Cell encapsulation system incorporating a pressurized gas solution displacement system

Abstract

The invention relates to a cell encapsulation system, the system comprising at least: two containers (11, 12), one of which is intended to hold a cell solution and the other a gelling solution; a milli-fluidic or micro-fluidic encapsulation device (13) connected to the containers to form cell microcompartments, the outer layer of which is the gelling solution and the core the cell solution; a collection tank (15) for the cell microcompartments containing a stiffening solution; each container being connected to an inlet of the encapsulation device by one or more distributors (14); and at least one first container (11) being connected to a solution displacement system (P11, P12) capable of introducing a pressurized gas into said container to generate a flow of the solution contained in said container towards the encapsulation device. Figure to be published with the abstract: Fig. 1

Description

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

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

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

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

[0004] A significant 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 numerous applications, particularly in the development of therapies. A particularly suitable technology is that described in application WO2018 / 096277, which consists of three-dimensional cell microcompartments for stem cell culture. This document describes a cell encapsulation device comprising a microfluidic or millifluidic injector, enabling the formation of cell microcompartments in the form of droplets whose The outer layer is formed by a solution containing alginate, and the core is formed by a solution of cells. These droplets are collected in a calcium bath, which stiffens their outer layer to form a shell.

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

[0006] It is known to place the solutions intended to form the microcompartments in containers and to connect these containers to the encapsulation device by flexible tubes in which the solutions move using peristaltic or diaphragm pumps. These pumps exert pressure on the flexible tubes, for example via rollers or rollers, which generates movement of the solutions contained in these tubes to move them to the encapsulation device. These pumps are preferred for the applications mentioned above because they do not require any contact between the solution in the tube and the external environment, thus maintaining a closed and sterile environment.

[0007] However, they present several disadvantages.

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

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

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

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

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

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

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

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

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

[0017] To this end, the invention relates to a cell encapsulation system, the system comprising at least: a. two containers, one of the containers being intended to hold a cell solution and the other of the containers being intended to hold a solution capable of gelling, b. a milli-fluidic or micro-fluidic encapsulation device connected to the containers and arranged to form cellular microcompartments, the outer layer of which is the gelling solution 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.

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

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

[0020] This type of system has the advantage of allowing precise control of the gas pressure and / or the flow rate of the gas introduced into the container, and therefore precise control of the flow rate of the solution to the encapsulation device. This system is thus capable of regulating this flow rate so that it is, for example, constant, regular, and without pulsations, regardless of the encapsulation conditions, particularly with regard to the nature of the solutions used, the pressure at the outlet of the encapsulation device, the volume of solution contained in each container, the desired production rate, or the condition of the distributors.Furthermore, given the absence of direct contact between an element of the solution displacement system and an element of the container as well as the solution contained therein, this system makes it possible to increase the lifespan of the distributors of the encapsulation system, and to implement simple solutions to guarantee a closed and sterile environment.

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

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

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

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

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

[0026] In the present invention, and without limitation, the pressurized gas may be one of the following gases or a combination of at least two of the following gases: air, oxygen, nitrogen, dinitrogen, carbon monoxide, carbon dioxide, nitric oxide, and nitrogen dioxide, hydrogen sulfide, ethylene, ozone, hydrogen, or any other inert gas suitable for a cell culture.

[0027] Advantageously, one of the containers contains the cell solution and the other of the containers contains the solution suitable for gelling.

[0028] 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 immersed in this solution.

[0029] If desired, the system may be provided to include a third container connected to an inlet of the encapsulation device by one or more distributors and intended to include, or including, an intermediate solution, such as an intermediate solution not containing a divalent cation such as Ca2+ Mg2+ to avoid too early crosslinking of the solution capable of gelling in the collection tank, preferably an isotonic solution not containing a divalent cation such as Ca2+ Mg2+ such as for example a sorbitol solution.

[0030] According to one embodiment, 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 embodiment, the intermediate solution may contain an extracellular matrix and / or an extracellular matrix substitute. Where applicable, the encapsulation device will be arranged to form, within the collection tank, cell microcompartments, the outer layer of which is the gel-ready solution, an intermediate layer forming a cell matrix or extracellular matrix substitute, and the core the cell solution. This cell matrix allows the cells in the cell solution to grow and multiply. For example, the extracellular matrix substitute may comprise a mixture of proteins and extracellular compounds necessary for cell culture, and more specifically for pluripotent cells.Preferably, the extracellular matrix or extracellular matrix substitute may comprise structural proteins, such as laminins containing the α1, α4 or α5 subunits, the [31 or

[32] subunits, and the λ or γ3 subunits, entactin, vitronectin, laminins, collagen, and growth factors, such as TGF-β and / or EGF. The extracellular matrix may be an aqueous solution and / or a hydrogel, preferably a hydrogel, different from the hydrogel forming the outer layer, such as, for example, a hydrogel comprising or composed of alginate, fibrin, laminin, fibronectin, etc. of entactin, hyaluronic acid, and / or collagen. It may also be an extracellular matrix or an extracellular matrix substitute such as Matrigel®. In the invention, the cell solution comprises a plurality of cells. The cells may be of any cell type. More preferably, the cells are selected from human, animal, and plant eukaryotic cells, and even more preferably pluripotent stem cells, progenitor cells, cells undergoing differentiation, and differentiated cells. Where appropriate, said pluripotent stem cells may be induced pluripotent stem cells (iPSCs), MUSE cells (Multilineage-differentiating Stress Enduring) found in the skin and bone marrow of adult mammals, or embryonic stem cells (ESCs).In a particular embodiment, and for legal or ethical reasons, stem cells are understood to exclude human embryonic stem cells or cells that have required the destruction of human embryos.

[0031] In one embodiment of the invention, the gelling solution comprises or is constituted by a hydrogel, such that the outer layer is a three-dimensional structure formed from a matrix of polymer chains swollen by a liquid, preferably water. For example, the gelling solution comprises or is constituted by alginate and, preferably, consists of alginate. In the context of the invention, "alginate" means linear polysaccharides formed from 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.

[0032] In the microcompartments obtained by means of the system according to the invention, the cells present in the internal part can be isolated and / or in the form of at least one layer and / or in the form of at least one three-dimensional aggregate and / or in the form of at least one three-dimensional cellular micro-tissue, optionally with at least one lumen.

[0033] According to one embodiment, at least one cellular microcompartment obtained by means of the system according to the invention comprises at least one cell layer and at least one lumen. When the microcompartment comprises at least one lumen, at least one cell layer, the intermediate solution layer of the inner part, and the outer layer are preferably successively organized around said lumen, this is referred to as a cyst-shaped conformation. Thus, according to one embodiment, at least one cellular microcompartment obtained by means of the system according to the invention comprises at least one cyst, the hollow center of which, or lumen, is Preferably aqueous. In the context of the invention, a "cyst" is understood to be a three-dimensional, monolayered arrangement of cells or an epithelial layer, spherical, surrounding a central lumen. This cystic conformation reduces the pressure exerted on the cells. This configuration also decreases cell mortality and increases the culture amplification factor. Consequently, it reduces the number of passages and dissociations required, and the culture time needed to reach the desired final cell count.

[0034] The cellular microcompartments obtained using the system according to the invention preferably comprise one or more cysts, and / or one or more tissues and / or micro-tissues and / or cell aggregates with or without lumen(s).

[0035] Advantageously, the system according to the invention is arranged so that each cellular microcompartment obtained by means of this system is closed. In one embodiment, the system according to the invention is arranged so that each cellular microcompartment obtained by means of this system has a spherical or teardrop shape. Preferably, the diameter of such a microcompartment is between 10 µm and 1 mm, more preferably between 50 µm and 700 µm, even more preferably greater than 200 µm, and preferably less than 600 µm.

[0036] 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.

[0037] In one embodiment of the invention, the encapsulation system comprises at least one organ capable of electrically charging at least one of the solutions with an electrical potential and the encapsulation device comprises a body arranged to form a concentric flow from the solutions supplied by the dispenser(s), of which an external flow is the solution capable of gelling and an internal flow is the cell solution, and a nozzle connected to the body to receive said concentric flow and forming the outlet of the encapsulation device, the encapsulation device being arranged to form, at the outlet of the nozzle, a concentric jet from the concentric flow so that this jet is fractionated into cellular microcompartments.

[0038] In this embodiment, the encapsulation device may be a microfluidic or millifluidic device capable of generating a concentric jet containing the cell solution at its center, optionally surrounded by the intermediate solution, itself optionally surrounded by the gelling solution. The increase in hydrodynamic instabilities within the jet forces it to fragment into droplets; this effect is known as Plateau-Rayleigh instability. These droplets, once immersed in the stiffening solution, form the cell microcompartments. Electrically charging at least one of the solutions passing through The encapsulation device improves the breaking of the jet into droplets. This technique is known as "electro-jetting." It should be noted that the relative sizes of the outer layer and the core of the microcompartments can be adjusted by modifying the flow ratios of the two solutions at the dispensers.

[0039] In the case of electro-jetting, an electric field-generating element, such as a metallic ring positioned downstream of the encapsulation device's outlet, may be added so that the jet or the cellular microcompartments pass through this ring. If necessary, the electric field-generating element may be connected to an electrical potential, for example, to ground. This electric field helps, in particular, to promote the dispersion of the cellular microcompartments.

[0040] In another embodiment of the invention, the encapsulation system comprises at least one component capable of electrically charging at least one of the solutions with an electrical potential, and the encapsulation device comprises a body arranged to form a concentric flow from the solutions supplied by the dispenser(s), of which an external flow is the solution capable of gelling and an internal flow is the cell solution, and a nozzle connected to the body for receiving said concentric flow and forming the outlet of the encapsulation device. The encapsulation device is arranged to form said cell microcompartments directly at the nozzle outlet from the concentric flow. The encapsulation device is thus of the "electro-dripping" type and forms the microcompartments one after the other directly from the nozzle, without a jet.

[0041] Regardless of the embodiment envisaged, the outlet of the encapsulation device may be positioned above the collection tank, so that the microcompartments fall by gravity into this collection tank. Advantageously, the collection tank and the encapsulation device are arranged at a distance from each other such that the cellular microcompartments formed by the encapsulation device pass through a gaseous volume, in particular air, defined by a closed and sterile enclosure before being collected by the collection tank.

[0042] In yet another embodiment, the encapsulation device comprises a body arranged to form a concentric flow from the solutions supplied by the dispenser(s), of which an external flow is the solution suitable for gelling and an internal flow is the cell solution, and a nozzle connected to the body for receiving said concentric flow and forming the outlet of the encapsulation device, the encapsulation device and the collection tank being arranged such that the nozzle of the encapsulation device is in contact with the stiffening solution contained in the collection tank and / or such that the nozzle of the encapsulation device is immersed in the stiffening solution contained in the collection tank, the encapsulation device being arranged to form, directly in the stiffening solution, said cellular microcompartments from the concentric flow.

[0043] Regardless of the embodiment considered, the body and / or the nozzle may be made of glass. Alternatively, the body and / or the nozzle may be made of polymer or metal. The body and the nozzle may be a single piece, or they may be manufactured separately and then assembled to form the encapsulation device.

[0044] Advantageously, the body comprises a first inlet connected to a first distributor to receive the cell solution and at least a second inlet connected to a second distributor to receive the solution suitable for gelling, as well as a single outlet connected to the nozzle, the body comprising a main channel including a substantially straight portion defining a central axis of the encapsulation device, the main channel connecting the first inlet to the single outlet and at least one secondary channel connecting the second inlet to the single outlet, said secondary channel subdividing into portions extending around the first channel, said subdivisions of the second channel joining at the single outlet in a single circular portion, concentric with the first channel, said single circular portion and the first channel joining to form the single outlet of the body.

[0045] In one embodiment of the invention, said first container is sealed by a cap adapted to ensure a seal between the container and the outside. The cap is provided with a gas inlet to which the solution displacement system is connected for introducing pressurized gas into said container, and a solution outlet through which extends a tube of the dispenser immersed in the solution contained in said container to receive the flow of this solution generated by the introduction of the pressurized gas. In this embodiment, the gas pressure is exerted directly on the solution contained in the container to generate the displacement of this solution towards the dispenser and the encapsulation device.The stopper ensures the sterility of the container and also provides protection against backflow of gas or solution into the solution transfer system, particularly if the pressure at the solution outlet is higher than that at the gas inlet, or in the event of a depressurization of the transfer system. The encapsulation system is thus sealed and sterile.

[0046] The cap may for example be made of a material chosen from: a metal, a polypropylene (PP), a polycarbonate (PC), a polymer or a cyclic olefin copolymer (COC), a polyetheretherketone (PEEK), a fluorocarbon (FC) or a perfluorocarbon (PFC), or a polyacetal and in particular a polyoxymethylene (POM) and more generally a thermoplastic material or a polymer. The cap may also be equipped with a sealing gasket intended to come into contact with an opening of the container intended to be closed by the cap, the gasket being made for example of a thermoplastic elastomer or silicone, a polydimethylsiloxane (PDMS), a natural rubber, a polyisoprene (IR), polybutadiene (BR), a polyurethane (PU), a polyacrylate (PA) or even an epoxy resin.

[0047] Advantageously, said first container may be a flexible bag, a syringe or a conical-bottom tube or a glass bottle or reservoir.

[0048] Preferably, the stopper is arranged so that the gas inlet is positioned above the solution contained in the container.

[0049] According to one example, the gas inlet may be formed on a side wall of the stopper and the solution outlet may be formed on a top wall of the stopper.

[0050] Advantageously, the solution displacement system is connected to the gas inlet of the stopper via a pressurized gas sterilization device. The sterilization device forms a sterilization barrier for the pressurized gas and optionally a backflow prevention barrier between the container and the solution displacement system. The sterilization device may include a gas-permeable, and preferably liquid-impermeable, filter, for example, formed by a sterile membrane having a plurality of holes with a diameter less than 0.3 µm, in particular 0.22 µm.

[0051] In another embodiment of the invention, the first container comprises a sub-container for holding said cell solution or said gelling solution, the first container being provided with a gas inlet to which is connected the solution displacement system for introducing pressurized gas into said container, and the distributor passes through said container to be connected to a solution outlet of the sub-container to receive the flow of solution generated by the introduction of pressurized gas into said container. For example, the container may be a pressurized and sterile chamber, and the sub-container may be formed by a flexible pouch arranged within this chamber.

[0052] In this embodiment, the gas pressure is exerted on the walls of the subcontainer and thus indirectly on the solution contained within the subcontainer, causing the solution to move towards the dispenser and the encapsulation device. Advantageously, the pressurized gas is not introduced into the subcontainer and therefore does not come into contact with the solution. This embodiment thus avoids the need to sterilize the pressurized gas.

[0053] Advantageously, it may be provided that the distributor includes, downstream of the first container, a check valve or a non-return valve.

[0054] Advantageously, each container and the encapsulation device together form a closed and sterile system. In the context of the present invention, and by way of non-limiting example, a "closed and sterile system" is understood to mean a system comprising a set of interconnected circulation circuits for one or more fluids, which together define an internal space in which the fluid(s) circulate. The system is arranged so that it is devoid of any interface between the internal space and the outside of the system, or only of sterilization interfaces between the internal space and the outside of the system. This feature thus makes it possible to meet the sterility requirements of the various applications for which the system according to the invention is intended.

[0055] In one embodiment, the solution transfer system is connected, directly or indirectly, to the first container via a pressurized gas sterilization device. Preferably, each container is connected to a solution transfer system, directly or indirectly, via a pressurized gas sterilization device. This sterilization device or devices form a sterilization barrier for the pressurized gas to ensure the sterility of the system according to the invention. The sterilization device may include a gas-permeable, and preferably liquid-impermeable, filter, in particular a hydrophobic one, for example, formed by a sterile membrane having a plurality of holes with a diameter of less than 0.3 µm, in particular 0.22 µm. This membrane, when hydrophobic, also prevents solution from flowing back into the solution transfer system.

[0056] In one embodiment of the invention, the encapsulation system includes a system for controlling the flow rate of pressurized gas introduced into the first container, and a sensor for the flow rate of pressurized gas introduced into said container and / or the flow of the solution contained in said container towards the encapsulation device, the control system being arranged to control the flow rate of pressurized gas introduced into said container as a function of the flow rate of pressurized gas and / or the flow of the solution measured by said sensor.

[0057] In the present invention, the term "control system" means a device or computer system designed to manage, regulate, and supervise the operations and processes of the encapsulation system by controlling and coordinating the actions of the various controllable elements of this system, such as the solution displacement system(s). The control system may be equipped with one or more processors, or even one or more microcontrollers, arranged to execute one or more computer programs to implement phases of a production cycle for a batch of microcellular compartments. The control system may also be embedded in a machine comprising the solution displacement system(s), the containers, the encapsulation device, the collection tank, and where applicable, the various stages and fluidic circuits provided downstream of the collection tank and enabling the collection of the cellular microcompartments, including the opening and closing valves of these fluidic circuits, or alternatively that the control system be remote from this machine while being connected to it by means of connection, wired or wireless.

[0058] In this embodiment, the control system is thus equipped with a control loop for continuously adjusting the flow rate of pressurized gas introduced into said container, for example by controlling a control valve provided on the pressurized gas inlet line, according to the flow rate of pressurized gas and / or the flow of the solution measured by said sensor

[0059] Advantageously, the sensor is arranged downstream of the first container, in the dispenser, to measure the flow rate of the solution contained in the first container towards the encapsulation device. This type of sensor, also called an "inline" sensor, could, for example, be a thermal mass flow sensor. Preferably, the sensor should be sterile to avoid contaminating the solution with which it is intended to come into contact.

[0060] Alternatively or cumulatively, the sensor may be a sensor arranged downstream of the first container, outside the dispenser, to measure the flow rate, within the dispenser, of the solution contained in the first container towards the encapsulation device. This type of sensor, also called an "online" sensor, may, for example, be an ultrasonic flow sensor, an electromagnetic flow sensor, or any other type of static flow sensor without contact with the fluid whose flow rate is to be measured. These non-invasive sensors offer the advantage of avoiding any contact with the solution and thus ensuring the sterility of the encapsulation system. This sensor may also be a scale or any other weighing device, particularly one capable of measuring the weight of the collection tank to deduce the flow rate of one or more solutions.

[0061] Alternatively or cumulatively, the sensor may be arranged downstream of the first container, in the solution displacement system, particularly on the inlet line, to measure the flow rate of pressurized gas introduced into the first container. In this embodiment, the sensor is referred to as "pre-line" and measures the flow rate of the gas itself, particularly in steady state, from which the control system can then deduce the flow rate of the solution exiting the container.

[0062] In one embodiment of the invention, each container is connected to a solution displacement system capable of introducing a pressurized gas into said container to generate a flow of the solution contained in said container towards the The encapsulation device, the encapsulation system comprising sensors for the flow rate of pressurized gas introduced into each container and / or the flow of the solution contained in each container towards the encapsulation device. Where applicable, the control system is arranged to control the flow rate of pressurized gas introduced into each container based on a combination of the pressurized gas flow rates and / or the flow rate of each solution measured by said sensors.

[0063] In one embodiment of the invention, the distributor connected to said first container includes a restrictor element arranged to prevent the flow of solution from the encapsulation device into said container. This restrictor element thus forms a backflow prevention device ensuring the sterility of the solution contained in the container. This restrictor element may be configurable by the control system to prevent or allow the flow of solution between the container and the encapsulation device, for example, comprising a pinch valve, a solenoid valve, a piezoelectric valve, or a mechanical valve. Alternatively, this restrictor element may be a passive element arranged to allow the flow of solution between the container and the encapsulation device in only one direction, for example, comprising a passive capillary-type filter.

[0064] It may be advantageous to provide that at least one distributor and in particular each distributor is provided with other elements, for example of the type of air presence detector, flow meter and / or pressure sensor.

[0065] In one embodiment of the invention, the encapsulation system includes a circuit for collecting the cellular microcompartments collected by the collection tank.

[0066] For example, it may be provided that the collection tank includes an outlet for withdrawing the stiffening solution and the cellular microcompartments from the collection tank to the collection circuit; and that the encapsulation system includes: a. A buffer stage comprising the collection circuit connected to the collection tank to receive the stiffening solution and the cellular microcompartments immersed in this solution; means for moving the stiffening solution in the collection circuit; and a first separation module suitable for receiving the stiffening solution collected by the collection circuit and arranged to separate and retain the cellular microcompartments from this solution; b. A harvesting stage comprising a second separation module adapted to receive the stiffening solution collected by the collection circuit and arranged to separate and retain the cellular microcompartments from this solution; a harvesting circuit connected to the second separation module, to a container containing a second solution and a harvesting container; and means of moving the second solution in the harvesting circuit.

[0067] In the present invention, the term "separation module" means a device capable of receiving a solution containing suspended particles, and capable of separating and retaining the particles from this solution while allowing the solution to flow through. By way of non-limiting example, this could be a membrane filter, a centrifugal filtration system, a tangential flow filtration system, or a decanter.

[0068] Where appropriate, the collection circuit may include a first valve suitable for preventing the passage of the stiffening solution from the collection circuit to the harvesting stage and the second separation module, and the control unit may be arranged to control the means of moving the collection circuit, the means of moving the harvesting circuit and the first valve so that the collection of the stiffening solution and the cell microcompartments immersed in this solution from the collection tank is continuous and so that the harvesting of the second solution and the cell microcompartments separated by the second separation module to the harvesting container is discontinuous.

[0069] According to these characteristics, a buffer stage is interposed between the collection tank and the harvesting stage. The valve then allows the buffer stage to be isolated from the harvesting stage, to simultaneously carry out collection from the collection tank to the buffer stage and harvesting with a change of medium in the harvesting stage, or conversely, to allow a simultaneous transfer from the buffer stage and from the collection tank, via the buffer stage, to the harvesting stage.

[0070] Thus, depending on the state of this valve, the means of movement of the collection circuit allow the transfer of the cell microcompartments from the collection tank and the stiffening solution to the first separation module and, conversely, the transfer of the cell microcompartments retained by the first separation module to the harvesting stage. For example, it could be envisaged that these cell microcompartments follow opposite paths in the same part of the collection circuit for each of these transfers, or that the collection circuit comprises distinct parts, each dedicated to one of these transfers.

[0071] The means of movement 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.

[0072] The various elements of the system can thus be controlled by the control system to define different phases during the production of a batch of microcompartments. In particular, the collection circuit and / or the harvesting circuit may be provided with one or more valves controllable by the control system to define, within this or these circuits, preferred paths according to given phases of a production cycle for a batch of microcompartments.

[0073] 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 collection tank, while simultaneously, the microcompartments contained in the separation module of the harvesting stage are discharged to the harvesting container via the second solution, thus effecting a change of medium. In a transfer phase, the valve is opened, and the microcompartments generated by the collection tank, as well as those contained in the separation module of the buffer stage, are transferred to the separation module of the harvesting stage via the recycling solution.

[0074] In other words, these characteristics therefore make it possible to ensure discontinuous filtration of the microcompartments, thanks to which it is possible to ensure continuity in the production of microcompartments by the collection tank while allowing, in a discontinuous manner, a change of medium of the microcompartments thus produced.

[0075] In one embodiment of the invention, the collection tank is formed by a lower part intended to receive the stiffening solution and on which the withdrawal outlet and the injection inlet are provided, and by an upper part forming a lid on which the encapsulation device is mounted. The lower part-upper part assembly thus defines a closed enclosure, suitable for the production of cell microcompartments with regard to the applicable sterility requirements.

[0076] In one embodiment of the invention, the collection tank is arranged so that the stiffening solution it contains is electrically connected to ground.

[0077] Thanks to this feature, the electrical charges carried by the cell microcompartments flow, in the stiffening solution, from these microcompartments to the bulk, thus preventing the accumulation of charges, electrically charging the solution itself, and creating repulsive effects between the microcompartments. It is therefore possible to increase the yield and quality of cell culture within the microcompartments.

[0078] The invention also relates to a method for producing cellular microcompartments implemented by means of a system according to the invention.

[0079] The present invention is now described by means of purely illustrative and in no way limiting examples of the scope of the invention, and from the accompanying drawings, in which the various figures represent:

[0080] [Fig-1] represents, schematically and partially, a view of a system cell encapsulation according to an embodiment of the invention;

[0081] [Fig.2] represents, schematically and partially, a cross-sectional view of an encapsulation stage of an encapsulation system according to a first embodiment of the invention;

[0082] [Fig.3] represents, schematically and partially, a cross-sectional view of an encapsulation stage of an encapsulation system according to a second embodiment of the invention.

[0083] In the following description, identical elements, by structure or by function, appearing on different figures retain, unless otherwise specified, the same references.

[0084] A cell encapsulation system according to one embodiment of the invention is shown in [Fig.1].

[0085] The system comprises two containers 11 and 12. A first container 11 contains a solution comprising a plurality of human pluripotent stem cells. A second container 12 contains a gelling solution, comprising, for example, a hydrogel such as alginate. The system may also include a third container containing an intermediate solution, for example, an isotonic solution such as sorbitol.

[0086] The system also includes a milli-fluidic or micro-fluidic encapsulation device 13 arranged to form, from the solutions of the containers 11 and 12, cellular microcompartments whose outer layer is the alginate solution and whose core is the cell solution.

[0087] The encapsulation device 13 has several inlets each connected to one of the containers 11, 12 via a distributor 14. Each container 11, 12 is also connected to a Pu, Pn solution displacement system capable of introducing a pressurized gas into this container 11, 12 to generate a flow of the solution contained in said container 11, 12 in the distributor 14 towards the corresponding inlet of the encapsulation device 13.

[0088] Each Pu,P[2] solution displacement system, also called a pressure pump, is thus equipped with a pressurized gas reservoir, an inlet line connecting the reservoir to the container 11, 12, and a regulator capable of controlling the pressure and / or flow rate of the pressurized gas.

[0089] The example in [Fig. 1] shows an embodiment in which each container 11, 12 is equipped with a dedicated pressure pump Pu, Pi2. It may be provided, in the variants not shown, where containers 11 and 12 share the same container. Each inlet line(s) may be equipped with a control valve, allowing the flow of gas to be authorized and prohibited towards container 11 or 12, or even controlling the gas flow rate, and / or a non-return valve, preventing gas from flowing back from container 11 or 12 to the gas tank.

[0090] Each pressure pump Pu, P[2] may include one or more separate gas reservoirs, which may, for example, be mixed, particularly in an adjustable manner, by a mixer, before entering the inlet line. Each reservoir may be equipped with a regulator for the pressure of the gas contained in that reservoir. The pressurized gas contained in these reservoirs may be one of the following gases or a combination of at least two of the following gases: air, oxygen, nitrogen, carbon monoxide, carbon dioxide, nitric oxide, and nitrogen dioxide, hydrogen sulfide, ethylene, ozone, hydrogen.

[0091] It should be noted that the dispenser 14 for dispensing the alginate solution is equipped, in the example described, with a device capable of electrically charging the alginate solution with an electrical potential. Alternatively, the alginate solution may be charged directly in its container 12, via an electrode immersed in the solution, or in the encapsulation device 13.

[0092] The encapsulation device 13 is a microfluidic device comprising a body, including the inlets, and a nozzle connected to a single outlet of the body and forming a single outlet of the device 13. The body and the nozzle may be made of glass or another material suitable for the pharmaceutical industry. The body and the nozzle may form a single piece or, conversely, be made separately and then assembled to form the encapsulation device 13.

[0093] In the described example, the body comprises a main channel including a substantially straight portion defining a central axis of the encapsulation device 13. This main channel connects the first inlet to the single output of the body. The body comprises a secondary channel connecting the second inlet to the single output. This secondary channel is subdivided into portions extending around the first channel, said subdivisions of the second channel joining at the single output of the body to form a single circular portion concentric with the first channel. This single circular portion and the first channel then join to form the single output of the body.

[0094] 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.

[0095] The nozzle thus receives the concentric flow. Given the flow rates of the solutions and the electrostatic force generated by the electrical charges carried by the alginate solution, the encapsulation device 13 generates, at the nozzle outlet, a concentric jet from the concentric flow. This concentric jet is fragmented, under the effect of Plateau-Rayleigh instability, into cellular microcompartments, the outer layer of which is the alginate solution and the core the cell solution.

[0096] The encapsulation device 13 is thus of the "electro-jetting" type. It should be noted that the relative sizes of the outer layer and the core of the microcompartments can be adjusted by modifying the flow ratios of the two solutions using the distributors 14, while the overall size of the microcompartments can be controlled by adjusting the overall flow rate of the solutions and the electrical potential of the alginate solution.

[0097] Alternatively, the flow rates of the solutions and the electrical potential may be sized so that the encapsulation device 13 is of the "electro-dripping" type, and thus forms the microcompartments one after the other directly from the nozzle.

[0098] Optionally, a grounded metal ring may be added downstream 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 promotes the dispersion of the cellular microcompartments, in the case of an "electro-jetting" type device.

[0099] With further reference to [Fig. 1], the system also includes a collection tank 15 arranged under the encapsulation device 13 and containing a first solution intended to collect the cellular microcompartments formed by this encapsulation device 13 and falling by gravity into the collection tank 15. The collection tank 15 and the encapsulation device 13 are arranged at a distance from each other such that the cellular microcompartments formed by the encapsulation device pass through a gaseous volume, in particular air, defined by a closed and sterile enclosure before being collected by the collection tank 15.

[0100] Alternatively, the encapsulation device 13 may be arranged so that a nozzle of the encapsulation device is immersed in the stiffening solution contained in the collection tank 15, the encapsulation device 13 being arranged to form, directly in the stiffening solution, said cellular microcompartments from the concentric flow.

[0101] The stiffening solution comprises a surfactant and a calcium salt enabling the crosslinking of an alginate solution, and thus enabling the stiffening of the outer layer of each cell microcompartment upon its immersion in the collection tank.

[0102] Each cellular microcompartment obtained by means of this first stage 1 is thus closed and, in the example described, has a spherical or elongated teardrop shape.

[0103] In one embodiment, the container comprising the cell solution and / or the container comprising the intermediate solution includes at least one extracellular matrix and / or an extracellular matrix substitute. In this case, the encapsulation device may form cellular microcompartments, the outer layer of which is a hydrogel derived from the alginate solution, the intermediate layer is a cellular matrix and / or an extracellular matrix substitute derived from the cell solution and / or the intermediate solution, and at least one layer and / or base and / or aggregate and / or cyst of cells derived from the cell solution. In this example, the electrically charged solution with said electrical potential may be the alginate solution and / or the sorbitol intermediate solution.

[0104] It will be noted that the collection tank 15 has a withdrawal outlet 151 for the stiffening solution and microcompartments immersed in this solution as well as an injection inlet 152 for the stiffening solution into the collection tank 15. These inlet and outlet 151 and 152 are connected to a collection circuit, which will be described later.

[0105] 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 hood on which the encapsulation device 13 and the metal ring 16 are mounted. The collection tank 153 - hood 154 assembly thus defines a closed enclosure, suitable for the production of cell microcompartments with regard to the sterility requirements that apply.

[0106] It may be provided that the lower part 153 of the collection tank 15 is arranged so that the stiffening solution it contains is electrically connected to ground.

[0107] In the example described, the containers 11 and 12, the dispensers 14, the encapsulation device 13 and the collection tank 15 form an encapsulation stage 1 intended for the generation of the cellular microcompartments of the production system.

[0108] The production system includes a second stage 2, called the buffer stage, connected to the first stage 1 via the draw-off outlet 151 of the collection tank, and a third stage 3, called the harvesting stage, connected to the second stage 2.

[0109] The second buffer stage 2 includes a collection circuit 21 connected to the withdrawal outlet 151 of the collection tank 15, as well as a pump 23 for withdrawing the stiffening solution and the microcompartments from the collection tank 15 to the collection circuit 21.

[0110] A first separation module 22 is positioned in the collection circuit 21 in order to receive the solution collected by the collection circuit 21.

[0111] The separation module 22 includes, for example, a filter having a first port and a second port, each connected to the collection circuit 21, on the side of the withdrawal outlet 151 and on the side of the injection inlet 152 of the collection tank 15.

[0112] 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.

[0113] 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.

[0114] It should be noted that the second buffer stage 2 also includes a replenishment container 24 containing a given quantity of the stiffening solution. This container 24 is connected to the reinjection loop of the collection circuit 21, via a pump 25.

[0115] The pumps 23 and 25 together form means for drawing and injecting the stiffening solution into the collection circuit 21, these means for drawing and injecting being reversible in order to move the stiffening solution in one direction or another in the collection circuit 21. These may include peristaltic pumps, or any other mechanism suitable for causing the movement of a fluid in a circuit.

[0116] The third harvesting stage 3 comprises a harvesting circuit 32 connected on the one hand to the collection circuit 21, between the dispensing outlet 151 and the first separation module 22, and on the other hand to various containers, namely a container 34 containing a second harvesting solution, a harvesting container 35 intended to receive the microcompartments, a container 36 containing a third rinsing solution, and a waste container 37. Various 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 container 34 to harvesting container 35.

[0117] In the example described, the second and third solutions may be identical and each may include a cell culture medium, and possibly a buffer solution and a ROCK inhibitor.

[0118] The third harvesting stage 3 also includes a pump 33, forming means of displacement, to circulate the different solutions of the production system in the harvesting circuit 32, according to the configurations mentioned above.

[0119] A second separation module 31, substantially identical to the first module 23, is positioned in the harvesting 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 front-end filtration type system.

[0120] The collection circuit 21 includes a valve 26 suitable for allowing or preventing the passage of a solution from the collection circuit 21 to the harvesting circuit 32. In the example described, the valve 26 is a solenoid valve.

[0121] In order to be able to carry out a production cycle of a batch of cellular microcompartments, the system includes a control system 4 capable of controlling the pumps 23, 25 and 33 and the valves 26, 32a, 32b, 34a, 35a, 36a and 37a, as well as the pressure pumps Pu and Pi2.

[0122] The control system 4 is provided with one or more microcontrollers (not shown), arranged to execute one or more computer programs to control and coordinate the pumps 23, 25 and 33 and the valves 26, 32a, 32b, 34a, 35a, 36a and 37a of the second and third stages 2 and 3, as well as the pressure pumps Pu and Pn of the first stage 1, in order to implement phases of a production cycle of a batch of microcellular compartments.

[0123] In the example described, the control system 4 can receive data from system sensors, such as flow rate data from solution sensors in the circuits of stages 1 to 3, images acquired by a camera representing the jet from the encapsulation device 13, and measurements of the weight of the collection tank 15 from one or more load cells. The control unit can thus execute one or more production cycle control programs capable of interpreting this data in real time and adjusting the system's operational parameters, such as the flow rate of the solutions from containers 11 and 12, based on this interpretation, or even stopping the production cycle.

[0124] It may also be envisaged 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.

[0125] In the example described, all the different elements shown in [Fig. 1], including the control system 4, form a single machine. In another example, the control system 4 can be located remotely and communicates with the other elements of the system via wireless or wired communication means.

[0126] We will now describe a phase of a production cycle of a batch of cell microcompartments, implemented by the control system 4, and in which cell microcompartments, generated by the first stage 1, are transferred from the collection tank 15 to the first separation module 22 to be stored there.

[0127] In this production phase, the control system 4 controls the first stage 1 to trigger the generation of a batch of cell microcompartments, each microcompartment from the encapsulation device 13 being collected in the collection tank 15 to be immersed in the stiffening solution in order to cause stiffening of its outer layer.

[0128] The first stage thus comprises one or more sensors measuring the flow rate of pressurized gas introduced into each container 11, 12 and / or the flow rate of the solution contained in each container 11, 12 towards the encapsulation device 13. The flow rates of pressurized gas and / or solution flow measured by said sensors are provided to the control system, which then controls the flow rate of pressurized gas introduced into each container 11, 12 according to these measured flow rates so that the flow of solutions from containers 11, 12 towards the encapsulation device 13 complies with flow rate requirements set according to the desired application. It is thus possible to control the relative sizes of the outer layer and the core of the microcompartments, the overall size of the microcompartments, and the production rate of the microcompartments at the outlet of the encapsulation device 13.

[0129] In the example of [Fig.1], these sensors are Cn and Ci2 flow rate sensors of the solution flowing in each dispenser 14 from each container 11, 12 to the encapsulation device 13.

[0130] More specifically, the control system 4 controls the flow rate of pressurized gas introduced into each container 11, 12 as a function of a combination of the flow rates of pressurized gas and / or the flow of each solution measured by said sensors.

[0131] Simultaneously, the control system 4 controls the pump 23 to draw, via the draw-off outlet 151, the stiffening solution contained in the collection tank 15, as well as the cellular microcompartments suspended in this solution, in the collection circuit 21 according to a first direction of movement.

[0132] The system 4 keeps the valve 26 closed, so as to cause a displacement of the stiffening solution and the cellular microcompartments only towards the first separation module 22.

[0133] 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.

[0134] The stiffening solution thus continues its journey via the reinjection loop of the collection circuit 21 towards the injection inlet 152 of the collection tank 15, to be recirculated in the tank and again in the collection circuit 21 during the entire production phase.

[0135] 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.

[0136] After a given time, determined so that a given quantity of cellular microcompartments are stored in the first separation module 22 and so that the immersion time of these cellular microcompartments in the stiffening solution is less than a given time, the system 4 controls the different elements of the system to implement a transfer phase, in which cellular microcompartments, both generated by the first stage 1 and stored in the first separation module, are transferred to the second separation module 31 to be stored there.

[0137] In this transfer phase, the system 4 controls the pump 25 so that stiffening solution contained in the replenishment container 24 is injected into the collection circuit 21. Part of this solution joins the reinjection loop and the collection tank 15 via the injection inlet 152, while another part joins the first separation module to be entered via the second port 222.

[0138] Pump 23 is kept active to continue drawing the stiffening solution and the micro-cell compartments from the collection tank. System 4 also controls valve 26 to allow the transfer of the solution from the second stage 2 to the third stage 3.

[0139] Therefore, the injection of the stiffening solution into the collection tank 15 is simultaneous with the withdrawal of the stiffening solution from the collection tank 15. The control system 4 thus controls the pumps 23 and 25 so that the flow rate of withdrawal of the stiffening solution from the collection tank 15 is identical to the injection flow rate of the stiffening solution into the collection tank.

[0140] For these purposes, the first stage includes means for measuring, directly or indirectly, the flow rate of the stiffening solution being drawn from the collection tank 15. In the example described, these measuring means include one or more load cells (not shown) through which the collection tank 15 is mounted on a support.

[0141] 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 relative to the flow rate of solution injected. The control system 4 thus links the injection flow rate of the stiffening solution into the collection tank 15 by the pump 25 to the withdrawal flow rate from the collection tank by the pump 23, so that the weight variations are substantially zero. In other words, the injection flow rate of the stiffening solution from the refill container 24 is adjusted to compensate for the withdrawal of this solution from the collection tank 15.

[0142] It may be conceived that the measuring means comprise, alternatively or cumulatively, means for measuring the flow rate of stiffening solution withdrawn at the withdrawal outlet, such as a flow meter.

[0143] 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.

[0144] Simultaneously, the part of the stiffening solution from the replenishment container 24 which enters through the second port of the filter of the first separation module, crosses in the opposite direction through the second compartment, the filter membrane and the first compartment to exit through the first port carrying with it the cellular microcompartments retained in this first compartment during the previous production phase.

[0145] 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 rejoin the stiffening solution and the objects immersed in this solution, transferred from the collection tank 15, the whole assembly passing into the collection circuit 32 of the third stage 3 through the valve 26.

[0146] The system 4 controls the valves 32a, 32b, 34a, 35a, 36a and 37a so that the collection circuit has the first configuration in which the stiffening solution can flow only towards the waste container 37. In the example described, all the valves are closed, except for the valve 37a connected to the waste container 37.

[0147] The system 4 controls the pump 33 so that the stiffening solution and the cellular microcompartments suspended in this solution, from the collection tank 15 and the first separation module 22, circulate in the harvesting circuit 32 towards the second separation module 31.

[0148] 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.

[0149] The stiffening solution thus continues its journey in the collection circuit 32 towards the waste container 37.

[0150] At the end of this transfer phase, the system 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 way to the previous production phase so that cellular microcompartments, generated by the first stage 1 and collected in the collection tank 15, are again stored in the first separation module 22.

[0151] Simultaneously, the system 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 and then to the waste container 37 to rinse the microcompartments 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 and then to the harvesting container 35 to harvest the microcompartments which are stored in this module 31.

[0152] It is thus understood that the rinsing and harvesting sub-phases allow for a change of medium in the cell microcompartments, while cell microcompartments continue to be generated by the first stage 1 and to be temporarily stored in the first separation module 22.

[0153] These production, transfer, rinsing and harvesting and collection phases can be iterated several times to complete a production cycle of a batch of cellular microcompartments.

[0154] It may be foreseen 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 sub-phases of final rinsing and final harvesting.

[0155] It should be noted that, for each of the stages 1, 2 and 3, the components of that stage defining the circulation circuits of the solutions and / or microcompartments form a closed and sterile system, devoid of any interface between the internal space of this system in which these solutions and / or microcompartments circulate and the outside of the system or only sterilization interfaces between this internal space and the outside of the system.

[0156] In connection with [Fig. 2], we will describe a first embodiment of a pressurized gas displacement system Pu, allowing us to define, for the first stage 1, a closed and sterile system. We may assume that the system described is identical for both the Pu and Pi2 systems, or that each of these systems is structurally or functionally distinct.

[0157] The container 11 comprises a flexible pouch, a syringe or a conical-bottom tube, sealed by a cap 111 suitable for ensuring a seal between the container 11 and the outside. The cap 111 may, for example, be made of a material chosen from: a metal, a polypropylene (PP), a polycarbonate (PC), a polymer or a cyclic olefin copolymer (COC), a polyetheretherketone (PEEK), a fluorocarbon (FC), a perfluorocarbon (PFC), or a polyoxymethylene (POM) and more generally a thermoplastic material or a polymer. The cap is equipped with a sealing gasket intended to come into contact with an opening in the container 11 intended to be closed by the cap, the gasket being for example made of a thermoplastic elastomer or silicone, a polydimethylsiloxane (PDMS), a natural rubber, a polyisoprene (IR), polybutadiene (BR), a polyurethane (PU), a polyacrylate (PA) or even an epoxy resin.

[0158] The cap 111 is provided with a gas inlet 112 to which the Pu solution displacement system is connected to introduce the pressurized gas into said container 11.

[0159] The gas inlet 112 is formed on a side wall of the plug 111, and has an elbow so that it opens into the enclosure of the container 11 above the solution contained in this container 11.

[0160] The pressure pump Pu is connected to the gas inlet 112 via a pressurized gas sterilization device 113, formed by a sterile membrane having a plurality of holes with a diameter of approximately 0.22 µm, thus being permeable to gas and impermeable to liquid. This membrane 113 forms a sterilization barrier for the pressurized gas and optionally acts as a backflow preventer between the container 11 and the pressure pump Pu.

[0161] The cap 111 is also provided with a solution outlet 114 through which extends a tube 141 of the dispenser 14 immersed in the solution contained in said container 11.

[0162] It is thus understood that when pressurized gas is introduced by the pressure pump Pu into the container 11 via the gas inlet 112, the gas pressure acts directly on the solution contained in the container 11 to generate a movement of this solution in tube 141 and therefore in distributor 14 towards encapsulation device 13.

[0163] The stopper 111 thus ensures the sterility of the container 11 and also provides protection against backflow of gas or solution towards the pressure pump Pu, particularly if the pressure at the solution outlet 114 is higher than that at the gas inlet 112, or if the pressure pump Pu is depressurized. The container 11, the dispenser 14, and the encapsulation device 13 thus form a closed and sterile SCS system.

[0164] In the example of [Fig.2], a "pre-line" sensor Cn, replacing or supplementing the sensor Cn of [Fig.1], is arranged on the injection line of the pressure pump Pu to measure the flow rate of pressurized gas introduced into the container 11. The control system 4 can then deduce the flow rate of the solution exiting the container 11 towards the encapsulation device and can therefore regulate the flow rate of pressurized gas supplied by the pump Pu.

[0165] Furthermore, the distributor 14 connecting the container 11 to the encapsulation device 13 includes a restricting element 142, namely a pinch valve, which prevents the flow of solution from the encapsulation device 13 to the container 11. This restricting element thus forms a backflow prevention means ensuring the sterility of the solution contained in the container 11.

[0166] In connection with [Fig.3], we will describe a second embodiment of a system of displacement to gas under pressure Pu, allowing to define, for the first stage 1, a closed and sterile system.

[0167] In this embodiment, the container 11 comprises a first flexible pouch 1111 inside which is arranged a sub-container, for example formed by a second flexible pouch 1112, containing said cell solution. The second flexible pouch 1112 is devoid of any fluid exchange interface with the first flexible pouch 1111.

[0168] The first flexible pouch 1111 is provided with a gas inlet 1113 to which the pressure pump Pu is connected to introduce the pressurized gas into the first flexible pouch 1111.

[0169] The distributor 14 passes through the first flexible bag 1111 to be connected to a solution outlet 1114 of the second flexible bag 1112.

[0170] It is thus understood that when pressurized gas is introduced by the pressure pump Pu into the first flexible pouch 1111, the gas pressure is exerted on the walls of the second flexible pouch 1112 and therefore indirectly on the solution contained in this second flexible pouch 1112. This pressure thus generates a displacement of this solution towards the outlet 1114, the distributor 14 and the device encapsulation 13. Given the absence of a fluid exchange interface between bags 1111 and 1112, the second bag 1112, the dispenser 14 and the encapsulation device 13 thus form a closed and sterile SCS system.

[0171] Similar to the embodiment of [Fig.2], the distributor 14 connecting the container 11 to the encapsulation device 13 includes a restricting element 142, namely a pinch valve, allowing to prohibit a flow of solution from the encapsulation device 13 to the container 11.

[0172] In the example of [Fig.3], the sensor Cn is a sensor for measuring the flow rate of the solution in the distributor 14.

[0173] It may be provided indifferently that the sensor Cn is an "inline" sensor arranged in the distributor, such as a thermal mass flow sensor, or an "online" sensor arranged outside the distributor, such as an ultrasonic flow sensor.

[0174] The preceding description clearly explains how the invention makes it possible to achieve the objectives it has set for itself, namely to offer an encapsulation system capable of responding to applications requiring small volumes, on the order of microlitres, as well as to applications requiring large volumes, on the order of ten litres; which does not substantially cause wear of the components conveying the solutions to the encapsulation device, nor disturbances, for example of the impulse type, in the flow of the solutions to the encapsulation device, and which makes it possible to produce microcompartments under conditions conforming to the sterility requirements of these applications.It is understood that these objectives are achieved by means of a pressurized gas displacement system allowing precise control of the gas pressure and / or the flow rate of the gas introduced into the container, and therefore allowing precise control of the flow rate of the solution towards the encapsulation device, increasing the lifespan of the distributors of the encapsulation system, and enabling the implementation of simple solutions to guarantee a closed and sterile environment.

[0175] In any event, the invention cannot be limited to the embodiments specifically described in this document, and extends in particular to all equivalent means and to any technically operative combination of these means.

Claims

Demands

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

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

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

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

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

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

7. An encapsulation system according to any one of the preceding claims, characterized in that it comprises a control system (4) for the flow rate of pressurized gas introduced into the first container (11), and a sensor (Cn, Ci2) for the flow rate of pressurized gas introduced into said container and / or the flow of the solution contained in said container towards the encapsulation device (13), the control system being arranged to control the flow rate of pressurized gas introduced into said container as a function of the flow rate of gas under pressure and / or flow of the solution measured by said sensor.

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

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

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

11. Encapsulation system according to any one of claims 7 to 10, wherein each container (11, 12) is connected to a solution displacement system (Pu, Pn) capable of introducing a gas under pressure into said container to generate a flow of the solution contained in said container towards the encapsulation device (13), characterized in that it comprises sensors (Cn, Ci 2) of the flow rate of gas under pressure introduced into each container and / or of the flow of the solution contained in each container towards the encapsulation device and in that the control system (4) is arranged to control the flow rate of gas under pressure introduced into each container as a function of a combination of the flow rates of gas under pressure and / or the flow of each solution measured by said sensors.

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

13. Encapsulation system according to any one of the preceding claims, characterized in that it comprises a collection circuit (21) for the cellular microcompartments collected by the collection tank (15).

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