System for producing objects in a solution with a change of environment

Abstract

The invention relates to a system for producing objects, comprising: a first stage (1) for generating objects; a second buffer stage (2) provided with a first separation module (22); a third harvesting stage (3) comprising a second separation module (31); a valve (26) capable of preventing the passage of a fluid from the second stage to the third stage and a control unit (4) arranged to control the elements of the system so that the collection of objects from the first stage to the separation module of the second stage is continuous and so that the harvesting of objects separated by the second separation module of the third stage is discontinuous. Figure to be published with the abstract: Fig. 1

Description

Title of the invention: System for producing objects in a solution with a change of medium

[0001] The invention relates to the field of production of objects involving a chemical and / or biotechnological process and requiring a change of production and / or culture medium, and in particular the field of encapsulation of cells in three-dimensional cell culture compartments.

[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 remaining too long in this calcium solution, which would be likely to degrade them.

[0007] However, it is not desirable to interrupt the injection of solutions into the encapsulation device, in particular in order to maintain a constant production flow to be able to produce a large quantity of microcompartments and to avoid having to implement injection re-initiation strategies during the production of a batch.

[0008] There is thus a need for a system for producing three-dimensional cell culture compartments which makes it possible to avoid stopping the production of a batch of compartments in order to change the compartment medium.

[0009] This need is more generally present in other fields than that of cell encapsulation, such as the production of pharmaceutical products such as granules, electronic components, etc., and can thus be generalized to the production of objects suspended in a solution and requiring a change of production and / or culture medium in a short time.

[0010] There is thus a need for a system for producing objects suspended in a solution, requiring a change of production and / or culture medium for the objects in a substantially short time with regard to their production, and which makes it possible to avoid stopping the production of a batch of objects to carry out this change of medium.

[0011] The present invention is placed in this context and aims to meet this need.

[0012] For these purposes, the invention relates to a system for producing objects, the system comprising at least: a. A first object generation stage comprising a collection tank containing a first solution intended to collect objects generated by said first stage; b. A second buffer stage comprising a collection circuit connected to the collection tank to receive the first solution and the objects immersed in this first solution; a first separation module capable of receiving the first solution collected by the collection circuit and arranged to separate and retain the objects from the first solution; and first means for moving the first solution in the collection circuit; c. A third harvesting stage comprising a second separation module capable of receiving the first solution collected by the harvesting circuit and arranged to separate and retain objects from the first solution; a harvesting circuit connected to the second separation module, to a container containing a second solution and to a harvesting container; and second means for moving the second solution in the harvesting circuit;

[0013] The system is characterized in that the collection circuit comprises a first valve capable of preventing the passage of the first solution from the collection circuit to the second separation module and a control unit arranged to control the first and second movement means and the first valve so that the collection of the first solution and the objects immersed in this first solution from the collection tank is continuous and so that the harvesting of the second solution and the objects separated by the second separation module to the harvesting container is discontinuous.

[0014] It is thus understood that the invention proposes to interpose a buffer stage between the object generation stage and the harvesting stage. The valve then makes it possible to isolate the buffer stage from the harvesting stage or, on the contrary, to authorize a transfer from the buffer stage to the harvesting stage. Depending on the state of this valve, the first movement means make it possible to transfer the objects from the collection tank and the first solution to the first separation module and, conversely, to transfer the objects retained by the first separation module to the harvesting stage.

[0015] It may be conceived that the first solution is used for the transfer of objects from the tank to the first separation module and for the transfer of objects from the first separation module to the collection stage, or that separate solutions may be used for each of these transfers. Similarly, it may be envisaged that the objects 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.

[0016] The second movement means make it possible to transfer the second solution contained in the container to the second separation module, then to transfer this second solution and the objects retained by the second separation module to the collection container.

[0017] The different elements of the system can thus be controlled by a control unit to define different phases during the production of a batch of objects. may in particular provide 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 routes according to given phases of a production cycle of a batch of object.

[0018] For example, in a collection phase, the valve is closed and the buffer stage can recover, via its separation module, objects continuously generated by the generation stage while, simultaneously, the objects 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 objects 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 first solution or another solution.

[0019] In other words, the invention therefore makes it possible to ensure discontinuous filtration of the objects, thanks to which it is possible to ensure continuity in the production of the objects by the generation stage while allowing, in a discontinuous manner, a change of medium of the objects thus produced.

[0020] In the present invention, the term "displacement means" means one or more mechanisms capable of causing a movement of a fluid in the collection circuit, respectively the harvesting circuit, in one direction and / or the other. By way of non-limiting example, the first and second displacement means may comprise pumps, in particular peristaltic pumps, or any other mechanism suitable for causing a movement of a fluid.

[0021] In the present invention, the term "valve" means one or more mechanical or electromechanical devices capable of authorizing, prohibiting, regulating or controlling the passage of a fluid in a circuit, in particular in a circuit connecting the second stage to the third stage and making it possible to transfer objects from this second stage to the third stage. By way of non-limiting example, the first valve may be a hydraulic, electric, pneumatic, ball, guillotine or membrane type valve.

[0022] 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.

[0023] In the present invention, the term "control unit" means a device or computer system designed to manage, regulate and supervise the operations and processes of the production system, by controlling and coordinating the actions of the different controllable elements of this system, such as the means of movement and the valves. The control unit may be equipped with one or more processors, or even one or more microcontrollers, designed to execute one or more computer programs to implement phases of a production cycle for a batch of objects. In particular, the control unit may be capable of receiving sensor data, interpreting this data in real time, and making automated decisions to adjust the operational parameters of the system. The control unit may also include a user interface allowing an operator to monitor production and, if necessary, intervene in this production.It may also be provided that the control unit is embedded in a machine comprising the first, second and third stages of the production system or, alternatively, that the control unit is remote from this machine while being connected to it by means of connection, wired or wireless.

[0024] In one embodiment of the invention, the control unit is arranged to control the first and second displacement means and the first valve so that: a. in a first collection and harvesting phase, the first solution contained in the collection tank and objects immersed in this first solution are transferred, via the collection circuit, to the first separation module and, simultaneously, the second solution contained in the container is transferred, via the collection circuit, to the second separation module and then transferred with said objects retained by the second separation module to the harvesting container; said first valve being closed to prevent the passage of the first solution from the collection circuit to the second separation module; b. in a second transfer phase, the first solution contained in the collection tank and objects immersed in this first solution are transferred, via the collection circuit, to the second separation module and, simultaneously, a portion of the first solution circulating in the collection circuit is transferred, via the collection circuit to the first separation module and then transferred with said objects retained by the first separation module to the second separation module; said first valve being open to allow the passage of the first solution from the collection circuit to the second separation module and the harvesting circuit.

[0025] In other words, the harvesting of the objects to the harvesting connector is carried out only during the harvesting phase, thus making it possible to carry out a change of medium during this phase. This change of medium can be carried out in one go, during the harvesting itself, or progressively, by carrying out one or more operations of rinsing the objects using different solutions. Simul Meanwhile, objects continue to be generated by the first generation stage and are temporarily stored in the first separation module. After a given time, long enough to complete the collection of the objects retained in the second separation module and short enough to prevent the objects retained in the first separation module from remaining suspended in the first solution for too long, the control unit opens the valve and controls the various elements of the collection and harvesting circuits to carry out a transfer phase.

[0026] During the transfer phase, the first solution circulates from the collection circuit to the harvesting circuit, transporting both the objects stored in the first separation module and the objects that the generation stage continues to produce to the second separation module.

[0027] Advantageously, the control unit is arranged to control the first and second displacement means and the first valve so that the first and second phases are iterated at least twice each, one after the other. In other words, the collection and transfer phases are repeated several times during a production cycle of a batch of objects.

[0028] It may be provided, where appropriate, that the control unit is arranged to control the first and second displacement means and the first valve, and where appropriate the elements of the collection and harvesting circuits, to implement initial phases preceding the first iteration of the transfer phase, and in particular at least one initial production phase in which only the first and second stages are in operation to store objects generated by the object generation stage in the first separation module and / or final phases following the last iteration of the transfer phase, and in particular a final harvesting phase in which only the third stage is in operation to collect, in the harvesting container, the objects stored in the second separation module.

[0029] According to an exemplary embodiment, the control unit is arranged to control the first and second movement means and the first valve so that: a. in the first collection and harvesting phase, the first solution contained in the collection tank and objects immersed in this first solution are transferred, via the collection circuit, to the first separation module in a first direction of movement in the collection circuit; b. in the second transfer phase, said portion of the first solution circulating in the collection circuit is transferred, via the collection circuit, to the first separation module, in a second direction of movement in the collection circuit opposite to the first direction of movement.

[0030] In the first phase of collection and harvesting, the submerged objects, or in suspension, in the first solution are thus retained by the first separation module, until the following transfer phase, in which these objects are transferred, in the opposite direction, with the first solution, to the second separation module, passing through the first valve.

[0031] In this embodiment, it may be provided that the first movement means are reversible and that the control unit is arranged to control the first movement means so that the transfer, in the collection phase, of the objects and the first solution from the collection tank to the first separation module and that the transfer, in the transfer phase, of the objects retained by the first separation module to the first valve are carried out at least in a common part of the collection circuit, in opposite directions.

[0032] In another embodiment, it may be provided that the collection circuit comprises valves and / or membrane filters controllable by the control unit and that the control unit is arranged to control these valves and / or these controllable membrane filters to configure the collection circuit so that it comprises, in the collection phase, a single sub-circuit from the collection tank to the first separation module and, in the transfer phase, two sub-circuits, one from the collection tank to the first valve and the other from the first separation module to the first valve.

[0033] Advantageously, the control unit is arranged to control the first and second movement means and the first valve so that: a. in the first collection and harvesting phase, the second solution contained in the container is transferred, via the harvesting circuit, to the second separation module, in a first direction of movement in the harvesting circuit; b. in the second transfer phase, the first solution circulating in the harvesting circuit is transferred, via the harvesting circuit, to the second separation module, in a second direction of movement in the harvesting circuit opposite to the first direction of movement.

[0034] In the first collection and harvesting phase, the second solution thus transports the objects retained by the second separation module to the harvesting container, while, in the second transfer phase, the objects immersed, or suspended, in the first solution are transported from the second stage in the opposite direction, by the first solution, to this second separation module. It may be provided that the third stage comprises a waste container connected to the harvesting circuit to receive the first solution after its passage through the second separation module.

[0035] According to an exemplary embodiment of the invention, each of the first and second separation modules comprise a filter provided with a first port and a second port each connected to the collection circuit, respectively to the harvesting circuit, and a filter membrane extending in 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 a solution transporting objects and penetrating 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 objects are retained in the first compartment, against the filter membrane.Conversely, a solution entering through the second port can then pass 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 objects retained in this first compartment.

[0036] Advantageously, each port can open into the filter in a direction 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 a plane of extension of the filter membrane, in particular in a direction tangential to said plane of extension.

[0037] 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 objects stuck in the module during the different phases.

[0038] 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.

[0039] According to another exemplary embodiment of the invention, each of the first and second separation modules comprises a counter-current centrifugation type filter. This type of filter makes it possible to trap objects immersed, or suspended, in a solution against a filter medium by a centrifugal effect, the solution circulating counter-currently in a rotating system.

[0040] According to yet another exemplary embodiment of the invention, each of the first and second separation modules comprises a frontal filtration type system.

[0041] In one embodiment of the invention, the collection tank comprises an outlet for drawing off the first solution and objects immersed in this first solution from the collection tank to the collection circuit; and an inlet for injecting the first solution into the collection tank. Where appropriate, the collection circuit comprises a reinjection loop connecting the drawing off outlet to the injection inlet and the control unit is arranged to control the first means for moving the first solution in the collection circuit to cause a transfer, via the reinjection loop, of the first solution drawn off via the drawing off outlet to the injection inlet. Preferably, the reinjection loop comprises a part of the collection circuit connecting the drawing off outlet to the first separation module and a part of the collection circuit connecting the first separation module to the injection inlet.

[0042] In this embodiment, the first solution withdrawn from the collection tank to transport the objects produced by the first stage is recirculated, via the reinjection loop, after passing through the first separation module, to the collection tank. It may be provided that the control unit is arranged to cause the transfer, via the reinjection loop, of the first solution withdrawn via the withdrawal outlet to the injection inlet only during the first collection and harvesting phase.

[0043] Advantageously, the second buffer stage comprises a replenishment container containing a given quantity of the first solution and connected to the reinjection loop of the collection circuit. Where appropriate, the first movement means are capable of causing a movement of the first solution contained in the replenishment container towards the reinjection loop and the control unit is arranged to control, during the first collection and harvesting phase, the first movement means to cause a transfer, via the reinjection loop, of the first solution contained in the replenishment container towards the injection inlet.It is thus possible to replenish the collection tank, during a collection and harvesting phase, with a given quantity of clean first solution, in particular in order to compensate for the loss of first solution due to the transfer of objects to the third stage during the second previous transfer phase.

[0044] In one embodiment of the invention, the third harvesting stage comprises a container containing a third rinsing solution and a waste container, each connected to the harvesting circuit. Where appropriate, the harvesting circuit comprises at least one second valve capable of defining a rinsing sub-circuit connecting the container containing the rinsing solution to the waste container via the second separation module and a harvesting sub-circuit containing connecting the container containing the second solution to the collection container via the second separation module. These different elements thus make it possible to implement, during the collection and harvesting phases, one or more sub-phases of rinsing the objects retained by the second separation module in order to harvest them, in particular to avoid weakening them when they are harvested using the second solution. It may be provided that the rinsing solution is identical to the second solution or that it is distinct from the second solution.

[0045] Advantageously, the control unit is arranged to control the first and second movement means and the second valve during the first collection and harvesting phase, so that: a. In a first rinsing sub-phase, the rinsing solution contained in the container is transferred, via the rinsing sub-circuit, to the second separation module, then to the waste container; b. In a second harvesting sub-phase, the second solution contained in the container is transferred, via the harvesting sub-circuit, to the second separation module and then transferred with said objects retained by the second separation module to the harvesting container.

[0046] In this example, the control unit therefore controls the second valve so that it alternately adopts, during the rinsing sub-phase and during the harvesting sub-phase, one or other of the configurations corresponding to the rinsing and harvesting sub-circuits. Preferably, the rinsing solution circulates in the harvesting circuit in the second direction of movement, so that the objects retained by the second separation module remain retained by the second separation module during the rinsing sub-phase.

[0047] It may be provided that the third stage comprises several containers each containing a rinsing solution and each connected to the harvesting circuit, the harvesting circuit comprising several valves capable of defining different rinsing sub-circuits each connecting one of these containers to the waste container via the second separation module. The control unit can thus control these valves to implement several successive rinsing sub-phases with each of the rinsing solutions before harvesting the objects retained in the second separation module.

[0048] In one embodiment, the first stage comprises: 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, in particular via one or more distributors, and arranged to form micro-compartments lular (MC) whose outer layer is the solution capable of gelling and the core the cell solution.

[0049] Where appropriate, the collection tank is arranged downstream of the encapsulation device to collect the cellular microcompartments formed by the encapsulation device and the first solution is arranged to cause stiffening of the outer layer of each cellular microcompartment during its immersion in this first solution, said cellular microcompartments whose outer layer is stiffened forming said objects

[0050] If desired, it may be provided that the first stage comprises a third container connected to the encapsulation device and 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 crosslinking of the hydrogel too early in the first stage, such as for example a sorbitol solution.

[0051] 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 an extracellular matrix and / or an extracellular matrix substitute. Where appropriate, the first stage will be arranged to form, in the collection tank, cellular microcompartments, the outer layer of which 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 of 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. specifically 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.

[0052] 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% of G and less than 40%, or even less than 20% of M, with an average molecular mass of 100 to 400 KDa and a total concentration of between 0.5% and 5% by mass.

[0053] In the microcompartments obtained by means of the first stage, 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.

[0054] According to a variant, at least one cellular microcompartment obtained by means of the first stage 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 increase the amplification factor of the culture. Consequently this . reduces the number of passages and dissociations required; reduces the culture time required to reach the final cell number required.

[0055] The cellular microcompartments obtained by means of the first stage preferably comprise one or more cysts, and / or one or more tissues and / or microtissues and / or cell aggregates with or without lumen(s).

[0056] Advantageously, the first stage is arranged so that each cellular microcompartment obtained by means of this first stage is closed. In one embodiment, the first stage 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.

[0057] In another embodiment, the first stage may be arranged so that each cellular microcompartment obtained by means of this first stage has an elongated shape, in particular an ovoid or tubular shape.

[0058] Advantageously, each container of the first stage may be a flexible bag, a syringe or a tube with a conical bottom. Advantageously still, 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.

[0059] In one embodiment, the encapsulation device may be a microfluidic type device capable of generating a concentric jet comprising the cell solution in the center, 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 first 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 makes it possible to improve 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 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.

[0060] In the case of electro-jetting, it may be possible to add a device for generating an electric field, such as a metal ring placed 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 device may be connected to an electric potential, for example to ground. This electric field helps in particular to promote the dispersion of the cellular microcompartments.

[0061] In one embodiment of the invention, the encapsulation device comprises a body arranged to form a concentric flow from the solutions supplied by the container(s) of the first stage, 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 splits into cellular microcompartments. The encapsulation device is thus of the “electro-jetting” type, the jet forming itself splits, 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 distributors.

[0062] 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.

[0063] 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.

[0064] 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 two parts: dividing into portions extending around the first channel, said subdivisions of the second channel joining at the single outlet into 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.

[0065] In one embodiment of the invention, the first solution comprises a surfactant and a calcium salt and the second solution comprises a cell culture medium. It may be provided that the second solution further comprises a buffer solution and a ROCK inhibitor.

[0066] The invention also relates to a method for producing objects implemented by means of a system according to the invention.

[0067] 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:

[0068] [Fig-1] represents, schematically and partially, a view of a system of production of objects according to an embodiment of the invention;

[0069] [Fig.2] represents, schematically and partially, a sectional view of the first stage of the object production system of [Fig.l];

[0070] [Fig.3] represents, schematically and partially, perspective and sectional views of the separation modules of the object production system of [Fig.l];

[0071] [Fig.4] represents, schematically and partially, a production cycle of objects implemented by the system of [Fig.l];

[0072] [Fig.5A] represents, schematically and partially, a view of the system of [Fig.l] in a first phase of a production cycle of a batch of objects;

[0073] [Fig.5B] represents, schematically and partially, a view of the system of [Fig.l] in a first phase of a production cycle of a batch of objects;

[0074] [Fig.5C] represents, schematically and partially, a view of the system of [Fig.l] in a first phase of a production cycle of a batch of objects;

[0075] [Fig.5D] represents, schematically and partially, a view of the system of [Fig.l] in a first phase of a production cycle of a batch of objects;

[0076] [Fig.5E] represents, schematically and partially, a view of the system of [Fig.l] in a first phase of a production cycle of a batch of objects;

[0077] [Fig.5F] represents, schematically and partially, a view of the system of [Fig.l] in a first phase of a production cycle of a batch of objects;

[0078] In the following description, elements that are identical, by structure or by function, appearing in different figures retain, unless otherwise specified, the same references.

[0079] [Fig.l] shows an object production system. In this exemplary embodiment, the object production system is a micro production system cellular compartments. It should be noted, however, that the principles and embodiments illustrated can be adapted and applied to other technical fields, and that the use of these figures is not limited to cell encapsulation.

[0080] The system comprises a first stage 1 for generating cellular microcompartments, which will be described with reference to [Fig.2].

[0081] The first stage 1 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. Provision may be made for the first stage 1 to comprise a third container comprising an intermediate solution, for example comprising an isotonic solution such as sorbitol.

[0082] The first stage 1 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.

[0083] For these purposes, 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 displacement 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 towards the inlet of the device 13 to which it is connected.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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 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.

[0090] 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.

[0091] In the case of an “electro-jetting” type device, it may be possible to add a metal ring connected to ground downstream of the outlet of the encapsulation device so that the jet or the cellular microcompartments pass through this ring. The electric field generated by this metal ring may make it possible to promote the dispersion of the cellular microcompartments, in the case of an “electro-jetting” type device.

[0092] The first stage 1 also 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.

[0093] The first solution comprises a surfactant and a calcium salt allowing the crosslinking of an alginate solution, and thus allowing the stiffening of the outer layer of each cellular microcompartment during its immersion in the collection tank.

[0094] Each cellular microcompartment obtained by means of this first stage 1 is thus closed and presents, in the example described, a spherical or elongated drop shape.

[0095] 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.

[0096] It will be noted that the collection tank 15 comprises a withdrawal outlet 151 for the first solution and microcompartments immersed in this first solution as well as an injection inlet 152 for the first solution into the collection tank 15.

[0097] 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.

[0098] 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 first solution and the microcompartments from the collection tank 15 to the collection circuit 21.

[0099] A first separation module 22 is positioned in the collection circuit 21 in order to receive the first solution collected by the collection circuit 21. The first separation module 22, which will be described later, can thus separate and retain the objects from the first solution.

[0100] 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.

[0101] It will be noted that the second buffer stage 2 also comprises a replenishment container 24 containing a given quantity of the first solution. This container 24 is connected to the reinjection loop of the collection circuit 21, via a pump 25.

[0102] The pumps 23 and 25 together form means for moving the first solution in the collection circuit 21, these moving means being reversible in order to move the first solution in one direction or another in the collection circuit. collection 21. This may include peristaltic pumps, or any other mechanism suitable for causing a fluid to move in a circuit.

[0103] 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.Various valves 32a, 32b, 34a, 35a, 36a, and 37a are positioned in the harvest circuit to define various configurations of the harvest 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 rinse solution can flow from the container 36 to the waste container 37, and a third configuration in which the second harvest solution can flow from the container 34 to the harvest container 35.

[0104] 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.

[0105] The third harvesting stage 3 also comprises a pump 33, forming second displacement means, for circulating the different solutions of the production system in the harvesting circuit 32, according to the configurations mentioned above.

[0106] A second separation module 31, substantially identical to the first module 22, is positioned in the harvesting circuit 32.

[0107] With reference to [Fig. 3], an exemplary embodiment of the separation module 22 will now be described, it being understood that the separation module 31 has substantially identical functions and structures. It will also be possible to provide for the structures of the separation modules 22 and 31 to be different from each other, and to use other types of filter than the one which will be described, such as a counter-current centrifugation type filter or a frontal filtration type system.

[0108] [Fig. 3] represents, on the left, a perspective view of the separation module 22 and, on the right, a sectional view of the separation module 22.

[0109] The separation module 22 comprises a filter 223 provided with a first port 221 and a second port 222 each connected to the collection circuit 21. More precisely, the first port 221 may be connected to the portion of the collection circuit 21 connected to the withdrawal outlet 151 of the collection tank 15 and the second port 222 may be connected to the portion of the collection circuit 21 connected to the injection inlet 152 of the collection tank 15.

[0110] A filter membrane 224 extends into the filter 223 to define two compartments, namely a first compartment 221a into which the first port 221 opens and a second compartment 222a into which the second port 222 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. It is thus understood that a solution transporting objects and penetrating through the first port 221 of the filter 223 can pass through the first compartment 221a, the filter membrane 224, and the second compartment 222a to exit through the second port 222, while the objects are retained in the first compartment 221a, against the filter membrane 224.Conversely, a solution entering through the second port 222 can then pass in the opposite direction through the second compartment 222a, the filter membrane 224 and the first compartment 221a to exit through the first port 221, taking with it the objects retained in this first compartment 221a.

[0111] As visible in [Fig.3], each port 221, 222 opens into the compartment 221a, 222a of the filter 223 in a direction parallel to an extension plane of the filter membrane 224.

[0112] It will be noted that the filter 223 comprises an inner envelope with edges having a toric shape and that the useful section of the filter membrane 224 has the shape of a circle or an ellipse.

[0113] Referring again to [Fig.l], 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.

[0114] 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.

[0115] 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.

[0116] 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 of 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 these real-time data, and to adjust the operational parameters of the system, such as the flow rate of solutions from containers 11 and 12, based on this interpretation, or even to stop the production cycle.

[0117] 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.

[0118] 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.

[0119] With reference to [Fig.4] and to [Fig.5A] to [Fig.5F], we will now describe different phases of a production cycle of a batch of cellular microcompartments, implemented by the control unit 4. [Fig.4] represents a succession of phases forming a production cycle, while each of [Fig.5A] to [Fig.5F] represents the state of the system during these different phases.

[0120] [Fig.5A] represents an initial production phase E1 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.

[0121] It may be provided that the production cycle includes initial phases preceding the initial production phase E1, such as priming phases of the collection and harvesting circuits 21 and 32 allowing the different solutions to circulate in these circuits.

[0122] In this initial production phase E1, 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 first solution in order to cause stiffening of its outer layer.

[0123] The control unit 4 controls the pump 23 to draw off, via the draw-off outlet 151, the first solution contained in the collection tank 15, as well as the cellular microcompartments suspended in this first solution, into the collection circuit 21 in a first direction of movement, identified by arrows on the circuit 21 in [Fig.5A].

[0124] Unit 4 keeps valve 26 closed, so as to cause movement of the first solution and the cellular microcompartments only towards the first separation module 22.

[0125] The first solution and the cellular microcompartments penetrate through the first port 221 of the filter 223 of this first module 22. The first solution passes through the first compartment 221a, the filter membrane 224, and the second compartment 222a to exit through the second port 222, while the cellular microcompartments are retained in the first compartment 221a, against the filter membrane 224.

[0126] The first 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 thus be recirculated in the tank and again in the collection circuit 21 during the entirety of this initial production phase.

[0127] In the example described, the pump 25 is inactive during the initial production phase E1, since the collection circuit 21 forms a closed circuit and there is therefore no need to replenish the collection tank 15 with the first solution. Alternatively, it could be conceived that the control unit activates this pump 25 during the initial production phase to supplement the collection tank 15 with a quantity of first solution from the replenishment container 24.

[0128] In this initial production phase El, the pump 33 is also inactive.

[0129] 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 first solution is less than a given time, the unit 4 controls the different elements of the system to implement a first iteration of a transfer phase E2. [Fig.5B] represents this transfer phase E2 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.

[0130] In this transfer phase, the unit 4 controls the pump 25 so that the first 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.

[0131] Pump 23 is kept active to continue withdrawing the first solution and the cellular microcompartments from the collection tank. Unit 4 also controls valve 26 to allow the transfer of the first solution from the second stage 2 to the third stage.

[0132] From then on, the first solution and the cellular microcompartments immersed in this first solution are transferred from the collection tank 15, via the collection circuit 21, to the third stage 3 through the valve 26.

[0133] Simultaneously, the part of the first solution from the replenishment container viewing 24 which enters through the second port 222 of the filter 223 of the first separation module 22 crosses in the opposite direction the second compartment 222a, the filter membrane 224 and the first compartment 221a to exit through the first port 221, taking with it the cellular microcompartments retained in this first compartment 221a during the initial production phase E1.

[0134] This first 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 first solution during the initial production phase. They thus join the first solution and the objects immersed in this first solution, transferred from the collection tank 15, the assembly passing into the harvesting circuit 32 of the third stage 3 through the valve 26.

[0135] Unit 4 controls valves 32a, 32b, 34a, 35a, 36a and 37a so that the harvesting circuit has the first configuration in which the first 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.

[0136] Unit 4 controls pump 33 so that the first solution and the cellular microcompartments suspended in this first 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.

[0137] The first solution and the cellular microcompartments enter through the first port 221 of the filter 223 of this second module 31. The first solution passes through the first compartment 221a, the filter membrane 224, and the second compartment 222a to exit through the second port 222, while the cellular microcompartments are retained in the first compartment 221a, against the filter membrane 224.

[0138] The first solution thus continues its journey in the collection circuit 32 towards the waste container 37.

[0139] At the end of a given time, determined so that the first separation module 22 can be emptied, the unit 4 controls the different elements of the system to implement a first iteration of a collection and harvesting phase E3.

[0140] In this collection and harvesting phase E3, the unit 4 closes the valve 26 again, so as to isolate the third stage 3 from the second stage 2 and controls the pumps 23 and 25 in a similar manner to the initial production phase E1 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.

[0141] Simultaneously, unit 4 controls the various elements of the system to implement a rinsing sub-phase E31, of this collection and harvesting phase E3. [Fig.5C] represents this rinsing sub-phase E31 in which the solution of Rinse water stored in the container 36 is circulated to the second separation module 31 to rinse the microcompartments stored there.

[0142] Unit 4 controls valves 32a, 32b, 34a, 35a, 36a and 37a so that the harvesting circuit 32 has the second configuration in which the third rinsing solution can flow into the harvesting circuit 32 from the container 36 to the waste container 37 via the second separation module, in a direction of movement identical to that of the first solution in the harvesting circuit 32. In the example described, all the valves are closed, with the exception of valve 36a connected to the container 36 and valve 32b.

[0143] Unit 4 controls pump 33 so that the third solution circulates from container 36 in harvesting circuit 32 to second separation module 31.

[0144] The third solution thus enters through the first port 221 of the filter 223 of this second module 31. The first solution passes through the first compartment 221a, the filter membrane 224, and the second compartment 222a to exit through the second port 222, rinsing the cellular microcompartments retained in the first compartment 221a, against the filter membrane 224.

[0145] The third solution finally continues its journey in the collection circuit 32 towards the waste container 37.

[0146] Although a single rinsing solution is used in the example described, it may be provided that the third stage 3 comprises several containers each containing a rinsing solution and each connected to the harvesting circuit 32, the harvesting circuit comprising several valves capable of defining different rinsing sub-circuits each connecting one of these containers to the waste container via the second separation module 31. The control unit 4 can thus control these valves to implement several successive rinsing sub-phases with each of the rinsing solutions before harvesting the microcompartments retained in the second separation module 31.

[0147] Following the rinsing sub-phase E31, the unit 4 controls the various elements of the system to implement a harvesting sub-phase E32 of this collection and harvesting phase E3. [Fig.5D] represents this harvesting sub-phase E32 in which the harvesting solution stored in the container 34 is circulated towards the second separation module 32 to harvest the microcompartments stored therein.

[0148] Unit 4 controls valves 32a, 32b, 34a, 35a, 36a and 37a so that the harvesting circuit has the third configuration in which the second harvesting solution can flow from container 34 to harvesting container 35 via the second separation module, in a direction of movement opposite to that of the first and third solutions in harvesting circuit 32. In the example described, only valves 34a, 35a, connected to containers 34 and 35, and valve 32a are open.

[0149] Unit 4 controls pump 33 so that the second solution circulates from container 34 in harvesting circuit 32 to second separation module 31, in this opposite direction of movement.

[0150] The third solution thus enters through the second port 222 of the filter 223 of the second separation module 31 and passes in the opposite direction through the second compartment 222a, the filter membrane 224 and the first compartment 221a to exit through the first port 221, taking with it the cellular microcompartments retained in this first compartment 221a during the transfer phase E2.

[0151] This third solution and these cellular microcompartments thus circulate in the harvesting circuit 32 in a direction of movement opposite to that of the first and third solutions in the harvesting circuit 32, until reaching the harvesting container 35.

[0152] It is thus understood that the rinsing and harvesting sub-phases E31 and E32 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.

[0153] At the end of a given time, long enough to complete the harvesting of the cellular microcompartments retained in the second separation module 31 and short enough to prevent the cellular microcompartments retained in the first separation module 22 from remaining too long in suspension in the first solution, the control unit 4 opens the valve 26 and controls the different elements of the system to operate a second iteration of the transfer phase E2.

[0154] It is thus understood that the control unit 4 repeats the transfer phases E2 and harvesting and collection E3 several times to carry out a production cycle of a batch of cellular microcompartments.

[0155] At the end of a given number of iterations of the transfer phases E2 and harvesting and collection E3, the unit 4 closes the valve 26 again, so as to isolate the third stage 3 from the second stage 2 and controls the valves 32a, 32b, 34a, 35a, 36a and 37a and the pump 33 in a similar manner to the rinsing sub-phases E31 and collection E32 so that the last series of cellular microcompartments stored in the second separation module 31 is rinsed and collected in the container 35.

[0156] [Fig.5E] represents the final rinsing sub-phase E41 in which the rinsing solution stored in the container 36 is circulated one last time to the second separation module 31 to rinse the microcompartments stored there.

[0157] [Fig.5F] represents the final harvest sub-phase E42 in which the solution of The harvest stored in the container 34 is circulated one last time to the second separation module 32 to collect the microcompartments stored there.

[0158] The preceding description clearly explains how the invention makes it possible to achieve the objectives it has set itself, namely to propose a system for producing objects suspended in a solution and in which it is possible to ensure continuity in the production of the objects while allowing, discontinuously, a change of medium for the objects thus produced. It is understood that these objectives are achieved using a buffer stage interposed between the object generation stage and the harvesting stage and a valve making it possible to isolate the buffer stage from the harvesting stage or, on the contrary, to allow a transfer from the buffer stage to the harvesting stage.

[0159] In any event, the invention cannot be limited to the embodiments specifically described in this document, and extends in particular to any equivalent means and to any technically effective combination of these means. It could be conceived that the same solution is used for the transfer of the objects from the tank to the first separation module and for the transfer of the objects from the first separation module to the harvesting stage, or that separate solutions could be used for each of these transfers. Similarly, it could be envisaged that the objects 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.

Claims

Claims

1. System for producing objects, the system comprising at least: a. A first stage (1) for generating objects comprising a collection tank (15) containing a first solution intended to collect objects generated by said first stage; b. A second buffer stage (2) comprising a collection circuit (21) connected to the collection tank to receive the first solution and the objects immersed in this first solution; a first separation module (22) capable of receiving the first solution collected by the collection circuit and arranged to separate and retain the objects from the first solution; and first means (23, 25) for moving the first solution in the collection circuit; c. A third harvesting stage (3) comprising a second separation module (31) capable of receiving the first solution collected by the harvesting circuit and arranged to separate and retain the objects from the first solution; a harvesting circuit (32) connected to the second separation module, to a container (34) containing a second solution and to a harvesting container (35); and second means (33) for moving the second solution in the harvesting circuit; characterized in that the collection circuit comprises a first valve (26) capable of preventing the passage of the first solution from the collection circuit to the second separation module and a control unit (4) arranged to control the first and second movement means and the first valve so that the collection of the first solution and of the objects immersed in this first solution from the collection tank is continuous and so that the harvesting of the second solution and of the objects separated by the second separation module to the harvesting container is discontinuous.

2. Production system according to the preceding claim, characterized in that the control unit (4) is arranged to control the first and second displacement means (23, 25, 33) and the first valve (26) so that: a. in a first phase of collection and harvesting (E3), the first solution contained in the collection tank (15) and objects immersed in this first solution are transferred, via the collection circuit (21), to the first separation module (22) and, simultaneously, the second solution contained in the container (34) is transferred, via the collection circuit (32), to the second separation module (31) then transferred with said objects retained by the second separation module to the collection container (35); said first valve (26) being closed to prevent the passage of the first solution from the collection circuit to the second separation module; b.in a second transfer phase (E2), the first solution contained in the collection tank (15) and objects immersed in this first solution are transferred, via the collection circuit (21), to the second separation module (31) and, simultaneously, a portion of the first solution circulating in the collection circuit (21) is transferred, via the collection circuit (21) to the first separation module (22) then transferred with said objects retained by the first separation module to the second separation module (31); said first valve (26) being open to allow the passage of the first solution from the collection circuit to the second separation module and the harvesting circuit.

3. Production system according to the preceding claim, characterized in that the control unit (4) is arranged to control the first and second displacement means (23, 25, 33) and the first valve (26) so that the first and second phases (E2, E3) are iterated at least twice each, one after the other.

4. Production system according to one of claims 2 to 3, characterized in that the control unit (4) is arranged to control the first and second displacement means (23, 25, 33) and the first valve (26) so that: a. in the first collection and harvesting phase (E3), the first solution contained in the collection tank (15) and objects immersed in this first solution are transferred, via the collection circuit (21), to the first se- separation (22) in a first direction of movement in the collection circuit; b. in the second transfer phase (E2), said part of the first solution circulating in the collection circuit (21) is transferred, via the collection circuit, to the first separation module (21), in a second direction of movement in the collection circuit opposite to the first direction of movement.

5. Production system according to one of claims 2 to 4, characterized in that the control unit (4) is arranged to control the first and second displacement means (23, 25, 33) and the first valve (26) so that: a. in the first collection and harvesting phase (E3), the second solution contained in the container (34) is transferred, via the harvesting circuit (32), to the second separation module (31), in a first direction of movement in the harvesting circuit; b. in the second transfer phase (E2), the first solution circulating in the harvesting circuit (32) is transferred, via the harvesting circuit, to the second separation module (31), in a second direction of movement in the harvesting circuit opposite to the first direction of movement.

6. Production system according to one of the preceding claims, characterized in that each of the first and second separation modules (22, 31) comprises a filter (223) provided with a first port (221) and a second port (222) each connected to the collection circuit (21), respectively to the harvesting circuit (32), and a filter membrane (224) extending in 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.

7. Production system according to one of the preceding claims, characterized in that the collection tank (15) comprises a withdrawal outlet (151) for the first solution and objects immersed in this first solution from the collection tank to the circuit of collection (21); and an injection inlet (152) for the first solution in the collection tank and in that the collection circuit comprises a reinjection loop connecting the withdrawal outlet to the injection inlet, the control unit (4) is arranged to control the first means of movement (23, 25) of the first solution in the collection circuit to cause a transfer, via the reinjection loop, of the first solution withdrawn via the withdrawal outlet to the injection inlet.

8. Production system according to one of the preceding claims, characterized in that the third harvesting stage (3) comprises a container (36) containing a third rinsing solution and a waste container (37), each connected to the harvesting circuit (32); characterized in that the harvesting circuit comprises at least one second valve (32a, 32b, 34a, 35a, 36a, 37a) capable of defining a rinsing sub-circuit connecting the container (36) containing the rinsing solution to the waste container (37) via the second separation module (31) and a harvesting sub-circuit containing connecting the container (34) containing the second solution to the harvesting container (35) via the second separation module (31).

9. Production system according to the preceding claim, characterized in that the control unit (4) is arranged to control the first and second moving means (23, 25, 33) and the second valve (32a, 32b, 34a, 35a, 36a, 37a) during the first collection and harvesting phase (E3), so that: a. In a first rinsing sub-phase (E31), the rinsing solution contained in the container (36) is transferred, via the rinsing sub-circuit, to the second separation module (31), then to the waste container (37); b. In a second harvesting sub-phase (E32), the second solution contained in the container (34) is transferred, via the harvesting sub-circuit, to the second separation module (31) and then transferred with said objects retained by the second separation module to the harvesting container (35).

10. Production system according to one of the preceding claims, wherein the first stage (1) comprises: a. two containers (11, 12), one of the containers being intended for

11. 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. Production system according to the preceding claim, wherein the first solution comprises a surfactant and a calcium salt and the second solution comprises a cell culture medium.

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