Cell encapsulation system comprising a collection tank

Abstract

The invention relates to a cell encapsulation system, the system comprising at least: two containers (11, 12) intended to contain a cell solution and a solution capable of gelling, an encapsulation device (13) arranged to form cellular microcompartments whose outer layer is the solution capable of gelling and the core the cell solution, a collection tank (15) containing a stiffening solution; a collection circuit (21) for the cellular microcompartments collected by the collection tank; characterized in that the collection tank comprises a withdrawal outlet (151) for the stiffening solution from the tank to the collection circuit; and an injection inlet (152) for the stiffening solution into the tank, said injection inlet being arranged so that the injection direction is substantially tangential to a wall of the tank. Figure to be published with the abstract: Fig. 1

Description

Title of the invention: Cell encapsulation system comprising a collection tank

[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 collection tank for collecting cell microcompartments generated by an encapsulation device.

[0002] Ex vivo cell culture is a field of growing interest, particularly in the medical and pharmaceutical fields. The cells cultured can be of any type. They can be differentiated cells with different phenotypes, progenitor cells, or stem cells. Pluripotent stem cells in particular are increasingly used. Indeed, in the context of research on genetic diseases, these cells can be used to design cellular models of these diseases. These cells can also be used to test the effects of new drugs, in order to understand their mechanism of action and their safety, or in genetic research, to study the regions of the genome that are involved in cell differentiation.Finally, in 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, the cultivation of cells in large quantities represents a significant challenge. The research subjects mentioned require a significant quantity of human pluripotent cells. Similarly, the success of cell therapy in humans is conditioned by the availability of industrial quantities of cells, and in particular human pluripotent stem cells.

[0004] An important advance in cell culture techniques is the introduction of three-dimensional culture systems. Three-dimensional cultures are indeed advantageously closer to natural in vivo systems, and can be used for many applications, particularly in the development of therapies. A particularly suitable technology is that described in application WO2018 / 096277, which consists of three-dimensional cellular microcompartments for the culture of stem cells. This document describes a cell encapsulation device comprising a microfluidic injector for forming cellular microcompartments in the form of drops, the outer layer of which is formed by a solution comprising alginate and the core of which is formed by a solution of cells. These drops 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 cultivated in a liquid medium, while the shell protects the cells from mechanical stresses linked to collisions or fusions during culture in liquid suspension.

[0006] It is then necessary to transfer the microcompartments immersed in the calcium solution to a harvesting medium, within a relatively short time after their formation, in particular to maintain an immersion time in the calcium solution that is substantially identical for all the microcompartments in a batch and to ensure homogeneity of production, and to prevent the microcompartments from being exposed to the calcium solution for too long, which would be likely to compromise the integrity of the encapsulated cells.

[0007] In this context, it is thus necessary to be able to quickly collect the microcompartments from the collection tank, once their outer layer has been stiffened, while ensuring that the flow rate of solution and microcompartments drawn from the collection tank is substantially stable, in particular so that the volume of solution remains constant during the production of a batch, to prevent the microcompartments from obstructing the draw-off outlet or accumulating in the collection tank and so that the immersion time of the microcompartments in the calcium solution is generally constant for the entire batch.

[0008] The present invention is placed in this context and aims to meet this need.

[0009] 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 capable of gelling, b. an encapsulation device connected to the containers and arranged to form cellular microcompartments (MC) whose outer layer is the solution capable of gelling and the core the cell solution, c. a collection tank containing a stiffening solution and arranged to collect the cellular microcompartments formed by the encapsulation device; d. a collection circuit for the cellular microcompartments collected by the collection tank;

[0010] The system according to the invention is characterized in that the collection tank comprises an outlet for drawing off the stiffening solution and the cellular microcompartments from the collection tank to the collection circuit; and an inlet for injecting the stiffening solution into the collection tank, said injection inlet being arranged so that the direction of injection of the stiffening solution into the tank is substantially tangential to a wall of the collection tank.

[0011] The invention thus proposes to inject stiffening solution simultaneously with the withdrawal of the stiffening solution and the cellular microcompartments from the collection tank to the collection circuit. It is thus possible to control the flow rate of the stiffening solution injected into the collection tank and / or the start of the stiffening solution withdrawn from the collection tank to ensure that the quantity of stiffening solution contained in the collection tank remains substantially constant during a production cycle of a batch of cellular microcompartments.

[0012] Furthermore, the direction of injection of the stiffening solution, with respect to the position of the withdrawal outlet, which may for example be positioned substantially in the center of the bottom of the tank, makes it possible to create a vortex of the stiffening solution in the collection tank, rotating around this withdrawal outlet. This vortex, associated with the control of the injection and / or withdrawal flow rate, makes it possible to ensure that the stiffening solution gradually and in a generally homogeneous manner moves the cellular microcompartments immersed in the tank, from the periphery of the tank towards the withdrawal outlet.The collection of the cellular microcompartments is done in a homogeneous manner, in a relatively short time, which prevents the microcompartments from obstructing the withdrawal outlet or accumulating in the collection tank and ensures that the immersion time of the microcompartments in the stiffening solution is generally constant for the entire batch.

[0013] Advantageously, one of the containers contains the cell solution and the other of the containers contains the solution capable of gelling.

[0014] Where appropriate, the collection tank is arranged downstream of the encapsulation device to collect the cellular microcompartments formed by the encapsulation device and the stiffening solution is arranged to cause stiffening of the outer layer of each cellular microcompartment when it is immersed in this solution.

[0015] If desired, it may be provided that the system comprises a third container connected to the encapsulation device and intended to contain, or comprising, an intermediate solution, such as an isotonic intermediate solution, preferably an isotonic solution not containing a divalent cation such as Ca2+ Mg2+ to avoid too early crosslinking of the hydrogel in the collection tank, such as for example a sorbitol solution.

[0016] According to one variant, the cell solution may contain culture medium and / or an extracellular matrix and / or an extracellular matrix substitute and / or an aqueous solution. According to another variant, the intermediate solution may contain a extracellular matrix and / or an extracellular matrix substitute. Where appropriate, the encapsulation device will be arranged to form, in the collection tank, cellular microcompartments whose outer layer is the solution capable of gelling, an intermediate layer forming a cellular matrix or extracellular matrix substitute and the core the cell solution. This cellular matrix allows the cells in the cell solution to grow and multiply. For example, the extracellular matrix substitute may comprise a mixture of proteins and extracellular compounds necessary for cell culture, and more particularly 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 β3 or β2 subunits, and the γt or γ3 subunits, entactin, vitronectin, laminins, collagen, as well as growth factors, such as TGF-beta 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 or consisting 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. Cells can be of any cell type.More preferably, the cells are chosen from human, animal and plant eukaryotic cells, even more preferably pluripotent stem cells, progenitors, cells undergoing differentiation and differentiated cells. Where appropriate, said pluripotent stem cells may be induced pluripotent stem (IPS) cells, MUSE (Multilineage-differentiating Stress Enduring) cells found in the skin and bone marrow of adult mammals, or embryonic stem (ES) cells. 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.

[0017] In an exemplary embodiment of the invention, the gelling solution comprises or consists of a hydrogel, such that the outer layer is a three-dimensional structure formed from a matrix of polymer chains swollen by a liquid, and preferably water. For example, the gelling solution comprises or consists of alginate and, preferably, consists of alginate. In the context of the invention, the term "alginate" means linear polysaccharides formed from [3-D-mannuronate (M) and [α-L-guluronate (G), salts and derivatives thereof. Advantageously, the alginate is a sodium alginate, composed of more than 60%, or even more than 80% G and less than 40%, or even less than 20% M, with an average molecular mass of 100 to 400 KDa and a total concentration of between 0.5% and 5% by mass.

[0018] 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 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.

[0019] According to a variant, at least one cellular microcompartment obtained by means of the system according to the invention comprises at least one layer of cells and at least one lumen. When the microcompartment comprises at least one lumen, at least one layer of cells, the intermediate solution layer of the internal part and the external layer are preferentially, are successively organized around said lumen. This is referred to as a cyst-shaped conformation. Thus, according to a variant, at least one cellular microcompartment obtained by means of 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" means a three-dimensional arrangement in a monolayer of cells or in an epithelial layer, spherical, surrounding a central lumen. This conformation in the form of cyst(s) makes it possible to reduce the pressures experienced by the cells.This configuration also allows to reduce cell mortality and to increase the amplification factor of the culture. Consequently, this allows to reduce the number of passages and dissociation necessary; to reduce the time in culture necessary to reach the final number of cells required.

[0020] The cellular microcompartments obtained by means of the system according to the invention preferably comprise one or more cysts, and / or one or more tissues and / or microtissues and / or aggregates of cells with or without lumen(s).

[0021] Advantageously, the system according to the invention is arranged so that each cellular microcompartment obtained by means of this staged 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 drop shape. Preferably, the diameter of such a microcompartment is between 10 qm and 1 mm, more preferably between 50 qm and 700 qm, even more preferably greater than 200 qm, preferably less than 600 qm.

[0022] 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.

[0023] Advantageously, each container of the system according to the invention can be a flexible bag, a syringe or a tube with a conical bottom. Advantageously, each container is connected to an inlet of the encapsulation device by one or more distributors, such as conduits, pipes or tubulars, a syringe pump or a peristaltic pump making it possible to distribute, continuously or by dose, the solution contained in this container towards said inlet of the encapsulation device.

[0024] In one embodiment, the encapsulation device may be a microfluidic type device capable of generating a concentric jet comprising in the center the cell solution, where appropriate surrounded by the intermediate solution, itself surrounded where appropriate by the solution capable of gelling. The increase in hydrodynamic instabilities in the jet forces the jet to fragment into drops, this effect being known as Plateau-Rayleigh instability.These drops, once immersed in the stiffening solution, form the cellular microcompartments. One of the solutions may be electrically charged. Charging at least one of the solutions passing through the encapsulation device improves the breaking of the jet into drops. This technique is notably called "electro-jetting". Alternatively, it may be provided that the drops form directly (without a jet) at the outlet of the encapsulation device, this technique being notably called "electro-dripping". Whatever the embodiment envisaged, the outlet of the encapsulation device may be arranged above the collection tank, so that the microcompartments fall by gravity into this collection tank.

[0025] In the case of electro-jetting, it may be possible to add an electric field generating member, such as a metal ring arranged downstream of the outlet of the encapsulation device so that the jet or the cellular microcompartments pass through this ring. If necessary, the electric field generating member may be connected to an electric potential, for example to ground. This electric field makes it possible in particular to promote the dispersion of the cellular microcompartments.

[0026] In one embodiment of the invention, the encapsulation device comprises a body arranged to form a concentric flow from the solutions provided by the container(s) of the system according to the invention, an external flow of which is the solution capable of gelling and an internal flow of which 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 being arranged to form, at the outlet of the nozzle, a concentric jet from the concentric flow so that this jet breaks up into cellular microcompartments. The encapsulation device is thus of the “electro-jetting” type, the jet forming itself, by Plateau-Rayleigh instability, into microcompartments. It will be noted that the relative sizes of the outer layer and the core of the microcompartments can be adjusted by modifying the flow rate ratios of the two solutions at the distributor level.

[0027] Alternatively, the encapsulation device comprises a body arranged to form a concentric flow from the solutions supplied by the distributor(s), an external flow of which is the solution capable of gelling and an internal flow of which 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 being arranged to form, directly at the outlet of the nozzle, said cellular microcompartments from the concentric flow. The encapsulation device is thus of the “electro-dripping” type, and thus forms the microcompartments one after the other directly from the nozzle.

[0028] Whatever the embodiment considered, it may be provided that the body and / or the nozzle are made of glass. Alternatively, the body and / or the nozzle may be made of polymer or metal. It may be provided that the body and the nozzle form a single piece or that the body and the nozzle are made separately and then assembled to form the encapsulation device.

[0029] Advantageously, the body comprises a first inlet connected to a first distributor for receiving the cell solution and at least one second inlet connected to a second distributor for receiving the solution capable of gelling, as well as a single outlet connected to the nozzle, the body comprising a main channel comprising a substantially rectilinear 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 being subdivided 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.

[0030] Advantageously, it may be provided that the system comprises, for each distributor and for at least one part of the collection circuit connected to the withdrawal outlet, a guide arranged to maintain said distributor and said part of the collection circuit in a given shape. In particular, it may be provided that the guide of said part of the collection circuit is arranged to maintain said part in the form of a siphon. This ensures that the directions of injection and withdrawal of the stiffening solution and that the directions of injection of the solutions into the encapsulation device remain generally constant, even in the event of mechanical disturbances of the system, such as vibrations or jolts or jerks generated by a pump of the system.

[0031] In one embodiment, the system comprises means for withdrawing and injecting the stiffening solution from and to the collection tank and a unit control means arranged to control said withdrawal and injection means so that the injection of the stiffening solution into the collection tank is simultaneous with the withdrawal of the stiffening solution from the collection tank.

[0032] In the present invention, the term "withdrawal and injection means" means one or more mechanisms capable of causing a movement of a fluid in the collection circuit, in one direction and / or the other. By way of non-limiting example, the withdrawal and injection means may comprise pumps, in particular peristaltic pumps, or any other mechanism suitable for causing a movement of a fluid. For example, it may be envisaged that the withdrawal and injection means comprise a pump for injecting the stiffening solution into the collection tank via the injection inlet and / or a pump for withdrawing the stiffening solution from the collection tank via the withdrawal outlet.

[0033] In the present invention, the term "control unit" means a device or a 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 withdrawal and injection means. It may be provided that the control unit is equipped with one or more processors, or even one or more microcontrollers, arranged to execute one or more computer programs in order to implement phases of a production cycle of a batch of object. It may in particular be provided that the control unit is capable of receiving sensor data, interpreting this data in real time, and making automated decisions to adjust the operational parameters of the system.It may also be provided that the control unit includes a user interface allowing an operator to monitor production and, if necessary, to intervene in this production. It may also be provided that the control unit is embedded in a machine comprising the containers, the encapsulation device, the collection tank and the collection circuit, or alternatively that the control unit is remote from this machine while being connected to it by connection means, wired or wireless.

[0034] In one embodiment of the invention, the collection circuit comprises a reinjection loop connecting the withdrawal outlet to the injection inlet, said withdrawal and injection means being capable of causing a movement of the stiffening solution in the reinjection loop. Where appropriate, the control unit is arranged to control said withdrawal and injection means to recirculate the stiffening solution drawn via the withdrawal outlet to the injection inlet.

[0035] In this embodiment, the stiffening solution drawn from the collection tank to transport the cellular microcompartments is recirculated, via the reinjection loop to the collection tank, to be injected there again. thus minimizes the amount of stiffening solution required for the production of a batch of microcompartments.

[0036] Alternatively, it may be provided that the collection circuit comprises a waste container and that the control unit is arranged to control said withdrawal and injection means to direct the stiffening solution withdrawn via the withdrawal outlet to the waste collector.

[0037] Advantageously, the reinjection loop comprises a separation module arranged to separate and retain the cellular microcompartments from the stiffening solution withdrawn via the withdrawal outlet.

[0038] In the present invention, the term "separation module" means a device capable of receiving a solution comprising objects in suspension, and capable of separating and retaining the objects from this solution while allowing this solution to flow. By way of non-limiting example, it could be a membrane filter, a centrifugal effect filtering system, a tangential filtration system, a decanter.

[0039] Preferably, the reinjection loop comprises a part of the collection circuit connecting the withdrawal outlet to the separation module and a part of the collection circuit connecting the separation module to the injection inlet.

[0040] According to an exemplary embodiment of the invention, the separation module comprises a filter provided with a first port and a second port each connected to the collection circuit, and a filter membrane extending into the filter to define two compartments, the membrane being capable of allowing the passage of a solution from one compartment to the other compartment and of preventing the passage of objects from one compartment to the other compartment. It is thus understood that the stiffening solution drawn from the collection tank and entering through the first port of the filter can pass through the first compartment, the filter membrane, and the second compartment to exit through the second port, while the cellular microcompartments that it transports are retained in the first compartment, against the filter membrane.Conversely, a solution entering through the second port can then cross in the opposite direction the second compartment, the filter membrane and the first compartment to exit through the first port, taking with it the cellular microcompartments retained in this first compartment.

[0041] Advantageously, each port can open into the filter in a direction that is not substantially orthogonal to a plane of extension of the filter membrane. Preferably, said direction is tangential or parallel to the plane of extension, which thus makes it possible to avoid creating an accumulation or damage to objects in the filter. As a variant, it may in particular be provided that the first port opens into the filter in a direction substantially orthogonal to the plane of extension of the filter membrane, and that the second port opens into the filter in a direction not substantially orthogonal to an extension plane of the filter membrane, in particular in a direction tangential to said extension plane.

[0042] According to one embodiment of the invention, the filter comprises an inner casing with edges having a rounded shape and the useful section of the filter membrane has the shape of a circle or an ellipse. This characteristic makes it possible to reduce the nooks inside the filter, thus limiting the accumulation of objects in the nooks and making it possible to avoid the loss of cellular microcompartments stuck in the module.

[0043] According to another embodiment of the invention, the filter has a toric shape and the useful section of the filter membrane has the shape of a circle or an ellipse. This toric shape makes it possible to increase a swirl effect in the filter.

[0044] According to another exemplary embodiment of the invention, the separation module comprises a counter-current centrifugation type filter. This type of filter makes it possible to trap the cellular microcompartments immersed, or in suspension, in the stiffening solution against a filter medium by a centrifugal effect, the solution circulating counter-currently in a rotating system.

[0045] According to yet another exemplary embodiment of the invention, the separation module comprises a frontal filtration type system.

[0046] Advantageously, the collection circuit comprises a replenishment container containing a stiffening solution and connected to the reinjection loop to inject said stiffening solution therein. Where appropriate, the control unit is arranged to control said withdrawal and injection means to direct the stiffening solution contained in the replenishment container to the injection inlet via the reinjection loop.

[0047] It is thus possible to replenish the collection tank with a given quantity of clean solution, in particular in order to compensate for the loss of solution which would be due to the transfer of the cellular microcompartments to a collection container.

[0048] In one embodiment of the invention, the control unit is arranged to control said withdrawal and injection means so that the withdrawal flow rate of the stiffening solution from the collection tank is identical to the injection flow rate of the stiffening solution into the collection tank.

[0049] Advantageously, the system according to the invention comprises means for measuring, directly or indirectly, the flow rate of withdrawal of the stiffening solution from the collection tank and the control unit is arranged to control the injection flow rate of the stiffening solution into the collection tank by said withdrawal and injection means to the withdrawal flow rate measured by said measuring means. In other words, it is ensured that the injection flow rate of the stiffening solution makes it possible to compensate for the withdrawal of this solution from the collection tank, including in the event of variations in the withdrawal flow rate which could be caused during certain phases of a production cycle.

[0050] According to an exemplary embodiment of the invention, said measuring means comprise means for weighing the collection tank. The weighing means may, for example, comprise one or more load cells via which the collection tank is mounted on a support. The measuring means thus make it possible to measure variations in the weight of the collection tank and therefore to deduce variations in the flow rate of solution withdrawn with respect to the flow rate of solution injected.

[0051] Alternatively, the measuring means may comprise means for measuring the flow rate of stiffening solution drawn off at the draw-off outlet, in particular a flow meter.

[0052] In one embodiment of the invention, the collection tank comprises a funnel-shaped bottom wall whose outlet orifice forms the withdrawal outlet of the collection tank. This shape thus makes it easier to move the cellular microcompartments suspended in the stiffening solution in the collection tank towards the withdrawal outlet.

[0053] Advantageously, the bottom wall may have a shape of symmetry of revolution around an axis passing through the draw-off outlet, in particular a conical shape towards the draw-off outlet positioned in the center of the bottom wall. Preferably, said bottom wall is smooth and has a substantially constant slope from its periphery towards the draw-off outlet.

[0054] Advantageously, the injection inlet of the stiffening solution into the collection tank is positioned above the bottom wall, said injection inlet being arranged so that the direction of injection of the stiffening solution into the tank is substantially tangential to a side wall of the collection tank. Where appropriate, said bottom wall extends from a lower periphery of the side wall.

[0055] In one embodiment of the invention, the injection inlet of the stiffening solution into the collection tank is formed by a nozzle comprising an inlet port located outside the collection tank and an outlet port opening into the collection tank, the nozzle having a reduction in its internal section from the inlet port to the outlet port. Said reduction may for example be formed by a projection of the internal section. This reduction in section makes it possible to increase the speed of the stiffening solution during its injection into the collection tank and thus to improve the vortex formed by the injection in the direction tangential to the wall of the collection tank.

[0056] In one embodiment of the invention, the collection tank is formed by a lower part intended to receive the stiffening solution and on which are provided the withdrawal outlet and the injection inlet and by an upper part forming a hood on which the encapsulation device is mounted. The collection tank - hood assembly thus defines a closed enclosure, suitable for the production of cellular microcompartments with regard to the sterility requirements which are imposed.

[0057] In one embodiment of the invention, the collection tank is arranged so that the stiffening solution which it contains is electrically connected to ground.

[0058] Thanks to this characteristic, the electrical charges carried by the microcompartments Cell compartments flow, in the stiffening solution, from these microcompartments towards the mass, which prevents the accumulation of charges, electrically charging the solution itself and creating repulsion effects between the microcompartments themselves. It is thus possible to increase the yield and quality of cell culture within the microcompartments.

[0059] Advantageously, the stiffening solution comprises a surfactant and a calcium salt.

[0060] The invention also relates to a production system comprising an encapsulation system according to the invention.

[0061] In one embodiment of the invention, the assembly formed by the containers intended to contain the cell solution and the solution capable of gelling, the encapsulation device and the collection tank forms a first stage for generating cellular microcompartments.

[0062] Advantageously, the production system according to the invention comprises: a. A second buffer stage comprising the collection circuit connected to the collection tank to receive the stiffening solution and the cellular microcompartments immersed in this solution; a first separation module capable of receiving the stiffening solution collected by the collection circuit and arranged to separate and retain the cellular microcompartments from this solution; b. A third harvesting stage comprising a second separation module capable of receiving the stiffening solution collected by the collection circuit and arranged to separate and retain the cellular microcompartments of this solution; a harvesting circuit connected to the second separation module, to a container containing a second solution and to a harvesting container; and means for moving the second solution in the harvesting circuit.

[0063] Where appropriate, the collection circuit may comprise a first valve capable of prevent the passage of the stiffening solution from the collection circuit to the third stage and the second separation module and the control unit can be arranged to control the means of withdrawal and injection of the collection circuit, the means of movement of the harvesting circuit and the first valve so that the collection of the stiffening solution and the cellular microcompartments immersed in this solution from the collection tank is continuous and such that the harvesting of the second solution and the cellular microcompartments separated by the second separation module to the harvesting container is discontinuous.

[0064] According to these characteristics, a buffer stage is interposed between the object generation stage and the harvesting stage. The valve then makes it possible to isolate the buffer stage from the harvesting stage, to simultaneously carry out a collection from the first stage to the second stage and a harvest with a change of medium in the third or, on the contrary, to authorize a simultaneous transfer from the second stage and from the first stage, via the second stage, to the third stage.

[0065] Thus, depending on the state of this valve, the withdrawal and injection means make it possible to transfer the cellular microcompartments from the collection tank and the stiffening solution to the first separation module and conversely to transfer the cellular microcompartments retained by the first separation module to the harvesting stage. For example, it may be envisaged that these cellular microcompartments follow reverse paths in the same part of the collection circuit for each of these transfers or that the collection circuit comprises separate parts each dedicated to one of these transfers.

[0066] The movement means make it possible to transfer, in the harvesting circuit, the second solution contained in the container to the second separation module, then to transfer this second solution and the microcompartments retained by the second separation module to the harvesting container, while carrying out a change of medium of these microcompartments using a second solution, more suitable for harvesting, than the stiffening solution.

[0067] The different elements of the system can thus be controlled by the control unit to define different phases during the production of a batch of objects. In particular, it may be provided that the collection circuit and / or the harvesting circuit are provided with one or more valves controllable by the control unit to define, within this or these circuits, preferential paths according to given phases of a production cycle of a batch of objects.

[0068] For example, in a collection phase, the valve is closed and the buffer stage can recover, via its separation module, microcompartments continuously generated by the generation stage while, simultaneously, the microcompartments contained in the separation module of the harvesting stage are evacuated to the harvesting container via the second solution, thus operating a change of medium. In a transfer phase, the valve is open, and the microcompartments generated by the generation stage as well as those contained in the separation module of the buffer stage are transferred to the separation module of the harvesting stage, via the recycling solution.

[0069] 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 the microcompartments by the generation stage while allowing, in a discontinuous manner, a change of medium of the microcompartments thus produced.

[0070] The invention also relates to a method for producing cellular microcompartments implemented by means of a system according to the invention.

[0071] The present invention is now described using examples which are purely illustrative and in no way limitative of the scope of the invention, and from the appended drawings, drawings in which the various figures represent:

[0072] [Fig.l] represents, schematically and partially, a view of a cell encapsulation system according to an embodiment of the invention;

[0073] [Fig.2] represents, schematically and partially, a sectional view of the encapsulation device of the system of [Fig.l];

[0074] [Fig.3] represents, schematically and partially, a top view of the system of [Fig.l];

[0075] [Fig.4] represents, schematically and partially, a sectional view of the system of [Fig.l]; and

[0076] [Fig.5] represents, schematically and partially, a view of a production system incorporating the system of [Fig.l].

[0077] In the following description, elements that are identical, by structure or by function, appearing in different figures retain, unless otherwise specified, the same references.

[0078] [Fig.l] shows a cell encapsulation system according to one embodiment of the invention.

[0079] The system comprises two containers 11 and 12. A first container 11 comprises a solution comprising a plurality of human pluripotent stem cells. A second container 12 comprises a solution capable of gelling, comprising for example a hydrogel such as alginate. It may be provided that the system comprises a third container comprising an intermediate solution, for example comprising an isotonic solution such as sorbitol.

[0080] The system also comprises an 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 the core the cell solution.

[0081] [Fig.2] represents a sectional view of the encapsulation device 13.

[0082] As shown in this [Fig.2], the encapsulation device 13 comprises several inlets each connected to one of the containers 11, 12 via a distributor 14. In the example described, each distributor 14 thus comprises a distribution circuit and a movement member, such as a syringe pump or a peristaltic pump, making it possible to distribute, continuously or by dose, the solution contained in a container 11, 12 to the inlet of the device 13 to which it is connected.

[0083] It should be noted that the dispenser 14 intended for dispensing the alginate solution is equipped, in the example described, with a member capable of electrically charging the alginate solution with an electrical potential. It may be provided, as a variant, that the alginate solution is charged directly into its container 12, via an electrode immersed in this solution.

[0084] The encapsulation device 13 is a microfluidic device which comprises a body, comprising the inlets, and a nozzle connected to a single outlet of the body and forming a single outlet of the device 13. It may be provided that the body and the nozzle 34 are made of glass or another material suitable for the pharmaceutical industry. It may be provided that the body and the nozzle form a single piece or, on the contrary, that they are made separately and then assembled to form the encapsulation device 13.

[0085] In the example described, the body comprises a main channel comprising a substantially rectilinear portion defining a central axis of the encapsulation device 13. This main channel connects the first inlet to the single outlet of the body. The body comprises a secondary channel connecting the second inlet to the single outlet. This secondary channel is subdivided into portions extending around the first channel, said subdivisions of the second channel joining at the single outlet of the body in a single circular portion, concentric with the first channel. This single circular portion and the first channel then join to form the single outlet of the body.

[0086] In other words, the body allows a concentric flow to be formed 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.

[0087] The nozzle thus receives the concentric flow. Taking into account the flow rates of the solutions, and the electrostatic force generated by the electrical charges carried by the alginate solution, the encapsulation device 13 thus generates, at the outlet of the nozzle, a concentric jet from the concentric flow. This concentric jet is split, under the effect of the Plateau-Rayleigh instability, into cellular microcompartments whose outer layer is the alginate solution and the core the cell solution.

[0088] The encapsulation device 13 is thus of the “electro-jetting” type. It will be noted that the relative sizes of the outer layer and the core of the microcompartments can be adjusted by modifying the flow rate ratios of the two solutions using the dis tributors 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.

[0089] As a variant, it may be possible to dimension the flow rates of the solutions as well as the electrical potential so that the encapsulation device 13 is of the type “electro-dripping”, and thus forms the micro-compartments one after the other directly from the nozzle.

[0090] As shown in [Fig.2], a metal ring 16, connected to ground by a connector 17, extends downstream of the outlet of the encapsulation device 13 so that the jet or the cellular microcompartments pass through this ring. The electric field generated by this metal ring makes it possible to promote the dispersion of the cellular microcompartments, in the case of an “electro-jetting” type device.

[0091] Referring again to [Fig.l], the system further comprises 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.

[0092] The stiffening solution comprises a surfactant and a calcium salt enabling an alginate solution to be crosslinked, and thus enabling the outer layer of each cellular microcompartment to become stiffer when it is immersed in the collection tank.

[0093] Each cellular microcompartment obtained by means of this first stage 1 is thus closed and has, in the example described, a spherical or elongated drop shape.

[0094] In a variant, the container comprising the cell solution and / or the container comprising the intermediate solution, comprises at least one extracellular matrix and / or an extracellular matrix substitute. In this case, the encapsulation device may form cellular microcompartments including the outer hydrogel layer originating from the alginate solution, an intermediate layer forming a cellular matrix and / or an extracellular matrix substitute originating from the cell solution and / or the intermediate solution and at least one layer and / or layer and / or aggregate and / or cyst of cells originating from the cell solution. In this example, it may be envisaged that the solution electrically charged with said electrical potential is the alginate solution and / or the intermediate sorbitol solution.

[0095] It will be noted that the collection tank 15 comprises 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 inlets and outlets 151 and 152 are connected to a collection circuit, which will be described later.

[0096] In the example described, the collection tank is formed by a lower part 153 containing the stiffening solution and on which the withdrawal outlet 151 and the injection inlet 152 are provided and by an upper part 154 forming a cover on which the encapsulation device 13 and the metal ring 16 are mounted. The collection tank 153 - cover 154 assembly thus defines a closed enclosure, suitable for the production of cellular microcompartments with regard to the sterility requirements which are imposed.

[0097] [Fig.3] and [Fig.4] respectively represent a top view and a side view of the lower part 153 of the collection tank 15.

[0098] The lower part 153 comprises a side wall 153a from which extends a funnel-shaped bottom wall 153b whose outlet orifice forms the withdrawal outlet 151 of the collection tank 15.

[0099] The injection inlet 153 is positioned above the bottom wall 153b, extending so that the injection direction of the stiffening solution into the tank 15 is substantially tangential to the side wall 153a of the collection tank. As shown in [Fig.3], this injection direction makes it possible to create a vortex of the stiffening solution in the collection tank 15, directed towards the draw-off outlet 151.

[0100] More precisely, the injection inlet 152 is formed by a nozzle 152, shown in enlargement at the bottom right of [Fig. 3]. This nozzle comprises an inlet port 152a located outside the collection tank 15, intended to receive the stiffening solution which must be injected, and an outlet port 152b opening into the collection tank.

[0101] The nozzle 152 has a reduction in its internal section from the inlet port 152a to the outlet port 152b, formed by a projection of the internal section. This reduction in section makes it possible to increase the speed of the stiffening solution during its injection into the collection tank 15 and thus to improve the vortex formed by the injection in the direction tangential to the wall of the collection tank.

[0102] The bottom wall 153b has a shape of symmetry of revolution, conical, around an axis passing through the draw-off outlet 151, which is located at the center of the bottom wall 153b. Furthermore, the bottom wall 153n is smooth and has a substantially constant slope from its periphery towards the draw-off outlet 151. The draw-off outlet 151, shown in enlargement at the bottom right of [Fig.4] is intended to be connected to a collection circuit.

[0103] It may be provided that the lower part 153 of the collection tank 15 is arranged so that the stiffening solution which it contains is electrically connected to ground.

[0104] [Fig.5] represents a schematic view of an in- production system incorporating the encapsulation system.

[0105] In the example described, the assembly shown in [Fig.l], namely the containers 11 and 12, the distributors 14, the encapsulation device 13 and the collection tank 15 forms a first stage 1 for generating the cellular microcompartments of the production system. In [Fig.5], only the collection tank 15 has been shown.

[0106] Referring again to [Fig.l], the production system comprises 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.

[0107] The second buffer stage 2 comprises a collection circuit 21 connected to the withdrawal outlet 151 of the collection tank 15, as well as a pump 23 for withdrawing the stiffening solution and the microcompartments from the collection tank 15 to the collection circuit 21.

[0108] A first separation module 22 is positioned in the collection circuit 21 in order to receive the solution collected by the collection circuit 21.

[0109] The separation module 22 comprises, for example, a filter provided with 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.

[0110] 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 compartment and of preventing the passage of solid objects from one compartment to the other compartment.

[0111] 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.

[0112] It will be noted that the second buffer stage 2 also comprises 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.

[0113] The pumps 23 and 25 together form means for withdrawing and injecting the stiffening solution into the collection circuit 21, these withdrawal and injection means being reversible in order to move the stiffening solution in one direction or another in the collection circuit 21. These may in particular be peristaltic pumps, or any other mechanism suitable for causing a movement of a fluid in a circuit.

[0114] The third harvesting stage 3 comprises a harvesting circuit 32 connected on the one hand to the collection circuit 21, between the withdrawal outlet 151 and the first separation module 22, and on the other hand to different 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 stage connecting it to the second stage 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.

[0115] In the example described, the second solution and the third solution may be identical and each comprise a cell culture medium, and possibly a buffer solution and a ROCK inhibitor.

[0116] The third harvesting stage 3 also comprises a pump 33, forming displacement means, for circulating the different solutions of the production system in the harvesting circuit 32, according to the configurations mentioned above.

[0117] A second separation module 31, substantially identical to the first module 23, is positioned in the collection circuit 32. It may be provided 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 frontal filtration type system.

[0118] The collection circuit 21 comprises a valve 26 capable of authorizing 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.

[0119] In order to be able to carry out a production cycle of a batch of cellular microcompartments, the system comprises a control unit 4 capable of controlling the pumps 23, 25 and 33 and the valves 26, 32a, 32b, 34a, 35a, 36a and 37a and the distributors 14.

[0120] The control unit 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 distributors 14 of the first stage 1, in order to implement phases of a production cycle of a batch of cellular microcompartments.

[0121] In the example described, the control unit 4 can receive data from sensors of the system, such as data from flow rate sensors of the solutions in the circuits from stages 1 to 3, images acquired by a camera and representing the jet coming from the encapsulation device 13, measurements of the weight of the collection tank 15 coming from one or more load cells. The control unit can thus execute one or more programs for controlling the execution of the production cycle capable of interpreting this data in real time, and adjusting the operational parameters of the system, such as the flow rate of the solutions coming from the containers 11 and 12, according to this interpretation, or even stopping the production cycle.

[0122] It may also be provided 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.

[0123] In the example described, all of the different elements represented in [Fig.l], including the control unit 4, form a single machine. In another example, the control unit 4 could be remote, which communicates with the other elements of the system via wireless or wired communication means.

[0124] We will now describe a phase of a production cycle of a batch of cellular microcompartments, implemented by the control unit 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.

[0125] In this production phase, the unit 4 controls the first stage 1 to trigger the generation of a batch of cellular 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.

[0126] The control unit 4 controls the pump 23 to draw off, via the draw-off outlet 151, the stiffening solution contained in the collection tank 15, as well as the cellular microcompartments suspended in this solution, into the collection circuit 21 in a first direction of movement.

[0127] Unit 4 keeps valve 26 closed, so as to cause movement of the stiffening solution and the cellular microcompartments only towards the first separation module 22.

[0128] 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.

[0129] The stiffening solution thus continues its journey via the reinjection loop of the collection circuit 21 to the injection inlet 152 of the collection tank 15, to be recirculated in the tank and again in the collection circuit 21 during the entirety of this production phase.

[0130] 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.

[0131] At the end of a given time, determined so that a given quantity of cellular microcompartments is 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 unit 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.

[0132] In this transfer phase, the unit 4 controls the pump 25 so that the stiffening solution contained in the replenishment container 24 is injected into the collection circuit 21. A portion of this solution joins the reinjection loop and the collection tank 15 via the injection inlet 152, while another portion joins the first separation module to be entered there via the second port 222.

[0133] Pump 23 is kept active to continue withdrawing the stiffening solution and the cellular microcompartments from the collection tank. Unit 4 also controls valve 26 to allow the transfer of the solution from the second stage 2 to the third stage 3.

[0134] 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 unit 4 thus controls the pumps 23 and 25 so that the withdrawal flow rate of the stiffening solution from the collection tank 15 is identical to the injection flow rate of the stiffening solution into the collection tank.

[0135] For these purposes, the first stage comprises means for measuring, directly or indirectly, the flow rate of withdrawal of the stiffening solution from the collection tank 15. In the example described, these measuring means comprise one or more load cells (not shown) via which the collection tank 15 is mounted on a support.

[0136] The measuring means thus make it possible to measure variations in the weight of the collection tank and therefore to deduce variations in the flow rate of solution withdrawn with respect to the flow rate of solution injected. The control unit 4 thus controls the injection flow rate of the stiffening solution into the collection tank 15 by the pump 25 to the flow rate of withdrawal from the collection tank by the pump 23, so that the variations in weight are substantially zero. In other words, the injection rate of the stiffening solution from the replenishment container 24 is adjusted to compensate for the withdrawal of this solution from the collection tank 15.

[0137] It may be conceived that the measuring means comprise, alternatively or cumulatively, means for measuring the flow rate of stiffening solution drawn off at the draw-off outlet, such as a flow meter.

[0138] From then on, 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.

[0139] Simultaneously, the part of the stiffening solution coming 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, taking with it the cellular microcompartments retained in this first compartment during the previous production phase.

[0140] 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 harvesting circuit 32 of the third stage 3 through the valve 26.

[0141] Unit 4 controls valves 32a, 32b, 34a, 35a, 36a and 37a so that the harvesting circuit has the first configuration in which the stiffening solution can flow only to the waste container 37. In the example described, all the valves are closed, with the exception of valve 37a connected to the waste container 37.

[0142] Unit 4 controls pump 33 so that the stiffening solution and the cellular microcompartments suspended in this solution, coming from the collection tank 15 and the first separation module 22, circulate in the collection circuit 32 towards the second separation module 31.

[0143] 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.

[0144] The stiffening solution thus continues its journey in the collection circuit 32 towards the waste container 37.

[0145] At the end of this transfer phase, the unit 4 can again close the valve 26, so as to isolate the third stage 3 from the second stage 2 and control the pumps 23 and 25 in a similar manner to the previous production phase so that microcoms cellular compartments, generated by the first stage 1 and collected in the collection tank 15, are again stored in the first separation module 22.

[0146] Simultaneously, the unit 4 can control the different elements of the third stage to implement rinsing sub-phases, using the rinsing solution stored in the container 36, which can be circulated to the second separation module 31 then to the waste container 37 to rinse the micro-compartments which are stored in this module 31, and harvesting sub-phases, using the harvesting solution stored in the container 34, which can be circulated to the second separation module 32 then to the harvesting container 35 to harvest the micro-compartments which are stored in this module 31.

[0147] It is thus understood that the rinsing and harvesting sub-phases thus make it possible to carry out a change of medium for the cellular microcompartments, while cellular microcompartments continue to be generated by the first stage E1 and to be temporarily stored in the first separation module 22.

[0148] These production, transfer, rinsing and harvesting and collection phases may be iterated several times to carry out a production cycle of a batch of cellular microcompartments.

[0149] It may be provided that the production cycle includes initial phases preceding the first production phase, such as priming phases of the collection and harvesting circuits 21 and 32 allowing the different solutions to circulate in these circuits, and final rinsing and final harvesting sub-phases.

[0150] The foregoing description clearly explains how the invention makes it possible to achieve the objectives it has set itself, namely to propose a system for encapsulating cells in cellular microcompartments, which makes it possible to quickly collect the microcompartments from a collection tank, once their outer layer has been stiffened, while ensuring that the flow rate of solution and microcompartments withdrawn from the collection tank is substantially stable, preventing the microcompartments from obstructing the withdrawal outlet or accumulating in the collection tank and ensuring that the immersion time of the microcompartments in the calcium solution is generally constant for the entire batch.It is understood that these objectives are achieved by means of a particular design of the collection tank, which notably integrates an injection inlet for a stiffening solution in an injection direction substantially tangential to a wall of the collection tank, which then makes it possible to create a withdrawal of the solution and cellular microcompartments in the form of a vortex.

[0151] 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 effective combination of these means.

Claims

Claims

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 capable of gelling, b. an encapsulation device (13) connected to the containers and arranged to form cellular microcompartments whose outer layer is the solution capable of gelling and the core the cell solution, c. a collection tank (15) containing a stiffening solution and arranged to collect the cellular microcompartments formed by the encapsulation device; d.a collection circuit (21) for the cellular microcompartments collected by the collection tank; characterized in that the collection tank comprises a withdrawal outlet (151) for the stiffening solution and the cellular microcompartments from the collection tank to the collection circuit; and an injection inlet (152) for the stiffening solution into the collection tank, said injection inlet being arranged so that the direction of injection of the stiffening solution into the tank is substantially tangential to a wall (153a, 153b) of the collection tank.

2. Encapsulation system according to the preceding claim, the system comprising means for withdrawing and injecting (23, 25) the stiffening solution from and to the collection tank (15) and a control unit (4) arranged to control said means for withdrawing and injecting so that the injection of the stiffening solution into the collection tank is simultaneous with the withdrawal of the stiffening solution from the collection tank.

3. Encapsulation system according to the preceding claim, characterized in that the collection circuit (21) comprises a reinjection loop connecting the withdrawal outlet (151) to the injection inlet (152), said withdrawal and injection means (23, 25) being capable of causing a movement of the stiffening solution in the reinjection loop and in which the control unit (4) is arranged to control said withdrawal and injection means for recirculating the stiffening solution withdrawn via the withdrawal outlet to the injection inlet.

4. Encapsulation system according to the preceding claim, characterized in that the reinjection loop comprises a separation module (22) arranged to separate and retain the cellular microcompartments from the stiffening solution withdrawn via the withdrawal outlet (151).

5. Encapsulation system according to one of claims 3 or 4, characterized in that the collection circuit comprises a replenishment container (24) containing a stiffening solution and connected to the reinjection loop to inject said stiffening solution therein, and in which the control unit (4) is arranged to control said withdrawal and injection means (23, 25) to direct the stiffening solution contained in the replenishment container towards the injection inlet (152) via the reinjection loop.

6. Encapsulation system according to one of claims 2 to 5, wherein the control unit (4) is arranged to control said withdrawal and injection means (23, 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.

7. Encapsulation system according to the preceding claim, characterized in that it comprises means for measuring, directly or indirectly, the flow rate of withdrawal of the stiffening solution from the collection tank (15) and in that the control unit (4) is arranged to control the flow rate of injection of the stiffening solution into the collection tank by said withdrawal and injection means (23, 25) to the withdrawal flow rate measured by said measuring means.

8. Encapsulation system according to the preceding claim, characterized in that said measuring means comprise means for weighing the collection tank (15).

9. Encapsulation system according to one of the preceding claims, characterized in that the collection tank (15) comprises a bottom wall (153b) in the shape of a funnel, the outlet orifice of which forms the withdrawal outlet (151) of the collection tank.

10. Encapsulation system according to the preceding claim, characterized in that the injection inlet (152) of the stiffening solution into the collection tank (15) is positioned above the bottom wall (153b), said injection inlet (152) being arranged so that the direction of injection of the stiffening solution into the tank is substantially tangential to a side wall (153a) of the collection tank.

11. Encapsulation system according to one of the preceding claims, in which the injection inlet (152) of the stiffening solution into the collection tank (15) is formed by a nozzle (152) comprising an inlet port (152a) located outside the collection tank and an outlet port (152b) opening into the collection tank, the nozzle having a reduction in its internal section from the inlet port to the outlet port.

12. Encapsulation system according to one of the preceding claims, characterized in that the collection tank (15) is formed by a lower part (153) intended to receive the stiffening solution and on which the withdrawal outlet (151) and the injection inlet (152) are provided and by an upper part (154) forming a cover on which the encapsulation device (13) is mounted.

13. Encapsulation system according to one of the preceding claims, characterized in that the collection tank (15) is arranged so that the stiffening solution it contains is electrically connected to ground.

14. Encapsulation system according to one of the preceding claims, characterized in that the stiffening solution comprises a surfactant and a calcium salt.

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