, degenerative diseases or diseases secondary to surgery or trauma.

For such purposes, the extracellular vesicles produced according to the present invention may be used as a vector or a carrier for delivering at least one therapeutic and/or imaging agent, for example, by way of administration to a subject in need thereof.

In preferred embodiments, the extracellular vesicles according to the invention may be obtained from physiologically relevant organoids/spheroids, and may be used, for example, in personalized medicine.

In some embodiments, the extracellular vesicles according to the invention may be obtained from THP-1 producer cells or lymphocytes, and may be used, for example, in immunotherapy and/or cancer therapy.

In other embodiments, the extracellular vesicles may be obtained from mesenchymal stem cells (MSC), and may be used in regenerative medicine for processes such as angiogenesis or wound healing.

In other embodiments, the extracellular vesicles can be used in the treatment of inflammatory diseases owing to their potential for anti-inflammatory effects, or in the treatment of tumors, infectious diseases, immunological diseases, metabolic diseases, cancer diseases, genetic diseases, degenerative diseases or diseases secondary to surgeries or trauma.

EXAMPLES

Preparation of cells grown on microcarrier beads

Cytodex 1 dextran microcamer beads (200 pm, GE Healthcare) were suspended in phosphate buffered saline (PBS) to obtain a bead density of 10 g/L and sterilized in an autoclave. The bead suspension in PBS was then re-diluted with Dulbecco's Modified Eagle Medium (DMEM) completed with 1 % of penicillin/streptomycin to obtain a bead density of 2.5 g/L. Then, the bead suspension was incubated at 37°C for 2 hours to ensure medium oxygenation.

After cell counting, cells were added to the bead/medium mix with a density of about 10 cells/bead, and submitted to 23 cycles of 3 minutes of gentle mixing (30 - 60 rpm) followed by 45 minutes of rest in order to ensure the cell adhesion. Fetal Bovin Serum (FBS) was added 1 hour after the beginning of these cycles in order to reach a 10% FBS concentration in the medium. The microcamer beads were then subjected to a gentle mixing (30 - 60 rpm) for 2 to 3 days before utilization.

Microwell spheroid generation

Spheroids in microwells were generated using an in-house 3D printed stamp with an array of micropillars (250 — 750 per stamp) with both a diameter and height of 200 pm. First, 4 mL of 2% agarose in PBS were added into each well of a 6-well plate. Then the 3D printed micropillar stamp was immediately used to print molds directly into the agarose, and left in place for 15 minutes for the solidification.

The stamp was then removed, revealing an array of molds with the dimensions of the micropillars. The 6-well plate was sterilized by UV light exposure for 30 minutes before cell seeding (1000 cells per well) through a single centrifugation step at 1200 rpm for 3 minutes. The seeded cells in the wells were placed in an incubator at 37°C for cell growth and spheroid formation and maturation.

Spheroids were collected either after 1 or 3 days of maturation, depending on the experimental requirements.

Spheroid generation on anti-adherent flasks

T75 cell culture flasks were pre-coated with 2% agarose in PBS to prevent cell adhesion. Prepared flasks were left at 4°C for 30 minutes to ensure complete agarose gelation.

Previously confluent cells in T75 flasks were detached and seeded at a 1 :1 ratio on the agarose-coated T75 flask and left to mature in an incubator at 37°C.

Cell interactions and aggregation while in forced suspension induced spheroid formation in the flask, and the generated spheroids were collected after either 1 or 3 days of maturation under these conditions.

Extracellular vesicle production in vessels of the invention

All vessels were custom made by 3D printing (capacity : 20 to 80 mL).

The vessels were coupled to an EC 45 motor (397172, Maxon) and an ESCON 50/5 Servocontroller (409510, Maxon). The setup was operated through the ESCON Studio software provided by the vendor (operating rotation speeds in the range of 100 to 2000 rpm). The controller was powered with a DC Power source, and connected to a waveform generator in order to control the frequency of rotation.

In the performed experiments, the rotation speed was set at 400, 600, 800, 1200, or 1600 rpm; and the frequency of reversal of rotational direction was set at 0.05 and 0.1 Hz.

The samples of single cells, microcamer beads or spheroids were, after counting, thoroughly washed in serum-free DMEM without phenol red and resuspended in an appropriate working volume and placed inside corresponding vessels.

Each vessel was sealed with a vented cap and the rotation system was placed inside the incubator at 37°C for the complete duration of the experiment. Collected supernatants containing the produced extracellular vesicles were analyzed for survival and morphology.

Extracellular vesicle production in spinner flasks

Cells grown on microcarrier beads were thoroughly washed in serum-free DMEM without phenol red and placed in a 500 mL spinner flask comprising a magnetic agitator and placed inside the incubator at 37°C.

The rotating speed of the magnetic agitator in the flask for extracellular vesicle production was set at 200 rpm at a final volume of 350 mL.

Extracellular vesicle quantification, qualitative analysis and purification

Collected samples from any production method as above were centrifuged at 2000 g for 10 minutes at 4°C and resuspended in fresh serum-free DMEM without phenol red. Nanoparticle Tracking Analysis of the processed sample suspension was performed with a NanoSight NS300 (manufactured by Malvern Panalytical) or a Videodrop to obtain the number and size distribution of the produced extracellular vesicles.

The samples were then filtered (Centricon 70 100kDa, manufactured by Merck Millipore) and purified on qEV columns (qEVoriginal 170 nm Gen 2 Column, manufactured by Izon Science) before cryo-TEM observation or western blot analysis.

Cell metabolic activity analysis

The alamarBlue™ metabolic assay (DAL1100, manufactured by Invitrogen) was used to assess cell viability through metabolic activity in collected cell and spheroid samples after rotation in the vessels or spinner flasks.

Following vendor instructions, the collected samples were stained in a 1 :10 reagent dilution in complete DMEM in 96-well plates and incubated at 37°C for 2 hours before analysis with an EnSight® multimode plate reader, using a fluorescence excitation and emission wavelengths of 570 and 585 nm, respectively.

Cytotoxicity analysis

The ToxiLight™ bioassay kit (LT017-117, manufactured by Lonza) was used to quantify cell damage due to the rotation in extracellular vesicle production.

Before centrifugation and processing of the collected samples from the vessels of the invention or spinner flasks, 20 pL of the supernatant was incubated with 100 pL of the AK (adenylate kinase) detection reagent for 5 minutes at room temperature, as per the vendor’s instructions.

A positive cell death control was setup separately using a ToxiLight 100% lysis reagent set following the instructions set by the vendor. The bioluminescent signals were then quantified using an EnSight multimode plate reader.

Western blot analysis

Standard Western blot procedures with anti-CD63, CD81 and anti-CD9 antibodies were used to detect exosomal protein markers, and with anti-Alix and anti-ferritin antibodies to detect cytosolic markers. Anti-14-3-3 antibody was used for contaminant soluble proteins. Anti-CD29, -CD44, -CD49e, -CD105, and - CD146 markers were used for mesenchymal proteins.

Example 1 : EV production in a vessel having a baffle structure comprising solid baffle plates from stem cells grown on beads

Vessels having the configuration shown in Fig. 3a and 3b (vessels having a baffle structure comprising solid baffle plates) were manufactured as described above in the section “Extracellular vesicle production in vessels of the invention”.

The experimental conditions were as follows:

- Producer cells: mouse mesenchymal stem cells (mMSCs) grown on microcarrier beads

- Concentration of beads: 6000 per mL

- Capacity of the vessel: 60 mL

- Rotation duration (step b)): 3 hours

- Rotation parameters: rotation speed of 0 to 1200 rpm; rotation frequency of 0.1 Hz

The EV production was performed under the above conditions, with varying rotation speeds, and was compared between the vessels of the invention and conventional spinner flasks with magnetic agitation at the highest regime, using the same cells grown on beads at the same densities.

The spinner flasks experiments were performed as described above in the section “Extracellular vesicle production in spinner flasks".

The metabolic activity of cells was also quantified after each rotation condition, as described above in the section “Cell metabolic activity analysis".

The quantification was performed using NTA.

Fig. 7a and Fig. 7b show, on the y-axis, the number of extracellular vesicles (EVs) produced per bead and the metabolic activity, respectively, as a function of the rotation speed (in rpm) on the x-axis, using the vessel of the vessel having a baffle structure comprising solid baffle plates (white circles) and a conventional spinner flask agitated at maximum agitation speed (grey circles on the right hand-side). The black bars represent the average value calculated over a number of experiments.

The number of produced extracellular vesicles per bead using the vessel of the invention at 600 rpm was comparable with the number of extracellular vesicles produced using the conventional spinner flaks, while the working volume was significantly decreased.

The use of the vessel of the invention at 800 rpm and 1200 rpm resulted in a higher yield of extracellular vesicles compared to the conventional spinner flaks, while the working volume was significantly decreased.

In addition, after each rotation experiment, the use of the vessel of the invention showed a better cell metabolic activity than when a spinner flask was used.

As shown in Fig. 7c, the protein expression by western blot confirmed the presence of both EV surface markers CD63 and CD9 as well as Alix and Ferritin (cytosolic proteins) in the EV extract prepared using the vessel of the invention.

However, these proteins were not observed (or rarely observed) in the EV extract prepared using the spinner flask.

In both cases, the non-EV marker 14-3-3 (markers of impurities and contaminants) was not observed.

Thus, Fig. 7c proves the preserved integrity of the extracellular vesicles prepared according to the present invention, and this integrity is better preserved than when a conventional spinner flak is used. The preserved integrity of the extracellular vesicles is also observed by transmission electron microscopy, as shown in Fig. 7d, demonstrating an intact morphological structure.

Example 2: EV production in a vessel having a baffle structure comprising solid baffle plates from mMSC spheroids and tumoroids

The same vessels were used as in Example 1 .

The experimental conditions were as follows:

- Producer cells: spheroids made of mMSCs and spheroids made of A- 673 cancer cells (tumoroids)

- Spheroid diameter: 80-150 pm

- Spheroid concentration: 100-200 per mL

- Number of cells per spheroid (average) : 500 (mMSC) and 2000 (A673).

- Rotation duration (step (b)): 1 , 2 or 3 hours - Rotation parameters: rotation speed of 800 rpm and rotation frequency of 0.1 Hz

The extracellular vesicles were produced using the vessel of the invention under the above conditions from mMSC spheroids and A673 spheroids, which were prepared in agarose microwells over 3 days of maturation, as described above in section “Microwell spheroid generation

The produced extracellular vesicles (EVs) were quantified by NTA.

Fig. 8a and Fig. 8b show, on the y-axis, the number of extracellular vesicles (EVs) per spheroid produced from mouse mesenchymal stem cells (mMSC) and the number of extracellular vesicles (EVs) per spheroid produced from A673 cancer cells, respectively, as a function of the rotation time (hours) on the x-axis, using the of the invention. The bars represent average values over a number of experiments. The white bars correspond to control samples without rotation. TO corresponds to the control at time 0.

Over all, a higher EV yield was observed using A673 spheroids as producer cells than mMSC spheroids because A673 cancer cells naturally produce more extracellular vesicles.

The EV yield in the vessel of the invention increased as the rotation time increased.

Example 3a: EV production in a vessel having a baffle structure comprising solid baffle plates from mMSC spheroids formed in the vessel

The same vessels were used as in Example 1 .

The experimental conditions were as follows.

- Producer cells: mouse stem cells (mMSC)

- Spheroid diameter: 80-150 pm

- Spheroid concentration: 100-200 per mL

- Number of cells per spheroid (average) : 500 to 1000

- Rotation duration: 2 hours or 4 hours

- Rotation parameters: rotation speed of 800 rpm and rotation frequency of 0.1 Hz for EV production, and rotation speed of 80 rpm for spheroid maturation

As producer cells, mMSC cells in suspension were directly supplied in the vessel, and were subjected to rotation at 80 rpm over 3 days, so as to form spheroids directly in the vessel.

Then, the rotation speed was increased to 800 rpm to trigger the production of extracellular vesicles under rotating stimulation for 2h or 4h. Imaging analysis (by LIVE/DEAD™ Cell Imaging Kit (R37601 , Invitrogen™) of the spheroids at 2h or 4h of rotating stimulation demonstrated that the spheroids themselves did not experience massive cell death as it could be the case for cells grown on beads.

The produced EVs were quantified by NTA.

The results are shown in Table 1 :

Table 1 : Number of extracellular vesicles produced from spheroids of stem cells (mMSC), matured directly inside the vessel

The production of extracellular vesicles per spheroid was impressively high, compared with the production per bead, which is usually at maximum half a million per bead, or much lower (for mMSC).

Thus, the present invention provides an all-in-one solution for producing extracellular vesicles from therapeutic cells in a physiological 3D configuration.

Example 3b: EV production in a vessel having a baffle structure comprising solid baffle plates from hMSC spheroids formed in the vessel

The EVs were produced in the same way as in Example 3a, except that certain conditions were changed as follows :

- Producer cells: human stem cells (hMSC)

- Rotation duration: 0 to 3 hours

- Rotation parameters for EV production: Rotation speed of 0 to 1600 rpm and rotation frequency of 0.2 Hz

The produced EVs were quantified by Videodrop. The cell death was also studied, as described in the section “Cytotoxicity analysis."

The results are shown in Fig. 13a, Fig. 13b and Fig. 13c.

Fig. 13a shows the production of EVs per producer cell across varying duration periods and different rotation speeds. It is to be noted that, compared to the numbers presented in Table 1 of Example 3a, Fig. 13a expresses the number of produced EVs per producer cell (instead of per spheroid) detected by Videodrop (instead of by NTA). The number of 13,000 EVs per producer cell in this Example is equivalent to 2x10⁷ EVs of Example 3a (considering that Videodrop detects approximately 4 times fewer objects than NTA, and that each spheroid contained about 500 cells).

According to the results of Fig. 13a, the number of EVs per producer cell increased with higher rotation speeds and longer rotation durations.

Fig. 13b shows that the rotation conditions can be readily adjusted so as to ensure a low impact on cell death.

As shown in Fig. 13c, the protein expression by western blot confirmed the absence of non-EV marker 14-3-3 in the EV extracts (EV1 to EV3), showing the samples’ purity. The presence of EV markers CD63 and CD81 and of the cytosolic EV marker Synt-1 in the EV extracts (EV1 to EV3) confirmed the integrity of the EVs prepared according to the present invention, using, as producer cells, hMSC spheroids formed in the vessel, as also evidenced by the control (2D and 3D).

Taken together, Example 3a shows that the present invention provides an all-in-one solution for producing EVs from therapeutic cells in a physiological 3D configuration.

Example 4a: EV production in a vessel having a baffle structure comprising solid baffle plates from spheroids formed outside the vessel

The same vessels were used as in Example 1 .

The experimental conditions were as follows:

- Producer cells: spheroids of hMSCs (human hTERT)

- Spheroid diameter : 80-250 pm

- Spheroid concentration: 50-250 per mL

- Rotation duration: 1 or 3 hours

- Rotation parameters: rotation speed of 0 to 1600 rpm and rotation freguency of 0.05 Hz

Spheroids were prepared on 2D agarose flasks over 1 -day maturation, as described in the section “Spheroid generation on anti-adherent flasks."

The guantification was performed using NTA.

Fig. 9a and Fig. 9b show, on the y-axis, the number of extracellular vesicles (EVs) per spheroid after 1-hour rotation and the number of extracellular vesicles (EVs) per spheroid after 3-hour rotation, respectively, as a function of the rotation speed (in rpm) on the x-axis, using a vessel of the invention. The black bars represent average values over a number of experiments.

According to the results shown in Fig. 9a and Fig. 9b, the EV production showed an increase as a function of rotation speed, with up to 20 million extracellular vesicles per spheroid after 3 hours of rotation at 1600 rpm. Fig. 9c demonstrates the presence of the EV surface marker CD63 in the EV extracts according to the present invention at both 800 rpm and at 1600 rpm. The level of CD63 detected was higher at 800 rpm than at 1600 rpm. The presence of the cytosolic EV marker Synt-1 was also observed. The absence of the non-EV marker 14-3-3 (markers of impurities and contaminants) in the EV extracts was also confirmed.

Thus, the results of Fig. 9c prove the preserved integrity of the extracellular vesicles prepared according to the present invention, even at a high rotation speed, such as 1600 rpm.

Importantly, when the cell metabolic activity and the cell death were studied after a 3-hour rotation step, as described in the section “Cell metabolic activity analysis" and “Cytotoxicity analysis," a low impact on the cell metabolic activity was observed even at higher rotation speeds. A similar low impact on cell death was also observed, although the cell death rate increased at 1600 rpm. See Table 2:

Table 2: Impact of rotation speed on the cell metabolic activity and viability.

Example 4b: EV production in a vessel having a baffle structure comprising solid baffle plates from spheroids formed outside the vessel

EVs were produced in the same way as in Example 4a, except that the rotation parameters were changed as follows:

- Rotation duration: 0 to 3 hours

- Rotation parameters: rotation speed of 0 to 1600 rpm and rotation freguency of 0.2 Hz

The produced extracellular vesicles were guantified by Videodrop.

The results are shown in Fig. 9d and Fig. 9e. The number of 20,000 EVs per producer cell in this Example is eguivalent to 2x10⁷ EVs of Example 4a (considering that Videodrop detects approximately 4 times less objects than NTA, and that each spheroid contained 200-300 cells).

These results show that the number of EVs per producer cell increased with higher rotation speeds and longer rotation durations (Fig. 9d), and that the rotation conditions can be readily adjusted so as to ensure a low impact on cell death (Fig. 9e).

Example 5a: EV production in a vessel having a baffle structure comprising solid baffle plates from single (individualized) cells

The same vessels were used as in Example 1 .

The experimental conditions were as follows.

- Producer cells: mouse mesenchymal stem cells (mMSC) and human mesenchymal stem cells (hMSC)

- Rotation duration : 3 hours

- Rotation parameters: rotation speed of 800 rpm and rotation freguency of 0.1 Hz

Mouse and human mesenchymal stem cells were detached and resuspended at 150 to 350,000 cells/mL and subjected to rotation under the above conditions.

The guantification was performed using NTA.

Fig. 10a shows the average number of extracellular vesicles (EVs) per cell from single (individualized) cells, using the vessel of the invention. The y-axis represents the number of extracellular vesicles per cell, and the x-axis represents the rotation speed in rpm. The two bars on the left correspond to the extracellular vesicles produced from mouse mesenchymal stem cells (mMSC) and the two bars on the right correspond to the extracellular vesicles produced from human mesenchymal stem cells (hMSC).

The results indicate that present invention provides increased EV yield even in the simplest, single-cell level configuration.

Example 5b: EV production in a vessel having a baffle structure comprising solid baffle plates from single (individualized) cells

EVs were produced in the same way as in Example 5a, using individualized hMSC cells as producer cells, except that the rotation parameters were changed as follows:

- Rotation duration: 0 to 3 hours

- Rotation parameters: rotation speed of 0 to 2000 rpm and rotation freguency of 0.2 Hz The produced extracellular vesicles were quantified by Videodrop.

The results are shown in Fig. 10b and Fig. 10c.

Fig. 10b illustrates that the number of EVs per producer cell increased with higher rotation speeds and longer rotation durations, although the number of EVs remained similar beyond the rotation speed of 800 rpm.

Fig. 10c demonstrates that the rotation conditions can be readily adjusted so as to ensure a low impact on cell death.

The EVs produced as above at 600 rpm and at 0.5 Hz for 2 hours were subjected to a western blot. The results are shown in in Fig. 10c.

Fig. 10c confirmed the absence of non-EV marker 14-3-3 in the EV extracts (EV), showing the sample’ purity. The presence of EV markers CD63 and CD81 and of the cytosolic EV marker Synt-1 (see higher exposition) in the EV extracts (EV) confirmed the integrity of the EVs prepared according to the present invention, using as producer cells individualized hMSCs.

Example 6: EV production in a vessel having a baffle structure comprising meshed baffle plates from spheroids

Vessels having the configuration shown in Fig. 4a to 4c (vessels having a baffle structure comprising meshed baffle plates) were manufactured as described above in the section “Extracellular vesicle production in vessels of the invention”.

The experimental conditions were as follows.

- Producer cells: spheroids of hMSCs

- Spheroid diameters: 80-250 pm

- Spheroid concentration: 100-200 per mL

- Capacity of the vessel: 60 mL

- Rotation duration: 1 or 3 hours

- Rotation parameters: rotation speed of 0 to 1600 rpm and rotation frequency of 0.05 Hz.

Fig. 11a and Fig. 11b show, on the y-axis, the number of extracellular vesicles (EVs) per spheroid after a 1 -hour rotation and the number of extracellular vesicles (EVs) per spheroid after a 3-hour rotation, respectively, as a function of the rotation speed (in rpm) on the x-axis. The black bars represent the average numbers of produced extracellular vesicles. The EV production showed a proportional increase with the rotation speed, showing a similar efficiency as with the vessel having a baffle structure comprising solid baffle plates.

The cell metabolic activity and the cell death were studied after the 3-hour rotation, as described in the section “Cell metabolic activity analysis" and “Cytotoxicity analysis," and demonstrated that there was a low impact on the cell metabolic activity and the cell viability as the rotation speed increased, although there was a slight increase of cell death as the rotation duration and rotation speed increased, like in Example 4 (data not shown).

Example 7: EV production in a vessel having a baffle structure comprising meshed baffle plates from spheroids in multi steps

The same vessels were used as in Example 6.

The experimental conditions were as follows:

- Producer cells: spheroids of hMSCs

- Spheroid diameters: 80-250 pm

- Spheroid concentration: 140±60 spheroids/mL

- Number of cells per spheroid (average) : 500 to 2000

- Capacity of the vessel: 60 mL

- Rotation and rest duration : 4 cycles of 1-hour rotation, and 30 minutes between each cycle (120 rpm)

- Rotation parameters: rotation speed of 800, 1200 or 1600 rpm and rotation freguency of 0.05 Hz

The guantification of EVs was performed using NTA.

The extracellular vesicles were produced under the above conditions. After the 1 -hour rotation, collected samples were centrifuged at 1200 g for 3 minutes at 4°C and the spheroids precipitate was immediately resuspended in fresh serum- free DMEM without phenol red in the vessel and placed in the incubator at 37°C for 30 min, under rotation, followed by the subseguent cycle of rotation. This was repeated four times.

The number of the produced extracellular vesicles at each cycle is shown in Fig. 12a.

Fig. 12a shows, on the y-axis, the number of extracellular vesicles (EVs) produced per spheroid as a function of the number of rotation cycles performed on the x-axis. The black circles represent the rotation at 800 rpm; the white circles represent the rotation at 1200 rpm; and the squares represent the rotation at 1600 rpm. The same depiction is also used in Fig. 12b and 12c.

Fig. 12a shows that, as more cycles were repeated, the EV production increased. The increase in the number of extracellular vesicles was more pronounced at a higher rotation speed.

Furthermore, the cell metabolic activity and the cell death were measured after each cycle, and are shown in Fig. 12b and Fig. 12c.

Fig. 12b and Fig. 12c show, on the y-axis, the metabolic activity and the cell death of cells, respectively, as a function of the number of rotation cycles performed on the x-axis.

Fig. 12b shows that there was a low impact on the metabolic activity at 800, 1200, and 1600 rpm, although the metabolic activity at 1600 rpm was slightly lower than at 800 and 1200 rpm.

Furthermore, the metabolic activity remained constant even as the number of cycles increased, indicating that the repeating the EV production from the same producer cells did not have a significant effect on the metabolic activity of the producer cells.

Fig. 13c shows that there was a low impact on the cell viability at 800, 1200, and 1600 rpm, although the cell death at 1600 rpm was slightly higher than at 800 and 1200 rpm.

The cell death remained relatively constant even as the number of cycles increased, indicating that the repeating the EV production from the same producer cells did not have a significant effect on the cell viability of the producer cells.

Example 8: Quality of EVs produced according to the invention

EVs were produced either from individualized hMSCs or hMSC spheroids formed in the vessel.

The EV production from hMSC spheroids formed in the vessel was performed in the same way as in Example 4a except that the rotation speed, the rotation frequency and the rotation duration were fixed to 600 rpm, 0.2 Hz, 2 hours respectively.

The EV production from individualized hMSCs was performed in the same way as in Example 5a except that the rotation speed, the rotation frequency and the rotation duration were fixed to 600 rpm, 0.2 Hz, 2 hours respectively.

As a control, EV production was also performed using the conventional method by way of 2D or 3D starvation. The quality of EVs thus produced was determined using the MACSPlex Exosome kit (Miltenyi Biotec, Auburn, CA) following the manufacturer's protocol.

The EVs mixed with specific antibody-coated beads were analyzed using a MACSQuant cytometer (Miltenyi Biotec) with MACSQuantify software.

Results are shown in Fig. 14a and Fig. 14b. The results indicate that the EVs produced according to the invention (either from individualized hMSCs (A), or from hMSC spheroids fired in the vessel (C1 , C2 and C3, corresponding to three independent production lines) have a comparable protein expression compared to the control (2D and 3D, corresponding to EVs produced by 2D starvation and by 3D starvation, respectively).

More specifically, for exosome-specific markers, markers CD63 and CD81 were present on the produced EVs produced according to the invention (A and C1 to C3), as observed in the classic starvation production conditions (2D and 3D). Marker CD9 was not present, as this marker is not expressed by hMSCs.

Regarding mesenchymal markers CD29, CD44, CD49e, CD105, and CD146, they were also well represented in EVs produced according to the invention.

Example 9: Neo-anqioqenic potential of EVs produced according to the invention The neo-angiogenic potential of EVs produced according to the invention was studied using human umbilical vein endothelial cells (HUVECs).

It is known that the treatment of HUVEC spheroids with a vascular endothelial growth factor (VEGF) or serum proteins such as FBS induces sprout formation.

HUVECs were cultured as 3D spheroids in agarose wells, as described in the section “ Microwell spheroid generation" , and were incorporated into a collagen matrix.

EVs were produced in the same way as in Example 3b (from hMSC spheroids formed inside the vessel), in Example 4 (from hMSC spheroids formed outside the vessel), in Example 5 (from individualized hMSC cell), except that the rotation parameters were as follows:

- Rotation duration : 2 hours

- Rotation speed: 600 rpm

- Rotation frequency: 0.2 Hz

As shown in Fig. 15a and in Fig. 15b, the endothelial spheroids were treated with EVs to observe sprout formation under different conditions, including 0% FBS (negative control); 10% FBS and 1 or 2 doses of VEGF (positive control); 1 or 2 doses of EVs produced by 2D or 3D starvation (control as a comparative example, “2D” and “3D”, respectively), and 1 , 2 or 4 doses of EVs produced according to the invention as above: EVs produced from individualized hMSC cell (A), EVs produced from hMSC spheroids formed outside the vessel (B), EVs produced from hMSC spheroids formed inside the vessel (C).

Fig. 15a shows that, in the presence of EVs produced according to the invention (A, B and C in different doses), comparable or even more prominent sprout formation was observed, compared to the treatment with 10% FBS or 2X/4X VEGF as well as the treatment with EVs produced by the conventional starvation (2D 1X or 2X).

Fig. 15b shows the number of the sprout measured by a conventional image analysis software. The results show that EVs produced according to the invention are, in all conditions (A, B and C in 1 , 2 and 4 doses), more efficient for the neo-vascularization than FBS or VEGF. In some cases, especially the EVs produced from hMSC spheroids formed outside the vessel (B) and inside the vessel (C) exhibited more sprout formation than with the EVs prepared by the conventional 2D or 3D starvation method (2D and 3D).

Taken together, these results show the potential of EVs produced according to the invention for neo-angiogenesis.

Example 10: Wound healing activity of EVs produced according to the invention

The wound hearing activity of EVs produced according to the invention was studied using Telomerized human fibroblasts (fHDF/TERT166) (HSFs).

EVs were produced in the same way as in Example 9: EVs from individualized hMSC cell (A), EVs from hMSC spheroids formed outside the vessel (B), EVs from hMSC spheroids formed inside the vessel (C).

HSFs were seeded into 96-well plates. A physical gap (scratch) was created within the monolayer, and the medium was replaced with a fresh media supplemented with the EVs produced as above. The closure of the gap by cell migration was monitored and guantified over time, using a conventional image software.

Overall, EVs produced according to the invention (A, B, C, see Fig. 16c) enhanced wound healing process. Specifically, EVs produced according to the invention (A, B, C) exhibited a significantly improved effect compared to the control (HSFs treated with FBS 1%, FBS 2%, and FBS 4%, see Fig. 16a) and a slightly improved effect compared to another control (HSFs treated with EVs prepared by the conventional 2D or 3D starvation method, 2D and 3D, see Fig. 16b). In addition, they exhibited a comparable effect with respect to the treatment with FBS 10% (Fig. 16a). Example 11 : Anti-inflammatory activity of EVs produced according to the invention The anti-inflammatory activity of EVs produced according to the invention was studied using a conventional nitric oxide (NO) measurement assay.

It is known that the treatment of mouse macrophage cells (RAW264.7) with lipopolysaccharide (LPS) induces formation of nitric oxide (NO), indicating an inflammatory reaction.

The mouse macrophage cells were treated with lipopolysaccharide (LPS), and then treated the EVs produced as above.

The results are shown in Fig. 17.

EVs produced according to the invention (A, B, C) significantly reduced NO formation in a dose-dependent manner, compared to the positive control (CTL +). Specifically, the EVs produced according to the invention at a dosage of 2X showed a comparable anti-inflammatory effect with respect to the treatment with a known anti-inflammatory inhibitor.

These results demonstrate that the EVs prepared according to the invention exhibit an anti-inflammatory activity.

Example 12: EV production in a vessel not in accordance with the invention

A vessel having the configuration shown in Fig. 18a and 18b was manufactured by 3D printing.

The vessel had a surface structure having 638 wells spaced approximately 1 .4 mm apart (density of about 0.65 wells/mm²). The dimensions of each well were as follows:

- Depth: 1 .25 mm

- Diameter: Entrance diameter of 0.53 mm and bottom diameter of 0.27 mm, thus the entrance having a slight flare on its surface.

- Angle: 42.5° relative to the axis of the tube.

Thus, the vessel did not have a baffle structure which deviates the flow of liquid within the vessel, in accordance with the invention, but had microwells on its internal surface for receiving cells.

The experimental conditions were as follows:

- Producer cells: hMSC

- Spheroid diameter: 80-150 pm

- Spheroid concentration: 100-200 per mL - Rotation duration: 0, 3, 6 and 24 hours for EV production and 48 hours for maturation

- Rotation parameters: rotation speed of 200 rpm and rotation frequency of 0.2 Hz for EV production, and rotation speed of 60 rpm for spheroid maturation

One million cells were seeded into the vessel and subjected to rotation at a speed of 200 rpm for 1 hour. The rotation speed was subsequently decreased to 60 rpm for a 48-hour maturation period, during which spheroids were formed in the wells of the tubes.

Following the maturation period, the production of EVs started at 200 rpm for a duration of 24 hours.

The results are shown in Fig. 18c.

The number of EVs produced per spheroid and per producer cell ranged from 1 to 2x10⁵ EVs per spheroid and 50 to 150 EVs per cell, respectively.

This represents a hundredfold reduction compared to the production yield observed in the production of EVs according to the invention (see for example Examples 3a and 3b).

This drastic reduction is believed to be due to the negligible stress experienced by the spheroids at the bottom of the well (where they are seeded and remain), which is insufficient to trigger a high-yield production of EVs achieved by the vessel of the invention.

These results indicate that the EV production using the vessel having a baffle structure of the invention provides a significantly improved yield compared to the vessel without such a baffle structure.

Claims

1. A method of producing extracellular vesicles from producer cells, comprising the steps of : a) placing producer cells in a liquid medium in a vessel comprising a baffle structure fixed inside the vessel; b) rotating the vessel so as to generate extracellular vesicles from the producer cells; and c) collecting the generated extracellular vesicles; wherein the step b) of rotating the vessel comprises repeatedly changing the rotational motion of the vessel.

2. The method of claim 1 or 2, wherein the vessel rotates around a rotation axis which is substantially vertically oriented.

3. The method of any one of claims 1 to 2, wherein the extracellular vesicles are generated from producer cells in the form of spheroids and/or organoids.

4. The method of claim 3, wherein:

- the method further comprises a preliminary step of growing spheroids and/or organoids outside of the vessel, and step a) of placing the producer cells in the vessel comprises supplying the grown spheroids and/or organoids to the vessel; or

- step a) of placing the producer cells in the vessel comprises supplying individualized cells to the vessel, and the method further comprising an intermediate step of generating spheroids and/or organoids from the individualized cells.

5. The method of any one of claims 1 to 4, wherein the step b) of rotating the vessel comprises periodically changing the rotational motion of the vessel, preferably step b) comprises repeatedly reversing the rotational direction of the vessel, repeatedly changing the rotational speed of the vessel, and/or intermittently rotating the vessel.

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

7. The method of any one of claims 1 to 6, wherein the step b) of rotating the vessel is carried out at a maximum rotational speed of 50 to 6000 rpm, preferably 600 to 1600 rpm.

8. The method of any one of claims 1 to 7, further comprising repeating cycles of at least steps b) and c), using the same producer cells.

9. A vessel (1 ) for producing extracellular vesicles from producer cells, wherein the vessel comprises a baffle structure (2) fixed inside the vessel, and a coupling (3) on an external surface of the vessel configured to be coupled to a rotating apparatus.

10. The vessel of claim 9, wherein the baffle structure (2) comprises one or more baffles (4) fixed to an internal surface of the vessel.

11. The vessel of claim 9 or 10, wherein the vessel (1 ) comprises a cylindrical inner wall (8) and a central axis.

12. The vessel of claim 11 , wherein the baffle structure (2) comprises a plurality of baffles (4) extending from the cylindrical inner wall (8) towards the central axis, and/or a plurality of baffles (4) extending from the central axis towards the cylindrical inner wall (8).

13. The vessel of any one of claims 10 to 12, wherein part or all of the baffles (4) are solid plates, or are meshed or perforated plates.

14. The vessel of any one of claims 10 to 12, wherein the baffle structure (2) comprises a plurality of plates (4’) fixed on struts (6), the struts (6) being preferably oriented parallel to the central axis of the vessel, wherein, preferably, differently oriented plates (4’) alternate along the struts (6).

15. A system for producing extracellular vesicles from producer cells, comprising a vessel according to one of claims 10 to 14, and a rotating apparatus, the vessel being configured to be rotationally fixed to the rotating apparatus by keying the coupling of the vessel to a corresponding coupling on the rotating apparatus.