High throughput production of extracellular vesicles in fluidic chip

Abstract

The present invention relates to a method for producing extracellular vesicles from producer cells in a fluidic chip, which comprises at least one first channel and at least one second channel, each channel having a longitudinal axis, an inlet and an outlet, wherein the outlet of the first channel is connected to the inlet of the second channel in a connection area, wherein the longitudinal axis of the first channel forms a non-zero angle with the longitudinal axis of the second channel in the connection area, the method comprising: passing a flow of liquid medium containing producer cells through the first channel, from the inlet to the outlet thereof; passing the flow of liquid medium through the second channel, from the inlet to the outlet thereof, thereby generating in the second channel a swirling flow extending from the inlet of the second channel, and collecting extracellular vesicles generated from the producer cells.

Description

[0001] HIGH THROUGHPUT PRODUCTION OF EXTRACELLULAR VESICLES IN FLUIDIC CHIP

[0002] TECHNICAL FIELD

[0003] The present invention relates to a method of producing extracellular vesicles from producer cells, and to a device for performing the method.

[0004] TECHNICAL BACKGROUND

[0005] 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. EVs 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, EVs 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.

[0006] For EVs to be used for clinical applications, 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 EVs from a 3D configuration, i.e., physiologically relevant, organoid-like settings.

[0007] Some methods for producing an increased yield of EVs, using a spinner flask bioreactor used for 3D cell culture, have been developed.

[0008] Document W02020 / 136362 discloses a fluidic system for producing extracellular vesicles from producer cells in suspension, comprising at least one container, a liquid medium contained in the container, producer cells in suspension, a liquid medium stirrer, means for controlling the speed of the stirrer suitable for the growth of the producer cells into suspension, characterized in that the means for controlling the speed of the stirrer, the stirrer and the shape and dimensions of the container are suitable for generating a turbulent flow of the liquid medium in the container for exerting shear stresses on the producer cells in order to carry out the production of extracellular vesicles, the Kolmogorov length of the flow being less than or equal to 50 pm. Document W02020 / 136361 discloses a fluidic system for loading a therapeutic or imaging agent into the lumen of extracellular vesicles from producer cells, comprising at least one container, a liquid medium contained in the container, producer cells, a liquid medium stirrer and means for controlling the speed of the stirrer suitable for the growth of the producer cells, wherein the shape of the stirrer and dimensions of the container are suitable for generating a turbulent flow of the liquid medium in the container for exerting shear stresses on the producer cells.

[0009] Document WO2019 / 002608 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.

[0010] In these documents, a stirrer or an agitator (also called as impeller) induces a turbulent flow to trigger the EV production. However, in these documents, the system is simply a repurposed commercial bioreactor, which raises several concerns. For example, 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. Moreover, a large production reactor, typically a reactor of 1 L, is employed, which requires a large number of cells (e.g., 100 x 106cells). This is not feasible to obtain EVs under physiological conditions in personalized medicine.

[0011] Several technologies for the analysis of EVs in a microfluidic device are also known.

[0012] For example, Jeong MH, et al., Adv Mater Technol. 2023 Apr. 6;8(7):2201412, proposes a non-contact microfluidic technique for measuring the stiffness of large EVs, in which stiffness distributions of large EVs derived from glioblastoma cell lines are compared to distinguish between EV populations.

[0013] However, none of them proposes EV production, let alone EV production from producer cells in a 3D configuration.

[0014] Max Piffoux et al., Adv. Biosys. 2017, 1 , 1700044, reviews different methods of EV production, and explains that EVs are produced by mechanical stress in a microfluidic chip mimicking vessel shear stress.

[0015] Hao R, et al., Mater Today Bio. 2022 Dec. 24; 18: 100527, proposes a microfluidic device for boosting EV secretion from mesenchymal stem cells derived from human fetal bone marrow. Nevertheless, the EV production in these microfluidic devices is deemed insufficient when evaluating the overall yield.

[0016] Thus, there is a need for a more efficient method for producing extracellular vesicles from minimal cell materials (a 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.

[0017] SUMMARY OF THE INVENTION

[0018] The present invention relates to the following items.

[0019] Item 1 . A method for producing extracellular vesicles from producer cells in a fluidic chip, wherein the fluidic chip comprises at least one first channel and at least one second channel, each channel having a longitudinal axis, an inlet and an outlet, wherein the outlet of the first channel is connected to the inlet of the second channel in a connection area, wherein the longitudinal axis of the first channel forms a non-zero angle with the longitudinal axis of the second channel in the connection area, the method comprising: a) passing a flow of liquid medium containing producer cells through the first channel, from the inlet to the outlet thereof; b) passing the flow of liquid medium through the second channel, from the inlet to the outlet thereof, thereby generating in the second channel a swirling flow extending from the inlet of the second channel, and c) collecting extracellular vesicles generated from the producer cells.

[0020] Item 1 a. A method for producing extracellular vesicles from producer cells in a fluidic chip, wherein the fluidic chip comprises at least one first channel and at least one second channel, each channel having a longitudinal axis, an inlet and an outlet, wherein the outlet of the first channel is connected to the inlet of the second channel in a connection area, wherein the longitudinal axis of the first channel forms a non-zero angle with the longitudinal axis of the second channel in the connection area, the method comprising: a) passing a flow of liquid medium containing producer cells through the first channel, from the inlet to the outlet thereof; b) passing the flow of liquid medium through the second channel, from the inlet to the outlet thereof, the flow of liquid medium at the inlet of the second channel being characterized by a Reynolds number of greater than 40, and c) collecting extracellular vesicles generated from the producer cells.

[0021] Item 2. The method according to Item 1 or 1 a, wherein the longitudinal axis of the first channel is substantially perpendicular to the longitudinal axis of the second channel in the connection area. Item 3. The method according to any one of Items 1 to 2, wherein the flow of liquid medium at the inlet of the second channel is characterized by a Reynolds number of greater than 40, or of greater than 50, or of greater than 60, or of greater than 70, or of greater than 80, or of greater than 90, of greater than 100, or of greater than 150, or of greater than 200, or of greater than 250, or of greater than 300, or of greater than 400.

[0022] Item 4. The method according to any one of Items 1 to 3, wherein the flow rate in the first channel is 5 mL / min or more, preferably 10 mL / min or more, more preferably 15 mL / min or more, and even more preferably 30 mL / min or more.

[0023] Item 5. The method according to any one of Items 1 to 4, wherein the fluidic chip comprises two second channels, the outlet of the first channel being connected to the inlets of the two second channels in the connection area, the longitudinal axes of the two second channels being preferably aligned in the connection area.

[0024] Item 6. The method according to any one of Items 1 to 5, wherein the fluidic chip comprises two first channels, the outlets of the first channels being connected to the inlet(s) of the second channel(s) in the connection area, the longitudinal axes of the two first channels being preferably aligned in the connection area.

[0025] Item 7. The method according to Item 6, wherein the liquid medium travels substantially at the same flow rate in the two first channels.

[0026] Item 8. The method according to any one of Items 1 to 7, wherein the extracellular vesicles are generated from producer cells in the form of spheroids and / or organoids.

[0027] Item 9. The method according to any one of Items 1 to 8, wherein at least steps a) and b) are performed in a closed system and the liquid medium is not exposed to an external environment.

[0028] Item 10. The method according to any one of Items 1 to 9, comprising repeating cycles of steps a) and b), using the same producer cells.

[0029] Item 11. The method according to Item 10, comprising reversing the direction of the flow of liquid medium, preferably comprising successively:

[0030] - flowing liquid medium through the first channel(s), from the inlet(s) thereof to the connection area, and then through the second channel(s), from the connection area to the outlet(s) thereof, thereby generating a swirling flow extending from the inlet the or each second channel; and

[0031] - flowing liquid medium through the second channel(s), from the outlet(s) thereof to the connection area, and then through the first channel(s), from the connection area to the inlet(s) thereof, thereby generating a swirling flow extending from the outlet the or each first channel.

[0032] Item 12. The method according to any one of Items 1 to 11 , wherein the duration of the method is from 1 to 5 hours, more preferably from 1 to 3 hours.

[0033] Item 13. The method according to any one of Items 1 to 12, wherein the pressure in the first and second channels is controlled in at least one of the steps a) and b).

[0034] Item 14. The method according to any one of Items 1 to 13, wherein the fluidic chip comprises a single first channel and two second channels, the outlet of the first channel being connected to the inlets of the two second channels in the connection area, the longitudinal axes of the two second channels being preferably aligned in the connection area.

[0035] Item 15. The method according to any one of Items 1 to 14, wherein the fluidic chip comprises two first channels, the outlets of the first channels being connected to the inlet(s) of the second channel(s) in the connection area, the longitudinal axes of the two first channels being aligned in the connection area.

[0036] Item 16. The method according to any one of Items 1 to 15, wherein the fluidic chip comprises two first channels and two second channels, wherein the outlets of the first channels are connected to the inlets of the second channels in the connection area, and wherein, preferably: the longitudinal axes of the first channels are aligned in the connection area; and / or the longitudinal axes of the second channels are aligned in the connection area; and / or the longitudinal axis of each first channel is substantially perpendicular to the longitudinal axis of each second channel, in the connection area.

[0037] Item 17. The method according to any one of Items 1 to 16, comprising repeatedly circulating the same liquid medium through the first channel(s), from the inlet(s) thereof to the connection area, and then through the second channel(s), from the connection area to the outlet(s) thereof, thereby generating a swirling flow extending from the inlet of the or each second channel.

[0038] Item 18. The method according to any one of Items 1 to 17, comprising, successively:

[0039] - flowing liquid medium through the first channel(s), from the inlet(s) thereof to the connection area, and then through the second channel(s), from the connection area to the outlet(s) thereof, thereby generating a swirling flow extending from the inlet of the or each second channel;

[0040] - flowing liquid medium through the second channel(s), from the outlet(s) thereof to the connection area, and then through the first channel(s), from the connection area to the inlet(s) thereof, thereby generating a swirling flow extending from the outlet of the or each first channel.

[0041] Item 19. The method according to any one of Items 1 to 18, wherein the fluidic chip is a microfluidic or millifluidic chip.

[0042] Item 20. The method according to any one of Items 1 to 19, wherein the fluidic chip is made of cyclic olefin copolymer or polydimethylsiloxane.

[0043] Item 21. The method according to any one of Items 1 to 20, wherein the method is carried out in a device comprising the fluidic chip and a flow controller system connected to the fluidic chip and configured for circulating a same liquid medium multiple times through the first channel and second channel.

[0044] Item 22. The method according to Item 21 , wherein the device is a closed system.

[0045] Item 23. The method according to Item 21 or 22, wherein the flow controller system comprises at least one reservoir and at least one pump.

[0046] Item 24. The method according to any one of Items 21 to 23, wherein the flow controller system comprises at least two reservoirs, one reservoir being connected to the inlet of the or each first channel, and another reservoir being connected to the outlet of the or each second channel.

[0047] Item 25. The method according to Item 23 or 24, wherein the or each reservoir is a syringe pump.

[0048] Item 26. The method according to any one of Items 21 to 25, wherein the device further comprises a pressure controller.

[0049] Item 27. A device for producing extracellular vesicles from producer cells, comprising:

[0050] - a fluidic chip comprising at least one first channel and at least one second channel, each channel having a longitudinal axis, an inlet and an outlet, wherein the outlet of the first channel is connected to the inlet of the second channel in a connection area, wherein the longitudinal axis of the first channel forms a non-zero angle with the longitudinal axis of the second channel in the connection area, and

[0051] - a flow controller system connected to the fluidic chip and configured for circulating a same liquid medium multiple times through the first channel and second channel.

[0052] Item 28. The device according to Item 27, wherein the longitudinal axis of the first channel is substantially perpendicular to the longitudinal axis of the second channel in the connection area.

[0053] Item 29. The device according to Item 27 or 28, wherein the device is a closed system. Item 30. The device according to any one of Items 27 to 29, wherein the fluidic chip comprises a single first channel and two second channels, the outlet of the first channel being connected to the inlets of the two second channels in the connection area, the longitudinal axes of the two second channels being preferably aligned in the connection area.

[0054] Item 31 . The device according to any one of Items 27 to 30, wherein the fluidic chip comprises two first channels, the outlets of the first channels being connected to the inlet(s) of the second channel(s) in the connection area, the longitudinal axes of the two first channels being preferably aligned in the connection area.

[0055] Item 32. The device according to any one of Items 27 to 31 , wherein the fluidic chip comprises two first channels and two second channels, wherein the outlets of the first channels are connected to the inlets of the second channels in the connection area, and wherein, preferably: the longitudinal axes of the first channels are aligned in the connection area; and / or the longitudinal axes of the second channels are aligned in the connection area; and / or the longitudinal axis of each first channel is substantially perpendicular to the longitudinal axis of each second channel, in the connection area.

[0056] Item 33. The device according to any one of Items 27 to 32, wherein the flow controller system is configured for repeatedly circulating the same liquid medium through the first channel(s), from the inlet(s) thereof to the connection area, and then through the second channel(s), from the connection area to the outlet(s) thereof, thereby generating a swirling flow extending from the inlet of the or each second channel.

[0057] Item 34. The device according to any one of Items 27 to 33, wherein the flow controller system is configured for, successively:

[0058] - o flowing liquid medium through the first channel(s), from the inlet(s) thereof to the connection area, and then through the second channel(s), from the connection area to the outlet(s) thereof, thereby generating a swirling flow extending from the inlet the or each second channel;

[0059] - o flowing liquid medium through the second channel(s), from the outlet(s) thereof to the connection area, and then through the first channel(s), from the connection area to the inlet(s) thereof, thereby generating a swirling flow extending from the outlet the or each first channel.

[0060] Item 35. The device according to any one of Items 27 to 34, wherein the flow controller system comprises at least one reservoir and at least one pump. Item 36. The device according to any one of Items 27 to 35, wherein the flow controller system comprises at least two reservoirs, one reservoir being connected to the inlet of the or each first channel, and another reservoir being connected to the outlet of the or each second channel.

[0061] Item 37. The device according to Item 35 or 36, wherein the or each reservoir is a syringe pump.

[0062] Item 38. The device according to any one of Items 27 to 37, further comprising a pressure controller.

[0063] Item 39. The device according to any one of Items 27 to 38, wherein the fluidic chip is a microfluidic or millifluidic chip.

[0064] Item 40. The device according to any one of Items 27 to 39, wherein the fluidic chip is made of cyclic olefin copolymer or polydimethylsiloxane.

[0065] Item 41 . The device according to any one of Items 27 to 40, which is configured for implementing the method of any one of Items 1 to 26.

[0066] Item 42. A method for producing extracellular vesicles from producer cells in a fluidic chip of the device according any one of Items 27 to 40, the method comprising: a) passing a flow of liquid medium containing producer cells through the first channel of the device, from the inlet to the outlet thereof; b) passing the flow of liquid medium through the second channel of the device, from the inlet to the outlet thereof, and c) collecting extracellular vesicles generated from the producer cells.

[0067] Item 43. The method of Item 42, wherein the method further comprises the additional features explicitly recited in any one of Items 2 to 26.

[0068] 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 device for performing the method.

[0069] The present inventors have discovered that the swirling flow generated in the fluidic chip as above can efficiently trigger the production of extracellular vesicles (EVs). In addition, the compact size of the fluidic chip, combined with its high yield of EVs produced per cell, allows for the generation of EVs with significantly small amounts of cellular material. This capacity distinguishes the invention from other existing EV production technologies on the market. Moreover, as the cells are believed to be trapped in the swirling flow and thus effectively subjected to high hydrodynamic stresses in the swirling flow, almost instantaneous EV production becomes possible with a brief exposure of cellular material to such stresses, greatly limiting the potential damage to the cells caused by the swirling flow.

[0070] In particular, the present invention may provide one or more of the following advantages:

[0071] - a comparable or even higher EV yield can be obtained compared with conventional repurposed bioreactors or microfluidic devices for EV production,

[0072] - a low-volume regime (working volume in the range of 0.5 to 10 mL) is possible, which is perfectly in line with the number of cells per unit associated with personalized medicine applications (for example, EVs can be produced from rare and fragile cell materials, such as patient- derived cells),

[0073] - precise control and continuous monitoring of EV production is possible through a live visualization of the method, leading to more robust methodology in terms of EV yield per cell,

[0074] - the device of the invention can be, compared to other EV production devices, produced at lower cost, making it affordable for a broader range of users,

[0075] - EVs can be produced from various cellular materials, including individual cells and cells in a 3D configuration such as organoids / spheroids, and

[0076] - mass production in both industrial and academic settings is also possible because the volume of the cellular material per fluidic chip can be easily increased as well as because parallelization can be easily achieved.

[0077] BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1A shows one example configuration of the first and second channels of the fluidic chip of the invention. Blank arrows indicate the direction of the flow of liquid medium. It is noted that the directions indicated by the blank arrows can be reversed.

[0079] Figure 1 B shows one example of the device of the invention.

[0080] Figure 2 shows ultrafast camera images illustrating the projected velocities of spheroids in the channels (longitudinal sections) of the fluidic chip of Figure 1 B at various Reynolds numbers (Re). The grayscale on the right indicates the projected particle velocity (mm / s). Figure 2A and Figure 2B correspond to the first channel and second channel, respectively (the liquid flowing from the first channel to the second channel). The numbers on the vertical axis indicate the position of the channels, with the number 6 corresponding to the cross junction. The grayscale on the right corresponds to the projected particle velocity in mm.s’1. Figure 2C corresponds to the second channel, showing the projected particle velocity of a single cell (at Re = 104.2). The numbers on the vertical axis indicate the position of the channels, with the number 6 corresponding to the cross junction. The grayscale on the right corresponds to the projected particle velocity in mm. s’1.

[0081] Figures 3A to 3C shows cryogenic transmission electron microscopy (cryoTEM) images of EVs. Figure 3A corresponds to EVs produced from THP-1 cells using the device of the present invention (Re = 104), as in Example 1 below; Figure 3B and Figure 3C correspond to EVs produced from hMSC spheroids using the device of the present invention at Re = 312.5 and at 416.7, respectively, as in Example 2B.

[0082] Figure 4 shows the number of EVs produced from hMSC spheroids, using the device of the invention, as tested in Example 2B below. The x-axis represents the Reynolds number (Re), and the y-axis represents the number of EVs per hMSC cell.

[0083] Figure 5 shows the number of EVs produced from hMSC spheroids, using the device of the invention (plot B) and a single-channel chip (plot C), as tested in Example 3 below. The plot A corresponds to the control in which EV production was induced by starvation. The three asterisks (***) represent a significant difference of p < 0.01 .

[0084] Figure 6 shows the cell integrity of the hMSC spheroids after EV production as tested in Example 2B. The x-axis represents the Reynolds number (Re), and the y-axis represents the percentage of total cells that are part of an intact spheroid.

[0085] Figure 7 shows the cell viability of the hMSC spheroids after the stimulation of EV production under different conditions, as tested in Example 2B below. The x-axis represents the Reynolds number (Re), and the y-axis represents and the y-axis represents the average cell viability (in %) of the hMSC spheroids.

[0086] Figures 8A, 8B and 8C show the expression of proteins of EVs, as tested in Example 5. Figures 8A and 8B shows the results of flow cytometry analysis, in which the x-axis represents protein markers detected, and the y-axis represents the expression normalized relative to the expression level of immunoglobulin (Ig); A1 and A2 correspond to the control (EVs produced by 2D starvation and 3D starvation, respectively); and B1 and B2 correspond to EVs produced by the device of the invention (at Re = 312.5 and at 416.7, respectively). Markers CD9 / CD63 / CD81 correspond to EV-specific membrane proteins (Fig. 8A) and markers CD29 / CD44 / CD49e / CD105 / CD146 correspond to proteins specific to the mesenchymal nature of EVs (Fig. 8B). Figures 8C shows the results of a western blot analysis, in which “1” corresponds to EVs produced by the device of the invention (Re = 208.3), “2” and “3” correspond to the control (EVs produced by 2D starvation and by 3D starvation, respectively). Markers CD63 / CD81 correspond to EV-specific membrane proteins; marker SDCBP corresponds to EV-specific cytosolic protein; and marker 14-3-3 corresponds to a non-EV marker (negative control). The number on the right represents the molecular weight (kDa). Figures 9A and 9B show the results of an analysis of healing properties of EVs, as tested in Example 6. Figure 9A shows the differential protein expression of the control (A1 , EVs produced by 2D starvation) and of EVs produced by the device of the invention (B1). The x-axis represents the Iog2(fold change) of the protein expression, and the y-axis represents the -log (adjusted p-value). The light grey circles correspond to proteins quantified in both conditions, and the dark grey circles correspond to proteins associated with the GO term “wound healing" (G0:0042060). Figure 9B shows the area coverage of a scratch wound in human skin fibroblasts over time in a wound healing assay, as tested in Example 6. 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 control in which HSFs are cultured alone or with 1 %, 2%, 4%, or 10% FBS; A1 and A2 correspond to another control (EVs produced by 2D starvation and 3D starvation, respectively); B1 and B2 correspond to EVs produced by the device of the invention (at Re = 312.5 and at 416.7, respectively).

[0087] Figure 9C shows the differential protein expression of the control (A1 , produced by 2D starvation) and of EVs produced by the device of the invention (B1 ), as tested in Example 5a. The circles in the left panel represent protein detected only in A1 , while the circles in the right panel represent proteins detected only in B1 . The number on the left axis of the left and right panels represents the number of peptides identified, normalized to a length of 100 amino acids. The circles in the center panel show the proteins detected in both A1 and B1 . The x- axis of the center panel represents the Iog2(fold change) of the protein expression, and the y-axis of the center panel represents the -log (adjusted p-value). Proteins shown in light gray (light grey circles) correspond to protein associated with mitochondria.

[0088] DESCRIPTION OF EMBODIMENTS

[0089] The invention will now be described in more detail without limitation in the following description. The term “extracellular vesicle (EV)” as used herein refers to a vesicle that is endogenously released by a producer cell in a constitutive or inducible manner. Examples thereof include, but are not limited to, exosomes, microvesicles and apoptotic bodies.

[0090] The term “cell” as used herein refers to the smallest fundamental structural and functional unit of living organisms, which can divide and multiply.

[0091] The term “producer cell” as used herein refer to a cell that is capable of secreting extracellular vesicles.

[0092] 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.

[0093] 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.

[0094] The term “ fluidic chip” as used herein refers to a platform for performing various tasks such as chemical analysis, biological assays, or medical diagnostics, allowing for manipulation of small volumes of liquids. The chip is characterized by three dimensions: length (dimension along the longest axis of the horizontal plane of the chip), width (dimension perpendicular to the length direction and along the horizontal plane of the chip), and thickness (dimension perpendicular to both of the length and width directions and along the vertical plane of the chip).

[0095] The term “microfluidic chip” as used herein means a chip in which the minimal channel or chamber dimensions are of the order of 1 to less than 1000 ppm. The term “millifluidic chip” herein means a chip in which the minimal channel or chamber dimensions are of the order of 1 to 10 mm.

[0096] The term “channel” means an elongated space such as a tube, duct, pipe, or conduit, along which fluids can flow. A channel is delimited by at least one inlet and at least one outlet.

[0097] The terms “first (channel)” and “second (channel)” as used herein are used for clarity and simplicity, and this designation does not limit the corresponding channel to a certain direction of the flow within the channels.

[0098] The term “longitudinal axis” as used herein refers to a fictive axis inside the channel along the general (average) direction of (laminar) flow within the channel. The longitudinal axis can also be generally defined as the line connecting the centroids of the transverse cross-sections of the channel (transverse meaning perpendicular to the (laminar) flow of liquid within the channel). The term “connection area” as used herein refers to a region where the fictive extension of the first channel and the fictive extension of the second channel intersect or connect. The connection area may be characterized by a spatial overlap or intersection of the fictive extension of the first channel and the fictive extension of the second channel.

[0099] The term “flow rate” as used herein refers to the in-chip flow rate at which a fluid flows within the channel(s) of the fluidic chip itself.

[0100] The term “swirling flow ” as used herein refers to a flow having a pattern of fluid motion characterized by a rotational or spinning or spiraling and / or helical movement around a fictive rotation axis. The term “vortex” can also be used to characterize a swirling flow.

[0101] By “swirling flow extending from the inlet of the second channel” is meant that there is a swirling flow, from the inlet of the second channel, up to a certain length along the second channel. This is different from the unresolved vortices which are generated in any turbulent flow and which may also be generated in the second channel, and which appear, disappear, move, increase or decrease in size, in an unpredictable manner over time and a very short time scale (of less than 0.3 second).

[0102] In some embodiments, the swirling flow is relatively stable at a substantially fixed position, such as substantially at the center of the channel along the second channel (relative to the transverse cross-section). The exact positioning, orientation and extent of the swirling flow may exhibit minor variations over time but overall stay relatively constant over a period of time of at least 0.3 second, or at least 0.5 second, or at least 1 second, or at least 5 seconds, or at least 30 seconds, or at least 2 minutes, or at least 5 minutes, or at least 10 minutes and preferably during substantially the entire time during which liquid flows from the first channel to the second channel.

[0103] In some cases, there is a single swirling flow extending from the inlet of the second channel. In other cases, there may be a plurality of swirling flows extending from the inlet of the second channel, such as 2 or 3 such swirling flows (during at least part of the time during which liquid flows from the first channel to the second channel). The rotation axes of these swirling flows may be parallel or not.

[0104] In some embodiments, the swirling flow may be present over substantially the entire time during which liquid flows from the first channel to the second channel. In other embodiments, the swirling flow may appear and disappear over time (for example because it may merge with another swirling flow or may break up into two or more swirling flow): in this case, the swirling flow must remain present for at least 0.3 second, or at least 0.5 second, or at least 1 second, or at least 5 seconds, or at least 30 seconds, or at least 2 minutes, or at least 5 minutes, or at least 10 minutes.

[0105] The presence of the swirling flow can be visualized in situ, if desired, for example using a microscopy technique, such as fluorescence microscopy or confocal microscopy, for example, as shown in Fig. 2. Alternatively, the presence of the swirling flow can be modeled and predicted using known numerical flow simulation techniques.

[0106] The generation and characterization of the abovementioned swirling flows has been described notably in Kang et al., Intracellular Nanomaterial Delivery via Spiral Hydroporation, ACS Nano 2020, 14, 3048-3058, in Zhang et al., Trapping region of impinging jets in a cross-shaped channel, AIChE Journal 2020, 66:e16822 and in Zhang et al., Experimental investigation of three-dimensional flow regimes in a cross-shaped reactor, Physics of Fluids, 31 , 034105 (2019).

[0107] The swirling flow generated in the second channel may rotate and extend along the longitudinal axis of the second channel. The rotation axis of the swirling flow may follow a substantially parallel trajectory with respect to the longitudinal axis of the second channel, or the rotation axis of the swirling flow may follow a non-parallel trajectory with respect to the longitudinal axis of the second channel, for example, the rotation axis of the swirling flow may be tilted by a certain degree with respect to the longitudinal axis of the second channel (preferably by less than 20°, or less than 10° relative to the longitudinal axis).

[0108] The swirling flow may be present at least at the inlet of the second channel, preferably along the entire second channel.

[0109] Device for producing extracellular vesicles from producer cells

[0110] The present invention provides a device for producing extracellular vesicles (EVs) from producer cells. The device comprises a fluidic chip comprising at least one first channel and at least one second channel and a flow controller system connected to the fluidic chip and configured for circulating a same liquid medium multiple times through the first channel and second channel.

[0111] Each channel has a longitudinal axis, an inlet and an outlet, wherein the outlet of the first channel is connected to the inlet of the second channel in a connection area, and wherein the longitudinal axis of the first channel forms a non-zero angle with the longitudinal axis of the second channel in the connection area. Each of the inlet of the first channel and the outlet of the second channel may correspond to a connection port or may represent a mere junction with another channel for other upstream / downstream fluidic operation.

[0112] In some embodiments, the length of each channel (along the longitudinal axis) may be from 100 pm to 50 mm, preferably from 1 mm to 30 mm, more preferably from 3 mm to 20 mm, or even more preferably from 4 mm to 20 mm. In some embodiments, the maximum dimension of each channel transverse to the longitudinal axis may be from 10 pm to 20 mm, preferably from 100 pm to 10 mm, more preferably from 500 pm to 5 mm.

[0113] As an example, especially when the producer cells are in the form of individualized cells (which will be described later), the length of each channel (along the longitudinal axis) may be preferably from 4 mm to 20 mm and the maximum dimension of each channel transverse to the longitudinal axis may be preferably 100 pm to 1 mm.

[0114] The shape of the transverse cross section of each channel may be appropriately chosen. For example, the shape of the transverse cross section of each channel may be a square, a rectangle, a trapezoid, a circle, an ellipse, a triangle, preferably a square or a rectangle. In the latter case, the transverse cross-section may have a width and a thickness corresponding to the two directions of the square or rectangle. The width may be from 10 pm to 20 mm, preferably from 500 pm to 10 mm, more preferably from 100 pm to 5 mm. The thickness may be from 10 pm to 20 mm, preferably from 500 pm to 10 mm, more preferably from 100 pm to 5 mm.

[0115] The transverse cross-sectional shape of each channel may remain constant throughout its entire length or vary along the longitudinal axis.

[0116] The fluidic chip may be a microfluidic or millifluidic chip.

[0117] In some embodiments, the fluidic chip may be made of cyclic olefin copolymer (COC) or polydimethylsiloxane (PDMS).

[0118] PDMS and COC provide an advantage of producing sterile, single-use disposable fluidic chips, thus allowing the resulting fluidic chips to adhere to GMP (Good Manufacturing Practice) standards, which are crucial for the medical and biotechnological industries. COC is more preferred because it exhibits favorable characteristics in terms of industrial-scale and large-scale production compared to PDMS.

[0119] The method of fabrication of such a fluidic chip is well known in the domain, for example, by a standard hot embossing process or a photolithography process. The hot embossing process with COC is preferred as it offers the necessary precision and scalability required for the intended purpose of the invention. The longitudinal axis of each channel can comprise one or more straight portions and / or one or more curved portions. In some embodiments, the longitudinal axis of each channel may be straight from the inlet to the outlet thereof. In other embodiments, the longitudinal axis of each channel may be nonstraight, i.e., segmented (for example, with breaks or interruptions in the path of the channel, resulting in distinct sections or segments separated by gaps or barriers) and / or curved with a certain degree of bending or curvature along the length thereof. In other embodiments, the longitudinal axis of the first channel may be straight while the longitudinal axis of the second channel may be non-straight, or the longitudinal axis of the first channel may be non-straight while the longitudinal axis of the second channel may be straight.

[0120] When at least one channel longitudinal axis is non-straight as above, it is understood that the features of the fluidic chip in the connection area such as the angle between the channels, which will be explained below, are not necessarily applicable outside the connection area. For example, it is possible that the longitudinal axes of the first channel and second channels outside the connection area (i.e., the longitudinal axis of the first channel close to the inlet thereof and the longitudinal axis of the second channel close to the outlet thereof) do not form the same non-zero angle as in the connection area.

[0121] In some embodiments, the non-zero angle formed between the longitudinal axis of the first channel and the longitudinal axis of the second channel in the connection area may be from 60° to 120°, more preferably 80° to 100°. For example, the non-zero angle may be from 60° to 80°, from 80° to 100°, or from 100° to 120°.

[0122] In preferred embodiments, the longitudinal axis of the first channel may be substantially perpendicular to the longitudinal axis of the second channel in the connection area, as shown in Fig. 1A.

[0123] Fig. 1A shows one example of first and second channels on the fluidic chip 10 of the invention.

[0124] In this example, the fluidic chip 10 comprises two first channels 1 and two second channels 2 (this configuration will be explained in more detail below). The first channels 1 each have a longitudinal axis, an inlet 3 and an outlet 4. The second channels 2 each have a longitudinal axis, an inlet 5 and an outlet 6. The outlets 4 of the first channels 1 are connected to the inlets 5 of the second channel 2 in a connection area 7 (a region outlined by a dashed line), wherein the longitudinal axis of each first channel 1 forms a non-zero angle with the longitudinal axis of each second channel 2 in the connection area 7. In this example, the longitudinal axes of the first channels 1 are substantially perpendicular to the longitudinal axes of the second channels 2 in the connection area.

[0125] The first channel(s) and the second(s) channel may extend in a two- dimensional (2D) or three-dimensional (3D) configuration.

[0126] For example, the first channel(s) and the second channel(s) may extend along the same two-dimensional plane. For example, the first channel may extend along the length or width direction of the fluidic chip while the second channel may extend along the length or width direction of the fluidic chip; or the first channel and the second channel may extend along the thickness direction of the fluidic chip.

[0127] As another example, the first channel and the second channel may extend along different two-dimensional planes. For example, the first channel may extend along the length or width direction of the fluidic chip while the second channel may extend along the thickness direction of the fluidic chip; or the first channel may extend along the thickness direction of the fluidic chip while the second channel may extend along the length or width direction of the fluidic chip.

[0128] In some embodiments, the fluidic chip may comprise a single first channel and two second channels. In this case, the outlet of the first channel may be connected to the inlets of the two second channels in the connection area.

[0129] The angle formed between the longitudinal axes of the two second channels in the connection area may be from 100° to 180°, more preferably 120° to 180°. For example, the angle may be from 100° to 120°, from 120° to 150°, or from 150° to 180°.

[0130] The longitudinal axes of the two second channels are preferably aligned in the connection area.

[0131] By “aligned in the connection area” is meant that the longitudinal axes are parallel or coincident, preferably coincident, in the connection area.

[0132] As an example, the connection area may form a T-junction, in which the two second channels connected in the connection area form the crossbar of the letter T (the horizontal stroke), and the first channel forms the stem that extends from the crossbar.

[0133] In other embodiments, the fluidic chip may comprise two first channels. The outlets of the first channels may be connected to the inlet(s) of the second channel(s) in the connection area.

[0134] The angle formed between the longitudinal axes of the two first channels in the connection area may be from 100° to 180°, more preferably 120° to 180°. For example, the angle may be from 100° to 120°, from 120° to 150°, or from 150° to 180°. Preferably, the longitudinal axes of the two first channels are aligned in the connection area.

[0135] As an example, the connection area may form a T-junction, in which the two first channels connected in the connection area form the crossbar of the letter T (the horizontal stroke), and the second channel forms the stem that extends from the crossbar.

[0136] In some embodiments, the fluidic chip may comprise two first channels and two second channels, wherein the outlets of the first channels may be connected to the inlets of the second channels in the connection area.

[0137] In preferred embodiments, the longitudinal axes of the two first channels are aligned in the connection area.

[0138] Additionally or alternatively, the longitudinal axes of the two second channels are preferably aligned in the connection area.

[0139] Additionally or alternatively, the longitudinal axis of each first channel is preferably substantially perpendicular to the longitudinal axis of each second channel, in the connection area.

[0140] As an example, the connection area may form a cross-junction, in which the two first channels and two second channels intersect at the connection area, as shown in Fig. 1A.

[0141] The fluidic chip comprising a single or two first channels and a single or two second channels are described above; however, it is understood that the fluidic chip may comprise three or more first channels and / or three or more second channels. As described above, the first channel(s) and the second(s) channel may extend in a two-dimensional (2D) or three-dimensional (3D) configuration. In addition, each of the first channels may extend along the same two-dimensional plane or may extend along different two-dimensional planes. Likewise, each of the second channels may extend along the same two-dimensional plane or may extend along different two-dimensional planes.

[0142] When more than one second channel is present, it is understood that a swirling flow may be present at least at the inlet of each second channel, preferably along the entire each second channel.

[0143] In some embodiments, the flow controller system of the device may comprise at least one reservoir and at least one pump. The pump may be for example a peristaltic pump. A reservoir and a pump may be integrated as a single mechanical component, for example as a syringe pump. Alternatively, use may be made of a pressure controller.

[0144] The reservoir may be connected to the inlet of the or each first channel and to the outlet of the or each second channel. In preferred embodiments, the flow controller system may comprise at least two reservoirs, and one reservoir may be connected to the inlet of the or each first channel, and another reservoir may be connected to the outlet of the or each second channel.

[0145] Preferably, the or each reservoir is a syringe pump.

[0146] In some embodiments, the reservoir(s) may be connected to the inlet of the or each first channel and / or to the outlet of the or each second channel via tubing.

[0147] The tubing may be, for example, silicon tubing.

[0148] The tubing may comprise at least two tubes, one tube connecting the reservoir to the inlet of the first channel and the other tube connecting the or another reservoir to the outlet of the second channel.

[0149] In some embodiments, especially when the flow controller system comprises two or more first channels and / or two or more second channels, the tubing may comprise three or more tubes, and the tubes may be connected to each other via a connector.

[0150] As an example, when the flow controller system comprises two first channels, the tubing may comprise one tube connected to the reservoir and two tubes connected to the inlets of the first channels, the three tubes being connected via a connector, as shown in Fig. 1 B.

[0151] Additionally or alternatively, when the flow controller system comprises two second channels, the tubing may comprise one tube connected to the or another reservoir and two tubes connected to the outlets of the second channels, the three tubes connected via a connector, as shown in Fig. 1 B.

[0152] The connector may be a T-connector, for example, made of polypropylene.

[0153] The pump may be attached to the tubing (for example, a peristaltic pump) or to the reservoir (for example, a syringe pump).

[0154] The pump may be controlled by a conventional automated system, for example, via a computer software.

[0155] Fig. 1 B shows one example of the device of the invention. The device comprises the fluidic chip 10 of Fig. 1 A, and a flow controller system 20 connected to the fluidic chip 10.

[0156] In this example, the flow controller system comprises two syringe pumps 8,9, connected to the fluidic chip via the silicon tubing 30.

[0157] One syringe 8 is connected to the inlets of the first channels 1 , for example through a main tube and two branch tubes, both branch tubes being connected to the main tube and each branch tube being connected to the respective inlet of one first channel 1. The other syringe 9 is connected to the outlets of the two second channels 2, for example through a main tube and two branch tubes, both branch tubes being connected to the main tube and each branch tube being connected to the respective outlet of one second channel 2.

[0158] The syringe pumps are controlled by an automated system connected to a computer software, which controls the flow rate within the fluidic chip.

[0159] The device may further comprise a pressure system 40 for assuring (substantially) the same hydraulic resistance in the tubing leading from the syringe pump 8 to the inlets of both first channels 1. The device may further comprise a pressure system 41 for assuring (substantially) the same hydraulic resistance in the tubing leading from the syringe pump 9 to the outlets of both second channels 2.

[0160] The flow controller system may be configured for repeatedly circulating the same liquid through the first channel(s) and through the second channel(s) in one direction.

[0161] For example, the flow controller system may be configured for repeatedly circulating the same liquid medium through the first channel(s), from the inlet(s) thereof to the connection area, and then through the second channel(s), from the connection area to the outlet(s) thereof, thereby generating a swirling flow extending from the inlet of the or each second channel.

[0162] In some embodiments, the flow controller system may be configured for repeatedly circulating the same liquid through the first channel(s) and through the second channel(s) in two opposite directions.

[0163] For example, as shown in Fig. 1 B, the flow controller system is configured for, successively:

[0164] - flowing liquid medium through the first channel(s), from the inlet(s) thereof to the connection area, and then through the second channel(s), from the connection area to the outlet(s) thereof, thereby generating a swirling flow extending from the inlet of the or each second channel;

[0165] - flowing liquid medium through the second channel(s), from the outlet(s) thereof to the connection area, and then through the first channel(s), from the connection area to the inlet(s) thereof, thereby generating a swirling flow extending from the outlet of the or each first channel.

[0166] In some embodiments, the device is a closed system. In other words, the circulation of the liquid medium is contained within the device and is not in contact with the external environment, such as air.

[0167] The components of the device as mentioned above, such as channels, tubing, pump(s), and reservoir(s), may be connected to each other in a sealed way, forming a closed loop. Such a closed system ensures sterility if needed and allows for the EV production without liquid-air interface. Such an interface is characterized by significantly higher hydrodynamic stresses than in the liquid, which is difficult to control and thus results in uncontrolled cell mortality. Thus, the closed system can reduce the mortality of the producer cells during the EV production.

[0168] Method for producing extracellular vesicles (EVs) from producer cells in a fluidic chip

[0169] The present invention also provides a method of producing extracellular vesicles from producer cells in a fluidic chip, wherein the fluidic chip comprises at least one first channel and at least one second channel, each channel having a longitudinal axis, an inlet and an outlet, wherein the outlet of the first channel is connected to the inlet of the second channel in a connection area, wherein the longitudinal axis of the first channel forms a non-zero angle with the longitudinal axis of the second channel in the connection area.

[0170] The method comprises: a) passing a flow of liquid medium containing producer cells through the first channel, from the inlet to the outlet thereof; b) passing the flow of liquid medium through the second channel, from the inlet to the outlet thereof, thereby generating in the second channel a swirling flow extending from the inlet of the second channel, c) collecting extracellular vesicles generated from the producer cells.

[0171] The method of the invention is performed ex vivo.

[0172] The fluidic chip may be as defined above.

[0173] The fluidic chip may constitute a device for producing extracellular vesicles from producer cells. The device may be as described above.

[0174] In some embodiments, the producer cells may be selected from human cells, animal cells, and combinations thereof.

[0175] 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.

[0176] 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.

[0177] In some embodiments, the producer cells may be animal cells, preferably murine cells, for example murine MSC (murine mesenchymal stem cells) cells.

[0178] 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, such as human mesenchymal stem cells (hMSC), genetically modified cells, human umbilical cord vein endothelial cells (HUVEC) or primary cells.

[0179] 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, human monocytes (THP-1 cells) or Raji cells.

[0180] 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.

[0181] 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.

[0182] The subject is preferably human but can also be an animal.

[0183] The producer cells may be either adherent to a culture medium or nonadherent to a culture medium (also referred to as suspension cells).

[0184] In the case of adherent producer cells, the culture medium can comprise microcarriers which themselves are suspended in a liquid culture medium.

[0185] 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.

[0186] 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.

[0187] 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.

[0188] 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 in the form of spheroids and / or organoids.

[0189] In some embodiments, when single cells are considered as producers, the concentration of the producer cells in the liquid medium is from 1 ,000 to 2 million cells per milliliter, preferably from 10,000 to 1 million cells per milliliter, more preferably from 50,000 to 500,000 cells per milliliter, even more preferably from 100,000 to 200,000 cells per milliliter.

[0190] 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.

[0191] In some embodiments, when producer cells are cell aggregates, encompassing spheroids and organoids, the aggregates have an average diameter of from 40 pm to 5 mm, preferably from 75 pm to 500 pm; the number of cells per aggregate is from 10 to 1 million, preferably from 100 to 50,000, more preferably from 150 to 1 ,000. The concentration of aggregates is from 10 to 20,000 aggregates per milliliter, preferably from 50 to 10,000 aggregates per milliliter, more preferably from 1 ,000 to 5,000 aggregates per milliliter.

[0192] 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.

[0193] In some embodiments, the extracellular vesicles may be generated from producer cells in the form of spheroids and / or organoids.

[0194] 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.

[0195] 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).

[0196] 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), Reswell Park Memorial Institute (RPMI 1640) medium, or serum-free media. The generation of the swirling flow can facilitate real-time tracking and visualization of producer cells during extracellular vesicle production, as shown in Fig. 2A, Fig. 2B, and Fig. 2C. Fig. 2A shows the parallel trajectories of the producer cells (spheroids) in the first channel with respect to the longitudinal axis of the channel, and Fig. 2B shows the generation of a swirling flow. Fig. 2C shows the path of a single spheroid traveling along the swirling flow. The generation of the swirling flow allows for the measurement of the velocities within the swirling flow and the actual stresses exerted on the producer cells, thereby resulting in precise control of production conditions.

[0197] In some embodiments, step c) of collecting the extracellular vesicles generated from the producer cells may be carried out by withdrawing the liquid medium including the producer cells from the fluidic chip (or from the device, particularly from the reservoir(s) if present), and separating the extracellular vesicles from the withdrawn liquid medium.

[0198] 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.

[0199] In other embodiments, especially when the method is performed using the device comprising a reservoir as described above, step c) may be carried out by withdrawing the liquid medium from the reservoir 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 reservoir itself is preferably carried out prior to withdrawing the liquid medium.

[0200] 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).

[0201] 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).

[0202] As explained above, the first channel(s) and the second channel(s) of the fluidic chip may extend in two-dimensional (2D) or three-dimensional (3D) configuration. For example, step a) of passing a flow of liquid medium through the first channel and step b) of passing the flow of liquid medium through the second channel may be carried along the same two-dimensional plane, for example, horizontally. Alternatively, step a) and step b) may be carried along different two- dimensional planes. For example, step a) of passing a flow of liquid medium through the first channel may be carried out horizontally while step b) of passing the flow of liquid medium through the second channel may be carried out vertically.

[0203] In some embodiments, the flow of liquid medium at the inlet of the second channel may be characterized by a Reynolds number of greater than 40, or of greater than 50, or of greater than 60, or of greater than 70, or of greater than 80, or of greater than 90, or of greater than 100, or of greater than 150, preferably greater than 200. For example, the Reynolds number may be from 40 to 1000, or from 100 to 750, or from 200 to 500.

[0204] The Reynolds number may be calculated according to the following formula: pLV / q, where p is the fluid density, L is the characteristic length of the system (in this case, the width of the channel), V is the flow speed of the fluid and q is the dynamic viscosity of the fluid. Without wishing to be bound by theory, at least in the conditions investigated by the present inventors, it is believed that when the Reynolds number exceeds a first critical value of approximately 37.9 in a channel having a width:thickness ratio of 1 , a swing motion is generated in the channel, resulting in the generation of a swirling flow. However, this first critical value can vary, depending on the overall geometry of the fluidic chip.

[0205] Without wishing to be bound by theory, at least in the conditions investigated by the present inventors, it is believed that, when the Reynolds number exceeds a second critical value higher than the first critical value, the production of EVs significantly increases. This second critical value has been found to be approximately equal to 200 in the conditions investigated by the inventors (in a channel having a width:thickness ratio of 1 ).

[0206] Therefore, in preferred embodiments, the flow of liquid medium at the inlet of the second channel is characterized by a Reynolds number from 200 to 500, preferably from 210 to 400, more preferably from 220 to 350, more preferably from 230 to 300.

[0207] The Reynolds number may be adjusted by controlling the flow rate of the flow of liquid medium.

[0208] In some embodiments, the flow rate in the first channel may be 5 mL / min or more, preferably 10 mL / min or more, more preferably 15 mL / min or more, and even more preferably 30 mL / min or more. For example, the flow rate in the first channel may be from 10 to 150 mL / min, preferably from 30 to 100 mL / min. In the above ranges, good EV production can be achieved while minimizing cellular damage. When the device comprises two or more first channels, the liquid medium may travel substantially at the same flow rate in the first channel(s).

[0209] When the fluidic chip comprises two first channels, step a) of passing a flow of liquid medium comprises passing two opposing flows of liquid medium each from the inlet to the outlet of each first channel and to the connection area, where the intersection of two opposing flows is established. Such intersection of two opposing flows in the connection area can facilitate the generation of the swirling flow in the second channel(s).

[0210] Preferably, the longitudinal axes of the two first channels are aligned in the connection area, and step a) of passing a flow of liquid medium comprises passing two diametrically opposing flows of liquid medium each from the inlet to the outlet of each first channel and to the connection area. The intersection of two diametrically opposing flows in the connection area can further facilitate the generation of the swirling flow in the second channel(s).

[0211] In some embodiments, the method may further comprise repeating cycles of steps a) and b), using the same producer cells.

[0212] The inventors have found that repeatedly subjecting cellular material to a brief but high hydrodynamic stress of a swirling flow can unexpectedly improve the efficiency of the EV production, even with substantial time gaps between each triggering event, while limiting the damage to the producer cells. The step of repeating cycles of steps a) and b) with the same producer cells thus can further reduce the amount of cellular material for the generation of EVs.

[0213] For example, at each cycle, the producer cells may be withdrawn from the fluidic chip and separated from the liquid medium (by way of, for example, centrifugation); and placed again in the fluidic chip together with fresh liquid medium.

[0214] Alternatively, especially when the method is performed using the device comprising a flow controller system, the liquid medium containing producer cells may be circulated multiple times in the fluidic chip, for example, by repeatedly circulating the same liquid medium through the first channel(s), from the inlet(s) thereof to the connection area, and then through the second channel(s), from the connection area to the outlet(s) thereof, thereby generating a swirling flow extending from the inlet of the or each second channel. In other terms, the liquid medium can be in a loop, being recycled from the outlet(s) of the second channel(s) back to the inlet(s) of the first channel(s).

[0215] In some embodiments, the method may comprise reversing the direction of the flow of liquid medium at each repeating cycle. In this case, the method may comprise successively: performing a cycle of steps a) and b); and a cycle of flowing liquid medium through the second channel(s), from the outlet(s) thereof to the connection area, and then through the first channel(s), from the connection area to the inlet(s) thereof, thereby generating a swirling flow extending from the outlet of the or each first channel; and optionally repeating these cycles.

[0216] Fig. 1 B shows an example of the method, in which the direction of the flow of liquid medium is reversed at each repeating cycle, using the device of the invention. In this example, as explained above, the two syringes 8, 9 are connected to the first channels 1 and second channels 2, respectively, of the fluidic chip 10. Reversing the direction of the flow of liquid medium in each repeating cycle may be carried out by alternating the sequence of pushing one syringe pump to inject the liquid medium while simultaneously pulling the other syringe pump. In this example, a flow of liquid medium containing producer cells is passed through the first channels 1 , from the inlets thereof to the outlets in the connection area, and then through the second channels 2, from the inlets in the connection area to the outlets thereof. This cycle corresponds to the sequence of pushing the syringe pump 8 and pulling the syringe pump 9. Then, the flow of liquid medium is passed through the second channels 2, from the outlets thereof to the connection area, and then through the first channels 1, from the connection area to the inlets thereof. This cycle corresponds to the sequence of pulling the syringe pump 8 and pushing the syringe pump 9.

[0217] The control of the syringe pumps and the control of the flow rates may be carried out in an automated system connected to a computer software.

[0218] In some embodiments, the duration of the method is from 1 to 5 hours, more preferably from 1 to 3 hours.

[0219] In some embodiments, the method is performed at 37°C. For example, when the method is performed using the device of the invention, all the components of the device are kept at 37°C.

[0220] In some embodiments, steps a) and b) are performed in a closed system and the liquid medium is not exposed to an external environment. Preferably, the method is performed in a closed system. As explained above, the method in such a closed system ensures sterility and allows for the EV production without liquidair interface, thereby reducing the mortality of the producer cells during the EV production.

[0221] In some embodiments, the method may further comprise a step of introducing a therapeutic agent or an imaging agent into the liquid medium.

[0222] The step of introducing a therapeutic agent or an imaging agent into the liquid medium may be performed before step a). 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.

[0223] 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.

[0224] Application of the produced extracellular vesicles

[0225] The present invention may also relate to extracellular vesicles produced by the method of the invention or using the device of the invention, as described above.

[0226] 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, wound healing therapies, 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.

[0227] 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.

[0228] 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.

[0229] 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.

[0230] In other embodiments, the extracellular vesicles may be obtained from mesenchymal stem cells (MSC), and may be used in wound healing therapies, in regenerative medicine or in the treatment of tumors, infectious diseases, inflammatory diseases, immunological diseases, metabolic diseases, cancer diseases, genetic diseases, degenerative diseases or diseases secondary to surgeries or trauma.

[0231] EXAMPLES Preparation of the device of the invention

[0232] A fluidic chip according to the invention was made with a 3D-printed mold using the DigitalWax 028J Plus 3D-printer and the DigitalWax DS3000 resin. The fluidic chip was designed to have two first channels (16 mm in length; 1 .2 mm in width; and 1.2 mm in height) and two second channels (16 mm in length; 1.2 mm in width; and 1 .2 mm in height), in which the longitudinal axes of the first channels are perpendicular to the longitudinal axes of the second channels. Unpolymerized polydimethylsiloxane (PDMS), prepared by combining SYLGARD™ 184 Silicone Elastomer Base and SYLGARD™ 184 Silicone Elastomer Curing Agent in a mass ratio of 4: 1 , was poured into the mold. To avoid the formation of air bubbles, the PDMS-filled mold was then degassed and then incubated at 70°C for 24 hours to polymerize PDMS. After polymerization, the PDMS layer was carefully unmolded, and 2-mm ports (inlets and outlets) were punched at the ends of the channels. The PDMS layer and a 50x50-mm glass slide were cleaned with isopropanol and surface-activated through plasma treatment using the Diener PICO plasma cleaner. The chip was then immediately assembled by placing the PDMS layer on the glass slide. Opposite ports of each channel were connected to each other using a silicone tube (length = 100 mm, inner diameter = 0.79 mm, outer diameter = 2.36 mm) and a polypropylene T-connector. Via the T-connector, the silicone tube was then connected to another silicone tube (length = 150 mm), which were connected on the other end to a needle (diameter = 1 .2 mm) of a syringe. All the connections were tightly secured with silicone coating (3140 RTV Coating, Dowsil). Hydraulic resistance of the system was tuned to ensure symmetrical flows in the fluidic chip, using a pressure system (3D-printed ring designed to exert pressure on the tubing outlets. A syringe pump connected to a computer running QmixElements software was utilized to impose repeated back and forth movements to the cell solution

[0233] Preparation of a single-channel device (comparative device)

[0234] A single-channel device was prepared in the same way as the fluidic chip of the invention as described above, except that the chip of the device had a single channel (32 mm in length; 1.2 mm in width; and 1.2 mm in height), and that the inlet and outlet of the single channel were connected to silicone tubes (length = 250 mm), which were each connected on the other end to a needle.

[0235] Cell culture

[0236] Human mesenchymal stem cells (hMSC), human monocytes (THP-1 ) and human skin fibroblasts (HSF) were cultured at 37°C and 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, 61965-026 for hMSC), in Reswell Park Memorial Institute medium (Gibco, 61870-010 for THP-1 ) or in PriGrow III medium (abm, TM003 for HSF) supplemented with 10% of heat-inactivated Fetal Bovine Serum (FBS) (Dutscher, S1900-500C for hMSC and THP-1 ; Gibco, A5209402 for HSF) and 1 % of penicillin and streptomycin (P / S) (Gibco, 15140-122).

[0237] Preparation of TH P1 -cells in suspension

[0238] Cells from passage 10-20 of THP-1 were collected and washed three times by centrifugation (300g, 4 minutes). Cell pellets were then resuspended in 220- nm-filtered DMEM without phenol red (Gibco, 31053-028) at a concentration of 106cells / mL before being used for EV production.

[0239] Preparation of spheroids in suspension

[0240] Cells from passage 10-20 of hMSC were detached from their culture flasks, centrifuged at 300g for 4 minutes and resuspended at a concentration of 650,000 cells / mL in DMEM supplemented with 10% FBS and 1 % P / S. Then 6-well plates containing the agarose microwells (a diameter of 28 mm and a height of 15 mm, produced by way of 3D-printing using the DigitalWax 028J Plus 3D printer and the DigitalWax DS3000 resin) were then rinsed with PBS, and 1 mL of the cell suspension was added to each well. Plates were left at rest 30 minutes at 37°C to allow cell sedimentation, then centrifuged at 200g for 2 minutes before being incubated at 37°C for 24 hours to generate mature hMSC spheroids. Following maturation and just before EV production, the hMSC spheroid mean diameter was measured as 107 pm. Then, hMSC spheroids were recovered from the microwell plates. Subsequently, the spheroid suspension underwent three washing steps through centrifugation (1000 RPM, 3 minutes) and resuspension in 220-nm- filtered DMEM without phenol red (Gibco, 31053-028). Spheroids were then resuspended in 220-nm-filtered DMEM without phenol red (Gibco, 31053-028) at a concentration of 2400 spheroids / mL.

[0241] Extracellular vesicle production by 2D or 3D starvation

[0242] For 2D starvation, after the culture reached confluency, hMSC stem cells were incubated in fresh serum-free medium in 150-cm2culture flasks for 72 hours at 37°C, 5% CO2. For 3D starvation, 4 mL of the spheroid suspension or of the cell suspension was placed in a 2% agarose-coated T25 culture flask and maintained at 37°C for 2 hours. Following the EV production in both 2D and 3D starvation, the EV-conditioned media was collected and then centrifuged at 2000 g and 4°C for 10 minutes before being stored at 4°C for further use. Extracellular vesicle production in the device of the invention or in single channel chip

[0243] A total of 4 mL of the prepared spheroid suspension or cell suspension was distributed into UV-sterilized 5 mL air-tight syringes (1005TLL, Hamilton) for inchip production flow rates below 15 mL / min, 10 mL air-tight syringes (1010TLL Hamilton) for flow rates between 15 mL / min and 30 mL / min, or 25 mL air-tight syringes (1025TLL, Hamilton) for flow rates exceeding 30 mL / min. These syringes were then connected to the needles and attached to a syringe pump (Base module 120 and Low Pressure Nemesys modules, Cetoni GmbH), controlled by the QmixElements software. Successive cycles of alternating back-and-forth movements of the syringe pumps were applied to the syringes, with flow rates ranging from 15 mL / min to 120 mL / min (corresponding to in-chip production flow rates ranging from 7.5 mL / min to 60 mL / min, respectively, and to in-chip Reynolds numbers ranging from 52.1 to 416.7, respectively). Following the EV production, spheroid suspensions were separately recovered and centrifuged at 200g for 3 minutes to separate spheroid pellets from the EV-conditioned media (CM), or cell suspension were separately recovered and centrifuged at 300g for 4 minutes to separate cell pellets from EV-conditioned media. EV CM were then centrifuged at 2000g and 4°C for 10 minutes before being stored at 4°C for further use. Spheroid pellets were resuspended in 400 pL of 220-nm-filtered DMEM without phenol red (Gibco, 31053-028) and stored at room temperature before further use.

[0244] In-chip particle tracking

[0245] The circulation of hMSC spheroids in the cross-slot device was conducted under an inverted microscope (Leica DM IRB) connected to a high-speed camera (Phantom VEO-E 310L). Spheroid trajectories were recorded at a 2.5X magnification, with recording speed ranging from 6400 to 9600 images per second, depending on the in-chip flowrate. The subsequent analysis of spheroid trajectories was performed using the TrackMate plugin in Fiji software. Particles were detected using the LoG detector method, with a quality threshold set at 10 and estimated object size of 0.025 mm. Particle tracking was then performed using the simple LAP Tracker method, with a linking max distance ranging from 0.05 mm to 0.15 mm depending on the in-chip flowrate, and a gap-closing max distance set as two times the linking max distance. Any incomplete or incorrect tracks were manually addressed by filling or correcting them using the Trackscheme tool. Spheroid velocities and residency times were then derived from the tracked coordinates using Matlab software. CryoTEM imaging

[0246] CryoTEM imaging of EV concentrated conditioned media and EV pools was performed using the Jeol JEM2100 transmission electronic microscope.

[0247] EV quantification by Nanoparticle Tracking Analysis (NTA)

[0248] EV samples were centrifuged at 2000 g and 4°C for 10 minutes before being diluted between approximately 2x107and 2x108EVs / mL. Nanoparticle Tracking Analysis was performed on the diluted samples using the NanoSight NS300 (Malvern) along with the NTA 3.4 software. During analysis, the camera level was set at 16, the flow rate was adjusted within the range of 20 of 30 pL / min and the detection threshold was set at 4.

[0249] Spheroid integrity analysis

[0250] 100 pL of Live / Dead reagent (Invitrogen 488 / 570) was combined with 50 pL of resuspended spheroid pellet in wells of a 96-well plate (Optiplate-96 F HB, Black, PerkinElmer) and left at room temperature. Following a 30-minute incubation period, spheroids were imaged in a fluorescence mode, at an excitation wavelength of 465 nm for Live (green) and of 525 nm for Dead (orange). Green and orange signals of each well were then superimposed in a single image, and images were binarized to separate spheroids and individualized cells from background using Fiji software. Binarization threshold was manually optimized for each image. Particles were then counted, measured, and discriminated between intact spheroids and individualized cells depending on their diameter. The discrimination threshold was calculated for each condition as half of the mean diameter of the spheroids, measured as described above.

[0251] Cell viability analysis

[0252] Toxilight toxicity assays were performed by combining 80 pL of Toxilight reagent (Lonza, LT107-217) with 20 pL of the previously collected conditioned media in wells of a 96-well plate (PerkinElmer, Optiplate-96 F HB, Black) and incubating the mixture at room temperature. Following a 15-minute incubation period, luminescence was measured using a plate reader (PerkinElmer, Ensight Multimode Plate Reader) with a measurement time of 0.1 seconds. A lysed sample of spheroids with the same spheroid concentration was used as a positive control, and 20 pL of DMEM without phenol red (Gibco, 31053-028) combined with 80 pL of Toxilight reagent was used as blank. Bead-based multiplex flow cytometry assay

[0253] EV pools were subjected to bead-based multiplex analysis by flow cytometry according to manufacturer instructions using anti-CD9 / CD63 / CD81 / and anti-CD89 / CD44 / CD49e / CD105 / CD146 (MACSPlex Exosome Kit, human, Miltenyi). The signals obtained were normalized relative to the expression level of immunoglobulin (Ig).

[0254] LC-MS / MS Analysis

[0255] The LC-MS / MS samples were prepared according to a standard protocol. Liquid chromatography (LC) was performed with a Vanquish Neo LC system (Thermo Scientific) coupled to an Orbitrap Astral mass spectrometer (MS), interfaced by a Nanospray Flex ion source (Thermo Scientific). Peptides were injected onto a C18 column (double nanoViper PepMap Neo, Thermo Scientific) at 50°C, and separated with a linear gradient from 100% buffer A (100% H2O + 0,1 % formic acid) to 28% buffer B (100% CH3CN + 0,1 % formic acid) at a flow rate of 300 nL min-1over 104 min. Peptides were analyzed in the MS applying a 2200 V spray voltage, funnel RF level at 40, and a heated capillary temperature set to 285°C. MS full scans (380-980 m z-1) were recorded in centroid mode using a resolution of 240000 at m / z 200, a normalized AGC target of 500%, and a maximum injection time of 5 ms. The fragment spectra were acquired in DIA mode, with a precursor mass range of 380-980 m z-1with 2 Da isolation windows (without overlap). Isolated precursors were fragmented in the HCD cell using 25% normalized collision energy, a normalized AGC target of 500%, and a maximum injection time of 3 ms. For identification, the data were searched against the Homo sapiens (UP000005640) Uniprot database using Pulsar search engine through Spectronaut v19 (Biognosys) by directDIA+, using default search settings. For protein quantification, ion XICs (sum of fragment peak areas) from proteotypic peptides shared between compared conditions (TopN matching) were used. Median and scale normalization at peptide level was applied on the total signal to correct the XICs for each biological replicate (N = 5). To evaluate the statistical significance of the change in protein abundance, a linear model (adjusted on peptides and biological replicates) was performed, and a two-sided T-test was then applied on the fold change. The p-values were then adjusted using the Benjamini - Hochberg FDR procedure. LFQ quantification was also performed following the algorithm, as described by J. Cox, et al., Mol. Cell. Proteomics 2014 (13), 2513. Proteins showing a Iog2(fold change) of_2 or more or -2 or less with an adjusted p-value of less than 0.05 were selected. Proteins with a Iog2(fold change) between -2 and 2 were considered as common to two conditions (control conditions and the conditions according to the invention).

[0256] Wound-healing assay

[0257] Human skin fibroblasts (HSFs) were cultured in 96-well plates (TPP, 92096) at 37°C, 5% CO2 until confluency. A scratch wound was made using a sterile 100-pL pipette tip and the medium was replaced with fresh serum-free culture medium, fresh serum-free culture media supplemented with 1 % to 10% FBS (1 %, 2%, 4%, 10%), fresh serum-free culture medium supplemented with EVs produced by starvation (109EVs / mL, either 2D or 3D), fresh serum-free culture medium supplemented with EVs produced in the cross-slot device (109EVs / mL, at a Reynolds of 312.5 or of 416.7). Cells were then incubated for 96 hours at 37°C; 5% CO2 and regularly imaged using a plate reader (PerkinElmer, Ensight Multimode Plate Reader). At each time point, total area covered by cells in the scratch wound was measured using Fiji software. Area coverage was then calculated as the area covered by the cells in the scratch wound divided by the total area of the wound.

[0258] Example 1 : EV production from THP-1 cells

[0259] The device having a configuration shown in Fig. 1 B was manufactured as described above, and EV production was performed as described above.

[0260] The experimental conditions were as follows:

[0261] - Producer cells: human monocytes (THP-1 ) in suspension

[0262] - Cell concentration: approximately 1.0x106cells / mL

[0263] - Flow rates (in chip): 2.5, 7.5 and 15 mL / min

[0264] - Duration: 1 hour (corresponding to approximately 60 seconds of high stress zone)

[0265] EV samples were then quantified as explained above in the section “EV quantification by Nanoparticle Tracking Analysis (NTA)".

[0266] The results are shown in Table 1 :

[0267] Table 1 : Number of EVs produced from THP-1 cells Thus, the number of EVs per cell was improved compared to the control (flow rate of 2.5 mL / min and Re of 17, at which no swirling flow is generated). In addition, the EV production increased as the Re increased.

[0268] In a conventional fluidic system with an agitator (for example, the system as described in W02020 / 136362), the system employs 50 times more cellular material (at the same cell concentration) in bioreactors, yielding around 10000 EVs per cell and per hour, which corresponds to approximately 200 EVs per minute of stimulation.

[0269] Moreover, considering the short residence time in the swirling flow in the method of the invention (hydrodynamic stresses were applied only for about 60 seconds to the cellular material at each flow rate), the virtual yield of 1350 to 1500 EVs per cell and per minute was achieved.

[0270] Overall, the above results demonstrated the potential of the method of the invention for achieving high-yield EVs production with minimal cellular material (especially cells in suspension) consumption.

[0271] Furthermore, the above results illustrate the possibility of triggering EV production within a relatively short time (60 seconds), which was previously unexpected.

[0272] The cryoTEM image analysis showed that the EVs exhibit intact structure and high membrane and intravesicular protein density (Fig. 3A).

[0273] Example 2A: EV production from hMSC spheroids

[0274] The same device as in Example 1 was used, and EV production was performed from spheroids of human mesenchymal stem cells (hMSC), as described above.

[0275] The experimental conditions were as follows:

[0276] - Producer cells: hMSC spheroids

[0277] - Cell concentration: approximately 2400 spheroids / mL

[0278] - Flow rates (in chip): 7.5 and 15 mL / min

[0279] - Duration: 1 hour and 2 hours (corresponding to approximately 60 seconds and 120 seconds of high stress zone, respectively)

[0280] EV samples were then quantified as explained above in the section “EV quantification by Nanoparticle Tracking Analysis (NTA)”.

[0281] The results are shown in Table 2:

[0282] Table 2 : Number of EVs produced from hMSC spheroids

[0283] The results show that the longer duration or a higher value of Reynolds number (Re) results in an increased number of produced EVs.

[0284] For comparison, the conventional technique of EV production by subjecting hMSC spheroids (having the same cell concentrations) to 72-hour 3D starvation in culture flasks produced approximately 1800 EVs per cell, corresponding to about 0.42 EVs per cell per minute of stimulation)

[0285] Example 2B: EV production from hMSC spheroids

[0286] EVs were produced in the same way as in Example 2A, except that the production duration was fixed at 2 hours and that the Reynolds number was varied. The results are shown in following Table 3 and in Fig. 4.

[0287] Table 3 : Number of EVs produced from hMSC spheroids

[0288] Fig. 4 shows that the number of EVs produced per hMSC cell generally increases with an increasing Reynolds number. In particular, the results demonstrates a critical Reynolds number between Re = 173.6 and Re = 208.3 above which the production of EVs is significantly increased. Beyond this critical Reynolds number, the increase in EV yield with a higher Re is even more pronounced.

[0289] The cryoTEM image analysis showed that the EVs produced at Re = 312.5 (Fig. 3B) and Re — 416.7 (Fig. 3C) exhibit intact structure and high membrane and intravesicular protein density. Example 3: EV production in the device of the invention and a single-channel device

[0290] EVs were produced in the same way as in Example 2A, except that the method was also performed in a single-channel device, that the production duration was fixed at 2 hours and that the Reynolds number was Re = 208.4. The results are shown in Fig. 5

[0291] Figure 5 shows that the number of EVs produced from hMSC spheroids is significantly higher when the device of the invention (plot B) is used, compared to when a single-channel chip (plot C) is used or compared to when EV production is induced by 3D starvation.

[0292] The above results show that the initiation of EV production was a result of the swirling flow and was not influenced by other processes within the fluidic chip device.

[0293] Example 4: Cell integrity and vitality after EV production

[0294] The EVs were produced in the same way as in Example 2B. The cell integrity and cell viability of the hMSCs following the EV production was evaluated, as described above in "Spheroid integrity analysis” for the cell integrity and “Cell viability analysis" for the cell viability.

[0295] Cellular aggregates are considered to be intact spheroids if their diameter is greater than half of the mean spheroid diameter measured before the EV production. The disruption or destruction of spheroids could occur due to the flux, especially at a higher Re.

[0296] Fig. 6 (cell integrity) shows that the spheroids are intact up to Re = 312.

[0297] Fig. 7 (cell vitality) shows that the EV production according to the invention does not impact the cellular viability for EV production at Re egual to or less than 138.9. The percentage of the viable cells decreased at Re = 416.7; however, 75% of cells were still found viable.

[0298] These results indicate that the invention can not only maintain cell viability but also preserve the integrity of spheroids.

[0299] Example 5: Analysis of EV-specific proteins

[0300] The EVs were produced in the same way as in Example 2B (Reynolds number = 208.3, 312.5 or 416.7).

[0301] EV-specific proteins were studied by flow cytometry, as described above in the section “Bead-based multiplex flow cytometry assay”. The results are shown in Fig. 8A and Fig. 8B. The results indicate that the EVs produced according to the invention (B1 and B2, with Re of 312.5 and of 416.7, respectively) have a comparable protein expression compared to the control (A1 and A2, corresponding to EVs produced by 2D starvation and by 3D starvation, respectively): for EV-specific membrane proteins (Fig. 8A), EVs are positive for CD63 and CD81 markers and negative for the CD9 marker; and for proteins specific to the mesenchymal nature of EVs (Fig. 8B), EVs are positive for CD29, CD44, CD49e and CD105 markers and negative for the CD146 marker.

[0302] More specifically, Fig. 8A shows that the EVs produced according to the invention are indeed EVs (and not pieces of cell membranes or apoptotic bodies, for example).

[0303] Fig. 8B shows that the EVs produced according to the invention have similar biological properties to the hMSCs from which they are derived. Additionally, the levels of the markers were higher in EVs generated from spheroids (A2, B1 and B2) compared to EVs produced in a 2D configuration.

[0304] A western blot analysis was also performed according to a standard protocol on the EV-specific proteins. The results are shown in Fig. 8C.

[0305] Fig. 8C confirms the results observed in Fig. 8A and Fig. 8B, showing the presence of EV-specific membrane markers, particularly CD63 and CD81 markers.

[0306] Fig. 8C also confirms the presence of the EV-specific cytosolic marker SDCBP under all tested conditions.

[0307] Example 5a: Analysis of the protein composition of EVs

[0308] The EVs were produced in the same way as in Example 2B (Reynolds number = 312.5), and the protein composition of the EVs was compared with that of the control (EVs produced by 2D starvation) by performing a comprehensive proteomic analysis, using quantitative label-free mass spectrometry on five independent biological replicates, as described in the section “LC-MS / MS Analysis".

[0309] A total of 3025 proteins were detected in both conditions with an adjusted p-value below 0.05 of the fold change. Of these 3025 proteins, 326 proteins were significantly enriched (Iog2 fold change > 2 and adjusted p-value < 0.05) in the control (EVs produced by 2D starvation), while 1248 proteins were enriched in the EVs produced according to the invention. A total of 25 proteins were exclusively quantified in the control while 46 proteins were exclusively quantified in the EVs produced according to the invention (data not shown). Moreover, among cytosolic proteins, EVs produced by 2D starvation were found to be significantly enriched in exosome-related proteins, while EVs produced according to the invention were significantly enriched in microvesicle- related proteins, suggesting different protein biogenesis pathways for the two different stimulations.

[0310] For instance, EVs produced by 2D starvation were found to be significantly enriched in proteins associated with exosome-related pathways such as the canonical ESCRT pathway, the syntenin-ALIX pathway, or pathways associated with CD63, floti II in 1 and 2 or RAB31 . Conversely, EVs produced according to the invention were significantly enriched in proteins associated with the microvesicle- related ARF6 pathways (data not shown).

[0311] Interestingly, among proteins detected in both conditions, 796 proteins were associated with the Gene Ontology (GO) term “extracellular vesicle” (GO: 1903561 , meaning that these proteins are related to the extracellular vesicle), including 261 proteins significantly enriched in the EVs produced according to the invention and 148 proteins significantly enriched in the control. Similarly, 44 proteins were associated with the GO term “vesicle budding from membrane" (G0:0006900, meaning that these proteins are related to the vesicle budding from membrane), including 16 proteins significantly enriched in the EVs produced according to the invention, and 6 proteins significantly enriched in the control (data not shown).

[0312] Further GO term analysis in the subset of proteins enriched in the EVs produced according to the invention exhibited an enrichment factor of 1 .97 for the GO term “extracellular vesicle" and of 3.53 for the GO term “vesicle budding from membrane".

[0313] Furthermore, Fig. 9C shows that EVs produced according to the invention were found to be significantly enriched in mitochondria-associated proteins compared to EVs produced by 2D starvation. In detail, 4 mitochondria-associated proteins were found to be significantly enriched (Iog2 fold change > 2 and adjusted p-value < 0.05) in the EVs produced by 2D starvation (A1 , left panel), while 98 mitochondria-associated proteins were found to be significantly enriched (Iog2 fold change > 2 and adjusted p-value < 0.05) in the EVs produced according to the invention (B1 , right panel).

[0314] Taken together, the above results indicate that EVs produced according to the invention have a distinct proteomic profile compared to the control (particularly EVs produced by 2D starvation). Example 6: Wound healing assay

[0315] The EVs were produced in the same way as in Example 2B (Reynolds number = 312.5 or 416.7).

[0316] The protein composition of the EVs according to the invention at Reynolds number of 312.5 was compared with that of the control (EVs produced by 2D starvation) by performing a proteomic analysis, as described above.

[0317] The results of the proteome analysis are shown in Fig. 9A.

[0318] According to Fig. 9A, among proteins quantified in both conditions (represented by light grey circles), 109 proteins were associated with the GO term “wound healing" (G0:0042060) which were detected at significant levels (represented by dark grey circles). In addition, a total of 31 proteins were significantly enriched (Iog2 fold change > 2 and adjusted p-value < 0.05) in the control (A1 ; EVs produced by 2D starvation), while 36 proteins were enriched in the EVs produced according to the invention (B1 ).

[0319] Further GO term analysis of the subset of proteins enriched in the EVs produced according to the invention exhibited an enrichment factor of 2.25 for the GO term “wound healing".

[0320] The above results show that, regarding proteins associated with the GO term “wound healing" (G0:0042060), EVs produced according to the invention have a distinct proteomic profile compared to the control (particularly EVs produced by 2D starvation).

[0321] A wound healing assay was also performed, as described above in the section “Wound-healing assay’’. The results are shown in Fig. 9B.

[0322] According to the results in Fig. 9B, the EVs produced from spheroids (A2, B1 and B2) exhibited superior healing capacities compared to EVs produced by 2D starvation, highlighting the relevance of 3D models. This includes EVs produced according to the invention at Re = 312.5, whose wound healing-related proteomic profile showed significant differences compared to the control, as explained above.

[0323] More specifically, the EVs produced according to the invention (B1 and B2 corresponding to Re of 312.5 and 416.7, respectively) exhibit increased area coverage rate over time compared to the control with human skin fibroblasts alone (FBS 0%) or cultured with different concentrations of FBS (FBS 1 %, FBS 2%, FBS 4% and FBS 10%). In addition, the EVs produced according to the invention exhibit comparable or even increased area coverage rate over time compared to the control with EVs produced by 2D starvation or 3D starvation (A1 and A2).

[0324] It is also to be noted that EVs produced according to the invention at low Reynolds (Re = 208) demonstrated similar healing abilities to EVs produced by 3D starvation (not shown). However, increasing the Reynolds number (Re = 313 and Re = 417) enhanced their wound healing capabilities as shown in Fig. 9B. Taken together, the results indicate that the EVs prepared according to the invention are of interest for the development of improved wound healing therapies.

Claims

CLAIMS1. A method for producing extracellular vesicles from producer cells in a fluidic chip, wherein the fluidic chip comprises at least one first channel and at least one second channel, each channel having a longitudinal axis, an inlet and an outlet, wherein the outlet of the first channel is connected to the inlet of the second channel in a connection area, wherein the longitudinal axis of the first channel forms a non-zero angle with the longitudinal axis of the second channel in the connection area, the method comprising: a) passing a flow of liquid medium containing producer cells through the first channel, from the inlet to the outlet thereof; b) passing the flow of liquid medium through the second channel, from the inlet to the outlet thereof, thereby generating in the second channel a swirling flow extending from the inlet of the second channel, and c) collecting extracellular vesicles generated from the producer cells.

2. The method according to claim 1 , wherein the longitudinal axis of the first channel is substantially perpendicular to the longitudinal axis of the second channel in the connection area.

3. The method according to claim 1 or 2, wherein the flow of liquid medium at the inlet of the second channel is characterized by a Reynolds number of greater than 40, or of greater than 100, preferably greater than 200.

4. The method according to any one of claims 1 to 3, wherein the fluidic chip comprises two second channels, the outlet of the first channel being connected to the inlets of the two second channels in the connection area, the longitudinal axes of the two second channels being preferably aligned in the connection area.

5. The method according to any one of claims 1 to 4, wherein the fluidic chip comprises two first channels, the outlets of the first channels being connected to the inlet(s) of the second channel(s) in the connection area, the longitudinal axes of the two first channels being preferably aligned in the connection area.

6. The method according to any one of claims 1 to 5, wherein at least steps a) and b) are performed in a closed system and the liquid medium is not exposed to an external environment.

7. The method according to any one of claims 1 to 6, comprising repeating cycles of steps a) and b), using the same producer cells.

8. The method according to claim 7, comprising reversing the direction of the flow of liquid medium, preferably comprising successively:- flowing liquid medium through the first channel(s), from the inlet(s) thereof to the connection area, and then through the second channel(s), from the connection area to the outlet(s) thereof, thereby generating a swirling flow extending from the inlet the or each second channel; and- flowing liquid medium through the second channel(s), from the outlet(s) thereof to the connection area, and then through the first channel(s), from the connection area to the inlet(s) thereof, thereby generating a swirling flow extending from the outlet the or each first channel.

9. A device for producing extracellular vesicles from producer cells, comprising:- a fluidic chip comprising at least one first channel and at least one second channel, each channel having a longitudinal axis, an inlet and an outlet, wherein the outlet of the first channel is connected to the inlet of the second channel in a connection area, wherein the longitudinal axis of the first channel forms a non-zero angle with the longitudinal axis of the second channel in the connection area, and- a flow controller system connected to the fluidic chip and configured for circulating a same liquid medium multiple times through the first channel and second channel.

10. The device according to claim 9, wherein the longitudinal axis of the first channel is substantially perpendicular to the longitudinal axis of the second channel in the connection area.

11. The device according to claim 9 or 10, wherein the device is a closed system.

12. The device according to any one of claims 9 to 11 , wherein the fluidic chip comprises two first channels and two second channels, wherein the outletsof the first channels are connected to the inlets of the second channels in the connection area, and wherein, preferably: the longitudinal axes of the first channels are aligned in the connection area; and / or the longitudinal axes of the second channels are aligned in the connection area; and / or the longitudinal axis of each first channel is substantially perpendicular to the longitudinal axis of each second channel, in the connection area.

13. The device according to any one of claims 9 to 12, wherein the flow controller system is configured for repeatedly circulating the same liquid medium through the first channel(s), from the inlet(s) thereof to the connection area, and then through the second channel(s), from the connection area to the outlet(s) thereof, thereby generating a swirling flow extending from the inlet of the or each second channel.

14. The device according to any one of claims 9 to 13, wherein the flow controller system is configured for, successively: o flowing liquid medium through the first channel(s), from the inlet(s) thereof to the connection area, and then through the second channel(s), from the connection area to the outlet(s) thereof, thereby generating a swirling flow extending from the inlet the or each second channel; o flowing liquid medium through the second channel(s), from the outlet(s) thereof to the connection area, and then through the first channel(s), from the connection area to the inlet(s) thereof, thereby generating a swirling flow extending from the outlet the or each first channel.

15. The device according to any one of claims 9 to 14, wherein the flow controller system comprises at least one reservoir and at least one pump.

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