Cell encapsulation system comprising solution dispensers

Abstract

The invention relates to a cell encapsulation system (1), the system comprising at least: a first container (11) intended to contain a solution of cells and a second container (12) intended to contain a solution capable of gelling, a millifluidic or microfluidic encapsulation device (20) connected to the containers for forming cellular microcompartments, of which the outer layer is the solution capable of gelling and the core is the solution of cells, wherein each container is connected to an inlet of the encapsulation device by a dispenser (31, 32) associated with a solution movement system (41, 42) capable of causing the solution contained in the container (11, 12) to flow through the dispenser toward the inlet of the encapsulation device (20), characterized in that the first dispenser (31) has at least one upwardly extending portion.

Description

Description Title of the invention: Cell encapsulation system comprising solution dispensers

[0001] The invention relates to the field of cell encapsulation in three-dimensional cell culture compartments. More specifically, the invention relates to a cell encapsulation system comprising a collection tank for collecting cell microcompartments generated by an encapsulation device.

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

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

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

[0005] The microcompartments thus formed allow the cells to be cultured in a liquid medium, while the shell protects the cells from mechanical stresses related to collisions or fusions during 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 via flexible tubing in which the solutions move using peristaltic or diaphragm pumps. These pumps exert pressure on the flexible tubing, for example via rollers or rollers, which generates movement of the solutions within the tubing, propelling them to the encapsulation device. These pumps are preferred for the applications mentioned above because they require no contact between the solution in the tubing and the external environment, thus maintaining a closed and sterile environment.

[0007] This type of architecture, however, presents several drawbacks. Firstly, the flexible tubes through which the solutions move are particularly thin and long. Secondly, the flow rate of the solutions to the encapsulation device, and therefore the velocity and volume of the microcompartments produced by this device, are difficult to control, especially at low flow rates, with this type of pump. These characteristics, particularly when combined, introduce significant variations in the control of solution movement within the tubes and thus lead to a problem in regulating the flow rate of each individual solution.These variations are likely to generate bubbles in the tubes, for example by cavitation, which are likely to create areas of cell concentration as well as foam, which would degrade the homogeneity of the number of cells in each microcompartment and therefore the quality of production.

[0008] This problem of synchronicity of the movement of the solutions is further aggravated by various factors, including the tolerance of the length of the tubes and the difference in viscosity of the solutions, the solution suitable for gelling being more viscous than the cell solution.

[0009] There is a need for a cell encapsulation system that overcomes the various disadvantages mentioned, and that, more specifically, prevents the appearance of bubbles in the tubes in which the solutions move and in the encapsulation device.

[0010] The present invention falls within this context and aims to address this need.

[0011] To this end, the invention relates to a cell encapsulation system, the system comprising at least: a. two containers, the first of which is intended to contain a cell solution and the second of which is intended to contain 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,

[0012] In the system according to the invention, each of the first and second containers is connected to an inlet of the encapsulation device by a first, respectively a second distributor associated with a first, respectively a second solution displacement system capable of causing a flow of the solution, contained in said container, in said distributor towards the inlet of the encapsulation device,

[0013] The system according to the invention is characterized in that the first distributor has at least one portion extending upwards.

[0014] The invention thus proposes to arrange the first dispenser so that the cell solution flows through the first dispenser from the first container to the inlet of the dedicated encapsulation device, moving upwards, at least along a portion of the dispenser. This allows for the removal of any bubbles present in the solution. This prevents cell accumulation at air-liquid interfaces that could be caused by bubbles in the dispenser, thereby improving the homogeneity of the cell count in each microcompartment and thus the quality of the production.

[0015] Advantageously, each dispenser in the encapsulation system can have at least one portion extending upwards. Specifically, each container in the encapsulation system can be arranged so that the connection between that container and its dispenser is located substantially below the inlet connecting that dispenser to the encapsulation device. According to these characteristics, each solution flows through its dispenser from its container to its dedicated inlet in the encapsulation device, moving upwards, at least along a portion of that dispenser. This allows for the removal of any air bubbles present in each solution during its flow through the associated dispenser.

[0016] Advantageously, one of the containers contains the cell solution and the other of the containers contains the solution suitable for gelling.

[0017] For example, the cell solution comprises a plurality of cells. The cells may be of any cell type. More preferably, the cells are chosen from human, animal, and plant eukaryotic cells, and even more preferably pluripotent stem cells, progenitor cells, cells undergoing differentiation, and differentiated cells. Where appropriate, these pluripotent stem cells may be induced pluripotent stem cells (iPSCs), MUSE (Multilineage-Differentiating Stress Enduring) cells 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, the stem cells are understood to exclude human embryonic stem cells or cells resulting from the destruction of human embryos.

[0018] In one embodiment, the gelling solution comprises or is composed of 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 composed of alginate and, preferably, consists of α-Iginate. In the context of the invention, "alginate" means linear polysaccharides formed from pD-mannuronate (M) and αL-guluronate (G), salts, and derivatives thereof. Advantageously, the alginate is sodium alginate, composed of more than 60%, or even more than 80%, of G and less than 40%, or even less than 20%, of M, with an average molecular weight of 100 to 400 kDa and a total concentration of between 0.5% and 5% by mass.

[0019] In the microcompartments obtained by means of the encapsulation system, 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 microtissue, possibly with at least one lumen.

[0020] According to one embodiment, at least one cellular microcompartment obtained using the system comprises at least one cell layer and at least one lumen. When the microcompartment comprises at least one lumen, at least one cell layer, an optional intermediate solution layer of the inner part, and the outer layer are preferentially arranged successively around said lumen, this is referred to as a cyst-like conformation. Thus, according to one embodiment, at least one cellular microcompartment obtained using the system comprises at least one cyst, the hollow center, or lumen, of which is preferentially aqueous. In the context of the invention, a "cyst" is understood to be a three-dimensional, spherical, monolayered arrangement of cells or an epithelial layer surrounding a central lumen. This cyst-like conformation reduces the pressures experienced by the cells.This configuration also reduces cell mortality and increases the culture amplification factor. Consequently, it reduces the number of passages and dissociations required, and the time in culture needed to reach the final cell count.

[0021] The cellular microcompartments obtained using the encapsulation system preferentially include one or more cysts, and / or one or more tissues and / or micro-tissues and / or cell aggregates with or without lumen(s).

[0022] Advantageously, the encapsulation system is arranged so that each cell microcompartment obtained by means of this system is closed. In one embodiment, the encapsulation system is arranged so that each cell 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.

[0023] In another embodiment, the encapsulation system 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.

[0024] In the context of the present invention, cellular tissues can be obtained from cells encapsulated in one or more three-dimensional closed microcompartments, obtained from an encapsulation system according to the invention. If applicable, the microcompartment(s) can be cultured in a culture medium within a closed chamber, in particular a bioreactor.

[0025] In the context of the present invention, a "container" means any closed, sterile receptacle designed to contain, store, or transport a solution in a controlled medical environment. The container may be rigid or flexible and may be of various shapes and sizes. By way of non-limiting example, examples of containers may include: flexible bags, syringes, conical-bottom tubes, bottles, glass containers, vials, ampoules, cartridges, flasks, and reservoirs. Each container may be provided with specialized interfaces, ports, or seals to facilitate aseptic flow of the solution it contains to the associated dispenser.

[0026] In the context of the present invention, a "dispenser" means any component designed to deliver, guide, or transfer a solution in a controlled medical environment, and in particular to maintain sterility and prevent contamination during the circulation of the solution. By way of non-limiting example, dispensers may include: tubes, conduits, tubing, pipes, cannulas, catheters, needles, capillaries, connectors, valves, tips, pipettes, and microfluidic channels. Each dispenser may be provided with filters and sensors to control and secure the circulation of the solution.

[0027] In the context of the present invention, a "displacement system" means any device designed to generate, control, or facilitate the flow of a solution contained in a container within a dispenser. By way of non-limiting example, examples of displacement systems may include: peristaltic pumps, diaphragm pumps, syringe pumps, pressurized gas displacement systems (also known as pressure pumps), piston pumps, gear pumps, centrifugal pumps, diaphragm pumps, gravity displacement systems, jet pumps, solenoid pumps, electrokinetic displacement systems, and microfluidic devices.

[0028] In the context of the present invention, and by way of non-limiting example, a "microfluidic device" means any device or combination of devices having one or more inlets and one or more outlets connected by a plurality of channels with a cross-section on the order of a hundred micrometers and capable of directing the flow of one or more fluids from the inlet(s) to the outlet(s). A "millifluidic device" also means any device or combination of devices having one or more inlets and one or more outlets connected by a plurality of channels with a cross-section on the order of a millimeter and capable of directing the flow of one or more fluids from the inlet(s) to the outlet(s).

[0029] We can foresee different ways of implementing the encapsulation device.

[0030] For example, the encapsulation device may include a body arranged to form a concentric flow from the solutions supplied by the containers, of which an external flow is the solution suitable for gelling and an internal flow is the cell solution, and a nozzle connected to the body to receive said concentric flow and forming the outlet of the encapsulation device.

[0031] If necessary, the encapsulation device can be arranged to form the micro-cellular compartments directly at the nozzle outlet from the concentric flow. In this embodiment, the encapsulation device forms the droplets one after another directly from the nozzle, either naturally or with the aid of external forces. The encapsulation device is thus of the "dripping" type, and forms the micro-compartments one after another directly from the nozzle, without a jet.

[0032] Alternatively, the encapsulation device can be arranged to form a concentric, or even continuous, jet at the nozzle outlet, such that the concentric flow is fragmented into cellular microcompartments. In this embodiment, the increase in hydrodynamic instabilities within the jet forces it to fragment into droplets at a certain distance from the nozzle outlet; this effect is known as the Plateau-Rayleigh instability. The encapsulation device is thus of the "jetting" type, forming the microcompartments by naturally fragmenting the jet or through the application of external forces.

[0033] Alternatively, the encapsulation device can be arranged to atomize the concentric flow at the nozzle outlet into a cloud of droplets forming the cellular microcompartments. In this embodiment, this atomization can occur naturally, depending on the flow rate of the solutions supplied by the dispenser(s) and / or the diameter of the nozzle outlet, or through external forces. The encapsulation device is thus of the "spraying" type, with each droplet forming a microcompartment.

[0034] Regardless of the embodiment envisaged, the encapsulation device may be equipped with one or more components capable of generating external forces to increase the hydrodynamic instabilities of the flow exiting the nozzle and / or to contribute to its fragmentation. These components may include, in particular, a component capable of electrically charging at least one of the solutions with an electrical potential, a component for generating an electric field located downstream of the nozzle outlet, an acoustic wave generator coupled to the nozzle and / or the body, a vibrating element coupled to the nozzle and / or the body, or a cutting element located downstream of the nozzle outlet.

[0035] In one embodiment, the encapsulation system includes at least one component capable of electrically charging at least one of the solutions with an electrical potential, and the encapsulation device includes 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 such that this jet is fractionated into cellular microcompartments.

[0036] 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, which may itself be 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 the gelling solution has cross-linked, form the cell microcompartments. Electrically charging at least one of the solutions passing through the encapsulation device enhances the fragmentation of the jet into droplets. This technique is notably called "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 distributors.

[0037] In the case of electro-jetting, an electric field-generating device, such as a metallic ring positioned downstream of the encapsulation device's outlet, can be added so that the jet or cell microcompartments pass through this ring. If necessary, the electric field-generating device can be connected to an electrical potential, for example, to ground. This electric field helps to promote the dispersion of the cell microcompartments.

[0038] In another embodiment, the encapsulation system includes at least one component capable of electrically charging at least one of the solutions with an electrical potential. 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. A nozzle connected to the body receives this concentric flow and forms the outlet of the encapsulation device. The encapsulation device is arranged to form droplets directly from the concentric flow at the nozzle outlet. Once the solution suitable for gelling has cross-linked, these droplets form the cell microcompartments. The encapsulation device is thus of the "electro-dripping" type and forms the droplets one after another directly from the nozzle, without a jet.

[0039] In yet another embodiment, the encapsulation device comprises a body arranged to form a concentric flow from the solutions supplied by the dispenser(s), of which an external flow is the solution suitable for gelling and an internal flow is the cell solution, and a nozzle connected to the body to receive said concentric flow and forming the outlet of the encapsulation device, and the encapsulation system also comprises an acoustic wave generator coupled to the nozzle and / or the body so that the encapsulation device is arranged to form, directly at the outlet of the nozzle, drops from the concentric flow.In this embodiment, the encapsulation device is of the "acousto-dripping" type, and forms the drops one after another directly from the nozzle under the effect of the acoustic waves emitted by the generator, the dimensions of the drops being determined according to the choice of the frequency and amplitude of the acoustic waves.

[0040] In yet another embodiment, the encapsulation device comprises a body arranged to form a concentric flow from the solutions supplied by the dispenser(s), of which an external flow is the solution suitable for gelling and an internal flow is the cell solution, and a nozzle connected to the body to receive said concentric flow and forming the outlet of the encapsulation device, and the encapsulation system also comprises a vibrating element coupled to the nozzle and / or the body so that the encapsulation device is arranged to form, at the outlet of the nozzle, a concentric jet from the concentric flow, said jet being broken into drops.In this embodiment, the encapsulation device is of the vibrating-jetting type and forms the droplets by breaking the concentric jet due to a Plateau-Rayleigh instability induced by the vibrations generated by the vibrating element. The dimensions of the droplets are determined by the choice of the frequency and amplitude of these vibrations. This vibrating element could, for example, be a piezoelectric actuator.

[0041] In yet another embodiment, the encapsulation device comprises a body arranged to form a concentric flow from the solutions supplied by the dispenser(s), where the outer flow is the solution suitable for gelling and the inner flow is the cell solution. A nozzle connected to the body receives this concentric flow and forms the outlet of the encapsulation device. The encapsulation system also includes a cutting element disposed downstream of the nozzle outlet, such that the encapsulation device is arranged to form a concentric jet from the concentric flow at the nozzle outlet, this jet being fractionated by the cutting element. In this embodiment, the encapsulation device is thus of the "jet cutting" type and forms the droplets by cutting the concentric jet with the cutting element.The cutting element could, for example, be a rotating blade, with the dimensions of the drops being determined according to the rotation speed and the dimensions of the rotating blade.

[0042] In another embodiment, the encapsulation device comprises a body arranged to form a concentric flow from the solutions supplied by the dispenser(s), with an outer flow being the solution suitable for gelling and an inner flow being the cell solution. A nozzle connected to the body receives this concentric flow and forms the outlet of the encapsulation device. The encapsulation system includes an electromechanical element coupled to the nozzle. The encapsulation device is arranged to form droplets directly from the concentric flow at the outlet of each nozzle, under the effect of a vibration applied by the electromechanical element. In this embodiment, the encapsulation device is thus of the "inkjet printing" type and forms the droplets one after another directly from each nozzle.The said electromechanical element could, for example, be a piezoelectric actuator, the dimensions of the drops being determined according to the choice of the frequency and amplitude of the vibrations applied by this element.

[0043] Regardless of the embodiment envisaged, the encapsulation system may be provided for, including a collection tank containing a stiffening solution and arranged to collect the cellular microcompartments formed by the priming encapsulation device. If necessary, the collection tank may be arranged downstream of the encapsulation device to collect the cellular microcompartments formed by the encapsulation device, the stiffening solution being arranged to cause stiffening of the outer layer of each cellular microcompartment upon its immersion in this solution.

[0044] For example, the outlet of the encapsulation device may be positioned above the collection tank, so that the microcompartments fall by gravity into the collection tank. Advantageously, the collection tank and the encapsulation device are arranged at a distance from each other such that the cellular microcompartments formed by the encapsulation device pass through a gaseous volume, such as air, defined by a closed and sterile enclosure before being collected by the collection tank.

[0045] In yet another embodiment, the encapsulation device comprises a body arranged to form a concentric flow from the solutions supplied by the dispenser(s), of which an external flow is the solution suitable for gelling and an internal flow is the cell solution, and a nozzle connected to the body to receive said concentric flow and forming the outlet of the encapsulation device, the encapsulation device and the collection tank being arranged so that the nozzle of the encapsulation device is in contact with a collection fluid, such as an oil, contained in a collection tank and / or so that the nozzle of the encapsulation device is immersed in a collection fluid contained in a collection tank, the encapsulation device being arranged to form, directly in the collection fluid, drops or a jet, which then break into drops, from the concentric flow.The gelling solution can then be cross-linked, for example via a stiffening solution, to form the cellular microcompartments.

[0046] Regardless of the embodiment considered, the body and / or nozzle may be made of glass. Alternatively, the body and / or nozzle may be made of polymer or metal. The body and nozzle may be a single piece, or they may be manufactured separately and then assembled to form the encapsulation device.

[0047] Advantageously, the body comprises a first inlet connected to the first dispenser to receive the cell solution and at least a second inlet connected to the second dispenser 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.

[0048] In another embodiment of the invention, the encapsulation device comprises a first body equipped with a nozzle, the first body being arranged to form a first jet from the cell solution supplied by the first dispenser at the nozzle outlet, this first jet being fragmented into droplets, and a second body equipped with a nozzle, the second body being arranged to form a second jet from the gelling solution supplied by the second dispenser at the nozzle outlet, the first and second bodies being arranged such that the droplets from the first jet interact with the second jet to form the cell microcompartments. In this embodiment, the first jet fragments into droplets by a Plateau-Rayleigh instability. These droplets encounter the continuous second jet, which then encapsulates these droplets, by the Marangoni effect, to form the cell microcompartments.It will then be possible to predict cross-linking of the outer layer of the microcompartments, formed by the solution capable of gelling, in a gaseous environment, such as in air, for example using ultraviolet radiation.

[0049] It may also be envisaged any combination of the embodiments described above, or even other embodiments of the encapsulation device allowing the generation of cellular microcompartments without departing from the scope of the present invention, and in particular encapsulation devices allowing the formation of drops one after the other at the outlet of the encapsulation device and equipped with a means of controlling the ejection of the drops and of controlling the dimensions of the drops during their ejection, encapsulation devices allowing the formation of a concentric jet at the outlet of the encapsulation device and equipped with a means of separating the jet, after its exit from the encapsulation device, into drops, or even encapsulation devices allowing the coating of drops or a jet from a first device with drops or a jet from another device.

[0050] In one embodiment, the system includes a third container for receiving a dilution solution, the third container being connected to a third distributor associated with a third solution displacement system capable of causing a flow of the dilution solution into the third distributor; and the first and third distributors join in a common portion connected to the inlet of the encapsulation device.

[0051] In the present invention, the dilution 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. If applicable, the encapsulation device will be arranged to form cell microcompartments, the outer layer of which is the gel-ready solution, an intermediate layer forming a cell matrix or extracellular matrix substitute, and the core the cell solution. This cell matrix allows the cells in the cell solution to grow and multiply. For example, the extracellular matrix substitute may 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 |3L or |32 subunits, and the θi 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, entactin, hyaluronic acid, and / or collagen. It may also be an extracellular matrix or an extracellular matrix substitute such as Matrigel®.

[0052] Advantageously, the first and third distributors can be joined at a T-connector, the common portion extending from the T-connector to the inlet of the encapsulation device. The delivery of cell and dilution solutions to the encapsulation device during a batch production step of microcompartments is thus carried out simultaneously by the first and third solution displacement systems, controlled by the control unit. The cell solution and dilution solution are mixed at the common portion before arriving together at the encapsulation device.

[0053] It may be envisaged that the connector may have another shape, in particular a Y shape, without departing from the scope of the present invention.

[0054] Alternatively, the system may be provided for without a container containing such a dilution solution, the first dispenser then being directly connected to the inlet of the encapsulation device intended to receive the cell solution.

[0055] In one embodiment, the first and third solution displacement systems are arranged on a portion of the first and third distributors, respectively, extending between the first and third containers and the junction between the first and third distributors, these portions being substantially vertical. This further enhances the bubble evacuation effect.

[0056] Preferably, the first dispenser extends almost exclusively upwards and / or horizontally to the common portion. This prevents bubbles from stagnating in the first dispenser, which could destabilize the flow of the cell solution and / or cause cell accumulation at the bubble sites.

[0057] Advantageously, the first distributor has a reduced cross-sectional area upstream of the junction between the first and third distributors. This reduced cross-sectional area can be achieved by locally restricting the internal diameter of the first distributor. This reduced cross-sectional area helps to smooth out flow oscillations that may be introduced by the first cell solution displacement system, particularly when it is a peristaltic pump.

[0058] Advantageously, each dispenser includes a fluid damper mounted on it downstream of the solution displacement system associated with that dispenser. For example, the fluid damper could consist of a tube, more rigid than the dispenser, connected to the dispenser, for example, by a Y-connector, and forming an air spring. This fluid damper smooths out flow oscillations that may be introduced by the solution displacement system, particularly when it is a peristaltic pump, and captures bubbles moving within the dispenser. The air spring could optionally be equipped with a pressure sensor.

[0059] Advantageously, the common portion includes a blender.

[0060] In the present invention, the term "mixer" means any component designed to combine, homogenize, or mix two or more solutions, in particular a cell solution and a dilution solution, in a controlled medical environment. The mixer can be configured to ensure homogeneous mixing while maintaining sterility and minimizing damage to cells or sensitive components of the solutions. By way of non-limiting example, examples of mixers may include: a passive linear mixer, e.g., chevron, helical, obstruction, or lamella mixer, or an active mixer.

[0061] For example, the mixer can be arranged so that the mixer outlet on the common section extends at least to the same height as the mixer inlet on the common section. This prevents bubbles from stagnating within the mixer.

[0062] Advantageously, the mixer is a linear passive type, with at least a portion of it extending upwards. Preferably, the mixer may be designed to extend obliquely. This solution allows the cell solution and the dilution solution to be mixed by circulating them upwards, thereby removing bubbles from the mixer. Alternatively, other configurations, including different shapes and arrangements of the mixer, may be considered to facilitate bubble removal.

[0063] In one embodiment of the invention, the encapsulation system includes a cooling unit arranged adjacent to a portion of the third dispenser and / or the third container. The cooling unit may be arranged to cool a cooling zone encompassing the third container, at least a portion of the third dispenser, and possibly the first dispenser.

[0064] If necessary, the cooling unit can be configured to cool a cooling zone extending from a point on the first distributor located downstream of a liquid presence sensor on the first distributor to a point on the common section located downstream of the junction of the first and third distributors. Therefore, this cooling zone advantageously extends to a portion of both the first and third distributors.

[0065] For example, the cooling unit could be a thermoelectric cooling unit such as a Peltier unit equipped with a fan. Alternatively, the cooling unit could be a liquid cooling unit, specifically a water cooling unit (or "watercooling") circulating behind the cooling zone.

[0066] In one embodiment of the invention, each dispenser is equipped with a liquid presence sensor positioned downstream of the container and upstream of the inlet of the encapsulation device connected by this dispenser. Each sensor may, in particular, be arranged between the container and the solution displacement system associated with the dispenser it equips.

[0067] In one embodiment of the invention, the encapsulation system comprises a container for holding an intermediate solution, said container being connected to an inlet of the encapsulation device by a distributor associated with a solution displacement system capable of causing the intermediate solution to flow from said distributor to the inlet of the encapsulation device. It should be noted that the encapsulation device preferably has an inlet dedicated to the first distributor, or its common portion, an inlet dedicated to the second distributor, and an inlet dedicated to the distributor of the intermediate solution.

[0068] In the invention, the intermediate solution may be a solution intended to avoid too early crosslinking of the solution capable of gelling, such as an intermediate solution not containing a divalent cation such as Ca2+ Mg2+ to avoid too early crosslinking of the hydrogel in the collection tank, preferably an isotonic solution not containing a divalent cation such as Ca2+ Mg2+ such for example a sorbitol solution.

[0069] In one embodiment of the invention, the encapsulation system includes a control unit arranged for, prior to a production step of a batch of cellular microcompartments: a. in a priming step of the first dispenser, control the first cell solution displacement system to circulate a portion of the cell solution into the first dispenser; b. in a priming step of the second dispenser triggered later than the priming step of the first dispenser, control the second system for displacing the solution capable of gelling to circulate a portion of said solution into the second dispenser.

[0070] In this embodiment, the encapsulation system is primed prior to a production phase according to a specific sequence. According to this sequence, the first dispenser is initially primed with a portion of the cell solution, which is moved by the first transport system towards the encapsulation device. During this priming step, the second transport system is inactive. Then, the second dispenser is primed with a portion of the solution capable of gelling. It should be noted that this priming step can be started while the previous priming step is still in progress; the priming of the first dispenser can continue until the priming of the second dispenser is complete.In this case, during the simultaneous implementation of the priming steps, the control unit is configured to simultaneously control the first and second displacement systems to circulate the cell solution in the first dispenser and the gel-ready solution in the second dispenser. The control unit may be designed to define separate flow rate commands for the first and second displacement systems, and in particular, a flow rate command for the second displacement system that is significantly higher than that of the first displacement system, for example, set at least three times, or even four times, the flow rate command of the first displacement system.

[0071] These priming steps synchronize the movement of solutions within the dispensers by initiating this movement upstream of the production phase. This allows for stable flow rates to be achieved at the start of the production phase, despite the length and small cross-section of the dispensers and the variations in solution viscosity. During the production phase, this prevents the formation of bubbles, both in the dispensers and in the encapsulation device, thus avoiding foaming and ensuring uniformity of the cell count within the microcompartments.

[0072] It should be noted that the control unit can be configured to, immediately or subsequently, control the solution handling systems following the priming steps, circulating these solutions through the dispensers to the encapsulation device to implement a production step for a batch of microcellular compartments. If necessary, the control unit can control each of the handling systems so that each solution flows to the encapsulation device at a rate specific to that production step, and in particular distinct from that used during the priming steps.

[0073] Advantageously, the priming steps can be triggered automatically, with each priming step being triggered at a precise time within a predetermined priming sequence and / or after a predetermined duration following the triggering of the previous priming step. Alternatively, the priming steps can be triggered semi-automatically, with the priming sequence being manually triggered by an operator of the encapsulation system, and each priming step in this sequence being triggered automatically from that manual triggering point onward. Alternatively, the priming steps can be triggered manually, with each priming step being manually triggered by an operator of the encapsulation system, for example, following an alert displayed on a screen of the encapsulation system informing them of the end of the previous priming step or an invitation to trigger a new priming step.

[0074] In the present invention, the term "control unit" refers to 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 solution displacement systems. The control unit 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 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 system's operational parameters.It can also be provided that the control unit includes a user interface allowing an operator to monitor production and, if necessary, intervene in it. It can also be provided that the control unit is integrated into a machine containing the containers, dispensers, and encapsulation device, or alternatively, that the control unit is located remotely from this machine but connected to it via wired or wireless means.

[0075] In an embodiment of the invention comprising the third container, the control unit is arranged to, in a first pre-priming step prior to the priming step of the first dispenser, control the first cell solution displacement system to circulate a portion of the cell solution in the first dispenser at least as far as the common portion. Furthermore, in the priming step of the first dispenser, the control unit is arranged to simultaneously control the first cell solution displacement system to circulate a portion of the cell solution in the first dispenser and the third dilution solution displacement system to circulate a portion of the dilution solution in the third dispenser.

[0076] In this example, during the first pre-priming step, the cell solution is moved through the first distributor to the junction with the third distributor, and even beyond into the common section. Then, once the pre-priming step is complete, the control unit can initiate the priming step of the first distributor, including the common section. This priming step is performed with a mixture of cell solution and dilution solution. This pre-priming step prevents the introduction of bubbles into the mixer, which could create areas of cell concentration at air-liquid interfaces.

[0077] In particular, it will be possible to anticipate that, in the priming stage of the first distributor, the control unit will define the same flow rate setpoint for the first and third displacement systems.

[0078] Advantageously, the control unit is arranged to, in a second pre-priming step triggered after the first pre-priming step, control the third dilution solution displacement system to circulate a portion of the cell solution from the third dispenser to the third container. Preferably, in the second pre-priming step, this portion of the cell solution is conveyed to the third container.

[0079] In other words, once the first pre-priming step is complete and the cell solution has been moved to the junction with the third distributor, or beyond into the common section, a portion of this cell solution is pumped back into the third distributor towards the third container. The priming step of the first distributor, including the common section, is thus performed after these pre-priming steps, with the cell solution in the first distributor and a mixture of cell solution and dilution solution in the third distributor. Since it is practically impossible, given the slow flow rate regulation, to synchronize the arrival of the cell and dilution solutions at the common section, these pre-priming steps prevent bubbles from being introduced into the third distributor, the mixer, and therefore the common section.

[0080] The control unit can be configured so that, in the second pre-priming stage, it continues to control the first cell solution displacement system to circulate a portion of the cell solution through the first distributor at least as far as the common section. If necessary, in the second pre-priming stage, the control unit can set a flow rate setpoint for the third displacement system that is significantly lower than that of the first displacement system, for example, 0.8 times the flow rate setpoint of the first displacement system. These features ensure the absence of air bubbles.

[0081] Advantageously, the control unit is arranged to control the third displacement system in the reverse direction of the first distributor's priming step, to circulate the aforementioned portion of the cell solution and the dilution solution through the third distributor to the common portion. Alternatively or cumulatively, a third pre-priming step, triggered after the second pre-priming step, may be provided, in which the control unit is arranged to control the third displacement system in the reverse direction of the second pre-priming step, to circulate the aforementioned portion of the cell solution and at least a portion of the dilution solution through the third distributor to the junction with the first distributor, or even beyond into the common portion.

[0082] In an embodiment of the invention in which a container is provided for holding an intermediate solution, the control unit is arranged to, in a priming step of said distributor triggered prior to the priming step of the first distributor, control said intermediate solution displacement system to circulate a part of said solution in said distributor.

[0083] In this embodiment, the invention aims to successively prime the distributor of the intermediate solution, then the first distributor of the cell solution, and finally the second distributor of the stiffening solution. If necessary, these priming steps may be preceded by pre-priming steps for the first and third distributors to enable priming of the first distributor at the point where it is shared with a mixture of cell solution and dilution solution. These features ensure that the different solutions arrive synchronously at the encapsulation device, or even that the stiffening solution arrives at the encapsulation device after the cell solution, or the cell solution-dilution solution mixture, itself arriving at the encapsulation device after the intermediate solution.

[0084] It should be noted that it is possible to start each priming step while the previous priming step is still in progress, with all priming steps continuing until the last priming step is completed. In this case, during the last priming step, the control unit is configured to simultaneously control all the movement systems to circulate the cell solution in the first dispenser, the gelling solution in the second dispenser, and the intermediate solution in its dispenser. The control unit can be configured to define separate flow rate commands for the movement systems, and in particular, a flow rate command for the intermediate solution movement system that is significantly higher than that of the first movement system, for example, set at least twice the flow rate command of the first movement system.

[0085] Advantageously, the control unit is arranged so that, when the priming step of the first distributor is triggered, it controls the intermediate solution displacement system to decrease the flow rate of said intermediate solution in said distributor.

[0086] In one embodiment of the invention, the encapsulation system comprises a collection tank arranged to collect the cellular microcompartments formed by the encapsulation device during priming steps, and a collection circuit for the cellular microcompartments collected by the tank. This collection circuit is arranged to switch between a collection circuit for the cellular microcompartments to a collection container and a discharge circuit for the collected cellular microcompartments to a waste container. If necessary, the control unit is arranged to switch the collection circuit to the discharge circuit after the priming step of the second dispenser.

[0087] In this example, the initial quantities of solutions, resulting from the priming steps and passing through the encapsulation device, are discharged into the waste container while the flow rates of the various transport systems stabilize. The control unit can be configured to switch the collection circuit to the harvesting circuit after a predetermined delay following the completion of the priming step or after receiving an instruction, for example, following manual input by an operator of the encapsulation system. More specifically, the encapsulation system can include equipment for monitoring the quality of encapsulation by the device, such as a camera filming the microcompartments formed by the device.The operator can thus control, for example, the trajectory of the jet formed by the encapsulation device, and issue an instruction to switch the collection circuit to the harvesting circuit once this trajectory seems satisfactory.

[0088] In one embodiment of the invention, the collection tank may be provided to include an outlet for withdrawing the stiffening solution and the cellular microcompartments from the collection tank to the collection circuit; and the encapsulation system may include: 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 suitable for receiving the stiffening solution collected by the collection circuit and arranged to separate and retain the cellular microcompartments of this solution; a harvesting circuit connected to the second separation module, to a container containing a second solution and to a harvesting container; and means for moving the second solution in the harvesting circuit.

[0089] In the present invention, the term "separation module" means a device capable of receiving a solution containing suspended particles, and capable of separating and retaining the particles from this solution while allowing the solution to flow through. By way of non-limiting example, this could include a membrane filter, a centrifugal filtration system, a tangential flow filtration system, or a decanter.

[0090] 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.

[0091] Based on these characteristics, a buffer stage is interposed between the collection tank and the harvesting stage. The valve then allows the buffer stage to be isolated from the harvesting stage, enabling simultaneous collection from the collection tank to the buffer stage and harvesting with a change of medium in the harvesting stage, or conversely, allowing simultaneous transfer from the buffer stage and from the collection tank, via the buffer stage, to the harvesting stage.

[0092] Thus, depending on the state of this valve, the means of movement within the collection circuit allow the transfer of the cellular microcompartments from the collection tank and the stiffening solution to the first separation module, and conversely, the transfer of the cellular microcompartments retained by the first separation module to the harvesting stage. For example, it could be possible for these cellular microcompartments to follow opposite paths within the same section of the collection circuit for each of these transfers, or for the collection circuit to have distinct sections, each dedicated to one of these transfers.

[0093] The means of movement allow the second solution contained in the container to be transferred in the harvesting circuit to the second separation module, and then this second solution and the microcompartments retained by the second separation module to be transferred to the harvesting container, while carrying out a change of environment of these microcompartments using a second solution, more suitable for harvesting, than the stiffening solution.

[0094] The various elements of the system can thus be controlled by the control unit to define different phases during the production of a batch of microcompartments. Specifically, the collection circuit and / or the harvesting circuit can be equipped with one or more valves controllable by the control unit to define, within this circuit or these circuits, preferred paths according to given phases of a production cycle for a batch of microcompartments.

[0095] For example, during the collection phase, the valve is closed, and the buffer stage can collect, via its separation module, microcompartments continuously generated by the collection tank. Simultaneously, the microcompartments contained in the separation module of the collection stage are discharged to the collection container via the second solution, thus effecting a change of medium. During the transfer phase, the valve is opened, and the microcompartments generated by the collection tank, as well as those contained in the separation module of the buffer stage, are transferred to the separation module of the collection stage via the recycling solution.

[0096] In other words, these characteristics therefore make it possible to ensure discontinuous filtration of the microcompartments, thanks to which it is possible to ensure continuity in the production of microcompartments by the collection tank while allowing, in a discontinuous way, a change of medium of the microcompartments thus produced.

[0097] In one embodiment of the invention, the encapsulation system includes at least one element capable of electrically charging at least one of the solutions with an electrical potential, and the control unit is arranged to, at the end of the priming step of the second distributor, control said element to electrically charge said solution with the electrical potential.

[0098] This electrical charging step of the solution can be triggered immediately after the priming step of the second distributor, after a delay following the end of the priming step of the second distributor or after an instruction received, for example following a manual entry of said instruction by an operator of the encapsulation system.

[0099] In an embodiment of the invention incorporating liquid presence sensors, during a verification step prior to the priming step of the first dispenser, and in particular prior to the pre-priming steps, the control unit is arranged to control each solution displacement system to circulate a portion of said solution through the associated dispenser to the liquid presence sensor. These steps ensure that all solutions are available for the priming steps, and where applicable, the pre-priming steps, and thus trigger these different steps if the various sensors confirm the presence of these solutions.

[0100] The invention also relates to a cell encapsulation system, the system comprising at least: a. two containers, the first of which is intended to contain a cell solution and the second of which is intended to contain 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, Each of the first and second containers is connected to an input of the encapsulation device by a first, respectively a second distributor associated with a first, respectively a second solution displacement system capable of causing a flow of the solution, contained in said container, in said distributor towards the input of the encapsulation device. characterized in that it comprises a control unit arranged for, prior to a production step of a batch of cellular microcompartments: c. in a priming step of the first dispenser, control the first cell solution displacement system to circulate a portion of the cell solution into the first dispenser; d. in a priming step of the second dispenser triggered later than the priming step of the first dispenser, control the second system for moving the solution capable of gelling to circulate a portion of said solution into the second dispenser.

[0101] The invention also relates to a method for priming a cell encapsulation system according to the invention.

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

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

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

[0105] [Fig. 2] represents, schematically and partially, a priming sequence of the input stage of the system of [Fig. 1];

[0106] [Fig. 3A] represents, schematically and partially, a view of the system of [Fig. 1] in a first step of the priming sequence of [Fig. 2];

[0107] [Fig. 3B] represents, schematically and partially, a view of the system of [Fig. 1] in a second step of the priming sequence of [Fig. 2];

[0108] [Fig. 3C] represents, schematically and partially, a view of the system of [Fig. 1] in a third step of the priming sequence of [Fig. 2];

[0109] [Fig. 3D] represents, schematically and partially, a view of the system of [Fig. 1] in a fourth step of the priming sequence of [Fig. 2];

[0110] In the description that follows, identical elements, by structure or by function, appearing on different figures retain, unless otherwise specified, the same references.

[0111] Figure 1 shows a cell encapsulation system according to one embodiment of the invention.

[0112] System 1 comprises four containers, 11 to 14. The first container, 11, contains a solution containing a plurality of human pluripotent stem cells. The second container, 12, contains a gel-forming solution, for example, a hydrogel such as alginate. The third container, 13, contains a diluent solution such as a culture medium and / or an extracellular matrix and / or an extracellular matrix substitute and / or an aqueous solution. The fourth container, 14, contains an intermediate solution, for example, an isotonic solution such as sorbitol.

[0113] In the example described, each of the containers 11 to 14 is formed by a flexible, sealed, and sterile pouch. Other types of containers, designed to contain, store, or transport a solution in a controlled medical environment, could be used without departing from the scope of the present invention.

[0114] System 1 also includes an encapsulation device 20 arranged to form, from the solutions in containers 11 to 14, cellular microcompartments, each organized in the form of a cyst. Each microcompartment thus comprises a central lumen, preferably aqueous, a core formed by the cell solution and the dilution solution, an intermediate layer formed by the intermediate solution, and an outer layer formed by the stiffening solution. The core, intermediate layer, and outer layer are successively arranged three-dimensionally around said lumen. Each cellular microcompartment obtained by means of the encapsulation device 20 may be spherical or teardrop-shaped, or alternatively, elongated, in particular ovoid or tubular.Cellular tissues can be obtained from cells encapsulated in one or more three-dimensional closed microcompartments, obtained from encapsulation system 1, after being cultured in a culture medium in a closed chamber, in particular a bioreactor.

[0115] In the example shown in [Fig. 1], the encapsulation device 20 is a microfluidic device comprising a body, including the inlets, and a nozzle connected to a single outlet on the body, forming a single outlet for the device 20. The body and nozzle may be made of glass or another material suitable for the pharmaceutical industry. The body and nozzle may be a single component, or alternatively, they may be manufactured separately and then assembled to form the encapsulation device 20.

[0116] In the example described, the body comprises a main channel including a substantially straight portion defining a central axis of the encapsulation device 20. This main channel connects a first inlet, intended to receive a mixture of the cell solution and the dilution solution, to the single outlet of the body. The body includes secondary channels connecting, respectively, a second inlet, intended to receive the intermediate solution, and a third inlet, intended to receive the stiffening solution, to the single outlet. Each secondary channel is subdivided into portions extending around the main channel, these subdivisions of each secondary channel converging at the single outlet of the body into a single circular portion, concentric with the main channel. The single circular portions and the main channel then merge to form the single outlet of the body.

[0117] In other words, the body allows the formation of a concentric flow from the solutions contained in containers 11 to 14, an external flow being formed by the solution capable of gelling, an intermediate flow being formed by the intermediate solution and an internal flow being formed by a mixture of the cell solution and the dilution solution.

[0118] In order to transport the solution contained in each container 11 to 14 to the dedicated inlet of the encapsulation device 20, the system 1 includes distributors 31 to 34, each distributor 31 to 34 connecting the container 11 to 14 to which it is associated with the inlet of the encapsulation device 20 dedicated to the solution contained in that container 11 to 14.

[0119] In the example described, each dispenser 31 to 34 is formed by a flexible tube. It is possible to use other types of containers designed to convey, guide, or transfer a solution in a controlled medical environment, and in particular to maintain sterility and prevent contamination during the circulation of the solution, without departing from the scope of the present invention.

[0120] For each container 11 to 14, the system 1 includes a solution displacement system 41 to 44, capable of causing a flow of the solution, contained in this container 11 to 14, in the distributor 31 to 34 associated with this container towards the inlet of the encapsulation device 20 dedicated to the solution contained in this container 11 to 14.

[0121] In the example described, each displacement system 41 to 44 includes a peristaltic pump. Other types of displacement systems, designed to generate, control, or facilitate the circulation of a solution contained in a container within a dispenser, may be used without departing from the scope of the present invention.

[0122] As shown in [Fig. 1], the distributors 31 and 33 join, using a T-connector, in a common portion 35 connected to the inlet of the encapsulation device 20 dedicated to the mixing of cell solution and dilution solution contained in containers 11 and 13.

[0123] The common portion 35 includes a mixer 35a, for mixing the cell solution and the dilution solution. In the example described, the mixer 35a is a linear passive mixer. Other types of mixers, designed to combine, homogenize, or mix two or more solutions, particularly a cell solution and a dilution solution, may be used in a controlled medical environment without departing from the scope of the present invention.

[0124] In the example described, in order to be able to evacuate any bubbles present in the solutions, each distributor 31 to 34 has at least one portion extending upwards.

[0125] More specifically, each container 11 to 14 is arranged so that the link of this container 11 to 14 to its distributor 31 to 34 is located substantially below the inlet linking this distributor 31 to 34 to the encapsulation device 20.

[0126] In addition, each pump 41 to 44 is arranged on a substantially vertical portion of the distributor 11 to 14 to which it is associated, each distributor 11 to 14 extending substantially only upwards and / or horizontally from this pump 41 to 44 to the inlet of the encapsulation device 20 to which it is connected.

[0127] Each solution then flows through its designated dispenser 31 to 34 from its container 11 to 14 to its inlet of the encapsulation device 20, generally moving upwards. This allows for the removal of any bubbles present in a solution and prevents bubbles from stagnating in dispensers 31 to 34, which could destabilize the flow of these solutions and / or cause cell accumulation at the bubble sites.

[0128] It should also be noted that mixer 35a extends obliquely, so that its outlet is positioned at a higher height than its inlet, which allows the cell solution and the dilution solution to be mixed by circulating them upwards, thus allowing the bubbles from mixer 35a to be removed.

[0129] In the example described, the distributor 31 includes a portion 311, provided between the pump 41 and the junction with the distributor 33, whose internal diameter is reduced compared to the rest of the distributor 31. This portion with a reduced cross-section 311 helps to smooth out flow oscillations that may be introduced by the pump 41.

[0130] Each dispenser 31 to 34 is equipped with a liquid presence sensor 51 to 54. Each sensor 51 to 54 is positioned downstream of the container 11 to 14 associated with the dispenser 31 to 34 and upstream of the solution displacement system 41 to 44.

[0131] In addition, the encapsulation system 1 includes a cooling unit 6 for cooling a cooling zone extending from a point on the first distributor 31 located downstream of the sensor 51 to a point on the common part 35 located downstream of the junction of the distributors 31 and 33.

[0132] Furthermore, the dispenser 32, intended for dispensing the alginate solution, is equipped, in the described example, with a component (not shown) 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. As a further alternative, the solution charged with said electrical potential may be the cell solution and / or the dilution solution and / or the intermediate solution.

[0133] In order to carry out a production cycle of a batch of cell microcompartments, the system 1 includes a control unit 4 capable of controlling the different elements of the encapsulation system and in particular the displacement systems 41 to 44. 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 41 to 44, in order to implement phases of a production cycle of a batch of cell microcompartments.

[0134] In the example described, the control unit 4 can receive data from system sensors, such as data from flow sensors of the solutions in the dispensers 31 to 34. The control unit can thus execute one or more control programs of the execution of the production cycle capable of interpreting this data in real time, and of adjusting the operational parameters of the system, such as the flow of the solutions from the containers 11 to 14 according to this interpretation, or even of stopping the production cycle.

[0135] It can also be foreseen that the control unit includes a user interface (not shown), such as a screen and a keyboard, allowing an operator to monitor the data from the sensors and / or the progress of the different phases of the production cycle, and to intervene in this production cycle, by manually modifying the operational parameters of the system or by interrupting it.

[0136] In the example described, all the different elements shown in [Fig. 1], including the control unit 4, form a single machine. In another example, the control unit 4 could be located remotely and communicate with the other system elements via wireless or wired communication.

[0137] In connection with [Fig. 2], we will now describe a priming sequence of the encapsulation system 1, implemented by the control unit 4 prior to a production cycle of a batch of cell microcompartments, and in the different solutions contained in the containers 11 to 14 are moved in the distributors 31 to 34 to the inlets of the encapsulation device 20 which are dedicated to them.

[0138] In a verification step 11, the control unit 4 controls each solution movement system 41 to 44 to circulate a portion of the solution contained in each container 11 to 14 through the associated dispenser 31 to 34 to the liquid presence sensor 51 to 54 of that dispenser. The detection of the solution by each presence sensor 51 to 54 then triggers the interruption of the corresponding movement system 41 to 44.

[0139] Figure 3A shows a view of system 1 at the end of this step El, with the progress of the solutions in distributors 31 to 34 shown in black lines.

[0140] Control unit 4 can thus confirm that all elements of system 1 are functional and that the boot sequence can continue. An alert regarding the proper functioning, or conversely, a failure of one of the elements of system 1, can be displayed on the user interface based on these detections.

[0141] In a first pre-priming step E21, following step El, the control unit 4 controls the cell solution displacement system 41 to circulate a portion of this cell solution in the distributor 31 to the common portion 35, beyond the junction of distributors 31 and 33. During this step E21, the other displacement systems 42 to 44 remain inactive.

[0142] Figure 3B shows a view of system 1 at the end of this step E21.

[0143] Then, in a second pre-priming step E22 triggered at the end of the first pre-priming step E21, the control unit 4 controls the displacement system 43 to circulate part of the cell solution in the dispenser 33 from the T-junction to the dilution solution container 13.

[0144] To keep the distributor 31 primed, in the second pre-priming step E22, the control unit 4 continues to control the displacement system 41 to circulate the cell solution through the distributor 31 to the T-junction. The control unit 4 then sets a flow rate setpoint for the displacement system 43 that is significantly lower than that of the displacement system 41, for example, set to 0.8 times the flow rate setpoint of the displacement system 41. This prevents air from entering the distributor 33.

[0145] Figure 3C shows a view of system 1 at the end of this step E22.

[0146] At the end of the second pre-priming step E22, the distributors 31 and 33 are pre-primed with the cell solution, part of this cell solution having been pumped into the distributor 33 from the T-junction to the container 13. This prevents air bubbles from being present in either of these distributors 31 and 33 and prevents air from being conveyed to the common portion 35 and its mixer 35a.

[0147] In steps E31, E32 and E33, the distributor 34, the common portion 35 and the distributor 32 are successively primed with respectively the intermediate solution from container 14, a mixture of cell solution and dilution solution from containers 11 and 13, and the stiffening solution from container 12.

[0148] More specifically, in step E31, control unit 4 controls the intermediate solution's displacement system 44 to circulate a portion of said solution from container 14 in dispenser 34 to the inlet of encapsulation device 20 dedicated to that solution. In this step E31, the other displacement systems 41, 42, and 43 remain inactive.

[0149] After a given delay, in a second step E32, the control unit 4 simultaneously controls the displacement system 41 to circulate part of the cell solution into the first dispenser 31 and the dilution solution displacement system 43 to circulate part of the dilution solution into the dispenser 33, these solutions being mixed in the mixer 35a and then circulating in the common portion 35 to the inlet of the encapsulation device 20 dedicated to this mixing of solutions.

[0150] In this step E32, the displacement system 42 remains inactive while the control unit 4 sets an identical flow setpoint for the displacement systems 41 and 43 and decreases the flow setpoint for the displacement system 42, for example by setting it to 2 times the flow setpoint of the displacement systems 41 and 43. If the flow setpoint of the displacement system 42 can be high in the priming step E31, in order to avoid flooding the encapsulation device, it can indeed be reduced during the priming step E32 in order to stabilize it.

[0151] It should be noted that, in this step 32, the displacement system 43 is controlled by the control unit 4 in the opposite direction to that of the second pre-priming step E22, to circulate the cell solution in the distributor 33 and then the dilution solution towards the common portion 35.

[0152] Finally, after a given delay, in a step E33, the control unit 4 controls the displacement system 42 of the solution suitable for gelling to circulate a part of said solution from the container 12 in the dispenser 32 to the inlet of the encapsulation device 20 dedicated to this solution.

[0153] In this step E33, the flow setpoints for the displacement systems 41, 43 and 44 remain identical, the control unit 4 setting for example a flow setpoint for the displacement system 42 at least 3 times, or even 4 times, the flow setpoint of the displacement systems 41 and 43.

[0154] Figure 3D shows a view of system 1 at the end of step E33.

[0155] According to these steps E31 to E33, the different solutions reach the encapsulation device 20 synchronously, thus preventing the stagnation of air bubbles in the distributors 31 to 34 and ensuring the stability of the encapsulation performed by the encapsulation device. Furthermore, the intervals between these steps allow stable pressures, and therefore stable flow rates, to be achieved within the distributors 31 to 34 as they are triggered. This prevents the formation of bubbles, both in the distributors and in the encapsulation device, despite the length and small cross-section of the distributors and the differences in solution viscosity.

[0156] It is conceivable that the priming steps 31 to 33 are triggered sequentially and automatically, each priming step E31 to E33 being triggered at a precise moment in a predetermined priming sequence and / or after a predetermined duration following the triggering of the preceding priming step. Alternatively, the priming steps E31 to E33 can be triggered semi-automatically, with the priming sequence being manually triggered by an operator of the encapsulation system via the user interface, and the priming steps E31 to E33 being triggered automatically, one after the other, starting from this manual triggering.Alternatively, the priming steps E31 to E33 can be triggered manually, with each priming step being manually initiated by an operator of the encapsulation system via the user interface, for example, following an alert displayed on a screen of the encapsulation system informing them of the completion of the previous priming step or a prompt to initiate a new priming step. According to this alternative, each distributor 31 to 34 can be equipped with a pressure sensor enabling the control unit 4 to detect the completion of each priming step E31 to E33.

[0157] With further reference to [Fig. 1], the system also includes a collection tank 21 arranged under the encapsulation device 20.

[0158] Following step E33, the control unit 4 continues to control the displacement systems 41 to 44 at the same flow rates. The solutions thus enter the encapsulation device 20 via the dedicated inlets, and the nozzle of the encapsulation device 20 receives the concentric flow formed by the body of the encapsulation device from these different solutions. Given the flow rates of the solutions and the electrostatic force generated by the electrical charges carried by the alginate solution, the encapsulation device 20 generates a concentric jet from the concentric flow at the nozzle outlet. This concentric jet is fragmented, due to Plateau-Rayleigh instability, into cellular microcompartments, the outer layer of which is the alginate solution and the core the cell solution.

[0159] The encapsulation device 20 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 solutions, 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.

[0160] As an alternative, it may be possible to size the flow rates of the solutions as well as the electrical potential so that the encapsulation device 20 is of the "electro-dripping" type, and thus forms the microcompartments one after the other directly from the nozzle.

[0161] A metallic ring, connected to ground and extending downstream of the encapsulation device 20's outlet, can be added so that the jet or cellular microcompartments pass through this ring. The electric field generated by this metallic ring promotes the dispersion of the cellular microcompartments, in the case of an "electro-jetting" type device.

[0162] The collection tank 21 contains a stiffening solution designed to collect the cell microcompartments formed by the encapsulation device 20 and falling by gravity. The stiffening solution comprises a surfactant and a calcium salt that cross-link the alginate solution, thereby causing the outer layer of each cell microcompartment to stiffen upon immersion in the collection tank.

[0163] The collection tank 21 includes a withdrawal outlet 22 for the stiffening solution and microcompartments immersed in this solution.

[0164] In the example described, the assembly represented in [Fig. 1], namely the containers 11 to 14, the dispensers 31 to 34, the displacement systems 41 to 44, the encapsulation device 20 and the collection tank 21, forms a first stage IA of generation of the cellular microcompartments of the encapsulation system 1.

[0165] The encapsulation system 1 includes a second stage IB, called the buffer stage, connected to the first stage 1 via the withdrawal outlet 22 of the collection tank 21, and a third stage IC, called the harvesting stage, connected to the second stage IB.

[0166] The second buffer stage IB includes a collection circuit connected to the withdrawal outlet 22 of the collection tank 21, allowing the stiffening solution and microcompartments to be withdrawn from the collection tank 21.

[0167] A first separation module (not shown) is positioned in the collection circuit to receive the stiffening solution and the microcompartments drawn from the collection tank 21. This first separation module is arranged to allow the passage of a solution and to prevent the passage of solid objects.

[0168] The third harvesting stage IC includes a harvesting circuit connected on one side to the buffer stage IB, and on the other side to various containers, including a harvesting container 71 intended to receive the microcompartments and a waste container 72. Different valves are positioned in the harvesting circuit to define different configurations of the harvesting circuit, including a configuration in which a solution can flow from an inlet of the third stage IC connecting it to the second stage IB towards the waste container 72 and a configuration in which a solution can flow towards the harvesting container 71.

[0169] A second separation module is positioned in the harvesting circuit of the third stage IC.

[0170] Finally, system 1 includes a valve 73 capable of allowing or preventing the passage of a solution from the collection circuit of the second stage IB to the collection circuit of the third stage IC.

[0171] With further reference to [Fig. 2], and in a step E41 implemented at the end of the priming step E33, the control unit 4 switches the valve 73 and the various circuits of stages IB and IC so that the stiffening solution flows in these circuits towards the waste container 72. The contents of the tank 21, resulting from the priming steps E31 to E33, are thus evacuated towards the waste container 72 while the flow rates of the various displacement systems are stabilized and a quality control of the jet formed by the encapsulation device 20 can be carried out.

[0172] In a step E42, following these various checks, the control unit can then control the charging element to electrically charge the solution suitable for stiffening with the electrical potential and the valve 73 and the various circuits of the stages IB and IC so that the stages IB and IC can collect and harvest cellular microcompartments in a later phase of a production cycle.

[0173] The preceding description clearly explains how the invention achieves its objectives, namely, to provide a cell encapsulation system in which the various solutions move synchronously through the tubes to the encapsulation device. This is particularly important to prevent the formation of bubbles in the tubes and the encapsulation device, which would create areas of cell concentration and foaming. It is understood that these objectives are achieved through various priming steps that synchronize the movement of the solutions in the dispensers. This initiation of movement occurs upstream of the production phase, thus ensuring stable flow rates at the start of this production phase.

[0174] 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. A cell encapsulation system (1), the system comprising at least: a. two containers (11, 12), the first of the containers (11) being intended to contain a cell solution and the second of the containers (12) being intended to contain a solution capable of gelling, b. a milli-fluidic or micro-fluidic encapsulation device (20) 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, in which each of the first and second containers is connected to an inlet of the encapsulation device by a first, respectively a second distributor (31, 32) associated with a first, respectively a second solution displacement system (41, 42) capable of causing a flow of the solution, contained in said container (11, 12), in said distributor towards the inlet of the encapsulation device (20), characterized in that the first distributor (31) has less than one portion extending upwards.

2. System (1) according to the preceding claim, characterized in that it comprises a third container (13) for receiving a dilution solution, the third container being connected to a third distributor (33) associated with a third solution displacement system (43) capable of causing a flow of the dilution solution into the third distributor; and in that the first and third distributors join in a common portion (35) connected to the inlet of the encapsulation device (20).

3. System (1) according to the preceding claim, characterized in that the first and third solution displacement systems (41, 43) are arranged on a portion of the first, respectively the third, distributor (31, 33) extending between the first, respectively the third container, and the junction between the first and third distributors, said portions being substantially vertical.

4. System (1) according to the preceding claim, characterized in that the first distributor (31) extends substantially only upwards and / or horizontally to the common portion (35).

5. System (1) according to any one of claims 2 to 4, characterized in that the first distributor (31) comprises a reduced cross-section portion (311) upstream of the junction between the first and third distributors (31, 33).

6. System (1) according to any one of claims 2 to 5, characterized in that the common portion (1) comprises a mixer (35a).

7. System (1) according to the preceding claim, characterized in that the mixer (35a) is arranged such that the outlet of the mixer on the common portion (35) extends at least to the same height as the inlet of the mixer on the common portion.

8. System (1) according to the preceding claim, characterized in that the mixer (35a) is a linear passive type mixer, at least a portion of the mixer extending upwards.

9. System (1) according to the preceding claim, characterized in that the mixer (35a) extends obliquely.

10. System (1) according to any one of claims 2 to 9, characterized in that it comprises a cooling unit (6) arranged opposite a part of the third distributor (33) and / or the third container (13).

11. System according to any one of the preceding claims, characterized in that each distributor (31, 32, 33, 34) is equipped with a liquid presence sensor (51, 52, 53, 54) in the distributor, positioned downstream of the container (11, 12, 13, 14) and upstream of the inlet of the encapsulation device (20) connected by this distributor.

12. System (1) according to any one of the preceding claims, characterized in that it comprises a container (14) intended to contain an intermediate solution, said container being connected to an inlet of the encapsulation device (20) by a distributor (34) associated with a solution displacement system (44) capable of causing a flow of the intermediate solution in said distributor towards the inlet of the encapsulation device (20).

13. System (1) according to any one of the preceding claims, characterized in that it comprises at least one element capable of electrically charging at least one of the solutions with an electrical potential.

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