Patent Application
20220313875
The invention relates to a system for the platelet release from a fluid comprising in particular megakaryocyte progenitors, megakaryocytic cells comprising cytoplasmic extensions. The invention also relates to an assembly provided with a plurality of systems such as this. Finally, the invention relates to a method for continuous release of platelets by means of such a system. The invention is more particularly adapted to the in vitro production of blood platelets on an industrial scale.
The in vitro production of blood platelets is meeting increasing needs in various medical applications. Currently, there are essentially two categories of systems intended to this specific purpose: the microfluidic systems and the “reservoir” systems. These categories of systems intended to release the platelets are derived from observations made in situ that show the need for a flow.
An example of a reservoir system is described in the document WO201909364 A1. The system is provided with means for stirring the fluid to be treated. It comprises a reservoir for the fluid comprising in particular the megakaryocytic cells and at least one means for stirring the fluid, the stirring causing the release of the platelets. This stirring is generated inside the reservoir itself, for example, by means of a vertical reciprocating paddle. The main disadvantage of such a system is that it is not adapted to operate under conditions which guarantee the quality of the platelets thus obtained, since it is not isolated from the external environment and the sources of contamination present in that environment, which makes it unsuitable for a medical use.
The microfluidic systems, as the name implies, are generally systems of micrometric dimensions (a few tens of microns to a few hundreds of microns at most) comprising a network of reservoirs and channels interconnected in a particular arrangement to perform different functions. Classically, they comprise a site dedicated to the culture of megakaryocytic cells or megakaryocytes, and optionally to the release of the platelets, the site being connected to a network of channels configured to extract/retrieve the platelets. These systems, most of which are biomimetic, seek to mimic the physiological environment to increase the platelet production.
Due to their small size, these microfluidic systems have two intrinsic limitations. The first is the low platelet flow rate that can be achieved by such systems, typically a few hundred microliters per hour (μL/h). As an example, a system with a flow rate of 200 μL/h would require 50,000 hours, or 5.7 years, to treat 10 L of fluid. It is then necessary to connect several systems in parallel to obtain a comparable total flow rate. However, in the above example the number of systems required would be very high, i.e. 50,000, and the complexity of the system would be further increased. At best such systems are only suitable for limited volumes of fluid samples.
In addition, the microfluidic devices described in the literature all implement a mechanism/method that “fix” the megakaryocytes and then extracts the platelets. This fixing phase is often based on the use of a drug or a chemical coating compound, which is a disadvantage.
More recently, a new method for releasing the platelets from megakaryocytic cells using pipetting has been developed. Such a method is described in the document Strassel et al. “Aryl hydrocarbon receptor-dependent enrichment of a high potential to produce propalets,” Blood, May 5, 2016, vol. 127, n° 18. In such a method, the process for releasing the platelets is a direct result of the pipetting. The pipetting is similar to a conventional pipetting in that a sample of a medium comprising megakaryocytic cells is taken by means of a pipette, except that the pipetting action is repeated as many times as necessary to create the stirring required to the release of the platelets. As a result of this method, platelets were detected in the treated fluid, validating the ability of pipetting to the release of the platelets, and with great ease of implementation. However, this method is not suitable for large-scale operation since the pipetting is by nature manual and therefore it is only suitable for treating limited volumes of fluid.
The invention allows to overcome the aforementioned disadvantages and to this end proposes a system for releasing the platelets from a fluid comprising, in particular, megakaryocytic cells comprising cytoplasmic extensions, said system comprising:
The system according to the invention is thus configured in such a way as to reproduce the pipetting process performed manually by means of a pipette, thus allowing a continuous and automatic release of the platelets.
A further noteworthy point is the fact that the invention is implemented on a large scale because the geometry of the system according to the invention allows large volumes of fluid to be treated at a high flow rate (of the order of several liters per hour), which makes it particularly suitable for use on an industrial scale.
In addition, the system allows to achieve a number of platelets released from the megakaryocytic cells at least equivalent to that obtained from the manual pipetting, making it a system with a particularly high platelet release efficiency. This is achieved mainly due to the synergy between the shape of the elements of the system, i.e. in particular the conicity of the first fluidic connecting element and the large dimensions of the reservoir, and the flow velocity generated by the pumping device. This allows for the generation of vortex disturbances that are significantly of the size of the megakaryocytes within the reservoir.
According to various characteristics of the invention which may be taken together or separately:
The invention further relates to a method for releasing platelets from a fluid comprising in particular megakaryocytic cells comprising cytoplasmic extensions, said method comprising the following steps, implemented by means of a system as previously described:
(100) providing a fluid comprising megakaryocytic cells suspended in said fluid, said megakaryocytic cells comprising cytoplasmic extensions,
(200) electrically powering the pumping device,
(300) controlling the programming system to initiate one or more platelet release sequences, the or each platelet release sequence being carried out so as to generate a continuous flow of the fluid between said injection orifice and said discharge orifice and vortex disturbances within the reservoir causing the fragmentation of the cytoplasmic extensions of the megakaryocytic cells.
Advantageously, during the step (200), a relative vacuum of between −10 kPa and −50 kPa is generated in the device.
Further objects, characteristics and advantages of the invention will become clearer in the following description, made with reference to the attached figures, in which:
With reference to
The fluid F in question is, for example, a culture medium containing a population of cells obtained from immortalized or non-immortalized strains cells at different stages of differentiation, including megakaryocyte progenitors, megakaryocytes. The megakaryocytes or megakaryocytic cells are large blood cells (up to 100 μm and 30 μm in culture) which, when mature, have long extensions called cytoplasmic extensions or proplatelets.
The mechanisms involved in the formation of the blood platelets are still the subject of much research. Among them, the platelet release occurs during a process of fragmentation of the Mk megakaryocytes and/or cytoplasmic extensions, Ck, into platelets. This is a highly coordinated in vivo process that occurs naturally in the blood due to the force of the blood flow. The megakaryocytes Mk play an essential role as they are precursor cells. However, the process of release of the platelets in the body remains poorly described and there are still many questions about transendothelial passage and the precise role of the blood flow in the formation of the platelets. This process was reproduced in vitro by means of microfluidic systems, which allowed some in vivo mechanisms to be corroborated by microfluidic experiments. This process can also be reproduced in vitro by means of a pipette manually or by means of devices such as those presented in the introductory part of this description, which also allows a better understanding of the mechanisms involved in the release of the platelets (Strassel et al. 2016). The production of platelets in vitro provides a better understanding of the mechanisms involved in the formation of the platelets. However, in general, the efficiencies of the release of the platelets are lower than those obtained in vivo. The device proposed by the invention allows to mimic the platelet release process by manual pipetting disclosed by Strassel et al., without reproducing the reciprocate movement generated during the pipetting which is replaced by a continuous movement using the system 1. The invention thus aims to reproduce the fragmentation process of the megakaryocytes Mk and/or cytoplasmic extensions Ck on an industrial scale and thus allows to treat large volumes of fluid F (several liters per hour).
The system 1 according to the invention comprises a device 2, a pumping device 60 and a programming system 70 which will be described in the following sections.
The device 2 comprises a reservoir 10 of platelet release, a first fluidic connecting element 20 and a second fluidic connecting element 30.
The reservoir 10 of platelet release (hereinafter referred to as “reservoir 10”) has millimeter to centimeter longitudinal dimensions, i.e., longitudinal dimensions between 1 mm and a few centimeters. For comparison, the smallest dimension of the reservoir 10 of the system 1 according to the invention is the largest dimension of a conventional microfluidic system. This makes it a larger reservoir and larger capacity than known microfluidic systems.
In the example embodiment shown in
The wall 12 comprises at least a first opening 16 and a second opening 18. The first opening 16 is dedicated to the fluidic connection with the first connecting element 20, while the second opening 18 is dedicated to the fluidic connection with the second connecting element 30. Preferably, they form the only openings in the wall 12, since the system 1 is closed.
The first fluidic connecting element 20 is a means for supplying the fluid F to the reservoir 10. In other words, it is an inlet for the fluid F within the reservoir 10. It is attached at the level of the first opening 16 of the reservoir 10. It comprises a connecting portion 26, a narrowing portion 24 and an orifice 22 of injection of the fluid in that order in the direction of flow direction of the fluid. The narrowing portion 24 thus opens onto the injection orifice 22. This being said, it should be noted that the connecting portion 26 is not indispensable as will be better understood in the following. Thus, in the example embodiment shown in
As mentioned earlier, the portion 24 is narrowing. The portion 24 is narrowing in that it narrows from the connecting portion 26 or, alternatively in the absence of a connecting portion 26, an end of the first connecting element 20 towards the orifice 22 of injection of the fluid. In other words, it narrows in the direction of the flow relative to a longitudinal axis X1 of said first connecting element 20 passing through the center of said orifice 22. The narrowing shape of said portion 24 allows the fluid F to be accelerated by the venturi effect. Incidentally, the fluid F entering the reservoir 10 therefore has a higher velocity compared to a fluid that would enter the system 1 through a portion 24 that does not have a flaring.
Advantageously, the narrowing of the narrowing portion 24 from the connecting portion 26 or, alternatively in the absence of a connecting portion 26, the end of the first connecting element 20 towards the orifice 22 of injection of the fluid may be substantially constant. The constancy of the narrowing allows to reduce the friction and further increase the acceleration of the fluid. In this regard, the narrowing portion 24 may be conical. Advantageously, the use of a conical portion 24 facilitates the connection with tubular, i.e. cylindrical, fluidic connecting means frequently sold commercially. By way of example, the portion 24 may have a pyramidal shape, a tetrahedral shape, etc. Most importantly, the narrowing portion 24 narrows from the connecting portion 26 of the first connecting element 20 toward the orifice 22 of injection of the fluid. This being the case, once discharged into the reservoir 10, the fluid F slows down substantially due to the difference in cross-section existing between the first fluidic connecting element 20, in particular the injection orifice 22, and the reservoir 10.
Indeed, the injection orifice 22 advantageously has an opening diameter of very small size compared to the diameter of the reservoir 10, in the case of spherical species. However, this diameter is greater than or equal to the size of the particles leaving the reservoir 10, i.e. the platelets P and other products, Dk, resulting essentially from the fragmentation of the megakaryocytes. Preferably, the opening diameter is less than or equal to 1 mm, whereas as we have seen, the reservoir 10 has longitudinal dimensions in the millimeter to centimeter range, and in any event much greater than those of the orifice 22. It should be noted at this point that the shape of the injection orifice 22 is not limiting. What is important here is the size of said injection orifice 22.
Thus, an abrupt cross-sectional enlargement exists between the injection orifice 22 and the reservoir 10. This abrupt enlargement corresponds to a singularity and gives rise to a singular flow profile of the fluid F. As mentioned previously, the fluid F is accelerated out of the narrowing portion 24 through the injection orifice 22, and then subjects a pressure drop, i.e. a slowing, as it passes from the injection orifice 22 to the reservoir, due to this singularity. The smaller in front of 1 the ratio, RE, of the diameter of the injection orifice 22 to the diameter or, more generally, the cross-sectional width of the reservoir 10, the greater the singularity and the greater the pressure drop, and vice versa as this ratio increases. An example of the system 1 according to the invention which has been implemented comprises a reservoir 10 with a diameter equal to 16 mm and an injection orifice 22 with a diameter equal to 0.8 mm (RE is therefore equal to 0.05). If such a system 1 allows to provide improved platelet release efficiencies, ratios RE of the opening diameter of the orifice 22 to the reservoir cross-sectional width of between 0.02 and 0.1 can also be envisaged for releasing platelets from a fluid F, always for an injection orifice 22 with an opening diameter of less than 1 mm. Advantageously, RE is between 0.04 and 0.08 an opening diameter of less than 1 mm. More advantageously, RE is between 0.04 and 0.06 an opening diameter of less than 1 mm. This being said, even more advantageously RE is equal to 0.05. In fact, in the latter case, the efficiency of release of the platelets efficiency is better.
However, as will be explained below, although the fluid F is slowed down on entering the reservoir 10 and maintains a laminar flow regime in the strict sense of the term—the Reynold's number varying between 0, at the center of a vortex whose size is close to that of the reservoir 10, and 1500, at the level of the injection orifice 22—it is not immobile within said reservoir 10 because of the displacement of fluid generated by the pumping device 60. The reservoir 10 is thus only a place of passage for the fluid F, and is not a place of storage of said fluid F, i.e. a place in which the fluid is brought to stagnate when the system 1 is in operation.
In this regard, it should be noted that in addition to the reservoir 10 of release of the platelets, the system 1 may also comprises a source reservoir 40 for the storage of the fluid F connected to the first fluidic connecting element 20 to supply the reservoir. The fluid F may therefore be stored in the source reservoir 40 prior to its passage through the first element 20, but this is not mandatory. The reservoir 40 is therefore the most upstream element of the system 1 in the direction of the flow.
In addition, the system 1 may also comprise a reservoir 50 of reception of the fluid F. The reservoir 50 of reception of the fluid is connected to the second fluidic connecting element 30 for collecting said fluid to be sucked from the reservoir. In contrast to the source reservoir 40, the receiving reservoir 50 is the furthest downstream element of the system 1 in the direction of the flow. The arrangement of the latter will be explained later with respect to the second fluidic connecting element 30.
The reservoir 10 can thus be seen as central in that the system 1 may comprise upstream of it the source reservoir 40 and downstream of it the receiving reservoir 50 in the direction of the flow.
The second fluidic connecting element 30 is, in turn, a means for discharging the fluid F outside the reservoir 10 and thus forms an outlet channel for the fluid F. It is in fluidic communication, via one of its ends, with the receiving reservoir 50. In addition, it is attached at the level of the second opening 18 of the reservoir. It comprises an orifice 32 of discharging the fluid, a discharging portion 34 and a connecting portion 36 in that order in the direction of flow of the fluid F. It is arranged similarly to the first fluidic connecting element 10 relative to the reservoir 10, although oriented differently, and has a similar structure (e.g., size of the discharge orifice 32, relative to the reservoir 10 of platelet release).
That being said, the geometry of the flow is different at the reservoir 10/discharge orifice 32 interface, which is mainly due to the type of singularity. In contrast to the injection orifice 22/reservoir 10 interface, the singularity lies in an abrupt narrowing of the cross-section in the direction of the flow since the diameter (or circumference of the reservoir 10 if applicable) is much larger than the diameter of the discharge orifice 32. Given the dimensions of connecting reservoir 10 and the discharge orifice 32, such a singularity would further increase the pressure drop subjected by the fluid F as it passes from the reservoir 10 to the second fluidic connecting element 30, but this is mitigated by the fluid displacement generated by the pumping device 60, as will be described in more detail below. This singularity allows to increase the residence time of the fluid within the reservoir 10 since the abrupt narrowing of the cross-section acts as a barrier for the fluid F which is prevented from exiting the reservoir 10 immediately if not directly from the reservoir. The direct consequence of this is that, as the residence time of the fluid F is increased, the formation of the turbulence or vortex disturbances within the reservoir 10 is favored.
Furthermore, the residence time of the fluid F within the reservoir 10 can be further advantageously increased by offsetting the injection orifice 22 with respect to the discharge orifice 32. Indeed, the injection orifice 22 and the orifice 32 although being carried by two axes and thus forming a plane, this plane is oblique, i.e. it is neither horizontal nor vertical, as illustrated in the example of
The discharge orifice 32 is thus separated by a non-zero distance, d, from the injection orifice 22 in a direction of axis Y, the axis Y being orthogonal to the longitudinal axes X1 and X2. Preferably, the distance, d, is between 3 and 4 mm. However, while said orifices 22, 32 may be separated by a distance d in the direction of axes Y, it is not excluded that they may be separated by a distance d′ in a direction of axis X in the same plane and/or in a plane XZ, the axis Z being orthogonal to the axis X in the direction of the cross-section (out of the plane of the figure). In this, the sectional view in
If the geometry of the flow depends substantially on the singularities previously described and thus on the shape of the elements, it also depends on the flow speed. The shape of the elements of the device 2 and the flow speed thus act synergistically to achieve such a flow geometry and allow continuous release of platelets P from the megakaryocytic cells Mk with an improved efficiency compared to the known devices. We will come back to this in the following.
In this regard and as previously mentioned, the system 1 is provided with a pumping device 60 (illustrated in
Alternatively, the pumping device 60 may be located upstream of the first fluidic connecting element 20 in the direction of the flow, preferably in the source reservoir 40. In this regard, a pumping device 60 by fluid transfer or an overpressure in the reservoir 40 may be provided. In this configuration, the fluid F being discharged in the desired flow direction, it passes successively through the narrowing portion 24, the injection orifice 22, the reservoir 10 and the discharge orifice 32, adopting a flow geometry resulting from each of the singularities. However, the use of a fluid transfer pumping device 60 (gear pump, peristaltic pump, etc.) could be detrimental to the release of the platelets as it would likely degrade the megakaryocytic cells before the process has even begun. The use of this type of pumping device 60 is not mandatory since an ejector type device could also be considered. In general, any type of pumping device 60 may be used, with the possible exception of a fluid transfer pump.
The pumping device 60 may be in fluidic communication with the reservoir 10 by means of the second fluid connecting element 30. Preferably, it is located in the receiving reservoir 50. By being arranged in this way, the pumping device 60 is therefore located downstream of the second fluidic connecting element 30 in the direction of flow of the fluid F. This allows full advantage to be taken of the singularities of the device 2 and improves the process of forming vortex disturbances within the reservoir 10, in particular its cavity 14.
In this regard, let us return to the discharge portion 34 of the second connecting element 30. Advantageously, it may be flared from the discharge orifice 32 but this is not essential, to allow the formation of vortex disturbances within the reservoir 10. It should be noted that such a flaring of the discharge portion 34 would be due to the fact that its cross-section enlarges from the orifice 32 of discharging the fluid in the direction of the pumping device 60, i.e. in the direction of the flow. The flaring of the discharge portion 34 allows the pumping device 60, when located downstream of the second connecting element 30, to move the fluid more efficiently. This is simply because the pumping would not be as efficient if the second connecting element 30 had a cross-sectional area equal to that of the discharge orifice 32 along its entire length, its length being defined as the distance between the discharge orifice 32 and another end of said second element 30. Indeed, as mentioned above, the discharge orifice 32 has a diameter of at most 1 mm. The flaring is therefore of no interest other than to allow more efficient suction.
The system 1 further comprises a power supply module 62 for the pumping device 60. The power supply module 62 allows the pumping device 60 to act as a motor and to move the fluid F. In addition, the power supply module 62 also allows the power delivered by the pumping device 60 to be adjusted and any other useful parameters that the person skilled in the art will appreciate. Moreover, preferably, the power supply module 62 is configured so that the power delivered by the pumping device 60 allows the displacement speed of the fluid F in the system 1 to be adjusted to obtain a flow rate of fluid F of between 37 mL/min and 120 mL/min or even more. However, this is not the only parameter influencing the flow rate of fluid, as we will see in the following. Moreover, the system 1 may be equipped with a flow meter for controlling the flow rate of fluid in the reservoir 10 as a function of the power delivered by the power supply module 62. As an example, such a flow meter could be placed between the source reservoir 40 and the reservoir 10. Furthermore, the power supply module 62 need not be located in close proximity to the pumping device 60. It can be deported.
The system 1 further comprises a programming system 70 configured to control the power supply module 62 for the pumping device 60 to implement one or more platelet release sequences carried out so as to generate a continuous flow of the fluid F between said injection orifice 22 and said discharge orifice 32 and vortex disturbances within the reservoir 10 causing the fragmentation of the cytoplasmic extensions Ck of the megakaryocytic cells Mk. The programming system 70 comprises at least one or more processors adapted to execute a program to implement said platelet release sequences. As will be discussed below, the platelet release sequences may vary in their duration, their number, etc. That said, it should be noted that while the power supply module 62 may be controlled by means of such a programming system 70 for an industrial application, it may just as well be controlled manually by an operator.
With reference to
In the second embodiment of the system 1 according to the invention illustrated in
An example of an embodiment of such an assembly is shown in
In this regard, it may also be stated that the reservoir or the reservoirs 10, 10′, the aforementioned assembling elements and the connecting elements 20, 20′, 30, 30′ may be manufactured by any suitable manufacturing method known to the prior art. In the present case, the devices 2, 2′ were manufactured by 3D printing and assembled by screwing. In this regard, the devices 2, 2′ may comprise fastening means to allow their assembly. Preferably, they are made from polyether imide (PEI) resins or photopolymerizable resins (such as those used in the dental orthoses). Preferably, it is a material that complies with the pharmacopoeia of the country in which the system 1 according to the invention is to be used. The material can be certified by the ANSM (Agence Nationale de Sécurité du Médicament et des Produits de Santé), the EMA (European Medicines Agency) in France, the PhEU (European Pharmacopoeia), the PMDA (Pharmaceutical and Medical Devices Agency) in Japan or the FDA (Food and Drug Administration) and/or the USP (United States Pharmacopoeia) in the United States, etc. For example, for use in the United States, the device may preferably be made of biocompatible materials classified as USP VI, where VI refers to the USP class. The USP class test is one of the most common test methods for determining the biocompatibility of the materials. There are six classes, VI being the most rigorous. The class VI tests are intended to certify that there are no harmful reactions or long-term physical effects caused by chemicals released from plastics. Because of these specificities and the fact that the devices 2′ are closed, they are particularly well adapted to a treatment of the fluid specific to a medical use.
Such a configuration is also advantageous in that it improves the efficiency of the system while being optimally arranged. Indeed, instead of providing a pumping device 60 and a power supply 62 per device 2, 2′, these elements are shared by two, three or even more devices 2, 2′. Indeed, while the pumping device 60 is in fluidic communication with the first device 2 by means of the second fluidic connecting element 30 of said first device 2, as seen previously, the other devices 2′ are connected directly to the first connecting element 20, 20′ of the device 2, 2′ which precedes them. In other words, only the first device 2 is connected to the pumping device 60.
Consider, for example, the first other device 2′ directly connected to the first device 2. The second fluidic connecting element 30′ of said first other device 2′ is in fluidic communication with said first fluidic connecting element 20 of said first device 2. Preferably, this fluidic communication is direct. It is therefore in indirect fluidic communication with the pumping device 60 via the first device 2. Now consider the second other device 2′ directly connected to the first other device 2′. The second fluidic connecting element 30′ of said second other device 2′ is in fluidic communication with said first fluidic connecting element 20′ of said first other device 2′. This also applies to the other successive devices 2′.
Other types of assemblies, i.e. connection of the pumping device 60 to the devices 2, 2′ can be envisaged to connect the devices 2, 2′ in series while respecting the inventive concept of the invention.
It should be noted that, similarly to what has been seen for the first embodiment, the system 1 according to this second embodiment comprises in addition to the pumping device 60, the power supply module 62 for the pumping device 60 and the associated programming system 70. The geometry of the flow is not changed in such a system 1. In fact, the flow speed is preserved since instead of generating the depression or a discharge in the circuit of a single device 2, it propagates in all the devices 2′.
It may then be necessary to adapt the suction or discharge power, depending on the pumping device 60 used, and more precisely to increase it so that a sufficient displacement of the fluid is obtained for the most distant device 2′. However, if the suction or discharge power is too high, the efficiency of the platelet release can be adversely affected, since the efficiency is only optimal within a defined range of fluid flow rate and thus suction or discharge power. It is therefore appropriate to make such an adaptation according to this constraint if the pumping device 60 used does not allow a constant pressure to be maintained in the system 1. Most of the pumping devices currently available on the market generally allow to avoid this problem.
In a similar manner to what has been mentioned above for the pumping device 60, the power supply module 62 and the programming system 70, it is also possible to provide only a single source reservoir 40 and a single receiving reservoir 50 for the entire system 1, whether it comprises one or, if applicable, a plurality of devices 2. Such an embodiment is, for example, illustrated in
In a third embodiment of the invention shown in
In any case, similarly to the alternative embodiment of
Furthermore, the system 1 of the present invention can be adapted to integrate additional functionality, again in keeping with the inventive concept of the invention. For example, a pipe with a shape in Y could be provided at the inlet of the reservoir 10, which would allow two types of fluid F to circulate and mix within the reservoir 10, which, as we have seen, is configured to generate vortex disturbances.
The invention further relates to a method 5 for releasing platelets P from a fluid F comprising megakaryocytic cells Mk comprising cytoplasmic extensions Ck. The method 5 according to the invention allows, as will be discussed in detail in the following sections, to fragment megakaryocytes, Mk, and/or cytoplasmic extensions, Ck, in order to release the platelets. In this regard, the method 5 is implemented by means of a system 1 as previously described.
With reference to
Considering the industrial quantities of fluid F that can be treated and incidentally of platelets that can be released, a preliminary cell culture step can be considered in order to obtain the desired quantity of fluid F and precursors (i.e. megakaryocytes). The purpose of this cell culture step is to allow the maturation of the megakaryocytes Mk and their multiplication, i.e. their proliferation. The document FR 3 039 166 describes the cell culture process in more detail.
For example, the megakaryocytes Mk used in the Fluid F are megakaryocytes derived from immortalized or non-immortalized CD34+ progenitors. The pre-cultivation step of such megakaryocytes Mk may comprise two phases lasting a total of 11-17 days. During a first phase lasting between 5 and 9 days, the megakaryocytes Mk are cultured with a mixture comprising, for example, the following agents: serum-free culture medium, a cytokine cocktail containing TPO, IL-6 and IL-9 and the AhR antagonist and the LDL (Low density lipoprotein intended to stimulate the proliferation of the cells CD34+ and to engage them in the pathway of the megakaryocytopoiesis). The AhR favorably stimulates the maturation of the megakaryocytes Mk. In a second phase of this culture step, which lasts approximately 6-8 days, the resulting cells are cultured with a mixture of SR1 and thrombopoietin, TPO, and optionally other agents such as listed above with reference to the first phase. It should be noted that the cells obtained comprise megakaryocytes but not exclusively. In addition, it should be noted that not all cells give rise to megakaryocytes. These phases can last more or less time depending on the number of cells desired, the agents used, etc.
This preliminary step will not be further described as it is not the subject of the present invention and does not form part of it, the invention being limited to propose a method for the release of the platelets from the fluid F and not a method comprising any culture step. In any case, at the end of this cell culture phase, proplatelet megakaryocytes are obtained, i.e. megakaryocytes provided with proplatelets. They are simply referred to as megakaryocytes Mk in this present invention for simplicity. Such cells are shown in
Referring again to
During the step 200, the power of the power supply module 62 for the pumping device 60 may be set so as to adjust the suction or discharge power. Specifically, the power should be adjusted so that the resulting flow rate of fluid F is between 37 mL/min and 120 mL/min or more. It should be noted that depending on the material of which the device of the system 1 according to the invention is made, the fluid flow rate can be significantly increased. This is the optimum flow rate range to achieve the highest platelet release efficiencies with the method according to the invention. The power to be applied is inherently dependent on the material used for the pumping device 60.
During a step 300, the programming system 70 is controlled to initiate one or more platelet release sequences as previously defined. Advantageously, the programming system 70 can control the power supply module 62. It also allows to control over the times and the number of platelet P release sequences. The platelet release sequences can last as long as the user wishes in order to treat the desired volume of fluid.
When the programming system 70 as described in the previous section is operated with the pumping device 62 powered, a depression or, depending on the configuration, a discharge is generated in the device or the devices 2, 2′, 2″. “depression” means that the pressure in the device or the devices 2, 2′, 2″ is lower than the atmospheric pressure, i.e. that it/they is/are subjected to a relative vacuum. The “discharge” corresponds to the fact of moving the fluid F by a discharge pump, for example an ejector, or a compressor. As a result of this depression or this discharge, a continuous flow of fluid F, i.e. a displacement takes place in the system 1 from the source reservoir 40 to the receiving reservoir 50. It should be noted that, preferably, the suction or discharge power of the pumping device 60 is kept constant during the method 5 so that the depression, where appropriate the discharge speed, and the flow rate of fluid F are also kept substantially constant. However, this does not prevent the power from being varied during the implementation of the method within limits that allow the optimum platelet release efficiency to be obtained.
As soon as the depression or the discharge is generated in the system or the systems 2, 2′, 2″, the fluid goes through different phases i.e. 302, 304 and 306, which occur continuously as long as the depression or the discharge is maintained, without further action on the programming system 70. These phases are the direct consequence of the fluid displacement in the system or the systems 2, 2′, 2″. A phase 302 corresponds to the phase before the fluid F enters the reservoir 10, a phase 304 corresponds to the phase when the fluid F is in the reservoir 10 and a phase 306 corresponds to the phase after the fluid F is sucked out of the reservoir 10. In other words, the phases 302, 304 and 306 occur in practice simultaneously since when a part of the fluid F is being treated, at the same time another portion of the fluid F is being treated in the reservoir 10 and another part is about to be treated upstream of the reservoir 10, i.e. in the first connecting element 20 or in the source reservoir 40, along the fluidic circuit.
During the phase 302, the fluid F is sucked through the first fluidic connecting element 20. As the pressure within the device is maintained constant, the speed of the fluid F increases as it passes through the narrowing portion 24, due to the very fact of the flaring of said narrowing portion 24. The fluid F is then discharged into the reservoir 10 via the injection orifice 22. The singularity due to the abrupt cross-sectional enlargement present at the injection orifice 22/reservoir 10 interface causes a slowing down of the fluid F when it enters the reservoir 10.
During the phase 304, the fluid F flows through the reservoir 10 being subjected to the depression or the discharge generated by the pumping device 60 within the limits of the cavity 14. Indeed, although the fluid has been slowed down by the singularity existing at the orifice 22/reservoir 10 interface, it remains in motion because of this depression or discharge. The fluid does not flow directly out of the reservoir, which allows the circulation of said fluid in the form of vortex disturbances. Indeed, the system 1 is configured so that the residence time of the fluid F in the reservoir 10 is sufficient to generate disturbances. As previously discussed, a sufficient residence time is achieved due to the singularity of the large size of the reservoir 10 relative to the discharge orifice 32. The residence time can also be increased, but to a lesser extent, by advantageously misaligning the injection orifice 22 with respect to the discharge orifice 32 in the path of the fluid F as explained below.
Under these conditions, vortex disturbances of approximately the same size as megakaryocytes are generated. They allow to fragment the megakaryocytes Mk and their cytoplasmic extensions Ck (proplatelets), resulting in the release of the platelets P. At the same time, other products Dk resulting from the fragmentation of megakaryocytes are produced. These products are bits of proplatelets, intact bodies of megakaryocyte Mk or pieces of cytoplasm. Since the fluid F is treated continuously over time for each platelet release sequence, the phenomena described above are reproduced continuously and the platelets are released continuously. In sum, while the fluid F entering the reservoir 10 comprises megakaryocytic cells Mk comprising cytoplasmic extensions, the fluid F exiting the reservoir 10 comprises essentially platelets P and the other products, Dk. In this regard, the use of a system 1 comprising devices 2, 2′ in series, as illustrated in
During the phase 306, the fluid F, loaded with platelets P and other products Dk resulting from the fragmentation of the megakaryocytes is sucked through the discharge orifice 32 and more generally through the second fluidic connecting element 30. It then reaches the receiving reservoir 50, if any, and the pumping device 60.
During a step 400, the sequence or the sequences are stopped by shutting down the power supply module 62 using the programming system 70.
These phases occur identically in any other devices 2′ and/or 2″ present in the system 1 according to the invention.
In order to release the platelets, the flow rate of fluid should advantageously be between 37 mL/min and 120 mL/min or more. These flow rate values are average values representing the average of six measurements.
This is due to the characteristics of the device or the devices 2,2′, 2″ used in the present example embodiment. Indeed, to adjust the flow rate, a compromise must be found between the suction or discharge power of the pumping device 60, the sizes of the injection orifices 22 and of the discharge orifices 32, but also the condition of the surface of the elements of the device or the devices 2, 2′, 2″ through which the fluid F actually transits, this within the limits of the invention. Concerning this last aspect, the more the surface condition (micro-roughness, roughness, etc.) will have the effect of exerting constraints on the fluid F and the more it will be slowed down and vice versa. In order to achieve an even higher platelet release efficiency, the surface condition can be adjusted, for example. This can be done by modifying the material used to manufacture the elements of the device or the devices 2, 2′, 2″ through which the fluid F flows, by using other manufacturing methods allowing a control of the structuring on a micrometric scale but also by infusing a compound into the chamber such as plasma or albumin.
| Number | Date | Country | Kind |
|---|---|---|---|
| FR1911303 | Oct 2019 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/078639 | 10/12/2020 | WO |
