FR3112147A1 - Method of calibrating a fluidic system for the production of extracellular vesicles and associated production fluidic system - Google Patents

Abstract

METHOD FOR CALIBRATION OF A FLUID SYSTEM FOR THE PRODUCTION OF EXTRACELLULAR VESICLES AND ASSOCIATED FLUID SYSTEM FOR PRODUCTION The subject of the present invention is a method for calibrating a fluid system (1) for the production of extracellular vesicles (EV) from cells cells (6), the method comprising the following steps of determining (E1) a first level of cellular impermeability (V1) depending on an initial number (N1) of producer cells prior to production of extracellular vesicles (EV) , for controlling (E2) at least a first production (PR1) of extracellular vesicles (EV) configured at a first agitation value (XCTL1) and at a first production duration (T1), at least a first measurement (E3) of cellular impermeability (Vt) of the cells of the first production (PR1) and comparison with a second predetermined level of cellular impermeability (V2) lower than said first level of impermeability (V1), recording (E7) of at least one agitation range (PA1) whose lower limit (Lim1) is equal to the first agitation value (XCTL1) for which the level of impermeability (Vt ) measured from the first production (PR1) is substantially equal to said second level of impermeability (V2). Figure for the abstract: figure 2

Description

Method for calibrating a fluidic system for the production of extracellular vesicles and associated fluidic production system

Field of invention

The field of the invention relates to a method for calibrating a fluidic system for the production of extracellular vesicles and a fluidic system for the production of extracellular vesicles. The invention also relates to a method for producing extracellular vesicles whose configuration parameters are obtained by the calibration method.

State of the art

Cells are known to release extracellular vesicles into their environment, for example, in vivo, into the biological fluids of an organism. Extracellular vesicles have been identified as effective ways to deliver drugs, in a personalized or targeted way, into the human body. They first exhibit native biocompatibility and immune tolerance. They can also internalize theranostic nanoparticles, making it possible both to map certain parts of the body and/or to deliver active principles with therapeutic functions. Extracellular vesicles also have an intercellular communication function: they make it possible, for example, to transport lipids, membrane and cytoplasmic proteins and/or nucleotides from the cell cytoplasm, such as mRNAs, microRNAs or long non-coding RNAs , between different cells.

In particular, the use of extracellular vesicles can solve known problems during the therapeutic use of cells, such as cell replication, differentiation, vascular occlusions, risks of rejection and storage and freezing difficulties. There is therefore an industrial need for the production of cell vesicles in sufficient quantities for therapeutic use, in particular as a replacement or in addition to cell therapies.

To this end, Piffoux et al. (Piffoux, M., Silva, AK, Lugagne, JB, Hersen, P., Wilhelm, C., & Gazeau, F., 2017, Extracellular Vesicle Production Loaded with Nanoparticles and Drugs in à Trade ‐ off between Loading, Yield and Purity: Towards a Personalized Drug Delivery System, Advanced Biosystems ) describe the comparison of different methods of producing extracellular vesicles.

A first method consists in producing extracellular vesicles from endothelial cells of the umbilical cord vein (HUVEC), by subjecting these cells to hydrodynamic stresses mimicking the stresses exerted under physiological conditions within blood capillaries or under pathological conditions. in blood vessel stenosis. These stresses are driven by the passage of producer cells through microfluidic channels. A microfluidic chip comprises two hundred channels in which the cells are transported in a laminar flow, to produce vesicles in a parallel fashion.

However, this method presents sizing problems: the quantities of vesicles produced by a microfluidic chip are not adapted to the quantities required for the aforementioned applications. In addition, the yield of extracellular vesicles produced per cell introduced into such a chip (approximately 2.10 ⁴ vesicles per cell) is much lower than the maximum theoretical yield of vesicles produced by a cell, for example of the order of 3.5.10 ⁶ vesicles per cell for a cell of the MSC type (acronym for Mesenchymal Stem Cell in English). Finally, this method requires compliance with the so-called GMP standards (acronym for Good Manufacturing Practices), necessary for the manufacture of drugs.

A second method commonly used in the literature and described by Piffoux et al. consists in cultivating HUVECs in a culture medium of the DMEM type (acronym for Dulbecco's Modified Eagle's Medium) without serum, for three days (technique known as starvation in English, or serum deficiency). The absence of serum leads to cellular stress triggering a release of vesicles by the producing cells. This method has a higher yield and makes it possible to produce a larger quantity of vesicles than the method using a microfluidic chip (approximately 4.10 ⁴ vesicles per producing cell). However, the calculated yield corresponds to a much longer production time than the production time of the previous method. This method does not make it possible to produce a quantity of extracellular vesicles sufficient for the aforementioned applications. Finally, this method does not make it possible to produce vesicles continuously because it induces cell death.

Watson et al. (Watson, DC, Bayik, D., Srivatsan, A., Bergamaschi, C., Valentin, A., Niu, G., ... & Jones, JC, 2016, Efficient production and enhanced tumor delivery of engineered extracellular vesicles , Biomaterials, 105, 195-205) describe a method for producing vesicles which makes it possible to increase the quantity of vesicles produced. This method consists in cultivating adherent cells of the HEK293 type in culture flasks, then in hollow fiber membranes (Hollow Fiber Membrane in English). The central passage of the hollow fibers makes it possible to convey the culture medium to the producing cells. The producer cells are first seeded around this passage, where they produce vesicles in an inter-fiber space. The liquid medium included in the inter-fiber space is collected three times a week, making it possible to produce approximately 3.10 ¹² vesicles in several weeks, for very large quantities of seeded cells, for example of the order of 5.10 ⁸ cells, causing a yield of approximately 6000 extracellular vesicles per cell and a very low purity ratio (for example 1.09.10 ⁹ particles per microgram of protein). However, this production is not high enough and too slow for the aforementioned applications. In addition, this method is described using producer cells corresponding to a cell line particularly resistant to culture in serum-free medium: this method may not be transposable to production of vesicles by producer cells such as stem cells, for example human, less resistant and particularly suitable for the targeted therapeutic applications.

It is also well known to those skilled in the art that the culture of cells in suspension in 3 dimensions (3D) requires the use of a method with low agitation in order not to induce the death of the cells which it is desired to culture. It is in particular known to those skilled in the art that the Kolmogorov length is a criterion which makes it possible to evaluate the turbulence created by the action of mixing and to determine when the turbulence is excessive for a 3D culture.

Furthermore, it is recalled that the applicants have filed patent applications FR3068361A1 and WO2019/002608A1 describing a fluidic system and a method for producing extracellular vesicles whose agitation control significantly increases the quantities of production of such vesicles and makes it possible to consider large-scale production. Nevertheless, the parameterization of a production depends on multiple parameters, first of all biological inherent to the cells, as well as parameters related to the fluidic system itself. In particular, significant production differences were observed from one fluidic system to another, making the task of configuring production protocols to optimize their performance problematic.

There is therefore a need to remedy the aforementioned problems.

The aim of the invention is to propose a protocol for the production of extracellular vesicles with improved yield compared to known production techniques. One objective is to optimize the calibration and configuration of a fluidic production system, the principle of which is based on the action of fluidic constraints applied by the fluctuations of the liquid around the cells by agitation and generation of a turbulent flow. , so as to increase the large-scale productivity of such a production system. One objective is to propose a protocol for calibrating the control parameters of such a system according to each type of producer cells likely to be used for the system. Another goal is the identification of an optimal agitation range for various producer cells. Another objective is to propose a process for the production of extracellular vesicles exploiting the maximum production potential of a producer cell and whose optimal control parameters can be identified from the calibration protocol according to the invention.

Presentation of the invention

More specifically, the invention relates to a method for calibrating a fluidic system for producing extracellular vesicles from producing cells comprising a stirrer suitable for generating a turbulent flow of the liquid medium in a container to exert fluidic stresses on the producer cells in order to achieve advantageous production of extracellular vesicles, the method comprising the following steps:
- the determination of a first level of impermeability depending on an initial number of producer cells prior to the production of extracellular vesicles,
- controlling at least a first production of extracellular vesicles configured at a first agitation value and at a first duration.

According to the invention, the method further comprises:
- at least one first measurement of impermeability of the cells of the first production and comparison with a second predetermined level of impermeability lower than said first level of impermeability, each measurement of cellular impermeability of a production of extracellular vesicles consisting in calculating an impermeability loss ratio depending on a number of impermeable producer cells of each said production compared to the initial number corresponding to the conditions prior to said production,
- the recording of at least one agitation range whose lower limit is equal to the first agitation value for which the measured level of impermeability of the first production is substantially equal to said second level of impermeability.

According to a variant, the calibration method also comprises the following steps:
- controlling at least a second production of extracellular vesicles configured at a second agitation value and at a second duration,
- a second measurement of impermeability of the producing cells of the second production and comparison with a third predetermined level of impermeability lower than said second level of impermeability,
- the recording of the agitation range delimited by the lower limit and an upper limit, said upper limit being equal to the second agitation value from which the measured impermeability level of the second production is substantially equal to the third level of waterproofing.

According to a first calibration variant, the first level is an initial ratio corresponding to a level of cellular impermeability at 100% and the second level of cellular impermeability and the third level of cellular impermeability delimit a range of cellular impermeability comprised between 95% and 60% of the cell impermeability loss ratio, or between 90% and 65%, preferably between 85% and 70%.

According to a second calibration variant, the first level is an initial ratio corresponding to a level of cellular impermeability at 100% and in which the second level of cellular impermeability and the third level of cellular impermeability delimit a range of cellular impermeability between 70% and 0% of the cell impermeability loss ratio, between 70% and 0%, between 70% and 5%, between 50% and 5%, between 35% and 0%, between 25% and 5% , or even between 15% and 5%.

Alternatively, the recording is configured to record a plurality of extracellular vesicle production shake ranges where each range is specifically associated with a specific production time value and a specific producer cell type.

According to a variant, each cell impermeability measurement calculates a number of impermeable producer cells at the end of the production of extracellular vesicles, or continuously according to a sampling rate during the production of extracellular vesicles.

The invention also provides a fluid system for producing extracellular vesicles from producing cells comprising at least:
- a stirrer adapted to the generation of a turbulent flow of the liquid medium in a container to exert fluid constraints on the producing cells in order to carry out the production of extracellular vesicles,
- means for controlling the production of extracellular vesicles according to configuration parameters comprising at least one level of agitation and a duration,
- means for measuring the level of cellular impermeability of the producing cells, the means for measuring the level of cellular impermeability are adapted to calculate a ratio of loss of impermeability depending on a number of impermeable cells of said production of extracellular vesicles compared to the number corresponding to the prerequisites for said production,
- and the control means are configured at a predetermined agitation value so that the cell impermeability level at the end of said production is less than or equal to a second predetermined cell impermeability level which is at least less than the first level of initial cell impermeability prior to said production, said first level being dependent on an initial number of impermeable producer cells prior to said production of extracellular vesicles.

According to a variant of the system, at least the agitation value and the duration are controlled so that the level of cellular impermeability at the end of said production is included in a range of cellular impermeability delimited by the second level of cellular impermeability predetermined level and a third predetermined cellular impermeability level at least lower than said second level.

According to a first mode of production, the first level is an initial ratio corresponding to a level of cellular impermeability at 100% and in which the level of cellular impermeability at the end of said production is between 95% and 60% of the ratio of loss of impermeability, or between 90% and 65%, preferably between 85% and 70%.

According to a second mode of production, the first level is an initial ratio corresponding to a level of cellular impermeability at 100% and in which the level of impermeability at the end of said production is between 70% and 0% of the loss ratio impermeability, between 70% and 5%, between 50% and 5%, between 35% and 0%, between 25% and 5%, or even between 15% and 5%.

According to a preferred variant, the system further comprises means for configuring a production of extracellular vesicles recording at least a first predetermined production configuration defined by a range of agitation and a predetermined duration, in which said range is obtained by the calibration method according to any one of the preceding embodiments, and in which the control means are configured to select the agitation value of a production included in said range to execute said production.

According to this last variant, either the production duration is equal to the predetermined duration before the production of the first predetermined production configuration, or the production duration is finalized when the measured cellular impermeability level is included in the impermeability range cellular delimited by the second level of cellular impermeability and the third level of cellular impermeability.

According to a variant, the configuration means comprise a plurality of stored production configurations, each configuration being specific to a production duration and to a type of producer cells.

The invention also relates to a method for the ex vivo production of extracellular vesicles comprising the following steps:
- in a first step, a liquid medium of producing cells is introduced into the container of the fluidic system. During this step, or before this step, the production process includes a control step during which the initial number of impermeable producer cells is determined,
- a second step for setting the planned production at least by a predetermined level of agitation and a production duration which may or may not be determined,
- a third step of producing extracellular vesicles at the level of agitation and at the production duration previously configured so that the level of cellular impermeability at the end of said production is less than or equal to a second predetermined level of impermeability which is at least lower than the first initial level of cellular impermeability prior to said production, and preferably comprised in the range of cellular impermeability delimited by the second predetermined level of cellular impermeability and the third level of predetermined cellular impermeability lower than the second level ,
- a step of continuous control either of the time elapsed since the instant of triggering of production, or of the level of impermeability of the producing cells during production,
- in case of detection of at least one of the two conditions, a fourth stage of collection of the liquid medium containing the extracellular vesicles.

According to a variant, the duration of production may condition the end of production, but this is not mandatory.

Preferably, the parameter setting provides for the selection of said configuration parameters in a configuration file saved in the memory of the means for configuring the fluidic system. This configuration file is obtained from the calibration method according to the invention.

According to a first production variant, the second level of cellular impermeability and the third level of cellular impermeability delimit a range of cellular impermeability comprised between 95% and 60% of the impermeability loss ratio, or comprised between 90% and 65%, preferably between 85% and 70%.

According to a second production variant, the second level of cellular impermeability and the third level of cellular impermeability delimit a range of cellular impermeability comprised between 70% and 0% of the impermeability loss ratio, between 70% and 0% , between 70% and 5%, between 35% and 0%, between 25% and 5%, or even between 15% and 5%.

According to a variant, the production time (T) can be between values of approximately 20 minutes to approximately 6 hours, even more preferentially from approximately 1 hour to approximately 6 hours, even more preferentially from approximately 2 hours to approximately 3 hours, or even more preferably be equal to around 2 hours or alternatively around 3 hours or alternatively around 4 hours.

Preferably, it is planned at the end of production to operate a separation of the extracellular vesicles and the cellular debris. However, this last operation is not mandatory.

The invention also relates to the extracellular vesicles produced by implementing the fluidic system according to the invention as described in the present application, and/or by the process for the ex vivo production of extracellular vesicles from producing cells according to the invention as described in this application.

According to one embodiment, the producer cells used in the context of the present invention are human cells, preferably healthy human cells, excluding human embryonic stem cells.

Alternatively, the producer cells used in the context of the present invention are pathological cells, for example cells derived from tissues and/or from cancerous lines such as HeLa cells.

According to one embodiment, the producer cells used in the context of the present invention are animal cells, preferably murine cells, for example murine MSC cells (murine mesenchymal stem cells).

According to a preferred characteristic, the producer cells used in the context of the present invention are non-adherent cells.

According to another preferred characteristic, the producer cells used in the context of the present invention are adherent cells detached from their culture support, for example by appropriate treatment which is chosen from an enzymatic treatment, a chemical treatment, a mechanical treatment or a combination of these means.

According to one embodiment, the producer cells used in the context of the present invention are stem cells, in particular induced pluripotent stem cells, multipotent cells. By way of example, said stem cells are chosen from among multipotent mesenchymal cells, genetically modified cells, umbilical cord vein endothelial cells (HUVEC) or primary cells in general.

According to another embodiment, the producer cells used in the context of the present invention are cell line cells, preferentially from the human line of monocytes or from the human line of cells of hematopoietic origin derived from B lymphocytes, more preferentially they are These are THP-1 cells or Raji cells.

According to another embodiment, the producer cells used in the context of the present invention are primary cells, for example red blood cells.

According to a preferred characteristic, the producer cells used in the context of the present invention are isogenic cells, that is to say they come from the subject for which the extracellular vesicles produced by said producer cells will be used, for example by administration or by ex vivo use.

According to another preferred characteristic, the producer cells used in the context of the present invention are cells not originating from the subject for which the extracellular vesicles produced by said producer cells will be used, for example by administration or by ex vivo use. According to a more preferred characteristic, the producer cells used in the context of the present invention are allogenic cells, that is to say from the same species as the species of the subject for which the extracellular vesicles produced by said producer cells will be used. Alternatively, the producer cells used in the context of the present invention are xenogenic cells, that is to say from a species different from the species of the subject for which the extracellular vesicles produced by said producer cells will be used.

For the producer cells, in particular the human cells, used in the context of the invention, the use of human embryonic stem cells is excluded.

According to a preferred characteristic, the concentration of the producer cells in the liquid medium of the container of the fluidic system is between 5.10 ⁴ and 9.10 ¹⁴ producer cells per liter of liquid medium, preferably between 5.10 ⁷ and 1.10 ¹⁴ producer cells per liter, preferentially between 5.10 ⁷ and 1.10 ¹³ per liter, even more preferably between 2.10 ⁸ and 1.10 ¹² per liter, even more preferably about 2.5.10 ⁸ per liter of said liquid medium. According to another preferred characteristic, the concentration of producer cells in the liquid medium of the container of the fluidic system is between 1.10 ⁸ and 1.10 ¹² producer cells per liter of liquid medium.

In particular, an object of the invention consists of the vesicles produced by the fluidic system according to the invention.

In particular, an object of the invention is the use of the vesicles produced by the fluidic system according to the invention to act on cells.

In particular, an object of the invention is the use of the vesicles produced by the fluidic system according to the invention for imaging purposes and/or for therapeutic purposes and/or for diagnostic purposes.

It is well known to those skilled in the art that the structure of extracellular vesicles varies according to the producer cells used and according to the method of obtaining used, in particular in terms of membrane markers and constituents present on these vesicles.

According to one embodiment, the extracellular vesicles according to the present invention have an average diameter of between 40 and 300 nm, preferentially between 45 and 90 nm, more preferentially between 50 and 65 nm, even more preferentially approximately 60 nm, said average diameter of the extracellular vesicles being measured by an interferometry method in combination or not with fluorescence, preferably said average diameter is measured using the ExoView™ R100 device marketed by the company NanoView Bioscience.

According to one embodiment, the extracellular vesicles according to the present invention have an average diameter of between 50 and 500 nm, preferably between 100 and 110 nm, more preferably between 105 and 109 nm, even more preferably around 106 nm or alternatively around 108 nm , said average diameter of the extracellular vesicles being measured by an individual particle tracking method (or NTA for Nanoparticle Tracking Analysis) for example with the NanoSight NS300 device marketed by the company Malvern Panalytical.

In particular, an object of the invention is the use of the vesicles produced by the fluidic system according to the invention to act on cells.

In particular, an object of the invention is the use of the vesicles produced by the fluidic system according to the invention for imaging purposes and/or for therapeutic and/or diagnostic purposes.

According to one embodiment, the stirrer of the fluidic system for producing extracellular vesicles from producing cells in suspension according to the invention consists of a blade. Alternatively, said stirrer consists of 2, 3, 4, 5, 6, 7, 8 or more than 8 blades.

According to a preferred characteristic, the at least one blade of said agitator is a vertical blade.

According to one embodiment, the stirrer of the fluidic system for producing extracellular vesicles from producing cells in suspension according to the invention is a stirrer of the propeller type, for example marine or propeller with profiled blades, or a turbine, for example Rushton, or a stirring anchor, or a barrier stirrer, or a helical ribbon propeller, or a paddle wheel, or a cogwheel, or a magnetic stirrer, or a combination of these stirrers.

In one embodiment, static structures may be present in the container, for example baffles, or even structures forming a partial barrier to liquid movement, such as those used in a static mixer.

According to one embodiment, the container of the fluidic system for the production of extracellular vesicles from producing cells in suspension according to the invention is a stirring flask with a capacity of 100 mL (for example the Spinner stirring flask Bellco device for cell suspensions, Bellco reference 505001), comprising a blade with a diameter of 3.8 cm and a working volume of less than 100 mL.

According to another embodiment, the container of the fluidic system for producing extracellular vesicles from producing cells in suspension according to the invention is a stirring flask whose structural characteristics (capacity, blade diameter and working volume) are all increased or decreased proportionally to those mentioned above for the 100 mL capacity stirring flask; according to another embodiment, said structural characteristics are all increased or decreased in a non-proportional manner compared to those mentioned above for the stirring flask with a capacity of 100 mL, in particular during a change of scale.

According to one embodiment, the container of the fluidic system for the production of extracellular vesicles from producing cells in suspension according to the invention is a stirring flask with a capacity of 500 mL (for example the Spinner stirring flask Bellco device for cell suspensions, Bellco reference 505005), comprising a blade with a diameter of 7.6 cm and a working volume of 200 mL to 500 mL, or a stirring flask whose structural characteristics (capacity, blade diameter and volume work) are all increased or decreased proportionally to those mentioned above for the stirring flask with a capacity of 500 mL, or a stirring flask whose said structural characteristics (capacity, blade diameter and volume work) are all increased or decreased in a non-proportional manner compared to those mentioned above for the 500 mL capacity stirring flask, in particular ment during a change of scale.

According to one embodiment, the container of the fluidic system for producing extracellular vesicles from producing cells in suspension according to the invention is a stirring flask with a capacity of 1000 mL (for example the Spinner stirring flask Bellco device for cell suspensions, Bellco reference 505010), comprising a blade with a diameter of 10.8 cm and a working volume greater than or equal to 300 mL and less than 1 L, or a stirring flask whose structural characteristics (capacity, diameter of the blade and working volume) are all increased or decreased proportionally with respect to those mentioned above for the stirring flask with a capacity of 1000 mL.

According to another embodiment, the container of the fluidic system for producing extracellular vesicles from producing cells in suspension according to the invention is a bioreactor whose working volume is between 400 and 1000 mL and the diameter of the blade is 6cm.

According to another embodiment, the container of the fluidic system for the production of extracellular vesicles from producer cells in suspension according to the invention is a bioreactor whose working volume and the diameter of the blade are increased or decreased proportionally by compared to the respective values of 400 mL and 6 cm.

According to another embodiment, the container of the fluidic system for the production of extracellular vesicles from producer cells according to the invention is a bioreactor with stirring means by blade, which the person skilled in the art can have or design.

According to another embodiment, the container of the fluidic system for the production of extracellular vesicles from producer cells according to the invention is a bioreactor whose working volume and the diameter of the blade are increased or decreased in a non-proportional manner with respect to to the respective values mentioned above, in particular during a change of scale.

According to another embodiment, the container of the fluidic system for producing extracellular vesicles from producing cells according to the invention is a bioreactor or a stirring flask, the geometric characteristics of which, the working volume, the type of mixer and its characteristics, and the mode of operation are chosen according to practices accessible to those skilled in the art.

The principle of the invention is based on the postulate that the optimal production levels of extracellular vesicles are located in areas related to the triggering of a drop in membrane impermeability. Indeed, we seek to cause sufficient fluidic stresses on the producing cells in order to bring them to their maximum vesiculation potential, a phase during which the producing cells draw on their internal lipid reserves.

In particular, two areas of loss of cellular impermeability that are interesting from a production yield point of view have been identified. A first zone corresponds to a zone of loss of reasonable cellular impermeability at the end of production while presenting interesting yields. A second zone corresponds to a higher production zone but in return which is associated with a sharp drop in the level of impermeability of the producing cells. The calibration process takes advantage of this postulate and makes it possible to establish a point of reference, independently of the cell type and the configuration of the production system, making it possible to detect zones of optimal production by an identification based on the calculation of a loss of impermeability ratio. In addition, the calibration process is based on a method for measuring the level of cellular impermeability at low cost and suitable for continuous monitoring of the level of impermeability of producing cells. Thanks to the calibration process, it is therefore possible to significantly improve the production yields of extracellular vesicles.

In addition, it is envisaged that the calibration method can be configured in memory of control means of the fluidic system in order to implement in an automated manner, prior to production of extracellular vesicles, a phase of calibration of the control parameters, in l occurrence the level of agitation of the liquid medium and the duration of production, to detect the optimal areas of production yield. It is also envisioned that the fluidic system is capable of configuring a plurality of optimal configurations, where each configuration depends on a specific cell type and production time. Another advantage of the invention is that it makes it possible to produce vesicles in an optimized manner in large quantities and in a system that complies with G.M.P (acronym for Good Manufacturing Practices) standards, necessary for the manufacture of drugs.

Other characteristics and advantages of the present invention will appear more clearly on reading the following detailed description comprising embodiments of the invention given by way of non-limiting examples and illustrated by the appended drawings, in which:

tricks

represents an example of a fluidic system suitable for implementing the calibration method according to the invention and suitable for the large-scale production of extracellular vesicles.

is a flowchart representing an embodiment of the method for calibrating a fluidic system according to the invention.

is a graph representing a first variant of optimum stirring range obtained by the calibration method according to the invention.

is a graph representing a second variant of optimum stirring range obtained by the calibration method according to the invention.

is a representative diagram of the fluidic system and configuration means whose control parameters are configured in accordance with the values obtained from the calibration.

is a flowchart depicting a method of producing extracellular vesicles ex vivo in accordance with the invention.

detailed description

The invention is a method for calibrating a fluidic system for producing extracellular vesicles from producing cells comprising a stirrer suitable for generating a turbulent flow of the liquid medium in a container to exert fluidic stresses on the producing cells in order to achieve the production of extracellular vesicles. The aim of the invention is to find an optimal agitation range in consideration of an extracellular vesicle production yield, a desired production duration and a given cell type.

Definitions

The term “extracellular vesicle” generally designates a vesicle released endogenously by a producer cell, the diameter of which is between 30 nm and 500 nm. An extracellular vesicle corresponds in particular to an exosome and/or a microvesicle and/or an apoptotic cellular body.

The term "producer cell" generally designates independently either a cell that adheres to a culture medium (culture medium also referred to as a micro-carrier), or a cell that is not adherent to a culture medium ( also referred to as a "suspension cell"), and can divide and multiply. In the case of adherent cells, these can be adherent on microcarriers themselves in suspension in the liquid culture medium. According to another aspect of the invention, the term "producer cells" designates human cells, excluding human embryonic stem cells, of animal or plant origin, bacteria or other microorganisms capable of secreting extracellular vesicles . According to another aspect of the invention, the term “producer cells” designates adherent cells detached from their culture support and placed in suspension. A gentle mixing created by the agitator allows the suspended producer cells as defined to remain in suspension in the liquid culture medium. According to another aspect of the invention, the term “producer cells” designates cellular aggregates. The term "cellular aggregates" designates an assembly of several producer cells which adhere to each other. A gentle mixing created by the agitator allows the suspended producer cells as defined to remain in suspension in the liquid culture medium.

The terms “microcarrier” and “microcarrier” designate a generally spherical matrix allowing the growth of adherent producer cells on its surface or inside and whose maximum characteristic size is between 50 μm and 500 μm, and preferentially between 100 μm and 300 µm. The microcarriers are generally beads whose density is chosen substantially close to that of the liquid culture medium of the producing cells. Thus, gentle mixing allows the beads to remain suspended in the liquid culture medium.

The term “fluidic stresses” designates the shearing and/or stretching stresses acting on the surface of the producing cells under the action of the turbulent fluid.

The term "agitator" must be understood in an extremely general sense, which is that of a means or a combination of means making it possible, by action on the liquid, to generate at least one flow, to promote the mixing of the liquid or to generate turbulence in this liquid.

The term "impermeable cell" should be understood in the sense of non-modification of the natural semi-permeability of the membrane of a cell; a cell loses its "impermeability" in the above sense if this cell allows dyes to pass into its intracellular medium which would be present in the extracellular medium, and which even in this case are normally not present in the intracellular medium (for example blue of Trypan)

The term “approximately”, or “substantially” placed in front of a number, signifies at most plus or minus 10% of the nominal value of this number.

The measurement of impermeable cell count can be subject to sampling errors or measurement errors which are generally related to the way of taking the sample and/or the principle of measurement. Those skilled in the art know how to obtain an indirect estimate of these errors by carrying out multiple measurements under the same conditions: any variations in measurements when the conditions are apparently the same are indicators of measurement errors. In this sense, the levels indicated for the cellular impermeability losses prescribed in the present application are naturally to be taken into account with an error bar which is a function of the effective errors estimated by according to the method above for the type of measurement carried out for the determination of the number of impermeable cells. Moreover, the present calibration method is based on the ratio between the number of impermeable cells at a time T and the number of initially impermeable cells. Measurement errors in both the numerator and the denominator of this ratio are to be taken into account in the error likely to affect this ratio. According to the calibration method according to the present invention, the errors in the determination of the above ratio are to be taken into account for the determination of the levels of impermeability.

The term "cell" designates the smallest fundamental structural and functional unit of living organisms, consisting of a protoplasm or cytoplasm, separated from the external environment by a membrane. In the context of the present invention, the term "cell" also encompasses red blood cells and platelets.

The term "healthy cells" refers to cells from healthy tissues, as opposed to cells from pathological tissues or organs, i.e. whose functions are impaired.

The expression "between X and Y" relates to the range of values between X and Y, the X and Y terminals being included in said range.

The term "immunotherapy" refers to the treatment of a disease by an intervention on the immune system.

The term "regenerative medicine" refers to all biomedical methods used for the replacement or regeneration of human tissues or organs for therapeutic purposes.

The term "subject" designates an animal, in particular a mammal which can be chosen from humans, rats, mice, pigs, chinchillas, guinea pigs, goats, sheep, cats, dogs, cow, monkey, male or female, regardless of age. Within the meaning of the present invention, a subject can be a patient, namely a person receiving medical care, undergoing or having undergone medical treatment, or monitored in the context of the development of a disease.

The term "cell therapy" refers to the use in a subject or patient of living somatic cells, manipulated or modified in their biological characteristics, to prevent, treat or alleviate certain pathologies.

Within the meaning of the present invention, the term “cellular debris” designates the cellular residues following the destruction of the cells, in particular fragments of the plasma membrane, elements of the cytosol, fragment of nucleus, nuclear envelope of intracellular organelle, and generally any protein or nucleoprotein assembly. The cellular debris can optionally be eliminated during a clarification step, if their characteristic size is such that a prefiltration or gentle centrifugation step makes it possible to eliminate them by separating these debris from the vesicles. A debris can in particular be a fragment of cell membrane whose membrane topology is not that of a closed globule as is the case for extracellular vesicles.

Surprisingly and unexpectedly, an excessive agitation value can be bad both in terms of the quantity of EVs produced by initial cells and also in terms of the quantity of cellular contaminants at the end of production.

As such, the production of vesicles according to certain preferred calibration modes of the invention can make it possible, after clarification, to obtain vesicles with an advantageous purity. Those skilled in the art are aware of various methods for defining certain types of purity. Furthermore, those skilled in the art know that all the types of impurities present in the solution which contains the vesicles do not necessarily present the same problem with respect to possible purification steps.

According to another advantageous mode of calibration according to the present invention, it is possible to seek to maximize the quantity of vesicles produced, to the detriment of a certain loss of purity. The complementary optimization finally chosen is naturally dependent on the steps possibly envisaged to carry out the purification of the interfering species with regard to in-vivo tests for example, both in terms of GMP qualification and possibly undesirable effects.

General architecture of the fluidic system

There schematically illustrates a fluidic system (1) for the optimized production of extracellular vesicles (EV). The fluid system (1) for producing extracellular vesicles (EV) is aimed at producing a large quantity of extracellular vesicles (EV) in a container (4). However, the invention is not limited to this embodiment and may comprise a series of containers (4) fluidically connected in parallel or in series.

The container (4) contains a liquid medium (5). The container (4) can in particular be a tank, a flange, for example made of glass or plastic, or any other container adapted to contain a liquid medium (5). The container can be flexible, or contain flexible parts. The volume of the container (4) is one of the factors making it possible to produce extracellular vesicles (EV) in large quantities: this volume can be between 50 mL and 500 L, preferentially between 100 mL and 100 L, and preferentially between 300 mL and 40 L. The volume of the container (4) illustrated schematically in the is 1 L. The container (4) typically comprises one or more gas inlets and one or more gas outlets, through which an atmosphere comprising air, O2, N2 and CO2 concentrations suitable for the cells can flow, for example example including 5% CO2. This atmosphere can come from a suitable gas injector/mixer or from a CO2 controlled atmosphere oven. A second pump (17) makes it possible to control this gaseous flow in the container (4). The container (4) also comprises an outlet (9) capable of comprising liquid medium (5) and extracellular vesicles (EV). This outlet can be supplemented with a means of separation and/or filtration of the cells in suspension making it possible not to recover cells in suspension outside the container (4). This outlet (9) allows the extracellular vesicles (EV) produced to be extracted from the container (4). The container (4) can also comprise at least one inlet (8) suitable for introducing the liquid medium (5) into the container (4).

The liquid medium (5) can generally be a saline solution, for example isotonic. Preferably, the liquid medium (5) is a liquid culture medium with the addition of compounds allowing the culture of the cells of interest, or a medium supplemented with serum or platelet lysate previously purified from extracellular vesicles or a serum-free medium, allowing no not contaminate the extracellular vesicles (EV) produced by the fluidic system (1) with proteins or other vesicles from a serum or platelet lysate. It can be used a liquid medium (5) without serum such as DMEM for example. The maximum volume of liquid medium (5) is determined in part by the container (4). This maximum volume can also be between 50 mL and 500 L, preferentially between 100 mL and 100 L, and more preferentially between 300 mL and 40 L. The minimum volume of liquid medium (5) contained by the container (4) is in part determined by the choice of the agitator (7) making it possible to agitate the liquid medium (5).

The fluidic system (1) also includes suspension producer cells (6), the term suspension producer cells including both suspension cells (non-adherent cells) and suspended cells (adherent cells). Extracellular vesicles (EV) are produced by the fluidic system (1) from these suspended producer cells (6). The producer cells in suspension (6) can be cultured, before the production of extracellular vesicles (EV) by the fluidic system (1) in a suitable cell culture medium. Thus, no transfer of cells is necessary between the culture of the producer cells in suspension (6) and the production of the extracellular vesicles (EV), which makes it possible to avoid any contamination and to simplify the process as a whole. The majority of the producer cells in suspension (6) are in suspension in a homogeneous manner in the medium, even if a minority proportion of producer cells in suspension (6) can be sedimented at the bottom of the container (4) or stuck to the wall of the container (4), for example by stirring the liquid medium (5). Preferably, the fluidic system (1) is adapted so as to generate gentle agitation making it possible to homogenize the producing cells (6) in the liquid medium (5) within the container (4), preferably before the production of the extracellular vesicles. In general, any type of producer cells (6) can be used, preferably non-adherent suspension producer cells (6).

The container (4) also includes a stirrer (7) for stirring the liquid medium (5). The agitator (7) can be a paddle wheel, the vanes of which are at least partially immersed in the liquid medium (5), and set in motion by any means known to those skilled in the art allowing, by any known means of the person skilled in the art to obtain a transmission of forces, for example and in a non-limiting way by magnetic and/or mechanical coupling. The agitator (7) can also be a liquid medium infusion system (5) at a rate sufficient to agitate the liquid medium (5) contained by the container, or a system with rotating walls (for example arranged on rollers) . The agitator (7) can alternatively be of the bottle roller or bottle roller type, orbital agitator for Erlenmeyers, with or without baffles (shaken flask), rocking agitator (wave), bioreceptacle with pneumatic agitation (air-lift) or a rotary stirrer with blades such as a stirrer of the marine propeller type, Rushton turbine, stirring anchors, barrier stirrer, helical ribbon propeller. A preferred rotary agitator is a vertical blade impeller. Finally, static structures may be present in the container, for example baffles, or even structures forming a partial barrier to liquid movement, such as those used in a static mixer, may of course also be used. The agitator (7) and the dimensions of the container (4) are adapted to control a turbulent flow of the liquid medium (5) in the container (4). By turbulent flow is meant a flow whose Reynolds number is greater than 2000. The Reynolds number can for example be calculated by the formula (IV). Preferably, the Reynolds number Re of the liquid medium flow (5) is greater than 7,000, preferably greater than 10,000 and preferably greater than 12,000.

Other stirrers (7) making it possible to control an at least partially turbulent flow according to the present invention are stirrers well known to those skilled in the art and capable of being installed in the system according to the present invention.

Typically, turbulent flow is induced and/or controlled by at least one agitator operational configuration parameter, referred to in the description as the agitation level configuration parameter. By way of non-limiting examples, the configuration parameter can be the speed of rotation of a stirrer, an injection rate in a static mixer, a shaking frequency, etc.).

Furthermore, it is common practice to establish a control of the level of agitation of a fluidic system by a parametric correspondence inherent to the turbulent fluid, which can for example be an energy injection rate, or even a length by Kolmogorov. This mode of controlling the level of agitation of a fluidic system is described in patent applications FR3068361A1 and WO2019/002608A1. Care is therefore taken to provide a description of the theoretical formulas below, the purpose of which is to establish this correspondence of calculation so that the person skilled in the art, through his general knowledge, can calculate the configuration parameter suitable for piloting of the chosen fluidic system.

Theoretical elements

The Kolmogorov length (or Kolmogorov dimension or Eddy length) is the length from which the viscosity of a fluid allows the kinetic energy of a flow of this fluid to be dissipated. In practice, the Kolmogorov length corresponds to the size of the smallest eddies in a turbulent flow. This length L _K is calculated in Kolmogorov's publication (Kolmogorov, AN, 1941, January, The local structure of turbulence in incompressible viscous fluid for very large Reynolds numbers , In Dokl. Akad. Nauk, SSSR, Vol. 30, No. 4, pp. 301-305) and described by the following formula (I):

[Math 1] (I)

where ν is the kinematic viscosity of the flowing liquid medium and ε is the average rate of energy dissipation into the fluid per unit mass (or rate of energy injection into the fluid).

Haruki Furukawa et al (Haruki Furukawa, Yoshihito Kato, Yoshiro Inoue, Tomoho Kato, Yutaka Tada, and Shunsuke Hashimoto, (2012) Correlation of Power Consumption for Several Kinds of Mixing Impellers, Int J. Chem. Eng. Article ID 106496, DOI : 10.1155/2012/106496), Zhou et al. (Zhou, G., Kresta, SM, 1996, Impact of tank geometry on the maximum turbulence energy dissipation rate for impellers , AIChE journal, 42(9), 2476-2490) describe the relationship between mean ε and the geometry of a container in which a liquid medium is agitated by a paddle wheel type agitator. This relationship is given by the following formula (II):

[Math 2] (II)

where N _p is the dimensioned power number (or Newton number) of the agitator in the liquid medium, D is the diameter of the agitator (in meters), N is the speed of rotation (in number of revolutions per second) and V is the volume of liquid medium (in cubic meters). This relation is used for the calculation of average ε corresponding to the geometry of a container and of a stirrer used for the implementation of the invention. The power number Np is given in a known way by the formula (III):

[Math 3] (III)

where P is the power provided by the stirrer, and ρ is the density of the liquid medium. Formula (III) can be adjusted as described in Haruki Furukawa et al (Haruki Furukawa, Yoshihito Kato ,Yoshiro Inoue, Tomoho Kato, Yutaka Tada,and Shunsuke Hashimoto,(2012) Correlation of Power Consumption for Several Kinds of Mixing Impellers,Int J. Chem. Eng. Article ID 106496, DOI: 10.1155/2012/106496), and Nienow et al. (Nienow, AW, & Miles, D., 1971, Impeller power numbers in closed vessels, Industrial & Engineering Chemistry Process Design and Development , 10(1), 41-43) or Zhou et al. (Zhou, G., Kresta, SM, 1996, Impact of tank geometry on the maximum turbulence energy dissipation rate for impellers , AIChE journal, 42(9), 2476-2490) as a function of the Reynolds number of the medium flow liquid. It is also possible to calculate the Reynolds number of the system by the following formula (IV):

[Math 4] (IV)

Alternatively, the person skilled in the art with his general knowledge and with alternative calculation methods can calculate the Kolmogorov length. In any case, the calculation presented above is only one way among many others known to those skilled in the art to calculate the Kolmogorov length.

The stirrer (7) used in the exemplary embodiments of the invention comprises a paddle wheel arranged in a container (4) and set in motion by a system for transmitting magnetic or mechanical forces. The speed of the paddle wheel in the liquid medium (5) causes a flow of the liquid medium (5). The agitator is adapted to control a flow which, given the dimensions of the container (4), can be partially turbulent. In the case of the agitator (7) illustrated in , several parameters make it possible to calculate a value representative of the turbulence of the liquid medium (5), in particular the kinematic viscosity v of the liquid medium (5), the dimensions of the container (4) and in particular the volume V of liquid medium (5 ) contained in the container (4), the power number Np corresponding to the submerged part of the paddle wheel or the blade, the diameter D of the agitator and in particular of the wheel, the speed N of rotation of the wheel. The user can thus calculate, as a function of these parameters, values representative of the turbulence of the flow, and in particular the Kolmogorov length LK, as given by equations (I), (II) and (III) .

In an exemplary embodiment of the fluidic system (1), the speed of rotation of the agitator (7) is capable of being controlled at 500 rpm (rotations per minute) for example, the diameter of a paddle wheel or the blade is 10.8 cm and the volume of liquid medium contained by the container (4) is 400 mL. The measured power number N _P of the paddle wheel or of the blade in the liquid medium (5), by the formula (III), is substantially equal to 3.2. The energy dissipated per unit mass ε, calculated by formula (II), is equal to 6.80.10 ^-1 J.kg ^-1 . The Kolmogorov length L _K calculated by formula (I) is thus equal to 11.0 μm.

In general, for production of extracellular vesicles, the fluidic system is controlled according to at least one parameter of the level of agitation of the stirrer (7) and a parameter of production duration. These two configuration parameters determine an extracellular vesicle (EV) production yield and are generally specific to the cell types of the producer cells (6). The calibration method according to the invention aims to identify an optimal production operating range dependent on a loss of impermeability of the producing cells (6).

Protocol production of extracellular vesicles (EV)

The extracellular vesicles (EV) are produced in a container (4) containing a liquid medium (5), for example without serum, of the producer cells (6) in suspension. The medium used before production for the culture of the producer cells (6) comprising serum, the container (4) is washed three to four times with liquid medium (5) DMEM without serum, each washing corresponding for example to a volume of about 400ml. The stirring of the liquid medium (5) is then controlled by the stirrer (7) so as to cause a turbulent flow in the container (4). The production of extracellular vesicles (EV) can be measured during production. For this purpose, the agitation can be continuous, intermittent, increasing or decreasing. The producer cells (6) are allowed to sediment at the bottom of the container (4), then a sample of liquid medium (5) comprising EV extracellular vesicles is taken. The sample is centrifuged at 2000 G for 10 minutes to remove cell debris. The supernatant is analyzed by an individual particle tracking method in order to count the number of extracellular vesicles (EV) and to deduce the concentration of extracellular vesicles (EV) in the samples. It can be verified that the concentration of extracellular vesicles (EV) at the start of shaking is close to zero or negligible.

The extracellular vesicles (EV) produced can also be observed and/or counted by cryo-transmission electron microscopy. For this purpose, a drop of 2.7 μL of solution comprising extracellular vesicles (EV) is deposited on a grid suitable for cryo-microscopy, then immersed in liquid ethane, resulting in an almost instantaneous freezing of said drop, avoiding the formation of ice crystals. The grid supporting the extracellular vesicles (EV) is introduced into the microscope and the extracellular vesicles (EV) are observed at a temperature of the order of -170°C.

Separation of extracellular vesicles

The extracellular vesicles (EV) produced in the container (4) are capable of being extracted from the container (4) through the outlet (9) of the container (4), suspended in the liquid medium (5). A filter (18) can be arranged at the outlet (9) so as to filter the producer cells (6) in suspension and the cellular debris during the extraction of extracellular vesicles (EV) from the container (4). A connector (13) is fluidically connected to the outlet (9), allowing the transport of the liquid medium (5) comprising the extracellular vesicles (EV) produced.

The fluidic system (1) may include an extracellular vesicle (EV) separator (15). The separator (15) comprises an inlet of the separator (10), into which the liquid medium (5) comprising extracellular vesicles (EV) from the container (4) can be conveyed directly or indirectly. The separator (15) can also comprise a first outlet (11) of the separator, through which the liquid medium (5) is capable of leaving the separator (15) with a concentration of extracellular vesicles (EV) smaller than at the inlet ( 10) of the separator (15), or even substantially zero. The separator (15) can also comprise a second outlet (12) from the separator (15), through which the liquid medium (5) is capable of leaving the separator (15) with a concentration of extracellular vesicles (EV) higher than at the inlet (10) of the separator (15).

In general, the separator (15) of extracellular vesicles (EV) can be fluidically connected to the container (4) so as to be capable of reintroducing a liquid medium (5) depleted in vesicles (EV) into the container (4), for example through the inlet (8) of the container (4). Thus, the production and/or the extraction of extracellular vesicles (EV) can be carried out continuously, at a substantially constant volume of liquid medium (5) in the container (4). According to an alternative embodiment, the fluidic system does not comprise an extracellular vesicle (EV) separator (15) or the fluidic system comprises an extracellular vesicle (EV) separator (15) which may or may not be fluidically connected, for example by via a closing means of said separator (15), to the container (4). Thus, the production and/or the extraction of extracellular vesicles (EV) can be carried out discontinuously or continuously depending on the opening or closing of the closing means arranged upstream of the separator (15 ).

In the case of discontinuous operation, the container containing the producing cells is agitated and the production time is preferably chosen for a time greater than 20 minutes.

The liquid can then be extracted from the container, and can be subjected to one or more subsequent purification steps, in particular to separate the vesicles from the producer cells. This separation can be carried out by means of techniques known to those skilled in the art, for example and taken without limitation, by acoustic techniques, filtration methods such as separation by tangential filtration, the use of rotary filters or any combinations of separating means.

In the case of continuous operation and according to a preferred embodiment, the separation system is internal to the container, the vesicles are gradually separated in a sub-compartment of the container. Various technical means are known to those skilled in the art for carrying out this type of separation, for example and without limitation, the use of rotary filters, or even acoustic means, or any combination of separation means. According to another embodiment, the separation system between cells and vesicles can involve a fluidic circuit designed to circulate the medium with the producing cells and the vesicles between the container on the one hand and a separation system external to the container on the other hand. 'somewhere else. This separation system external to the container may involve techniques known to those skilled in the art, for example, and without limitation, tangential filtration, or acoustic separation, or even any combination of means. known separations. At the outlet of the separation system, the liquid depleted in vesicles is reinjected into the container, so that the production of vesicles by the producing cells can continue in the container.

In the exemplary embodiment of a fluidic system (1) illustrated in the , the liquid medium (5) can be extracted from the container (4) by a first pump (16), via a connector (13), so as to transport the liquid medium (5) into a collector (19). Another first pump (16') makes it possible to convey the liquid medium (5) contained in the collector (19) to the inlet (10) of the separator (15), via another connector. The first outlet (11) of the separator (15) is connected to the container (4) via a connector, so as to reintroduce liquid medium (5) depleted in extracellular vesicles (EV) into the container (4). The second outlet (12) of the separator (15) is connected to the collector (19) via a connector, so as to enrich the liquid medium (5) contained in the collector (19) with extracellular vesicles (EV). Alternatively, the inlet (10) of the separator (15) can be directly connected to the outlet (9) of the container (4) (or via a first pump (16)). The first outlet (11) of the separator (15) is connected to the container (4) and the second outlet (12) of the separator (15) is connected to the collector (19). Several separators can also be arranged in series to vary the degree of separation into extracellular vesicles (EV) in the liquid medium (5), and/or in parallel to adapt the flow rate of liquid medium (5) in each separator (15) at the flow rate of a first pump (16).

Principle of production optimization by identification of an operating range dependent on a loss impermeability producer cells

Measure impermeability

A normal cell needs selective membrane permeability in order to let in some compounds needed by the cell and eject others, including maintaining osmolarity by releasing certain ions to the exclusion of others. In the present invention, a cell is considered to be impermeable if the selective membrane permeability is maintained. The impermeability measurement implemented in the calibration method according to the invention consists in estimating an impermeability loss ratio (RPI) of the producer cells (6) depending on a number of impermeable producer cells Nn of a production of extracellular vesicles relative to the number of impermeable producer cells corresponding to the initial conditions prior to said production N1. The monitoring of this ratio (RPI) consists for example of calculating the formula RPI=Nn/N1 for each production and makes it possible to detect an optimal production range. Within the meaning of the description, a high level of cellular impermeability corresponds to a ratio close to 100%, while a low level of cellular impermeability corresponds to a ratio close to 0%.

For the production of vesicles, in a very surprising and unusual way, whether during production, or even at the end of production, it has been identified that it is very advantageous not to be interested in the number of impermeable cells reported the number of total cells of the same sample and as measured on a single sample or a continuous or discontinuous measurement of a single medium element. It has been determined that it is critical to relate the number of vesicles produced on the one hand, and the ratio between the number of impermeable producer cells at a time T during production, relative to the number of impermeable cells at the start of the production.

The measurement of this ratio is particularly advantageous for the production of extracellular vesicles under the action of mechanical stresses by turbulent agitation, because it makes it possible to identify the phase of the production process where the process of reduction in the number of cells is observed. impermeable producers, indicating a process of alteration of the membrane porosity. The measurement of membrane impermeability coupled with the control of the ratio of the number of impermeable cells relative to the number of initial impermeable cells has the advantage of identifying at least the lower limit, and preferably both the lower limit and the upper limit, an optimum range of agitation level for extracellular vesicle production.

In addition, the impermeability measurement is advantageous, because it is low cost, easy to implement and provides a unit count measurement of the number of producer cells so as to be able to observe the evolution of the number of impermeable producer cells during extracellular vesicle production. The impermeability measurement of the cells can be carried out continuously in an automated manner according to a sampling rhythm by a measuring means integrating the fluidic system or else manually by an operator by taking a sample and appropriate measuring means. The measuring means are capable of carrying out continuous measurements according to a predetermined sampling interval, which may be between one minute and several hours for example, depending on the control protocol, preferably one minute, 10 minutes, 15 minutes, 30 minutes, 45 minutes, or even 1 hour, or 2 hours.

For example, the impermeability measurement can be based on counting the number of viable cells, this measurement consisting in detecting a non-selective porosity of the membrane of a cell. We know of compounds that are able to enter a cell only if the cell membrane has lost its selective permeability, for example trypan blue. Using this kind of compounds placed in the presence of cells in solution, the measurement of the number of viable cells per unit volume can determine the absence of these compounds inside the cell. This counting on a sample taken of the order of 100 microliters can be done in a few minutes with a tabletop device, such as the NucleoCounter® (NC-200™ marketed by the company Chemometec). According to this non-limiting technique, the sample taken (after possibly adding certain reagents) is introduced into a disposable cassette containing DAPI and Acridine Orange, said cassette then being placed in the device. Finally, a file is automatically saved on a computer containing the following non-limiting data: estimation of the number of viable cells per millilitre, and error bar, estimation of the number of non-viable cells per millilitre, error bar, estimation of debris, etc. .

Other impermeability measurement techniques are known to those skilled in the art, some of which are integrated into the fluidic system itself and which is capable of automatically exploiting this data in real time according to a continuous sampling rate. The impermeability data can be recorded in the memory of production configuration means of a control station for the purposes of calibration and/or control of production of extracellular vesicles.

A person skilled in the art, through his general knowledge, knows how to adapt the technique for measuring impermeability in order to calculate and compare different levels of impermeability representative of a ratio of loss of impermeability. This ratio, expressed as a percentage or by a representative variable of a measurable physical quantity, depends on a number of impermeable cells of a production of extracellular vesicles compared to the number corresponding to the conditions prior to said production, and is used to implement the calibration method according to the invention. Preferably, the impermeability measurement implemented for the calibration method consists in calculating unitarily the number of impermeable cells of a production of extracellular vesicles relative to the number of impermeable cells corresponding to the conditions prior to said production.

For example, a standard impermeability measurement technique for a measurement of permeability alteration is the measurement technique known in English as "Dye exclusion" where a healthy cell does not let in all the components present, and in particular excludes the entry of certain dyes inside the cell so as to maintain the dyes in the extra-cellular environment. Another similar method involves the use of two dyes, one suitable for penetrating normal cells without any problem so as to mark all the cells (for example acridine orange (“permeable cell”) or DAPI (partial cell permeability) , and another dye adapted to enter only a cell with altered permeability (for example Trypan blue). Mention may be made, for example, of the article by authors Simon Johnson, et al . (Simon Johnson, Vy Nguyen, David Coder, Assessment of Cell Viability , 2013, CurrentProtocols), describing conventional dye alternatives. We also know methods by flow cytometry, or by automated imaging, such as the NucleoCounter, or by in-situ microscopy targeting the liquid medium of the system. fluidic adapted to detect alterations in shape without the use of dye, or even by automated microscopy working on a flow of liquid medium extracted from the container in a continuous manner according to a rhythm sampling. Alternatively, another impermeability measurement technique can be the monitoring of the dielectric properties of the producing cells. The principle is based on the capacitive properties in the electrical sense of a cell. This technique consists in measuring a variable representative of a change in permittivity of the liquid medium by frequency scanning. Mention may be made of the publication “ A novel approach for using Dielectric Spectroscopy to Predict Viable Cell Volume (VCV) in early Process Development ”, C. Justice et. al, 2011 , ELSEVIER , describing the implementation of such a method. Alternatively, another technique is based on the detection in the liquid medium of proteins which are normally maintained in the cell. The enzymatic activity assay is a possible method and can be implemented by the ToxiLight ^TM kit marketed by the company Fonza for example. Finally, another alternative method can be associated with the detection of an apoptosis process. The outer cell membrane exhibits polarity-like properties, that is, the lipid composition of the inner face is different from the lipid composition of the outer face. In the event of apoptosis, the cell membrane ends up losing this polarity, and certain lipids which are generally little or not exposed to the outside become detectable on the external surface of the cell.

Measurement of concentration extracellular vesicles

To detect and measure the concentration of these vesicles, there are a certain number of techniques requiring specific measuring means. As such, the "NTA" (Nanoparticle Tracking Analysis, for example the NanoSight NS300 device marketed by the company Malvern Panalytical) is a device that measures the concentration of these vesicles in a liquid in an approximate size range of 10 nanometers to 1000 nanometers.

Other techniques for measuring the concentration of impermeable cells are known to those skilled in the art who will know how to choose the technique adapted to the fluidic system used to set up monitoring of a production yield. Optionally, it is envisaged that the measurement of the concentration of impermeable cells can be carried out in continuous monitoring mode by measurement means integrated into the system.

Production control parameter

The fluidic system (1) is controlled during the implementation of the calibration method according to at least the parameter of the level of agitation of the stirrer (7) and a parameter of production duration. These two configuration parameters determine an extracellular vesicle production yield and the optimal values are specific to the cell type of the producer cells. The agitation level parameter can be the rotation speed of an agitator, an injection rate in a static mixer, or a shaking frequency, for example. This agitation level parameter is controlled by the control means of the fluidic system. The control of the level of agitation may be dependent on other parameters depending on the system used. The calibration method according to the invention provides for the recording of all the controllable parameters likely to act on the level of agitation of the production of extracellular vesicles of the fluidic system during the detection of levels of impermeability sought for identify the optimal range.

It is specified that the level of agitation must be interpreted as an energy injection rate in the general sense, which rate depends in particular on the speed of the agitator, the type of agitator as well as the shape and dimensions of the container. of the fluidic system.

Moreover, when the agitation level parameter is a speed of rotation of an actuator of the fluidic system, a lower limit of the agitation level corresponds to low values of rotation speed of an agitation range. An upper limit corresponds to a high rotational speed value of an agitation range. Conversely, when the agitation level parameter corresponds to a Kolmogorov length L _k , a lower limit of the agitation level corresponds to a high value L _k of an agitation range. An upper limit of the agitation level corresponds to a low value Lk of an agitation range.

In an exemplary embodiment of the fluidic system (1) where the stirrer comprises a paddle wheel, the speed of rotation of the stirrer (7) can be controlled within a stirring range generally between 0 rpm and 800 rpm. Preferably, the agitation range can be between 30 rpm and 500 rpm, preferably between 40 rpm and 400 rpm, preferably between 200 rpm and 500 rpm and preferably between 250 rpm and 400 rpm. The calibration method according to the invention aims to establish an optimal range of the level of agitation in the range operable by the fluidic system.

The production time parameter is critical and will be set to specifically controlled values during the extracellular vesicle production phases of the calibration process. By way of non-limiting example, the predetermined production times can be between values of approximately 20 minutes to approximately 6 hours, even more preferentially from approximately 1 hour to approximately 6 hours, even more preferentially approximately 2 hours to approximately 3 hours, or even more preferably be equal to around 2 hours or alternatively around 3 hours or alternatively around 4 hours.

The calibration method according to the invention aims to detect a loss of impermeability characteristic of an optimal extracellular vesicle production yield specifically in these production duration ranges in order to establish an optimal agitation range, for one or each specific production times mentioned above and a given cell type.

Description p process d and calibration allowing the determination of an optimal range of agitation

In we now describe a preferred embodiment of the calibration method according to the invention consisting in identifying, independently of the type of cell and of the fluidic system model, an optimal range of agitation parameters from the detection of a loss of impermeability of producer cells, wherein said loss is characteristic of optimum vesicle production efficiency. The calibration method preferably applies to producing cells in suspension due to a more stable vesicular behavior under the action of fluidic stresses. However, the possibility that the calibration method also applies to producer cells adherent to microcarriers is not ruled out.

The calibration process consists of finding at least the lower limit, and preferably both the lower limit and the upper limit, of an optimal agitation range for a given cell type and a chosen production time. For each limit of the agitation range, the calibration method can provide for one or more productions of extracellular vesicles necessary for the detection of a level of loss of cellular impermeability characteristic of a limit of the optimal range.

The calibration method comprises a first phase of searching for the lower limit (Lim1) of the agitation range comprising the steps (E1), (E2) and (E3) repeatable until the detection (CD1) of the level of loss impermeability sought at the end of a production, and then optionally a second phase of searching for the upper limit (Lim2) of the agitation range comprising steps (E4), (E5) and (E6) repeatable up to the detection (CD2) of the level of loss of impermeability sought at the end of a production.

According to a variant of research as described by the , the agitation values of each production of extracellular vesicles (E2) and (E5), are chosen successively within the operable range of the fluidic system according to a dichotomy type methodology. Those skilled in the art, through their general knowledge, will be able to apply other methodologies for researching the agitation value in order to detect the level of loss of impermeability sought at the end of production.

Step E1: D determination of a first level of impermeability reference initial before production of extracellular vesicles (EV)

More specifically, the calibration process comprises a first step (E1) of determining an initial level of cellular impermeability (V1) from which a loss of impermeability will be characterized. The level (V1) depends on an initial number (N1) of producer cells prior to the production of extracellular vesicles (EV). In general, the level (V1) depends on a measured physical quantity which is specific to the impermeability measurement technique, the value of which depends on the initial number of producing cells. Preferably during this step (E1), the number of impermeable cells is calculated individually in order to establish the reference allowing the loss of impermeability to be identified. For the implementation of the calibration method, several levels of impermeability (V1), (V2) and (V3) are used as predetermined reference levels to identify a loss of impermeability characteristic of a range of production of extracellular vesicles of optimum performance.

The initial level of impermeability (V1) can in one embodiment be determined on a sample of cells not subjected to a stirring action. However, according to a preferred mode of the calibration method, the initial impermeability measurement (V1) is carried out after a “gentle” stirring phase for control purposes. This phase prior to the production phase is carried out for a predetermined duration Tinit, for example between 5 minutes and 6 hours, even more preferentially from approximately 1 hour to approximately 6 hours, even more preferentially approximately 2 hours to approximately 3 hours, or even more preferably be equal to approximately 2 hours or alternatively approximately 3 hours or alternatively approximately 4 hours. In a preferred mode, it is not necessarily necessary to carry out a long step (i.e. more than a few minutes) for the determination of the level of initial impermeability, if it has previously been established that the gentle agitation used does not modify not significantly the level of impermeability. In a preferred embodiment, it is also possible to use a small sample of cells placed in a small container adapted to the volume of the sample and kept under gentle agitation. It is then possible to check that the gentle agitation on this small sample has not significantly modified the level of impermeability of the cells after a time T. In addition, this phase prior to the production phase is carried out at a negligible agitation value or much less than a production agitation value (for example, for a rotary agitator as described in at a blade rotation speed value of between 10 rpm and 50 rpm for the predetermined durations (Tinit) mentioned previously. These control parameters of the preliminary phase (E1) are chosen according to the appropriate production configuration to determine the initial level of impermeability (V1) so as not to cause any membrane alteration of the producing cells.

The initial level of impermeability (V1) is determined by means of the fluidic system (1) of the , either from the volume of liquid medium (5) containing the producer cells present in the container (4), or from a volume of liquid medium from the same culture of producer cells intended to be used for one or more productions extracellular vesicles. As we will see in the remainder of the description, the calibration process provides for at least one, and very certainly, several phases of production of extracellular vesicles necessary for the search for each of the limits (Lim1), (Lim2) of the range of optimal stirring.

The initial cell impermeability level (V1) of each production can be determined from a single and same culture medium supplying all the productions necessary for the calibration process, in a methodology for example in which the productions of extracellular vesicles are performed simultaneously on multiple fluidic systems to speed up the research process. Alternatively, for each production of extracellular vesicles necessary to identify the limits of the optimal agitation range, the calibration process includes a step of determining the initial level of impermeability (V1) of the fresh producer cells at each production.

Step E2: P production PR1 extracellular vesicles of a duration T1

Then, the method comprises, from the same liquid medium of producing cells for which the initial level of impermeability (V1) has been determined, the control (E2) of at least a first production (PR1) of extracellular vesicles (EV) configured to a first agitation value (XCTL1) and to a first predetermined duration (T1). According to the calibration process, the agitation value (XCTL1) of a production (E2) remains constant throughout the production period (T1). The first agitation value (XCTL1) of the production (E2) can be set to different values during the process of finding the lower limit (Lim1).

According to a variant of the dichotomy search methodology, the first value of the agitation level (XCTL1) is chosen at the maximum agitation value operable by the fluidic system, or generally at a high value of the operable range. A first agitation value search range is then defined by the minimum limit and the maximum limit operable by the fluidic system.

The first predetermined duration (T1) is for example between 20 minutes and 6 hours, even more preferentially from approximately 1 hour to approximately 6 hours, even more preferentially approximately 2 hours to approximately 3 hours, or even more preferentially to be equal at about 2 hours or alternatively about 3 hours or alternatively about 4 hours. As is well known to those skilled in the art, certain cell types are more or less sensitive to the action of fluidic stresses. It is therefore considered that the duration (T1) is a function of the cell type.

Step E 3: M measure d u level d 'impermeability

The calibration method then comprises at least a first measurement (E3) of cell impermeability (Vt) of the cells of the first production (PR1). The impermeability measurement is carried out continuously during production (PR1), or only at the end of production (PR1) when the duration (T1) has elapsed.

According to the invention, each measurement of impermeability (E3) of a production of extracellular vesicles consists in calculating a level of impermeability (Vt) equal to an impermeability loss ratio (RPI) depending on the number of impermeable producer cells of each said production (E3) with respect to the initial number (N1) of impermeable cells corresponding to the conditions prior to said production (E3).

More specifically, the impermeability loss ratio (RPI) is equal to the following relationship: RPI=100*Nn/N1, where Nn is the number of impermeable producing cells at the end of a production, and N1 is the initial number of impermeable producer cells prior to said production. It is specified that the first initial level (V1) is an initial ratio corresponding to a level of impermeability of 100%, where V1=100*N1/N1.

The calibration method includes a comparison step between the level of impermeability (Vt) at the end of the production step (E2), and a second level of impermeability (V2) whose value is strictly lower than the first level (V1 ). The second level (V2) is specifically chosen to delimit an optimal extracellular vesicle production range in terms of yield and where V2=100*N2/N1.

In the event of detection that the impermeability level (Vt) obtained at the end of the production step (E2) differs from the predetermined impermeability level (V2), whether it is higher or lower, the calibration method provides for repeat in a loop at least the steps of production of extracellular vesicles (E2) and measurement of impermeability (E3) with a new sample of fresh cells, that is to say which have not been subjected to production agitation , but also step (E1) if the determination of an initial level (V1) is once again necessary for each production as is the case in . At each search loop, the agitation value (XCTL1) is modified to a new agitation value specific to each new production, until the detection of a level of impermeability (Vt) approximately equal to the level (V2 ).

For example, according to the dichotomous search method, at each agitation value search loop, if the ratio (RPI) is lower than the level (V2), the agitation level value (XCTL1) for the production of extracellular vesicles of the next search loop is set to a lower agitation level. If the impermeability loss ratio (RPI) is higher than the level (V2), the agitation level (XCTL1) for extracellular vesicle production of the next search loop is increased. The agitation level of the following loop is chosen at an intermediate value, preferably the middle value of the search range of the previous loop. The search loops are repeated in this manner until the measured level of impermeability of a measurement step (E3) is approximately equal to the predetermined level of impermeability (V2) which is sought.

Finally, the search phase for the lower limit (Lim1) ends in the event of detection, an event illustrated by the reference (CD1) in , that the level of impermeability (Vt) obtained at the end of the production step (E2) is substantially equal to the level of impermeability (V2), that is to say with a margin on the ratio which is dependent the margin of error characteristic of the type of measurement carried out as defined in the present application. In this case, the method comprises the recording, in an electronic memory of the fluidic system or the control station of the fluidic system, of a value of the level of agitation (XCTL1) which was configured during the production step (E2) of the last search loop. The lower limit (Lim1) of the optimal agitation range is equal to the value of the agitation level (XCTL1). It is recalled that the range of agitation (PA1) is specific at least to one cell type (CEL1) and to a given production duration (T1).

FIGS. 3A and 3B are graphs representing values of the loss of impermeability ratio (Vt) at the end of production plotted in a continuous dotted line, on the left ordinate axis, as a function of configurable values of the level of agitation (XCTL) for the production of extracellular vesicles, on the x-axis. In this example, the impermeability loss ratio (Vt) is equal to Nn/N1. The quantity of extracellular vesicles (QEV) is plotted in a broken dotted line, the values of which are represented on the right y-axis. It is the amount of extracellular vesicles produced for each production of a duration (T1) as a function of the agitation value (XCTL). The amount of vesicle (QEV) is a yield variable expressed as the number of extracellular vesicles per number of initial impermeable cells.

In a first variant of optimum agitation range (PA1), illustrated by the , the impermeability level (V2) is an impermeability loss ratio (RPI) whose value is equal to or less than 95%, and by way of example is equal to 95%. Alternatively, (V2) can be equal to either 90% or 85%. This value of loss of membrane impermeability of the producing cells marks the lower limit (Lim1) of an agitation range for which a fraction of producing cells loses its impermeability or is no longer taken into account in the measurements, for example because that they remain attached to the wall of the reactor, but this again avoids in this specific area the excessive presence of cellular debris making it difficult to collect the extracellular vesicles in the liquid medium. This impermeability loss ratio value marks the triggering of a process of alteration of the membrane porosity of a fraction of the producing cells which, however, does not prevent the collection of the vesicles after production.

The calibration method then provides, for this first variant of agitation range, to seek the upper limit (Lim2) of the agitation range corresponding to a predetermined level of impermeability (V3) at the end of production of extracellular vesicles which can reach a value of the loss of impermeability ratio of approximately 60% for example. Therefore, the method further provides for a second phase of searching for the upper limit (Lim2) executed by the steps (E4), (E5) and (E6), then finally the recording (E7) of the lower limits (Lim1) and upper (Lim2) of the optimum stirring range (PA1).

In a second variant of optimal agitation range (PA1), illustrated by the , the impermeability level (V2) is an impermeability loss ratio whose value targets a higher loss range, for values of the ratio (RPI) equal to or less than 70%, and by way of example (V2 ) can be equal to either 70% as shown in , either at 35%, or possibly up to a value of 25%. This second variant aims to delimit the optimum range of agitation where a loss of the number of higher producing cells is accepted in order to obtain a higher quantity of extracellular vesicles.

For this second variant of agitation range, the calibration method then provides for finding the upper limit (Lim2) of the agitation range corresponding to a predetermined level of impermeability (V3) at the end of the production of extracellular vesicles which can reach a ratio value of 5% for example, or even 0%. Therefore, the method further provides for a second phase of searching for the upper limit (Lim2) executed by the steps (E4), (E5) and (E6), then finally the recording (E7) of the lower and upper limits (Lim1 ) and (Lim2) of the optimal stirring range.

We now describe the second phase of searching for the upper limit (Lim2) of the agitation range during which the method comprises the steps (E4), (E5) and (E6) repeatable until the detection (CD2) of the level of loss of impermeability sought at the end of a production. Steps (E4), (E5) and (E6) are executed similarly to steps (E1), (E2) and (E3) respectively except that the level of impermeability sought is the predetermined level (V3) of value strictly below the level (V2). Moreover, identically to the first phase of research, the second agitation value (XCTL2) of each production of extracellular vesicles (E5) can be modified successively within the operable range of the fluidic system according to a dichotomy type methodology. Those skilled in the art, through their general knowledge, will be able to apply other methodologies for researching the agitation value in order to detect the level of loss of impermeability sought at the end of production.

Step E4: Determination of a first level of initial impermeability before production of extracellular vesicles (EV)

The calibration method includes the step (E4) of determining the initial level of impermeability of a new culture of producer cells. This step is executed identically to step (E1). It is carried out on a liquid medium of fresh producer cells to determine an initial number, always referenced (N1), of impermeable cells prior to the production of extracellular vesicles.

Step E 5: PR2 production of extracellular vesicles with a duration T2

Next, the calibration method comprises, using the same liquid medium of producing cells for which the initial level of impermeability (V1) has been determined, the control (E5) of a production of extracellular vesicles configured at a second level of agitation (XCTL2) whose value is greater than the agitation value (XCTL1), and over a duration (T2), preferably equal to the duration (T1). The level of agitation (XCTL2) is higher because this research phase consists in obtaining, at the end of production, a lower level of impermeability (V3) compared to the level (V2). According to the calibration process, the agitation value (XCTL2) of a production (E5) remains constant throughout the production period. The loss of impermeability being an essentially irreversible process, if an impermeability level obtained is close to zero then the value to be retained for the level of agitation according to the present invention is the lowest agitation value (XCTL) for which we obtain a level of impermeability close to zero.

According to the dichotomy search methodology variant, the agitation value (XCTL2) is chosen at the maximum agitation value operable by the fluidic system. Thus, for this second search phase, a first agitation value search range is then defined by the value (XCTL1) and the maximum limit operable by the fluidic system.

The predetermined duration (T2) is for example between 20 minutes and 6 hours, even more preferentially from approximately 1 hour to approximately 6 hours, even more preferentially approximately 2 hours to approximately 3 hours, or even more preferentially to be equal to about 2 hours or alternatively about 3 hours or alternatively about 4 hours. As is well known to those skilled in the art, certain cell types are more or less sensitive to the action of fluidic stresses. It is therefore considered that the duration (T2) is a function of the cell type.

Step E 6: Measurement of the level of impermeability

Again, following the production of extracellular vesicles, the calibration process includes a measurement (E6) of the impermeability (Vt) of the cells of this latter production (E5). The impermeability measurement (E6) is carried out continuously during production, or only at the end of production when the duration (T2) has elapsed.

According to the invention, each measurement of impermeability (E6) of a production of extracellular vesicles consists in calculating a level of impermeability (Vt) equal to an impermeability loss ratio (RPI) depending on the number of impermeable producer cells of each said production (E5) with respect to the initial number of impermeable cells (N1) corresponding to the conditions prior to said production (E5).

The calibration method includes a comparison step between the level of impermeability (Vt) at the end of the production step (E5), and the third level of impermeability (V3) whose value is strictly lower than the second level (V2 ). The third level (V3) is specifically chosen to delimit the upper limit of the range of production of optimal extracellular vesicles in terms of yield and where V3=100*N3/N1.

If it is detected that the impermeability level (Vt) obtained at the end of the production step (E5) differs from the predetermined impermeability level (V3), whether it is higher or lower, the calibration method provides for repeating in a loop at least the steps of production of extracellular vesicles (E5) and measurement of impermeability (E6), but also step (E4) if the determination of an initial level (V1) is once again necessary to each production, with new agitation values specific to each production, until the detection of a level of impermeability (Vt) approximately equal to the level (V3).

For example, according to the dichotomous search method, at each agitation value search loop, if the ratio (RPI) is lower than the level (V3), the agitation level value (XCTL2) for the production of extracellular vesicles of the next search loop is set to a lower agitation level. If the ratio (RPI) is higher than the level (V3), the level of agitation (XCTL2) for the production of extracellular vesicles of the next search loop is increased. The agitation level of the following loop is chosen at an intermediate value, preferably the middle value of the search range of the previous loop. The search loops are repeated in this manner until the measured level of impermeability of a measurement step (E6) is approximately equal to the predetermined level of impermeability (V3) which is sought.

Finally, the search phase for the upper limit (Lim2) ends in the event of detection, an event illustrated by the reference (CD2) in , that the level of impermeability (Vt) obtained at the end of the production step (E6) is substantially equal to the level of impermeability (V3), that is to say with a margin on the ratio dependent on the margin of error characteristic of the type of measurement carried out as defined in the present application, said margin of error typically being between 0% and 35%, preferably between 5% and 30%. For the first optimal stirring range variant (PA1), illustrated by the , the level of impermeability (V3) is a ratio of loss of impermeability whose value is equal to 60%, or 65%, or 70%. This value associated with the upper agitation limit prevents excessive presence of cellular debris and contaminants in the liquid medium.

For the second optimal stirring range variant (PA1), illustrated by the , the impermeability level (V3) is an impermeability loss ratio whose value is equal either to 0%, or preferably to 5%. This last value indicates the end of the zone with the optimal yield. This value increases the presence of cellular debris and contaminants in the liquid medium but makes it possible to obtain higher production yields and nevertheless retains an advantage of use for certain uses despite a greater presence of contaminants.

Step E 7: Optimum shaking range recording

Then, the calibration method includes the step (E7) of recording, in an electronic memory of the fluidic system or the control station of the fluidic system, the value of the level of agitation (XCTL1) which was configured during the production step (E2) which made it possible to reach the predetermined level (V2) at the end of vesicle production, as well as the value of the agitation level (XCTL2) which was configured during the production step (E5) which made it possible to reach the predetermined level (V3) at the end of vesicle production. The lower limit (Lim1) of the optimal stirring range (PA1) is equal to the stirring value (XCTL1) and the upper limit (Lim2) is equal to the stirring value (XCTL2). It is recalled that the stirring range (PA1) is specific to at least one cell type (CEL1) and to the production duration chosen for (T1) and (T2) of the respective production phases (E2), (E5). This data is saved in a fluidics system configuration file.

Finally, for the first optimal stirring range variant (PA1) described in , the second level of impermeability (V2) and the third level of impermeability (V3) define a range of impermeability between 95% and 60% of the impermeability loss ratio. Alternatively, the agitation range (PA1) is between 90% and 65% impermeability loss ratio, preferably between 85% and 70%, with an approximate margin depending on the margin of error characteristic of the type of measurement carried out as defined in the present application.

For this domain of agitation, the producing cells begin to lose their function of membrane selection while not being totally damaged, which again avoids in this specific domain the presence of a large number of cellular debris and/or contaminants making difficult the task of collecting extracellular vesicles in the liquid medium and/or purifying them. This range of loss of impermeability marks the triggering of a process of rapid alteration of the producer cells identified by a drop in the number of impermeable producer cells compared to the number calculated under the initial conditions for production. Moreover, as illustrated by the , measurements of extracellular vesicle (QEV) concentrations show that the production yield is particularly high.

In the second optimal stirring range variant (PA1) described in , the second level of impermeability (V2) and the third level of impermeability (V3) define a range of impermeability between 70% and 0% of the impermeability loss ratio. Alternatively, the impermeability range is between 70% and 5%, 50% and 5%, 35% and 0%, preferably between 25% and 5%, or even between 15% and 5%, with a approximate margin of a few percent.

For these areas of loss of impermeability between 70% and 5%, the production yields of extracellular vesicles (QEV) are maximal as illustrated by the . The significant alteration of membrane porosity can lead to the presence of intracellular proteins normally held captive in producing cells. These proteins will mix with the extracellular vesicles produced in the fluid that contains the producing cells. However, for this area the level of impurity of the liquid medium can be reduced by a suitable method. It is only envisaged that this level of impurity remains acceptable and alters little or not at all the therapeutic effects in certain uses of extracellular vesicles. Beyond this zone, too high levels of agitation cause destructive fluidic stresses of the producing cells, reducing the production yields of extracellular vesicles (EV).

Variants of the calibration process

The calibration method according to the invention makes it possible to obtain an optimal stirring range specific to each cell type and to each production duration. The durations of each production of steps (E2) and (E5) of the calibration process are equal or during the same calibration process. However, the possibility of seeking several optimal agitation ranges for different production time values is not ruled out. Lower optimal agitation levels were observed for longer production times.

Similarly, the agitation level (XCTL1) and (XCTL2) of each production (E2) and (E5) is controlled at a constant value throughout the duration of the respective production.

Alternatively, the impermeability measurements (E3) and (E6) are performed only once the production steps (E2) and (E5) are completed respectively. In another variant, the measurements (E3) and (E6) are performed continuously during the production steps (E2) and (E5) respectively.

Furthermore, the describes a calibration method in which the lower limit (Lim1) is sought by the steps (E1), (E2) and (E3) prior to the search for the upper limit (Lim2) carried out by the steps (E4), (E5 ) and (E6) of the calibration method. The variant of the method where the upper limit (Lim2) is sought before the lower limit (Lim1), or else simultaneously, is not ruled out.

The calibration process can comprise a search loop (E2), (E3), or several search loops (E2), (E3) before finding the value (XCTL1) for which the level of impermeability (Vt) is equal at (V2). Similarly, the calibration process can include a search loop (E5), (E6) or several search loops before finding the value (XCTL2) for which the level of impermeability (Vt) is equal to (V3).

In , we have described a mode of research of the lower and upper limits by a dichotomy type approach. Other search modes are applicable by those skilled in the art.

R presentation of the fluidic system and the means of configuration and control of production

We now describe a system and method for the ex vivo production of extracellular vesicles using a fluidic system for producing extracellular vesicles from producer cells (6). Such a fluidic system (1) has already been described in and preferably the system is specially configured by production parameters obtained by the calibration method according to the invention to carry out the method for producing extracellular vesicles.

In , in addition to the fluidic system (1) represented this time briefly, the control means (20) of said system (1) are described, allowing optimal large-scale production. These piloting means (20) comprise piloting means (22) of a production according to configuration parameters comprising at least one level of agitation (XCTL) and a production duration (T), measurement means ( 23) the level of cellular impermeability (Vt) of the producing cells during production or after production, and preferably means for configuring (21) a production of extracellular vesicles recording one or more predetermined production configurations (PA1) , (PA2) and (PA3), where each production configuration is defined by an optimal agitation range and a predetermined duration. Each production configuration is specific to a production duration and to a type of producer cells.

The control means (20) are configured to select the agitation value of a production included in one of said agitation ranges to execute a production of extracellular vesicles.

More precisely, the configuration means (21) can be configuration files saved in the memory of a software production program and the control means (22) can comprise a control program, automatic or manual, of the configuration parameters of the level of agitation (XCTL) and duration of production (T) and measurement of the level of cell impermeability (Vt) in order to control the level of impermeability of the producing cells at the end of production, as well as the concentration of the vesicles extracellular, in accordance for example with the techniques described previously in the description.

Furthermore, the configuration means (21) are specifically provided for executing the calibration method according to the invention by means of a software calibration program stored in memory. Preferably, each predetermined production configuration (PA1), (PA2), (PA3) has been obtained by the calibration method according to the invention.

In accordance with the invention, the means (23) for measuring the level of cellular impermeability (Vt) are suitable for calculating a ratio of loss of impermeability depending on a number of impermeable cells of said production of extracellular vesicles with respect to the number corresponding to the prerequisites for said production. In this way, the system (1) is specifically configured to detect a drop in the number of producer cells and the triggering of a phase of impaired membrane selection function. This phase is particularly interesting because it indicates a precise zone of high yield for the production of extracellular vesicles.

Furthermore, in accordance with the invention, the control means (22) are configured during production of extracellular vesicles at a predetermined agitation value (XCTL) so that the level of impermeability (Vt) at the end of said production is less than or equal to a second predetermined level of impermeability (V2) which is at least less than the first initial level of impermeability (V1) prior to said production, said first level (V1) being dependent on an initial number ( N1) of producer cells prior to said production of extracellular vesicles (EV). The end of production control according to the second level (V2) corresponding to a predetermined impermeability loss ratio according to the invention marks the start of a high-yield production zone.

The production fluidic system provides at least two production modes. In a first mode, the production duration is equal to the predetermined duration before production. This duration is recorded in the predetermined production configuration of the system, for example by the configurations (PA1), (PA2), and (PA3). The production time can be between values of about 20 minutes to about 6 hours, even more preferably from about 1 hour to about 6 hours, even more preferably about 2 hours to about 3 hours, or even more preferably be equal to approximately 2 hours or alternatively approximately 3 hours or alternatively approximately 4 hours.

In a second mode, the production time is finalized when the level of impermeability (Vt) measured is included in a range of impermeability delimited by the second level of impermeability (V2), and preferably both by the second and the third level of impermeability (V2) and (V3). According to this second mode, the duration of production can be preconfigured without necessarily strictly conditioning the end of production in order to leave an operating margin of production taking into account the vagaries of the biological conditions of production. The production duration is for example framed by limits around this preconfigured duration. When the level of impermeability (Vt) measured continuously according to a sampling rate on the fluidic system, the control means (22) are adapted to shorten the production time if the level of impermeability (Vt) is reached before the preconfigured time, but also to extend the production time until the level of impermeability (Vt) reaches a value within the range of optimal impermeability. In both cases, the production duration will be between the limits around the preconfigured duration.

The impermeability level (V3) has the advantage of limiting cellular debris at the end of production and also of limiting the quantity of impurities at the end of production. However, the possibility of a therapeutic use of extracellular vesicles is not ruled out even in the presence of impurities and small cellular debris, which makes it possible to operate production at a loss of impermeability ratio equal to approximately 5%, or even 0%. It is also envisaged during the production process the implementation of one or more techniques of separation by clarification of the cellular debris and the vesicles allowing the collection of the extracellular vesicles only and of the possible impurities.

This clarification step consists in removing the cells, the debris, and in general we seek to eliminate anything that is of a characteristic size larger than typically 0.5 to 1 micrometer. It is known in fact that the vesicles which it is sought to obtain have characteristic sizes in the range 10 nm to 500 nm, more preferably in the range 20 nm to 300 nm.

From a clarified liquid containing vesicles, those skilled in the art may wish to carry out one or more purification steps, very generally with the limitation that the more one seeks to purify, the lower the yield and/or the The vesicles themselves may possibly be altered during the purification process(es).

According to a particular embodiment, one or more stages of partial elimination of residual impurities can be carried out at the end of the production process.

The fluidic system for producing extracellular vesicles is specifically configured in accordance with the calibration protocol according to the invention. According to a first mode of production, the first level (V1) is an initial ratio corresponding to a level of impermeability at 100% and the level of impermeability (Vt) at the end of said production is between 95% and 60% of impermeability loss ratio, or between 90% and 65%, preferably between 85% and 70%. This mode of production is advantageous because this range of loss of impermeability marks the triggering of a process of rapid alteration of the producing cells identified by a drop in the number of impermeable producing cells compared to the number calculated under the initial conditions for production. . In addition, measurements of extracellular vesicle (EQV) concentrations showed that the production yield is high specifically in this area.

According to a second mode of production, the first level (V1) is an initial ratio corresponding to a level of impermeability at 100% and in which the level of impermeability (Vt) at the end of said production is between 70% and 0 % of the loss of impermeability ratio, between 70% and 5%, between 35% and 0%, between 25% and 5%, or even between 15% and 5%. This second mode of production makes it possible to achieve a higher yield compared to the first mode of production. Although the significant alteration of membrane porosity can lead to the presence of intracellular proteins normally held captive in producing cells, this second mode remains of interest. Indeed, for this area the impurity level of the liquid medium can be reduced by a suitable method. It is only expected that this level of impurity will remain acceptable and will alter the therapeutic effects little or not at all in certain intended uses of the extracellular vesicles.

Description of a p process of production extracellular vesicles

There describes an embodiment of the method for the ex vivo production of extracellular vesicles in accordance with the invention by means of the fluidic system described in And . This method can be executed by the means for configuring (20) the fluidic system by means of a production program.

At a first step (E20), a liquid medium of producer cells is introduced into the container of the fluidic system where it is expected to generate a turbulent flow of the liquid medium to exert fluidic stresses on the producer cells in order to achieve the production of vesicles extracellular. During this step, or before this step, the production process includes a control step during which the initial level of impermeability (V1) of the producing cells is determined. Preferably, this step consists of calculating an initial number of impermeable producer cells (N1) prior to the planned production of extracellular vesicles.

In a second step (E21), the production process includes a production parameterization step planned at least by a predetermined level of agitation (XCTL), and a production duration (T). The duration (T) may condition the end of production, but this is not mandatory. Preferably, this step provides for the selection of said configuration parameters in a configuration file saved in the memory of the configuration means of the fluidic system. This configuration file is obtained from the calibration method according to the invention.

The production time (T) can be between values of approximately 20 minutes to approximately 6 hours, even more preferably from approximately 1 hour to approximately 6 hours, even more preferably approximately 2 hours to approximately 3 hours, or even more preferably be equal to about 2 hours or alternatively about 3 hours or alternatively about 4 hours.

In a third step (E23), the production process comprises a step of producing extracellular vesicles at the agitation level and at the production duration previously configured so that the level of impermeability (Vt) at the end of said production is less than or equal to a second predetermined level of impermeability (V2) which is at least less than the first initial level of impermeability (V1) prior to said production, and preferably included in the range of impermeability delimited by the second level d predetermined impermeability (V2) and the third predetermined impermeability level (V3) lower than the second level (V2). The levels (V2) and (V3) are equal to the predetermined levels configured in the production system as described in And in accordance with the two production methods described above.

To this end, the production process provides for a continuous control step (CD3) either of the time elapsed since the instant of production initiation, or of the level of impermeability of the producing cells during production.

In the event of detection of at least one of the two conditions, the production process comprises a fourth step (E24) of collection of the liquid medium containing the extracellular vesicles. Preferably, it is planned at the end of production to operate a separation of the extracellular vesicles and the cellular debris. The latter may in particular concern a separation of extracellular vesicles and clarifiable compounds such as the cells themselves, various debris, a DNA fraction, etc. However, this last operation is not mandatory.

During the execution of the production process, it is possible that steps to control the concentration of extracellular vesicles be carried out in order to control the production yields. However, these steps are not mandatory if the configuration parameters were obtained by the calibration process.

Apps and u use s envisaged extracellular vesicles produced by the fluidic system and the production process according to the invention not

The present invention also relates to the extracellular vesicles produced by the fluidic system according to the invention as described in the present application, and/or obtained thanks to the process for the ex vivo production of extracellular vesicles from producing cells according to the invention such as described in the present application, for their use in immunotherapy, in regenerative medicine, as an alternative or in addition to cell therapy, as a vector for delivering at least one therapeutic and/or imaging agent and/or in the treatment tumours, infectious diseases, inflammatory diseases, immunological diseases, metabolic diseases, cancerous diseases, genetic diseases, degenerative diseases or diseases secondary to surgery or trauma.

The present invention also relates to a method of treatment in immunotherapy, in regenerative medicine, as an alternative or in addition to cell therapy, as vectors of at least one therapeutic and/or imaging agent, and/or treatment tumours, infectious diseases, inflammatory diseases, immunological diseases, metabolic diseases, cancerous diseases, genetic diseases, degenerative diseases or diseases secondary to surgeries or traumas, involving administration to a subject in need thereof of extracellular vesicles produced by the fluidic system according to the invention as described in the present application, and/or obtained thanks to the process for the ex vivo production of extracellular vesicles from producer cells according to the invention as described in the present application .

The present invention also relates to the use of the extracellular vesicles produced by the fluidic system according to the invention as described in the present application, and/or obtained thanks to the process for the ex vivo production of extracellular vesicles from producing cells according to the invention as described in the present application, with a view to the manufacture of a medicament used in immunotherapy, in regenerative medicine, as an alternative or in addition to cell therapy, as a vector of at least one therapeutic agent and/ or imaging, and/or used in the treatment of tumors, infectious diseases, inflammatory diseases, immunological diseases, metabolic diseases, cancerous diseases, genetic diseases, degenerative diseases or diseases secondary to surgeries or traumas.

According to one embodiment, the therapeutic use of extracellular vesicles, the extracellular vesicles for their therapeutic use, the methods of treatment or the use of the extracellular vesicles for their use for the manufacture of a medicament, as described above above, involves the ex vivo administration and/or use of said extracellular vesicles. The administration can for example be parenteral or enteral, such as an injectable administration (in particular intravenous, intramuscular, subcutaneous, intraspinal, etc.), oral buccal, cutaneous, local, vaginal, rectal, ocular, auricular, etc.

According to a preferred characteristic, the invention relates to the extracellular vesicles according to the invention, as described in the present application, obtained from THP-1 producing cells or lymphocytes, for their use for example in immunotherapy and/or oncology .

According to another preferred characteristic, the invention relates to the extracellular vesicles according to the invention, as described in the present application, obtained from producer cells which are mesenchymal stem cells (MSC), for their use in regenerative medicine or in the treatment of tumors, infectious diseases, inflammatory diseases, immunological diseases, metabolic diseases, cancerous diseases, genetic diseases, degenerative diseases or diseases secondary to surgery or trauma.

According to another preferred characteristic, the invention relates to the extracellular vesicles according to the invention, as described in the present application, obtained from any type of cell or from red blood cells, and loaded with at least one therapeutic agent , for use in delivering the at least one therapeutic agent into the body of a subject.

According to another preferred characteristic, the invention relates to the use of extracellular vesicles according to the invention, as described in the present application, obtained from any type of cell or from red blood cells, and loaded with at least an imaging agent, to perform a medical imaging examination.

According to another preferred characteristic, the invention relates to the use of extracellular vesicles according to the invention, as described in the present application, obtained from any type of cell or from red blood cells, and loaded with at least a therapeutic agent and at least one imaging agent, for monitoring the distribution of said extracellular vesicles in the body of a subject by medical imaging and delivering the at least one therapeutic agent in the body of said subject.

Claims (12)

Method for calibrating a fluidic system (1) for the production of extracellular vesicles (EV) from producer cells (6) comprising a stirrer (7) suitable for generating a turbulent flow of the liquid medium in a container to exert fluidic constraints on the producer cells (6) in order to achieve the production of extracellular vesicles (EV), the method comprising the following steps:
- the determination (E1) of a first level of cellular impermeability (V1) depending on an initial number (N1) of impermeable producer cells prior to the production of extracellular vesicles (EV),
- the control (E2) of at least a first production (PR1) of extracellular vesicles (EV) configured at a first agitation value (XCTL1) and at a first production duration (T1),
the method being characterized in that it further comprises:
- at least one first measurement (E3) of impermeability (Vt) of the cells of the first production (PR1) and comparison with a second predetermined level of impermeability (V2) lower than said first level of impermeability (V1), each measurement of cellular impermeability of a production of extracellular vesicles (PR1) consisting in calculating an impermeability loss ratio (RPI) depending on a number of impermeable producer cells of each said production with respect to the initial number (N1) corresponding to the prerequisites for said production,
- recording (E7) of at least one agitation range (PA1) whose lower limit (Lim1) is equal to the first agitation value (XCTL1) for which the level of impermeability (Vt) measured from the first production (PR1) is substantially equal to said second level of cellular impermeability (V2). Calibration method according to claim 1 further comprising the following steps:
- the control (E5) of at least a second production (PR2) of extracellular vesicles configured at a second agitation value (XCTL2) and at a second production duration (T2),
- a second measurement (E6) of impermeability (Vt) of the producing cells of the second production (PR2) and comparison with a third predetermined level of cellular impermeability (V3) lower than said second level of impermeability (V2),
- recording (E7) of the agitation range (PA1) delimited by the lower limit (Lim1) and an upper limit (Lim2), said upper limit (Lim2) being equal to the second agitation value (XCTL2) from which the measured level of cellular impermeability (Vt) of the second production is substantially equal to the third level of impermeability (V3). Calibration method according to claim 2, in which the first level (V1) is an initial ratio corresponding to a level of cellular impermeability at 100% and in which the second level of impermeability (V2) and the third level of impermeability (V3) delimit a range of cellular impermeability comprised between 95% and 60% of the impermeability loss ratio (RPI), or comprised between 90% and 65%, preferably comprised between 85% and 70%. Calibration method according to claim 2, in which the first level (V1) is an initial ratio corresponding to a level of cellular impermeability at 100% and in which the second level of impermeability (V2) and the third level of impermeability (V3) delimit a range of cellular impermeability between 70% and 0% of the impermeability loss ratio (RPI), between 70% and 0%, between 70% and 5%, between 35% and 0%, between 25% and 5%, or even between 15% and 5%. Calibration method according to any one of claims 1 to 4, in which the recording is configured to record a plurality of agitation ranges (PA1, PA2, PA3) of production of extracellular vesicles where each range is associated specifically with a specific production time value and to a specific producer cell type. Calibration method according to any one of Claims 1 to 5, in which each measurement (E3, E6) of cellular impermeability calculates a number of impermeable producer cells at the end of the production of extracellular vesicles, or continuously according to a rhythm sampling during production of extracellular vesicles. Fluidic system (1) for producing extracellular vesicles (EV) from producer cells (6) comprising at least:
- a stirrer (7) suitable for generating a turbulent flow of the liquid medium in a container to exert fluidic stresses on the producing cells (6) in order to carry out the production of extracellular vesicles (EV),
- means (22) for controlling a production of extracellular vesicles according to configuration parameters comprising at least one level of agitation (XCTL) and a production duration (T),
- means (23) for measuring the level of cellular impermeability (Vt) of the producing cells (6),
characterized in that the means (23) for measuring the level of impermeability (Vt) are suitable for calculating an impermeability loss ratio (RPI) depending on a number of impermeable cells of said production of extracellular vesicles with respect to the number corresponding to the conditions prior to said production, and in that the control means (22) are configured at a predetermined agitation value (XCTL) so that the level of impermeability (Vt) at the end of said production is lower or equal to a second predetermined level of impermeability (V2) which is at least lower than the first initial level of impermeability (V1) prior to said production, said first level being dependent on an initial number (N1) of impermeable producer cells prior to said production of extracellular vesicles (EV). Fluidic system according to Claim 7, in which the control means (22) comprising at least one agitation value and a duration are configured so that the level of impermeability (Vt) at the end of said production is included in a range of impermeability delimited by the second predetermined level of impermeability (V2) and a third predetermined level of impermeability (V3) lower than the second level (V2). Fluidic system according to any one of Claims 7 to 8, further comprising means for configuring (21) a production of extracellular vesicles recording at least a first predetermined production configuration defined by an agitation range (PA1) and a predetermined duration (T1), in which said range (PA1) is obtained by the calibration method according to any one of Claims 1 to 6, and in which the control means (22) are configured to select the agitation value of a production included in said range (PA1) to execute said production. Fluidic system according to claim 9, in which the control means (22) are configured either so that the production duration is equal to the predetermined duration before the production of the first predetermined production configuration, or so that that the duration of production is finalized when the level of impermeability (Vt) measured is included in the range of impermeability delimited by the second level of impermeability (V2) and the third level of impermeability (V3). A fluidic system according to claim 9 or 10, wherein the configuration means (21) comprises a plurality of stored production configurations (PA1, PA2, PA3), each configuration being specific to a production time and to a type of producing cells . Extracellular vesicles produced by the fluidic system according to any one of claims 7 to 11.