System for encapsulating cells comprising a system for displacing a solution using pressurised gas

Abstract

The invention relates to a system for encapsulating cells, the system comprising at least: two containers (11, 12), one of which is intended to comprise a solution of cells and the other a solution capable of gelling, a millifluidic or microfluidic encapsulation device (13) connected to the containers in order to form cellular microcompartments, wherein the outer layer comprises the solution capable of gelling and the core the cell solution, a collection vessel (15) for collecting the cellular microcompartments and containing a stiffening solution; each container being connected to an inlet of the encapsulation device by one or more distributors (14), and at least one first container (11) of the containers being connected to a solution displacement system (P11, P12) capable of introducing a pressurised gas into the container in order to generate a flow of the solution contained in the container towards the encapsulation device.

Description

Description Title of the invention: Cell encapsulation system comprising a pressurized gas solution displacement system

[0001] The invention relates to the field of cell encapsulation in three-dimensional cell culture compartments. More specifically, the invention relates to a cell encapsulation system comprising a solution displacement system using pressurized gas.

[0002] Ex vivo cell culture is a field of growing interest, particularly in the medical and pharmaceutical sectors. The cells cultured can be of any type, including differentiated cells with various phenotypes, progenitor cells, and stem cells. Pluripotent stem cells, in particular, are increasingly used. Indeed, in research on genetic diseases, these cells can be used to design cellular models of these diseases. They can also be used to test the effects of new drugs, to understand their mechanism of action and safety, or in genetic research, to study regions of the genome involved in cell differentiation.Finally, in the field of cell therapy, pluripotent stem cells can be used to differentiate into specific cells that can be used to replace damaged or missing cells in the body, such as heart, pancreatic, or liver cells.

[0003] In these various applications, culturing cells in large quantities presents a significant challenge. The research topics mentioned require a substantial quantity of human pluripotent cells. Similarly, the success of cell therapy in humans depends on the availability of industrial quantities of cells, particularly human pluripotent stem cells.

[0004] A significant advancement in cell culture techniques is the introduction of three-dimensional culture systems. Three-dimensional cultures are indeed more advantageously similar to natural in vivo systems and can be used for numerous applications, particularly in the development of therapies. A particularly suitable technology is that described in application WO2018 / 096277, which consists of three-dimensional cell microcompartments for stem cell culture. This document describes a cell encapsulation device comprising a microfluidic or millifluidic injector, enabling the formation of cell microcompartments in the form of droplets. The outer layer of these droplets is formed by a solution containing α-IgNate, and the core is formed by a cell solution. These droplets are collected in a calcium bath, which stiffens their outer layer to form a shell.

[0005] The microcompartments thus formed allow the cells to be cultured in a liquid medium, while the shell protects the cells from mechanical stresses related to collisions or fusions during liquid suspension culture.

[0006] It is known to place the solutions intended to form the microcompartments in containers and to connect these containers to the encapsulation device via flexible tubing in which the solutions move using peristaltic or diaphragm pumps. These pumps exert pressure on the flexible tubing, for example via rollers or rollers, which generates movement of the solutions within the tubing, propelling them to the encapsulation device. These pumps are preferred for the applications mentioned above because they require no contact between the solution in the tubing and the external environment, thus maintaining a closed and sterile environment.

[0007] However, they have several drawbacks.

[0008] On the one hand, the flow rate of the solutions to the encapsulation device, and therefore the speed and volume of microcompartments produced by this device, are difficult to control, especially at low flow rates, with this type of pump. However, it is necessary to maintain a constant flow rate of the solutions in order to control, among other things, the microcompartment production rate, the size of the microcompartments at the outlet of the encapsulation device, and the integrity and shape of the microcompartments when they are collected and dispersed in the calcium bath.

[0009] However, the flow generated by this type of pump is naturally pulsating. These pulsations are therefore likely to disrupt the flow of solutions towards the encapsulation device, and thus generate losses in quality and quantity in the production of the microcompartments.

[0010] This flow control problem is further subject to a reproducibility problem with regard to the applications envisaged, which may require very different flow rates and therefore impose very different constraints on volumes, speeds, pressures, and tube diameters.

[0011] Furthermore, these pumps can only provide stable pressures of around 2 to 3 bar in flexible tubing. Their use is therefore limited to low flow rates and low-viscosity solutions.

[0012] Finally, given the operation of these pumps, the variability of production conditions and / or the nature of the solutions used, the flexible tubes are subject to wear which may disrupt the flow of solutions and therefore conflicts with the need for constant flow, beyond the consequences that this wear may have on the integrity of the tubes, the sterility of the production system and the cost of replacing the tubes.

[0013] To overcome these drawbacks, it is common practice to replace peristaltic or diaphragm pumps with syringe pumps. This type of pump solves some of the problems mentioned above, particularly in terms of flow control and wear.

[0014] However, these pumps are only suitable for small production volumes, below one liter, and can therefore only be used for low-flow applications. Furthermore, they require a complex environment to ensure that the encapsulation system is sealed and sterile, given the contact between the syringe plunger and the solution. This complexity is particularly evident during the loading of solutions into syringes to ensure sterile filling.

[0015] Finally, the flow of the solution caused by this type of pump may be subject to pulsations due to the sliding of the piston in the syringe, and in particular to the presence of air bubbles which may appear when filling the syringe with the solution.

[0016] There is therefore a need for a cell encapsulation system capable of meeting the needs of applications requiring small volumes, on the order of microliters, as well as applications requiring large volumes, on the order of tens of liters; which does not cause significant wear on the components conveying the solutions to the encapsulation device, nor disturbances, for example of the impulse type, in the flow of solutions to the encapsulation device, and which allows the production of microcompartments under conditions conforming to the sterility requirements of these applications.

[0017] For these purposes, the invention relates to a cell encapsulation system, the system comprising at least: a. two containers, one of the containers being intended to contain a cell solution and the other of the containers being intended to contain a solution suitable for gelling, b. a milli-fluidic or micro-fluidic encapsulation device connected to the containers and arranged to form cell microcompartments, the outer layer of which is the solution suitable for gelling and the core the cell solution.

[0018] The system is characterized in that each container is connected to an inlet of the encapsulation device by one or more distributors, and in that at least one first container of the containers is connected to a solution displacement system capable of introducing a pressurized gas into said container to generate a flow of the solution contained in said container towards the encapsulation device.

[0019] The invention thus proposes to use a pressurized gas displacement system, also called a pressure pump, to move a solution from one of the containers to the encapsulation device. The pressurized gas can, for example, be injected directly into the container to increase the pressure within it and force the solution contained therein to flow towards a container outlet connected to the dispenser, where the pressure is lower. Alternatively, the pressurized gas can be injected into the container to exert pressure on a sub-container arranged within the container and containing the solution, thereby forcing this solution to flow towards a container outlet connected to the dispenser, where the pressure is lower.

[0020] This type of system offers the advantage of precise control over the gas pressure and / or flow rate introduced into the container, thus enabling precise control of the solution flow rate to the encapsulation device. This system is therefore capable of regulating this flow rate to ensure it is constant, regular, and free of pulsations, regardless of the encapsulation conditions. These conditions include the nature of the solutions used, the pressure at the encapsulation device outlet, the volume of solution in each container, the desired production rate, and the condition of the distributors.Furthermore, given the absence of direct contact between an element of the solution displacement system and an element of the container as well as the solution contained therein, this system makes it possible to increase the lifespan of the distributors of the encapsulation system, and to implement simple solutions to guarantee a closed and sterile environment.

[0021] Furthermore, this system is capable of meeting different volume requirements, and therefore of meeting different applications, including: a. research and development applications, requiring less than one milliliter of solution volume in the container, and in which different parameters are tested, such as solutions, cells, solution concentrations, flow rates, and microcompartment dimensions, in order to identify production conditions suitable for a given application; b. small-scale production applications, requiring between one milliliter and one liter of solution volume in the container, allowing for a small volume of microcompartments in order to test cell culture in a bioreactor, to conduct research on genetic diseases, or to test the effects of new drugs; c.large-scale production applications, requiring at least one liter of solution, or even several tens of liters, in the container, in order to produce complete batches of microcompartments intended for cell therapies.

[0022] Preferably, each container is connected to a solution displacement system capable of introducing a pressurized gas into said container to generate a flow of the solution contained in said container towards the encapsulation device.

[0023] It may be foreseen that the distributor(s) connecting each container to an inlet of the encapsulation device shall include a conduit, a pipe or a tube.

[0024] In the context of the present invention, and by way of non-limiting example, a "microfluidic device" means any device having one or more inlets and one or more outlets connected by a plurality of channels with a cross-section on the order of tens or hundreds of micrometers and capable of directing the flow of one or more fluids from the inlet(s) to the outlet(s). A "millifluidic device" also means any device having one or more inlets and one or more outlets connected by a plurality of channels with a cross-section on the order of millimeters and capable of directing the flow of one or more fluids from the inlet(s) to the outlet(s).

[0025] In the context of the present invention, and by way of non-limiting example, a "solution displacement system for introducing a gas under pressure into a container" is understood to mean a combination of a pressurized gas reservoir, an inlet line connecting the reservoir to the container and capable of allowing or preventing the flow of gas from the reservoir to the container, and a regulator capable of controlling the pressure and / or flow rate of the pressurized gas. The system may comprise one or more separate gas reservoirs, which may, for example, be mixed, particularly in an adjustable manner, before entering the inlet line. Each reservoir may be equipped with a regulator for the pressure of the gas contained in that reservoir.The system may include one or more inlet lines, formed by tubes, each connected to the same container or each connected to a separate container, for example, to allow the solutions from these containers to flow simultaneously. The inlet line may be equipped with a control valve, allowing the flow of gas to be authorized or prohibited to the container, or even allowing the gas flow rate to be controlled. The inlet line may also be equipped with a check valve, preventing gas from flowing back from the container to the tank.

[0026] The system includes various types of valves to regulate the flow of pressurized gas. These valves may include, but are not limited to, proportional valves that allow for precise and continuous flow control. Proportional valves can be used to finely adjust the flow of pressurized gas introduced into the containers and thus regulate the flow of solutions from the dispensers to the encapsulation device.

[0027] Advantageously, a control system can be used to pilot the proportional valve, generating initial pressurization of the container to a predefined maximum pressure, followed by fine-tuning. This approach allows for a rapid response to changes in demand while maintaining precise flow control.

[0028] Advantageously, the proportional valve may have a narrow cross-section for the passage of pressurized gas, thus enabling fine regulation. If necessary, the inlet line may include a secondary bypass line for this proportional valve, preferably with a larger cross-section than the proportional valve itself, and also equipped with a control valve to allow and prevent gas flow to the container via this secondary bypass line.

[0029] Thus, the control system can be configured to, in an initial container pressurization phase, operate the proportional valve and the control valve to allow gas to flow to the container only via the secondary bypass line. This secondary bypass line accelerates the container pressurization to the predefined maximum pressure. Then, in a second regulation phase, the control system can be configured to operate the valve. proportional and said control valve to allow only the passage of gas through the proportional valve. The proportional valve thus enables fine regulation.

[0030] Advantageously, the inlet line may include a second secondary bypass line for the proportional valve, preferably with a larger cross-section than the proportional valve itself, and opening to ambient air. This second secondary bypass line thus allows for faster depressurization of the container.

[0031] In the present invention, and without limitation, the pressurized gas may be one of the following gases or a combination of at least two of the following gases: air, oxygen, nitrogen, dinitrogen, carbon monoxide, carbon dioxide, nitric oxide, and nitrogen dioxide, hydrogen sulfide, ethylene, ozone, hydrogen, or any other inert gas suitable for a cell culture.

[0032] Advantageously, one of the containers contains the cell solution and the other of the containers contains the solution suitable for gelling.

[0033] If desired, the system may include a third container connected to an inlet of the encapsulation device by one or more distributors and intended to contain, or containing, an intermediate solution, such as an intermediate solution not containing a divalent cation such as Ca2+ Mg2+ to avoid too early crosslinking of the solution capable of gelling, preferably an isotonic solution not containing a divalent cation such as Ca2+ Mg2+ such as for example a sorbitol solution.

[0034] In one variation, the cell solution may contain culture medium and / or an extracellular matrix and / or an extracellular matrix substitute and / or an aqueous solution. In another variation, the intermediate solution may contain an extracellular matrix and / or an extracellular matrix substitute. If applicable, the encapsulation device will be arranged to form cell microcompartments, the outer layer of which is the gel-ready solution, an intermediate layer forming a cell matrix or extracellular matrix substitute, and the core the cell solution. This cell matrix allows the cells in the cell solution to grow and multiply. For example, the extracellular matrix substitute may include a mixture of proteins and extracellular compounds necessary for cell culture, particularly for pluripotent cells.Preferably, the extracellular matrix or extracellular matrix substitute may comprise structural proteins, such as laminins containing the α1, α4, or α5 subunits, the α1 or β2 subunits, and the λ1 or γ3 subunits, entactin, vitronectin, laminins, collagen, and growth factors, such as TGF-β and / or EGF. The extracellular matrix may be an aqueous solution and / or a hydrogel, preferably a hydrogel, different from the hydrogel forming the outer layer, such as, for example, a hydrogel comprising [missing information]. composed of alginate, fibrin, laminin, fibronectin, entactin, hyaluronic acid, and / or collagen. It may also be an extracellular matrix or an extracellular matrix substitute such as Matrigel®. In the invention, the cell solution comprises a plurality of cells. The cells may be of any cell type. More preferably, the cells are selected from human, animal, and plant eukaryotic cells, and even more preferably pluripotent stem cells, progenitor cells, cells undergoing differentiation, and differentiated cells. Where appropriate, said pluripotent stem cells may be induced pluripotent stem cells (iPSCs), MUSE cells (Multilineage-Differentiating Stress Enduring) found in the skin and bone marrow of adult mammals, or embryonic stem cells (ESCs).In a particular embodiment, and for legal or ethical reasons, stem cells are understood to exclude human embryonic stem cells or cells that have required the destruction of human embryos.

[0035] In one embodiment of the invention, the gelling solution comprises or is constituted by a hydrogel, such that the outer layer is a three-dimensional structure formed from a matrix of polymer chains swollen by a liquid, preferably water. For example, the gelling solution comprises or is constituted by alginate and, preferably, consists of alginate. In the context of the invention, "alginate" means linear polysaccharides formed from α-3-D-mannuronate (M) and α-L-guluronate (G), salts and derivatives thereof. Advantageously, the alginate is sodium alginate, composed of more than 60%, or even more than 80%, of G and less than 40%, or even less than 20%, of M, with an average molecular weight of 100 to 400 kDa and a total concentration of between 0.5% and 5% by mass.

[0036] In the microcompartments obtained by means of the system according to the invention, the cells present in the internal part can be isolated and / or in the form of at least one layer and / or in the form of at least one three-dimensional aggregate and / or in the form of at least one three-dimensional cellular micro-tissue, optionally with at least one lumen.

[0037] According to one embodiment, at least one cellular microcompartment obtained using the system according to the invention comprises at least one cell layer and at least one lumen. When the microcompartment comprises at least one lumen, at least one cell layer, the intermediate solution layer of the inner part, and the outer layer are preferentially arranged successively around said lumen, this is referred to as a cyst-like conformation. Thus, according to one embodiment, at least one cellular microcompartment obtained using the system according to the invention comprises at least one cyst, the hollow center, or lumen, of which is preferentially aqueous. In the context of the invention, a "cyst" is understood to be a three-dimensional, spherical, monolayered arrangement of cells or an epithelial layer surrounding a central lumen. This cyst-like conformation reduces the pressures experienced by the cells. This configuration also allows for decreased cell mortality and increased culture amplification. Consequently, it reduces the number of passages and dissociations required, and the time in culture needed to reach the desired final cell count.

[0038] The cellular microcompartments obtained using the system according to the invention preferably comprise one or more cysts, and / or one or more tissues and / or micro-tissues and / or cell aggregates with or without lumen(s).

[0039] Advantageously, the system according to the invention is arranged so that each cellular microcompartment obtained by means of this system is closed. In one embodiment, the system according to the invention is arranged so that each cellular microcompartment obtained by means of this system has a spherical or teardrop shape. Preferably, the diameter of such a microcompartment is between 10 µm and 1 mm, more preferably between 50 µm and 700 µm, even more preferably greater than 200 µm, and preferably less than 600 µm.

[0040] In another embodiment, the system according to the invention can be arranged so that each cellular microcompartment obtained by means of this system has an elongated shape, in particular an ovoid or tubular shape.

[0041] In one embodiment, the encapsulation device comprises a body arranged to form a concentric flow from the solutions supplied by the dispenser(s), where the outer flow is the solution suitable for gelling and the inner flow is the cell solution, and a nozzle connected to the body to receive said concentric flow and forming the outlet of the encapsulation device. The encapsulation device is arranged to form, directly at the nozzle outlet, said cell microcompartments from the concentric flow. In this embodiment, the encapsulation device forms the droplets one after another directly from the nozzle, either naturally or under the influence of external forces. The encapsulation device is thus of the "dripping" type, and forms the microcompartments one after another directly from the nozzle, without a jet.

[0042] In another embodiment, the encapsulation device comprises a body arranged to form a concentric flow from the solutions supplied by the dispenser(s), of which an external flow is the solution suitable for gelling and an internal flow is the cell solution, and a nozzle connected to the body to receive said concentric flow and forming the outlet of the encapsulation device. The encapsulation device is arranged to form, at the nozzle outlet, a concentric jet, in particular a continuous one, from the concentric flow such that this jet is fragmented into micro-cell compartments. In this embodiment, the increase in hydrodynamic instabilities within the jet forces it to fragment into droplets at a certain distance from the nozzle outlet; this effect is known as the Plateau-Rayleigh instability.The encapsulation device is thus of the "jetting" type, and forms the microcompartments by fragmentation of the jet naturally or under the input of external forces.

[0043] In yet another embodiment, the encapsulation device comprises a body arranged to form a concentric flow from the solutions supplied by the dispenser(s), of which an external flow is the solution suitable for gelling and an internal flow is the cell solution, and a nozzle connected to the body to receive said concentric flow and forming the outlet of the encapsulation device. The encapsulation device is arranged to atomize said concentric flow at the nozzle outlet into a cloud of droplets forming the cell microcompartments. In this embodiment, this atomization may occur naturally, in particular depending on the flow rate of the solutions supplied by the dispenser(s) and / or the diameter of the nozzle outlet, or by means of external forces. The encapsulation device is thus of the "spraying" type, each droplet forming a microcompartment.

[0044] In these embodiments, the encapsulation device may be a micro-fluidic or milli-fluidic type device capable of generating a concentric flow comprising at the center the cell solution, optionally surrounded by the intermediate solution, itself optionally surrounded by the solution capable of gelling.

[0045] In one embodiment of the invention, the encapsulation system includes at least one component capable of electrically charging at least one of the solutions with an electrical potential.

[0046] For a jetting-type encapsulation device, electrically charging at least one of the solutions passing through the device improves the jet's breakup into droplets. This technique is known as electro-jetting. It should be noted that the relative sizes of the outer layer and the core of the microcompartments can be adjusted by modifying the flow ratios of the two solutions at the dispensers.

[0047] For a jetting-type encapsulation device, electrically charging at least one of the solutions passing through the encapsulation device allows control over the size of the droplets formed at the nozzle exit, resulting in significantly more monodisperse droplets and reducing the risk of coalescence between droplets. The encapsulation device is thus of the "electro-dripping" type.

[0048] In the case of electro-jetting or electro-dripping, an electric field generating device, such as a metallic ring positioned downstream of the encapsulation device's outlet, can be added so that the jet or cell microcompartments pass through this ring. If necessary, the electric field generating device can be connected to an electrical potential, for example, to ground. This electric field helps to promote the dispersion of the cell microcompartments.

[0049] Alternatively, one of the solutions can be electrically connected to ground, and the device generating an electric field can be connected to a non-zero electrical potential. This voltage reversal makes it possible, for example, to avoid charging one of the solutions with a non-zero electrical potential, in order to avoid passing such an electrically charged solution through an encapsulation device that cannot withstand an electrical potential, particularly due to the presence of a sensitive sensor.

[0050] In yet another embodiment, the encapsulation system also includes an acoustic wave generator coupled to the nozzle and / or the body so that the encapsulation device is arranged to form droplets directly from the concentric flow at the nozzle outlet. In this embodiment, the encapsulation device is thus of the "acousto-dripping" type, and forms the droplets one after another directly from the nozzle under the effect of the acoustic waves emitted by the generator, the dimensions of the droplets being determined according to the choice of the frequency and amplitude of the acoustic waves.

[0051] In yet another embodiment, the encapsulation system also includes a vibrating element coupled to the nozzle and / or the body such that the encapsulation device is arranged to form, at the nozzle outlet, a concentric jet from the concentric flow, said jet being fragmented into droplets. In this embodiment, the encapsulation device is thus of the "vibrating-jetting" type and forms the droplets by breaking the concentric jet through a Plateau-Rayleigh instability induced by the vibrations generated by the vibrating element, the dimensions of the droplets being determined according to the choice of the frequency and amplitude of these vibrations. Said vibrating element could, for example, be a piezoelectric actuator.

[0052] In yet another embodiment, the encapsulation system also includes a cutting element disposed downstream of the nozzle outlet, such that the encapsulation device is arranged to form a concentric jet from the concentric flow at the nozzle outlet, this jet being fractionated by the cutting element. In this embodiment, the encapsulation device is thus of the "jet cutting" type and forms the droplets by cutting the concentric jet with the cutting element. The cutting element could, for example, be a rotating blade, the dimensions of the droplets being determined according to the rotational speed and the dimensions of the rotating blade.

[0053] In another embodiment, the encapsulation system comprises an electromechanical element coupled to the nozzle. The encapsulation device is arranged to form droplets directly at the outlet of each nozzle from the concentric flow under the effect of a vibration applied by the electromechanical element. In this embodiment, the encapsulation device is thus of the "inkjet printing" type and forms the droplets one after another directly from each nozzle. This electromechanical element could, for example, be a piezoelectric actuator, the dimensions of the droplets being determined according to the chosen frequency and amplitude of the vibrations applied by this element.

[0054] Regardless of the embodiment envisaged, the encapsulation system can be provided for, including a collection tank containing a stiffening solution and arranged to collect the cellular microcompartments formed by the encapsulation device. If necessary, the collection tank can be arranged downstream of the encapsulation device to collect the cellular microcompartments formed by the encapsulation device, the stiffening solution being arranged to cause stiffening of the outer layer of each cellular microcompartment upon immersion in this solution.

[0055] For example, the outlet of the encapsulation device may be positioned above the collection tank, so that the microcompartments fall by gravity into the collection tank. Advantageously, the collection tank and the encapsulation device are arranged at a distance from each other such that the cellular microcompartments formed by the encapsulation device pass through a gaseous volume, such as air, defined by a closed and sterile enclosure before being collected by the collection tank.

[0056] In yet another embodiment, the encapsulation device is arranged so that its nozzle is in contact with a collection fluid, such as oil, contained in a collection tank, and / or so that its nozzle is immersed in a collection fluid contained in a collection tank. The encapsulation device is arranged to form droplets or a jet directly in the collection fluid, which then break into droplets from the concentric flow. The solution suitable for gelling can then be cross-linked, for example, with a stiffening solution, to form the cellular microcompartments.

[0057] Regardless of the embodiment considered, the body and / or nozzle may be made of glass. Alternatively, the body and / or nozzle may be made of polymer or metal. The body and nozzle may be a single piece, or they may be manufactured separately and then assembled to form the encapsulation device.

[0058] Advantageously, the body comprises a first inlet connected to a first dispenser for receiving the cell solution and at least a second inlet connected to a second dispenser for receiving the solution suitable for gelling, as well as a single outlet connected to the nozzle, the body comprising a main channel including a substantially straight portion defining a central axis of the encapsulation device, the main channel connecting the first inlet to the single outlet and at least one secondary channel connecting the second inlet to the single outlet, said secondary channel subdividing into portions extending around the first channel, said subdivisions of the second channel joining at the single outlet in a single circular portion, concentric with the first channel, said single circular portion and the first channel joining to form the single outlet of the body.

[0059] In another embodiment of the invention, the encapsulation device comprises a first body equipped with a nozzle, the first body being arranged to form, at the nozzle outlet, a first jet from the cell solution supplied by the first dispenser, this A first jet breaks into droplets, and a second body is equipped with a nozzle. The second body is arranged to form a second jet at the nozzle outlet from the gelling solution supplied by the second dispenser. The first and second bodies are arranged so that the droplets from the first jet interact with the second jet to form the cellular microcompartments. In this embodiment, the first jet breaks into droplets by a Plateau-Rayleigh instability. These droplets encounter the continuous second jet, which then encapsulates them, by the Marangoni effect, to form the cellular microcompartments.It will then be possible to provide for crosslinking of the outer layer of the microcompartments, formed by the solution capable of gelling, in a gaseous environment, such as in air, for example using ultraviolet radiation, and / or using a third jet containing a crosslinking solution capable of interacting with the drops formed by the first and second jets and / or using a crosslinking solution contained in a collection tank in which the drops formed by the first and second jets are immersed.

[0060] It may also be envisaged any combination of the embodiments described above, or even other embodiments of the encapsulation device allowing the generation of cellular microcompartments without departing from the scope of the present invention, and in particular encapsulation devices allowing the formation of drops at the outlet of the encapsulation device and equipped with a means of controlling the ejection of the drops and of controlling the dimensions of the drops during their ejection, encapsulation devices allowing the formation of a concentric jet at the outlet of the encapsulation device and equipped with a means of separating the jet, after its exit from the encapsulation device, into drops, or even encapsulation devices allowing the coating of drops or a jet from a first device with drops or a jet from another device.

[0061] In one embodiment of the invention, the first container is sealed by a cap adapted to ensure a seal between the container and the outside. The cap is provided with a gas inlet to which the solution displacement system is connected for introducing pressurized gas into the container, and a solution outlet through which extends a tube of the dispenser immersed in the solution contained in the container to receive the flow of this solution generated by the introduction of the pressurized gas. In this embodiment, the gas pressure is exerted directly on the solution contained in the container to generate the displacement of this solution towards the dispenser and the encapsulation device.The stopper ensures the sterility of the container and also provides protection against backflow of gas or solution into the solution transfer system, particularly if the pressure at the solution outlet is higher than the pressure at the gas inlet, or if the transfer system depressurizes. The encapsulation system is thus sealed and sterile.

[0062] The cap could, for example, be made from a material chosen from: a metal, a Polypropylene (PP), polycarbonate (PC), cyclic olefin (COC) polymer or copolymer, polyetheretherketone (PEEK), fluorocarbon (FC) or perfluorocarbon (PFC), or polyacetal, including polyoxymethylene (POM), and more generally, a thermoplastic material or polymer. The cap may also be fitted with a sealing gasket designed to come into contact with an opening in the container intended to be closed by the cap; the gasket being made, for example, of a thermoplastic elastomer or silicone, polydimethylsiloxane (PDMS), natural rubber, polyisoprene (IR), polybutadiene (BR), polyurethane (PU), polyacrylate (PA), or epoxy resin.

[0063] Advantageously, the first container may be a flexible bag, a syringe, a conical-bottom tube, or a bottle or reservoir made of glass, polycarbonate, polypropylene, or any other suitable material for containing one of the aforementioned solutions. If the first container is a flexible bag, it may be provided that this bag is positioned within a rigid container designed to hold the bag in place during the procedure.

[0064] Preferably, the stopper is arranged so that the gas inlet is positioned above the solution contained in the container.

[0065] For example, the gas inlet could be formed on a side wall of the stopper and the solution outlet could be formed on a top wall of the stopper.

[0066] Advantageously, the solution displacement system is connected to the gas inlet of the stopper via a pressurized gas sterilization device. This sterilization device forms a barrier to sterilize the pressurized gas and potentially prevents backflow between the container and the solution displacement system. The sterilization device may include a gas-permeable, and preferably liquid-impermeable, filter, for example, formed by a sterile membrane with a plurality of holes with a diameter less than 0.3 µm, in particular 0.22 µm.

[0067] In one embodiment, the cell encapsulation system comprises a mother container containing the cell solution or the gelling solution, and a feeding circuit connecting the mother container to the first container, which is a daughter container, to supply the first container with the solution. The feeding circuit is controllable to regulate the flow of the solution from the mother container to the first container. This embodiment allows for a solution reservoir supplying the first container, which can be a bottle or a single-use reservoir. It should be noted that this solution is particularly advantageous when the mother container is intended to hold the cell solution. Indeed, it avoids pressurizing the entire cell solution, which could damage the cells, during batch production.

[0068] For example, said supply circuit may include a pinch valve and / or a pump to control the flow of said solution from the mother container to the daughter container.

[0069] Advantageously, the encapsulation system can be designed to include two daughter containers, each connected to the parent container by a dedicated, controllable power supply circuit. Each daughter container is connected to the same input of the encapsulation device. For example, each daughter container could be connected to the same input of the encapsulation device via a distributor, with the distributors joining at a common point, for example, via a T-connector, connected to said input of the encapsulation device.

[0070] Where appropriate, each daughter container is connected to a dedicated solution displacement system, this system being capable of introducing a pressurized gas into said container to generate a flow of the solution contained in said daughter container towards the encapsulation device.

[0071] Preferably, a control system for the encapsulation system can alternately control each of the movement systems and supply circuits, for example, to simultaneously cause the solution to flow from the mother container to one of the daughter containers and the solution from the other daughter container to flow back to the encapsulation device. This makes it possible to achieve large-scale production at a constant flow rate while maintaining a closed, sterile, single-use system.

[0072] In another example of implementation, the first container contains the entirety of said cell solution or said gelling solution intended for the production of a batch.

[0073] In another embodiment of the invention, the first container comprises a sub-container for holding the cell solution or the gelling solution. The first container is equipped with a gas inlet to which the solution displacement system is connected for introducing pressurized gas into the container. The distributor passes through the container and is connected to a solution outlet of the sub-container to receive the flow of solution generated by the introduction of pressurized gas into the container. For example, the container may be a pressurized, sterile chamber, and the sub-container may be formed by a flexible pouch arranged within this chamber, for example, a three-dimensional pouch that generally conforms to the internal shape of the container, or a two-dimensional pouch, generally flat when empty, positioned inside the container.

[0074] In this embodiment, the gas pressure is exerted on the walls of the subcontainer and thus indirectly on the solution contained within it, causing the solution to move towards the dispenser and the encapsulation device. Advantageously, the pressurized gas is not introduced into the subcontainer and therefore does not come into contact with the solution. This embodiment thus eliminates the need to sterilize the pressurized gas.

[0075] Advantageously, it can be provided that the distributor includes, downstream of the first container, a check valve or a non-return valve.

[0076] Advantageously, each container and the encapsulation device together form a closed and sterile system, particularly for single use. In the context of the present invention, and by way of non-limiting example, a "closed and sterile system" is understood to mean a system comprising a set of interconnected circulation circuits for one or more fluids, together defining an internal space in which the fluid(s) circulate. The system is arranged so that it has no interface between the internal and external spaces of the system, or only sterilization interfaces between the internal and external spaces of the system. This feature thus makes it possible to meet the sterility requirements of the various applications for which the system according to the invention is intended.

[0077] The closed, sterile system may include pre-sterilized, single-use components. These single-use components may include, but are not limited to, plastic parts, flexible bags, tubing, connectors, filters, sensors, valves, solution displacement systems, or any other element suitable for maintaining system sterility. The components may be sterilized by various methods, such as steam or autoclave sterilization, gamma sterilization, ethylene oxide sterilization, electron beam sterilization, sanitization, or any other appropriate sterilization method. These single-use elements, such as containers or the encapsulation device, may be replaced between each production batch to maintain system sterility.This single-use configuration can facilitate the maintenance of sterile conditions, reduce the risks of cross-contamination between production batches, and simplify cleaning and validation procedures.

[0078] In one embodiment, the solution transfer system is connected, directly or indirectly, to the first container via a pressurized gas sterilization device. Preferably, each container is connected to a solution transfer system, directly or indirectly, via a pressurized gas sterilization device. This sterilization device or devices form a sterilization barrier for the pressurized gas to ensure the sterility of the system according to the invention. The sterilization device may include a gas-permeable, and preferably liquid-impermeable, filter, in particular a hydrophobic one, for example, formed by a sterile membrane having a plurality of holes with a diameter less than 0.3 µm, in particular 0.22 µm. This membrane, when hydrophobic, also prevents backflow of solution into the solution transfer system.

[0079] In one embodiment of the invention, the encapsulation system includes a system for controlling the flow rate of pressurized gas introduced into the first container, and a sensor for the flow rate of pressurized gas introduced into said container and / or the flow of the solution contained in said container towards the encapsulation device, the control system being arranged to control the flow rate of pressurized gas introduced into said container as a function of the flow rate of pressurized gas and / or the flow of the solution measured by said sensor.

[0080] In the present invention, the term "control system" means a device or computer system designed to manage, regulate, and supervise the operations and processes of the encapsulation system by controlling and coordinating the actions of the various controllable elements of this system, such as the solution displacement system(s). The control system may be equipped with one or more processors, or even one or more microcontrollers, arranged to execute one or more computer programs to implement phases of a production cycle for a batch of microcellular compartments.It may also be envisaged that the control system is embedded in a machine comprising the solution displacement system(s), the containers, the encapsulation device, the collection tank, and where applicable, the various stages and fluidic circuits provided downstream of the collection tank and enabling the collection of the cellular microcompartments, including the opening and closing valves of these fluidic circuits, or alternatively that the control system is remote from this machine while being connected to it by means of connection, wired or wireless.

[0081] In this embodiment, the control system is thus equipped with a control loop enabling continuous adjustment of the flow rate of pressurized gas introduced into said container, for example by controlling a control valve provided on the pressurized gas inlet line, according to the flow rate of pressurized gas and / or the flow of the solution measured by said sensor

[0082] Advantageously, the sensor is positioned downstream of the first container, within the dispenser, to measure the flow rate of the solution from the first container to the encapsulation device. This type of sensor, also called an "inline" sensor, could, for example, be a thermal mass flow sensor. It is preferable that this sensor be sterile to avoid contaminating the solution with which it is intended to come into contact.

[0083] Alternatively, or cumulatively, the sensor could be positioned downstream of the first container, outside the dispenser, to measure the flow rate of the solution from the first container to the encapsulation device within the dispenser. This type of sensor, also called an "online" sensor, could be, for example, an ultrasonic flow sensor, an electromagnetic flow sensor, or any other type of static flow sensor that does not contact the fluid whose flow rate is to be measured. These non-invasive sensors offer the advantage of avoiding any contact with the solution and thus ensuring the sterility of the encapsulation system. The sensor could also be a scale or other weighing device, specifically designed to measure the weight of the collection tank to determine the flow rate of one or more solutions.

[0084] Alternatively or cumulatively, the sensor can be arranged downstream of the first container, in the solution displacement system, particularly on the inlet line, to measure the flow rate of pressurized gas introduced into the first container. In this In this embodiment, the sensor is called "pre-line" and measures the flow rate of the gas itself, particularly in steady state, the control system then being able to deduce the flow rate of the solution exiting the container.

[0085] Alternatively or cumulatively, the sensor could be a weight sensor for the first container, such as a scale or any other weighing device. In this embodiment, the control system can thus deduce the flow rate of the solution exiting the container from the container's weight, and in particular from the container's weight loss over time.

[0086] In one embodiment of the invention, each container is connected to a solution displacement system capable of introducing a pressurized gas into said container to generate a flow of the solution contained in said container towards the encapsulation device. The encapsulation system includes sensors for the flow rate of pressurized gas introduced into each container and / or the flow rate of the solution contained in each container towards the encapsulation device. If necessary, the control system is arranged to control the flow rate of pressurized gas introduced into each container based on a combination of the pressurized gas flow rates and / or the flow rate of each solution measured by said sensors.

[0087] In one embodiment of the invention, the distributor connected to said first container includes a restrictor element arranged to prevent the flow of solution from the encapsulation device into said container. This restrictor element thus forms a backflow prevention device ensuring the sterility of the solution contained in the container. This restrictor element may be configurable by the control system to prevent or allow the flow of solution between the container and the encapsulation device, for example, comprising a pinch valve, a solenoid valve, a piezoelectric valve, or a mechanical valve. Alternatively, this restrictor element may be a passive element arranged to allow the flow of solution between the container and the encapsulation device in only one direction, for example, comprising a passive capillary filter.

[0088] It may be advantageous to provide that at least one distributor, and in particular each distributor, is equipped with other elements, for example, air presence detectors, flow meters and / or pressure sensors.

[0089] In one embodiment of the invention, the encapsulation system comprises a collection tank containing a stiffening solution and arranged to collect the cellular microcompartments formed by the encapsulation device; and a circuit for collecting the cellular microcompartments collected by the collection tank.

[0090] For example, the collection tank could be designed to have an outlet for drawing off the stiffening solution and the cellular microcompartments from the collection tank to the collection circuit; and the encapsulation system could include: a. A buffer stage comprising the collection circuit connected to the collection tank to receive a. A harvesting stage comprising a second separation module comprising a second separation module comprising a second separation module comprising a second separation module comprising a second separation module comprising a second separation module comprising a second solution ...

[0091] In the present invention, the term "separation module" means a device capable of receiving a solution containing suspended particles, and capable of separating and retaining the particles from this solution while allowing the solution to flow through. By way of non-limiting example, this could include a membrane filter, a centrifugal filtration system, a tangential flow filtration system, or a decanter.

[0092] Where appropriate, the collection circuit may include a first valve suitable for preventing the passage of the stiffening solution from the collection circuit to the harvesting stage and the second separation module, and the control unit may be arranged to control the means of moving the collection circuit, the means of moving the harvesting circuit and the first valve so that the collection of the stiffening solution and the cell microcompartments immersed in this solution from the collection tank is continuous and so that the harvesting of the second solution and the cell microcompartments separated by the second separation module to the harvesting container is discontinuous.

[0093] Based on these characteristics, a buffer stage is interposed between the collection tank and the harvesting stage. The valve then allows the buffer stage to be isolated from the harvesting stage, to simultaneously collect from the collection tank to the buffer stage and harvest with a change of medium in the harvesting stage, or conversely, to allow a simultaneous transfer from the buffer stage and from the collection tank, via the buffer stage, to the harvesting stage.

[0094] Thus, depending on the state of this valve, the means of movement within the collection circuit allow the transfer of the cellular microcompartments from the collection tank and the stiffening solution to the first separation module, and conversely, the transfer of the cellular microcompartments retained by the first separation module to the harvesting stage. For example, it is possible to have these cellular microcompartments follow opposite paths within the same section of the collection circuit for each of these transfers, or to have the collection circuit comprise distinct sections, each dedicated to one of these transfers.

[0095] The means of movement allow the second solution contained in the container to be transferred in the harvesting circuit to the second separation module, and then this second solution and the microcompartments retained by the second separation module to be transferred to the harvesting container, while carrying out a change of environment of these microcompartments using a second solution, more suitable for harvesting, than the stiffening solution.

[0096] The various elements of the system can thus be controlled by the control system to define different phases during the production of a batch of microcompartments. Specifically, the collection circuit and / or the harvesting circuit can be equipped with one or more valves controllable by the control system to define, within this circuit or these circuits, preferred paths according to given phases of a production cycle for a batch of microcompartments.

[0097] For example, during the collection phase, the valve is closed, and the buffer stage can collect, via its separation module, microcompartments continuously generated by the collection tank. Simultaneously, the microcompartments contained in the separation module of the collection stage are discharged to the collection container via the second solution, thus effecting a change of medium. During the transfer phase, the valve is opened, and the microcompartments generated by the collection tank, as well as those contained in the separation module of the buffer stage, are transferred to the separation module of the collection stage via the recycling solution.

[0098] In other words, these characteristics therefore make it possible to ensure discontinuous filtration of the microcompartments, thanks to which it is possible to ensure continuity in the production of microcompartments by the collection tank while allowing, in a discontinuous way, a change of medium of the microcompartments thus produced.

[0099] In one embodiment of the invention, the collection tank is formed by a lower part intended to receive the stiffening solution and on which the dispensing outlet and injection inlet are provided, and by an upper part forming a lid on which the encapsulation device is mounted. The lower and upper parts together thus define a closed enclosure, suitable for the production of cell microcompartments with regard to the applicable sterility requirements.

[0100] In one embodiment of the invention, the collection tank is arranged so that the stiffening solution it contains is electrically connected to ground.

[0101] Thanks to this characteristic, the electrical charges carried by the cell microcompartments flow, within the stiffening solution, from these microcompartments to the bulk, thus preventing the accumulation of charges, electrically charging the solution itself, and creating repulsive effects between the microcompartments. This makes it possible to increase the yield and quality of cell culture within the microcompartments.

[0102] The invention also relates to a method for producing microcompartments. cellular systems implemented by means of a system according to the invention.

[0103] The present invention is now described by means of purely illustrative and in no way limiting examples of the scope of the invention, and from the accompanying drawings, in which the various figures represent:

[0104] [Fig. 1] represents, schematically and partially, a view of a cell encapsulation system according to an embodiment of the invention;

[0105] [Fig. 2] represents, schematically and partially, a cross-sectional view of an encapsulation stage of an encapsulation system according to a first example of an embodiment of the invention;

[0106] [Fig. 3] represents, schematically and partially, a cross-sectional view of an encapsulation stage of an encapsulation system according to a second embodiment of the invention;

[0107] [Fig. 4] schematically and partially represents a cross-sectional view of an encapsulation stage of an encapsulation system according to a third embodiment of the invention; and

[0108] [Fig. 5] represents, schematically and partially, a cross-sectional view of an encapsulation stage of an encapsulation system according to a fourth embodiment of the invention.

[0109] In the description that follows, identical elements, by structure or by function, appearing on different figures retain, unless otherwise specified, the same references.

[0110] [Fig. 1] shows a cell encapsulation system according to one embodiment of the invention.

[0111] The system comprises two containers, 11 and 12. The first container, 11, contains a solution containing a plurality of human pluripotent stem cells. The second container, 12, contains a gel-forming solution, such as a hydrogel like alginate. The system may also include a third container containing an intermediate solution, such as an isotonic solution like sorbitol.

[0112] The system also includes a milli-fluidic or micro-fluidic encapsulation device 13 arranged to form, from the solutions of the containers 11 and 12, cellular microcompartments whose outer layer is the alginate solution and whose core is the cell solution.

[0113] The encapsulation device 13 has several inlets each connected to one of the containers 11, 12 via a distributor 14. Each container 11, 12 is also connected to a Pu, P solution displacement system 12 capable of introducing a pressurized gas into this container 11, 12 to generate a flow of the solution contained in said container 11, 12 in the distributor 14 towards the corresponding inlet of the encapsulation device 13.

[0114] Each Pu, P12 solution displacement system, also called a pressure pump, is thus equipped with a pressurized gas tank, an inlet line connecting the reservoir to container 11, 12, and a regulator capable of controlling the pressure and / or flow of the pressurized gas.

[0115] The example in [Fig. 1] shows an embodiment in which each container 11, 12 is equipped with a dedicated pressure pump Pu, P12. In other, unshown variations, containers 11, 12 may share a single container. Each inlet line(s) may be equipped with a control valve, allowing the flow of gas to be authorized and prohibited to the container 11, 12, or even to control the gas flow rate, and / or with a non-return valve, preventing gas from flowing back from container 11, 12 to the gas tank.

[0116] Each Pu, P12 pressure pump may include one or more separate gas reservoirs, which may, for example, be mixed, particularly in an adjustable manner, by a mixer, before entering the inlet line. Each reservoir may be equipped with a pressure regulator for the gas contained within it. The pressurized gas contained in these reservoirs may be one of the following gases or a combination of at least two of the following gases: air, oxygen, nitrogen, carbon monoxide, carbon dioxide, nitric oxide, nitrogen dioxide, hydrogen sulfide, ethylene, ozone, hydrogen.

[0117] It should be noted that the dispenser 14, intended for dispensing the alginate solution, is equipped, in the example described, with a device capable of electrically charging the alginate solution with an electrical potential. Alternatively, the alginate solution may be charged directly in its container 12, via an electrode immersed in the solution, or in the encapsulation device 13.

[0118] The encapsulation device 13 is a microfluidic device comprising a body, including the inlets, and a nozzle connected to a single outlet of the body, forming a single outlet of the device 13. The body and nozzle may be made of glass or another material suitable for the pharmaceutical industry. The body and nozzle may be a single component or, alternatively, manufactured separately and then assembled to form the encapsulation device 13.

[0119] In the example described, the body has a main channel comprising a substantially straight portion defining a central axis of the encapsulation device 13. This main channel connects the first input to the single output of the body. The body has a secondary channel connecting the second input to the single output. This secondary channel is subdivided into portions extending around the first channel, these subdivisions of the second channel converging at the single output of the body into a single circular portion, concentric with the first channel. This single circular portion and the first channel then merge to form the single output of the body.

[0120] In other words, the body allows the formation of a concentric flow from the solutions supplied by the containers 11, 12 via the distributors 14, an external flow being formed by the alginate solution and an internal flow being formed by the cell solution.

[0121] The nozzle thus receives the concentric flow. Given the flow rates of the solutions, and the The electrostatic force generated by the electrical charges carried by the alginate solution causes the encapsulation device 13 to generate, at the nozzle outlet, a concentric jet from the concentric flow. This concentric jet is fragmented, under the effect of Plateau-Rayleigh instability, into cellular microcompartments whose outer layer is the alginate solution and whose core is the cell solution.

[0122] The encapsulation device 13 is thus of the "electro-jetting" type. It should be noted that the relative sizes of the outer layer and the core of the microcompartments can be adjusted by modifying the flow ratios of the two solutions using the distributors 14, while the overall size of the microcompartments can be controlled by adjusting the overall flow rate of the solutions and the electrical potential of the alginate solution.

[0123] As an alternative, it may be possible to size the flow rates of the solutions as well as the electrical potential so that the encapsulation device 13 is of the "electro-dripping" type, and thus forms the microcompartments one after the other directly from the nozzle.

[0124] Optionally, a grounded metal ring can be added downstream of the encapsulation device 13's outlet so that the jet or cell microcompartments pass through it. The electric field generated by this metal ring promotes the dispersion of the cell microcompartments, in the case of an "electro-jetting" type device.

[0125] With further reference to [Fig. 1], the system also includes a collection tank 15 arranged under the encapsulation device 13 and containing a first solution intended to collect the cellular microcompartments formed by this encapsulation device 13 and falling by gravity into the collection tank 15. The collection tank 15 and the encapsulation device 13 are arranged at a distance from each other such that the cellular microcompartments formed by the encapsulation device pass through a gaseous volume, in particular air, defined by a closed and sterile enclosure before being collected by the collection tank 15.

[0126] Alternatively, the encapsulation device 13 may be arranged so that a nozzle of the encapsulation device is immersed in the stiffening solution contained in the collection tank 15, the encapsulation device 13 being arranged to form, directly in the stiffening solution, the said cellular microcompartments from the concentric flow.

[0127] The stiffening solution includes a surfactant and a calcium salt that cross-links an alginate solution, thereby causing stiffening of the outer layer of each cell microcompartment when immersed in the collection tank.

[0128] Each cellular microcompartment obtained by means of this first stage 1 is thus closed and presents, in the example described, a spherical or elongated teardrop shape.

[0129] In one variant, the container containing the cell solution and / or the container containing the intermediate solution, includes at least an extracellular matrix and / or a extracellular matrix substitute. In this case, the encapsulation device can form cellular microcompartments, the outer layer of which is a hydrogel derived from the alginate solution, an intermediate layer forming a cellular matrix and / or an extracellular matrix substitute derived from the cell solution and / or the intermediate solution, and at least one layer and / or base and / or aggregate and / or cyst of cells derived from the cell solution. In this example, the electrically charged solution with said electrical potential could be the alginate solution and / or the sorbitol intermediate solution.

[0130] It should be noted that the collection tank 15 has a withdrawal outlet 151 for the stiffening solution and microcompartments immersed in this solution, as well as an injection inlet 152 for the stiffening solution into the collection tank 15. These inlet and outlet 151 and 152 are connected to a collection circuit, which will be described later.

[0131] In the example described, the collection tank is formed by a lower part 153 containing the stiffening solution and on which are provided the withdrawal outlet 151 and the injection inlet 152 and by an upper part 154 forming a hood on which is mounted the encapsulation device 13 and the metal ring 16. The collection tank 153 - hood 154 assembly thus defines a closed enclosure, suitable for the production of cell microcompartments with regard to the sterility requirements which are required.

[0132] It can be provided that the lower part 153 of the collection tank 15 is arranged so that the stiffening solution it contains is electrically connected to ground.

[0133] In the example described, containers 11 and 12, dispensers 14, encapsulation device 13 and collection tank 15 form an encapsulation stage 1 intended for the generation of cellular microcompartments of the production system.

[0134] The production system includes a second stage 2, called the buffer stage, connected to the first stage 1 via the withdrawal outlet 151 of the collection tank, and a third stage 3, called the harvesting stage, connected to the second stage 2.

[0135] The second buffer stage 2 includes a collection circuit 21 connected to the withdrawal outlet 151 of the collection tank 15, as well as a pump 23 allowing the stiffening solution and microcompartments to be withdrawn from the collection tank 15 to the collection circuit 21.

[0136] A first separation module 22 is positioned in the collection circuit 21 in order to receive the solution collected by the collection circuit 21.

[0137] The separation module 22 includes, for example, a filter having a first port and a second port, each connected to the collection circuit 21, on the side of the withdrawal outlet 151 and on the side of the injection inlet 152 of the collection tank 15.

[0138] A filter membrane extends within the filter to define a first compartment, into which the first port opens, and a second compartment, into which the second port opens. The membrane is capable of allowing the passage of a solution from one compartment to the other and preventing the passage of solid objects from one compartment to the other. compartment to the other compartment.

[0139] Downstream of the first separation module, the collection circuit 21 connects the first separation module 22 to the injection inlet 152 of the collection tank 15, to form a reinjection loop connecting the withdrawal outlet 151 to the injection inlet 152.

[0140] It should be noted that the second buffer stage 2 also includes a replenishment container 24 containing a given quantity of the stiffening solution. This container 24 is connected to the reinjection loop of the collection circuit 21, via a pump 25.

[0141] The pumps 23 and 25 together form means for drawing and injecting the stiffening solution into the collection circuit 21, these means of drawing and injecting being reversible in order to move the stiffening solution in one direction or another in the collection circuit 21. These may include peristaltic pumps, or any other mechanism suitable for causing the movement of a fluid in a circuit.

[0142] The third harvesting stage 3 includes a harvesting circuit 32 connected on the one hand to the collection circuit 21, between the draw-off outlet 151 and the first separation module 22, and on the other hand to various containers, namely a container 34 containing a second harvesting solution, a harvesting container 35 intended to receive the microcompartments, a container 36 containing a third rinsing solution and a waste container 37.Different valves 32a, 32b, 34a, 35a, 36a and 37a are positioned in the harvesting circuit to define different configurations of the harvesting circuit, including a first configuration in which a solution can flow from an inlet of the third floor connecting it to the second floor to the waste container 37, a second configuration in which the third rinsing solution can flow from the container 36 to the waste container 37 and a third configuration in which the second harvesting solution can flow from the container 34 to the harvesting container 35.

[0143] In the example described, the second and third solutions may be identical and each include a cell culture medium, and possibly a buffer solution and a ROCK inhibitor.

[0144] The third harvesting stage 3 also includes a pump 33, forming means of movement, to circulate the different solutions of the production system in the harvesting circuit 32, according to the configurations mentioned above.

[0145] A second separation module 31, substantially identical to the first module 23, is positioned in the harvesting circuit 32. It may be envisaged that the structures of the separation modules 22 and 31 are different from each other, and other types of filter may be used, such as a counter-current centrifugation type filter or a front-end filtration type system.

[0146] The collection circuit 21 includes a valve 26 capable of allowing or preventing the passage of a solution from the collection circuit 21 to the harvesting circuit 32. In the example described, the valve 26 is a solenoid valve.

[0147] In order to be able to carry out a production cycle for a batch of cell microcompartments, the system includes a control system 4 capable of controlling pumps 23, 25 and 33 and valves 26, 32a, 32b, 34a, 35a, 36a and 37a, as well as pressure pumps Pu and P 12.

[0148] The control system 4 is provided with one or more microcontrollers (not shown), arranged to execute one or more computer programs to control and coordinate the pumps 23, 25 and 33 and the valves 26, 32a, 32b, 34a, 35a, 36a and 37a of the second and third stages 2 and 3, as well as the pressure pumps Pu and P12 of the first stage 1, in order to implement phases of a production cycle of a batch of microcellular compartments.

[0149] In the example described, the control system 4 can receive data from system sensors, such as flow rate data from solution sensors in the circuits of stages 1 to 3, images acquired by a camera representing the jet from the encapsulation device 13, and weight measurements of the collection tank 15 from one or more load cells. The control unit can thus execute one or more production cycle control programs capable of interpreting this data in real time and adjusting the system's operational parameters, such as the flow rate of solutions from containers 11 and 12, based on this interpretation, or even stopping the production cycle.

[0150] It can also be foreseen that the control unit includes a user interface (not shown), such as a screen and a keyboard, allowing an operator to monitor the data from the sensors and / or the progress of the different phases of the production cycle, and to intervene in this production cycle, by manually modifying the operational parameters of the system or by interrupting it.

[0151] In the example described, all the different elements shown in [Fig. 1], including the control system 4, form a single machine. In another example, the control system 4 could be located remotely and communicate with the other system elements via wireless or wired communication.

[0152] We will now describe a phase of a production cycle of a batch of cellular microcompartments, implemented by the control system 4, and in which cellular microcompartments, generated by the first stage 1, are transferred from the collection tank 15 to the first separation module 22 to be stored there.

[0153] In this production phase, the control system 4 controls the first stage 1 to trigger the generation of a batch of cell microcompartments, each microcompartment from the encapsulation device 13 being collected in the collection tank 15 to be immersed in the stiffening solution in order to cause stiffening of its outer layer.

[0154] The first stage thus includes one or more sensors for the flow rate of pressurized gas introduced into each container 11, 12 and / or the flow rate of the solution contained in each container 11, 12 towards the encapsulation device 13. The flow rates of pressurized gas and / or the flow rate of the solutions measured by said sensors are provided to the control system, which then controls the flow rate of pressurized gas introduced into each Containers 11 and 12 are adjusted based on these measured flow rates so that the flow of solutions from containers 11 and 12 to the encapsulation device 13 complies with flow rate guidelines established for the desired application. This allows control of the relative sizes of the outer layer and the core of the microcompartments, the overall size of the microcompartments, and the production rate of the microcompartments exiting the encapsulation device 13.

[0155] In the example of [Fig. 1], these sensors are Cn and C12 flow rate sensors of the solution flowing into each dispenser 14 from each container 11, 12 to the encapsulation device 13.

[0156] More specifically, the control system 4 controls the flow rate of pressurized gas introduced into each container 11, 12 according to a combination of the flow rates of pressurized gas and / or the flow of each solution measured by said sensors.

[0157] Simultaneously, the control system 4 controls the pump 23 to draw, via the draw-off outlet 151, the stiffening solution contained in the collection tank 15, as well as the microcellular compartments suspended in this solution, into the collection circuit 21 in a first direction of movement.

[0158] System 4 keeps valve 26 closed, so as to cause a displacement of the stiffening solution and the cellular microcompartments only towards the first separation module 22.

[0159] This solution and the cellular microcompartments enter through a first port of the filter of this first module 22. The solution passes through the first compartment, the filter membrane, and the second compartment to exit through the second port, while the cellular microcompartments are retained in the first compartment, against the filter membrane.

[0160] The stiffening solution thus continues its journey via the reinjection loop of the collection circuit 21 towards the injection inlet 152 of the collection tank 15, to be recirculated in the tank and again in the collection circuit 21 during the entire production phase.

[0161] In the example described, the pump 25 is inactive during the production phase, since the collection circuit 21 forms a closed circuit and there is therefore no need to replenish the collection tank 15 with stiffening solution.

[0162] After a given time, determined so that a given quantity of cellular microcompartments are stored in the first separation module 22 and so that the immersion time of these cellular microcompartments in the stiffening solution is less than a given time, the system 4 controls the different elements of the system to implement a transfer phase, in which cellular microcompartments, both generated by the first stage 1 and stored in the first separation module, are transferred to the second separation module 31 to be stored there.

[0163] In this transfer phase, system 4 controls pump 25 so that the solution stiffening contained in the replenishment container 24 is injected into the collection circuit 21. Part of this solution joins the reinjection loop and the collection tank 15 via the injection inlet 152, while another part joins the first separation module to be entered via the second port 222.

[0164] Pump 23 remains active to continue drawing the stiffening solution and the micro-cell compartments from the collection tank. System 4 also controls valve 26 to allow the transfer of the solution from the second stage 2 to the third stage 3.

[0165] Therefore, the injection of the stiffening solution into the collection tank 15 is simultaneous with the withdrawal of the stiffening solution from the collection tank 15. The control system 4 thus controls the pumps 23 and 25 so that the withdrawal rate of the stiffening solution from the collection tank 15 is identical to the injection rate of the stiffening solution into the collection tank.

[0166] For these purposes, the first stage includes means for measuring, directly or indirectly, the flow rate of the stiffening solution being drawn from the collection tank 15. In the example described, these means of measuring include one or more load cells (not shown) via which the collection tank 15 is mounted on a support.

[0167] The measuring devices thus allow for the measurement of variations in the weight of the collection tank and therefore the deduction of variations in the flow rate of solution withdrawn relative to the flow rate of solution injected. The control system 4 therefore links the injection rate of the stiffening solution into the collection tank 15 by pump 25 to the withdrawal rate from the collection tank by pump 23, so that the weight variations are essentially zero. In other words, the injection rate of the stiffening solution from the refill container 24 is adjusted to compensate for the withdrawal of this solution from the collection tank 15.

[0168] It can be conceived that the means of measurement include, alternatively or cumulatively, means of measuring the flow rate of stiffening solution withdrawn at the withdrawal outlet, such as a flow meter.

[0169] Therefore, the stiffening solution and the cellular microcompartments immersed in this solution are transferred from the collection tank 15, via the collection circuit 21, to the third stage 3 through the valve 26.

[0170] Simultaneously, the part of the stiffening solution from the replenishment container 24 which enters through the second port of the filter of the first separation module, passes in the opposite direction through the second compartment, the filter membrane and the first compartment to exit through the first port carrying with it the cellular microcompartments retained in this first compartment during the previous production phase.

[0171] This solution and these cellular microcompartments thus circulate in the collection circuit 21 in a second direction of movement opposite to the first direction of movement of the stiffening solution during the previous production phase. They thus join the stiffening solution and the objects immersed in this solution, transferred from the collection tank 15, the whole passing into the collection circuit 32 of the third stage 3 through the valve 26.

[0172] System 4 controls valves 32a, 32b, 34a, 35a, 36a and 37a so that the collection circuit has the first configuration in which the stiffening solution can flow only towards the waste container 37. In the example described, all valves are closed, except for valve 37a connected to the waste container 37.

[0173] System 4 controls pump 33 so that the stiffening solution and the microcellular compartments suspended in this solution, from the collection tank 15 and the first separation module 22, circulate in the harvesting circuit 32 towards the second separation module 31.

[0174] Similar to the operation of the first module 23 during the production phase, the solution passes through the filter while the cellular microcompartments are retained in the first compartment of the second module.

[0175] The stiffening solution thus continues its journey in the collection circuit 32 towards the waste container 37.

[0176] At the end of this transfer phase, system 4 can again close valve 26, so as to isolate the third stage 3 from the second stage 2 and control pumps 23 and 25 in a similar way to the previous production phase so that cellular microcompartments, generated by the first stage 1 and collected in the collection tank 15, are again stored in the first separation module 22.

[0177] Simultaneously, system 4 can control the different elements of the third stage to implement rinsing sub-phases, using the rinsing solution stored in container 36, which can be circulated to the second separation module 31 and then to the waste container 37 to rinse the microcompartments that are stored in this module 31, and harvesting sub-phases, using the harvesting solution stored in container 34, which can be circulated to the second separation module 32 and then to the harvesting container 35 to harvest the microcompartments that are stored in this module 31.

[0178] It is thus understood that the rinsing and harvesting sub-phases allow for a change of medium in the cellular microcompartments, while cellular microcompartments continue to be generated by the first stage 1 and to be temporarily stored in the first separation module 22.

[0179] These production, transfer, rinsing, harvesting, and collection phases can be iterated several times to complete a production cycle of a batch of cellular microcompartments.

[0180] It can be anticipated that the production cycle includes initial phases preceding the first production phase, such as priming phases for the collection circuits and harvesting 21 and 32 allowing the different solutions to circulate in these circuits, and sub-phases of final rinsing and final harvesting.

[0181] It should be noted that, for each of the stages 1, 2 and 3, the components of this stage defining circuits for the circulation of solutions and / or microcompartments form a closed and sterile system, devoid of any interface between the interior space of this system in which these solutions and / or microcompartments circulate and the exterior of the system or only sterilization interfaces between this interior space and the exterior of the system.

[0182] In connection with [Fig. 2], we will describe a first embodiment of a pressurized gas displacement system Pu, allowing us to define, for the first stage 1, a closed and sterile system. We can assume that the system described is identical for both the Pu and P12 systems, or that each of these systems is structurally or functionally distinct.

[0183] The container 11 includes a flexible pouch, a syringe or a conical-bottom tube, sealed by a cap 111 suitable for ensuring a seal between the container 11 and the outside. The cap 111 may, for example, be made of a material chosen from: a metal, a polypropylene (PP), a polycarbonate (PC), a polymer or a cyclic olefin copolymer (COC), a polyetheretherketone (PEEK), a fluorocarbon (FC), a perfluorocarbon (PFC), or a polyoxymethylene (POM) and more generally a thermoplastic material or a polymer. The cap is equipped with a sealing gasket intended to come into contact with an opening in the container 11 intended to be closed by the cap, the gasket being for example made of a thermoplastic elastomer or silicone, a polydi-methylsiloxane (PDMS), a natural rubber, a polyisoprene (IR), polybutadiene (BR), a polyurethane (PU), a polyacrylate (PA) or even an epoxy resin.

[0184] The cap 111 is provided with a gas inlet 112 to which the Pu solution displacement system is connected to introduce the pressurized gas into said container 11.

[0185] The gas inlet 112 is formed on a side wall of the plug 111, and has an elbow so that it opens into the enclosure of the container 11 above the solution contained in this container 11.

[0186] The pressure pump Pu is connected to the gas inlet 112 via a pressurized gas sterilization device 113, consisting of a sterile membrane with a plurality of holes approximately 0.22 µm in diameter, making it permeable to gas and impermeable to liquid. This membrane 113 forms a sterilization barrier for the pressurized gas and potentially acts as a backflow preventer between the container 11 and the pressure pump Pu.

[0187] The cap 111 is also provided with a solution outlet 114 through which extends a tube 141 of the dispenser 14 immersed in the solution contained in said container 11.

[0188] It is thus understood that when pressurized gas is introduced by the pressure pump Pu into the container 11 via the gas inlet 112, the gas pressure acts directly on the solution contained in the container 11 to generate a displacement of this solution in tube 141 and therefore in distributor 14 towards encapsulation device 13.

[0189] The cap 111 ensures the sterility of the container 11 and also provides protection against backflow of gas or solution into the pressure pump Pu, particularly if the pressure at the solution outlet 114 is higher than that at the gas inlet 112, or if the pressure pump Pu depressurizes. The container 11, the dispenser 14, and the encapsulation device 13 thus form a closed and sterile system (SCS).

[0190] In the example of [Fig. 2], a "pre-line" sensor Cn, replacing or supplementing the sensor Cn of [Fig. 1], is arranged on the injection line of the pressure pump Pu to measure the flow rate of pressurized gas introduced into the container 11. The control system 4 can then deduce the flow rate of the solution exiting the container 11 towards the encapsulation device and can therefore regulate the flow rate of pressurized gas supplied by the pump Pu.

[0191] Furthermore, the distributor 14 connecting the container 11 to the encapsulation device 13 includes a restricting element 142, namely a pinch valve, which prevents the flow of solution from the encapsulation device 13 to the container 11. This restricting element thus forms a backflow prevention device ensuring the sterility of the solution contained in the container 11.

[0192] In connection with [Fig. 3], we will describe a second embodiment of a pressure gas displacement system Pu, allowing us to define, for the first stage 1, a closed and sterile system.

[0193] In this embodiment, the container 11 comprises a first flexible pouch 1111 inside which is arranged a sub-container, for example formed by a second flexible pouch 1112, containing said cell solution. The second flexible pouch 1112 lacks any fluid exchange interface with the first flexible pouch 1111.

[0194] The first flexible pouch 1111 is equipped with a gas inlet 1113 to which the pressure pump Pu is connected to introduce pressurized gas into the first flexible pouch 1111.

[0195] The distributor 14 passes through the first flexible bag 1111 to be connected to a solution outlet 1114 of the second flexible bag 1112.

[0196] It is thus understood that when pressurized gas is introduced by the pressure pump Pu into the first flexible bag 1111, the gas pressure is exerted on the walls of the second flexible bag 1112 and therefore indirectly on the solution contained in this second flexible bag 1112. This pressure thus generates a displacement of this solution towards the outlet 1114, the distributor 14 and the encapsulation device 13. Given the absence of a fluid exchange interface between the bags 1111 and 1112, the second bag 1112, the distributor 14 and the encapsulation device 13 thus form a closed and sterile SCS system.

[0197] Similar to the embodiment of [Fig. 2], the distributor 14 connecting the container 11 to the encapsulation device 13 includes a restricting element 142, namely a pinch valve, allowing to prohibit a flow of solution from the encapsulation device 13 to the container 11.

[0198] In the example of [Fig. 3], the Cn sensor is a sensor used to measure the flow rate of the solution in the distributor 14.

[0199] It can be provided that the Cn sensor is either an "inline" sensor arranged in the distributor, like a thermal mass flow sensor, or an "online" sensor arranged outside the distributor, like an ultrasonic flow sensor.

[0200] In connection with [Fig. 4], we will describe a third embodiment of a pressurized gas displacement system Pu,

[0201] In this embodiment, the displacement system Pu includes a compressor 51 and an inlet line 52 connecting this compressor to the container 11.

[0202] The inlet line 52 is equipped with a control valve, namely a proportional valve 53, which controls the flow of pressurized gas generated by the compressor 51 towards the container 11. The proportional valve 53 is controlled by a control system 4, in particular to finely regulate the flow of the solution contained in the container 11 towards the encapsulation device.

[0203] The inlet line 52 also includes two secondary lines 54 and 55 bypassing the proportional valve 53, one looping back onto the inlet line 52 to bypass the valve 53 for pressurizing container 11 and the other opening into the ambient air to bypass the valve 53 for depressurizing container 11.

[0204] Each secondary line 54 and 55 has a larger pressurized gas flow section than valve 53, and is also equipped with a control valve (not shown) to allow and prohibit the flow of pressurized gas through this secondary bypass line.

[0205] The control system 4 can thus be arranged to, in a first phase of pressurization of container 11, control the proportional valve 53 and the control valve of the secondary line 54 in order to allow only the passage of pressurized gas to container 11 via the secondary line 54. The secondary line 54 thus makes it possible to accelerate the pressurization of container 11 towards a predefined maximum pressure.

[0206] Then in a second phase of regulation, the control system 4 can be arranged to control the proportional valve 53 and the secondary line control valve 54 in order to allow only the passage of gas through the proportional valve 53. The proportional valve thus allows fine regulation of the pressure of the container 11 and therefore of the flow of the solution contained in the container 11 towards the encapsulation device.

[0207] It should be noted that, similarly, control system 4 can bypass proportional valve 53 with secondary line 55 in order to achieve rapid depressurization from container 55.

[0208] It should be noted that this Pu pressurized gas displacement system can be used interchangeably for the different embodiments previously described.

[0209] [Fig. 5] shows a cell encapsulation system according to another embodiment of the invention.

[0210] In this example, the system comprises two pressurized gas displacement systems Pu and two containers 11, each similar to that of the embodiment of [Fig. 2],

[0211] On the other hand, the system includes a mother container 6, containing the cell solution or said solution capable of gelling, the containers 11 thus being daughter containers.

[0212] This mother container 6 is connected to each daughter container 11 by a dedicated supply circuit 61, the plug 111 of each container 11 being provided with an additional inlet 115 to receive the solution flowing from the mother container 6 through this supply circuit 61.

[0213] Each supply circuit 61 also includes a means 62 for controlling the flow of the solution from the mother container 6 to the daughter container 11, such as a pinch valve and / or a pump.

[0214] Furthermore, a tube 141 of a distributor 14 is immersed in the solution contained in each daughter container 11 and extends through a solution outlet 114 of the stopper 11 to a T-connector 143. The distributors 14 of each daughter container 11 thus join together at a common portion 144 connected to an inlet of the encapsulation device 13.

[0215] Note that a pinch valve 145 is positioned on each distributor 14, upstream of the T-connector 143.

[0216] In this embodiment, a control system (not shown) of the encapsulation system can then alternately control each of the pressurized gas displacement systems Pu and control means 62 of the supply circuits 61, to simultaneously cause a flow of the solution from the mother container 6 to one of the daughter containers 11 and a flow of the solution contained in the other of the daughter containers 11 to the encapsulation device 13. It is thus possible to achieve large-scale production, at a constant flow rate, while maintaining a closed and sterile single-use system.

[0217] The preceding description clearly explains how the invention achieves its objectives, namely, to provide an encapsulation system capable of meeting the needs of both small-volume applications, on the order of microliters, and large-volume applications, on the order of tens of liters; that does not substantially cause wear on the components conveying the solutions to the encapsulation device, nor any disturbances, such as impulses, in the flow of solutions to the encapsulation device; and that enables the production of microcompartments under conditions that meet the sterility requirements of these applications. It is understood that these objectives are achieved by means of a pressurized gas displacement system allowing precise control of gas pressure and / or gas flow introduced into the container, and therefore allowing precise control of the flow of the solution to the encapsulation device, increasing the life of the distributors of the encapsulation system, and enabling the implementation of simple solutions to guarantee a closed and sterile environment.

[0218] In any event, the invention cannot be limited to the embodiments specifically described in this document, and extends in particular to all equivalent means and to any technically operative combination of these means.

Claims

Demands

1. Cell encapsulation system, the system comprising at least: a. two containers (11, 12), one of the containers being intended to contain a cell solution and the other of the containers being intended to contain a solution suitable for gelling, b. a milli-fluidic or micro-fluidic encapsulation device (13) connected to the containers and arranged to form cell microcompartments, the outer layer of which is the solution suitable for gelling and the core the cell solution, characterized in that each container is connected to an inlet of the encapsulation device by one or more distributors (14), and in that at least a first container (11) of the containers is connected to a solution displacement system (Pu, P12) suitable for introducing a pressurized gas into said container to generate a flow of the solution contained in said container towards the encapsulation device.

2. Encapsulation system according to the preceding claim, characterized in that it comprises at least one element capable of electrically charging at least one of the solutions with an electrical potential and in that the encapsulation device (13) comprises a body arranged to form a concentric flow from the solutions supplied by the dispenser(s) (14), of which an external flow is the solution capable of gelling and an internal flow is the cell solution, and a nozzle connected to the body for receiving said concentric flow and forming the outlet of the encapsulation device, the encapsulation device being arranged to form, at the outlet of the nozzle, a concentric jet from the concentric flow such that this jet is fractionated into cellular microcompartments.

3. Encapsulation system according to any one of the preceding claims, characterized in that said first container (11) is sealed by a cap (111) suitable for ensuring sealing between the container enclosure and the outside, the cap being provided with a gas inlet (112) to which is connected the solution displacement system (Pu) for introducing the gas under pressure into said container and a solution outlet (114) through which extends a tube (141) of the distributor (14) immersed in the solution contained in said container to receive the flow of this solution generated by the introduction of the gas under pressure.

4. Encapsulation system according to the preceding claim, characterized in that the solution displacement system (Pu) is connected to the gas inlet (112) of the stopper (111) via a pressurized gas sterilization device (113).

5. An encapsulation system according to claim 1 or 2, characterized in that the first container (11) comprises a sub-container (1112) for holding said cell solution or said gelling solution, in that the first container is provided with a gas inlet (1113) to which the solution displacement system (Pu) is connected for introducing pressurized gas into said container, and in that the distributor (14) passes through said container to be connected to a solution outlet (1114) of the subcontainer to receive the flow of the solution generated by the introduction of the pressurized gas into said container.

6. Encapsulation system according to any one of the preceding claims, characterized in that each container (11, 12) and the encapsulation device (13) together form a closed and sterile system (CSS).

7. Encapsulation system according to any one of the preceding claims, characterized in that it comprises a control system (4) for the flow rate of pressurized gas introduced into the first container (11), and a sensor (Cn, C12) for the flow rate of pressurized gas introduced into said container and / or the flow of the solution contained in said container towards the encapsulation device (13), the control system being arranged to control the flow rate of pressurized gas introduced into said container as a function of the flow rate of pressurized gas and / or the flow of the solution measured by said sensor.

8. Encapsulation system according to the preceding claim, characterized in that the sensor (Cn) is a sensor arranged in the distributor (14) to measure the flow rate, in the distributor, of the solution contained in the first container (11) towards the encapsulation device (13).

9. Encapsulation system according to claim 7, characterized in that the sensor (Cn) is a sensor arranged outside the distributor (14) to measure the flow rate, in the distributor, of the solution contained in the first container (11) towards the encapsulation device (13).

10. Encapsulation system according to claim 7, characterized in that the sensor (Cn) is a sensor arranged in the solution displacement system (Pu) to measure the flow rate of pressurized gas introduced into the first container (11).

11. Encapsulation system according to any one of claims 7 to 10, wherein each container (11, 12) is connected to a solution displacement system (Pu, P12) capable of introducing a gas under pressure into said container to generate a flow of the solution contained in said container towards the encapsulation device (13), characterized in that it comprises sensors (Cn, C12) of the flow rate of gas under pressure introduced into each container and / or of the flow of the solution contained in each container towards the encapsulation device and in that the control system (4) is arranged to control the flow rate of gas under pressure introduced into each container as a function of a combination of the flow rates of gas under pressure and / or the flow of each solution measured by said sensors.

12. Encapsulation system according to any one of the preceding claims, characterized in that the distributor (14) connected to said first container (11) comprises a restrictor element (142) arranged to prohibit a flow of solution from the encapsulation device (13) to said container.

13. An encapsulation system according to any one of the preceding claims, characterized in that it comprises a collection tank (15) containing a stiffening solution and arranged to collect the cellular microcompartments formed by the encapsulation device; and a collection circuit (21) for the cellular microcompartments collected by the collection tank (15).

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