METHOD FOR RELEASE OF PLATELETS IN TURBULENT FLOW AND PLATELET RELEASE SYSTEM FOR CARRYING OUT THE METHOD - Patent application

Abstract

The invention relates to a method for releasing platelets from megakaryocytes contained in a fluid (F) using a system (1). The system (1) used comprises an inner cylinder (11) with a cylindrical wall (12) and a hollow outer cylinder (13) located radially outside the inner cylinder and having a cylindrical wall (14), both cylinders being separated by a space (15) intended to receive a fluid (F). The method comprises a step (100) of feeding a fluid (F) containing megakaryocytes into the space (15) and a step (200) of generating a turbulent flow of the fluid (F) at least partially in the space (15) by rotating the inner cylinder (11) about its axis while the outer cylinder (13) is stationary or by moving the two cylinders (11, 13) in opposite directions about their axes, thereby obtaining a second fluid (F') enriched with platelets.

Description

[Technical field]

[0001] The present invention relates in particular to a method for releasing platelets from a fluid containing megakaryocyte progenitor cells, the method being carried out by a platelet releasing system designed for this purpose. The invention further relates to such a system for releasing platelets. The invention is particularly suitable for the in vitro production of platelets on an industrial scale and for the production of extracellular vesicles. [Background technology]

[0002] Until now, the general solution to perform in vitro platelet release techniques using miniaturized systems, usually measuring tens of micrometers to centimeters, has been to reproduce the natural platelet release mechanism that occurs in the human body, hence these methods are called biomimetics.

[0003] The mechanisms of platelet release in the human body are still being studied extensively. Platelet release is known to occur during the process of megakaryocyte fragmentation and / or cytoplasmic extenders becoming platelets. This highly regulated in vivo process occurs naturally in blood when the forces of blood flow drive cytoplasmic extenders through the endothelial barrier and then platelet precursors mature and enter the pulmonary microcirculation. Megakaryocytes play an important role as progenitor cells. These mechanisms operate at a very slow rate in the human body, and platelet production in conventional biomimetic microfluidic reactors also occurs at a relatively slow rate.

[0004] US Patent No. 5,399,666 proposes a microfluidic bioreactor with a structure with micropore walls that mimic endothelial passages as fluid circulates inside it. In normal operation, the fluid flow state is laminar and the shear rate measured at the height of the micropore walls corresponds to physiological shear rates. The processing time is usually several hours. US Patent No. 5,399,666 is similar, in which a membrane structure plays the role of the micropore walls of US Patent No. 5,399,666.

[0005] In recent years, efforts have been made to industrialize the method of releasing platelets, leading to the development of new types of bioreactors. Patent document 3 discloses an example of a bioreactor intended for this type of use. The proposed system aims to increase the number and lifespan of healthy platelets. It comprises a reservoir for a fluid containing megakaryocytes and at least one means for generating turbulence in the reservoir so that the platelets are released. In one embodiment, this means consists of a blade moving back and forth along the reservoir. The disadvantage of such a system lies in the fact that the blade is highly likely to collide with the megakaryocytes contained in the fluid, which damages a significant proportion of the megakaryocyte progenitor cells obtained during the use of the system. In fact, the presence of the blade in the volume through which the fluid moves is already a cause of collisions with the megakaryocyte progenitor cells. The movement of the blade is then supposed to give a stronger impact on the megakaryocyte progenitor cells. Thus, a large proportion of the obtained platelets may have a significantly reduced activity or even be absent. Furthermore, the cover 1a of the culture / release reservoir passes through the axis, allowing the blade to move back and forth. Such an arrangement would likely not maintain sterility within the reservoir.

[0006] US Patent No. 5,999,663 discloses another method for releasing platelets from megakaryocytes, which was realized by the team of US Patent No. 5,999,663. The bioreactor includes a blade that moves back and forth and up and down so as to stir the culture solution containing megakaryocytes. As in the method described in US Patent No. 5,999,663, the movement of the blade in the space where the fluid circulates where the platelet release occurs may cause collisions with megakaryocyte progenitor cells, which may reduce the activity of the platelets released from the megakaryocyte progenitor cells. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO2014 / 107240A1 [Patent Document 2] WO2018 / 165308A1 [Patent Document 3] WO2019 / 09364A1 [Patent Document 4] EP3372674A1 (equivalent to WO2017 / 077964) Summary of the Invention

[0008] The present invention aims to overcome the above-mentioned drawbacks and for this purpose proposes a method for releasing platelets from megakaryocytes contained in a fluid, the method being carried out by a system comprising two concentric cylinders, an inner cylinder with a cylinder wall and a hollow outer cylinder located radially outside the inner cylinder, the outer cylinder comprising a base cylinder wall at the level at which the second cylinder closes, the cylinder(s) being separated by a space free of mechanical parts, the space intended to receive a fluid, the method then comprising the following steps: - (100) supplying a fluid containing megakaryocytes to said space; - (200) rotating the inner cylinder about its axis while the outer cylinder is stationary, or alternatively moving the two cylinders in opposite directions about their axes, generating at least partially a turbulent fluid flow in said space to obtain a second fluid enriched with platelets; Includes.

[0009] The platelet release method under turbulent flow conditions used in the present invention can prevent platelet degradation due to the structure of the platelet release system, thus significantly improving the platelet release efficiency and foreseeing industrial application. The concentric cylinders of the system used in the method according to the present invention are separated by a space free of mechanical parts so that platelets are released into the fluid when one or more cylinders move about their axis without affecting other parts or elements of the system located in the inter-cylinder space.

[0010] Moreover, since the cylinder(s) are concentric, a certain air gap is guaranteed along the walls. The air gap is the distance between the two walls. It is a parameter that can be manipulated to better control the flow conditions in the system to allow the release of platelets. At the same time, the value of the air gap itself can further improve the efficiency of the platelet release. It is also possible to influence the speed of movement of one or more cylinders in order to obtain turbulent conditions of the fluid. In this way, when carrying out the method, the operating parameters can be adjusted to obtain conditions specific to turbulent flow conditions.

[0011] According to the various aspects of the invention, the following aspects are taken into consideration, either jointly or individually:

[0012] - If the inner cylinder is moved while the outer cylinder is stationary, Reynolds number Re i (see formula 1) is set to 1400 or more, and the inner cylinder is moved.

number

[0013] - When the two cylinders move in opposite directions, the inner cylinder and the outer cylinder, respectively, Reynolds number Re greater than 1000 i (See Equation 2) Reynolds number Re greater than 1000 o (see Equation 3)

number

number

[0014] - the two cylinders are separated by a distance d less than 5 mm; - The two cylinders are separated by a distance d between 2mm and 4mm; - the two cylinders are separated by a distance d of about 3 mm; - in step 100, continuously supplying a fluid to the space, the system including an inlet for filling the space with said fluid and an opening for discharging the platelet-enriched fluid, said opening being located at the level of the base; - the residence time of the fluid in said space during step 200 is between 4 minutes and 6 minutes, preferably about 5 minutes; - the inner cylinder and the outer cylinder include other peripheral inner walls forming a first pattern and a second pattern, respectively, the first pattern and the second pattern being nested within one another such that the other walls of the inner cylinder and the other walls of the outer cylinder are concentric and at least partially opposed to one another; - This is also how extracellular vesicles are released.

[0015] According to an alternative embodiment of the invention, the method of the invention relates to a method for releasing platelets from megakaryocytes contained in a fluid, said method being carried out by a system comprising two parallel planar walls separated by a space without mechanical parts, said space intended to receive a fluid, said walls being movable, said method comprising the following steps: (100) Supplying a fluid containing megakaryocytes to the space; (200) moving one of the (two) planar walls in its plane while the other wall is stationary, or moving the two walls in opposite directions, each wall moving in its own plane, to generate at least partially a turbulent fluid flow in the space to obtain a second fluid enriched with platelets; Includes.

[0016] According to the various aspects of the invention, the following aspects are taken into consideration, either jointly or individually: That is: - the first planar wall is a wall of the first conveyor belt and the second planar wall is a wall of the second conveyor belt; - If two walls move in opposite directions, they move in opposite directions with the same speed; - The two walls move at a speed of about 1 meter per second; - If two walls are moving in opposite directions, they move with a speed difference of up to 10% relative to the average value; - Extracellular vesicles are also released. [Brief description of the drawings]

[0017] [Figure 1a] FIG. 1 shows a schematic representation in perspective of a platelet release system which allows the implementation of a method according to a first embodiment of the invention. [Figure 1b] FIG. 1 shows a schematic representation in cross-section of a platelet release system allowing the implementation of a method according to a first embodiment of the invention. [Figure 2a] FIG. 2 shows a schematic diagram of the platelet release system of FIG. 1 in a top view, including the velocity distribution between the inner and outer cylinders as they rotate. The lower diagram shows the radial velocity distribution of the fluid in space. [Figure 2b] FIG. 2 is a schematic diagram of the platelet release system of FIG. 1 in a top view, including the velocity distribution between the inner and outer cylinders as the inner cylinder rotates. The lower diagram shows the radial velocity distribution of the fluid in space. [Figure 3a]2 is a schematic diagram illustrating an alternative embodiment of the platelet release system shown in FIG. 1 in perspective view. [Figure 3b] 2A-2C are schematic cross-sectional views of an alternative embodiment of the platelet release system shown in FIG. 1; [Figure 4a] FIG. 2 shows a schematic representation in perspective view of a platelet release system which allows the implementation of a method according to a second embodiment of the invention. [Figure 4b] FIG. 2 shows a schematic representation in cross-section of a platelet release system allowing the implementation of a method according to a second embodiment of the invention. [Figure 5a] FIG. 4C is a schematic diagram of an alternative embodiment of the platelet release system shown in FIGS. 4a and 4b. [Figure 5b] FIG. 4C is a schematic diagram of an alternative embodiment of the platelet release system shown in FIGS. 4a and 4b. [Figure 6] This is a diagram based on Andereck's (1986) diagram. [Figure 7] FIG. 1 shows the count of double positive platelets for a reference sample and also for samples processed with the method according to the invention using the platelet release system according to the embodiment shown in FIGS. 1a and 1b with reverse rotation of the cylinder and an air gap of 3 mm, 4 mm and 5 mm (from left to right). [Figure 8] FIG. 1 shows the number of double positive platelets for a reference sample (left dots) and for samples treated with the method according to the invention using the platelet release system shown in FIGS. 1a and 1b with an air gap of 3 mm, when the cylinders rotate in opposite directions (middle dots) and when only the inner cylinder rotates (right dots). [Figure 9] FIG. 1 shows an example of a two-cylinder device for carrying out the method of the present invention. [Figure 10]a) Number of released CD41+ and CD42+ platelets for passage of 5,000 beads in flow cytometry, b) platelet count per cell on day 7 of culture, and c) platelet count per CD34+ plated. The white columns represent results obtained with a pipette, the grey columns represent results obtained with a configuration in which the two cylinders rotate in opposite directions, and the black columns represent results obtained with a configuration in which only the inner cylinder rotates. [Figure 11] Figure 11 shows the number of platelets expressing the major glycoproteins on their surface depending on the release mode. Figure 11(a) compares the percentage of positive platelets in a configuration with two counter-rotating cylinders (grey column) with a configuration with only the inner cylinder rotating (black column). Figure 11(b) compares the configuration with two counter-rotating cylinders (grey column) with a configuration using a pipette (white column). Figure 11(c) compares the configuration with only the inner cylinder rotating (black column) with a configuration using a pipette (white column). [Figure 12] Figure 1. Pre-activated (resting) state of released platelets and their activation performance when placed in the presence of an antagonist (thrombin): the white columns represent the results obtained with a pipette, the grey columns represent the results obtained with a configuration in which the two cylinders rotate in opposite directions, and the black columns represent the results obtained with a configuration in which only the inner cylinder rotates. The figures on the left correspond to the results analyzed for the GPIIb-IIIa pre-activation marker, and the figures on the right correspond to the results analyzed for the expression of P-selectin on the platelet surface. [Figure 13] 1 shows the platelet recirculation phenomenon (ratio of platelet count to platelet count over 3 minutes as a function of time) for cultured platelets in vivo in mice obtained using the method of the invention in the case of only the inner cylinder rotating (square on the left) compared to a mixture of platelet concentrate obtained using the method of the invention (solid circle on the left) and cultured platelets, and in the case of the two cylinders rotating in opposite directions (square on the right) compared to a mixture of platelet concentrate (solid circle on the right). [Figure 14]14A-14B show immunofluorescence images of released platelets. Immunofluorescence images of platelets released by manual pipetting (FIG. 14a), by the method of the present invention when only the inner cylinder rotates (FIG. 14b), and by the method of the present invention when the two cylinders rotate in opposite directions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present invention relates to a method for releasing platelets from a fluid F containing megakaryocytes, and a system 1 for carrying out said method, as illustrated in Figures 1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b, 5a and 5b.

[0019] The fluid of interest F is a medium containing a cell population obtained from immortalized or non-immortalized cell lines at various stages of differentiation, including, for example, megakaryocyte progenitor cells, megakaryocytes. Megakaryocytes, or megakaryocytic cells, are large blood cells (up to 100 μm, 30 μm in culture) with long extensions that, upon maturation, are called cytoplasmic extensions, or platelet precursor cells.

[0020] The mechanisms involved in platelet formation are still the subject of much research. Among them, platelet release is known to occur during the process of fragmentation of megakaryocytes Mk and / or cytoplasmic extension bodies Ck to become platelets. This is a highly regulated in vivo process that occurs naturally in blood by the force of blood flow. Megakaryocytes Mk play a key role since they are progenitor cells. However, the process by which platelets are released in the body is still not fully understood, and many questions remain about the precise role of blood flow in endothelial crossing and platelet formation. This process has been reproduced in vitro using microfluidic systems, which has allowed certain in vivo mechanisms to be supported by microfluidic experiments. This process can also be reproduced in vitro using a manual pipette or the device introduced in the introduction of this specification, allowing a deeper understanding of the mechanisms involved in platelet release (Strassel et al., "Aryl hydrocarbon receptor-dependent enrichment of a high potential to produce propalets", Blood, 5 May 2016, vol. 127, n18). Producing platelets in vitro allows a better understanding of the mechanisms involved in platelet formation. However, the efficiency of platelet release is often lower than that obtained in vivo. Therefore, the present invention aims to reproduce the fragmentation process of megakaryocyte Mk and / or cytoplasmic extension body Ck for a method of releasing platelets in turbulent flow conditions.

[0021] Before describing the invention in more detail, it should be noted that in addition to enabling the release of platelets, the invention also allows the release of other functional cytoplasmic elements from the platelets thus released, such as extracellular vesicles. Currently, extracellular vesicles are recognized as vectors of biological material that allow the transfer of biological material contents between cells. Extracellular vesicles are nanovesicles that originate from the cell membrane and are secreted physiologically into the extracellular medium. They consist of several subtypes (exosomes, microvesicles, apoptotic bodies) and vary in size from 30 nm to 1 μm. By way of comparison, platelets typically have a diameter of 3.5 μm to 5 μm.

[0022] The principles behind this technique for releasing platelets are introduced in the following paragraphs. There are many ways to generate turbulence, but the method described in this invention uses a mechanism similar to that used to generate instabilities in a system of two concentric cylinders. These mechanisms, which underlie the operation of turbomachines and the formation of planets, are commonly used in large-scale systems. It should be remembered that in the field of the invention, despite the greater adoption of pre-industrial bioreactors, there is a general trend towards using small-scale systems such as those described in the introduction of this specification.

[0023] See the paper by C. Andereck et al., "Flow regimes in a circular Couette system with independently rotating cylinders," J. Fluid Mech. 1986, and the paper by Grossmann et al., "High-Reynolds Number Taylor-Couette Turbulence," Annu. Rev. Fluid Mech., 2016. In what follows, we focus more specifically on the phase diagram of Andereck et al., which is given in these publications and reproduced in FIG. 6 of the present application. The phase diagram of Andereck et al. shows the various flow conditions that can occur in a system of two concentric cylinders where the angular velocity, and therefore the Reynolds number, can be varied to generate turbulence. The Andereck phase diagram is plotted on the horizontal axis as the Reynolds number of the inner cylinder (in some publications, the Reynolds number is also called the Reynolds number). i or R i ), the Reynolds number of the outer cylinder (Re o or R o ) are shown. Thus, we define the flow states according to the parameters mentioned above, and each type of flow is i , Re o )

[0024] The inventors of the present invention have demonstrated that a platelet release phenomenon exists from a fluid F containing megakaryocyte cells under turbulent flow conditions, and further, that the platelet release phenomenon is significantly improved in certain highly specific areas of the Andereck diagram.

[0025] Reynolds number Re of the inner cylinder in absolute value i (see formula 4) is advantageously greater than or equal to 500, and the Reynolds number Re of the outer cylinder o These flow conditions ("spiral turbulence" or "featureless turbulence" in the Andereck diagram) occur when (see equation 5), advantageously, is greater than or equal to 1000.

number

number

[0026] The other of these conditions corresponds to a turbulent Taylor vortex flow ("turbulent Taylor vortex" in the Andereck diagram), henceforth referred to as "vortex". This condition corresponds to a configuration in which the outer cylinder is stationary and the inner cylinder rotates about its axis. To achieve the turbulent Taylor vortex condition, the Reynolds number Re of the inner cylinder must be i (see equation 6) is preferably 1400 or greater.

number

[0027] These are the conditions under which we have demonstrated an improvement in the platelet release process in a system of two circular concentric cylinders, where platelet release occurs as soon as the flow conditions become turbulent, thus increasing the Re i and Re o It will be appreciated that the range of will be greater than the ranges stated above.

[0028] As will become clear from the following paragraphs, the inventors of the present invention have demonstrated that platelet release can be achieved not only by a system having two circular concentric cylinders, but also by other systems, all of which have in common with the system described above two substantially parallel walls separated by a predefined distance obtained in experiments.

[0029] 1a and 1b, an example of an embodiment of a system 1 includes two concentric cylinders 11, 13 separated by a space 15 that is free of mechanical parts. The system 1 includes an inner cylinder 11 and a hollow outer cylinder 13 located radially outside the inner cylinder 11, the inner cylinder 11 and the outer cylinder 13 being concentric. By "concentric cylinders" it is meant that the inner cylinder 11 and the outer cylinder 13 are coaxial, i.e. the axes of the inner cylinder 11 and the outer cylinder 13 are coincident.

[0030] The inner cylinder 11 corresponds to a solid body including two parallel bases 11a, 11b of substantially circular shape separated by a distance h corresponding to the height of the inner cylinder 11. Along its height h, the inner cylinder 11 defines an outer side surface, hereinafter referred to as the "first wall 12". Incidentally, the first wall 12 has a cylindrical shape.

[0031] The outer cylinder 13 is a hollow solid including a base 13a that is substantially circular in shape, from which extends a second wall 14 that is cylindrical in shape, similar to the first wall 12. Thus, in the embodiment shown, the outer cylinder 13 is externally delimited by the base 13a and the second cylindrical wall 14, which delimit a cylinder housing 16 having an opening at the level of its upper surface, where the terms "upper" and "lower" are not intended to be limiting, but are merely intended to facilitate understanding of the invention with reference to the figures.

[0032] As previously indicated, the housing 16 accommodates the inner cylinder 11 so that it can be arranged concentrically with the outer cylinder 13. However, the inner cylinder 11 does not occupy the entire housing 16, since a part of the housing 16 is left to form a space 15. The space 15 separates the first cylindrical wall 12 from the second cylindrical wall 14, and more broadly the inner cylinder 11 from the outer cylinder 13, so that the space 15 has the shape of a hollow cylinder bounded on the inside by the inner cylinder 11 and on the outside by the outer cylinder 13. The space 15 thus forms a reservoir capable of accommodating a fluid F containing megakaryocyte cells, which are precursor cells of platelets. Moreover, according to the invention, the space 15 is free of mechanical parts. The advantages of this arrangement are explained below.

[0033] As can be seen from the previous paragraph, the first cylindrical wall 12 is spaced from the second wall 14 by a constant distance d. Similarly, the base 11a of the first cylinder, which is parallel to the base 13a of the outer cylinder 13, is also spaced from the outer cylinder by a distance d', which is not necessarily the same as the distance d. In the remainder of the description, the distance d separating the first cylindrical wall 12 from the second wall 14 will be called the "air gap". The air gap is set during the design of the system 1. It is constant in order to control the flow conditions in the space 15. This means that if the air gap is set by the dimensions of the first cylinder 11 and the second cylinder 13, the optimum air gap is not selected according to said dimensions, but rather the dimensions of the inner cylinder 11 and the outer cylinder 13 are selected according to the desired air gap (but not solely). Significant improvements in the efficiency of platelet release from fluid F have been demonstrated depending on the selected gap and further depending on the velocity of either or both of first cylindrical wall 12 and second cylindrical wall 14 .

[0034] It should be noted that the rotation speed v of one or both of the cylinders depends on the dimensions of the inner cylinder 11 and the outer cylinder 13. By definition, the linear speed v of each of the inner cylinder 11 and the outer cylinder 13 depends on the dimensions of the cylinders, more specifically, their respective diameters. Therefore, the dimensions of the inner cylinder 11 and the outer cylinder 13 are selected according to the desired air gap, but can also be selected taking into account the rotation speed of the inner cylinder 11 and the outer cylinder 13. For example, if a person skilled in the art has a choice of system for driving the cylinders, specifically a limit on the driving force, he or she must take this new constraint into account when designing the system 1.

[0035] The system 1 comprises a frame, not shown in Figures 1a and 1b (but shown in Figure 9), which allows the first cylinder 11 and second cylinder 13 to be positioned as described above, so that although the inner cylinder 11 appears to be suspended in Figures 1a and 1b, it is in fact held in its intended position by the frame, and similarly for the outer cylinder 13.

[0036] In fact, the movement of the inner cylinder 11 and the outer cylinder 13 displaces the fluid F contained in the space 15. In this respect, the system 1 also comprises a system (not shown) for driving the inner cylinder 11 and the outer cylinder 13. The driving system comprises a power supply system, one or more motors and a speed control system for controlling the motor or motors. This driving system allows a defined speed to be applied to each of said cylinders 11, 13 individually. The method according to the invention can now be implemented in various embodiments, which are described in more detail below. For example, the driving system can consist of a magnetic driving system rotated by an electric motor controlled by a speed regulation system.

[0037] According to a first embodiment of the method according to the present invention, after the space 15 is filled with the fluid F containing the megakaryocytes, the inner cylinder 11 and the outer cylinder 13 move in opposite directions about their axes so as to generate a turbulent flow of the fluid F in the space 15. That is, the inner cylinder 11 and the outer cylinder 13 perform a rotational movement, and the rotation direction of the inner cylinder 11 is opposite to that of the outer cylinder 13.

[0038] Figure 2a shows the distribution of velocities in the space 15 when the inner cylinder 11 and the outer cylinder 13 are moving in opposite directions. The system 1 shown in Figure 2a is an enlarged top view of the system 1 shown in Figures 1a and 1b. Although the first wall 12 and the second wall 14 appear flat, they are cylindrical. Furthermore, the illustrated system 1 is not to scale, but rather the dimensions have been chosen merely to better illustrate the local flow conditions in the space 15. In the following, the flow velocity of the fluid F is denoted as v, which is measured in the vicinity of each wall 12, 14, i.e. in the area RP, respectively. 12 , R.P. 14 At the height of the space 15, the flow velocity v of the fluid F is relatively fast, and in the central region RC of the space 15, the flow velocity v is almost zero. 12 and Area RP 14 The flow of the fluid F at the wall RP is turbulent. Therefore, on average, the fluid F is radially static between the first wall 12 and the second wall 14, but is radially static at the wall RP. 12 and Wall RP 14 There is a lot of turbulence in the region close to

[0039] Advantageously, turbulence is generated by having inner cylinder 11 and outer cylinder 13 each with a Reynolds number greater than 1000. However, this is a preferred embodiment and is not a prerequisite for obtaining platelet release.

[0040] It is convenient if the inner cylinder 11 and the outer cylinder 13 are separated by a gap d of less than 5 mm. Since there is a space 15, it is not zero. Within this value range (0 mm < d ≤ 5 mm), the platelet release efficiency can be improved. However, outside this range, especially when d > 5 mm, the platelet release efficiency is very low. Therefore, compared with a reference sample with very low platelet release, if the gap value is in the range of 0 mm < d ≤ 5 mm, the platelet release efficiency can be improved.

[0041] In practice, since the gap d is related to the rotation speed v of the inner cylinder 11 and the outer cylinder 13, it is convenient to define the system 1 with a predetermined pair (d, v). The rotation speed of the cylinder is itself linked to the Reynolds number of the cylinder. Each shape (i.e., dimension) of the system 1 is related to one or more optimal pairs (d, v), that is, pairs that can generate a turbulent flow capable of optimizing the release of platelets from the fluid F. However, the optimal rotation speed v essentially depends on the shape of the system 1, while the optimal gap does not depend on this shape. Therefore, as described above, after determining the optimal gap d that can achieve the highest platelet release efficiency through experiments, the shape of the system 1 is selected considering the gap. Therefore, as shown in FIGS. 1a and 1b, it will be clear that there are many different shapes in the system 1 that can satisfy this constraint. When selecting a shape, the type of system available for driving the inner cylinder 11 and the outer cylinder 13 is also taken into account. A user who wants to manufacture the system 1 but only has a drive system for rotating a small system will be restricted by the amount of fluid F to be processed.

[0042] According to an example of an embodiment of the system 1, the inner cylinder 11 has an outer diameter of 2 cm to 6 cm, the outer cylinder 13 has an inner diameter of 2 cm to 6 cm, and the height h of the outer cylinder 13 is 5 cm to 6 cm. As described above, in the case where such a system 1 has a gap in the range of 0 mm < d ≤ 5 mm and the inner cylinder 11 and the outer cylinder 13 move in opposite directions at a rotation speed v of 0.5 m / s to 2 m / s, an improvement in platelet release efficiency can be achieved. For example, in the system 1 where the outer diameter of the inner cylinder 11 is 27 mm, the inner diameter of the outer cylinder 13 is 33 mm, and the height of the outer cylinder 13 is 60 mm, when the gap is in the range of 0 mm < d ≤ 5 mm and the inner cylinder 11 and the outer cylinder 13 move in opposite directions at a rotation speed v of 0.8 m / s to 1.5 m / s respectively, an improvement in platelet release efficiency can be achieved.

[0043] It is more convenient if the first cylinder 11 and the second cylinder 13 are separated by a gap d of 2 mm to 4 mm. This configuration is advantageous in that when this method is executed, the platelet release efficiency is further improved compared to the method implemented in the system 1 where the gap is outside this range. Taking the system 1 where the outer diameter of the inner cylinder 11 is 2 cm to 6 cm, the inner diameter of the outer cylinder 13 is 2 cm to 6 cm, and the height h of the outer cylinder 13 is 5 cm to 6 cm as an example, the optimal rotation speed v of the inner cylinder 11 and the outer cylinder 13 is 0.9 m / s to 1.2 m / s, and the cylinders move in opposite directions respectively.

[0044] It is extremely convenient if the first cylinder 11 and the second cylinder 13 are separated by a gap d of 3 mm. Due to this gap, in the configuration shown in FIGS. 1a and 1b, better platelet release efficiency can be obtained compared to the case where the method according to the present invention is implemented with a gap outside this range. FIG. 7 shows such an optimal state in the configuration where the two cylinders rotate in opposite directions. Taking the system 1 where the outer diameter of the inner cylinder 11 is 2 cm to 6 cm, the inner diameter of the outer cylinder 13 is 2 cm to 6 cm, and the height h of the outer cylinder 13 is 5 cm to 6 cm as an example, the optimal rotation speed v of the inner cylinder 11 and the outer cylinder 13 is about 1 m / s respectively, and the cylinders move in opposite directions respectively.

[0045] According to a variant of the first embodiment of the method according to the invention, a first step of the method consists in filling the space 15 with a fluid F, and a second step of the method consists in rotating the first wall 12 while the second wall 14 remains stationary, it being understood that the inner cylinder 11 rotates about its axis while the outer cylinder 13 remains stationary. Under this condition, turbulent flow conditions are obtained in the space 15. Figure 8 shows that when only the inner cylinder 11 rotates about its axis (right sample), the platelet release efficiency is further improved compared to a configuration in which both cylinders 11, 13 rotate in opposite directions.

[0046] FIG. 2b shows the distribution of the velocity of the fluid F between the first wall 12 and the second wall 14 when only the first wall 12 is rotating. The system 1 shown in FIG. 2b is an enlarged top view of the system 1 shown in FIGS. 1a and 1b. As shown in this figure, when only the inner cylinder 11 is rotating, the inventors have observed that the velocity v of the fluid F gradually decreases as it moves away from the first wall 12. That is, the velocity v of the fluid F is highest at the height of the first wall 12, gradually decreases as it approaches the second wall 14, and becomes very slow at the height of the second wall 14. This velocity distribution is substantially different from that found in a configuration with counter-rotating two cylinders 11, 13. To compensate for this radial velocity change, a fluid circulation can be used as described below.

[0047] It would be advantageous to generate perfect turbulence to generate turbulent vortices that enhance platelet release. To achieve such flow conditions with System 1, the inner cylinder 11 must rotate independently about its axis and be driven at a very high angular velocity, corresponding to a Reynolds number of the inner cylinder 11 of more than 1400. This flow condition is characterized by low turbulence with the presence of a high turbulence phenomenon called a vortex, also referred to in the scientific literature as a "roller", along the height of the cylinder, about its axis of rotation.

[0048] Similar to the first embodiment in which the two cylinders 11 and 13 rotate, the gap is preferably in the range of 0 mm < d ≤ 5 mm, more preferably 2 mm ≤ d ≤ 4 mm, and most preferably d = 3 mm. The same effects as in the first embodiment were observed. However, the optimal rotation speed v associated with each of these preferred implementations is substantially different in that only the inner cylinder 11 rotates.

[0049] Consider the system 1 where the outer diameter of the inner cylinder 11 is 2 cm to 6 cm, the inner diameter of the outer cylinder 13 is 2 cm to 6 cm, and the height h of the outer cylinder 13 is 5 cm to 6 cm. In such a system 1, the optimal rotation speed v of the inner cylinder 11 is 0.8 m / s to 2.5 m / s when the gap d is 0 mm (excluding 0 mm) to 5 mm, 1.2 m / s to 2.2 m / s when the gap d is 2 mm to 4 mm, and further 1.6 m / s to 1.9 m / s when the gap is about 3 mm.

[0050] Furthermore, if the flow state of the fluid F in the space 15 can cause the release of platelets, it is also important that the produced platelets exhibit behavior similar to that of in-vivo platelets in terms of their biochemical activity. This is achieved by having no mechanical parts in the space 15 containing only the fluid F. Different from the systems known in the prior art where a blade moving in a reservoir containing a fluid causes agitation in the reservoir, in the present invention, the turbulent flow is generated by the moving speed of the inner cylinder 11 and, if necessary, the outer cylinder 13. The absence of movable mechanical parts in the space 15 prevents collisions with megakaryocytes and can maintain the biochemical activity of the released platelets. Inactive platelets or platelets with reduced activity cannot participate in the mechanisms involved in maintaining the structural integrity of blood vessels, primary hemostasis, or procoagulation. Similarly, the biochemical activity of extracellular vesicles released from platelets can be maintained thereby.

[0051] The system 1 shown in Figures 1a and 1b can range in size from a few centimeters to tens of centimeters. One solution to increase the amount of fluid processed while maintaining a high platelet release efficiency is to increase the size of the inner cylinder 11 and the outer cylinder 13. However, to achieve this solution in the above configuration, the dimensions of the inner cylinder 11 and the outer cylinder 13 would need to be significantly increased in order to keep the air gap between 0 mm (exclusive) and 5 mm.

[0052] In this context, the inventors of the present invention have implemented a system 1 as shown in Figures 3a and 3b. In this embodiment, the system 1 comprises an inner cylinder 11 and an outer cylinder 13 like the system 1 shown in Figures 1a and 1b, and differs therefrom only in the following features:

[0053] As shown in figures 3a and 3b, the inner cylinder 11 and the outer cylinder 13 each comprise a further wall 122, 142. In particular, the wall 122, 142 is added to the first wall 12 and the second wall 14 of said cylinders, respectively. That is to say, the inner cylinder 11 comprises the first wall 12 and the further wall 122, and the outer cylinder 12 comprises the second wall 14 and the further wall 142. The first wall 12 and the further wall 122 form a first pattern 120, and the second wall 14 and the further wall 142 form a second pattern 140.

[0054] The first pattern 120 and the second pattern 140 are nested such that the first wall 12 and the further wall 122 on the one hand and the second wall 14 and the further wall 142 on the other hand are parallel to each other and at least partially face each other. The system 1 thus obtained comprises an alternation of parallel cylindrical walls, the outermost wall being the second wall 14, followed by the first wall 12, followed by the further first wall 142, followed by the further first wall 122, followed by the further second wall 142, and so on. The first wall 12 is therefore at least partially facing the second wall 14 and the first further wall 142. The expression "at least partially facing" means that the first wall 12 is not facing the second wall 14 and the first further wall 142 over its entire height. Similar to the configuration shown in Figures 1a and 1b, there is an air gap d' between the base 13a of the outer cylinder 13 and the "base" 11a of the inner cylinder 11. However, it should be noted that the base 11a of the system 1 shown in Figures 3a and 3b is not continuous due to the empty space between the walls that is left for the fluid F to permeate. Thus, in this configuration, the base 11a of the inner cylinder 11 is discontinuous.

[0055] This configuration is particularly advantageous since it allows to increase the amount of fluid F to be processed without excessively increasing the dimensions of the system 1. By arranging the walls 12, 14, 122, 142 alternately, a large number of intermediate spaces are formed between the central axis of the system 1 and the second wall 14. All of these are spaces into which the fluid F can enter. In the exemplary embodiment shown in Fig. 3a and Fig. 3b, the system 1 includes the first wall 12 and two other walls 122, and the second wall 14 and two other walls 142. That is, four additional spaces are added around the circumference. The number of the other walls is not limited to the exemplary embodiment shown in Fig. 3a and Fig. 3b. It is therefore understood that other walls 122, 142 can also be provided, and that with n (n being a strictly positive natural number) other walls 122 and n other walls 142, there are 2xn additional spaces, i.e. in addition to the space 15. Incidentally, the larger the diameter of the inner cylinder 11 and the outer cylinder 13, the more additional space can be created and the larger the amount of fluid that can be processed.

[0056] For all spaces formed between the walls 12, 14, 122, 142, it is desirable to match the gap between two consecutive spaces of the system 1 in order to keep the same Reynolds in the various spaces of the system 1. This is because it is preferable to match the gap so that the rotational angular velocity is constant in the radial direction and the linear velocity is different for each space 15, and the platelet release efficiency is improved uniformly throughout the system 1. Therefore, the gap d between the second wall 14 and the first wall 12 is different from the gap d between the first wall 12 and the other first wall 142, as well as the gap d between the other first wall 142 and the other first wall 122.

[0057] The optimal pair (d,v), i.e. the pair that allows to generate a turbulence optimizing the release of platelets from the fluid F, is the same as seen before. At this stage, it is noted that the method according to the invention can be carried out according to the two previously described embodiments, using the system 1 in the configuration shown diagrammatically in figures 3a and 3b. It is thus possible to rotate the inner cylinder 11 and the outer cylinder 13 in opposite directions, as well as to rotate only the inner cylinder 11. Whatever the configuration considered, the optimal pair (d,v) is the same, as is the platelet release efficiency obtained. It should be noted that, compared to the configuration shown in figures 1a and 1b, the configuration shown in figures 3a and 3b advantageously allows a significant increase in the amount of fluid F to be processed.

[0058] In addition to the gap d and the rotation speed v of the inner cylinder 11 and the outer cylinder 13, it is also possible to influence the platelet release phenomenon by a third parameter, namely the residence time of the fluid in the reservoir. Just to be clear, in the system 1 illustrated in Figures 1a and 1b, the space 15 forms the reservoir for the fluid F, whereas in the system 1 illustrated in Figures 3a and 3b, it is not only the space 15 that forms the reservoir for the fluid F, but also all the spaces formed between the parallel cylindrical walls. In order to vary the residence time of the fluid in both cases, the system 1 can be provided with an inlet orifice (not shown) at the height of the frame and an outlet orifice (not shown) at the height of the base 13a of the outer cylinder, allowing the fluid F to penetrate and leave the reservoir, respectively.

[0059] Moreover, when the device comprises an inlet orifice and an outlet orifice, it is possible to set up a process of circulating the fluid F in the reservoir, assuming any configuration, i.e. even if the two cylinders 11, 13, inner and outer, rotate in opposite directions, or even if only the inner cylinder 11 rotates. Fluid circulation involves the constant withdrawal of fluid already treated and extracted from the reservoir, replacing it with untreated fluid from the reservoir. Naturally, such circulation is advantageously carried out in a closed loop, so that the fluid F is not exposed to external media and sources of contamination. Circulation thus makes it possible to turn a batch processing method into a continuous processing method, which is particularly advantageous for industrial applications.

[0060] The fluid residence time is more precisely controlled by acting on the opening diameter, or more generally on the opening cross section of the outlet orifice. This allows for continuous processing of the fluid F containing megakaryocytes, a further step towards the industrialization of the method according to the invention. In this configuration, the dimensions of the system 1 are less critical, since the fluid F is processed continuously and several liters of fluid can be processed in just one hour. This means that the fluid residence time must be selected to optimize the platelet release efficiency.

[0061] The residence time of the fluid F in the reservoir is advantageously between 4 and 6 minutes, preferably about 5 minutes. If the residence time of the fluid in the reservoir is less than 5 minutes, the platelet release efficiency is not optimal. However, if the residence time of the fluid in the reservoir exceeds 5 minutes, no additional benefits are observed. That is, the platelet release efficiency is not increased because the fluid F remains in the reservoir for more than 5 minutes. Therefore, it is not necessary for the residence time to exceed 5 minutes. This was observed in both implementations of the method described above, i.e., in the system 1 shown in Figures 1a and 1b or in the system 1 shown in Figures 3a and 3b, where the inner cylinder 11 and the outer cylinder 13 rotate in opposite directions, and also in the system 1 shown in Figures 1a and 1b or in the system 1 shown in Figures 3a and 3b, where only the inner cylinder 11 rotates.

[0062] See figures 4a and 4b. In a second embodiment according to the invention, this is implemented by a system 1 comprising two conveyor belts 17, 18. Each of the conveyor belts 17, 18 comprises an endless strip 17a, 18a arranged closely around two guide means 17b, 18b. The guide means 17b, 18b are roller-like. These guide means are spaced apart by a sufficient distance to tension the endless strips 17a, 18a so that they completely conform to the outer contour of the guide means 17b, 18b at the side edges of each conveyor belt 17, 18 and so that outside the areas where the endless strips conform to the outer contour of the guide means, the endless strips 17a, 18a have a relatively wide flat surface. By "wide" we mean a flat surface of a few centimetres up to a few tens of centimetres.

[0063] A first conveyor belt 17, referred to as the "upper belt", includes a first planar wall 12 corresponding to one of the planar surfaces of the upper belt 17, and a second conveyor belt 18, referred to as the "lower belt", includes a second planar wall 14 corresponding to one of the planar surfaces of the lower belt 18. The upper belt 17 and the lower belt 18 are superimposed, i.e. the upper belt 17 is arranged opposite the lower belt 18. Also, similar to the first embodiment (FIGS. 1a-3b), the upper belt 17 is arranged parallel to the lower belt 18 such that the first wall 12 and the second wall 14 are parallel and at least partially opposite each other. It should be noted that the terms "upper" and "lower" are not intended to be limiting, but are merely intended to facilitate understanding of the invention with reference to the figures.

[0064] The upper belt 17 is spaced from the lower belt by a non-zero distance d, which forms a space 15 between the two belts. The space 15 located between the upper belt 17 and the lower belt 18 is similar to the space 15 located between the inner cylinder 11 and the outer cylinder 13 in the first embodiment of the system 1. However, like the first embodiment, it can contain a fluid F, but unlike the first embodiment, it does not form a closed reservoir for the fluid F. Indeed, in the embodiment shown in Figures 4a and 4b, the system 1 includes a reservoir 20 in which the conveyor belts 17, 18 are arranged. This reservoir contains the aforementioned fluid F. Furthermore, the system 1 also includes a frame (not shown) that allows the conveyor belts 17, 18 to assume the positions shown in Figures 4a and 4b.

[0065] The endless strips 17a, 18a are movable. In this respect, the guide means 17b, 18b are guided in rotation about their respective pivot axes A17, A'17, A18, A'18 by a drive system. Like the drive system of the first embodiment, this includes a power supply system, one or more motors, as well as a speed regulation system used to control the motor or motors. This drive system allows a defined speed to be applied to said guide means 17b, 18b individually. The guide means 17b, 18b can be rotated in either direction about their respective pivot axes, thus causing the endless strips 17a, 18a to move in a given direction.

[0066] The method according to the invention can therefore be carried out either by rotating the endless strips 17a, 18a in the opposite direction and moving the first and second walls 12, 14, respectively, in their planes, or by rotating only one of the endless strips 17a, 18a and moving one of the first and second walls 12, 14 in their planes. And the skilled person will understand that in this way, as in the first embodiment, turbulent flow conditions suitable for the release of platelets from the megakaryocytes contained in the fluid F can be generated in the space 15. To further improve the platelet release efficiency, it is sufficient to select an optimal gap d. At the same time, it is sufficient to select the rotation speed v of the guide means in an appropriate range, i.e. in a speed range in which the flow conditions of the fluid F allow the optimal release of the platelets. The rotation speed is selected to achieve turbulent flow conditions, taking into full account the dimensions of the conveyor belts 17, 18, since it depends on this parameter.

[0067] It is advantageous if the upper conveyor belt 17 and the lower conveyor belt 18 move in opposite directions at the same speed. In this configuration, the fluid is further sheared, even though it remains stationary on average between the first wall 12 and the second wall 14. Moreover, in this configuration, the optimal residence time is then the same as in the embodiment with the inner cylinder 11 and the outer cylinder 13, i.e. a residence time of 4 to 6 minutes, preferably about 5 minutes, should be sufficient to obtain an optimal platelet release efficiency. However, this is not mandatory, since a difference in the rotation speed between the conveyor belts 17, 18 allows for a continuous supply of the fluid F to be envisaged.

[0068] Alternatively, the upper conveyor belt 17 and the lower conveyor belt 18, and thus the two walls 12, 14, move at a speed difference of at most 10% relative to the average value, allowing flexibility in the design of the device 1 while maintaining adequate shear of the fluid.

[0069] This second embodiment of the system 1 operates similarly to the first embodiment of the system 1, except for the difference in the calculation of the Reynolds number of the conveyor belt. Indeed, when the diameter of a cylinder approaches a very large value or approaches infinity, the cylindrical wall of this cylinder can be considered identical to a plane wall. For example, if we consider the system 1 used in the first embodiment, where the dimensions of the cylinders 11, 13 are known to vary between a few centimeters and a few tens of centimeters, a very large cylindrical wall would have a diameter of at least about one meter. Locally, such a wall can therefore be considered as flat. In the second embodiment of the system 1, the first wall 12 and the second wall 14 of the conveyor belts 17, 18 can be considered as the walls of a cylinder of very large diameter. This explains why the phenomena associated with turbulence found in connection with the system 1 according to the first embodiment can be reproduced by the system 1 with two conveyor belts, in a configuration with two rotating cylinders 11, 13 as well as in a configuration with only the inner cylinder 11 rotating.

[0070] Moreover, it is possible to provide more conveyor belts, as can be seen in Fig. 5a. In the variant shown in Fig. 5a, an additional pair of conveyor belts 17', 18' is also provided in the system 1. These are superimposed on the upper conveyor belt 17 and the lower conveyor belt 18, so that a system 1 with four conveyor belts and three spaces 15 is obtained. Each of these spaces 15 constitutes a turbulent flow area. The number of conveyor belts is not limiting and more belts can be envisaged, provided that the dimensions of the reservoir 20 are suitable to accommodate the number of envisaged conveyor belts. The more conveyor belts there are, the higher the amount of fluid F that can be processed. This example embodiment should be compared with the example embodiment of the system 1 shown in Figs. 3a and 3b, where the spaces 15 are significantly expanded by a set of other walls 122, 142 in each cylinder.

[0071] In the illustrated embodiment shown in Fig. 5b, the upper conveyor belt 17 and the lower conveyor belt 18 are larger. This allows the length of the space 15 to be increased, and therefore the amount of fluid F to be processed to be increased. However, this option may be more restrictive than the previous one, insofar as the movements associated with the larger guide means 17b, 18b may pose technical and / or economic constraints.

[0072] Materials and methods of the present invention The device shown in FIG. 9 was used to carry out the method according to the invention and the experimental results are shown in the following figures and further discussed in the following paragraphs.

[0073] [Megakaryocyte differentiation in culture] The hematopoietic progenitor cells CD34 were isolated from leukocyte reduction filters (TACSI, Terumo BCT, Zaventem, Belgium) used in the preparation of unstable blood products. + The cells contained in the filter were eluted and then incubated with anti-CD34 bound to magnetic beads (CD34 MicroBead Kit UltraPure, Miltenyi Biotec, Bergisch Gladbach, Germany). + These cells were identified by magnetic sorting using antibodies against CD34 + The cells were then seeded in Serum-Free Expansion Medium (SFEM) supplemented with 20 μg / mL human low-density lipoprotein (LDL), a cocktail of cytokines (IL-6, IL-9, SCF, TPO) (CC220, Stemcell Technologies, Vancouver, BC, Canada), and 1 μM SR1 (Stemcell Technologies). On day 7, the cells were harvested, washed, and seeded in StemSpan SFEM containing 1 μM SR1, 50 ng / mL TPO, and 20 μg / mL LDL for an additional 6 days (Strassel et al., 2016). Cultures were incubated at 37°C under normoxic conditions and 5% CO2 atmosphere.

[0074] [Release of cultured platelets] After 13 days in culture, megakaryocytes displayed cytoplasmic extensions (bodies) that were termed platelet precursor cells. The medium containing the platelet precursor megakaryocytes was placed in a Couette device and platelets were released in one of the configurations defined below.

[0075] Two configurations of the Couette device were tested: - A "two-rotor" configuration in which the device's two rotors (the inner cylinder 11 and the outer cylinder 13) rotate in opposite directions - A "rotor-up" configuration in which only the inner rotor 11 rotates. They determined these configurations based on flow mapping studied for a fluid between two closely spaced walls moving relative to each other.

[0076] The number of released platelets was estimated using tubes containing calibration beads (BD Trucount™, BD Biosciences, San Jose, USA). Platelet counts after passing 5,000 beads were shown, followed by platelet counts per cell at D7 and CD34 counts at D0. + The cell seeding number was reduced.

[0077] [Morphological investigation (Fig. 14)] After fixation with paraformaldehyde, platelets were cytospun, permeabilized with 0.1% Triton X-100, and incubated with anti-β1-tubulin antibody (1 μg / mL, Eurogentec, Liege, Belgium), followed by GAM-488 secondary antibody (10 μg / mL) and anti-GPIIbIIIa mAb (10 μg / mL) diluted in PBS with 1% BSA (bovine serum albumin). Cells were then mounted in Mowiol (Mountant, Permafluor, Thermo Fisher Scientific, UK) and observed under a confocal microscope (TCS SP8, Leica Microsystems, Rueil-Malmaison, France) equipped with an oil objective (F-type immersion, ne23=1.5180, ve=46, Leica Microsystems). Data were acquired using LASAF software, version 1.62 (Leica Microsystems).

[0078] [In vitro functionality study] Activation Ability (Figure 12) For the activation assay, platelet suspensions were labeled with antibodies against GPIIb-IIIa and GPIbα and incubated for 10 min at room temperature. Three analysis tubes were prepared containing either anti-CD62P antibody, PAC1 antibody (anti-GPIIb-IIIa activated form), or isotope control. After 10 min incubation at room temperature, 10 μg / mL of 10 μM TRAP (for activated tubes) or PBS (for non-activated tubes) was added. The percentage of activated platelets was analyzed by flow cytometry.

[0079] Glycoprotein Expression (Figure 11) For glycoprotein expression assays, platelet suspensions were labeled with antibodies against GPIIb-IIIa, GPIbα, GPIbβ, GPV, GPIX, GPVI, and CD9. After 30 min of incubation at room temperature, samples were analyzed by flow cytometry. Fluorescence Minus One (FMO) was performed to determine the analytical window when using multiple fluorochromes.

[0080] [In vivo functionality study (Figure 13)] Recirculation after transfusion After adding 0.5 μM iloprost (Ilomedine 0.1 mg / 1 ml, Bayer AG, Germany) to the medium containing the platelet precursor megakaryocytes, the cultured platelets were harvested. Platelets were then centrifuged and resuspended in Tyrode's buffer containing albumin as described in the literature (Hechler et al., 2013; Strassel et al., 2016). After a final washing step, platelets were transferred to a storage solution consisting of one-third plasma and two-thirds Intersol (Fresenius Kabi, Homburg, Germany) as an additive. This storage solution is used for the storage and transfusion of native platelets.

[0081] Cultured from MCP (mixed platelet concentrate) or native 5 × 10 7 Aliquots containing 100 μg of washed human platelets were injected via the retroorbital vein into NSG mice (NOD.Cg-Prkdc scid, Il2rg tm1Wjl / SzJ) that had previously been macrophage-depleted (by injection with clodronate liposomes on day 1).

[0082] Circulating human and mouse platelets were analyzed by flow cytometry in whole blood samples taken at 3, 6, 10, 15, 30, 120, 240, 1400, 2800, and 4320 min after transfusion. Each blood sample was analyzed by flow cytometry after labeling with the RAM.1-568 antibody, which recognizes human and mouse GPIbβ, and the ALMA17-488 antibody, which recognizes only human GPIIb-IIIa (Do Sacramento et al., 2020). To make the results comparable, the ratio of human platelet count to total platelet count was arbitrarily set to 1 after 3 min of the first stage of the analysis. [Explanation of symbols]

[0083] 1 System 11 Inner Cylinder 12 Cylindrical wall of inner cylinder or flat wall of conveyor belt 13 Outer Cylinder 13a Base of outer cylinder 14 Cylindrical wall of an outer cylinder or flat wall of a conveyor belt 15 Space

Claims

1. A method for releasing platelets from megakaryocytes contained in a fluid (F), the method being carried out by a system (1) comprising two concentric cylinders (11, 13): an inner cylinder (11) with a cylindrical wall (12) and a hollow outer cylinder (13) located radially outside the inner cylinder (11), the outer cylinder (13) having a cylindrical wall (14) with a base (13a) at the level at which the second cylinder is closed, the two cylinders (11, 13) being separated by a space (15) free of mechanical parts, the space (15) intended to receive the fluid (F), The method comprises the following steps: (100) A step of supplying a fluid (F) containing megakaryocytes to the space (15); (200) generating turbulent flow of fluid (F) at least partially in said space (15) by rotating said inner cylinder (11) about its axis while said outer cylinder (13) is stationary or by moving said two cylinders (11, 13) in opposite directions about their axes, to obtain a second fluid (F') enriched with platelets; A method comprising:

2. When the inner cylinder (11) is moved while the outer cylinder (13) is stationary, the inner cylinder has a Reynolds number Re ｉ (see equation 1) is moved to set it to 1400 or more, [0010] In the formula, r ｉ is the radius of the inner cylinder (11), ω ｉ 2. The method of claim 1, wherein: θ is the angular velocity of the inner cylinder (11), d is the distance separating the inner cylinder (11) and the outer cylinder (13), and θ is the dynamic viscosity of the fluid between the inner and outer cylinders.

3. When the two cylinders (11, 13) are moved in opposite directions, the inner cylinder (11) and the outer cylinder (13) each have a Reynolds number Re greater than 1000. ｉ (see Equation 2), and a Reynolds number Re greater than 1000 ｏ (see equation 3), [0025] [0030] In the formula, r ｉ is the radius of the inner cylinder (11), r ｏ is the radius of the outer cylinder (13), ω ｉ is the angular velocity of the inner cylinder (11), ω ｏ 2. The method of claim 1, wherein: θ is the angular velocity of the outer cylinder (13), d is the distance separating the inner cylinder (11) and the outer cylinder (13), and θ is the dynamic viscosity of the fluid between the inner and outer cylinders.

4. 2. The method according to claim 1, wherein the two cylinders (11, 13) are separated by a distance (d) of less than 5 mm.

5. The method according to claim 1, wherein the two cylinders (11, 13) are separated by a distance (d) between 2 mm and 4 mm.

6. 2. The method according to claim 1, wherein the two cylinders (11, 13) are separated by a distance (d) of about 3 mm.

7. 2. The method according to claim 1, wherein in step (100), the fluid (F) is continuously supplied to the space (15), the system (1) comprising an inlet (2) for filling the space (15) with the fluid (F) and an opening (3) for discharging a fluid enriched with platelets (F'), the opening (3) being located at the height of the base (13a).

8. The method according to claim 7, wherein the residence time of the fluid (F) in the space (15) during step (200) is between 4 minutes and 6 minutes, preferably about 5 minutes.

9. 2. The method of claim 1, wherein the inner cylinder (11) and the outer cylinder (13) include other peripheral inner walls (122, 142) forming a first pattern (120) and a second pattern (140), respectively, the first pattern and the second pattern (120, 140) being nested within one another such that the other walls (122) of the inner cylinder (11) and the other walls (142) of the outer cylinder (13) are concentric and at least partially opposed to one another.

10. A method for releasing platelets from megakaryocytes contained in a fluid (F), the method being carried out by a system (1) comprising two parallel planar walls (12, 14) separated by a space (15) without mechanical parts, the space (15) being designed to receive the fluid (F), the planar walls (12, 14) being movable, The method includes the following steps: (100) A step of supplying a fluid (F) containing megakaryocytes to the space (15); (200) generating turbulent flow of fluid (F) at least partially in said space (15) by moving one of said planar walls (12, 14) in its plane while the other of said planar walls (12, 14) is stationary or by moving said two planar walls (12, 14) in opposite directions and each in its plane, thereby obtaining a second fluid (F') enriched with platelets; A method comprising:

11. 11. The method of claim 10, wherein the first planar wall (12) is a wall of a first conveyor belt and the second planar wall (14) is a wall of a second conveyor belt.

12. 11. The method of claim 10, wherein when the two walls (12, 14) are moved in opposite directions, the two walls (12, 14) move in opposite directions at the same speed.

13. 13. The method of claim 12, wherein the two walls (12, 14) move at a speed of about 1 meter per second.

14. 11. The method according to claim 10, wherein when the two walls (12, 14) move in opposite directions, the two walls (12, 14) move with a maximum speed difference of 10% relative to an average value.

15. The method for releasing platelets according to any one of claims 1 to 14, wherein extracellular vesicles are additionally released.

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