High-yield extracellular vesicle production from microorganism producer cells under rotating motion in baffled vessel

Abstract

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

Description

[0001] HIGH-YIELD EXTRACELLULAR VESICLE PRODUCTION FROM MICROORGANISM PRODUCER CELLS UNDER ROTATING MOTION IN BAFFLED VESSEL

[0002] TECHNICAL FIELD

[0003] The present invention relates to a method of producing extracellular vesicles from microorganism producer cells, and to a vessel 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. Extracellular vesicles are endogenously released by cells in a constitutive or inducible manner.

[0006] Many studies of EV production are based on the use of relatively large eukaryotic cells (such as animal cells or plant cells) as producer cells. However, EV production from smaller producer cells, such as microorganism producer cells, has been drawing recent attention. For example, it is now established that both gram-positive and gram-negative bacteria release spherical, lipid-based nanovesicles ranging in size of from 20 to 600 nm. These nanovesicles play an important role in communication pathways during the development of human pathologies and infectious diseases, stimulate the host immune response to infections and support microbiome homeostasis, and also influence cancer and antibiotic resistance disseminations. Consequently, such nanovesicles are emerging as promising candidates as therapeutic nanovectors.

[0007] Most of the current methods for the production of EVs rely on low-yield, time-consuming protocols, such as spontaneous EV release in a complete culture medium, resulting in a low EV production of less than 500 extracellular vesicles per cell over days. Serum starvation or oxygen deprivation are alternative methods, but the increase in EV production is about 2-fold, producing EVs in the range of 1000 per cell, in 2 to 3 consecutive days. Cell activation can also trigger EV release, such as with TNF-a-induced vesiculation, but again the yield is only 2-fold higher than that of spontaneous release. A recent trend based on a low- stress, hollow-fiber bioreactor achieves an increased EV yield of 2000-10000 EVs / cell; however, the production time is rather long, at over 10 days.

[0008] Several bioreactors for cell culture in the 3D configuration are known. For example, document JP200722203 discloses a cell-culturing and centrifuging tube comprising a separation tube and a stopper for sealing the upper opening of the separation tube, an opening in the stopper, a culture gas- penetrable nonwoven fabric to cover the opening, and at least one baffle on the inner wall surface of the separation tube, which is integrally formed with the separation tube.

[0009] There also exist commercially available systems for 3D cell culture, such as ClinoStar system and CERO 3D.

[0010] However, none of them proposes using the bioreactors for EV production.

[0011] Some methods for increasing the yield of extracellular vesicles, using a spinner flask bioreactor used for 3D cell culture, have also been developed.

[0012] For example, document FR3091296 discloses a fluidic system for loading a therapeutic or imaging agent into the lumen of extracellular vesicles (extracellular vesicles) from producer cells, comprising at least one vessel, a liquid medium contained by the vessel, producer cells, a liquid medium agitator and agitator speed control means adapted for the growth of the producer cells.

[0013] Document FR3091295 discloses a fluidic system for producing extracellular vesicles from suspended producer cells, comprising at least one vessel, a liquid medium contained by the vessel, suspended producer cells, a liquid medium agitator, agitator speed control means adapted for growth of the suspended producer cells.

[0014] Document FR3068361 discloses a fluidic system for producing extracellular vesicles from producer cells, including at least one container, a liquid medium contained by the container and producer cells, which also includes microcamers suspended in the liquid medium, the majority of producer cells being adherent to the surface of the microcarriers, and a liquid medium agitator, the agitator and the dimensions of the container being adapted to control a turbulent flow of the liquid medium in the container.

[0015] Document FR3112147 discloses a method for calibrating a fluidic system for producing extracellular vesicles from producer cells.

[0016] However, in these documents, the system is simply a repurposed commercial bioreactor, and is not designed specifically for EV production. In addition, the technique disclosed in the above documents cannot easily be adapted to EV production from microorganism producer cells, because of the size of the microorganism producer cells, the energy required to overcome the membrane barrier (such as the thick membranes of bacteria), and the requirement of the growth of the producer cells on 200-pm polymeric beads. It is known that a smaller vortex size ( / .e., smaller Kolmogorov length) can trigger a higher release of EVs, but smaller Kolmogorov lengths at the scale of the size of the microorganism is achievable only through higher rotation speeds. Thus, to manipulate individual cells and accommodate the small size of microorganisms such as bacteria, the rotational speed would need to be increased to over 2000 rpm, which is unattainable with current bioreactors.

[0017] Document CN113092642A discloses a rapid extraction device for EVs, comprising a precipitation assembly, a ring hoisting device and a chromatography assembly, wherein the ring hoisting device comprises a bracket, rotary generators and clamping rings.

[0018] However, the system disclosed in this document is designed for EV extraction (purification and separation) from a solution containing EVs but is not designed for EV production.

[0019] Document EP4015622A1 discloses a device for seeding cells which comprises a container with a wall, a bottom and a lid, wherein the container comprises a structured surface, and the structured surface is equipped to receive cells.

[0020] However, possibly due to the negligible stress experienced by the cells at the bottom of the well on the structured surface (where cells are seeded and remain), the resulting EV yield is not sufficient.

[0021] Most of the current methods for the EV production from microorganism producer cells rely on simple collection and purification of spontaneously released EVs from a culture broth. This method requires large amounts of cell materials and laboratory consumables, and is labor-intensive and time-consuming.

[0022] More recent approaches for increased yield include the use of detergents, toxic metabolites and antibiotics; the use of an enzymatic solution; increased temperature, or mechanical disruptions such as sonication. However, these methods result in the loss of important microbial antigens or the release of unsafe non-membrane components in the EV formulation. Genetic manipulations can increase the yield up to 200-fold, for example, in E. coir, yet, this method requires an additional gene disruption step.

[0023] In addition, all of the above methods suffer from limitations in terms of yield: if the number of EVs required for successful clinical translation is similar to that of eukaryotic cell-derived EVs, these existing methods are still inadequate.

[0024] Thus, there is a need for a more efficient method for producing extracellular vesicles from minimal materials (low volume) of microorganism producer cells.

[0025] SUMMARY OF THE INVENTION

[0026] The present invention relates to the following items. Item 1. A method of producing extracellular vesicles from microorganism producer cells, comprising the steps of : a) placing microorganism producer cells in a liquid medium in a vessel comprising a baffle structure fixed inside the vessel; b) rotating the vessel so as to generate extracellular vesicles from the microorganism producer cells; and c) collecting the generated extracellular vesicles; wherein the step b) of rotating the vessel comprises repeatedly changing the rotational motion of the vessel.

[0027] Item 2. The method of Item 1 , wherein the vessel rotates around a rotation axis which is substantially vertically oriented.

[0028] Item 3. The method of Item 1 or 2, wherein the microorganism producer cells are selected from bacteria, yeast, archaea, fungi, algae and combinations thereof.

[0029] Item 4. The method of Item 1 or 2, wherein the microorganism producer cells are bacteria.

[0030] Item 5. The method of Item 3 or 4, wherein the bacteria are selected from Escherichia coli BL21 (E. coli BL21 ), Bacillus subtilis (B. subtilis), Escherichia coli Nissle 1917 (E. coli Nissle 1917 /

[0031] Item 6. The method of any one of Items 1 to 5, wherein the extracellular vesicles are generated from microorganism producer cells in the form of individualized cells suspended in the liquid medium.

[0032] Item 7. The method of any one of Items 1 to 6, wherein the step b) of rotating the vessel comprises periodically changing the rotational motion of the vessel.

[0033] Item 8. The method of Item 7, wherein the frequency of changing the rotational motion of the vessel is from 0.001 to 10 Hz, preferably from 0.01 to 5 Hz, more preferably 0.1 to 1 Hz.

[0034] Item 9. The method of any one of Items 1 to 8, wherein the step b) of rotating the vessel is carried out at a maximum rotational speed of 50 to 8000 rpm, preferably 600 to 4000 rpm.

[0035] Item 10. The method of any one of Items 1 to 9, wherein step b) comprises repeatedly reversing the rotational direction of the vessel.

[0036] Item 11. The method of any one of Items 1 to 10, wherein step b) comprises repeatedly changing the rotational speed of the vessel.

[0037] Item 12. The method of any one of Items 1 to 11 , wherein step b) comprises intermittently rotating the vessel. Item 13. The method of any one of Items 1 to 12, further comprising a step of introducing a therapeutic agent or an imaging agent into the liquid medium.

[0038] Item 14. The method of any one of Items 1 to 13, wherein step c) is carried out by withdrawing the liquid medium including the producer cells from the vessel, and separating the extracellular vesicles from the withdrawn liquid medium, preferably by centrifugation.

[0039] Item 15. The method of any one of Items 1 to 13, wherein step c) is carried by withdrawing the liquid medium from the vessel without substantially withdrawing the producer cells, and separating the extracellular vesicles from the withdrawn liquid medium; wherein, preferably, decantation and / or centrifugation in the vessel is carried out prior to withdrawing the liquid medium.

[0040] Item 16. The method of any one of Items 1 to 15, further comprising repeating cycles of at least steps b) and c), using the same microorganism producer cells.

[0041] Item 17. The method of Item 16, wherein, at each cycle:

[0042] - the producer cells are withdrawn from the vessel, separated from the liquid medium, and placed again in the vessel together with a fresh liquid medium; or

[0043] - the liquid medium is withdrawn from the vessel, the producer cells substantially remaining within the vessel, and a fresh liquid medium is added to the vessel.

[0044] Item 18. The method of Item 17, further comprising a time interval of rest between two subsequent cycles, wherein the producer cells are kept in the vessel without rotation of the vessel.

[0045] Item 19. The method of any one of Items 1 to 18, further comprising a preliminary step of culturing the microorganism producer cells with an antimicrobial substance, preferably the microorganism producer cells comprising bacterial cells and the antimicrobial substance being an antibiotic.

[0046] Item 20. The method of any one of Items 1 to 19, wherein the baffle structure comprises one or more baffles fixed to an internal surface of the vessel.

[0047] Item 21 . The method of any one of Items 1 to 20, wherein the vessel comprises a cylindrical inner wall and a central axis.

[0048] Item 22. The method of Item 21 , wherein the baffle structure comprises one or more pairs of baffles, each pair of baffles comprising two diametrically opposed baffles relative to the central axis.

[0049] Item 23. The method of any one of Items 1 to 22, wherein the baffle structure comprises a plurality of plates fixed on struts, the struts being preferably oriented parallel to the central axis of the vessel, wherein, preferably, differently oriented plates alternate along the struts.

[0050] Item 24. The method of Item 21 , wherein the baffle structure comprises a plurality of baffles or a plurality of plates fixed on struts extending from the cylindrical inner wall towards the central axis.

[0051] Item 25. The method of Item 24, wherein the baffles or the plurality of plates fixed on struts do not extend to the central axis.

[0052] Item 26. The method of Item 24, wherein the baffles or the plates fixed on struts extend to the central axis, thereby dividing the inside of the vessel into a plurality of compartments, the compartments being in fluid communication with one another.

[0053] Item 27. The method of Item 21 , wherein the baffle structure comprises a plurality of baffles or a plurality of plates fixed on struts extending from the central axis towards the cylindrical inner wall.

[0054] Item 28. The method of any one Items 21 to 27, wherein part or all of the baffles are solid plates, or are meshed or perforated plates.

[0055] Item 29. The method of Item 28, wherein the part or all of the plates are oriented substantially parallel to the central axis of the vessel.

[0056] Item 30. The method of Item 28 or 29, wherein part or all of the plates are oriented at an angle from 10° to 80°, preferably from 30° to 60° relative to the central axis of the vessel.

[0057] Item 31 . The method of any one of items 1 to 30, wherein the vessel further comprises a coupling on an external surface of the vessel configured to be coupled to a rotating apparatus.

[0058] Item 32. The method of Item 31 , wherein the coupling comprises one or more grooves or ridges on an external surface of the vessel.

[0059] Item 33. The method of Item 31 or 32, wherein the vessel further comprises a closing cap.

[0060] 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 microorganism producer cells.

[0061] This is achieved because the method comprises placing microorganism producer cells in a liquid medium in a vessel comprising a baffle structure inside the vessel, and rotating the vessel so as to generate extracellular vesicles from the producer cells, wherein the step of rotating the vessel comprises repeatedly changing the rotational motion of the vessel.

[0062] The step of rotating the vessel eliminates the need to use an impeller or an agitator. In this case, the vessel diameter determines the production volume, satisfying the need for miniaturization, which has been difficult to achieve with a conventional stirred tank with an impeller or an agitator.

[0063] The present inventors have discovered that, by repeatedly changing the rotational motion of the vessel with a baffle structure, the flow inside the rotating vessel is disrupted, resulting in significantly higher EV production in comparison with the conventional EV production by simultaneous EV release and with the production from the same cells in a stirred tank (spinner flask) bioreactor operating at maximum regime.

[0064] In particular, the present invention provides the following advantages:

[0065] - a higher EV yield can be obtained compared with conventional simultaneous EV release from microorganism producer cells;

[0066] - upscaling can be easily performed, with a volume of up to 10 L for massive production from microorganism producer cells, such as bacterial cells;

[0067] - control of the flow conditions and thus the turbulent regime, is easier, allowing a more robust methodology in terms of the EV yield per cell;

[0068] - EV production can be easily coupled and streamlined with conventional microbial culture.

[0069] BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 shows one example of a vessel of the invention.

[0071] Figure 2a shows a top view of an example of a vessel having a baffle structure comprising solid baffle plates of the invention.

[0072] Figure 2b shows a diagonal top view of the vessel shown in Figure 2a, cut along a plane parallel to the central axis of the vessel.

[0073] Figure 3a shows a top view of another example of a vessel with a baffle structure comprising solid baffle plates of the invention.

[0074] Figure 3b shows a diagonal top view of the vessel shown in Figure 3a, cut along a plane parallel to the central axis of the vessel.

[0075] Figure 4a shows a top view of another example of a vessel with a baffle structure comprising meshed baffle plates of the invention.

[0076] Figure 4b shows a side view of the vessel shown in Figure 4a, cut along a plane parallel to the central axis of the vessel.

[0077] Figure 4c shows a diagonal top view of the vessel shown in Figure 4a, cut along a plane parallel to the central axis of the vessel.

[0078] Figure 5a shows a top view of another example of a vessel with a baffle structure comprising plates fixed on struts of the invention. Figure 5b shows a side view of the vessel shown in Figure 5a, cut along a plane parallel to the central axis of the vessel.

[0079] Figure 5c shows a diagonal top view of the vessel shown in Figure 5a, cut along a plane parallel to the central axis of the vessel.

[0080] Figure 6a shows a top view of another example of a vessel with a baffle structure comprising plates fixed on struts of the invention.

[0081] Figure 6b shows a side view of the vessel shown in Figure 6a, cut along a plane parallel to the central axis of the vessel.

[0082] Figure 6c shows a diagonal top view of the vessel shown in Figure 6a, cut along a plane parallel to the central axis of the vessel.

[0083] Figure 7a shows the increase of production of extracellular vesicles (EVs) over the control (by spontaneous release production) as a function of the rotational speed (in rpm), using a vessel having baffles of meshed plates (squares, M) and using a vessel having baffles of solid plates (circles, S), as tested in Example 1 below. The white squares or circles correspond to the producer cells subjected to a 24-hour maturation; and the grey squares or circles correspond to producer cells subjected to a 48-hour maturation. The y-axis represents the fold increase over the control. The x-axis represents the rotational speed of the vessel.

[0084] Figure 7b shows transmission electron microscopy images of EVs produced by the conventional simultaneous release (control), as tested in Example 1 below.

[0085] Figure 7c shows transmission electron microscopy images of EVs produced according to the invention, as tested in Example 1 below.

[0086] Figure 8a shows a fold increase of EVs over the control as a function of the rotational speed (in rpm), using the method of the invention and E. coli BL21 strains as producer cells, as tested in Example 1a below. The y-axis represents the fold increase over the control, and the x-axis represents the rotational speed.

[0087] Figure 8b shows the level of GFP fluorescence of EVs suspensions collected after using the method of the invention (from FRB-GFP-expressing E. coli strains) as a function of the rotational speed (in rpm), as tested in Example 1a below. The y-axis represents the level of GFP fluorescence (arbitrary units), and the x-axis represents the rotational speed. CTL corresponds to the control.

[0088] Figure 9a shows a fold increase of EVs over the control as a function of the rotational speed (in rpm), using the method of the invention and E. coli Nissle 1917 strains as producer cells, as tested in Example 2a below. The y-axis represents the fold increase over the control, and the x-axis represents the rotational speed. Figure 9b shows transmission electron microscopy images of the produced EVs, as tested in Example 2a below.

[0089] Figure 9c shows the number of EVs produced by the method of the invention, as tested in Example 2b below. The y-axis represents the number of EVs produced (particles / mL of the liquid medium), and the x-axis represents the rotational duration (6 h or 24 h). For each rotational duration, “C” (light grey circles) corresponds to the control, and “1” (light grey diamonds), “2” (grey diamonds), “3” (medium grey diamonds), “4” (dark grey diamonds), and “5” (black diamonds) represent EVs produced at a rotational speed of 500 rpm, 800 rpm, 1500 rpm, 2500 rpm, and 3500 rpm, respectively. The bars indicate the mean value for each corresponding condition. # indicates significance compared to the control, with p- values as follows : p < 0.05 (#), p < 0.01 (##), p < 0.001 (###), and p < 0.0001 (####). * indicates significance between two conditions connected by a bracket, with p-values as follows: p < 0.05 (*) and p < 0.01 (**).

[0090] Figure 10 shows a fold increase of EVs over the control as a function of the rotational speed (in rpm), using the method of the invention and B. subtilis strains as producer cells, as tested in Example 3 below. The y-axis represents the fold increase over the control, and the x-axis represents the rotational speed.

[0091] Figure 11a shows a fold increase of EVs over the control as a function of the rotational speed (in rpm), in the presence or absence of doxorubicin, as tested in Example 4a below. The y-axis represents the fold increase over the control, and the x-axis represents the rotational speed. 1 corresponds to the presence of doxorubicin and 2 corresponds to the absence of doxorubicin.

[0092] Figure 11b shows the number of EVs produced by the method of the invention, as tested in Example 4b below. The y-axis represents the number of EVs produced (particles / mL of the liquid medium), and the x-axis represents the doxorubicin concentration (pM). The left, middle and right sections correspond respectively to the rotational duration of 3h, 5h and 24h. For each rotational duration, “C” (light grey circles) corresponds to the control; “1” (dark grey circles) represents EVs produced at a rotational speed of 800 rpm; and “2” (black circles) represents EVs produced at alternating rotational speeds between 800 rpm and 1500 rpm (5 hours at 800 rpm followed by 3 hours at 1500 rpm). The bars correspond to the average value for each corresponding condition.

[0093] Figure 12 compares EV production between the method of the invention and a conventional spinner flask method, as tested in Example 5 below. Figure 12a shows results obtained using a Videodrop device while Figure 12b shows results obtained using a NanoSight NS 300. In each figure, the y-axis represents the number of EVs produced (particles / mL of the liquid medium), and the x-axis represents the production time (j.e., rotational duration for the method of the invention and stirring duration for the spinner flask method). For each production time, “C” (light grey boxplot), “1” (grey boxplot), “2” (medium grey boxplot), “3” (dark grey boxplot) correspond, respectively, to the control, a method using a 150- mL spinner flask, a method using a 350-mL spinner flask, and the method of the invention. * indicates significance between two conditions connected by a bracket, with p-values as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) and p < 0.0001 (****)

[0094] Figure 13 compares the optical density (OD) of the liquid medium after EV production using the method of the invention and the conventional spinner flask method, as tested in Example 5 below. The y-axis represents the OD, and the x- axis represents the production time (i.e., rotational duration for the method of the invention and stirring duration for the spinner flask method). The light grey circle, grey square, dark grey triangle, and black diamond correspond, respectively, to the control, a method using a 150-mL spinner flask, a method using a 350-mL spinner flask, and the method of the invention.

[0095] Figure 14a shows the results of a 3D Principal Component Analysis (PCA) of the protein composition dataset obtained from EVs produced using the method of the invention, as tested in Example 6. Light grey circles represent the control, grey circles represent the method of invention at a rotational speed of 500 rpm, and black circles represent the method of the invention at a rotational speed of 3500 rpm. The “Dim 1" axis, the “Dim 2’ axis, and the “Dim 3” axis correspond to Principal Component 1 (PC1 ), PC2, and PC3, respectively.

[0096] Figure 14b shows the results of the clustering of the protein composition dataset obtained from EVs produced using the method of the invention, as tested in Example 6. Labels “C-1” to “C-8” correspond to 8 control samples, labels “1 -1” to “1 -6” correspond to 6 samples of EVs produced using the method of the invention at a rotational speed of 500 rpm, and “2-1” to “2-6” correspond to 6 samples of the EVs produced using the method of the invention at a rotational speed of 3500 rpm.

[0097] DESCRIPTION OF EMBODIMENTS

[0098] The invention will now be described in more detail without limitation in the following description.

[0099] The term “extracellular vesicle” as used herein refers to a vesicle that is endogenously released by a producer cell in a constitutive or inducible manner. An extracellular vesicle generally has a diameter of from 30 nm to 500 nm. Examples thereof include, but are not limited to, exosomes, microvesicles and apoptotic bodies.

[0100] The term “cell” as used herein refers to the smallest fundamental structural and functional unit of living organisms, which can divide and multiply.

[0101] The term “producer cell” as used herein refer to a cell that is capable of secreting extracellular vesicles.

[0102] The term “vessel” as used herein refers to any type of container for containing liquid medium, such as a tube or a tank.

[0103] The term “vertical” or “vertically” as used herein refers to a direction which is perpendicular to the plane of the horizon and parallel to the direction of gravity.

[0104] Method for producing extracellular vesicles (EVs) from microorganism producer cells

[0105] The present invention provides a method of producing extracellular vesicles from microorganism producer cells.

[0106] By “microorganism producer cells" is meant producer cells that make up a microorganism, an organism of microscopic size, that can be seen individually under a microscope. The microorganism in question may be preferably unicellular.

[0107] The method comprises: a) placing microorganism producer cells in a liquid medium in a vessel comprising a baffle structure inside the vessel, b) rotating the vessel so as to generate extracellular vesicles from the microorganism producer cells; and c) collecting the generated extracellular vesicles, wherein the step b) of rotating the vessel comprises repeatedly changing the rotational motion of the vessel.

[0108] The method of the invention is performed ex vivo.

[0109] The vessel will be described in detail later. The vessel may be rotated owing to a rotating apparatus, which will be also described later.

[0110] By rotating the vessel, a flow of liquid medium within the vessel is achieved. Preferably, no addition of liquid medium and no withdrawal of liquid medium takes place during the rotation. Preferably, the vessel does not comprise a feeding line and / or a collecting line flu idical ly connected to the internal space of the vessel.

[0111] Preferably, the microorganism producer cells move freely under the effect of the flow of liquid medium. In other terms, preferably, the producer cells are comprised in the bulk of the liquid medium, and are not fixed to a wall of the vessel.

[0112] The rotation of the vessel is preferably such that the flow of liquid with the vessel is turbulent. Preferably, a turbulent regime characterized by a Reynolds number of greater than 1 ,000 or 2,000, or of greater than 7,000, preferably greater than 10,000 and most preferably greater than 12,000 or greater than 15,000 or greater than 20,000. At a point in time when there is a stationary regime, i.e. the average velocity of liquid in the vessel is equal to the velocity of the vessel, the global Reynolds number can be overall estimated as Re = R x V / v, wherein R is the internal radius of the vessel, V is the velocity of the vessel peripheral wall and v is the kinematic viscosity of the liquid. At a point in time when there is a transitional regime, i.e. a change in speed of rotation or a reversal of the rotational direction, the Reynolds number can be locally estimated as Re = W x V' / v, wherein W is a characteristic dimension of a baffle element (such as the radial length of a baffle plate), V’ is the relative velocity between the liquid and the baffle element, and v is the kinematic viscosity of the liquid. Preferably, the global Reynolds number and / or a local Reynolds number as defined above is within one of the ranges cited above during at least part of the duration of step b).

[0113] The microorganism producer cells may move relative to the vessel at a velocity which can reach a maximum value of at least 50 mm / s, at least 100 mm / s, at least 125 mm / s, at least 250 mm / s, at least 500 mm / s.

[0114] Preferably, there is no impeller in the vessel nor any other rotating or agitation element (such as a magnetic agitator), so that the flow of liquid is solely effected by the rotation of the vessel itself.

[0115] During step b), the vessel is preferably closed (such as by using the closure element described above). The vessel may be substantially filled with liquid medium during this step, i.e. may comprise no (or substantially no) gaseous headspace. In alternative variations, a gaseous headspace may be present. Preferably, the volume of gaseous headspace in the vessel, if present, may be less than 60%, or less than 40%, or less than 20%, or less than 10%, or less than 5%, or less than 2%, or less than 1 %, relative to the volume of the vessel.

[0116] In some embodiments, the microorganism producer cells may be prokaryotic or eukaryotic, preferably prokaryotic.

[0117] In some embodiments, the microorganism producer cells may be singlecelled. The microorganism producer cells may form a colony of single cells, or even a bacterial biofilm.

[0118] In some embodiments, the microorganism producer cells may be selected from bacteria, yeast, archaea, fungi, algae, and combinations thereof, preferably bacteria. In some embodiments, the microorganism producer cells may be Gramnegative bacteria, Gram-positive bacteria, or the combination thereof.

[0119] By “Gram-negative bacteria" is meant bacteria that have a thin layer of peptidoglycans in between two lipidic membranes (an inner membrane and an outer membrane). By “Gram-positive bacteria" is meant bacteria that have only one membrane that is surrounded by a thicker layer of peptidoglycans.

[0120] For example, Gram-negative bacteria include Escherichia coli (E. coli), Neisseria meningitidis (N. meningitidis). Gram-positive bacteria include Bacillus subtilis (JB. subtilis).

[0121] The microorganism producer cells may be either adherent to a culture medium or non-adherent to a culture medium (also referred to as suspension cells), preferably 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.

[0122] In some embodiments, when single cells are considered as producers, the concentration of the producer cells in the liquid medium in the vessel when the extracellular vesicles are generated is from 1 million to 10 billion, preferably 10 million to 10 billion, preferably 100 million to 10 billion, preferably 100 million to 1 billion, more preferably 100 million to 5000 million, further more preferably 100 million to 1000 million cells per milliliter.

[0123] 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 and within these vesicles.

[0124] In some embodiments, the extracellular vesicles produced according to the present invention have an average diameter of from 20 to 500 nm, preferably from 20 to 150 nm, more preferably from 20 to 100 nm.

[0125] 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).

[0126] The liquid medium used in the invention for the production of extracellular vesicles may be a conventional liquid medium, such as Luria-Bertani (LB) broth supplemented with antibiotics (for example, kanamycin for the E. coli BL21 strain, spectinomycin for the E. coli Nissle strain).

[0127] The vessel rotates around a rotation axis. It is noted that “rotating around a rotation axis" differs from orbital motion, such as a movement of a vessel, for example, on an orbital shaker, in which vessel follows in a circular trajectory without rotating around a rotation axis. Most preferably, when the vessel has a central axis (which will be defined later), the rotation axis corresponds to the central axis of the vessel.

[0128] In some preferred embodiments, the rotation axis is substantially vertically oriented (in parallel to the direction of gravity).

[0129] In some embodiments, direct visualization of flow trajectories of the liquid within the vessel is carried out during the rotation. In such a configuration, it is possible to track the movement of (e.g. fluorescent) beads (or of cells or particles tied to such beads or labeled with fluorescent markers) in a plane of the rotating vessel, as it rotates, with an ultrafast camera. This can provide thorough analysis of the shear stress experienced by the cells during the rotation, resulting in a better control of the flow conditions.

[0130] In some embodiments, step b) of rotating the vessel is carried out at a maximum rotational speed of 50 to 8000 rpm, preferably 500 to 5000 rpm, preferably from 1500 to 5000, preferably from 2500 to 3500. In some embodiments, the maximum rotational speed may vary from 50 to 100 rpm; or from 100 to 200 rpm; or from 200 to 300 rpm; or from 300 to 600 rpm; or from 600 to 1000 rpm; or from 1000 to 1600 rpm; or from 1600 to 4000 rpm; or from 4000 to 8000 rpm.

[0131] Step b) comprises repeatedly changing the rotational motion of the vessel. This means that the vessel does not rotate at a constant speed during the entirety of step b). The speed of rotation of the vessel changes multiple times during step b).

[0132] In some embodiments, step b) may comprise periodically changing the rotational motion of the vessel. This means that a certain pattern of rotational motion is repeated multiple times with a certain frequency.

[0133] The frequency of changing the rotational motion of the vessel may be from 0.001 to 10 Hz, preferably from 0.01 to 5 Hz, more preferably 0.1 to 1 Hz.

[0134] For example, step b) may comprise repeatedly (e.g. periodically) reversing the rotational direction (from clockwise to counterclockwise and conversely).

[0135] Alternatively or additionally, step b) may comprise repeatedly (e.g. periodically) changing the rotational speed of the vessel.

[0136] Alternatively or additionally, step b) may comprise intermittently rotating the vessel. This means that there are resting sequences within step b) when the vessel does not rotate. Rotating sequences alternate with resting sequences. Successive rotating sequences may be characterized by the same rotational direction or by different rotational directions. The duration step b) may be for example from 1 hour to 24 hours, e.g., approximately 1 , 2, 3, 4, 5, 5 to 8, 8 to 10, 10 to 12, 12 to 15, 15 to 20, 20 to 24 hours.

[0137] In some embodiments, the method may further comprise a preliminary step of culturing microorganism producer cells to a stationary phase (maturation). By “stationary phase" is meant a phase in which the growth rate of a population of the microorganism slows down significantly, which occurs after the exponential growth phase (log phase).

[0138] In some embodiments, the preliminary step may be performed inside the vessel. In this case, the preliminary step may comprise rotating the vessel, preferably at a maximum speed of rotation which is less than a maximum speed of rotation during step b).

[0139] The rotational speed during the preliminary step may be from 20 to 300 rpm, preferably from 50 to 150 rpm. During this preliminary step, the rotation motion may remain constant. Alternatively, repeated changes of rotational motion may take place, as describe above in connection with step b).

[0140] In other embodiments, the microorganism producer cells may be cultured outside the vessel, following a conventional cell culture protocol.

[0141] In some embodiments, the preliminary step may further comprising culturing the microorganism producer cells (either inside or outside the vessel) with an antimicrobial substance.

[0142] The antimicrobial substance may be a detergent, a toxic metabolite, an antibiotic, and combinations thereof.

[0143] Examples of the antibiotic include doxorubicin.

[0144] In some embodiments, the microorganism producer cells may comprise bacterial cells and the antimicrobial substance may be an antibiotic, such as doxorubicin.

[0145] In some embodiments, step c) of collecting the generated extracellular vesicles may be carried out by withdrawing the liquid medium including the microorganism producer cells from the vessel, and separating the extracellular vesicles from the withdrawn liquid medium.

[0146] 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.

[0147] In other embodiments, step c) may be carried by withdrawing the liquid medium from the vessel without substantially withdrawing the microorganism producer cells, and separating the extracellular vesicles from the withdrawn liquid medium, by any separation method as described above, preferably centrifugation.

[0148] In this case, decantation and / or centrifugation in the vessel itself is preferably carried out prior to withdrawing the liquid medium.

[0149] 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).

[0150] 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).

[0151] In some embodiments, the method may further comprise repeating cycles of at least step b) of rotating the vessel and step c) of collecting the generated extracellular vesicles, using the same microorganism producer cells.

[0152] For example, at each cycle, the microorganism producer cells may be withdrawn from the vessel; separated from the liquid medium (by way of, for example, centrifugation); and placed again in the vessel together with fresh liquid medium.

[0153] Alternatively, at each cycle, the liquid medium may be withdrawn from the vessel, the microorganism producer cells substantially remaining within the vessel (by way of, for example, centrifugation), and fresh liquid medium may be added to the vessel.

[0154] The method may further comprise a time interval of rest between two successive cycles.

[0155] The microorganism producer cells may be kept in the vessel without rotation of the vessel during the time interval of rest.

[0156] The microorganism producer cells may be also kept in a conventional cell culture apparatus, such as a cell culture flask, without rotation during the time interval of the rest.

[0157] The microorganism producer cells may be also kept in the vessel in rotation (this may enable cell growth during the time interval of the rest). If the vessel rotates during rest, the rotation may be constant. The rotational speed (if constant) or the maximum rotational speed (if not constant) is less than the rotational speed during the cycles of step b). The rotational speed during any rest step may be from 20 to 200 rpm, preferably from 50 to 150 rpm.

[0158] For example, the method may comprise 2, 3, 4, 5, 6, 7, 8, 9 or 10 cycles. The duration of each cycle (optionally step a) and steps b) and c)) may be from 1 hour to 24 hours, e.g., approximately 1 , 2, 3, 4 or 5, 5 to 8, 8 to 10, 10 to 12, 12 to 15, 15 to 20, 20 to 24 hours. The duration of the time interval of rest between successive cycles may be from 30 min to 24 hours, for example, approximately 30 min, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 10 hours, 15 hours, 20 hours, 24 hours.

[0159] In some embodiments, the method may further comprise a step of introducing a therapeutic agent or an imaging agent into the liquid medium.

[0160] The step of introducing a therapeutic agent or an imaging agent into the liquid medium may be performed before, during and / or after the steps a) to c).

[0161] The therapeutic agent can be any agent that can prevent, inhibit, or arrest the symptoms and / or progression of an infectious, an autoimmune disease, a fibrotic disease, an inflammatory disease, a neurodegenerative disease, or a heart and vascular disease, cancer or any other disease disclosed below. The therapeutic agent may be, for example, doxorubicin.

[0162] 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. The imaging agent may be, for example, a fluorescent marker.

[0163] Vessel for producing extracellular vesicles from producer cells

[0164] The method of the present invention may be performed in a vessel for producing extracellular vesicles (EVs) from microorganism producer cells. The vessel has an internal space for holding liquid medium. The vessel comprises a baffle structure inside the vessel, i.e. within the internal space. Preferably, the vessel comprises a coupling on an external surface configured to be coupled to a rotating apparatus as will be described in more detail below.

[0165] By “baffle structure" is meant one or more elements which are fixed within the vessel and which deviate the flow of liquid within the vessel. The baffle structure generally promotes turbulent flow in the vessel. By “fixed within the vessef’ is meant that the baffle structure is physically secured or anchored inside the vessel, preferably immobilized within the vessel. Preferably, the baffle structure remains stationary relative to the vessel during its rotation. In other words, when the vessel is rotated, the baffle structure rotates together with the vessel but does not rotate independently within the vessel.

[0166] Baffles, together with the tube geometry and rotating motors (speed), allow, through a combined effect, the Kolmogorov lengths to be reduced at the scale of microorganisms, thereby generating an adequate turbulent flow at this scale, particularly at the bacterial scale. Fig. 1 shows one example of a vessel of the invention.

[0167] The vessel 1 comprises a baffle structure inside the vessel (not shown in Fig. 1), which will be explained in detail later, and a coupling 3 on an external surface of the vessel configured to be coupled to a rotating apparatus.

[0168] The vessel may be made of a suitable material for the production of extracellular vesicles, e.g., a biocompatible resin, a biocompatible polymer, or a metal.

[0169] The vessel may be manufactured by 3D printing, injection molding, blow molding, or compression molding, preferably by 3D printing.

[0170] In some embodiments, the vessel has not been subjected to (or has not undergone) any surface treatment.

[0171] Preferably, and as illustrated, the internal space of the vessel is structurally delimited by a base and a peripheral wall extending from the base. The base is preferably substantially planar (flat). Preferably, the baffle structure does not cover the entirety of the peripheral wall. Preferably, the peripheral wall is smooth, i.e. does not include wells.

[0172] Preferably, the shape of the vessel is substantially cylindrical (or at least the shape of the internal space of the vessel is substantially cylindrical). In this case, the peripheral wall is a cylindrical inner wall, as shown on Fig. 1. The central axis of the vessel can then be defined as the axis of the cylinder. Preferably, the axis is perpendicular to the base and the cylinder is a right cylinder. Preferably, the cylinder is a circular cylinder, more preferably a right circular cylinder. In alternative embodiments, the peripheral wall may be in the shape of a non-circular cylinder. For example, if the base is substantially in the shape of a polygon (such as a square or a rectangle), the peripheral wall may be in the shape of a cylinder composed of a number of planar sections joined along respective edges thereof (such as four planar sections).

[0173] The capacity volume of the vessel may be suitably adjusted depending on, for example, the working volume, and the target number of the extracellular vesicles to be produced.

[0174] In some embodiments, the vessel may have a capacity of from 10 mL to 10 L. For example, the capacity of the vessel may be from 10 mL to 5 L, from 10 mL to 1 L, from 10 mL to 500 mL, or from 10 mL to 250 mL.

[0175] The diameter of the vessel may be suitably adjusted to accommodate different working volumes. For example, the internal diameter of the vessel may be from 2 to 25 cm. For example, the diameter of the vessel may be from 2 to 20 cm, from 3 to 15 cm, or from 3 to 10 cm. The height (maximal dimension in the direction parallel to the central axis) of the vessel may be from 1 to 30 cm, preferably from 2 to 20 cm, and more preferably from 3 to 10 cm.

[0176] The vessel may be provided with a closure element, such as a cap. In this case, the vessel may comprise a neck at the top part of the vessel (opposite the base at the bottom part), which is designed to receive the cap to seal the vessel.

[0177] The size and shape of the neck may vary depending on the type of vessel and the cap. Generally, the neck has a smaller diameter than the rest of the vessel (also referred to as “body” of the vessel), having a shoulder (transition between the body and the neck), which may be a curved or sloping part of the vessel where the diameter changes from the body to the neck. The cap may be fixed to the vessel by a threaded engagement, by a bayonet connection, by friction fitting, by a magnetic connection or the like.

[0178] The coupling may have any suitable geometry, provided that the geometry allows the vessel to be rotatably fixed to the rotating apparatus.

[0179] In some embodiments, the coupling may comprise one or more grooves or ridges on the external surface of the vessel.

[0180] For example, as shown in Fig. 1 , the coupling 3 may comprise a ridge (a raised or protruding line) running along the external surface of the vessel in the direction parallel to the central axis of the vessel (on the surface across from the peripheral wall in the internal space). The same is possible with a groove running along the external surface of the vessel in the direction parallel to the central axis of the vessel.

[0181] Alternatively or additionally, the coupling 3 may be provided on a bottom external surface of the vessel (across from the base in the internal space).

[0182] The baffle structure may extend from the peripheral wall of the vessel, towards the central axis of the vessel.

[0183] In some embodiments, the baffle structure may comprise one or more baffles. By “baffle" is meant a wall, which can be either substantially planar or curved, and preferably is substantially planar. The baffles are fixed in the internal space of the vessel, to an internal surface of the vessel. Preferably, they are fixed to the base and / or to the peripheral wall and are preferably integrally formed with the base and / or the peripheral wall. Each baffle acts as an obstacle to the flow of liquid within the vessel and is configured to deflect such flow of liquid. In the present text, when A is said to be “fixed to” B, it may mean “indirectly fixed to” ( / .e. A is fixed to B via an intermediate element); or, more preferably, “directly fixed to” ( / .e. A is fixed to B without any intermediate element between A and B). For example, the baffle structure may comprise one baffle, two baffles, three baffles, four baffles, five baffles, or six baffles, seven baffles, eight baffles, nine baffles or ten baffles which is / are fixed to an internal surface of the vessel.

[0184] When the baffle structure comprises two or more baffles, the baffles may be regularly spaced within the vessel or may be non-regularly spaced within the vessel.

[0185] In some embodiments, the baffles may be fixed on the internal surface of the vessel symmetrically relative to the central axis of the vessel.

[0186] The baffles may have either the same dimensions (e.g., height, thickness, length), or different dimensions from each other.

[0187] For example, the height of each baffle may be from 1 to 25 cm, preferably from 1 to 15 cm, and more preferably from 1.5 to 6 cm. The term “height” for a baffle as used herein refers to the maximum dimension of the baffle in the direction parallel to the central axis.

[0188] The length of each baffle may be from 1 to 12.5 cm, preferably from 1 to 5 cm, and more preferably from 1 to 2.5 cm. The term “length” for a baffle as used herein refers to the maximum dimension of the baffle perpendicular to the central axis (and preferably parallel to the base of the vessel).

[0189] The thickness of each baffle may be from 0.1 to 5 cm, preferably from 0.1 to 2.5 cm, and more preferably from 0.1 to 0.5 cm. The term “thickness” for a baffle as used herein refers to the dimension which is smaller than the maximum dimensions in the orthogonal directions and which is perpendicular to the length direction (and preferably parallel to the base of the vessel).

[0190] In some embodiments, the baffle structure comprises one or more pairs of baffles. Each pair of baffles may comprise two diametrically opposed baffles relative to the central axis of the vessel.

[0191] Preferably, the two baffles of each pair are identical in terms of dimensions.

[0192] For example, the baffle structure may comprise one pair, two pairs, three pairs, four pairs, or five pairs of the diametrically opposed baffles.

[0193] When the baffle structure comprises two or more pairs of baffles (for example, n pairs), the pairs may be arranged such that the baffles (for example, 2n baffles) are spaced apart equidistantly from each other. Alternatively, the pairs may be arranged such that the baffles (for example, 2n baffles) are spaced apart non-equidistantly from each other.

[0194] In some embodiments, the pairs of baffles may be symmetrically arranged relative to the central axis of the vessel.

[0195] The two baffles in each pair may have either the same dimensions among all pairs, or different dimensions among pairs. In some embodiments, the baffle structure may comprise a plurality of baffles extending from the peripheral wall towards the central axis.

[0196] The baffles may be directly joined together. For example, the baffles may extend to the central axis and be joined at the central axis. In this case, the baffle structure may divide the internal space of the vessel into a plurality of compartments, which are in fluid communication with one another.

[0197] Alternatively, the baffles are not directly joined together (but are only indirectly joined via the base or the peripheral wall). In particular, the baffles may not extend up to the central axis, thus leaving an unobstructed central space in the vessel. In this case, the baffles may extend a different distance towards the central axis, or the baffles may extend the same distance towards the central axis.

[0198] In other embodiments, the baffle structure may comprise a plurality of baffles extending from the central axis of the vessel towards the peripheral wall of the vessel.

[0199] In some embodiments, the baffles do not extend to the peripheral wall of the vessel, thus leaving an unobstructed peripheral space between the baffles and the peripheral wall.

[0200] Part of or all of the baffles may be plates or walls, i.e. substantially flat elements. These elements may have one dimension, namely the thickness, which is much smaller (such as at least 10 times or 100 times smaller) than the maximum dimensions in the orthogonal directions.

[0201] The plates or walls are preferably substantially planar but may alternatively be curved.

[0202] When a plate or wall is substantially planar, the thickness is the dimension of the plate perpendicular to the main plane of the plate or wall. The thickness may vary or be constant across the structure. If it varies, then any thickness values are meant to designate the average thickness.

[0203] The plates or walls may be solid plates or walls, or may be meshed or perforated plates or walls. The term “meshed plate” as used herein refers to a plate made from interlocking wires or struts (also referred to as a “grid”). The term “perforated plate” as used herein refers to a plate comprising openings or holes.

[0204] In the case of meshed or perforated plates or walls, the mesh size (size of the openings / holes), the uniformity of the openings / holes, the arrangement of the openings / holes can be adjusted suitably.

[0205] For example, the size of the openings / holes (for example, a diameter in the case of circular openings / holes or a diagonal in the case of polygonal openings / holes) in a meshed or perforated plate or wall may be from 0.1 to 5 cm, preferably from 0.1 to 1 cm, and more preferably from 0.1 to 0.5 cm. The plates or walls may have an outer shape (perpendicular to the direction of the thickness) which may be substantially square, rectangular, triangular, trapezoidal, diamond, pentagonal, hexagonal, octagonal, more generally polygonal, or which may be at least partly curved. The plates or walls may in particular be substantially perpendicular to the base and may be oriented substantially parallel to the central axis of the vessel.

[0206] Alternatively, the plates or walls may be oriented substantially perpendicular to the central axis of the vessel.

[0207] The plates or walls may also be oriented at an angle from 10° to 80°, preferably from 30° to 60° relative to the central axis of the vessel, and / or relative to the base.

[0208] For example, the plates or walls may be oriented at an angle of approximately 10°, 20°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 70°, or 80° relative to the central axis of the vessel and / or relative to the base.

[0209] All angle values in the present text are absolute values.

[0210] All baffles may be oriented similarly or not.

[0211] In some embodiments, the baffle structure may comprise a plurality of plates fixed on struts, the struts being preferably oriented parallel to the central axis of the vessel. Preferably, differently oriented plates alternate along the struts.

[0212] Below, several possible designs of the vessel of the invention will be explained more in detail by reference to the drawings, but it is understood that the design of the vessel can be configured and further modified to achieve optimal flow conditions within the vessel.

[0213] Making reference to Fig. 2a and Fig. 2b, the vessel 1 may be substantially cylindrical in shape and may comprise a baffle structure 2 inside the vessel and a coupling 3.

[0214] The vessel may further comprise a neck T at the top part of the vessel and a body 1”. The body 1” comprises the base 7 and peripheral wall 8 as described above in connection with Fig. 1

[0215] The baffle structure 2 may comprise three pairs of baffles 4, each pair of baffles comprising two diametrically opposed baffles relative to the central axis of the vessel. A different number of pairs of baffles is of course possible.

[0216] In this example, the baffle structure 2 comprises six baffles 4 in total fixed to an internal surface of the vessel (the peripheral wall 8 and / or the base 7), and the three pairs are arranged such that the six baffles are regularly spaced apart along the circumference of the cylinder. All of the baffles may be solid plates as shown, but the baffles may be also meshed or perforated plates, or a combination of solid plates, meshed plates and perforated plates.

[0217] The plates may have, but are not limited to, a rectangular shape.

[0218] The baffles may extend from the peripheral wall 8 towards the central axis, without extending to the central axis (in other words, the baffles do not reach all the way to the central axis of the vessel and leave an unobstructed central space).

[0219] The two baffles 4 in each pair may be identical within the pair, but the dimensions may differ from pair to pair. For example, the baffles may be plates having the same thickness and the same length, but may differ in height among the pairs. The term “height” as used herein (for a plate, baffle or other structure) refers to the maximum dimension of the plate, baffle or other structure in the direction parallel to the central axis.

[0220] The term “length” as used herein (for plate, baffle or other structure) refers to the maximum dimension of the plate, baffle or other structure perpendicular to the central axis (and preferably parallel to the base 7).

[0221] Alternatively, the two baffles 4 in each pair may have, among the pairs, the same height, length, and thickness (identical among pairs); or the same thickness but different heights and lengths; or the same height and thickness but a different length; or the same height and length but a different thickness; or the same height but different thickness and length; or the same length but different height and thickness; or different height, length, and thickness.

[0222] The planes of part of or all of the plates may be oriented substantially parallel to the central axis of the vessel.

[0223] For example, as shown in Fig. 2a and 2b, the planes of all of the plates may be oriented substantially parallel to the central axis of the vessel.

[0224] As a general remark, in all embodiments described therein, rounding or fillets may be provided at each edge or corner in order to avoid sharp edges which may induce local high shear areas which may damage any biological material present in the vessel.

[0225] In the example of Fig. 2a and 2b, the edges of the baffles 4 facing towards the central axis and preferably running parallel to the central axis are rounded.

[0226] The vessel shown in Fig. 3a and Fig. 3b is the same as the vessel of the example shown in Fig. 2a and Fig. 2b, except that there is in addition a fillet in the area where the baffles 4 are connected to the peripheral wall 8 and to the base 7, allowing for a smoother transition from each baffle to the peripheral wall 8 and the base 7. The arrangement, the orientation, and the dimension of the baffles may be as defined above.

[0227] The vessel 1 shown in Fig. 4a to Fig. 4c is the same as the vessel of the examples shown in Fig. 2 and Fig. 3, except for the baffle structure.

[0228] The baffle structure 2 may comprise six baffles 4 and a central strut 5. The central strut may be aligned with the central axis of the vessel and may be fixed to the base 7 of the vessel.

[0229] As shown in Fig. 4a to Fig. 4c, the baffles may be meshed plates having the same dimensions, but it is understood that they may be also perforated plates and / or may have different dimensions from each other. The plates are preferably substantially planar.

[0230] The baffles 4 may be fixed to the central strut 5 and extend from the central strut 5 towards the cylindrical inner wall. If planar, the baffles may be substantially perpendicular to the base 7.

[0231] The central strut 5 may have a height which is longer than the height of the body 1” of the vessel (the height in in the direction parallel to the wall of the vessel). In other terms, the central strut 5 may extend from the base 7 up to the area of the neck T. The top edge of each baffle ( / .e. the edge opposite the base 7) may be oriented at an angle different from 90°, such as approximately 45°, relative to the central axis (the central strut 5). As a result, the planes of the plates may have a trapezoid outer shape (see Fig. 4b).

[0232] However, it is understood that the central strut 5 may have a shorter height, and the planes of the baffles may also have another outer shape, such as a rectangle.

[0233] The plates may be regularly spaced apart around the central axis.

[0234] In Fig. 4b and Fig. 4c, the baffles do not extend to the peripheral wall 8, leaving an unobstructed space between each baffle and the peripheral wall 8. Alternatively, the baffles may divide the inside of the vessel into a plurality of compartments by extending from the central strut 5 to the peripheral wall 8. The compartments may be in fluid communication with one another through, for example, the openings of the meshed or perforated plates.

[0235] In some embodiments, the vessel does not comprise a central strut. In this case, the baffles 4 may extend from the central axis of the vessel towards (or to) the cylindrical inner wall (and they may be fixed together along the central axis) or may extend from of the cylindrical inner wall of the vessel towards (or to) the central axis. In some other embodiments, the baffles 4 may be fixed on the base 7 of the vessel, leaving an unobstructed peripheral space between the baffles and the peripheral wall and / or an unobstructed central space in the vessel.

[0236] The baffles 4 may be oriented radially relative to the central axis of the vessel.

[0237] Making now reference to the embodiment shown in Fig. 5a to 5c, the vessel 1 of this example is the same as the vessel of the examples shown in Fig. 2 to Fig. 4, except for the baffle structure.

[0238] As shown in Fig. 5a, the baffle structure 2 comprises a plurality of plates 4’ fixed on multiple struts 6.

[0239] The plurality of plates 4’ fixed on multiple struts 6 act as an obstacle to the flow of liquid within the vessel and is configured to deflect such flow of liquid.

[0240] The struts 6 are preferably cylindrical in shape and extend along respective strut axes. Their cross-section (perpendicular to the strut axis) may be circular, polygonal or other. In the illustrated embodiments, the cross-section is starshaped. Such a non-circular shape may further improve flow characteristics within the vessel.

[0241] The struts 6 are preferably parallel and are preferably oriented parallel to the central axis of the vessel.

[0242] The struts 6 on which the plates 4’ are fixed may be fixed on the base 7 of the vessel (see Fig. 5b and Fig. 5c). They may be arranged in arrays, each array supporting a different plurality of plates. The structure composed of an array of struts and the plurality of plates supported by this array can be referred to as a stack 2’.

[0243] The number of struts 6 may be adjusted depending on the number or the dimension of the plates 4’. For example, the struts 6 in each array may be arranged in three rows, each row having five struts, as shown in Fig. 5c. As other examples, the struts may be arranged in one, two, three, four or five rows, with two, three, four, five, six, seven, eight, nine or ten struts per row, in each array. The rows may for example be oriented radially relative to the central axis of the vessel.

[0244] The plates are preferably planar. The planes of each plate may be oriented at an angle from 10° to 80°, preferably from 30° to 60° relative to the central axis of the vessel; and / or may be oriented at an angle from 10° to 80°, preferably from 30° to 60° relative to the base 7 of the vessel.

[0245] For example, as shown in Fig. 5c, the plane of each plate facing the central axis of the vessel may be oriented approximately at an angle from 45° relative to the central axis of the vessel. The plates 4’ may be also differently oriented, and the differently-oriented plates 4’ may alternate along the struts 6.

[0246] Merely by way of example, successive plates along each array of struts may have a symmetrical orientation, relative to a plane between these successive plates (which is preferably parallel to the base 7 of the vessel). These differently- oriented plates 4’ may alternate along the struts, as shown in Fig. 5b and Fig. 5c, forming a zig zag pattern.

[0247] In some embodiments, the plates 4’ may extend from the peripheral wall towards the central axis.

[0248] For example, the plates 4’ may extend to the central axis and be joined at the central axis. In this case, the baffle structure may divide the internal space of the vessel into a plurality of compartments, which are in fluid communication with one another. Alternatively, the plates 4’ are not directly joined together (but are only indirectly joined via the base or the peripheral wall). In particular, the plates 4’ may not extend up to the central axis, thus leaving an unobstructed central space in the vessel. In this case, the plates 4’ may extend a different distance towards the central axis, or the plates 4’ may extend the same distance towards the central axis.

[0249] The baffle structure 2 may comprise one or more baffle stacks 2’.

[0250] The baffle stacks 2’ may comprise successive plates 4’ along each array of struts 6.

[0251] The stacks 2’ may be spaced apart equidistantly from each other. Alternatively, the stacks 2’ may be spaced apart non-equidistantly from each other. They may in particular be regularly spaced around the central axis.

[0252] In some embodiments, the stacks 2’ may be symmetrically arranged relative to the central axis of the vessel (by pairs).

[0253] The stacks 2’ may have either the same dimensions, or different dimensions from each other.

[0254] The baffle structure 2 may comprise one or more pair of stacks 2’, for example, three pairs of stacks 2’, each pair comprising two diametrically opposed stacks relative to the central axis of the vessel.

[0255] The pairs of stacks 2’ may be arranged such that all the stacks are spaced apart equidistantly from each other.

[0256] For example; the baffle structure 2 may comprise three pairs of stacks 2’, thus in total six stacks 2’, and the three pairs may be arranged such that the six stacks are spaced apart equidistantly from each other.

[0257] The stacks 2’ in each pair may have the same dimensions within the pair, but may have different dimensions from one pair to the other. Alternatively, the stacks 2’ in each pair may have different dimensions.

[0258] An unobstructed area around the central axis may be present. In some cases, the plates 4’ may be fixed to the peripheral wall 8. In other cases, an unobstructed space may be present in an annular area between the stacks 2’ and the peripheral wall 8.

[0259] Making reference to Fig. 6a to 6c, the vessel 1 of this example is the same as the vessel of the examples shown in Fig. 5a to 5c, with the following modifications.

[0260] As shown in Fig. 6a, in addition to the vessel shown Fig. 5a to 5c, the vessel further comprises a central strut 5.

[0261] The central strut 5 is preferably aligned with the central axis of the vessel and may be fixed to the base 7 of the vessel.

[0262] The plates 4’ may be fixed to the struts 6 of one array and to the central strut 5.

[0263] Each stack 2’ may extend from the central strut towards or to the peripheral wall 8. In some cases, the plates 4’ may be fixed to the peripheral wall 8. In other cases, an unobstructed space may be present in an annular area between the stacks 2’ and the peripheral wall 8.

[0264] System for producing extracellular vesicles (EVs) from microorganism producer cells

[0265] The method of the present invention may be also performed in a system for producing extracellular vesicles (EVs) from microorganism producer cells.

[0266] The system comprises a vessel as defined above, and a rotating apparatus.

[0267] The vessel may be configured to be rotationally fixed to the rotating apparatus by keying the coupling of the vessel to a corresponding coupling on the rotating apparatus. If the coupling of the vessel comprises grooves, the coupling on the rotating apparatus may comprise corresponding ridges. If the coupling of the vessel comprises ridges, the coupling on the rotating apparatus may comprise corresponding grooves.

[0268] In some embodiments, the rotating apparatus may comprise a rotating element such as a cup configured for receiving the vessel. The coupling on the rotating apparatus may be present on an internal surface of the cup which is in contact with an external surface of the vessel.

[0269] Alternatively or in combination, the rotating apparatus may comprise a securing mechanism for maintaining the vessel rotatably fixed within the rotating element, comprising for example tightening means using screws, or a friction fit engagement.

[0270] In some embodiments, the outer shape of the vessel is non circular and the cup has a corresponding shape, thus ensuring that the vessel is rotationally fixed relative to the cup without any requirement for respective couplings on the external surface of the vessel and on the internal surface of the cup. For example, the vessel may comprise one or more planar external surfaces in addition to the base (e.g. the vessel may have an overall cuboid shape), or may comprise an ellipsoidal surface. In this case, the cup has a complementary shape so as to ensure contact between one or more external surfaces of the vessel and one or more internal surfaces of the cup.

[0271] In some embodiments, the rotating apparatus may comprise a drive mechanism and a control unit for controlling the drive mechanism.

[0272] The rotating element (e.g. cup) may be actuated by the drive mechanism. The rotating apparatus may comprise a stationary part which may include the drive mechanism and the control unit.

[0273] Preferably, the control unit may be configured for implementing the rotation of the vessel as required by the method of the invention (which will be explained below).

[0274] The control unit may comprise one or more processors coupled to a storage medium, as well as a computer program comprising instructions stored thereon, for performing the various steps described in more detail below. The control unit may receive input from sensors in or associated with the rotating apparatus and / or input from the user. The control unit may process the input data and, as a result, provide instructions to the drive mechanism. In some embodiments, part of or all of the control unit may be provided not in the rotating apparatus itself but in a separate computing device.

[0275] Application of the produced extracellular vesicles

[0276] The present invention may also relate to the use of EVs produced as above for imaging purposes, for therapeutic purposes, for diagnostic purposes, for preventive purposes, for nutritional purposes, and / or for wellness purposes, such as personalized medicine; immunotherapy; regenerative medicine; cell therapy; 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; and / or microbiota-related uses. 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.

[0277] In particular, microbial EVs are promising candidates as therapeutic EVs: they have the capability to trigger immune responses that could confer protection, leading to the proposition of using them as vaccines (e.g. for meningitis, pneumonia or tuberculosis).

[0278] Interestingly, research indicates that EVs derived from E. coli have a tropism for tumoral tissue and can induce long-term antitumoral immune responses leading to tumor eradication, paving the way for their use in cancer immunotherapy.

[0279] Furthermore, the extracellular vesicles produced according to the present invention may be used in a probiotic and / or a prebiotic for microbiota-related use. The microbiota-related uses may include, for example, preventive, nutritional, and / or wellness-related interventions, aimed at enhancing the beneficial functions of the microbiota. Particularly, the communication between the gut microbiota and the host is also mediated by vesicles secreted by the microbiota itself, which have the capability to enter systemic circulation and disseminate to distant organs and tissues.

[0280] EXAMPLES

[0281] Extracellular vesicle production in vessels of the invention

[0282] All vessels were custom made by 3D printing (capacity: 20 to 80 mL).

[0283] The vessels were coupled to stepper motors, which are easily controllable, robust, and cost-effective.

[0284] In the performed experiments, the frequency of reversal of rotational direction was set at 0.4 Hz.

[0285] The microorganism producer cells were grown, as a preculture, overnight according to a conventional method (at 37°C under shaking at 220 rpm) in appropriate medium. In Examples 1 and 2a, prior to the EV production, the preculture was diluted and grown to a stationary phase at 37°C under shaking at 220 rpm (maturation), and then transferred to the vessel. In Example 2b, the preculture was diluted to an optical density at 600 nm (ODeoo) of 0.1 and directly placed transferred the vessel.

[0286] Each vessel was sealed with a vented cap and the rotation system was placed inside the incubator at 37°C for the complete duration of the experiment (3h). Bacterial growth before and after extracellular vesicle production (at Oh and at 3h, respectively in Examples 1 and 2a; and at 6h and at 24h, respectively in Example 2b) was assessed by measuring the optical density (OD) at 600 nm of the culture in each condition using the EnSight Plate Reader and by bacterial count.

[0287] Extracellular vesicle production by spontaneous release (control)

[0288] Bacteria were cultured in Erlenmeyer flasks under 220 rpm orbital shaking at 37°C for a period of time. The bacteria and debris were then removed and the supernatant containing EVs was concentrated.

[0289] FRB-GFP protein overexpression

[0290] A small cytoplasmic protein FRB (rapamycin-binding domain of mTOR) was tagged with a green fluorescent protein (GFP) in E. coli B21 strains by conventional genetic manipulation. In order to trigger the expression of the recombinant FRB-GFP, isopropyl [3-D-1 thiogalactopyranoside (IPTG, Sigma- Aldrich) at a concentration of 0.5 mM was added during the 24h maturation time, when the OD600nm measured is in between 0.4 and 0.6 of both control and rotation cultures.

[0291] The measurement of this protein in the produced EVs was carried out using an EnSight Multi Mode Plate Reader (PerkinElmer) in fluorescence mode at A = 510 nm after bacteria removal from the suspension by centrifugation.

[0292] Extracellular vesicle quantification, qualitative analysis and purification

[0293] Collected samples from any production method as above were centrifuged at 17,000 g for 20 minutes at 4°C and the supernatant containing the EV suspension was collected. Nanoparticle Tracking Analysis of the processed sample suspension was performed with a Videodrop (manufactured by Myriade) or with a NanoSight NS 300 to obtain the number and size distribution of the produced extracellular vesicles.

[0294] The final yields were calculated, after a 3h production, by subtracting the yield of the control condition from the yield under rotation, and then dividing the difference by the resulting subtraction of the yield of the control condition at Oh from the yield of the control condition after 3h of production, thereby obtaining the fold change relative to the control.

[0295] The samples were then filtered (Centricon Plus 70 100kDa, manufactured by Merck Millipore), purified on qEV columns (qEVoriginal / 70 nm Gen 2 Column, manufactured by Izon Science) and concentrated (Amicon Ultra-4 10kDa, manufactured by Sigma) before cryo-TEM observation.

[0296] Example 1 : EV production from gram-negative bacterial cells in a vessel having baffles of solid plates and in a vessel having baffles of meshed plates

[0297] Vessels having the configuration shown in Fig. 3a and 3b (vessels having baffles of solid plates) and the configuration shown in Fig. 4a to 4c (vessels having baffles of meshed plates) were manufactured as described above in the section “Extracellular vesicle production in vessels of the invention”.

[0298] The experimental conditions were as follows:

[0299] - Microorganism producer cells: E. coli (BL21 strain)

[0300] - Capacity of the vessel: 60 mL

[0301] - Maturation time: 24 hours or 48 hours

[0302] - Rotational duration (step b): 3 hours

[0303] - Rotational speed: 500 to 4000 rpm

[0304] EV production was performed under the above conditions, with varying rotational speeds, and compared between the vessels of the invention and a conventional culture method (see the section ” Extracellular vesicle production by spontaneous release (control)"), using the same cells.

[0305] The results are shown in Fig. 7a. Overall, at least a 5-fold increase, sometimes even a 10-fold increase, was obtained over the control. In addition, the results also show the dependence of EV yield on the rotational speed and initial bacterial maturation culture time. Generally, an increased EV yield was obtained with a higher rotational speed and a longer maturation time.

[0306] The increase in the number of EVs in the production according to the invention compared to the control was also gualitatively evident in electron microscopy images (Fig. 7b for the control, and Fig 7c for the EVs produced according to the invention). Importantly, the EVs produced according to the invention possess physical integrity (Fig 7c), indicating the adeguacy of the turbulent flow conditions to induce non-damaging bacteria stimulation.

[0307] Example 1 a: Cytoplasmic protein transport and transfer through EVs

[0308] The cytoplasmic protein transport and transfer through the produced EVs were also tested. The overexpression of FRB-GFP protein was performed in E. coli cells (see the section “FRB-GFP protein overexpression"). Then, EV production was performed in the same way as in Example 1 , except that the maturation time was fixed to 24 hours. Fig. 8a shows that the EV yield consistently exceeded a 10-fold increase compared to the control at a high rpm (> 1500 rpm), and EV production increased with the rotational speed, as in Example 1.

[0309] In addition, Fig. 8b shows that the level of GFP fluorescence associated with FRB proteins in the suspension of produced EVs was higher than in the control (CTL), and that the level of GFP fluorescence increased as the rotational speed increased.

[0310] The subsequent confocal imaging of tumoroids incubated with these GFP fluorescent EVs demonstrated the co-localization of the EVs with a model tumoroid (images not shown).

[0311] These results prove the possibility of fluorescent cytoplasmic protein transport and transfer through the produced EVs.

[0312] Example 2a: EV production from gram-negative bacterial cells in a vessel having a baffle structure

[0313] The same experimental conditions as in Example 1 were used, except that the vessels having the configuration shown in Fig. 3a and 3b were used; the microorganism producer cells were changed to E.coli Nissle 1917 strain; and the maturation time was fixed to 24 hours.

[0314] The results are shown in Fig. 9a. Consistent with the results of Example 1 , the dependence of the EV yield on the rotational speed was again observed. Moreover, the EV yield increased impressively by over 25-fold at high rotational speed (> 1500 rpm), compared to the control (Fig. 9a). The produced EVs were also physically intact, as observed by cryo-electron microscopy (Fig. 9b).

[0315] Example 2b: EV production from gram-negative bacterial cells in a vessel having a baffle structure

[0316] The same experimental conditions as in Example 1 were used, except for the following changes:

[0317] - Microorganism producer cells: E. co / / (Nissle 1917 strain)

[0318] - Vessel configuration: as shown in Fig. 3a and Fig. 3b

[0319] - Capacity of the vessel: 0 to 50m L

[0320] - Initial optical density of the culture: OD = 0.1

[0321] - Rotational duration (step b): 6 to 24 hours

[0322] - Rotational speed: 500 to 3500 rpm

[0323] The number of EVs produced was counted using a Videodrop device (see the section “Extracellular vesicle quantification, qualitative analysis and purification") and was subjected to a two-way ANOVA test to assess statistical significance. The results were compared between the vessels of the invention and a conventional culture method (see the section "Extracellular vesicle production by spontaneous release (control)"), using the same cells.

[0324] The results are shown in Fig. 9c. Overall, the number of produced EVs was at least 3 times higher, in some cases 5 times higher, than the control. Consistent with previous results, EV production increased with the rotational speed.

[0325] Example 3: EV production from gram-positive bacterial cells in a vessel having a baffle structure

[0326] The same experimental conditions as in Example 2a were used, except that the microorganism producer cells were changed to a B. subtilis strain.

[0327] The fold increase in EV yield over the control, normalized by the OD values, is shown in Fig. 10. The results show a 10-fold increase in the EV yield in comparison with the control, and an increased EV yield at a higher rotational speed.

[0328] Example 4a: Dual production stimulation with antibiotics and a turbulent flow

[0329] The same experimental conditions as in Example 1 a were used, except that doxorubicin (antibiotic) was added at the start of the maturation at a final concentration of 50 pM.

[0330] The fold increase of EV yield was calculated based on the control condition which did not receive any hydrodynamic stimulation (grown in Erlenmeyer flask) or doxorubicin incubation.

[0331] Fig. 11a shows that EV production increased in the presence of doxorubicin, potentially exceeding more than 25 times in comparison with the control.

[0332] Example 4b: Dual production stimulation with antibiotics and a turbulent flow

[0333] The same experimental conditions as in Example 2b were used, except that doxorubicin (an antibiotic) was added at the start of the maturation at a final concentration of 10 pM or 50 pM.

[0334] The number of EVs produced was counted as in Example 2b, as shown in Fig. 11b

[0335] Fig. 11b shows that EV production increased in the presence of doxorubicin, potentially reaching the EV concentrations up to 15 times higher than that those observed without doxorubicin incubation (0 pM). EV production increased with doxorubicin concentration. Consistent with previous results, EV production also increased with rotational speed and rotational duration. Example 5: Comparative experiments using a spinner flask method

[0336] EV production using the method of the invention was compared with EV production using a conventional stirred tank (spinner flask).

[0337] For EV production by the method of the invention, a vessel as shown in Fig. 3a and Fig. 3b was used, and the rotational speed was set at 1500 rpm.

[0338] For EV production with spinner flasks, spinner flasks having a capacity of 150 mL and 350 mL were used, and the rotational speed of the stirrer (or impeller) inside the flask was set at 200 rpm, which is a common attainable rotational speed in the conventional spinner flasks.

[0339] The rest of the conditions (microorganism producer cells, initial OD, and so on) were the same as in Example 2b.

[0340] Spinner flasks are generally known for allowing a massive EV production from eukaryotic cells cultured on microcamers by introducing a turbulent flow, which in turn generates small stress vortices. Such spinner flasks are known, for example, in WO 2020 / 136361 A1 and WO 2020 / 136362 A1.

[0341] The number of EVs produced was counted using a Videodrop device and a NanoSight NS 300 (see the section “Extracellular vesicle quantification, qualitative analysis and purification") and compared with the control (see the section "Extracellular vesicle production by spontaneous release (control)").

[0342] The results are shown in Fig.12a and Fig.12b.

[0343] The results show that EV production increased impressively, potentially reaching EV concentrations up to 15 times higher compared to production under the control condition (no hydrodynamic stimulation in Erlenmeyer flask) and production using spinner flasks.

[0344] Thus, when using non-eukaryotic cells (in this case, bacterial cells) as producer cells, EV production using spinner flasks resulted in very low yield, comparable to that of the control condition, regardless of the volume of the spinner flask.

[0345] This lower EV yield obtained by the spinner flask method can be explained by the Kolmogorov equations, which describe the energy dissipation of turbulent flow through a cascade of vortices of decreasing sizes. The vortex size ( / .e., Kolmogorov length) is thus a key parameter in turbulence-triggered EV production: smaller vortices trigger a significantly higher release of EVs but require an increased rotation speed. Indeed, smaller Kolmogorov lengths at the scale of the bacterial cell size, are achievable only at higher rotation speeds, which remain unattainable with conventional spinner flasks. The optical density (OD) at 600 nm of the liquid medium in each condition was also measured using a EnSight Plate Reader.

[0346] The results are shown in Fig. 13.

[0347] Fig. 13 shows a higher optical density, indicating an enhanced and faster bacterial growth, in the condition of the method of the invention (black diamond), compared to the control (light grey circle), and two conditions with a 150-mL spinner flask (grey square) and a 350-mL spinner flask (dark grey triangle).

[0348] Example 6: Proteome studies of produced EVs

[0349] EVs were produced under same experimental conditions as in Example 2a, except that the rotational speed was set at 500 rpm and at 3500rpm.

[0350] Proteins were extracted from the produced EVs using the Micro BCA protein assay kit following a standard protocol. The extracted protein contents of the EVs were analyzed by Liquid Chromatography (LC) with a Vanquish Neo LC system coupled to an Orbitrap Astral mass spectrometer (MS) to obtain proteomics data.

[0351] The differences in protein composition among the different conditions were analyzed using principal component analysis (PCA), using the myProMS v3.10 open software, using total protein abundance for proteins with three or more distinct peptides. PCA is a statistical method to simplify a large number of variables (in this case, variances in terms of the protein composition) and reduce them to a few new variables (called principal components) that capture the main patterns of variation in the data.

[0352] The first three principal components (PC1 , PC2, and PC3) were used for visualization in a 3D scatter plot, as shown in Fig. 14a.

[0353] Fig. 14a shows that PC1 (“Dim 1") explained 53.58% of the variance, and PC2 (“Dim 2”) and PC3 (“Dim 3”) explained 16.36% and 7.22% of the variance, respectively.

[0354] Fig. 14a also shows that the EVs produced using the method of the invention were grouped separately from the control, clearly indicating distinct EV populations between EVs produced by the method of the invention and EVs produced under the control condition (in Erlenmeyer flasks without any hydrodynamic stimulation).

[0355] Clustering analysis was performed on the same proteomics data, using total protein abundance for proteins with three or more distinct peptides. The clustering analysis partitioned the protein composition variables into different groups: proteins clustering within the same group show high similarity, and proteins clustering in different groups show low similarity. The results, as shown in Fig. 14b, demonstrate that EVs produced under the control condition (no hydrodynamic stimulation) were clustered together on one side of the dendrogram while EVs produced by the method of the invention (with hydrodynamic stimulation) were clustered together on the other side, thereby confirming the results of the PCA.

[0356] Taken together, these results suggest that the method of the invention can imprint a specific proteome signature on the produced EVs.

Claims

37CLAIMS1. A method of producing extracellular vesicles from microorganism producer cells, comprising the steps of : a) placing microorganism producer cells in a liquid medium in a vessel comprising a baffle structure fixed inside the vessel; b) rotating the vessel so as to generate extracellular vesicles from the microorganism producer cells; and c) collecting the generated extracellular vesicles; wherein the step b) of rotating the vessel comprises repeatedly changing the rotational motion of the vessel.

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

3. The method of claim 1 or 2, wherein the microorganism producer cells are selected from bacteria, yeast, archaea, fungi, algae and combinations thereof, preferably bacteria.

4. The method of claim 3, wherein the bacteria are selected from Escherichia coli B21 (E. coli B21), Bacillus subtilis (B. subtilis), Escherichia coli Nissle 1917 (E. co / / Nissle 1917).

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

6. The method of claim 5, wherein the frequency of changing the rotational motion of the vessel is from 0.001 to 10 Hz, preferably from 0.01 to 5 Hz, more preferably 0.1 to 1 Hz.

7. The method of any one of claims 1 to 6, wherein the step b) of rotating the vessel is carried out at a maximum rotational speed of 20 to 8000 rpm, preferably 600 to 4000 rpm.

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

389. The method of any one of claims 1 to 8, further comprising a preliminary step of culturing the microorganism producer cells with an antimicrobial substance, preferably the microorganism producer cells comprising bacterial cells and the antimicrobial substance being an antibiotic.

10. The method of any one of claims 1 to 9, wherein the baffle structure comprises one or more baffles fixed to an internal surface of the vessel.

11. The method of any one of claims 1 to 10, wherein part or all of the baffles are solid plates, or are meshed or perforated plates.

12. The method of any one of claims 1 to 11 , wherein the baffle structure comprises a plurality of plates fixed on struts, the struts being preferably oriented parallel to the central axis of the vessel, wherein, preferably, differently oriented plates alternate along the struts.

13. The method of any one of claims 1 to 13, wherein the vessel comprises a cylindrical inner wall and a central axis.

14. The method of claim 13, wherein the baffle structure comprises a plurality of baffles or a plurality of plates fixed on struts extending from the cylindrical inner wall towards the central axis, and / or extending from the central axis towards the cylindrical inner wall.

15. The method of any one of claims 1 to 14, wherein the vessel further comprises a coupling on an external surface of the vessel configured to be coupled to a rotating apparatus.

16. Use of the extracellular vesicles produced by the method of any one of claims 1 to 15 for imaging purposes, for therapeutic purposes, for diagnostic purposes, for preventive purposes, for nutritional purposes, and / or for wellness purposes.

Images